Cell Adhesion and Migration in Skin Disease (J Barker)

CELL ADHESION AND MIGRATION IN SKIN
DISEASE
Cell Adhesion and Communication
A series of books encompassing monographs on classes of adhesion molecules and
monographs giving a broader functional synopsis on adhesion molecules of a particular
system.
Edited by Steven Pals
Volume 1
Cell Adhesion Molecules in Cancer and Inflammation
edited by A.A.Epenetos and M.Pignatelli
Volume 2
The Laminins
edited by P.Ekblom and R.Timpl
Volume 3
Tenascin and Counteradhesive Molecules of the Extracellular Matrix
edited by K.L.Crossin
Volume 4
Adhesion Molecules and Chemokines in Lymphocyte Trafficking
edited by A.Hamann
Volume 5
Cell Adhesion and Communication Mediated by the CEA Family Basic and Clinical
Perspectives
edited by C.P.Stanners
Volume 6
Ig Superfamily Molecules in the Nervous System
edited by P.Sonderegger
Volume 7
Epithelial Morphogenesis in Development and Disease
edited by W.Birchmeier and C.Birchmeier
Volume 8
Cell Adhesion and Migration in Skin Disease
edited by J.Barker and J.McGrath
This book is part of a series. The publisher will accept continuation orders which may
be cancelled at any time and which provide for automatic billing and shipping of each title
in the series upon publication. Please write for details.
CELL ADHESION AND
MIGRATION IN SKIN
DISEASE
Edited by
Jonathan Barker
and
John McGrath
St.Thomas’ Hospital, London, UK
harwood academic publishers

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British Library Cataloguing in Publication Data
Cell adhesion and migration in skin disease.—(Cell adhesion and
commnunication; v. 8)
1. Skin—Diseases—Cytopathology 2. Cell adhesion 3. Cell adhesion—
Molecular aspects
I.Barker, J. (Jonathan) II. McGrath, John
616.5′07
ISBN 0-203-30459-4 Master e-book ISBN
ISBN 0-203-34336-0 (Adobe eReader Format)

ISBN: 90-5823-067-8 (Print Edition)
ISSN: 1023–7046
CONTENTS
Preface to the Series vii

Preface ix
Contributors x
1. Introduction 1
John A.McGrath
CELL-CELL ATTACHMENT
2. The Cornified Cell Envelope 8
Jorge Frank and Angela M.Christiano
3. Keratin and Keratin Disorders 26
Laura D.Corden and W.H.Irwin McLean
4. Desmosomes 56
David R.Garrod, Martyn A.J.Chidgey, Alison J.North, Sarah K.Runswick and
Chris Tselepis
CELL-MATRIX ATTACHMENT
5. Protein-Protein Interactions at the Dermal-Epidermal BMZ 88
M.Peter Marinkovich
6. Biology and Pathology of Hemidesmosomes 106
Leena Pulkkinen and jouni Uitto
7. Dermal-Epidermal Adhesion 131
Leena Bruckner-Tuderman
LEUKOCYTE TRAFFICKING IN SKIN DISEASES

8. Introduction 164
Jonathan N.W.N.Barker
9. Skin Homing Lymphocytes 170
Conrad Hauser and René Moser
vi
10. T-cell Accessory Molecules 202

Ralf W.Denfeld and Jan C.Simon
11. Animal Models of Skin Inflammation 221
Benjamin E.Rich and Thomas S.Kupper
12. Langerhans Cell Migration 241
Georg Stingl and Dieter Maurer
13. Leukocyte Adhesion and Accessory Molecules as Therapeutic Targets for 251
Inflammatory Skin Diseases
Kimberly E.Foreman and Brian J.Nickoloff
Index 264
PREFACE TO THE SERIES
The development and normal functioning of all multicellular organisms is governed to a

large part by the interactions cells undergo with neighbouring cells-and with their
acellular environment. Many of these interactions are mediated by cell-cell adhesion
molecules and by extracellular matrix components and their cellular receptors, that is by
molecules which establish direct cell-cell and cell-matrix contacts. These molecules are
particularly important for determining whether a cell remains where it is or moves
elsewhere and, if a cell moves, where it goes and when it stops migrating. These ness and
metastasis. Moreover, recent advances in the field show that most, if are of course key
events during normal development, but they play equally crucial roles in adult physiology
and pathology, such as the extravasation of white blood cells, inflammatory processes and
wound healing, tumour invasive-not all, cell adhesion molecules are capable of triggering
intracellular events, in the same way as diffusible growth and differentiation factors and their
cellular receptors do. It is thus hardly surprising that clinicians are devoting increasing
attention to the molecular mechanisms underlying cell adhesion, and that cell adhesion
molecules are not being considered as suitable targets for drug development.
This book series is aimed at scientists, both in academia and in industry, at graduate
students planning to move into the area, at the clinician, who wants to become familiar
with a field with many clinical implications, and at scientists already working in the field,
who want to keep abreast with the recent developments outside their own speciality. Hence,
each volume of the series reviews a particular segment of the field and provides a critical
assessment of recent discoveries and future developments. Each volume has a volume
editor, who is an expert in the field and invites contributors to cover the different aspects
of the topic. By keeping the number of contributors to each volume small, we hope to avoid
overlaps and redundancies, common pitfalls of multi-author volumes. By looking at the
previous volumes, I have the impression that we have been successful.
Previous volumes of the series addressed the role of cell adhesion in selected
physiological and pathological phenomena or concentrated on important structural
families. The volumes on Cell Adhesion Molecules in Cancer and Inflammation and on Adhesion
Molecules and Chemokines in Lymphocyte Trafficking are examples of the first, those on The
Laminins and on the CEA Family are examples of the second kind.
Whilst the previous volume, Epithelial Morphogenesis in Development and Disease, focused
on the epithelia, whose development and integrity relies heavily on cell adhesion
phenomena, this volume addresses this crucial phenomena in an especially important
viii
epithelium, the epidermis of the skin. Together the two books will constitute an
invaluable source of information for scientists and clinicians interested in skin biology and
diseases.
PREFACE
Cell adhesion and migration play a critical role in many cutaneous diseases. These include
blistering disorders both inherited and acquired, and inflammatory conditions, such as
eczema and psoriasis. In recent years, considerable advances have been made in our
understanding of the molecular basis of cell adhesion and migration in the skin and the
precise role of many adhesive proteins have been defined.
In this book, we bring together a series of articles written by international experts,
describing cell-cell, and cell-matrix adhesion and the biological processes involved in
maintaining integrity of the skin and in the orchestration of inflammatory events.
In the first part of the book, the molecular mechanisms involved in epidermal cell-cell
attachment and keratinocyte structural integrity are discussed in detail. Emphasis is placed
on the cornified cell envelope, intermediate filaments and desmosomes. Abnormalities of
these structures, for example as a result of genetic mutation or disruption by
autoantibodies, lead to specific blistering skin diseases such as epidermolysis bullosa
simplex or pemphigus.
In the second part of the book, the molecular basis for securing adhesion between the
epidermis and the dermis via the intricate network of macromolecules that comprise the
cutaneous basement membrane zone is discussed. The importance of perturbations in
these adhesion complexes is highlighted by diseases such as junctional epidermolysis
bullosa, resulting from inherited abnormalities in genes encoding structural components of
hemidesmosomes and anchoring filaments and bullous pemphigoid resulting from
autoimmune assault against a hemidesmosomal collagen.
In the third part of the book, the molecular events responsible for accumulation of
inflammatory cells into skin are discussed. Leukocyte adhesion molecules play a critical
role in the control of lymphocyte recruitment into skin and as such are attractive targets
for therapeutic intervention in designing new strategies to combat common inflammatory
skin diseases including psoriasis and eczema. Indeed, clinical trials are presently under way
using biotechnological approaches to anti-leukocyte adhesion molecule therapy and the
rationale for such studies is discussed.
It is hoped that the high quality chapters from international experts and the layout of
this book will appeal to basic scientists and clinicians working in skin biology. Further we
believe the book will also be of invaluable assistance to industry by providing information
on important molecular targets in bullous and inflammatory skin diseases.
CONTRIBUTORS
Jonathan N.W.N. Barker

St. John’s Institute of Dermatology
St. Thomas’ Hospital
Lambeth Palace Road
London SE1 7EH
UK
Leena Bruckner-Tuderman
Department of Dermatology
University of Münster
Von-Esmarch-Str. 56
48149 Münster
Germany
Martyn A.J.Chidgey
School of Biological Sciences
3.239 Stopford Building
University of Manchester
Oxford Road
Manchester M13 9PT
UK
Angela M.Christiano
College of Physicians and Surgeons
Columbia University
630 West 168th Street, VC-1526
New York, NY 10032
USA
Laura D.Corden
Epithelial Genetics Group
Department of Molecular and
Cellular Pathology
Ninewells Medical School
University of Dundee
xi
Dundee DD1 9SY

UK
Ralf W. Denfeld
Albert-Ludwigs-Universität
Freiburg
Germany
Kimberly E.Foreman
Department of Pathology
Skin Cancer Research Laboratories
Cardinal Bernardin Cancer Center
Loyola University Medical Centre
Maywood, IL
USA
Jorge Frank
College of Physicians and Surgeons
Columbia University
630 West 168th Street, VC-1526
New York, NY 10032
USA
David R.Garrod
Oxford Road
Manchester M13 9PT
UK
Conrad Hauser
Allergy Unit
Division of Immunology and Allergy, and Department of Dermatology
(DHURDV)
University Hospitals
1211 Geneva 14
Switzerland
Thomas S.Kupper
Division of Dermatology
Harvard Skin Disease Research Center
Brigham and Women’s Hospital
Boston, MA 02115
USA
M.Peter Marinkovich
xii
MSLS Building
Rm. p208
Stanford University School of Medicine
Stanford, CA 94305
USA
Dieter Maurer
Division of Immunology, Allergy and Infectious Diseases (DIAID)
University of Vienna Medical School
Vienna
Austria
John A.McGrath
St. John’s Institute of Dermatology
St. Thomas’ Hospital
Lambeth Palace Road
London, SE1 7EH
UK
W.H.Irwin McLean
Epithelial Genetics Group
Department of Molecular and
Cellular Pathology
Ninewells Medical School
University of Dundee
Dundee DDI 9SY
UK
René Moser
Institute for Biopharmaceutical
Research Inc.
9545 Waengi
Switzerland
Brian J.Nickoloff
Department of Pathology
Skin Cancer Research Laboratories
Cardinal Bernardin Cancer Center
Loyola University Medical Centre
Maywood, IL
USA
Alison J.North
Oxford Road
Manchester M13 9PT
xiii
UK
Leena Pulkkinen
Department of Biochemistry and
Molecular Pharmacology
Thomas Jefferson University
Philadelphia, PA 19107
USA
Benjamin E.Rich
Division of Dermatology
Harvard Skin Disease Research Center
Brigham and Women’s Hospital
Boston, MA 02115
USA
Sarah K.Runswick
Oxford Road
Manchester M13 9PT
UK
Jan C.Simon
Albert-Ludwigs-Universität Freiburg
Germany
Georg Stingl
Division of Immunology, Allergy and
Infectious Diseases (DIAID)
University of Vienna Medical School Vienna
Austria
Chris Tselepis
Oxford Road
Manchester M13 9PT
UK
Jouni Uitto
Department of Dermatology and
Cutaneous Biology
xiv
Jefferson Medical College

Thomas Jefferson University
Philadelphia, PA 19107
USA
1.
INTRODUCTION
JOHN A.McGRATH
One of the main functions of human skin is to act as a protective barrier against the external
environment. Subcutaneous fat and dermal connective tissue help cushion and disperse
mechanical trauma, but the actual barrier principally depends on the integrity of the
epidermis, including the stratum corneum, as well as a complex network of adhesive
junctions that link adjacent keratinocytes to one another as well as basal keratinocytes to
the underlying dermis.
Cell adhesion in skin involves contributions from several structural proteins,
glycoproteins and lipids. Many of these are organised into specific adhesion complexes,
the main types of which consist of hemidesmosomes (linking basal keratinocytes to cell
matrix) and desmosomes (adhesion between adjacent keratinocytes) (Figure 1.1). Other
epithelial anchorage sites include focal contact complexes, adherens junctions, gap
junctions and tight junctions. Each of these is composed of a dense network of assorted
structural macromolecules specific to each type of junction. Although these junctional
components have a predominant role in securing cell adhesion, their function is by no
means static. Many proteins or glycoproteins are redistributed or differentially expressed
under certain circumstances, such as during cell migration in wound repair or even during
the process of normal keratinocyte terminal differentiation. In addition to this dynamic
structural modulation, many junctional macromolecules also have roles in cell signalling
and cell regulation. Thus, cell adhesion is not a rigid phenomenon. Rather, the adhesive
components of these junctional complexes are intricately involved in several aspects of
keratinocyte cell biology and physiology.
In recent years, considerable information about both the structural composition of cell
adhesion junctions and the function of individual components of the various complexes
has started to emerge. This influx of information has come from several sources including
immunoelectron microscopy studies, clinical observations in patients with acquired or
inherited skin fragility disorders, gene targeting experiments, transfection of mutant
constructs, and analysis of specific protein-protein interactions, for example using the
yeast-two-hybrid system.
Studies of human blistering skin diseases have been particularly helpful in providing new
data about the biological mechanisms of cell adhesion (Figure 1.2). In the early 1990s,
assessment of skin biopsies from patients with the Dowling-Meara form of epidermolysis
bullosa simplex revealed that the clumped aggregates within keratinocytes were keratin
intermediate filaments, composed of keratin 14 and 5 (Ishida-Yamamoto et al., 1991).
2 JOHN A.MCGRATH
Figure 1.1 Ultrastructural appearances of cell adhesion junctions in human epidermis. (a):
hemidesmosomes (arrow) at the dermal-epidermal junction (×40,000); (b): a desmosome (arrow)
between adjacent keratinocytes (×70,000).
Subsequently, pathogenetic missense mutations were demonstrated in the corresponding

genes, KRT14 and KRT5, in DNA from patients with this autosomal dominant skin-
fragility syndrome. Thus, epidermolysis bullosa simplex was established as the first keratin
gene disorder and the pathological importance of cytoskeletal integrity to cell adhesion
was established. In subsequent years, keratin gene defects have been delineated in a large
number of other skin diseases, many involving abnormalities of cell adhesion (Irvine and
McLean, 1999). To date, mutations have been described in 15 different epithelial keratins
as well as two hair keratins. The associated diseases include bullous congenital
ichthyosiform erythroderma (epidermolytic hyperkeratosis), ichthyosis bullosa of
Siemens, epidermolytic palmoplantar keratoderma, diffuse and focal non-epidermolytic
palmoplantar keratoderma, pachyonychia congenita and monilethrix.
Further insights into cell adhesion have also been established from studies of diseases
affecting keratinocyte terminal differentiation. Specifically, some cases of the autosomal
dominant mutilating keratoderma, Vohwinkel’s syndrome, and the disorder, progressive
symmetric erythrokeratoderma, have been shown to result from mutations in the gene
encoding loricrin, the major component of the cornified cell envelope (Maestrini et al.,
1996; Ishida-Yamamoto et al., 1998). Immunoelectron microscopy studies have revealed
INTRODUCTION 3
that the mutant loricrin fails to be incorporated into the cell membrane/envelope but
instead has an intranuclear localisation. Desquamation normally involves alterations in cell
adhesion through degradation of lamellated lipid and loss of residual desmosomes but this
is perturbed by the presence of a dominant-negative loricrin mutation. This process may
also be disrupted by abnormalities in keratinocyte transglutaminase, mutations in which may
lead to the autosomal recessive disorder, lamellar ichthyosis.
Studies of skin diseases involving pathological changes in desmosomes have also
provided significant information about keratinocyte adhesion. Desmosomes comprise a
collection of transmembranous glycoproteins (desmogleins and desmocollins) and several
intracellular proteins that link the cell membrane to the cytoskeleton, including
plakoglobin, desmoplakin and plakophilin 1 (Smith and Fuchs, 1998). The acquired
blistering skin disease, pemphigus, involves pathogenic autoantibodies directed against
conformational epitopes on some of the desmosomal cadherins leading to a loss of cell-
cell adhesion and acantholysis. Studies of the different subtypes of pemphigus and the
distribution of target epitopes in skin and mucosal epithelium have provided further new
insights into cell adhesion (Amagai, 1999). For example, patient sera containing anti-
desmoglein 3 antibodies alone lead to a predominantly mucosal form of pemphigus
vulgaris with limited skin involvement. Sera containing both anti-desmoglein 3 and anti-
desmoglein 1 antibodies may lead to pemphigus vulgaris, with disrupted desmosomal
adhesion in both skin and mucosa. In contrast, the presence of desmoglein 1 antibodies
alone results in pemphigus foliaceous with skin blisters but no mucosal involvement.
Thus, targeting of particular desmosomal cadherins by autoantibodies compromises cell
adhesion with different clinical consequences. Naturally occurring mutations in the
desmoglein 1 gene have also been described. Specifically, an amino-terminal deletion in
desmoglein 1 has been shown to underlie some cases of striate palmoplantar keratoderma,
an autosomal dominant genodermatosis (Rickman et al., 1999). This condition does not
lead to blistering, but keratinocyte terminal differentiation is affected through perturbed
desmosome physiology leading to hyperkeratosis of the palms and soles. This
genodermatosis may also result from haploinsufficiency in the desmosomal plaque
protein, desmoplakin (Armstrong et al., 1999). In such patients, cell adhesion is disrupted
in that electron microscopy reveals that some of the desmosomes are small with widening
of intercellular spaces between keratinocytes and disruption of the keratin filament
network which is compacted in a perinuclear distribution. Similar ultrastructural features
are also seen in patients who have complete ablation of plakophilin 1 (McGrath et al.,
1997). In addition to skin fragility with loss of cell-cell adhesion, these patients show
features of a hypohidrotic ectodermal dysplasia with abnormalities of hair, nails, teeth and
sweating. Such changes arise because plakophilin 1 not only has a role as a structural
component of desmosomes but, in addition, as a member of the armadillo gene/protein
family, it also contributes to epidermal development and morphogenesis. Components of
other epithelial junctions may also have broad biological significance. For example, in gap
junctions, mutations in some of the connexin genes (connexins 26 and 31) may either
affect keratinocyte terminal differentiation and lead to keratoderma or the mutations may
result in non-syndromic deafness, or even clinical overlap between the two depending on
the type or combination of connexin mutations (Kelsell et al., 1997; Richard et al., 1998).
4 JOHN A.MCGRATH
Figure 1.2 Inherited and acquired blistering skin diseases provide insights into the mechanisms of
epidermal cell adhesion, (a): blisters and erosions in a 40-year-old male with inherited mutations in
the type XVII collagen gene (COL17A1) resulting in complete ablation of the protein; (b): extensive
conjunctival scarring in a 65-year-old male with acquired autoantibodies to the α3 chain of laminin
5; (c) erosions and scarring on the dorsum of the hand of a 48-year-old male resulting from
autoantibodies against the NC-1 domain of type VII collagen; (d) prominent sacral erosions in a 72-
year-old female with an inherited nonsense/missense combination of mutations in the LAMB3 gene
of laminin 5; (e) blisters, erosions, scars and milia on the knees of a 14-year-old girl with an
inherited homozygous nonsense mutation in the type VII collagen gene, COL7A1; (f) tense blisters
and urticated erythema on the lower leg of a 62-year-old female with acquired autoantibodies
against the NC16A domain of type XVII collagen.
INTRODUCTION 5
Cell adhesion between basal keratinocytes and the underlying dermis also involves
interactions between multiple structural macromolecules, many of which may be targets
for autoantibodies in acquired blistering skin diseases or subjects of inherited mutations in
the genes encoding these proteins in genetic blistering skin disorders. Both types of
abnormality have offered fresh insights into the structural dynamics of cell adhesion as
well as the pathology of certain skin diseases (Borradori and Sonnenberg, 1999). For
example, autoantibodies against type XVII collagen (also known as the 180-kDa bullous
pemphigoid antigen) lead to blister formation through the lamina lucida and the clinical
disorder, bullous pemphigoid. Mutations in the corresponding gene, COL17A1, result in
the autosomal recessive genodermatosis, non-Herlitz junctional epidermolysis bullosa.
Similar paradigms for acquired-inherited disorders of cell adhesion exist for other
hemidesmosome-associated components, including laminin 5 and type VII collagen and,
to a lesser extent, plectin and α6β4 integrin (McGrath and Eady, 1998). These diseases
provide information about the particular roles of individual structural proteins in cell
adhesion. In addition, they offer clues to the relative contributions of specific proteins
involved in cell adhesion. For example, total ablation of laminin 5, resulting from
inherited nonsense mutations on both alleles of LAMA3, LAMB3 or LAMC2 (the three genes
encoding laminin 5), results in the lethal, Herlitz form of junctional epidermolysis
bullosa, in contrast to the milder, non-Herlitz phenotype resulting from complete
ablation of type XVII collagen (Jonkman, 1999). Just as some components of desmosomes
have roles beyond simple mechanical adhesion, the same is true for hemidesmosomes. For
example, the α6β4 integrin not only helps secure adhesion between the hemidesmosome
and epidermal basement membrane, but it is also able to transduce signals from the
extracellular matrix to the cell interior and thereby modulate keratinocyte processes such
as differentiation, proliferation and cytoskeletal organisation.
The aim of this first section of the book (chapters 2–8) is to examine the mechanics of
keratinocyte cell adhesion in more detail. Specifically, the chapters cover the topics of the
cornified cell envelope, the keratin intermediate filaments, desmosomes,
hemidesmosomes, dermal adhesion, and protein-protein interactions. The goal is to
outline the complexity and intricacy of the biology of keratinocyte cell adhesion as well as
to demonstrate the rapidly evolving state of knowledge in this field and its relevance to
skin scientists and dermatologists.
REFERENCES
Amagai M. Autoimmunity against desmosomal cadherins in pemphigus. J Dermatol Sci 20: 92–102,
1999.
Armstrong DKB, McKenna KE, Purkis PE et al.Haploinsufficency of desmoplakin causes a striate
type of palmoplantar keratoderma. Hum Mol Genet 8:143–148, 1999.
Borradori L, Sonnenberg A. Structure and function of hemidesmosomes: more than simple adhesion
complexes. J Invest Dermatol 112:411–418, 1999.
Irvine AD. McLean WHI. Human keratin diseases: the increasing spectrum of disease and subtlety of
the phenotype-genotype correlation. Br J Dermatol 140:815–828, 1999.
6 JOHN A.MCGRATH
Ishida-Yamamoto A, McGrath JA, Chapman SJ, Leigh IM, Lane EB, Eady RAJ. Epidermolysis
bullosa simplex (Dowling-Meara) is a genetic disease characterized by an abnormal keratin
filament network involving keratins K5and K14. J Invest Dermatol 97:959–968, 1991.
Ishida-Yamamoto A, Takahashi H, lizuka H. Loricrin and human skin diseases: molecular basis of
loricrin keratodermas. Histol Histopathol 13:819–826, 1998.
Jonkman MF. Hereditary diseases of hemidesmosomes. J Dermatol Sci 20:103–121, 1999.
Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM. Connexin
26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387:80–83, 1997.
Maestrini E, Monaco AP, McGrath JA, Ishida-Yamamoto A, Camisa C, Hovnanian A, Weeks DE,
Lathrop M, Uitto J, Christiano AM. A molecular defect in loricrin, the major component of
the cornified cell envelope, underlies Vohwinkel’s syndrome. Nat Genet 13: 70–77, 1996.
McGrath JA, McMillan JR, Shemanko CS, Runswick SK, Leigh IM, Lane EB, Garrod DR, Eady
RAJ. Mutations in plakophilin 1 result in ectodermal dysplasia-skin fragility syndrome. Nat
Genet 17:240–244, 1997.
McGrath JA, Eady RAJ. Molecular basis of blistering skin diseases. Hosp Med 59:28–32, 1998.
Richard G, Smith LE, Bailey RA, Itin P, Hohl D, Epstein EH Jr, DiGiovanna J.J, Compton JG, Bale
SJ. Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis. Nat Genet
20:366–369, 1998.
Rickman L, Simrak D, Stevens HP, Hunt DM, King IA, Bryant SP, Eady RAJ, Leigh IM, Arnemann
J, Magee AI, Kelsell DP, Buxton RS. Amino-terminal deletion in a desmosomal cadherin
causes the skin disease striate palmoplantar keratoderma. Hum Mol Genet 8:971–976, 1999.
Smith EA, Fuchs E. Defining the interactions between intermediate filaments and desmosomes. J
Cell Biol 141:1229–1241, 1998.
CELL—CELL ATTACHMENT
2.
THE CORNIFIED CELL ENVELOPE
JORGE FRANK AND ANGELA M.CHRISTIANO
SUMMARY
The skin is the largest organ of the human body, and is the home of constant cycles of
exquisitely regulated cell migration, differentiation, and regeneration. In stratifying
squamous epithelia, such as the skin, the cornified cell envelope (CE), a highly insoluble
peripheral layer of crosslinked proteins, replaces the plasma membrane in a complex and
sequential fashion from precursor proteins initially dispersed in the cytoplasm. The
insolubility of the CE is based on the presence of N-ε(γ-glutamyl) lysine isodipeptide
cross-links formed by epidermal transglutaminases. Because of its mechanical resilience
and impenetrability, the CE provides the human skin with a protective barrier against the
environment. Here, we provide an overview of the different components of the CE and
their properties and function within this complex scaffolding of cross-linked proteins.
INTRODUCTION
The human body is continuously exposed to a variety of external environmental
aggressors. The first and most important barrier of defense and protection against these
hazards is provided by the skin, in particular by the epidermis, the outermost skin layer.
The epidermis consists of four different cell layers, the basal layer (stratum basalis), the
spinous layer (stratum spinosum), the granular layer (stratum granulosum), and the
cornified layer (stratum corneum). Additionally, a transitional cell layer (stratum lucidum)
can be differentiated. This stratum lucidum is located between the stratum granulosum
and the stratum corneum and, thus, separates the “living” parts of the epidermis from the
“dead” epidermal layers. The epidermal network consists of a variety of different cell
types, the most important of which is the epidermal keratinocyte, and in recent years, a
large number of inherited disorders involving the keratinocyte have been described
(Figure 2.1). Through a complex and highly structured cycle of differentiation,
keratinocytes are responsible for constructing a protective barrier against the
environment. The last step in keratinocyte differentiation is characterized by the
formation of cornified cells, so called corneocytes. These corneocytes are enucleated,
THE CORNIFIED CELL ENVELOPE 9
Figure 2.1 A summary of recent progress in the molecular basis of genodermatoses. The inherited
skin disorders involving specific skin layers are indicated on the left, and the genes proteins are
listed on the right (BPAG1, bullous pemphigoid antigen 1; BPAG2, bullous pemphigoid antigen 2;
EB, epidermolysis bullosa; EHK, epidermolytic hyperkeratosis; HD1, plectin; PPK, palmoplantar
keratodermas; SPRRs, small proline-rich proteins; VS, Vohwinkel’s syndrome). Reproduced with
permission from Ref. 79.
flattened polyhedrons, which consist of a stabilized array of keratin filaments encased within
the cornified cell envelope (CE) (1).
The CE represents the most insoluble component of stratified squamous epithelial cells
and appears as an electron dense and homogeneous band, initially about 15 nm in
thickness, replacing the plasma membrane in the uppermost cell layers of squamous
epithelia in vertebrates (2, 3). Later, it gradually increases its thickness and rigidity, and in
cornified cells, the CE is about 20 nm thick (3). CE formation occurs as a complex but
highly structured sequence of events, involving the sequential deposition of distinct
proteins (2). These proteins are cross-linked by the formation of Nε-(γ-glutamyl) lysine
isodipeptide and disulfide bonds. Nε-(γ-glutamyl) lysine cross-linkage is catalyzed by
distinct isoforms of transglutaminases (TG), TG1 and TG 3, and the formation of disulfide
bonds is catalyzed by sulfhydryl oxidase (2, 3). Apart from the TGs, the best characterized
10 JORGE FRANK AND ANGELA M.CHRISTIANO
proteins of the CE are loricrin, involucrin and the small proline-rich proteins (SPRRs),
which are all encoded by genes localized within a 2 megabase region on human
chromosome 1q21, known as the epidermal differentiation complex (EDC) (Figure 2.2)
(4, 5). Further components of the CE include elafin (ELA; synonym cysteine-rich protein
or skin-derived antileukoproteinase, SKALP), profilaggrin (PFN), trichohyalin (THH),
cystatin A (CYA; synonym keratolinin), the S100 protein family (S100A1–S100A11),
envoplakin (EPL), periplakin (PPL), and corneodesmosin (CDE) (1–3, 5–9). Here, we
discuss the major components of the CE and their properties and function within this
complex scaffolding of cross-linked proteins.
MAJOR COMPONENTS OF THE CORNIFIED CELL ENVELOPE
Transglutaminases
The TGs constitute a family of calcium- and thiol-dependent enzymes, catalyzing the
formation of Nε-(γ-glutamyl) lysine isodipeptide bonds and N1,N8-bis(γ-glutamyl)
spermidine bonds (3). In humans, the TG family consists of six known members, and at
least two of these, TG1 and TG3, are believed to play an important role in the formation
and maintenance of the CE (10).
TG1 is an approximately 92 kDa protein, consisting of 817 amino acids. It is thought to
function in a plasma membrane-bound form via its amino terminal region, and also as a
soluble form in the cytoplasm (3). The major expression sites are in the upper spinous and
granular layers (11), although expression of TG1 can also be detected in undifferentiated
epidermal basal cells (12). The gene encoding human TG1 is located on human
chromosome 14q 11.2 (13).
TG3 is a 77 kDa soluble pro-enzyme, consisting of 692 amino acids. The intact TG3
protein can be subdivided into two globular domains, a 50 kDa amino-terminus
containing catalytic regions, and a 27 kDa carboxy-terminus. These two domains are
separated by a flexible hinge, corresponding to the cleavage site for conversion to the
active protein (14, 15). TG3 is exclusively expressed in the stratum granulosum and the
expression is regulated by calcium and the Sp1 and ets transcription factors (16). The gene
coding for TG3 has been mapped to human chromosome 20q 11.2 (13, 17).
Loricrin
Loricrin is a 26 kDa protein that is located in the stratum granulosum of the epidermis and
on the inner surface of purified/sonicated CEs (18, 19). The protein consists of 315
amino acids, is rich in glycine (55%), serine (22%), and cysteine (7%), and is highly
insoluble. The insolubility is attributed to the high content of glycine residues and the
formation of intra- and intermolecular disulfide bonds (1, 3). Loricrin contains repeats of
a unique, highly flexible glycine-rich sequence, held in place by aromatic amino acid
interactions, and referred to as a “glycine-loop” (1, 3). The amino- and carboxy-terminal
ends of loricrin are rich in glutamine and lysine residues (3). Loricrin is the major
Figure 2.2 The epidermal differentiation complex (EDC). The three gene families residing in the
EDC region on human chromosome 1q21 are indicated to the right of the diagram. The loricrin,
involucrin and SPRR gene families are shown as purple rectangles, the trichohyalin and filaggrin gene
families are shown in green, and the S100 gene family is shown in black. Genomic distances are
represented in kilobases (kb). Reproduced with permission from Ref. 79.
component of the CE, comprising about 75% of the total CE mass, or 85–95% of the
cytoplasmic two-thirds of the CE (20). Using recombinant loricrin molecules, it was
demonstrated that TG1 is involved in establishing inter-molecular cross-links resulting in
very large loricrin oligomers, whereas most of the cross-links formed by TG3 are intra-
molecular bridges (20). Loricrin is mainly expressed in the granular layers of the
epidermis, and its expression is upregulated by calcium, cell density, and phorbol ester,
and downregulated by retinoids (3). The loricrin gene is a single copy gene and resides
within the EDC on human chromosome 1q21 (4,21).
Involucrin
Involucrin is an acidic, water-soluble, rod-shaped protein of 68 kDa and consists of 585
amino acids, rich in glutamine and glutamic acid (3). The protein can be subdivided into
amino- and carboxy-terminal flanking domains, showing homology to those of loricrin,
and a central domain containing 39 repeats of a consensus decapeptide (22). All 39
repeats contain, on average, 3 glutamine residues, each of them representing a potential
cross-linking site (1). Involucrin is ubiquitously synthesized by all stratified squamous
epithelia and is expressed in the spinous layer of normal epidermis (3) and in cells of the
inner root sheath of the hair follicle (23). Recent studies in transgenic mice demonstrated
that over-expression of involucrin in mouse epidermis is associated with a delay in hair
ingrowth, alterations in hair appearance, and changes in the appearance of the epidermis
(24). Involucrin is believed to act as a major initial component of the CE, functioning as a
scaffold onto which other proteins are incorporated (3). However, not all involucrin
protein is incorporated into the CE, and it is conceivable that it might exhibit other
specific biological functions in the cytoplasm (3). Involucrin expression is positively
regulated by phorbol ester, calcium, vitamin D, and hydrocortisone, and negatively
regulated by retinoids (3). Involucrin is encoded by a unique gene on human chromosome
1q21 within theEDC (4,21).
Small Proline-rich Proteins

The SPRRs are small (about 10 to 30 kDa), basic, soluble, proline-rich proteins (3).
Members of this family of proteins have also been named pancornulins and cornifin (25, 26).
To date, three classes of SPRRs have been distinguished, SPRR1, SPRR2, and SPRR3 (2).
They contain a variable number of repeating elements in their central portions with a
common sequence motif (XKXPEPXX) and end domains revealing high homology with
the amino- and carboxy-terminal domains of loricrin and involucrin (2). Cross-linking
occurs at these amino- and carboxy-terminal regions, suggesting that SPRRs may function
as molecular cross-bridges connecting two proteins (6, 27). Expression of SPRRs is
distinctively regulated in various stratified epithelia (2, 3). In normal human epidermis,
SPRR1 is mainly expressed in the skin appendages and SPRR2 is expressed in the stratum
granulosum, whereas SPRR3 is absent (3). The expression of SPRR2 and SPRR3 is
downregulated by retinoic acid (2). Calcium, interleukin-1 and interleukin-3 upregulate
the expression SPRR1, and whereas TGF-β results in downregulation. Human SPRR
genes constitute a multigene family clustered within a 300 kb DNA region in the EDC on
chromosome 1q21, close to the genes encoding for loricrin and involucrin (4, 28). This
gene cluster contains two known SPRR1 genes, eight SPRR2 genes, and a single SPRR3
gene (28).
Elafin
Elafin is a basic, 6 kDa protein derived from the 12 kDa precursor preproelafin (1,3, 29, 30).
Preproelafin consists of 117 amino acids; the initiation methionine, a putative 25-amino
acid signal peptide (“pre” sequence), a 35-amino acid “pro” sequence, and the 57 amino
acids that constitute the mature elafin protein (31). Elafin is a potent inhibitor of elastase
and proteinase-3, both derived from polymorphonuclear leukocytes (3). It has a high
content of cysteine (14%), glycine (13%), and proline (12%), and its structure suggests
the potential to form disulfide bonds (1). Elafin contains an internal segment that is a
highly reactive TG substrate. This sequence consists of a series of six amino acids repeated
five times (VKGQDP) (32). In normal adult epidermis, there is only little, if any,
expression of elafin, but it is highly expressed in differentiated keratinocytes in various
pathological conditions, including psoriatic epidermis (3). Further, it is transiently
expressed during fetal and neonatal development of the epidermis. The expression of
elafin is induced by interleukin Iβ and TNFα (33). The gene encoding elafin has been
mapped to chromosome 20q12–q13 (34). In contrast to other CE precursors, the elafin
gene contains a signal sequence and proelafin is stored in secretory vesicles and secreted
extracellularly (35).
Cystatin A
Cystatin A is a 12 kDa, lysine-rich protein, belonging to the evolutionarily related family
of cystatins which inhibit cysteine proteinases (3, 36). Phosphorylated cystatin A is a
natural substrate of epidermal TG and a constituent of the CE (6, 37). It functions as a
bacteriostatic barrier (38) and is expressed in the cells of the epidermal spinous layer (39).
Cystatin A is upregulated by calcium and forskolin, a cAMP elevating agent (40). The
human cystatin A gene is localized on human chromosome 3cen-q21 (41).
S100 Protein Family

The S100 proteins are a group of small (10–12 kDa), acidic proteins that form homo- and
heterodimers and bind calcium via two EF hand motifs (27, 42). They share a common
sequence and structure and are expressed in a tissue- and differentiation-specific manner
(42). Some of the S100 proteins are expressed in the skin, and S100A10 (calpactin light
chain) as well as S100A11 (S100C, calgizzarin) are precursors of the CE (27). Since S100
proteins have no known enzymatic activity, they are thought to function in a calmodulin-
like manner to regulate calcium-dependent cell signaling, proliferation, and morphology
(27, 42). Recent studies revealed that S100A10 binds to annexin II to form a tetramer,
called calpactin 1 (27). S100A11 interacts with annexin I in a calcium-dependent manner,
an interaction that requires the 12 first amino acids of the annexin I amino terminus (27).
The genes encoding the proteins of the S100 family reside in the EDC on chromosome
1q21 (21).
Profilaggrin/Filaggrin
Profilaggrin is a highly phosphorylated protein component of the keratohyalin granules of
mammalian epidermis in the stratum granulosum (43). It consists of 10 or more tandemly
repeated filaggrin units plus an amino- and a carboxy-domain (43, 44). The amino
terminus of profilaggrin reveals a high degree of homology to the small calcium-binding

SI00 proteins in that it contains two alpha-helical EF-hand calcium-binding motifs (43). It
is supposed to play an important role in the differentiation of the epidermis by
autoregulating its own processing in a calcium-dependent manner or by participation in
the transduction of calcium signalling in epidermal cells. Profilaggrin is processed into the
intermediate filament-associated filaggrin by specific dephosphorylation and proteolysis
during terminal differentiation of epidermal cells (43). Filaggrin is a histidine-rich basic
protein that aggregates keratin filaments of terminally differentiating cells of mammalian
epidermis (45). The class of filaggrins shows wide species variations and their aberrant
expression has been implicated in a number of keratinizing disorders (45). The human
filaggrin gene has been mapped to the EDC on chromosome 1q21 and encodes a protein of
324 amino acids (45). The human filaggrin gene repeats show a considerable degree of
polymorphism (45). While all repeats are of the same size (324 amino acids), sequences
obtained display a high degree of variation (10–15%), in most cases attributable to single
base changes (46).
Trichohyalin
Trichohyalin is a protein that associates in regular arrays with keratin intermediate
filaments of the inner root sheath cells of the hair follicle, the medulla layer of the hair
follicle, the granular layer of the epidermis, and is a known substrate of transglutaminases
(47, 48). It is a high molecular weight (248 kDa) insoluble α-helix-rich constituent of the
CE that forms rigid structures as a result of postsynthetic modifications by two Ca2+ -
dependent enzymes, TGs (protein cross-linking) and peptidyl-arginine deaminase
(conversion of arginines to citrullines with loss of organized structure) (47, 48).
Trichohyalin contains one of the highest contents of charged residues of any protein.
Several defined domains of trichohyalin (domains 2–4, 6, and 8) are almost entirely α-
helical, configured as a series of peptide repeats of varying regularity, and are thought to
form a single-stranded α-helical rod stabilized by ionic interactions between successive
turns of the α-helix (47). The protein is similar in structure to involucrin, although several
times longer. Involucrin and trichohyalin may serve as scaffold proteins in the organization
of the CE of terminally differentiating cells or even anchor the cell envelope to the keratin
intermediate filament network (47, 48). Additionally, trichohyalin possesses a pair of
functional calcium-binding domains of the EF hand type at its amino terminus that may be
involved in its calcium-dependent postsynthetic processing during terminal differentiation
(47). By in situ hybridization, the gene coding for trichohyalin has been localized in the
EDC on chromosome 1q21 (49, 50).
Envoplakin
In 1984, Simon and Green reported the identification of two membrane-associated
proteins with molecular masses of 195 and 210 kDa that become incorporated into the CE
on TG activation (51). Recently, overlapping cDNA clones encoding the 210 kDa protein
were sequenced, and the name “envoplakin” was proposed for this precursor of the CE
(52). Envoplakin shows homology to the intermediate filament-associated proteins

desmoplakin I, bullous pemphigoid antigen I, and plectin (52). All four proteins are
characterized by a similar domain structure with an amino-terminal globular domain, a
central rod domain, and a carboxy-terminal globular domain. The COOH termini of the
plakin family members contain a variable number of tandem repeats that are predicted to
be organized into discrete subdomains consisting of α-helices separated by β-turns (52),
and first described for desmoplakin I (53). By in situ hybridization, the envoplakin gene
was mapped to human chromosome 17q25 (54).
Periplakin
The second membrane-associated protein identified by Simon and Green in 1984 has a
molecular mass of 195 kDa (51). It is expressed in keratinizing and non-keratinizing
stratified squamous epithelia and in a number of other epithelia, and was designated
“periplakin” (55, 56). Periplakin, like envoplakin, also belongs to the plakin family, whose
members are responsible for the maintenance of skin integrity and that of other tissues
(57). Like other plakins, periplakin contains a globular amino-terminal domain, consisting
of bundled antiparallel -helices, and a central coiled-coil rod domain (55). However, the
carboxy-terminal domain of periplakin differs from that of the other plakins in that it lacks
any of the sequence-related subdomains (A, B or C) that consist of helices separated by β-
turns (55). The primary sequences of the amino- and carboxy-termini of periplakin show
more than 20% sequence identity to the corresponding regions of desmoplakin I, bullous
pemphigoid antigen I, plectin, and envoplakin (55). Periplakin is most closely related to
envoplakin, and detailed sequence analyses of the two proteins revealed that in particular
their rod domains are more closely related to each other than to those of the other plakin
family members (55). This suggested that both proteins could form two-stranded parallel
homodimers or heterodimers, which would be stabilized by extensive interchain ion
pairing (55). The human periplakin gene consists of 1756 amino acids and was recently
mapped to human chromosome 16pl3 (56).
Corneodesmosin
Corneodesmosin is a basic, phosphorylated and glycosylated protein with a molecular
weight of 52–56 kDa (58). In humans, Corneodesmosin is exclusively expressed in
cornified squamous epithelia, like the epidermis, hard palate, and the inner root sheath of
the hair follicle (58). It is synthesized at the latest stage of keratinocyte differentiation and
persists between the cells of the stratum corneum until desquamation occurs (59). By
immunofluorescence and immunoelectron microscopy, Corneodesmosin was
demonstrated to be bound to the CE (58). Recently, a gene designated “S” (because it was
identified in skin) was identified in the class I region of the human major
histocompatibility (HLA) complex on chromosome 6p21.3 (60). This gene is expressed at
high levels as 2.2 kb- and 2.6 kb mRNAs in human skin, and in situ hybridization revealed
that its expression is restricted to the differentiating keratinocytes in the granular layers of
the epidermis (60). Later, it became obvious that the product encoded by this gene is, in
fact, Corneodesmosin (58) and that the protein contains a high percentage of serine,
glycine, and proline residues (60). Although there are significant similarities with the
proteins encoded by loricrin, keratin 1, and keratin 10, all major components of the
stratum granulosum, the S gene is structurally unrelated to other known genes of the HLA
complex (60), and, in a study investigating the potential role of the S gene in psoriasis
vulgaris, several genetic intragenic polymorphisms have been identified (61).
MOLECULAR BASIS OF DISEASES AFFECTING THE CE
Lamellar Ichthyosis and Transglutaminase Mutations

Lamellar ichthyosis (LI) (OMIM accession numbers 242300 and 601277) is a genetic skin
disorder, inherited in an autosomal recessive fashion (62). Clinically, ichthyotic lesions are
present at birth and almost always involve the entire skin surface. The children are usually
born encased in a collodion membrane that desquamates during the first 10 to 14 days of
life. The skin is erythematous and covered with large scales, fissures of the hands and feet,
scarring alopecia, dystrophic nails, and ectropion are common symptoms. Because of an
obstruction of the eccrine glands, the patients do not sweat normally, which can lead to
hyperpyrexia (63). Recently, it was demonstrated that there exist at least three genetically
distinct forms of recessively inherited LI characterized by typical pathology of the skin,
with direct sequelae of primary cutaneous lesions (for example ectropion, joint
contractures, and digital necrosis) and without in volvement of other organ systems (64, 65).
The form that maps to chromosome 14q 11 has been designated LI Type 1. This form of LI
is characterized by mutations in TG1, one of the major components of the CE (64, 66–74).
A second form, LI Type 2, maps to chromosome 2q33–q35 (65); however, no candidate
gene for LI Type 2 has been identified so far in this chromosomal region. A third form of
LI, LI Type 3, has been mapped to chromosome 20q l l.2 in the region of the TG3 gene
(13, 17). A large number of mutations in the TG1 gene in LI have enabled genotype/
phenotype correlation in families with lamellar ichthyosis (73). Linkage analyses using
microsatellite markers spanning the region of the TG1 gene confirmed genetic
heterogeneity. In patients not linked to the TG1 locus, the second region identified on
chromosome 2q33–q35 and the third candidate region on chromosome 20 were
excluded, suggesting the existence of at least four loci for lamellar ichthyosis (73).
Vohwinkel’s Syndrome and Loricrin Mutations

Vohwinkel’s syndrome (OMIM number 124500), first reported in 1929, is an autosomal
dominant palmoplantar is keratoderma with pseudoainhum, and sometimes associated
with deafness (75, 76) (figure 3). In an extended Vohwinkel’s syndrome family, Maestrini
et al. demonstrated linkage to the EDC on chromosome Iq21, the calculated maximum
multipoint lod score being 14.3 (75), and a mutation consisting of a 1 bp insertion after
nucleotide 730 (730insG) was detected in the loricrin gene (77). This additional G
nucleotide was found in an area of six subsequent wild-type G nucleotides (codons 230
Figure 2.3 Clinical presentation of Vohwinkel’s syndrome, (a) Characteristic honeycomb

keratoderma on the palm, (b) Pseudoainhum is present on the third, fourth and fifth digits, (c)
Bilateral autoamputation of the fifth toes in an affected individual. Reproduced with permission from
Ref. 79.
and 231), introducing a frameshift mutation at codon 232 that altered the terminal 84
amino acids of the encoded protein and resulted in a delayed termination codon, thereby
extending the mutant protein by 22 amino acids. Affected individuals were heterozygous
for the mutation, which was not found in unaffected members of the family or in
unrelated, unaffected control individuals (77). The authors noted that replacement of the
fourth glycine-rich domain and the carboxy-terminal glutamine/ lysine-rich domain,
which is believed to be involved in normal protein cross-linking would be expected to
alter the function of the protein and to impair cross-linking by Tgs (77). Since loricrin
monomers become cross-linked to each other as well as to other proteins by isopeptide
bonds, the defect would be expected to have a dominant-negative effect, in accordance
with the autosomal dominant inheritance of Vohwinkel syndrome (77). The results of
immunoelectron microscopy studies suggested that the mutant loricrin protein is
abnormally or less efficiently incorporated into the CE and accumulates in intranuclear
granules. Detection of this mutation in the loricrin gene was the first evidence for a
molecular defect in a gene of the EDC/CE underlying a human disease (75). It was
reported for a second time in a seemingly unrelated Vohwinkel’s syndrome family (78). In
this study, a lack of linkage to chromosome Iq21 was demonstrated in a third VS family,
suggesting genetic heterogeneity within VS (78).
Progressive Symmetric Erythrokeratoderma and Loricrin

Mutations
The clinically and genetically heterogeneous group of disorders known collectively as the
erythrokeratodermas (EKs) is characterized by widespread erythematous plaques, either
stationary or migratory, associated with features that include palmoplantar keratoderma
(79). One type, known as progressive symmetric erythrokeratoderma (PSEK; OMIM
number 602036), and inherited in an autosomal dominant fashion, shows incomplete
penetrance and variable expression (80). The disease is characterized by erythematous and
hyperkeratotic plaques, and only approximately 30 cases have been reported since the
initial description by Darier in 1911 (80, 81). Non-migratory erythematous plaques
develop shortly after birth and are distributed symmetrically over the body surface, in
particular on the extremities, the buttocks, and sometimes the face, together with
palmoplantar keratoderma. A second major clinical subtype of erythrokeratoderma is EK
variabilis (EKV; OMIM number 133200), first described by Mendes da Costa in 1925,
and characterized by relatively fixed hyperkeratotic patches on erythematous areas, and by
capriciously formed outlines, like the boundary lines of seacoasts on maps (82). Like
Vohwinkel syndrome and PSEK, EKV is also inherited as an autosomal dominant trait
with incomplete penetrance. The condition is usually evident at birth or within the first
year of life, and skin lesions predominantly affect the face, buttocks, and extremities. The
erythematous areas move from hour to hour, and palmoplantar keratoderma is generally
present. The major feature distinguishing PSEK and EKV is the sharply outlined,
geographic regions of migratory erythema in EKV. The questions of whether these two
disorders are in fact one disease was raised by MacFarlane et al. in 1991 when they
reported studies of two sisters, one of them apparently suffering from EKV and the other
one showing clinical symptoms of PSEK (83). The genetic locus for EKV maps to
chromosome 1p36.2–p34.
In 1997, Ishida-Yamamoto et al. demonstrated one family with PSEK had a mutation in
the loricrin gene on chromosome 1q21 (80), suggesting that PSEK and EKV are
genetically distinct disorders. They reported a 1-bp insertion of a C following nucleotide
709 (709insC) in the loricrin gene, resulting in a frameshift and a delayed termination
codon in the loricrin mRNA (80). The frameshift caused replacement of the carboxy-
terminal 91 amino acids and extended the coding sequence by an additional 65
nucleotides. The wild-type loricrin polypeptide consists of 315 amino acid, so this
mutation replaces the carboxy-terminal third of loricrin with missense amino acids and
removes approximately one third of the glutamine and lysine residues involved in
isodipeptide cross-link formation (80). The similarities between this disorder and
Vohwinkel’s syndrome include palmoplantar hyperkeratosis with a honeycomb
appearance, pseudoainhum, and ichthyotic lesions on other body areas, although the PSEK
cases revealed significantly more widespread and striking erythematous hyperkeratotic
plaques (80). Ishida-Yamamoto et al. noted that the PSEK mutation involves the insertion
of a C after a stretch of 4 consecutive C nucleotides and is located only 21-bp upstream of
the site of the loricrin mutation causing Vohwinkel syndrome (80), which consists of a 1-
bp insertion of a G after a stretch of 6 consecutive G nucleotides (78).
POSSIBILITIES OF FUTURE GENE THERAPY FOR DISORDERS

OF THE CE
The skin represents an accessible somatic tissue for gene transfer and, depending on
therapeutic goals, a variety of cutaneous gene delivery approaches are currently available
(84–86). Recently, corrective gene transfer was demonstrated in LI, a disfiguring skin
disease characterized by abnormal epidermal differentiation and defective cutaneous
barrier function (84). LI is associated with a decreased activity of keratinocyte TG1, the
enzyme necessary for normal formation of the cornified epidermal barrier. Using LI as a
prototype for therapeutic cutaneous gene delivery, Choate et al. developed a human skin/
immunodeficient mouse xenograft model to correct the molecular, histological and
functional abnormalities of skin derived from LI patients in vivo. After retroviral
transduction of TG1 into TG1-deficient primary keratinocytes, they regenerated
engineered human LI epidermis on immunodeficient mice. The engineered LI epidermis
displayed normal TG1 expression in vivo, in contrast to unengineered LI epidermis where
TG1 was absent. Epidermal architecture was also normalized by TG1 restoration, as was
expression of the epidermal differentiation marker filaggrin. Engineered LI skin
demonstrated restoration of cutaneous barrier function to levels seen in epidermis
regenerated by keratinocytes from patients with normal skin, indicating functional
correction of the proposed primary pathophysiologic defect in LI. These results confirm a
major role for TG1 in epidermal differentiation, and demonstrate a potential future
approach to therapeutic gene delivery in human skin (84). In 1997, the same group of
researchers examined the regeneration of skin from TG1-deficient patients with LI on
nude mice by direct injection of naked plasmid DNA (85). Regenerated LI patient skin
receiving repeated in vivo injections with a TG1 expression plasmid displayed restoration
of TG1 expression in the correct tissue location in the suprabasal epidermis. However,
unlike LI skin regenerated from keratinocytes first transduced with a retroviral expression
vector for TG1 prior to grafting, directly injected LI skin displayed a non-uniform TG1
gene expression pattern (85). Further, direct injection failed to correct the central
histologie and functional abnormalities of the disease. These data demonstrate that partial
restoration of gene expression can be achieved via direct injection of naked DNA in
human skin, but underscore the need for new advances to achieve efficient and sustained
plasmid-based gene delivery to the skin.
Recently, another group of researchers has also demonstrated successful corrective
gene transfer in the human skin disorder X-linked ichthyosis (XLI) (OMIM number
308100) (86). XLI is characterized by loss of function of the steroid sulfatase arylsulfatase
C (STS). Freiberg et al. developed a model of corrective gene delivery to human skin in
vivo (86). A retroviral expression vector was produced and utilized for STS gene transfer
into primary keratinocytes from XLI patients. Transduction was associated with
restoration of full-length STS protein expression as well as steroid sulfatase enzymatic
activity in proportion to the number of proviral integrations in XLI cells. Transduced and
uncorrected XLI keratinocytes, along with normal controls, were then grafted onto
immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI
keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with
defective skin barrier function, effectively recapitulating the human disease. Transduced
XLI keratinocytes from the same patients, however, regenerated an epidermis
histologically indistinguishable from that formed by keratinocytes from patients with
normal skin (86). STS expression in transduced XLI epidermis was demonstrated in vivo
by immunostaining as well as a normalization of histologie appearance at 5 weeks post-
grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function
parameters to normal. Thus, these findings demonstrate corrective gene delivery in
human XLI patient skin tissue at both molecular and functional levels and provide a model
of human cutaneous gene therapy (86).
Collectively, these data support the notion that the epidermis is an attractive site for
therapeutic gene delivery due to its accessibility and the potential for delivering
polypeptides to the systemic circulation. A number of obstacles, however, have emerged
in attempts at cutaneous gene delivery, and central among these is the inability to sustain
therapeutic gene production. The challenges for the future of gene therapy in the skin
have been identified in these early studies, and provide a foundation of knowledge for the
growth of this exciting and important field.
CONCLUSIONS
In recent years, chromosomal mapping and cloning of the genes encoding the different
protein components of the CE, as well as the identification of TG1 and loricrin mutations
underlying diseases affecting the CE, has greatly enhanced our understanding of the
structure and organization of this important structure of the skin. However, more
discoveries will be made before the mysteries of this complex protein cross-linked
treasury are solved. The number of newly identified precursors of the CE continues to
grow, and elucidation of their relevance to the maintenance of skin integrity, inherited
skin disorders and approaches to gene therapy is our challenge for the future.
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3.
KERATINS AND KERATIN DISORDERS
LAURA D.CORDEN AND W.H.IRWIN MCLEAN
MOLECULAR ASPECTS OF KERATIN INTERMEDIATE

FILAMENTS
The Intermediate Filament Family of Cytoskeletal Proteins

In addition to the actin microfilament and microtuble cytoskeletal systems found in all
mammalian cells, there is a third cytoskeletal network comprised of filaments which are
intermediate in size between the other two systems and which are therefore termed
intermediate filaments (Figure 3.1 & 3.2). Lazarides described five classes of intermediate
filament within higher eukaryotic cells, distinguishable biochemically and
immunologically: keratin, desmin, vimentin, neurofilaments and glial filaments
(Lazarides, 1980). The current classification of the different types of intermediate
filament is shown in Table 3.1, below.
THE MOLECULAR STRUCTURE OF INTERMEDIATE

FILAMENTS
All intermediate filaments have a diameter of approximately lOnm (Lazarides, 1980) and
share a common structure composed of a central a-helical rod domain of approximately
310 amino acids (Geisler and Weber, 1982; Hanukoglu and Fuchs, 1982; Hanukoglu and
Fuchs, 1983; Lewis et al., 1984; Quax et al., 1983; Steinert et al., 1983; Tyner et al.,
1985; Weber and Geisler, 1982), corresponding to 46nm in length (reviewed in (Steinert
and Bale, 1993; Steinert and Parry, 1985)). The domain structures of type I and II
keratins, typical of all intermediate filament types, are shown in Figure 3.3. The rod
domain is interspersed by three non α-helical regions which are predicted to take the form
of β-turns (Hanukoglu and Fuchs, 1983; Steinert et al., 1983), and divide the rod
into four sub-domains termed 1A, 1B, 2A and 2B (Steinert and Parry, 1985)
(Figure 3.3). The four helical domains are reasonably constant in size between all
intermediate filament types, approximately 30–50, 95, 35 and 95 amino acid residues in
length respectively (Hanukoglu and Fuchs, 1983).
KERATINS AND KERATIN DISORDERS 27
Table 3.1 Types of intermediate filament
Figure 3.1 Epithelial cell line PtK2 transfected with a human K17 cDNA clone in eukaryotic
expression plasmid pCR3.1, stained with monoclonal antibody F3 against K17 and FITC secondary
antibody. This confocal scanning laser microgaph shows the typical dense cytoplasmic meshwork of
keratin filaments seen in mammalian epithelial cells. Photograph provided by Drs Frances J.D.Smith
and Seana P.Covello, Epithelial Genetics Group, Thomas Jefferson University, Philadelphia.
The non-helical linker regions possess different net charges, implying that ionic
interactions between these regions on opposite molecules might play a part in stabilizing
the filament (North et al., 1994). The neutral/basic LI linker separates the 1A and 1B
subdomains of the rod (Figure 3.3) and consists of between 8–12 residues dependent
upon filament type. The acidic linker LI2 separates the rod domain between the IB region
and 2A (Figure 3.3). This linker is variable in length (16–22 residues) but shows some
degree of sequence conservation between the intermediate filament types. The acidic
linker L2, separating the 2A and 2B subdomains of the rod, is the shortest of the three
linkers. It is uniform in length, consisting of 8 residues and shows sequence conservation
28 LAURA D. CORDEN AND W.H. IRWIN MCLEAN
Figure 3.2 Transmission electron micrograph of human epidermis, showing keratin filament
bundles (black arrows) in the cytoplasm of epidermal keratinocytes. Also seen are desmosomes
(white arrow), transmembrane complexes which connect the keratin networks of neighbouring
cells. Magnification 35,000× approx. figure kindly provided by Dr John A.McGrath, St John’s
Institute of Dermatology, St Thomas’s Hospital, London.
(North et al., 1994). The linker regions are thought to impart flexibility on the rod
domain (Steinert, 1993). The 2B domain contains a “stutter”, which is a highly conserved
discontinuity in the regularity of heptad periodicity in the coiled-coil, of unknown
function (North et al., 1994; Steinert and Parry, 1985). The a-helical conformation is
slightly distorted in the region of the “stutter”, which could represent a point of weakness
within the coiled-coil, enabling it to bend (North et al., 1994). Alternatively, this helix
inversion could be necessary to allow filament packing. These hypotheses cannot be
formally proven without X-ray crystallographic data.
At the amino terminal end of the central rod domain is a region termed the helix
initiation motif and at the carboxy terminal end of the rod, the helix termination motif
(Steinert, 1993) (red areas, Figure 3.3). These regions show striking sequence
conservation among and between intermediate filament types (Conway and Parry, 1988).
Conservation of these sequences indicates that they must play an important role in
filament assembly (Steinert et al., 1993b; Wilson et al., 1992) (Figure 3.3). This is
supported by the identification of mutations in these regions which have dramatic effects
on filament assembly and function, as discussed below.
The central a-helical region is flanked by a variable domain V1 at the amino-terminus
and V2 domain at the carboxy-terminus (Hanukoglu and Fuchs, 1982; Weber and
Geisler, 1982) (Figure 3.3). These domains are highly variable both in length and
sequence, accounting for size differences specific for each keratin polypeptide. These
domains usually contain tandem peptide repeats and are rich in serines and glycines
(Korge et al., 1992; Steinert et al., 1985a). The type II keratins also possess short,
complex sequences, H1 and H2 (homology) subdomains, which lie between the rod
domain and the V1 and V2 domains, respectively (Steinert et al., 1985a). Mutations in the
H1 domain of type II keratins have been shown to cause skin disease, as discussed below.
keratins: Protein Domain Structure
Figure 3.3 Schematic diagram showing the protein domain organisation of the type I and type II
keratins, a structure which is similar for all intermediate filament types. The a-helical coiled-coil rod
domain, which is responsible for heteropolymerisation, consists of 4 subdomains, 1A, 1B, 2A and
2B, separated by non-helical linkers L1, L12 and L2. In helix 2B, is a stutter or discontinuity in the
coiled-coil heptad repeats (S). The rod domain is flanked by globular V1 and V2 domains, which
vary greatly in size and amino acid composition between keratins compared to the rod domains,
which are well conserved in size and sequence. The most highly conserved sequences are the helix
boundary motifs (shaded red), at the beginning and end of the rod domain and which are thought to
be involved in molecular overlap interactions in filament assembly. Type II keratins have additional
conserved features within their variable domains: the homology subdomains H1 and H2, which
probably play a role in polymerisation; and the ISIS box, which is thought to be a site of interaction
of keratins with other protein complexes.
In contrast to the rod domain, the head and tail regions show great variability not only
between individual keratins, but also differ between the intermediate filament types
(Geisler and Weber, 1982; Hanukoglu and Fuchs, 1983), reviewed in (Steinert and
Parry, 1985; Steinert et al., 1985b). It has been proposed that these differences specify the
function of the intermediate filament in which they are found (Steinert et al., 1985b).
Experiments carried out to examine filament formation in vitro using the rod domain of
desmin, have shown that this domain alone is incapable of forming lOnm filaments,
indicating that the head and tail domains are required for polymerisation into mature
filaments (Geisler et al., 1982). Studies involving expression of deleted keratin proteins,
where either the head or tail domain had been deleted from one or both keratins, have
revealed that one member of the heterotypic keratin pair requires an intact head and tail
domain for normal filament assembly and elongation to occur (Lu and Lane, 1990). These
studies showed that homotypic head/tail interactions of like polypeptides played an
important role in filament elongation. More recent investigations have pointed to a role
for the type II head domain in elongation and lateral alignments/registration of
neighbouring molecules, possibly mediated by HI, whereas the tail domains of type I and
II keratins have been implicated in filament stabilization (Hatzfeld and Burba,
1994; Steinert and Parry, 1993; Wilson et al., 1992). An evolutionarily conserved
nonapeptide motif within the head domain of vimentin (also found in desmin and
peripherin) has been shown to be necessary in assembly of soluble tetramers into
filaments (Herrmann et al., 1992).
The amino acid sequence of the rod domain of an intermediate filament shows over most
of its length, a heptad periodicity of hydrophobic residues at positions a and d of every
seven (abcdefg). This periodicity is indicative of a protein that forms coiled-coil polymers
(Crick, 1953); reviewed in (Cohen and Parry, 1986). A single polypeptide chain, which is
predicted to assume a-helical structure is generally unstable in an aqueous environment
due to the presence of hydrophobic or apolar residues within the molecule, as reviewed in
(Cohen and Parry, 1986; Conway and Parry, 1991). Dimerisation of two a-helical
molecules involves twisting of the two polypeptide chains together, placing the
hydrophobic residues in a position protected from the aqueous environment, thus forming
a stable coiled-coil molecule. Residues in positions e and g are charged and thought to be
involved in interchain ionic interactions which stabilize the arrangement of two a-helices
within the coiled-coil (Cohen and Parry, 1986). The polar hydrophilic residues b, c and f are
located at the periphery of the coiled-coil, in contact with the aqueous environment. The
importance of the stability of interations within the coiled-coil has been demonstrated in
experiments where head and tail deleted keratin polypeptides were expressed within
cultured fibroblasts. Interactions within the heterotypic coiled-coil protected the
individual type I or type II keratin from proteolysis which occurs when a single type
keratin is expressed alone. Keratins deleted in the head and/or tail regions were capable of
resisting proteolysis in combination with a keratin of the opposite type (Lu and Lane,
1990).
Keratins: the Type I and Type II Intermediate Filaments

Studies on sheep wool proteins suggested that there were two groups of keratins (Parry et
al., 1977). Fuchs and colleagues described the existence of two different classes of keratin
genes (Fuchs et al., 1981), the type I and II intermediate filaments which constitute the
largest fraction of the intermediate filament protein family, with >30 different human
keratins described to date (Quinlan et al., 1994). Keratins contribute to the intermediate
filament cytoskeleton within epithelial cells, forming a basket-like network around the
nucleus in eukaryotic cells extending to the cell periphery, where they contact the plasma
membrane through interaction with specific protein junctions, the hemidesmosomes (on
the basal cell surface) and the desmosomes (at intercellular membrane sites). It is through
interaction with the desmosomes that the intermediate filaments link the cells into a three
dimensional structure, increasing mechanical integrity of individual cells and the tissue as
a whole.
The desmosomal component desmoplakin has been shown to associate directly through
its carboxy terminal tail with the amino terminal head domain of several type II epidermal
keratins (Kouklis et al., 1994). This association was not found with the simple epithelial
type II keratins, the type I keratins or vimentin (Kouklis et al., 1994). It is the amino
terminus of desmoplakin that targets the protein to the desmosome and the carboxy
terminus which attaches intermediate filaments to the desmosomal plaque (Stappenbeck et
al., 1993; Stappenbeck et al., 1994). Phosphorylation of a serine residue within the
carboxy terminus of desmoplakin has been found to be likely to negatively regulate its
interaction with keratin intermediate filament networks (Stappenbeck et al., 1994).
Keratins are obligate heteropolymers (Hatzfeld and Franke, 1985; Lee and Baden,
1976; Steinert et al., 1976) and the first step in keratin filament assembly has been shown
to be the formation of a coiled-coil type I/II heterodimer (Figure 3.4) (Hatzfeld and
Weber, 1990; Steinert, 1990). Thus the obligatory heteropolymer step in keratin
intermediate filament assembly occurs at the dimer stage and not during tetramer
formation (Hatzfeld and Weber, 1990). This heterotypic association appears to be
mediated by the α-helical rod domains, as deletions of the head and tail domains do not
prevent this association (Lu and Lane, 1990). Within the coiled-coil dimer, the two
molecules are parallel and in axial register, as reviewed in (Cohen and Parry, 1990;
Steinert, 1993). Chemical cross-linking studies using the rod domain of desmin, capable of
homopolymerisation, indicated that intermediate filament structure is based on a
tetramer, composed of a dimer of two double stranded coiled-coils (Geisler and Weber,
1982). The tetramer is considered the most stable intermediate filament oligomeric
species in vitro (Quinlan et al., 1984).
The precise manner in which tetramers associate to form mature 1Onm filaments is
not known with certainty, however the free tetramer is composed of two coiledcoil
dimers which are thought to exist in an anti-parallel and staggered arrangement (Geisler et
al., 1985; Meng et al., 1996; Steinert, 1993; Steinert et al., 1993a; Steinert et al., 1994;
Stewart et al., 1989) (see Figures 3.3 & 3.4). This arrangement within the filament raises
the possibility of a large number of interactions between adjacent molecules (Figure 3.4).
Elongation involves end to end interactions of additional tetramers, with an overlap
between the first 10–11 residues of the 1A region of one dimer and the last 10–11
residues of the 2B region of the other (emphasising the importance of the helix initiation
and termination motifs) (Steinert, 1993; Steinert et al., 1993a; Steinert et al., 1994)
(Figure 3.4). Higher orders of filament assembly have not been conclusively deduced,
although protofilaments (2–3nm) and protofibrils are thought to assemble by longitudinal
extension of laterally associated tetramers and “half-filaments” by longitudinal extension
of oligomers containing 12–20 laterally associated dimers, as reviewed by (Lane, 1993;
Quinlan et al., 1994; Steinert, 1993).
Accuracy of the type I/II keratin pairing is thought to be important in terms of three-
dimensional quality of the keratin filament network within the cell (Lu and Lane, 1990).
Type I keratins are acidic (K9–K20) and their genes have been found to map to
chromosome 17q (Milisavljevic et al., 1996; Rosenberg et al., 1988), except for K18
which maps to the type II (K1–K8) keratin gene cluster (Waseem et al., 1990; Yoon et al.,
1994) on chromosome 12q (Rosenberg et al., 1991). The keratins are designated as type I
Figure 3.4 Potential domain alignments within keratin intermediate filaments, adapted from Steinert,
1993a; Steinert et al., 1993b; see also Meng et al., 1996.
Cross-linking experiments (Steinert et al., 1993) have established four modes of alignment of two
nearest neighbour molecules in the keratin intermediate filament. (A12) the two molecules are
aligned antiparallel and in almost axial register. (A11) the two molecules are arranged anti-parallel
to each other and staggered, bringing 1B sub-domains into approximate alignment. (A22) the two
molecules are again anti-parallel and staggered to bring the 2B sub-domains into close alignment.
Alignments A11 and A22 reveal the fourth mode (ACN), here two similarly directed molecules
overlap so that the first 10 amino acid residues of 1A overlap with the last 10 amino acid residues of
sub-domain 2B on the other molecule (revealing the importance of these conserved regions).
Intermediate filament protein domain interactions (vimentin) were studied using the two-hybrid
system (Meng et al., 1996). These studies indicated that the 1B sub-domains interact strongly with
one another and that the 2B domains also interact strongly with each other. The A11 and A22
modes of alignment were considered to reveal true domain interactions, whereas the A12 and ACN
modes may describe adjacent molecules within the filament, but not interacting molecules (as no
interaction was found between different constructs containing the 1A or 2B domains).
or II according to their apparent molecular weight and isoelectric point in 2D gel
electrophoresis (Moll et al., 1982). The differences in size between the type I and II
keratins result from differing lengths of the amino and carboxy terminal regions of the
proteins, rather than from significant insertions or deletions within the central rod domain
(Hanukoglu and Fuchs, 1983).
Keratins are generally expressed in specific pairs depending upon cell type,
developmental stage, growth environment and differentiation or disease state of the cell
(Lazarides, 1980; Sun et al., 1983; Sun et al., 1985). The tissue-specific expression
Table 3.2 Cytokeratin expression patterns
*indicates that mutations in this keratin have been found to cause human disease.
patterns of human keratins are shown in Table 3.2. For example, keratins K5 and K14 are
expressed in the basal cells of the epidermis (Moll et al., 1982; Purkis et al., 1990),
although K15 may also be expressed to a lesser extent in these cells (Moll et al., 1982). As
cells leave this layer and undergo differentiation, expression of K5 and K14 is down-
regulated, with increased expression of the keratin pair K1 and K10. In conjunction with
K1/K10 expression, K2e (the epidermal form of K2) is expressed in the more superficial
suprabasal cells (Collin et al., 1992). The amount of K1 and K10 within the cell increases
as it becomes more suprabasal (Coulombe, 1993). In addition to the keratins discussed
above, other keratins are also expressed in the epidermis, with the particular keratin
expressed being dependent upon tissue location. K9 is a major keratin expressed
suprabasally within palmar and plantar skin (Langbein et al., 1993). Keratins K6 and K16
are also found in sole and palm skin and are expressed in regions of the skin which are
actively proliferating, for example during wound healing or in psoriasis (Weiss et al.,
1984). The anterior corneal epithelium, a non-cornified stratified squamous epithelium,
expresses K3 and K12 predominantly, with low levels of K5 and K14 expression within
the basal cells (Sun et al., 1984). The non-stratified oesophageal mucosa specifically
expresses K4 and K13 (Moll et al., 1982). The expression of two major epidermal
keratins in human skin is shown in Figure 3.5.
MEDICAL ASPECTS OF KERATIN INTERMEDIATE FILAMENTS
Mutations in Keratin Genes Cause a Wide Range of Human

Diseases
Since 1991, mutations have been found in 15 different epithelial keratin genes and two
hair keratin genes causing a wide range of human diseases affecting the epidermis and
various other epithelia. In each case the phenotype observed has been dependent upon the
expression pattern of the defective keratin. All these disorders display keratinocyte
fragility, demonstrating that a major function of keratin filaments is to enable epithelial
cells to resist physical damage.
Figure 3.5 Indirect immunoperoxidase staining of human epidermis using antibodies against (a)
keratin 1 (LH1) and (b) keratin 14 (RCK107). K14 expression is confined to the basal cell
compartment (b), whereas K1 expression is found in all other (suprabasal) cell layers of the epidermis.
The precise reasons for these striking comparmentalised keratin expression patterns in differentiated
epithelia still remain a mystery but are presumed to indicate tissue-specific and/or differentiation-
specific functions of the keratin cytoskeleton. Photographs courtesy of Declan P.Lunny, CRC Cell
Structure Research Group, Department of Anatomy and Physiology, University of Dundee.
Epidermolysis Bullosa Simplex

Epidermolysis bullosa (EB) is a clinically and genetically heterogeneous group of inherited
disorders characterised by skin blistering. There are three main forms of EB: junctional,
dystrophic and simplex which were originally classified according to the level within the
skin where the blister occurs (Fine et al., 1991). These can now be better classified according
to which of the 10 known EB genes are involved, as reviewed by (Uitto et al., 1997). In
EB simplex (EBS), the split is intraepidermal and cytolysis occurs within the basal
keratinocyte, usually midway between the nucleus and the hemidesmosomes. Mutations
in the K5 and K14 genes have been shown to underlie this form of EB. The identification
of these mutations confirmed that the primary function of keratin filaments is structural,
since these mutations lead to fragility of the tissue in which the mutant keratin is
expressed.
Figure 3.6 Clinical appearance of the phenotypes arising from mutations in three major epidermal
keratins, (a) The Dowling-Meara form of epidermolysis bullosa simplex (EBS-DM)—widespread
epidermal blistering caused by mutations in or near the helix boundary motifs of basal cell keratins
K5 or K14. (b) The thickened, hyperkeratotic skin of a patient with bullous congenital ichthyosiform
erythroderma (BCIE), which can be caused by mutations in K1 or K10, the keratins expressed in all
suprabasal layers of the epidermis. (c) Ichthyosis bullosa of Siemens (IBS), a mild epidermolytic
ichthyosis caused by mutations in K2e, a keratin expressed only in high suprabasal layers of the
epidermis. Thanks to Dr Alan D.Irvine, Department of Dermatology, Royal Victoria Hospital,
Belfast; Dr Marion White, Department of Dermatology, Aberdeen Royal Infirmary, Aberdeen; and
Prof Irene M.Leigh, Academic Department of Dermatology, The Royal London School of Medicine
and Dentistry, London, for providing these figures.
There are three main forms of EBS (Fine et al., 1991) which generally exhibit autosomal
dominant inheritance. The Dowling-Meara form (EBS-DM) is considered to be the most
severe, with blisters clustered at any body site as a result of mild physical trauma (Fine et
al., 1991). The clinical appearance of EBS-DM is shown in Figure 3.6. A widespread
distribution but less severe blistering is associated with the Köbner form of EBS (EBS-K).
The mildest variant, Weber-Cockayne EBS (EBS-WC), causes blistering localized to the
hands and feet, the major trauma sites of the epidermis (Fine et al., 1991). Electron
Figure 3.7 Transmission electron micrograph of epidermis from a patient with bullous congenital
ichthyosiform erythroderma (BCIE), the phenotype due to K1 or K10 mutations shown in
Fig. 3.6b. The basal cells at the bottom of the figure, in contact with the basement membrane
(arrowheads), do not express K1 or K10 and therefore have normal appearing keratin filaments
(compare Fig. 3.2). In contrast, all the upper suprabasal cells can be seen to contain dense filament
aggregates due to the mutant keratin expression (arrows) and consequently are undergoing
cytolysis. The scale bar represents 5µm. Thanks to Prof Robin A.J.Eady and Dr Akemi Ishida-
Yamamoto, St John’s Institute of Dermatology, St Thomas’s Hospital, London for providing this
illustration.
microscopy of biopsy material from EBS-DM patients revealed keratin filament collapse
within basal keratinocytes, leading to the formation of electron-dense aggregates within
the cytoplasm. Immuno-electron microscopy revealed that these aggregates contained K5
and K14 (Ishida-Yamamoto et al., 1991), suggesting these keratins as candidate genes for
EBS. Expression of mutant keratins in cultured cells had been shown to produce similar
keratin aggregates (Albers and Fuchs, 1987; Albers and Fuchs, 1989). Filament
aggregation can also be seen by EM in many other keratin disorders. Typical tonofilament
aggregates are shown in Figure 3.7, in the skin of a patient carrying a K1 mutation.
Expression of a truncated K14 gene in the skin of transgenic mice where more than
30% of the rod domain and all of the V2 domain had been replaced by a lacZ fusion
protein, led to a phenotype resembling EBS (Vassar et al., 1991). Like EBS-DM, electron
microscopy showed the presence of electron dense aggregates within basal keratinocyte
cytoplasm (Vassar et al., 1991). Mice in which only 50 amino acids had been removed
from the carboxy terminus of the protein displayed no skin blistering or basal cell
cytolysis, although keratin aggregates were seen in some basal cells. This showed that
phenotypic severity can depend on the particular mutation involved (Vassar et al., 1991).
Further transgenic experiments revealed that different K14 mutations could generate
phenotypes of mild and severe blistering in mice (Coulombe et al., 1991b).
Linkage analysis provided further support to K5 and K14 as candidate genes for EBS.
Genetic linkage mapped the EBS-K gene to the type I keratin gene cluster on chromosome
17q and later, the causative mutation in this family (L384P) was discovered within the 2B
segment of the K14 rod domain (Bonifas et al., 1991a; Bonifas et al., 1991b). Mutations
encoding different amino acid substitutions within the same codon in K14 (R125H/
R125C) were also identified in EBS-DM cases (Coulombe et al., 1991a). These mutations
involved the 125th amino acid, located within the highly conserved helix initiation motif
of the K14 polypeptide (Coulombe et al., 1991a). Arginine 125 is the 10th codon within
the 1A domain and contains a CpG sequence which is conserved in most type I keratins
(McLean and Lane, 1995). Many mutations have now been found to affect analogous
residues in different keratins, showing this to be a mutation hot spot. The first mutation in
K5 was revealed in a large family with EBS-DM. This mutation was found within the helix
termination motif of the K5 helix 2B domain (Lane et al., 1992). Mutations in the K5 and
K14 genes associated with EBS-WC soon followed (Chan et al., 1993; Humphries et al.,
1993; Rugg et al., 1993). Mutations in the milder forms of EBS have been found to occur
outside the highly conserved helix boundary motifs and filaments examined by electron
microscopy appear normal (McLean and Lane, 1995). To date, there have been numerous
reports of mutations in the K5 and K14 genes in dominant EBS families, reviewed
(Corden and McLean, 1996). Pre-natal diagnosis has now been carried out for the
Dowling-Meara form of EBS, using PCR amplification and sequencing of genomic DNA
(Rugg et al., 1997).
More recently a heterozygous point mutation, P25L, was found in the non-helical V1
domain of K5 in individuals from a number of unrelated families with EBS with mottled
pigmentation (EBS-MP) (Irvine et al., 1997b; Uttam et al., 1996). Although keratin
aggregates are not characteristic of this disorder, organelle distribution was reportedly
aberrant in the basal keratinocytes from affected individuals (Uttam et al., 1996). Filament
assembly studies carried out in vitro with the mutant K5 revealed subtle effects on the
length of the 10nm keratin filaments. Expression of the mutant K5 within transfected
PtK2 cells which express K8 and K18, produced keratin filament networks indistinguishable
from wild-type (Uttam et al., 1996). How a mutation within this domain acts to weaken
the keratin filaments is unknown but it indicates a subtle, previously undetected role for
the V1 domain in filament dynamics. It is also unknown how this mutation leads to
pigmentary changes, however the slight change in the keratin network within
keratinocytes may influence longevity of the melanin granules, or possibly efficiency of
melanin transfer from the melanocytes (Uttam et al., 1996).
Recessive Forms of EBS—Nature’s Gene Knockout

Experiments
Reports of recessive EBS are less common than those exhibiting autosomal dominant
inheritance, due to the requirement of two mutant alleles in order for the phenotype to
be displayed. One inbred kindred has been described with a very mild recessive form of
Weber-Cockayne EBS (Hovnanian et al., 1993). Sequence analysis of affected individuals
revealed a homozygous point mutation (E144A) within the 1A domain of K14, outside the
helix initiation motif. Heterozygous carriers of this mutation were clinically normal.
There has been no other report of a mild recessive form of EBS such as this to date. This
may be due to the mild phenotype which may seldom present clinically and/or the rarity
of recessive EBS.
Following this paper describing a kindred with a mild form of recessive EBS, came two
reports of recessive EBS where affected individuals had severe generalised blistering due
to complete ablation of K14 expression (Chan et al., 1994; Rugg et al., 1994). In both
cases electron microscopy revealed a lack of tonofilament bundles within the basal
keratinocyte cytoplasm of affected individuals. K5 was detectable by
immunohistochemistry, immuno-electron microscopy and immunoblotting, but K14
expression was completely absent. These patients were clinically described as EBS-
Köbner, however disease severity was comparable to EBS-DM.
In one kindred, the affected individual was the offspring from a consanguineous
marriage, with a homozygous 2-nucleotide deletion mutation (313de12). This mutation
led to the formation of a premature termination codon, with predicted truncation of K14
within the V1 domain (Rugg et al., 1994). Nonsense-mediated decay of K14 mRNA
within the patient’s skin is thought to have rendered the message unde tec table.
Simultaneously, there was a report of a child from a consanguineous marriage with a
homozygous nonsense mutation (Y204X) in K14 (Chan et al., 1994). This mutation led to
a premature termination codon within the 1B domain of K14 and expression of the K14
protein was again undetectable. The phenotype of the affected individual in this second
kindred was very similar to the affected individual described by Rugg and colleagues.
These two reports described the first “knockouts” of a major epidermal keratin in
humans, however, K14 was subsequently ablated in mice by gene targeting. Lack of K14
in homozygous mice was found to be fatal by three months of age. Fatality was not caused
by the skin blistering itself which recovered at the time of hair growth, but by
oesophageal damage (Lloyd et al., 1995). From these results in K14 “knockout” mice,
prognosis for the recessive EBS patients (Chan et al., 1994; Rugg et al., 1994) was
expected to be poor. Two other severe recessive EBS families have since been described with
the causative mutation again leading to ablation of K14 expression, due to a homozygus
splicing mutation (Jonkman et al., 1996) and a homozygous nonsense mutation (Corden et
al., 1998). In the family described by Jonkman and colleagues, (Jonkman et al., 1996),
there were two elderly affected persons. This showed that the prognosis for K14
knockout in humans is much better than in mice, illustrating that caution should be used
in predicting human phenotypes based on mouse models. There have been no recessive
mutations published in the K5 gene to date.
In all of the above kindreds affected by severe, generalised recessive EBS, the
heterozygous individuals do not display the disease phenotype. This indicates that
expression from one normal allele is sufficient for normal keratin function. In terms of
gene therapy for such recessive forms of EBS, this implies that introduction of one normal
allele into the keratinocytes of the homozygous affected individuals should be sufficient to
rescue the disease phenotype.
Mutations in K1 and K10 Cause The Skin Thickening

Disease BCIE
Bullous congenital ichthyosiform erythroderma (BCIE), also known as epidermolytic
hyperkeratosis (EH), was the second disease affecting the skin for which mutations in
keratin genes were found. BCIE is characterised by blistering and erythroderma, which
develop in infancy, and the onset of severe generalised epidermolytic hyperkeratosis in
adulthood. The skin of an adult with BCIE is shown in Figure 3.6b. By EM, the basal cells
of the epidermis appear normal in comparison to EBS skin, however there is suprabasal
cytolysis, as shown in Figure 3.7. Tonofilament aggregates within the suprabasal cells of
patients affected by BCIE were found to label with K1 and K10 antibodies (Ishida-
Yamamoto et al., 1992). Transgenic mice were engineered, expressing a mutant K10 gene
with a phenotype resembling BCIE/EH (Fuchs et al., 1992) and genetic linkage of this
disorder to the type II keratin gene cluster on chromosome 12q was demonstrated
(Bonifas et al., 1992; Compton et al., 1992).
Subsequently, mutations were found in the K1 and K10 genes (Cheng et al., 1992;
Chipev et al., 1992; Rothnagel et al., 1992). Like those found to cause EBS-DM, these
mutations clustered in or near the highly conserved helix boundary motifs of K1 and K10.
Further mutations have been published within the helix boundary motifs in association
with BCIE/EH, reviewed by (Corden and McLean, 1996) and PCR-based prenatal diagnosis
has been carried out (Rothnagel et al., 1994a).
BCIE can also occur as a nevoid condition, where epidermolytic hyperkeratosis follows
the lines of Blaschko. Heterozygous K10 point mutations, similar to other mutations
causing classic BCIE, have been detected in the affected skin of several nevoid cases.
However no mutation was found within unaffected skin from these individuals, indicating
that this condition may be explained by a post-zygotic mutation occurring early in
development (Moss et al., 1995; Paller et al., 1994). It is important to note that the
offspring of individuals with epidermal nevi may suffer from generalised BCIE/EH, if the
cell line containing the keratin mutation contributes to the germline of the parent.
It is not yet understood why a defective keratin cytoskeleton should lead to
hyperkeratosis (Williams and Elias, 1993), although it is thought that the cytolysis of
suprabasal cells leads to cytokine release, which causes over-proliferation of the basal cell
layer beneath (Stoof et al., 1994). All of the keratin diseases reported to date involve some
degree of hyperkeratosis, although less evident in EBS where the basal cell compartment
is destroyed through cytolysis. Nevertheless, palmoplantar hyperkeratosis can accompany
EBS-DM (Chan et al., 1996) and epidermolytic hyperkeratosis of other body sites has been
seen in recessive EBS (Jonkman et al., 1996). It has previously been reported that a K5/
K14 network is a pre-requisite for the formation of a normal K1/K10 network, which
might explain how mutations in K5/K14 could have a detrimental effect in suprabasal
cells (Kartasova et al., 1993).
Alternate Phenotypes Caused by K1 Mutation

One family with diffuse non-epidermolytic palmoplantar keratoderma (NEPPK) has been
reported with a missense mutation outside the K1 rod domain (Kimonis et al., 1994). This
mutation occurs within the ISIS box, a 22 amino acid motif conserved within the V1
domain of several type II keratins (Kimonis et al., 1994). The lysine to isoleucine
substitution generated by this mutation, occurs in an invariant lysine residue within the
ISIS box consensus sequence. This lysine residue in the type II keratins has been found to
be frequently involved in the formation of intermolecular cross-links during cornified cell
envelope formation (Marekov and Steinert, 1995). The ISIS box sequence within the
amino terminal head of type II keratins is thought to be involved in binding of the
desmoplakin tail (Kouklis et al., 1994), and so the K73I mutation may act by interfering with
the interaction of K1 with other molecules.
Ichthyosis Bullosa of Siemens and K2e Mutations

Ichthyosis bullosa of Siemens (IBS) is a type of mild epidermolytic ichthyosis localised
mainly on the flexures (Siemens, 1937). The outer layers of the epidermis undergo a
characteristic moulting/shedding, not seen in patients with EH/BCIE (Siemens, 1937). In
a number of kindreds, IBS was found to be linked to the type II keratin gene cluster on
chromosome 12q (Steijlen et al., 1994). Tonofilament aggregation and cytolysis are both
characteristic of this disorder, but these phenomena are restricted to the upper spinous
and granular layers of the epidermis, unlike BCIE (McLean et al., 1994). K2e expression
was found to closely mirror the distribution of affected cells in IBS (Collin et al., 1992),
which suggested that this was a candidate gene for this disease. Mutations were initially
found in the K2e gene in six families affected with IBS, although two of the families were
mis-diagnosed as having EH (Rothnagel et al., 1994b). These mutations affected codon
493 within the helix termination motif of K2e (Rothnagel et al., 1994b). Five of the
mutations were identical (E493K), and are likely to represent a hot spot for mutations
within this gene, involving a CpG dinucleotide. Further cases of this E493K mutation have
been found, as well as other mutations in both helix boundary motifs, reviewed by
(Corden and McLean, 1996).
K9 Mutations Cause EPPK—Fragility of Palm and Sole Skin

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant skin disorder
characterised by diffuse thickening of the epidermis on the entire palmar and plantar
surfaces, bounded by erythematous margins (Vörner, 1901). The expression of K9
specifically within the suprabasal keratinocytes of the palm and sole led to this gene becoming
the candidate for EPPK (Langbein et al., 1993). The gene for EPPK was initially mapped
to the type I keratin gene cluster on 17q12–21 in a large German kindred (Reis et al.,
1992). Reis and colleagues then went on to discover three different mutations within the
K9 gene, all in the helix initiation motif (Reis et al., 1994). One mutation was found in
five unrelated kindreds (R162W), the type I keratin CpG hotspot mutation in the helix
initiation motif (McLean and Lane, 1995). Further mutations have been found within the
K9 gene, all involving the 1A rod domain, as reviewed (Corden and McLean, 1996).
Pachyonychia—Thick Nails due to Mutations in K6a, K6b,

K16 & K17
Pachyonychia congenita (PC) is a group of autosomal dominant ectodermal dysplasias
characterised by hypertrophie nail dystrophy with various constellations of other
ectodermal abnormalitites. There are two main sub-types of PC (McKusick, 1998). The
Jadassohn-Lewandowsky variant or type 1 (PC-1) involves pachyonychia in conjunction
with severe non-epidermolytic palmoplantar hyperkeratosis, hyperhidrosis, infrequent
blistering, oral leucokeratosis and follicular keratoses (Jadassohn and Lewandowsky,
1906). The Jackson-Lawler form or PC-2, has minor oral involvement but displays multiple
pilosebaceous cysts (Jackson & Lawler, 1951). This form of the disease may also be
associated with pili torti (twisted hairs), natal teeth (some teeth prematurely erupted at
birth) and other ectodermal abnormalities (McLean et al., 1995). The fingernails of a
patient with PC-1, carrying a mutation in K6a, are shown in Figure 3.8.
Genetic linkage to the type I keratin gene cluster was obtained in a large Scottish
kindred with PC-2 (Munro et al., 1994). The mutation in this family was found to be a
heterozygous missense mutation N92D in the K17 gene (McLean et al., 1995). A missense
mutation L130P was simultaneously reported in the K16 gene, in an individual with
sporadic PC-1 (McLean et al., 1995). Both of these mutations occur within the highly
conserved helix initiation motif of the specific type I keratin. Further K17 mutations have
been reported in PC-2 kindreds and also in families diagnosed with steatocystoma
multiplex (Covello et al., 1998; Smith et al., 1997). Kl6 mutations have been found in
families suffering from focal non-epidermolytic palmoplantar keratoderma (FNEPPK) and
extremely subtle nail changes were found in the affected individuals (Shamsher et al.,
1995). Mutations were subsequently reported in PC-1 cases in K6a, the type II keratin
partner of K16 (Bowden et al., 1995; Smith et al., 1998b).
One surprising result has emerged from the study of PC-2. K17 was not known to be
specifically expressed in conjunction with any other keratin and was thought to
polymerise with K5 in basal cells and K6 or another type II protein, when expressed
suprabasally (Lane, 1993). Thus, there was no type II keratin in which mutations could be
predicted to produce a PC-2 phenotype and many PC-2 families were shown to have K17
defects, further implying genetic homogeneity. However, a PC-2 family was recently
identified where no K17 mutation could be found, but genetic linkage was obtained to
markers in the type II keratin cluster and subsequently, a mutation was identified in K6b
(Smith et al., 1998a). The PC-2 phenotype in the K6b family was very similar to
individuals carrying K17 mutations. K17 and K6b were shown to be co-expressed in many
epidermal appendages, confirming that these proteins are expression partners in general,
although they differed in a few places (Smith et al., 1998a). Thus, the study of this keratin
disorder revealed the previously undiscovered role of K6b in epithelial biology.
There are at least six highly homologous copies of the K6 gene (K6a–f), at least four of
which are expressed (Takahashi et al., 1995). To date, mutations are known in only two
of these genes, K6a and K6b. It is not yet known what phenotypes might arise from
mutations within the remaining expressed K6 isogenes.
K4 and K13 Mutations Cause Fragility of Mucosal Tissues

White sponge nevus of Cannon (WSN) is an autosomal dominant disorder which affects
non-cornifying stratified squamous epithelia (McKusick, 1998). White “spongy” plaques
are found in the mouth, as seen in Figure 3.8, and to a lesser extent in the oesophagus
and anogenital mucosa (Jorgenson and Levin, 1981). As K4 and K13 are specifically
expressed within these tissues, they became the candidate genes for this disorder (Moll et
al., 1982). Two Scottish families were found to carry a mutation in K4 (Rugg et al., 1995)
and the complementary mutation in K13 was published simultaneously (Richard et al.,
1995). Mutations in these two genes were the first found to affect a tissue other than the
epidermis and its appendages.
K18 Mutation May Cause Liver Disease Predisposition

Expression of K18 carrying a missense mutation in the 1A domain was found to produce
filament aggregation (Ku et al., 1995). Transgenic mice expressing mutant K18 developed
chronic hepatitis and hepatocyte fragility (Ku et al., 1995). These experiments indicated
that liver defects within humans might also be caused by mutations in either of the simple
epithelial keratins, K8 or K18. One heterozygous K18 mutation has been published,
H127L, which affects the last amino acid of the L1 domain and is believed to predispose
towards or be responsible for the development of cryptogenic cirrhosis within the patient
(Ku et al., 1997). Mutations in this domain, which is less well conserved at the sequence
level and whose function is currently unclear, have not been reported in any other keratin
disorder. However, in vitro assembly studies with this K18 mutant showed defective
filaments by electron microscopy, giving some indication that this mutation may be
pathogenic (Ku et al., 1997).
Monilethrix—Beaded Hair Due to Mutations in Hair

Keratins
Monilethrix is an autosomal dominant hair disorder showing variable phenotype (Winter
et al., 1997a; Winter et al., 1997b). Hairs from affected individuals have a characteristic
beaded appearance and so trichocyte hair keratins were good candidate genes. The first
monilethrix mutations were found in the type II hair cortex keratin, hHb6. Again, these
mutations involved amino acid substitution in the helix termination motif (Winter et al.,
1997b). A mutation in another type II hair keratin, hHbl, also in the helix termination
Figure 3.8 Clinical appearance of three diverse disorders of differentiation-specific keratins, (a)
The hypertrophie nail dystrophy which is a major characteristic of pachyonychia congenita type
PC-1 and PC-2, seen here in a PC-1 patient with a K6a mutation. Mutations in four keratins, K6a,
K6b, K16 and K17 produce variants of this disease, (b) The buccal hyperkeratosis seen in white
sponge nevus syndrome (WSN), due to mutations in mucosal keratins K4 or K13. (c)
Retroillumination slit lamp photo-graph of microcysts within the corneal epithelium (arrows) in a
patient with Meesmann’s corneal dystrophy (MCD), caused by mutations in keratins K3 or K12.
Thanks to Dr Kevin McKenna, Department of Dermatology, Craigavon Area Hospital, Northern
Ireland; Dr Colin Munro, Department of Dermatology, Southern General Hospital, Glasgow and Drs
Ole and Beate Swensson, Departments of Dermatology and Ophthalmology, Christian-Albrechts-
Universität, Kiel, Germany for kindly providing these illustrations.
motif, has been described in a family with monilethrix (Winter et al., 1997a), as well as
further mutations in hHb6 (Korge et al., 1998).
Focusing on K3 and K12 Mutations in Meesmann’s Corneal

Dystrophy
The anterior epithelium of the cornea is the outermost protective layer of the eye and
consists of about four layers of stratified, non-cornified keratinocytes which
predominantly express cornea-specific keratins K3 and K12 (Schermer et al., 1986).
Given that mutations in epidermal keratins produce skin fragility disorders, it was logical
to think that mutations in K3 or K12 would produce a corneal fragility disease. At the end
of 1996, our group considered the corneal dystrophies, of which there are many types
(Grayson, 1983), as candidates for K3 and K12 mutations. However, Meesmann’s
corneal dystrophy (MCD), a clinically mild disorder characterised by fragility of the
anterior corneal epithelium (Meesmann and Wilke, 1939), seemed to be the best
candidate for a variety of reasons. Firstly, it is autosomal dominant, like most of the
keratin diseases. Secondly, MCD affects only the anterior corneal epithelium, the single
tissue where K3 and K12 are specifically expressed. Thirdly, there had been a number of
ultrastructural reports of intracellular aggregates of unknown composition in MCD
corneocytes (Tremblay and Dube, 1982). Although there had been no suggestion in the
literature that these were keratin aggregates, this was nevertheless a valuable clue. The
clinical appearance of an MCD cornea, myriads of microcysts seen by slit-lamp
examination, is shown in Figure 3.8. Analysis of two Northern Irish families and a large
pedigree representing the descendants of Meesmann’s original German family gave
positive genetic linkage to the keratin loci on chromosomes 12 and 17 (Irvine et al.,
1997a). Mutations were found in the helix boundary motifs of both K3 and K12 in these
initial families (Irvine et al., 1997a). Further K12 mutations have also been reported in
Japanese MCD kindreds (Nishida et al., 1997). Overall, these results revealed the role of
K3 and K12 in maintaining the structural integrity of corneal keratinocytes as well as the
underlying molecular pathology in MCD.
THE FUTURE OF KERATIN GENETICS RESEARCH
More Keratins, More Associated Molecules, More Diseases

Since the discovery of the first human keratin mutations at the end of 1991, a large part of
the mutation-based research has been focused on the identification of further keratin
diseases and in determining the complete range of mutations which are pathogenic in
keratins. There are two main outcomes from this research. Firstly, there are now 17
keratins known in human disease and this list will probably continue to grow until there is
a genetic disease association for all keratins. Secondly, certain molecular features of
keratins have come to light as being structurally important due to the emergence of
mutation clusters in these regions, as shown in Figure 3.9. This has been particularly
surprising in some cases, such as the mutations in the linker region L12 of K5 and K14 in
mild forms of EBS (Rugg et al., 1993); the discovery of the ISIS motif due to NEPPK
mutation in K1 (Kimonis et al., 1994); and mutations at the start of the V1 domain of K5
in EBS-MP (Irvine et al., 1997b; Uttam et al., 1996). Further mutation clusters may
emerge with the study of more mutations, particularly in EBS, which might shed light on
the structural interactions of intermediate filament molecules. It is interesting to note that
outside of K5 and K14 in EBS, most mutations in all other keratins have been found only
in or near the helix boundary motifs, reviewed in (Corden and McLean, 1996). One
explanation for this anomaly is that subtle mutations in the keratins of basal cells are not
tolerable compared to other epithelia, perhaps because other epithelia express more
keratin pairs, which might have a dilution effect of the mutant polypeptide. Alternatively,
it may well be that mild mutations in other keratins produce more subtle phenotypes
which have not yet been identified clinically.
Another area for future research in this field is the study of pathogenic mutations in
keratin-associated proteins. There are many molecules involved in the anchorage of the
intermediate filament cytoskeleton to transmembrane plaques and other subcellular
systems, as discussed elsewhere in this book. Of these, only two molecules have so far
been shown to have human disease associations, plectin and plakophilin 1 (McGrath et al.,
1997; McLean et al., 1996; Smith et al., 1996). In both cases, invaluable information on
the biological role of these molecules has been a direct consequence of the disease studies.
Although plectin is very widely expressed in both epithelial and non-epithelial tissues, loss
of the protein in humans produces only skin blistering and muscle disease, showing that its
function is vital only in the context of these tissues. Similarly, through the discovery of
plakophilin 1 mutations in a human ectodermal dysplasia, this protein has been shown to
be a key player in mediating the connection of keratins to the cytoplasmic side of the
desmosomal plaque. Future years will undoubtedly see the discovery of human mutations
in further keratin-associated proteins.
Prevention and Therapy of Keratin Diseases

Although the study of keratin diseases has been valuable in understanding the basic biology
of these protein systems, this work has also enhanced patient care for these disorders
through the application of molecular diagnostic techniques. Some keratin diseases, such as
MCD or WSN are so mild in their effects that demand for prenatal testing is low. In other
disorders with more devastating phenotypes, such as EBS-DM and BCIE, affected families
do request prenatal testing. In these cases, knowledge of the mutation in a particular
family allows prenatal diagnosis by chorionic villus biopsy to be done at a very early stage
of pregnancy. These preventative measures for severe keratin disorders are of no use in
prevention of the sporadic mutations which account for many cases of keratin disease and
so there will always be a need for improved treatment. Gene therapy is made doubly
difficult in the case of keratins since most mutations are dominant-acting, so that gene
Keratin Dominant-Negative Mutation Clusters
Figure 3.9 Clustring of dominant-negative mutations in all keratin diseases within the type I and II protain domain structures. actual numbers of mutations
per domain are shown, as of mid-1998. Red bar indicate mutations affecting the helix boundery motif sequences, which can be seen to be mutation
hotspots. Mutations outside these highly conserved regions have been shown to result in milder desease phenotypes.
deactivation rather than replacement therapy must be developed. Despite these

difficulties, some research groups are currently engaged in the search for novel gene
therapy agents for dominant genetic diseases (Millington-Ward et al., 1997; Montgomery
and Dietz, 1997). The increasing body of knowledge of both the genetics and the basic
biochemistry of keratins, together with advances in gene therapy for other genetic
diseases, should lead to improved treatment for keratin disorders in the new millenium.
ACKNOWLEDGEMENTS
Thanks to our many friends who provided figures. Original work from this group was
supported by grants from The Wellcome Trust (037444/A/93/Z, to E.B. Lane,
I.M.Leigh and R.A.J.Eady) ; Cancer Research Campaign (grant SP2060, to E.B.Lane);
The Dystrophic Epidermolysis Bullosa Research Associations (DEBRA) of U.K. and
America (WHIM); and by the U.S. Public Health Service, National Institutes of Health
(grant PO1-AR38923, to J.Uitto). The authors are currently supported by a Wellcome
Trust Senior Research Fellowship (to WHIM) and grants from DEBRA UK (WHIM).
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4.
DESMOSOMES
DAVID R.GARROD, MARTYN A.J.CHIDGEY, ALISON J.NORTH,
SARAH K.RUNSWICK AND CHRIS TSELEPIS
INTRODUCTION
Desmosomes are punctate, adhesive intercellular junctions that bind cells together and
provide membrane anchoring points for the intermediate filament cytoskeleton. They are
circular membrane domains of up to 0.5 µm in diameter. The intercellular material or
desmoglea occupies a region of about 30 nm wide between the apposed plasma
membranes. It has a highly organised ultrastructure consisting of an electron-dense mid
line from which cross-bridges extend to the plasma membranes. Close to the cytoplasmic
faces of the plasma membranes are dense outer plaques. These are approximately 20 nm
in thickness and are the most consistent and easily-recognisable feature of these junctions.
At about 50 nm from the plasma membranes are less dense inner plaques that appear to
associate with the intermediate filaments. Thus the total thickness of a desmosome from
one inner plaque to the other is approximately 130 nm. Desmosomes may be thought of
as the “scaffold couplings” that link the intermediate filament cytoskeleton throughout a
tissue.
Widely distributed, desmosomes are especially abundant in stratified epithelia where
strong intercellular adhesion is required to resist external friction. They are present in
almost all epithelia and also in cardiac muscle, the arachnoid and pia of the meninges, and
the follicular dendritic cells of the lymphoid system.
Desmosomes are macromolecular complexes consisting of at least six interacting
proteins (Figure 4.1, Table 4.1). Their adhesion molecules are the desmocollins (Dscs)
and desmogleins (Dsgs), members of the cadherin family of calcium-dependent adhesion
molecules. These transmembrane proteins have their extracellular domains in the
desmoglea and their cytoplasmic domains in the outer dense plaque. The outer plaque
contains two proteins that are members of the armadillo family named after the Drosophila
segment polarity signalling gene. These are plakoglobin (Pg), also known as γ catenin and
present in other intercellular junctions, and plakophilin (Pp). The outer and inner plaques
are bridged by the plakin family member desmoplakin (Dp), which mediates interaction
between the adhesive domain and the intermediate filaments (reviewed by Schwartz et al.,
1990; Buxton and Magee, 1992; Legan et al., 1992; Garrod, 1993; Garrod et al., 1996;
Green and Jones, 1996; Chidgey, 1997; Garrod et al., 1998; Burdett, 1998).
D.R.GARROD ET AL. 57
Figure 4.1 Cartoon showing major components of desmosomes. ML=mid-line, PM=plasma

membranes OP=outer plaque, IP=inner plaque, IF=intermediate filaments. The numbers at the
bottom of the picture indicate distances in nanometres from the plasma membranes of the various
plaque components as determined by quantitative measurements from immunogold labelling with
domain-specific antibodies. Dsc=desmocollin, a=“a” form, b=“b” form; Dsg=desmoglein;
DP=desmoplakin, PG=plakoglobin; PP=plakophilin. N=N-terminus; C=C-terminus. Only one half
of the desmosome is shown: it is symmetrical about ML. The intercellular space is not drawn to
scale as it would be almost 50% wider if shown at the same magnification as the plaque region.
The most recent additions to the tally of desmosomal components are the two plakin
family members, envoplakin and periplakin (Ruhrberg et al., 1996; .
Ruhrberg et al., 1997; Ruhrberg and Watt, 1997), which although not exclusively
epidermal, appear to be of considerable importance in cornified envelope formation.
DESMOSOMAL GLYCOPROTEINS
Desmosomal Glycoproteins and Cell Adhesion

Desmosomes have long been regarded as points of strong adhesion between cells. Apart
from their very “solid” appearance by electron microscopy, this view has been based on
their particular abundance in stratified epithelia, such as epidermis, which are subject to
substantial frictional forces, and to the loss of adhesion (acantholysis) that occurs in the
group of autoimmune epidermal blistering diseases known as pemphigus, where loss of
adhesion is caused by antibodies against desmosomal glycoproteins (Stanley, 1993; Stanley
and Kárpáti, 1994). Recent experimental demonstrations of the importance of
desmosomal adhesion in the epidermis and the heart have been achieved, respectively, by
dominant negative and null mutations of the pemphigus vulgaris antigen Dsg3 (Allen et
58 D.R.GARROD ET AL.
al., 1996; Koch et al., 1997) and plakoglobin (Ruiz et al., 1996; Bierkamp et al., 1996).
Furthermore, the first human desmosomal mutation, effectively a Pp1 knockout, gives
rise to loss of adhesion within the epidermis (McGrath et al., 1997).
It has always been assumed that desmosomal adhesion is mediated by the major
desmosomal glycoproteins, Dsc and Dsg. Two early pieces of evidence supporting this
view were, firstly, that the glycoproteins were greatly enriched in isolates of desmosomal
cores (Gorbsky and Steinberg, 1981) and, secondly, that inhibition of desmosome
assembly could be obtained in MDBK cells using high concentrations of Fab’ fragments of
anti-Dsc antibodies (Cowin et al., 1984). The adhesive roles of these glycoproteins
seemed even more likely when it was shown that both were members of the cadherin
family of calcium-dependent adhesion molecules (Holton et al., 1990; Koch et al., 1990).
Despite all these indications, it has proved extremely difficult to provide experimental
proof of the adhesive roles of Dsc and Dsg. Classical cadherins generally participate in
homophilic, calcium-dependent cell-cell adhesion (Nose et al., 1990; Shapiro et al., 1996;
Nagar et al., 1996). Such homophilic adhesion by Dsc or Dsg is, however, negligible
(Amagai et al., 1994a; Chidgey et al., 1996; Kowalcyzk et al., 1996). Recent work by
three groups has now provided definitive evidence for mediation of cell-cell adhesion by
desmosomal glycoproteins and some indication of how it may operate (Chitaev and
Troyanovsky, 1997; Marcozzi et al., 1998; Tselepis et al., 1998).
The human fibrosarcoma cell line HT1080 adheres by means of adherens junctions
mediated by N-cadherin, but also synthesises Dsg2 (Sacco et al., 1995). Chitaev and
Troyanovsky (1997) utilized transfection of these cells with bovine Dscl and human Pg to
study desmosomal adhesion. Marcozzi et al., (1998) and Tselepis et al., (1998) used
multiple transfection of the non-adhesive L929 mouse fibroblast line to generate
adhesion. These studies suggest a number of novel conclusions about desmosomal
adhesion, as follows:-
(i)
Both Dsc and Dsg are required for intercellular adhesion.
Previous work had shown that expression of single desmosomal glycoproteins in L929
cells did not generate adhesion (Amagai et al., 1994b; Chidgey et al., 1996; Kowalcyzk et
al., 1996). Both Marcozzi et al., (1998) and Tselepis et al., (1998) were able to generate
extensive aggregation of these cells by simultaneous expression of Dsc, Dsg and Pg. These
results suggest that both Dsc and Dsg are required in order to generate adhesion.
(ii)
Dsc and Dsg can interact heterophilically.
Chitaev and Troyanovsky (1997) elegantly demonstrated heterophilic interaction between
Dsc and Dsg. They transfected one population of Dsg-expressing HT1080 cells with Dsc
which was truncated cytoplasmically to remove the Pg binding domain. Another
population of the same cells was transfected with myc-tagged Pg. The two cell
populations were co-cultured, then solubilized and immimoprecipitated with anti-Dsc
Table 4.1 The Principal Components of Desmosomes
For further details, other components and references, see text.

antibody. It was found that myc-Pg co-precipitated with Dsc, a result explicable only in
terms of interaction between Dsc and Dsg on adjacent cells. By contrast, Marcozzi et al.
(1998) were unable to precipitate Dsc-Dsg complexes, and Tselepis et al. (1998) found no
convincing evidence for adhesive interaction between L cells expressing the different
individual glycoproteins.
(iii)
Cytoplasmic interaction with Pg is required for adhesion
Marcozzi et al. (1998) demonstrated that adhesion between L929 cells expressing both
desmosomal glycoproteins took place when the cells were further transfected with Pg.
Tselepis et al. (1998) demonstrated co-localization of Dsc, Dsg and Pg at the cell surface
in transfected L929 cells, but detected no evidence of junction assembly by electron
microscopy.
(iv)
Different glycoprotein isoforms and desmosomal glycoproteins from different
species interact
Dsc and Dsg each occur as three different isoforms which are the products of different
genes. These isoforms are called Dsc1, 2 and 3, and Dsgl, 2 and 3. Dsc2 and Dsg2 are
ubiquitous and occur in all desmosome-bearing tissues, but Dscl and 3 and Dsgl and 3
occur principally in stratified epithelia (Legan et al., 1994; Nuber et al., 1995; Schäfer et
al., 1994). Marcozzi et al. (1998) used the human Dsgl and Dsc2 isoforms in their
transfection experiments showing that these can participate in mutual interaction. Chitaev
and Troyanovsky (1997) demonstrated interaction between human Dsg2 and bovine Dscl
indicating that not only different isoforms but also proteins from different species can
interact. The latter result is consistent with previous studies showing desmosome
formation between cells of a variety of different species and phyla including anuran
amphibians, birds, and mammals (Overton, 1977; Mattey and Garrod, 1985). North et al.
(1996) showed that where different desmocollin isoforms are expressed in the same cells
in epidermis, they occur in the same individual desmosomes. It is likely that desmoglein
isoforms are similarly distributed (Shimizu et al. 1995). In view of the above experiments,
it is probable that different glycoprotein isoforms interact in vivo.
(v)
Cell adhesion recognition (CAR) sites function in desmosomal glycoprotein
interaction
E- and N-cadherin possess tripeptide sequences (the CAR sites) in their N-termi nal
regions that have been shown to be involved in adhesive binding (Blaschuk et al., 1990). In
these molecules the tripeptide sequence is -histidine-alanine-valine- (HAV). The
desmosomal cadherins have similarly located, related sequences, which have previously
been referred to as putative CAR sites. In bovine Dscl and Dsgl these sites are YAT and
RAL, respectively. Tselepis et al. (1998) showed that 10mer peptides centred around
these putative CAR sites blocked desmosomal adhesion. The peptides were effective at
concentration of 0.5 mM. Furthermore, the Dsc peptide and the Dsg peptide were
equally effective in inhibiting adhesion, and either peptide alone was as effective as the
two together. This demonstrates that the CAR sites are functionally important in adhesion
mediated by desmosomal glycoproteins and supports the involvement of heterophilic Dsc-
Dsg interaction.
(vi)
The “b” form of Dsc is not essential for adhesion
All Dscs occur as two alternatively spliced variants with different sized cytoplasmic
domains, that of the “a” form being longer than the “b”. The “a” and “b” forms occur with
approximately equal stoichiometry in all desmosome-containing cells and tissue so far
examined. Troyanovsky et al. (1993) provided evidence that the cytoplasmic domain of
the “a” form contains the signals necessary for plaque assembly. Smith and Fuchs (1998)
showed that the three major plaque constituents Pg, Pp and Dp can all interact with “a”
form cytoplasmic domain. To obtain adhesion of L cells, Tselepis et al., (1998) expressed
both the “a” and “b” forms of bovine Dscl together with Pg and Dsg1. However, Marcozzi
et al. (1998) were able to obtain apparently similar adhesion using only the “a” form of
Dscs. It therefore appears that the “b” form is not essential for development of adhesion.
However, the “b” form is present in all desmosome-containing cells and therefore may
have an essential function, possibly in regulating the binding affinity and organisation of
desmosomes.
(vii)
Adhesion between desmosomal glycoproteins is at least partially calcium
dependent
Both Chitaev and Troyanovsky (1997) and Tselepis et al. (1998) showed that the adhesion
generated in their systems was partially abolished by removal of calcium, so that some
residual adhesion occurred even in the absence of calcium. Although desmosomal
assembly is calcium dependent (Hennings and Holbrook 1983, Watt et al., 1984; Mattey
and Garrod, 1986a), desmosomes can also exist in the calcium independent state (Mattey
and Garrod, 1986b). It is thus not entirely surprising to find that interaction between
desmosomal glycoproteins was not completely abolished by calcium removal.
(viii)
Clustering of Dsc and Dsg is dependent on the extracellular domains
Utilizing Dsc mutants where either the cytoplasmic domain or the N-terminal region of
the extracellular domain were deleted, Chitaev and Troyanovsky (1997) showed that
mutual clustering of Dsc and Dsg was dependent on the extracellular domain. Thus
clustering took place after deletion of the entire cytoplasmic domain but was abolished by
deletion of the N-terminal region of the extracellular domain.
(ix)
Classical cadherin-mediated adhesion is not required to initiate adhesive
interactions of desmosomal glycoproteins
The HT1080 cells used by Chitaev and Troyanovsky (1997) form adherens junctions via N-
cadherin. Their results are consistent with previous studies suggesting that cellular
interaction via cadherin is essential to general desmosomal adhesion and that there is cross
talk between adherens junctions and desmosomes (Wheelock and Jensen, 1992; Amagai
et al., 1995a; Lewis et al., 1997). It was possible therefore that adhesive interaction
generated by desmosomal glycoproteins in L929 cells was dependent on up-regulation of
a cadherin (Tselepis et al., 1998). Two lines of evidence suggest that this is not the case.
Firstly, adhesion was blocked by desmosomal glycoprotein CAR pep tides, but not by
corresponding conventional cadherin peptides. Furthermore, no up-regulation of
expression of cadherin or β-catenin could be detected by immunoblotting in transfected
L929 cells exhibiting desmosomal adhesion. Thus, adhesion between desmosomal
glycoproteins does not require prior interaction by conventional cadherins or adherens
junctions.
The results of these studies represent a substantial advance in our understanding of the
mechanism of desmosomal adhesion. However, many issues remain to be resolved. Like
conventional cadherins, the extracellular domains of the desmosomal cadherins consist of
five subdomains (EC1 to 5) each of approximately 100 amino acids. On the basis of
binding studies between recombinant proteins of different lengths, Chitaev and
Troyanovsky (1997), proposed a model for interaction in which the extracellular domains
of the molecules on apposing cells overlap by 3 EC domains. This is different from the
results of structural studies of E- and N-cadherins (Shapiro et al., 1996; Naga et al., 1996)
where interaction was shown to be between the N-terminal EC1 domains. These are the
domains that contain the HAV sites which are located on the faces of the molecules that
interact in adhesion (Shapiro et al., 1996). The demonstration that the desmocollin and
desmoglein CAR sites are involved in adhesion (Tselepis et al., 1998) suggests that
interaction between desmosomal glycoproteins may resemble that seen between
conventional cadherins. In addition, the distance between the desmosomal plasma
membranes (25 to 35 nm) appears to correspond well with an interaction between the
apposed EC1 domains. Furthermore, although our pep tide inhibition studies on
transfected cells showed that both Dsc and Dsg peptides were equally effective in
inhibiting adhesion, and also as effective as the two peptides combined (Tselepis et al.,
1998), provisional experiments with desmosome-forming cells indicate that both peptides
are required to block desmosome assembly (Runswick, O’Hare, Streuli and Garrod,
unpublished). These results are not entirely consistent with the involvement of
heterophilic interaction between Dsc and Dsg in desmosome formation. It may be that the
interactions seen in transfected cells, which do not form desmosomes even though they
adhere to each other (Tselepis et al., 1998), do not mimic precisely the interaction seen in
whole desmosomes.
Cells in stratified squamous epithelia, transitional epithelia and squamous cells
carcinomas possess an additional desmosomal glycoprotein, the E-48 antigen. This 20- to
22 kDa, glycophospatidyl inositol anchored protein has been shown to function in cell-cell
adhesion by monoclonal antibody inhibition and transfection studies (Schrijvers et al.,
1991; Brakenhoff et al., 1995).
Cytoplasmic Interactions of Desmosomal Glycoproteins

In in vitro overlay assays Dsc la has been shown to bind to Pp1, Pg and the Dp N-terminus, ,
while Dsgl binds Pp1 and Pg, the former more weakly and the latter more strongly than
Dscla (Smith and Fuchs, 1998). The interactions of Pg with the cytoplasmic tails of the
desmosomal glycoproteins have been studied in detail by Troyanovsky and colleagues,
using expression studies in epithelial cells (principally A431) with chimeric proteins
consisting of the gap junction protein connexin 32 and the cytoplasmic domain of either
Dsc1a or Dsg1. These constructs target the desmosomal glycoprotein C-termini to the
plasma membrane as part of a gap junction-like structure, and the association of cytoplasmic
proteins Dp, Pg and keratin filaments (KF) can be studied by immunofluorescence,
immunoprecipitation and electron microscopy. The first use of this technique showed that
the Dscla cytoplasmic domain would support Pg, Dp and KF association and plaque
formation, and that Dsgl had a dominant negative effect leading to disruption of
endogenous desmosomes (Troyanovsky et al., 1993).
Deletion mutagenesis of the Dscla tail localised the site for Pg interaction to 37 amino
acids at the extreme C-terminus, and this deletion also abolished KF association with the
plaque (Troyanovsky et al., 1994a). Dp association was localised to a 10 amino acid region
of the Dscla cytoplasmic tail close to the plasma membrane. The dominant negative effect
of the Dsgl cytoplasmic domain was not abolished by deletion of 262 amino acids from the
extreme C-terminus, but further deletion of 41 amino acids abolished both the dominant
negative effect and Pg binding, thus localising the Pg binding domain to this 41 amino acid
stretch (Troyanovsky et al., 1994b). This Pg binding region of Dsgl corresponds to a
region that is conserved in other cadherins, including Dscla, where it located at the
extreme C-terminus (Chitaev et al., 1996). More detailed analysis of Dsg-Pg binding by
alanine scanning mutagenesis showed that a series of large hydrophobic amino acids in the
conserved region are involved in Pg interaction (Chitaev et al., 1998). Similarly it was
shown that nine hydrophobic amino acids within the arm repeats 1–3 of Pg are involved in
Dsg-Pg interaction (Chitaev et al., 1998). Chitaev et al. (1998) found a 1:1 stoichiometry
of Pg-Dsg binding, in contrast to the suggestion from immunoprecipitation data of a 6:1
Pg:Dsg binding ratio by Kowalczyk et al. (1996). If correct, the latter suggestion might
provide an explanation for the dominant negative effect of the Dsgl cytoplasmic domain,
since overexpression would be expected to sequester substantial amounts of Pg making
insufficient availability for junctional assembly. A further possibility for the dominant
negative effect of the Dsgl tail arose from the demonstration that expression in A431 cells
of chimeras of E-cadherin extracellular domain and Dsg1 tail resulted in a 3-fold decrease
inE-cadherin bound to plakoglobin and a 5- to 10-fold reduction in the steady state levels
of endogenous desmosomal cadherins, Dsg2 and Dsc2 (Norvell and Green, 1998).
Binding of Pp to desmosomal glycoproteins (Smith and Fuchs, 1998) is clearly of
functional importance since the human mutations described by McGrath et al. (1997)
result in weakened desmosomal adhesion as well as detachment of Dp and KF from the
desmosomal plaque.
Desmosomal Glycoproteins in Epidermis

The different isoforms of the desmosomal glycoproteins have distinct patterns of
expression in epidermis and other stratified epithelia (Arnemann et al., 1993; Legan et al.,
1994; King et al., 1995; Yue et al., 1995; North et al., 1996). In bovine epidermis, Dsc1 is
associated with the upper, terminally-differentiating layers but is absent from the basal
layer and the bases of the deep rete ridges. Dsc2 is most strongly expressed in cells
immediately above the basal layer that have begun terminal differentiation. This region of
greatest Dsc2 expression corresponds with the location in rete ridges of transit amplifying
cells, the most rapidly proliferating keratinocyte population (Lavkar and Sun, 1983). Dsc3
is strongly present in the basal layer and gradually fades in the suprabasal layers (Legan et
al., 1994; Yue et al., 1995; North et al., 1996). In human epidermis the expression
patterns of Dscs are similar (Arnemann et al., 1993; King et al., 1995; 1996). The desmoglein
patterns in epidermis are similarly differentiation-related with Dsg3 being most strongly
expressed in the basal regions and Dsg1 increasing in expression suprabasally (Arnemann
et al., 1993; Shimizu et al., 1995; Amagai et al., 1996). Curiously, Dsg2 appears to be
expressed predominantly in the basal layer (Theis et al., 1993).
An intriguing pattern of desmosomal glycoprotein expression is also found in mammary
epithelium. Here Dsc1 and Dsg1 are not expressed. Dsc2 and Dsg2 are present in both
lumenal and myoepithelial cells but Dsc3 and Dsg3 are confined to the latter (Runswick et
al., 1996), thus again occupying a basal location. Preliminary studies also reveal
interesting Dsc distributions in corneal epithelium. Dscl is absent from this non-
keratinizing epithelium, Dsc2 is expressed throughout, and Dsc3 is confined to the basal
and suprabasal limbal region where the corneal stem cells are believed to reside (Messent
et al., 2000).
Quantification of the distribution of Dscl and Dsc3 isoforms by immunogold labelling of
desmosomes at different levels in the epidermis, revealed that they are expressed in a
reciprocally graded manner, with Dsc3 decreasing suprabasally from the basal layer and
Dsc1 increasing (North et al., 1996). An inversely graded pattern of Dsg3 and Dsg1
expression in human epidermis has also been observed (Shimizu et al., 1995). It may be
inferred that different Dsg isoforms are also present in the same junctions, since at
intermediate levels of epidermis all desmosomes can be labelled using sera specific for
either Dsg1 or Dsg3.
These reciprocally-graded expression patterns suggest two conclusions. Firstly, since
their molecular composition changes, desmosomes in epidermis are presumably
continually turning over as the cells ascend through the layers. At present the functional
significance of this is not clear. It may be that changes in cell-cell adhesive properties are
important in regulating epidermal morphology and differentiation. Secondly, expression

of the different glycoprotein isoforms appears to be linked. All six human Dsc and Dsg
genes are located at chromosome 18q12, the mouse desmoglein genes are closely linked
in the proximal region of chromosome 18 and the bovine desmocollin genes cluster on
chromosome 24q21/q22 (King et al., 1995; Arnemann et al., 1992; Wang et al., 1994;
Amagai et al., 1995b; Simrak et al., 1995; Buxton et al., 1994; Solinas-Toldo et al., 1995).
This clustering may be required for the regulation of desmosomal glycoprotein gene
expression. The functional significance of these remarkable desmosomal glycoprotein
distributions in epidermis is an important area for future investigation.
During development of the mouse embryo three of the six desmosomal cadherin
isoforms (Dsc1, Dsc3 and Dsg1) have so far been shown to be first expressed in the
developing epidermis. In the mouse embryo, desmosomes first form at the 32-cell stage in
a trophectoderm-specific location coincident with the onset of cavitation and blastocoel
formation (Fleming et al., 1991). The only Dsc isoform present at this stage is Dsc2.
Embryonic Dsc2 gene expression first occurs at the 16-cell stage and is specific to the
trophectoderm lineage (Collins et al., 1995). Dsc2 proteins appear immediately after
detection of its mRNA and coincides with initial desmosome assembly (Fleming et al.,
1991; Collins et al., 1995). Presumably the Dsg isoform present is the ubiquitous Dsg2,
though this has not been directly demonstrated. Desmosome formation between
trophectoderm cells may be regulated by transcription of the glycoprotein genes although
studies on the Dsg genes will be required to confirm this view.
The desmocollin isoforms Dsc1 and Dsc3 are not expressed in the early mouse
embryo. Dscl is first detected in the epidermis of the external nares at E13.5 (King et al.,
1996; Chidgey et al. 1997). Dsc3 is initially expressed 12 hours earlier in nasal epidermis,
whisker and the most mature vibrissa follicles (Chidgey et al., 1997). Both genes are up-
regulated in the general body epidermis at E14.5. At the same time both isoforms are
expressed in the suprabasal layers of the newly stratified epidermis. However, by day 18.5
the region of maximum Dsc3 expression becomes basal whilst that of Dscl remains
suprabasal (King et al., 1996; Chidgey et al., 1997). Thus the adult pattern of Dsc
expression (see North et al., 1996) is established, simultaneously with the onset of the adult
pattern of differentiation including the basal location of cell proliferation and the
formation of stratum corneum. We have suggested the ratio of Dsc1 to Dsc3 expression
at different levels in the epidermis is fundamental to establishing this pattern of
differentiation (Chidgey et al., 1997). However, Dsg isoforms are also likely to play a role
in this process. It has recently been shown that the Dsgl gene is first expressed at day 13.5
and shows a regional pattern of induction similar to that of Dsc1, being present in the
outer cell layers of stratifying epidermis (King et al., 1996). An in situ hybridisation study
of desmosomal glycoprotein gene expression during mouse development concluded that
the DSC and DSCG genes are transcribed in hierarchical, overlapping temporal and
spatial patterns, and that the spatial order in which they are expressed correlates with the
physical order of the genes at the desmosomal cadherin locus (King et al., 1997)
Denning et al., (1998) investigated the regulation of Dsg isoform expression in cultured
human keratinocytes, HaCaT cells and squamous carcinoma cell lines. In particular they
showed that down-regulation of PKC by long term treatment with phorbol ester or
bryostatin 1 led to decreased expression of Dsgl and Dsg3 but not Dsg2. These results are
consistent with a role for PKC activation in regulation of the expression of Dsgl and Dsg3
in substantial cells.
Adams et al. (1998) expressed a 4.2kb region of the human DSG1 promotor linked to
the lacZ reporter gene in transgenic mice. They found that this region does not contain all
the regulatory elements necessary for correct expression of the gene but might have
elements that regulate activity during hair growth.
Desmosomal glycoproteins in the epidermis are the target antigens in the autoimmune
blistering disease pemphigus. There are two main forms of the disease, pemphigus
foliaceous (PF) and pemphigus vulgaris (PV). Both are associated with formation of
epidermal blisters: in PV these tend to be flaccid and fluid-filled, whereas PF the blisters
burst readily and become encrusted. PV, but not PF, is also characterised by mucous
membrane lesions, particularly of the oral mucosa. Histologically PV blisters show loss of
adhesion between keratinocytes or acantholysis immediately above the basal layer while in
PF this occurs in the granular layer or directly below it. Thus the blister roof is much
thicker in PV than in PF which accounts for the different characteristics of the blisters.
Pemphigus is characterised by the presence of circulating autoantibodies to the
desmosomal glycoproteins (Dsg1 in PF and Dsg3 in PV) (reviewed by Stanley, 1993;
Stanley and Kárpáti, 1994). These autoantibodies, which become bound in the epidermis,
have been shown to be pathogenic by a number of criteria: they induce acantholysis of
cultured human keratinocytes (Schiltz and Michel, 1976; Hashimoto et al., 1983); they
cause epidermal acantholysis when injected into new born mice (Anhalt et al., 1982;
Roscoe et al., 1985), even in the absence of complement (Anhalt, et al., 1986); their
activity in the new born mouse assay is abrogated by prior incubation with recombinant
Dsgl (Amagai et al., 1994a; 1995c).
The binding of PV and PF antisera to the extracellular regions of epidermal
desmosomes appears to occur in a reciprocally graded fashion; in PV autoantibodies bind
more extensively to basal cells whereas in PF autoantibodies bind more extensively to the
granular layer (Shumizu et al., 1995). Thus the location of greatest binding corresponds
approximately with the level of acantholysis. Because autoantibody binding is clearly not
precisely localized to the level of acantholysis we have suggested that acantholysis occurs at
the weakest points in the system, where keratinocyte adhesion is most reduced
(Domchowski et al., 1995). The mechanism by which acantholysis occurs is not clear.
Targeted disruption of the Dsg3 gene in mice produces epidermal and mucosal lesions
closely resembling those seen in PV blisters (Koch et al., 1997). This would be consistent
with a direct effect on desmosomal adhesion by PV antibodies. However, activation of
proteases that cleave adhesion molecules is also a possibility (Hashimoto et al., 1983). It
has also been shown that pemphigus sera cause transient changes in cytoplasmic calcium
concentration, protein kinase C relocation and activation, and phospholipase C activation
in keratinocytes (Esaki et al., 1995; Seishima et al., 1995; Osada et al., 1997). These
changes may generate an adhesion-disrupting signal.
Further examination of the Dsg3 −/− mouse indicates a role for Dsg3 in anchoring
hairs into follicles at the telogen phase of the hair cycle, since young mice lose hair during
this phase (Koch et al., 1998). A histochemical study of human hair folicles shows that the
desmosomal glycoproteins have distinct distribution patterns leading to the conclusion

that compositionally different desmosomes are present in various compartments of the
follicle (Kruzen et al., 1998).
ORGANISATION OF THE DESMOSOMAL PLAQUE

The cytoplasmic domains of the Dscs and Dsgs are located in the outer dense cytoplasmic
plaque of the desmosome (Figure 4.1, OP) (North et al., 1999). Here they associate with
at least three other proteins, Pg, Pp and Dp. Pg and Pp are members of the armadillo
family of junctional/signalling molecules, which both participate in the organisation of
intercellular junctions and, as complexes with transcription factors, enter the nucleus to
regulate gene activity. Pg contributes to the structure of both desmosomes and adherens
junctions (Cowin et al., 1986), and has been shown to cause axis duplication in Xenopus
development (Karnovsky and Klymkowsky, 1995), while Pp has both junctional and
nuclear location (Mertens et al., 1996; Schmidt et al., 1997). The possibility that
desmosomal components can also regulate gene expression and cellular differentiation is
an important area for current and future investigation. Dp appears to provide a molecular
link between the adhesive components of the desmosome and the intermediate filament
cytoskeleton (Stappenbeck and Green, 1992; Bornslaegar et al., 1996). Its C-terminus is
located in the desmosomal inner plaque (IP, Figure 4.1), into which KF insert (Miller et
al., 1987; North et al., 1999).
Plakoglobin and Plakophilin

Pg and Pp belong to the armadillo family which includes the adherens junction-associated
proteins β-catenin and p120cas, and the tumour supressor protein APC (for reviews see
Peifer, 1995; Gumbiner, 1995; Klymkowsky and Parr, 1995; Barth et al., 1997; Cowin
and Burke, 1996; Huber et al., 1996). Pg occurs as a single isoform, which is a universal
component of the cytoplasmic plaques of desmosomes, whereas the two isoforms of Pp
have restricted tissue distributions.
Members of the armadillo family interact with several different proteins. These
interactions appear to be mediated by overlapping regions of a centrally located group of
so-called armadillo repeats (arm motifs) (Figure 4.2), each consisting of 42 amino acids
(Peifer, 1995; Klymkowsky and Parr, 1995). Pg contains 13 consecutive arm repeats
flanked by distinct N-terminal and C-terminal domains (Riggleman et al., 1989). It shares
60–70% sequence identity with armadillo and β-catenin. The Pps consist of 9 arm repeats
followed by a C-terminal extension of 13 (Pp 1) or 11 (Pp 2) amino acids and no N-
terminal flanking domain (Hatzfeld et al., 1994; Heid et al., 1994; Mertens et al., 1996).
The Pps show amino acid identity of 33% to p120 with which they form a sub-group, but
less than 25% to Pg, β-catenin and armadillo (Cowin and Burke, 1996).
Pg interacts with the cytoplasm domains of Dsc and Dsg, and the classical cadherins
(Korman et al., 1989; Kowalczyk et al., 1994; Mathur et al., 1994; Peifer et al., 1992; Roh
and Stanley, 1995; Troyanovsky et al., 1994a,b; Butz and Kemler, 1994; Jou et al., 1995;
Knudsen and Wheelock, 1992; Piepenhagen and Nelson, 1993). It binds with high affinity
to Dsg, with lower affinity to Dsc “a” and only weakly to E-cadherin (Chitaev et al.,
1996). It also forms cytoplasmic complexes with APC and β-catenin, the latter being a
component of the adherens junction (Rubinfeld et al., 1995). Deletion analyses show that
the central arm repeats mediate association with classical cadherins, whereas the flanking
repeats are essential for desmosomal cadherin binding (Mathur et al., 1994; Troyanovsky
et al., 1994a, b; Roh and Stanley, 1995; Chitaev et al., 1996; Troyanovsky et al., 1996;
Wahl et al., 1996; Witcher et al., 1996). Competition for binding domains on Pg may
determine which complexes it forms. For example, the Dsg binding domain in the first
three arm repeats overlaps the β-catenin binding domain (Sacco et al., 1995; Witcher et
al., 1996; Chitaev et al., 1996; Wahl et al., 1996; for review see Cowin and Burke,
1996), and may regulate whether Pg associates with desmosomes or with classical
cadherins in adherens junctions. Chitaev et al. (1998) have identified, by alanine scanning
mutagenesis, nine hydrophobic amino acids in arm repeats 1–3 of Pg that are required for
binding to Dsg and Dsc.
Human, bovine, murine and rat Pg sequences are available (Franke et al., 1989, Cowin
et al., 1986; Butz et al., 1992; Hiipakka, 1996). A variant of Pg from a human transitional
carcinoma cell line contains a 120bp deletion corresponding to fourth arm repeat,
believed to result from alternative splicing (Ozawa et al., 1995a). This variant binds with
lower affinity to E-cadherin, Dsg2 and APC than full-length Pg, but both forms bind a-
catenin equally (Ozawa et al., 1995b). Where the alternative form is expressed in vivo is
not clear.
Since Pg is common to both adherens junctions and desmosomes it may have a
regulatory function injunction assembly. One such role may be segregating components
between these junctions. The defective heart muscle of Pg −/− mice (Ruiz et al., 1996;
Bierkamp et al., 1996) has no desmosomes and Dp becomes merged with the components
of adherens junctions into extended junctions, while Dsg2 is scattered over the surface of
the cells. The force exerted by contraction of heart muscle may cause intermixing of
junctional components. Discrete but abnormal desmosomes are present in epithelia of Pg
−/− mice. Thus in null mice surviving until birth, abnormal structure of epidermal
desmosomes was accompanied by blistering and subcorneal acantholysis (Bierkamp et al.,
1996) These desmosomes lacked either the inner or outer plaque, or both, and
intermediate filament attachment was impaired.
Deletion of Pg and yeast two hybrid analysis suggests that the N-terminus of Dp binds
to Pg at a site located in the central arm repeats (Palka and Green, 1997; Kowalczyk et
al., 1997). Co-expression of a chimera of the E-cadherin extracellular domain and the
Dsgl cytoplasmic domain with Pg and truncated Dp (containing the N-terminus) in L929
and COS cells produced plaque-like junctional structures at points of cell-cell contact
(Kowalczyk et al., 1997). Pg, therefore, acts as a link between the cytoplasmic domain of
Dsg and Dp. Whether it also links Dsc and Dp is not yet clear.
Pg, β-catenin and armadillo share substantial sequence homology in the arm repeats
but the N- and C-termini are divergent, possibly conferring specific functions on the
different molecules. To address this, mutant Pgs lacking either or both N- and C-termini
were expressed in A-431 cells (Palka and Green, 1997). N-terminal deletion did not
affect desmosome assembly or morphology but Pg lacking the C-terminus was
Figure 4.2 Comparsion of the domain structures of human plakglobin (adapted from Wahl et al., 1996) and human plakophilins 1a, 1b, 2a & 2b. The
imperfect armadilo repeats are shown as nymbered boxes. The domains of plakoglobin necessary for association with desmosomal cadherins, classical cadherins,
α-catenin, APC and desmoplakin as determined by deletion analyses are indicated. The fourth armadillo repeat which is lacking in the alternative form of
plakoglobin due to a 120bp deletion is shown (hatched box). The plakophilin la mutations (Q304X in arm repeat 1 and 1132ins28 in arm repeat 3) which
result in ectodermal dysplasia/skin fragility syndrome are shown. The additional 21 amino acids and 44 amino acids inserts found in the alternative splice
forms, plakophilin 1b and 2b, are indicated (solid box). Incomplete carboxyl-terminal armadillo repeats are shown (patterned boxes). Numbers represent
amino acid residues. Diagram is not to scale.
incorporated into abnormally long desmosomes, possibly indicating that the C-terminus is
involved in limiting desmosome length. The C-terminus may mask sites which bind other
desmosomal components. Consistent with this a sequence of amino acids in arm repeat 13,
which interacts with and masks cadherin binding sites located further upstream
(Troyanovsky et al., 1996). The C-terminus may mask cryptic binding sites for Dp, so
limiting over-recruitment of components and desmosome length (Palka and Green,
1997). It should be remembered, however, that normal desmosomes appear to fuse when
brought together by low calcium medium-induced contraction of MDCK cells (Mattey
and Garrod,1986b). Such large desmosomes do not disperse when normal extracellular
calcium concentration and cell morphology are restored (unpublished observations).
Expression of N-terminally-truncated Pg in both Xenopus and cultured epithelial cells
increased the cytosolic levels of endogenous and ectopic Pg (Rubenstein et al., 1997; Palka
and Green, 1997). This may reflect enhanced stability of Pg in the non-cadherin associated
pool. Stabilisation of β-catenin depends upon dephosphorylation of a serine/threonine
glycogen synthase kinase 3β (GSK3β) consensus site in the N-terminus (Peifer et al.,
1994b; Rubenstein et al., 1997). GSK3β is the vertebrate homologue of Drosophila Zeste-
white-3, a segment polarity gene that is negatively regulated by wingless leading to
dephosphorylation, stabilisation and increased cytoplasmic levels of armadillo. Pg also
contains a GSK3β consensus site in its N-terminus, deletion of which generates increased
cytosolic levels in Xenopus embryos (Rubenstein et al., 1997). Overexpression of β-catenin
or Pg suggests that these molecules exert signalling effects. Micro-injection of β-catenin
or Pg mRNA into Xenopus embryos causes duplication of the body axis, replicating the
effect of Wnt-1 overexpression (Funayama et al., 1995; Karnovsky and Klymkowsky,
1995). The balance between cadherin-bound and unbound pools of β-catenin and Pg may
be crucial for signalling during development. The architectural HMG box transcription
factor, LEF-1/XTCF-3 interacts with β-catenin (Behrens et al., 1996; Huber et al., 1996;
Molenaar et al., 1996) and Pg (Huber et al., 1996). In mammalian cells, β-catenin/LEF-1
or Pg/LEF-1 complexes translocate to the nucleus. The former complex binds to the E-
cadherin promoter and may regulate E-cadherin transcription thus affecting assembly of
intercellular junctions.
Pg also forms complexes with the tumour suppresser protein APC (Rubinfeld et al.,
1995). This complex targets proteins for ubiquitination and degradation, thus reducing
their levels. APC competes with cadherins for the association with Pg and β-catenin (for
review see Polakis, 1995) and may thus regulate the levels of cytosolic Pg. Mutant forms
of APC, unable to down-regulate β-catenin and Pg, are associated with colonie
hyperplasia. Hence Pg may be a key regulator in cell adhesion, differentiation and
proliferation. It is not clear how the diverse functions of Pg are regulated, but modulation
of tyrosine phosphorylation may be important. Growth factor-dependent tyrosine
phosphorylation of Pg correlates with a more invasive cell state (Shibamoto et al., 1994)
and may regulate junction assembly.
Additionally, it has been reported that the assembly and formation of adherens junctions
precedes desmosome assembly (O’Keefe et al., 1987; Wheelock and Jensen, 1992; Lewis
et al., 1997). Studies of the role of Pg injunction assembly suggest that association of Pg
with a classical cadherin is necessary before desmosomes can assemble (Lewis et al.,
1997). However, it has also been shown that Pg preferentially assembles into desmosomes
rather than adherens junctions (Näthke et al., 1994; Adams et al., 1996). Furthermore,
desmosomal adhesion can form in the absence of classical cadherin-mediated cell
interactions (Tselepis et al., 1998). The events leading to junction formation are thus
complex with Pg playing an important modulatory role. Steric hindrance between binding
partners, molecular conformation, levels of expression and variation in binding kinetics
between cadherin cytoplasmic domains may be mechanisms for controlling Pg complex
formation.
Pp, formerly designated “band 6” in enriched desmosome preparations from bovine
nasal epidermis (Skerrow and Matoltsy, 1974), occurs as two isoforms, Pp1 and Pp2.
Sequences are available for human and bovine Pp1 (Heid et al., 1994; Hatzfeld et al., 1994).
Alternative splicing of exon 7 which encodes 21 amino acids at the beginning of arm
repeat 4 of the human Ppl gene results in two variants, 1a (726 amino acids) and 1b (747
amino acids) (Schmidt et al., 1997). Pp2 occurs as two variants, 2a (837 amino acids) and
2b (881 amino acids), through alternative splicing of an exon between the second and
third arm repeats. Pp1 and 2 are widely expressed proteins that occur in the nuclei of
both epithelial and non-epithelial cells (Mertens et al., 1996; Schmidt et al., 1997). The
nuclear function of Pps, if any, is presently unknown.
The desmosomal complement of Pps is cell-type specific. Thus, in desmosomes of the
suprabasal layers of stratified epithelia, only Pp1 is present, whereas in simple epithelia or
myocardial cells only Pp2 can be detected. In other epithelia, both Pp1 and 2 can coexist
in desmosomes (Heid et al., 1994; Mertens et al., 1996; Schmidt et al., 1997). Only Pp 1a
localises to desmosomes, whereas the 1b variant is restricted to nuclei, probably because
the additional sequence in the 1b variant somehow restricts its distribution (Schmidt et al.,
1997).
Pp1 has a major role in maintaining desmosome function in epidermis. The first-
described human desmosomal mutations result in functional knockout of Pp1 (McGrath et
al., 1997). In the affected individual, absence of Pp1 results in skin fragility and
ectodermal dysplasia, affecting skin, hair and nails. No abnormalities in other organs have
been detected. Mutational analysis revealed different nonsense mutations in both of the
Pp1 alleles, each causing premature termination of translation. These mutations would
result in severely truncated forms of Pp1 and immunostaining was indeed absent from
epidermis. Desmosomes in the affected skin are small and greatly reduced in number,
lacking inner plaques and IF attachment. Large intercellular spaces indicate weakened
desmosomal adhesion. Dp immunostaining was diffuse rather than cell-peripheral,
whereas staining for other desmosomal components appeared normal. This suggests that
Pp1 links Dp to the desmosomal cadherins and is important for desmosomal adhesion.
Desmoplakin
DpI and II are major components of the desmosomal plaque, extending across both outer
and inner plaque domains (North et al., 1999) (Figure 4.1). Dp is also present in
complexus adherentes junctions in vascular endothelial cells (Schmelz and Franke, 1993;
Schmeltz et al., 1994; Valiron et al. 1996). Dps are constitutive desmosomal components
except that DpII is absent from cardiac muscle (Angst et al., 1990). Derived by alternative
splicing from a single gene, DpI and DpII have predicted MWs of 332 and 260 kDa,
respectively (see Bornslaeger et al., 1994 for review). DpI is a homodimer with a central
α-helical coiled-coil rod domain flanked by globular end domains (Bornslaeger et al.,
1994). By rotary shadowing, this rod domain is ~130 nm in length, while that of DpII is ~
43 nm (O’Keefe et al., 1989). However, simultaneous immunogold labelling of both
globular end domains in desmosomes of bovine nasal epi dermis showed the length of Dp,
measured perpendicular to the plasma membrane, to be only ~40 nm (North et al.,
1999). Thus DpI may be folded or coiled in tissue. The rod domain of DpI is characterized
by periodic distribution of charged residues indicating that it may aggregate with itself or
with similar proteins to form higher order filamentous structures (Stappenbeck and
Green, 1992).
The Dp C-terminal domain consists of three 176 amino acid subdomains, A, B and C,
plus a 68 residue “tail” at the C-terminus. Each subdomain is composed of a 38 residue
repeat motif with the same periodicity of acidic and basic residues as the 1B rod domain of
IF proteins, suggesting that DP could interact with the latter (Bornslaeger et al., 1994).
The N-terminal domain consists of a series of shorter heptad repeats which are predicted
to form compact bundles (Virata etal, 1992).
By transfection of specific Dp domains into cultured epithelial cells, Green and
colleagues have demonstrated that the C-terminus mediates interactions with IF
(Stappenbeck and Green, 1992; Stappenbeck et al., 1993; see also below). At least two C-
terminal subdomains are involved in this binding. An additional 48–68 residue region at
the C-terminus is critical for keratin, but not vimentin, association (Stappenbeck et al.,
1993). The rod domain apparently mediates aggregation of desmoplakin molecules, thus
contributing to desmosomal plaque architecture (Stappenbeck and Green, 1992;
Bornslaeger et al., 1996): yeast two-hybrid studies indicate that the carboxyl portion of
the rod contains a site essential for dimerization (Meng et al., 1997). The N-terminal
domain targets Dp to the outer plaque (Stappenbeck et al., 1993; Bornslaeger et al., 1996)
and clusters plakoglobin-desmosomal cadherin complexes into discrete plasma membrane
domains (Kowalczyk et al., 1997). Consistent with this, immunogold labelling shows the
Dp N-terminus to lie in the outer plaque and the C-terminus in the zone of IF attachment
(North et al., 1999) (Figure 4.1). Smith and Fuchs (1998) have shown that targeting of Dp
to desmosomes is disrupted by deletion of only about 10% of its N-terminal sequence,
and that only 86 residues of the N-terminal residues are sufficient for desmosomal
targeting. Transfection of cells with Dp lacking the C-terminus (Bornslaeger et al., 1996),
causes formation of abnormal junctions in which desmosomal (Dp, desmosomal
cadherins) and adherens junction (E-cadherin, α- and β-catenin) components merge,
suggesting that the C-terminal domain may be involved in segregating desmosomes from
other junctions.
In endothelial cells, molecular interactions and junctional associations of Dp differ from
those in epithelial cells. Kowalczyk et al. (1998) have shown that a Dp amino terminal
peptide is recruited into cell junctions in association with VE-cadherin, plakoglobin and β-
catenin, suggesting the formation of junctional complexes which associate with both the
actin and intermediate filament cytoskeletons.
Desmoplakin Homologues: Envoplakin, Periplakin and

Plectin
Dp belongs to a protein family, recently termed the plakin family (Uitto et al., 1996;
Ruhrberg and Watt, 1997), which includes plectin, envoplakin, periplakin and BPAG1
(bullous pemphigoid antigen 1 or BP230), which is a component of hemidesmosomes. All
have predicted structures consisting of a rod domain of variable length flanked by globular
end domains (reviewed by Green and Jones, 1996; Ruhrberg and Watt, 1997). The C-
terminal domains have different numbers of conserved subdomains, which may be
involved in binding to different types of IFs (Stappenbeck et al., 1993). The plakins are
predicted to form homodimers or heterodimers via the heptad repeats of the central rod.
Envoplakin (Ruhrberg et al., 1996), a precursor of the cornified envelope of epidermis,
is expressed in stratified squamous epithelia, but not in simple epithelia or in non-
epithelial cells. With a molecular mass of 210 kDa, it is derived from a 6.5 kb mRNA
transcribed from a single copy gene. Its up regulation during terminal differentiation and
its colocalisation with Dp at desmosomes and on keratin filaments suggests a role in
linking the cornified envelope to the desmosome-IF network.
Periplakin (Ruhrberg et al., 1997), a 195 kDa protein, associates with the desmosomal
plaque and with keratin filaments in the differentiated layers of the epidermis. Like
envoplakin it was originally identified as a cornified envelope precursor (Simon and
Green, 1984). Periplakin and envoplakin co-immunoprecipitate and immunolocalisation
indicates that they form a network radiating out from desmosomes (Ruhrberg et al., 1996;
Ruhrberg et al., 1997). Together the proteins may provide a scaffolding onto which the
cornified envelope is assembled (Ruhrberg et al., 1997).
Plectin is a 300 kDa multifunctional IF-associated protein which is widely distributed
among tissues, and which forms homotetramers up to 200 nm long and 2–3 nm wide
(Foisner and Wiche, 1987, 1991; Svitkina et al., 1996). Believed to be a versatile
cytoplasmic cross-linker, it interacts with multiple proteins, crosslinking IFs and
connecting them to microtubules, actin microfilaments and membrane adhesion sites
including desmosomes and hemidesmosomes (Foisner and Wiche, 1991; Svitkina et al.,
1996; Eger et al., 1997). Plectin binding to DP has been demonstrated in vitro (Eger et al.,
1997) and a cytokeratin binding site has been identified in the C-terminus (Nikolic et al.,
1996). A number of alternatively spliced plectin variants may be involved in these diverse
interactions (McLean et al., 1996; Elliott et al., 1997). The disease muscular dystrophy
associated with epidermolysis bullosa simplex (MD-EBS) has been linked to plectin
mutations (Gache et al., 1996; McLean et al., 1996; Pulkinnen et al., 1996; Smith et al.,
1996). Electron microscopy of patients epidermis showed disruption of the inner plaques
of hemidesmosomes and keratin filament detachment. However, no disruption of
desmosomes was observed and desmosomes were unaffected in plectin-deficient mice
(Andrä et al., 1997) suggesting that plectin is not indispensable in desmosomes.
Intermediate Filament Attachment and Accessory Plaque

Proteins
The principal desmosomal component involved in mediating interactions between IFs and
the desmosomal plaque is Dp. Cell transfection experiments using mutant desmoplakin
constructs have shown that the C-terminus of desmoplakin can interact with both keratin
and vimentin IFs (Stappenbeck and Green, 1992; Stappenbeck et al., 1993) in a
phosphorylation-dependent manner (Stappenbeck et al., 1994). Direct interactions
between Dp and IF proteins have also been demonstrated in vitro (Kouklis et al., 1994;
Meng et al., 1997). Specific IF types interact with DP via distinct sequences in different IF
domains (Meng et al., 1997). Thus type II epidermal keratins bind to the Dp C-terminus via
sequences in the N-terminal head domain of a single keratin polypeptide chain, while the
interaction of Dp with simple epithelial keratins requires the presence of both type I and
type II partner proteins, indicating the importance of the tertiary structure of the α-
helical coiled coil (Meng et al., 1997). The interactions of the Dp C-terminus with type III
IF proteins (e.g. vimentin) is weaker than with epidermal keratins (Meng et al., 1997),
but may be strengthened in vivo by the formation of coiled-coil Dp dimers, since strong
alignment of Dp with vimentin IF in transient transfections has been shown to require the
Dp rod domain (Stappenbeck and Green, 1992; Stappenbeck et al., 1993). The
physiological relevance of Dp-IF binding has been confirmed by the expression of Dp N-
terminal polypeptides in stably transfected cell lines: these polypeptides act in a dominant
negative manner to produce disrupted Junetional structures which lack associated keratin
filaments (Bornslaeger et al., 1996).
Further members of the plakin family are strong candidates for a role in IF-desmosome
attachment, both by their localisation to the region where IF insert into the plaque and by
their homology to Dp. Plectin has been reported to bind both Dp and IF and by
immunoelectron microscopy lies further from the desmosomal membrane than Dp: it could
thus function as a linker between them (Eger et al., 1997). Intermediate filament
Associated Protein (IFAP) 300, a vimentin IF-binding protein, localises to desmosomes
and hemidesmosomes at the region where IF attach to the inner plaque and has been
demonstrated to bind keratins in vitro (Skalli et al., 1994): the relationship of this protein
to plectin remains unclear. The sequence similarity of envoplakin and periplakin to Dp,
together with their localisation to desmosomes and along keratin IF, may also indicate an
involvement in anchoring filaments to desmosomes (Ruhrberg et al., 1997). Alternatively
these proteins may mediate cross-linking and stabilisation of desmosome-associated IF,
rather than themselves anchoring the filaments. A further component which may be
particularly important in this latter role, namely the organisation and/or stabilisation of a
mature desmosome-IF network is pinin (originally known as the O8L antigen; Ouyang
and Sugrue, 1992). This 140kDa desmosomal accessory protein is located at the periphery
of the plaque of only mature desmosomes of epithelia, its temporal appearance here
apparently correlating with the establishment of a highly-organised desmosome- IF
complex (Ouyang and Sugrue, 1996). However, reservations about a desmosomal role
for pinin have recently been expressed (Brandner et al., 1997).
A number of other desmosomal components located nearer to the plasma membrane may
also be involved in IF attachment. Visualisation of isolated Dsg1 tails by rotary shadowing
has revealed a head portion of ~ 4 nm diameter, presumed to reside near to the membrane,
and a thin tail some 19 nm long (Rutman et al., 1994). It has been proposed that this long
tail portion, which contains basic residues, could stretch across the plaque and thus be
favourably located to bind IF (Nilles et al., 1991). Such binding has not been
demonstrated. Pp1 has been demonstrated to bind cytokeratins in vitro (Kapprell et al.,
1988; Hatzfeld et al, 1994; Smith and Fuchs, 1998) yet by immunogold labelling lies
within the outer, rather than inner, plaque domain (North et al., 1999, Figure 4.1).
Desmocalmin (bovine form; the human form is known as keratocalmin (Fairley et al.,
1991) is a 240 kDa calmodulin-binding plaque protein which is expressed only in stratified
epithelia (Tsukita and Tsukita, 1985). Like Pp1, desmocalmin is localised immediately
subjacent to the plasma membrane yet binds to polymerised keratin IF in vitro (Tsukita and
Tsukita, 1985).
How can the in vitro binding results be reconciled with apparent physical separation of
Pp1 and desmocalmin from the inserting IF? Two possible explanations are either that the
in vitro binding to keratin IF of some of the above proteins is not physiologically relevant,
or that the keratins may actually approach nearer to the membrane than previously
supposed. Leloup et al. (1979) suggested that the traversing filaments were unraveled
protofilaments from terminating IF. Recent expression studies have shown that DP
proteins containing both the rod and C-terminal domains aggregate with IF proteins to
form structures that resemble the 4–5 nm traversing filaments of the inner plaque
(Stappenbeck and Green, 1992). This meshwork is not seen with Dp alone, suggesting
that IF may be present in the plaque as an anastomosing network of fine filaments
associated with Dp (Bornslaeger et al., 1994).
CONCLUSION
Experimental confirmation of the adhesive role of desmosomal glycoproteins and recent
discoveries on the interactions between molecules in the desmosomal plaque have greatly
increased our understanding of these junctions. However, much more detailed knowledge
is required, as well as a clearer insight into how they relate to desmosome structure.
Further important issues are the process of desmosome assembly and breakdown, and
the dynamic regulation of desmosomal adhesion. These topics have been reviewed
recently (Garrod et al., 1996; 1998; Burdett, 1998). These reviews indicate the likelihood
that both “inside-out” and “outside-in” signalling are involved in the regulation of
desmosomal adhesion.
A much greater understanding of the function of desmosomes in the epidermis is
required. An important question relates to the function of the differential expression of
desmosomal glycoproteins and plakophilins in epidermis. Do the patterns represent
differential adhesion in different epidermal layers and, if so, how is this important in
relation to epidermal structure and morphogenesis? Alternatively, could these patterns
somehow generate signals that provide positional information to regulate epidermal
differentiation?
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Wahl, J.K., Sacco, P.A., McGranahan-Sadler, T.M., Sauppe, L.M., Wheelock, M.J., & Johnson
K.R. (1996). Plakoglobin domains that define its association with the desmosomal cadherins
and the classical cadherins: identification of unique and shared domains. J. Cell Sci. 109:
1143–1154.
Wang, Y.M., Amagai, M., Minoshimia, S., Sakai, K., Green, K.J., Nishikawa, T. & Shimizu, N.
(1994). The human genes for desmogleins (DSG1 and DSG3) are located in a small region on
chromosome 18ql2. Genomics 20:492–495.
Watt, F.M., Mattey, D.L. & Garrod, D.R. (1984). Calcium-induced reorganization of desmosomal
components in cultured human keratinocytes. J. Biol. 99:2211–2215. Wheelock, M.J.
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Witcher, L.L., Collins, R., Puttagunta, S., Mechanic, S.E., Munson, M., Gumbiner, B. & Cowin,
P. (1996). Desmosomal cadherin binding domains of plakoglobin. J. Biol. Chem. 271:
10904–10909.
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CELL—MATRIX ATTACHMENT
5.
PROTEIN-PROTEIN INTERACTIONS AT
THE DERMAL-EPIDERMAL BMZ
M.PETER MARINKOVICH
Specific protein-protein interactions are required for the assembly and continued integrity
of all basement membranes. In the highly specialized dermal-epidermal basement
membrane zone (BMZ) an intricate and precise organization of multiple components is
required to maintain dermal-epidermal cohesion. Disruption of this organized array of
components, either by inherited (epidermolysis bullosa) (Marinkovich et al., 1999) or
acquired (autoimmune bullous) disease (Lin et al., 1997), can result in extensive skin and
mucosal blistering.
ULTRASTRUCTURAL ORGANIZATION OF THE DERMAL-

EPIDERMAL BMZ
BMZ ultrastructure has traditionally been visualized by standard transmission electron
microscopy (Figure 5.1a). Through this technique, it can be appreciated that at the
superior end of the dermal-epidermal BMZ, intermediate filaments insert upon electron
dense condensations of the keratinocyte plasma membrane termed hemidesmosomes.
Anchoring filaments appear as thin threadlike structures (Ellison and Garrod, 1984) which
extend from the extracellular surface of hemidesmosomes, span the electron lucent space
in the BMZ termed the lamina lucida, and appear to connect hemidesmosomes to the
electron dense portion of the extracellular BMZ termed the lamina densa.
Anchoring fibrils are banded structures which extend perpendicularly from the lamina
densa into the adjacent superficial papillary dermis. Anchoring fibrils either extend back to
reinsert onto the lamina densa or connect with electron dense structures of the papillary
dermis termed anchoring plaques (Keene et al., 1987). In connecting to these structures,
anchoring fibrils entrap interstitial collagen and other dermal molecules and help to firmly
attach the lamina densa onto the papillary dermis.
Newer techniques in the analysis of tissues by electron microscopy combine high
pressure, cryopreservation and freeze substitution to yield improved preservation of tissue
architecture (Hippe-Sanwald, 1993). During the initial cryopreservation step, water in
tissues is converted to noncrystaline ice, through the combined application of very low
temperature and very high pressure. Subsequently, absolute acetone or methanol with
aldehyde or osmium is substituted for the ice, and crosslinking occurs at very low
PROTEIN-PROTEIN INTERACTIONS AT THE DERMAL-EPIDERMAL BMZ 89
Figure 5.1 Ultrastructural appearance of the dermal-epidermal basement membrane. Human

neonatal foreskin was examined by transmission electron microscopy using standard fixation (A), or
high pressure freeze substitution (B). Note absence of anchoring fibrils and lamina lucida structures
in B. Magnification: A, 72, 660; B, 55,650. Micrographs provided by Douglas Keene, Shriners
Hospital, Portland Oregon.
temperatures. This technique greatly reduces the loss of soluble substances such as
proteoglycans.
Recent studies with high-pressure freeze substitution electron microscopy have
demonstrated that dermal-epidermal BMZ ultrastructure seen with conventional fixation
may be artifactual (Keene and McDonald, 1993) (Keene et al., 1997). While
hemidesmosomes and intermediate filaments are clearly visible with these techniques,
anchoring fibrils and the lamina lucida appear absent (Figure 5.1b). Instead, a continuous
felt-like matrix extends from the basal keratinocyte plasma membrane into the papillary
dermis.
These studies suggest that the lamina lucida seen after conventional fixation may arise
as a dehydration artifact caused by a shrinking away of the basal cell plasma membrane
from the underlying dermis after water removal. Likewise, the lamina densa, anchoring
filaments and anchoring fibrils may appear due to artifactual precipitation resulting from
removal of proteoglycans and other soluble substances during conventional fixation
methods. These studies provide additional insight into BMZ organization.
INTERMEDIATE FILAMENT-HEMIDESMOSOME
INTERACTIONS
Basal keratinocyte intermediate filaments contain the acidic keratin 14 and the basic
keratin 5. Initially these keratins combine together as polar coiled-coil dimers. The dimers
go on to form staggered anti-parallel tetramers and then undergo additional lateral and
end to end associations to form 2 to 3 nm diameter apolar protofilaments and 4 to 5 nm
90 M.PETER MARINKOVICH
diameter protofibrils. finally two to four protofilaments associate into a coiled coil lattice
to form 10 nm diameter intermediate filaments (Fuchs and Cleveland, 1998).
Recent studies have suggested that the nonhelical keratin 5 head domain may be
important in the anchoring of intermediate filaments to other structures (Smith and
Fuchs, 1998). The desmosomal components desmoplakin, plakophilin 1 and plakoglobin,
all appear to interact with this domain. Intermediate filaments through the keratin 5 head
domain may also associate with hemidesmosomal components although this remains to be
proven. The keratin 5 head domain is also suspected to be involved with the binding of
intermediate filaments to melanosomes (Uttam et al., 1996) as mutations which affect this
domain are associated with epidermolysis bullosa simplex with mottled pigmentation.
BPAG1
Hemidesmosomes contain two plaque proteins, including BPAG1 (BP230) and plectin
(HD1). BPAG1 is a 230 kD protein which consists of a central rod flanked by carboxyl
(C) and amino (N) terminal globular domains (Tanaka et al., 1991) (Sawamura et al.,
1991) (Tamai et al., 1995) (Tang et al., 1996). BPAG1 has significant homology both to
desmoplakin as well as to plectin in its two C terminal repeating globular domains. This
homologous region contains the keratin binding region.
BPAG1 localizes to the inner plaque on the cytoplasmic surface of the hemidesmosome
and functions in the connection between hemidesmosomes and intermediate filaments.
BPAG1 negative transgenic mice lack a hemidesmosomal inner plaque and the connection
between hemidesmosomes and intermediate filaments is severed, creating a cytoplasmic
zone of mechanical fragility just above the hemidesmosomes, which results in intra-
epidermal blistering (Guo et al., 1995). In these mice neither hemidesmosome stability or
cell substratum adhesion appear to be weakened. Thus BPAG1 does not appear vital for
hemidesmosome or BMZ assembly.
BPAG1 also exists as an alternatively spliced neural isoform termed BPAG1n
(dystonin). As a result of the ablation of the domains common for both BPAG1 and BPAGln,
BPAG1 knockout mice also develop severe dystonia and sensory nerve degeneration
typical of dystonia musculorum (dt/dt) mice (Bowling et al., 1997). BPAG1 is also a
major autoantigen targeted by sera of most patients with the blistering disease, bullous
pemphigoid (Stanley, 1991).
PLECTIN
Plectin is a 500 kD cytoskeletal linker protein (Liu et al., 1996). Plectin associates with a
variety of types of cytoskeletal structures including keratin, vimentin, neurofilaments,
microfilaments, and microtubules. It shows a very widespread expression in the majority
of tissues and cell types studied. In stratified epithelial cells, plectin promotes cell-cell
adhesion by linking keratin containing intermediate filaments and desmosomes, while
plectin’s role in promoting dermal-epidermal cohesion lies in its ability to link keratin into
the hemidesmosomal plaque.
Figure 5.2 Anchoring filaments: central organizers of specialized basement membrane assembly.
Four anchoring filament proteins, α6β4 integrin, collagen XVII, laminin 5 and laminin 6 are
depicted schematically on the left. Important structural domains and proposed functions are listed
to the right.
Rotary shadowing electron microscopic studies have shown that plectin, like many
BMZ proteins, assumes a dumbbell shape and contains a central 200 nm helical coiled-coil
rod, which is flanked at either end by large globular domains (Wiche, 1998). Gel
permeation studies which predicts plectin’s molecular weight to be 1100 kD, suggest that
plectin exists as a dimer in solution.
Immuno-gold electron microscopy studies have demonstrated localization of plectin
with intermediate filaments (Foisner et al., 1995). The C terminal globular domain of
plectin contains 6 repeating domains containing approximately 300 amino acids each.
Each domain contain multiple copies of a tandemly repeated 19 amino acid motif. A 50
amino acid segment bridging repeat domains 5 and 6 contains a basic amino acid residue
cluster located in a bipartite nuclear localization sequence that functions as the
intermediate filament binding site (Nikolic et al., 1996). Near this site of domain 6 is a
phophorylation site for p34cdc2 protein kinase that may be involved with dissociation of
plectin from intermediate filaments (Malecz et al., 1996).
Plectin’s interaction with hemidesmosomes studied by immuno-gold electron
microscopy with domain specific antibodies (Foisner et al., 1994) reveal a lack of
orientation of plectin’s association between intermediate filaments and hemidesmosomal
plaque. In this study, the same domains were seen to insert both into keratin filaments as
well as hemidesmosomal plaque. These studies suggest that two binding sites of plectin
exist which each are capable of interacting with other hemidesmosomal elements. These
findings are consistent with direct ligand binding studies (Rezniczek et al., 1998) which
demonstrate that both the N and C terminal globular domains of plectin are capable of
binding to β4 integrin. Furthermore, plectin is capable of associating with two different
regions of the β4 integrin cytoplasmic domain. The significance of these multiple
interactions remains to be elucidated.
Plectin has been recently shown to exist in multiple isoforms generated by alternative
splicing of the amino termini (Elliott et al., 1997). At least eight isoforms have been
detected thus far. Isoform expression levels as shown by RNAase protection assays,
demonstrate a diversity of different isoform expression in individual tissues. The splicing
diversity affects both coding and noncoding regions and may affect plectin function and/
or expression. Other molecules, including HD1 (Heida et al., 1992) and IFAP 300 (Skalli
et al., 1994), which show similarities as well as differences with plectin in terms of tissue
distribution as well as other parameters may represent some of these alternatively spliced
plectin isoforms, however this remains to be determined.
Plectin deficient patients show a blistering phenotype with separation of the skin
occurring above the level of the hemidesmosome (Smith et al., 1996) (Gache et al., 1996)
(McLean et al., 1996) (Chavanas et al., 1996). Hemidesmosomes are intac suggesting that
plectin is not essential for hemidesmosome formation. Plectin deficient patients have been
shown to have an associated muscular dystrophy, which accounts for the name,
epidermolysis bullosa simplex with muscular dystrophy. Premature stop codon mutations
of the plectin gene were found to be associated with the majority of these patients,
although in one case, a three amino acid deletion was found in the N terminal globular
region. This region, which is involved with the interaction of plectin with β4 integrin, is
likely to be important in plectin function. The mechanism as to how a lack of plectin in
muscle leads to muscular dystrophy remains to be determined. Plectin knockout mice

(Andrä et al., 1997) also demonstrate a blistering phenotype similar to epidermolysis
bullosa patients described above, but show more extensive muscle pathology.
α6β4 INTEGRIN
Hemidesmosomes also contain the transmembrane proteins collagen XVII and α6β4
integrin (Figure 5.2). The cytoplasmic portions of these molecules make up part of the
hemidesmosome dense plaque whereas the extracellular portions of these molecules make
up portions of the anchoring filament and may contribute to the structure known as the
subbasal dense plate which underlies hemidesmosomes in the lamina lucida region.
α6β4 integrin is an essential and central component of the hemidesmosome
(Sonnenberg et al., 1991). Like all of the members of the family of molecules known as
integrins (Hynes, 1992), it consists of two transmembrane subunits assembled together in
a noncovalent fashion (Tamura et al., 1990) (Sonnenberg et al., 1990). The extracellular
domains of these subunits combine together to form a ligand binding site, whereas the
intracellular domains interact with other hemidesmosomal components. β4 integrin is
only known to combine with the α6 subunit, whereas the α6 subunit can combine with β4
or β1 subunits. The α6β1 or α6β4 integrin combinations have been shown to act as
receptors for laminin, and α6β4 integrin has been shown to act as a receptor for laminin 5.
β4 integrin contains an especially large approximately 1000 amino acid cytoplasmic
domain, which consists of two pairs of fibronectin repeats separated by a connecting
segment. Replacement of β4 integrin deletion constructs in β4 integrin negative EB-PA
keratinocytes revealed a 27 amino acid region of the connecting segment was required for
hemidesmosome assembly (Niessen et al., 1997). This region of β4 integrin is critical in
localizing plectin to hemidesmosomes, and a direct interaction between β4 integrin
cytoplasmic domain and plectin has been demonstrated (Rezniczek et al, 1998).
β4 integrin directly interacts with collagen XVII (Schaapveld et al., 1998) (Borradori et
al., 1997) and influences its localization to hemidesmosomes. The sequences on β4
integrin which appear to mediate this interaction lie on the C terminal section of the
connecting segment and the third fibronectin repeat. In addition to binding collagen XVII
and plectin, β4 integrin cytoplasmic domain, under some circumstances may fold over
and bind to itself. Ligand binding studies show that the C terminal 85 amino acids of β4
integrin can interact with and bind the connecting segment and regions N terminal to it.
This folding appears to influence the affinity of β4 integrin for plectin and may be involved
in hemidesmosome assembly/disassembly.
β4 integrin contains a tyrosine activation motif (TAM) in its connecting segment.
Mutations involving the tyrosines in this motif have been shown to have variable effects. In
one study, these mutations appeared to prevent the incorporation of β4 integrin into
hemidesmosomes (Mainiero et al., 1995).In another study, TAM mutations appeared to
have only a minor effect on the localization of collagen XVII to hemidesmosomes
(Borradori et al., 1997).
As mentioned above, α6β4 integrin appears to play a central role in hemidesmosome
formation in that transgenic mice lacking the β4 integrin show skin devoid of
hemidesmosomes as well as blistering and severe deficits in cell adhesion (Bowling et al.,
1996). Patients with absence of the β4 (Vidal et al., 1995) or 6 integrin (Ruzzi et al.,
1997) due to underlying gene mutations demonstrate very rudimentary hemidesmosome
formation at best, widespread blistering and pyloric atresia (epidermolysis bullosa with
pyloric atresia, EB-PA).
COLLAGEN XVII
Collagen XVII (BP180/BPAG2) is another transmembrane component of
hemidesmosomes. It is collagenous protein with a type II transmembrane orientation
(Giudice et al., 1992). Based on electron microscopy and crosslinking studies, collagen
XVII appears to assemble into a homotrimer (Schumann and Bruckner-Tuderman, 1996)
and contains three main regions; an intracellular N terminal globular head, a central rod
and an extracellular flexible tail (Hirako et al., 1996). The C terminal portion of the
molecule appears to be involved with polarization of the collagen XVII molecule to the
basal surface of keratinocytes. As mentioned above the cytoplasmic domain of collagen
XVII colocalizes and binds with the β4 integrin in the hemidesmosome (Borradori et al.,
1998) (Aho and Uitto, 1998). A subset of epidermolysis bullosa patients who lack
collagen XVII expression also show a lack of localization of BPAG1 into
hemidesmosomes. Analysis of deleted collagen XVII cDNA constructs suggests that the C
terminal half of the collagen XVII cytoplasmic domain is required for BPAG1 localization
into hemidesmosomes. Thus the collagen XVII endodomain has two important functions:
ligation of BPAG1 (which links intermediate filaments to the hemidesmosome) and
ligation of the β4 integrin endodomain (which serves to correctly localize collagen XVII to
the hemidesmosome).
Collagen XVII assists in promoting epidermal adhesion, as evidenced by the widespread
blistering that occurs in patients with generalized atrophie benign epidermolysis bullosa
(GABEB) who lack this molecule. Interestingly, retrovirally mediated collagen XVII gene
replacement in primary GABEB keratinocytes corrected adhesion defects in vitro and
corrected blistering defects in vivo after grafting of these cells onto immunodeficient mice
(Seitz et al., 1999).
The large non-collagenous region of collagen XVII near the transmembrane region,
termed the NC16A domain may be involved with association of collagen XVII with 6
integrin in keratinocytes in vitro, indicating that this portion of the molecule may be
necessary for binding to α6β4 integrin (Hopkinson et al., 1995). This portion of the
molecule also contains the epitopes recognized by bullous pemphigoid autoantibodies (Liu
et al., 1995). The central rod and flexible tail region of collagen XVII contains a number
of interrupted collagenous domains which assemble into a triple helix. The most distal
extracellular portion of this molecule contains epitopes recognized by autoantibodies
from some patients with cicatricial pemphigoid (Balding et al., 1996). Based on rotary
shadowed electron microscopic measurements, the extracellular portions of collagen
XVII may extend up to 193 nm. It is therefore likely that collagen XVII extends the full
length of the lamina lucida and inserts into the lamina densa. This is consistent with the
observation that cicatricial pemphigoid autoantibodies recognize autoepitopes in the lamina

densa region of the BMZ (Bedane etal, 1997).
The anchoring filament component, LAD-1 is a 120 kD protein expressed by
keratinocytes (Marinkovich et al., 1996) which represents the nondegraded form of the
previously identified 97 kDa linear IgA bullous dermatosis autoantigen (Zone et al.,
1990). The mAb 123 which recognizes LAD-1, induces deepithelialization of human skin
in situ, suggesting that LAD-1 plays an important cohesive role in the dermal-epidermal
BMZ (Marinkovich et al., 1996). A previously described 125 kD component of anchoring
filaments which appears to be the bovine analog of LAD-1, was found to be the first
protein to be expressed during the earliest phases of wound healing in an organ culture
model (Klatte et al., 1989).
Recent evidence implicates LAD-1 as a proteolytic fragment of the collagen XVII
exodomain. Linear IgA bullous dermatosis autoantibodies, as well a group of monoclonal
antibodies have been shown to react with LAD-1 extracted from skin and keratinocyte
medium, but do not show significant reaction with full length collagen XVII (Klatte et al.,
1989) (Marinkovich et al., 1996) (Pas et al., 1997) (Zone et al., 1998). These observations
were the main evidence which supported LAD-1 being a novel protein. Recent peptide
sequencing studies has shown that LAD-1 contains homologous sequences with collagen
XVII (Zone et al., 1998). Northern blot analysis has failed to confirm any alternatively
spliced variants of collagen XVII (Schacke et al., 1998) and an absence of both LAD-1 and
collagen XVII in patients with generalized atrophie benign epidermolysis bullosa
(Marinkovich et al., 1997) suggests that LAD-1 probably is not a separate gene product. With
these possibilities ruled out, the most likely hypothesis appears to be that LAD-1 may
indeed be a proteolytic fragment of collagen XVII, with proteolysis inducing a group of
neoepitopes. As these neoepitopes include those recognized by linear IgA bullous
dermatosis autoantibodies, and by mAb 123 which induces skin de-epidermalization in
situ, the proteolysis of collagen XVII appears to be an important event from a disease
standpoint.
N terminal sequencing of processed collagen XVII isolated from skin suggests that the
cleavage occurs in the NC16A domain (Zone et al., 1998). Processed collagen XVII
appears to exist as a triple helix (Balding et al., 1997), and contains domains which are
collagenase sensitive but pepsin resistant (Schacke et al., 1998). In one study, proteolytic
cleavage of a bacterial fusion protein containing the NC16a and its proximal collagenous
domain was demonstrated using purified gelatinase B (Stahle-Backdahl et al., 1994),
however cleavage of native collagen XVII has not been demonstrated with this enzyme. In
another study, inhibition of furin served to inhibit collagen XVII processing (Schacke et
al., 1998). A furin consensus site is present in the NC16a region of collagen XVII and it is
possible that this enzyme in a membrane associated form may be involved in collagen
XVII processing. Alternatively, it is known that furin proteolytically activates membrane
type metalloproteinase 1 and that this enzyme activates gelatinase A. Thus it is also
possible that either of these two enzymes may be responsible for collagen XVII
processing.
LAMININS OF THE DERMAL-EPIDERMAL BMZ

In addition to α6β4 integrin and collagen XVII, anchoring filaments contain the molecules
laminin 5 (Verrando et al., 1988) (Rousselle et al., 1991) (Carter et al., 1991) and laminin
6 (Marinkovich et al., 1992a) (Figure 5.2). Laminins are family of hetero trimeric BMZ
proteins which contain a, β and γ chains (Burgeson et al., 1994). Laminin 5 contains α3,
β3, and γ2 chains. All previously described laminins were shown to have three short arms
and one long arm, forming a cross shape as shown by rotary shadowing analysis. In
contrast, laminin 5 appears as a dumbbell shape which is the result of severe truncations
of the short arms. Each of these short arm reductions occur primarily as a result of
truncations of the genes coding for these subunits, however some of this reduction occurs
post-translationally as extracellular processing events on the α3 and γ2 short arms
(Marinkovich et al., 1992b). Because of these short arm truncations, laminin 5 cannot
polymerize or bind to nidogen. Instead laminin 5 forms a disulfide bonded complex with
laminin 6 (Champliaud et al., 1996).
The importance of laminin 5 in mediating epithelial adhesion is best illustrated by the
widespread blistering, mucosal erosions and extracutaneous epithelial adhesion defects
that occur in patients with Herlitz junctional epidermolysis bullosa, where laminin 5
expression is absent (Meneguzzi et al., 1992) (Marinkovich et al., 1993) due to underlying
gene mutations (Uitto et al., 1995). Laminin 5 is initially synthesized and secreted by
keratinocytes in an unprocessed form with an α3 chain of 200 kD, a β3 chain of 140 kD,
and the γ2 chain of 155 kD (Marinkovich et al., 1992b). The domains I and II in all three
chains are believed to adopt a helical coil conformation, account for the rod-like central
portion of the laminin molecule, and are essential for laminin assembly, βγ dimers
accumulate intracellularly and assembly of these dimers with the α chain appears to be the
rate limiting step in laminin assembly (Matsui et al., 1995). Shortly after secretion, the α3
chain becomes processed from 200 to 165 kD. In addition, the γ2 chain is processed from
155 to 105 kD by the enzyme BMP-1 (Lee et al., 1997). These processing steps occur
both in vitro in cultured keratinocytes, as well in vivo in human tissues. An additional
processing step predominantly occurs in vivo, which results in the conversion of the a
three chain from 165 to 145 kD (Marinkovich et al., 1992b).
Analysis of laminin 5 extracted from human tissue shows that the former two steps
occur to completion while the latter processing step occurs in approximately half of the
laminin 5 molecules. In addition, further processing via MMP-2, of the γ2 chain from 105
kD to 80 kD, at the boundary of domains III and II; occurs in certain proliferative states,
such as squamous cell carcinoma and the developing mammary gland (Giannelli et al.,
1997). This processing which has only been demonstrated in rat tissues, appears to
facilitate a promigratory epithelial phenotype. It remains to be shown whether this
processing step occurs in human tissues.
The laminin 3 chain contains an especially large C terminal globular domain which
consists of five repeating EGF-like segments. The G domain of laminin 5 α3 chain
contains the major cell binding activity, which provides laminin 5 the ability to serve as
the major attachment ligand for a number of epithelial cell types including keratinocytes
and squamous carcinoma cells. Processing of the α3 chain from 200 to 165 kD appears to
occur near the junction of the G3 and G4 domains, and laminin 5 containing processed
165 kD α3 chain supports cell attachment (Rousselle and Aumailley, 1994) (Rousselle et al.,
1995). These observations make it clear that the cell binding activity and the binding sites
for integrins α3β1 and α6β4 are located somewhere in the G1 to G3 domains. Further
confirming this, an antibody, termed BM165, which recognizes the G1 domain, has been
shown to inhibit the attachment of keratinocytes to underlying culture substrate and to
interfere with the attachment of epidermis to dermis (Rousselle et al., 1991).
Laminin 5 additionally has heparin binding properties contained within the G4 and 5
domains (Sung et al., 1997). The significance of the heparin binding is as yet unclear. It is
possible that, prior to processing and insertion into the BMZ, laminin 5 may bind to BMZ
heparan sulfate proteoglycan, also known as perlecan, or alternatively cell surface heparan
sulfate proteoglycan, syndecans, may bind to laminin 5 and laminin 6 to influence BMZ
formation and cell migration. The G5 domain has been shown to have a promigratory
activity (Sung et al., 1997) and removal of this domain through processing appears to
decrease the ability of laminin 5 to support cell migration and increase the ability of
laminin 5 to support hemidesmosome formation (Goldfinger et al., 1998).
Laminin 6, the other known anchoring filament laminin, contains α3, β1 and γ1 chains
(Marinkovich et al., 1992a), and based on sequence analysis, it has been hypothesized that
one of the unpaired cysteine residues in the laminin 6 α3 chain domain 3 EGF1 region
binds to an unpaired cysteine residue in the laminin 5 β3 domain 6 region (Champliaud et
al., 1996). The α3 and γ2 chains of laminin 5 are consistently and completely processed
when laminin 5 is complexed with laminin 6, and these processing steps are probably
necessary in the covalent association of laminin 5 and laminin 6. Laminin 5 has also been
shown to complex with another component, laminin 7, which contains the β2 laminin chain,
however laminin 7 does not appear to be present in skin to any significant extent.
Additionally, the N terminal portion of the α3 chain exists in two spliced variant forms (Ryan
et al., 1994). The functional significance of this alternative splicing is not yet clear. As
laminin 6 also contains the α3 G domain, it can also theoretically combine with basal cell
surface receptors. This suggests that the laminin 5, laminin 6 heterodimer contains two cell
binding regions per dimeric complex.
Laminins-5 and 6 function as autoantigens in a subset of patients with the blistering
disease cicatricial pemphigoid (Domloge-Hultsch et al., 1992). The autoantibodies
recognize the processed laminin 5 α3 chain (Kirtschig et al., 1995) and thus the
autoantibody resides on this chain must reside somewhere between the N terminus and G
domain 3 of this chain. As the autoantibodies disrupt skin cohesion, it is possible that
autoepitope site(s) are located in G domains 1–3 near the cell binding region.
The current data suggest that laminin 5 is oriented in the BMZ with its C terminal G
domain located adjacent to the basal cell surface and the N terminal short arms facing the
lamina densa complexing with laminin 6. Laminin 6, located in the lower lamina lucida
and lamina densa forms interactions with other BMZ components through linkage of its γ1
chain with nidogen (Mayer et al., 1993). Through this interaction, the divalent nidogen
can link laminin-6 and the anchoring complex with its other known BMZ ligands, collagen
IV and perlecan (Dziadek et al., 1985). In addition to its interaction with laminin 6,
laminin 5 also is capable of binding with the NC1 domain of collagen VII (Chen et al.,
1997) (Rousselle et al., 1997) determined by solid phase binding assays and rotary
shadowing analysis.
It is not entirely clear whether laminin 5 can simultaneously associate with both
collagen VII and laminin 6. Even if laminin 5 in incapable of such a dual association, only
approximately half of the laminin 5 purified from tissue is complexed with laminin 6. This
would leave a significant portion of laminin 5 available to bind to collagen VII. The above
model predicts that the N terminal globular short arm domains are probably the regions
of laminin 5 that come in contact with collagen VII and thus these regions probably
contain the binding sites. Preliminary studies suggest that collagen XVII exodomain binds
to laminin 5, (D. Reddy, manuscript in preparation) indicating another possible important
interaction of laminin 5 with the extracellular BMZ. Due to its multiple interactions,
laminin 5 is a molecule of central importance in the assembly of extracellular components
of the dermal-epidermal BMZ.
The dermal-epidermal BMZ contains at least one other large laminin in addition to
laminin 6 which contains β1 and γ1 chains as well as an a chain recognized by mAb 4C7
(Engvall et al., 1986). While it was originally believed that mAb 4C7 recognized laminin
a1 chain, recent evidence has shown that this mAb instead recognizes laminin a5 chain
(Tiger et al., 1997). Therefore, it is likely that the dermal-epidermal BMZ contains the
laminin combination, a5β1γ1, which has been termed laminin 10. Laminin 1 (a1β1γ1), in
contrast, does not appear to be a significant constituent of the dermal-epidermal BMZ.
ANCHORING COMPLEX—BMZ ASSOCIATIONS

All BMZs contain some type of laminin, collagen IV, nidogen and perlecan, a large
heparan sulfate proteoglycan (Martin and Timpl, 1987). A number of studies have
demonstrated that these purified components are capable of assembling into structures
resembling the lamina densa of the basement membrane (Yurchenco and O’Rear, 1994).
This occurs through a number of interactions, which have been extensively studied.
Collagen IV, through a quaternary association via its 7-A regions, and a binary association
through its NC-1 domain, has the ability to self-polymerize into an extended network
which probably represents the central feature of the lamina densa (Yurchenco, 1994).
Laminins, as discussed below, also are capable of self-polymerizing and also contribute to
the self assembly of the BMZ. Linkages of collagen IV, laminins and perlecan with the 150
kD linker protein nidogen (Dziadek et al., 1985) are additional mechanisms which underly
the self assembly of the lamina densa/ lamina lucida in all BMZs.
In specialized BMZs, such as the one at the dermal-epidermal junction, the assembly of
the ubiquitous BMZ structures into a lamina densa/lamina lucida, and the assembly of
anchoring complex structures may occur independently. Impairment of anchoring
complex, but not lamina densa/lamina lucida formation is seen in knockout murine or EB-
PA patient skin which lacks a6β4 integrin (Bowling et al., 1996) (Smith, 1993). In
contrast, impairment of lamina densa/lamina lucida formation, but not anchoring
complex formation is seen in the skin of α3 integrin knockout mice which lacks the a3β1
integrin (Taverna et al., 1998). These studies suggest that both α6β4 and α3β1 integrin
perform important complementary functions in the development of the dermal-epidermal
BMZ and that development of the ubiquitous and specialized portions of the dermal-
epidermal BMZ may occur via separate mechanisms.
A primary mode of linkage of the specialized components of the anchoring complex
with the rest of the ubiquitous BMZ structures probably occurs through association
between the 4th EGF repeat on domain III of the γ1 chain (Mayer et al., 1993) of laminin 6
with nidogen. Through this linkage with nidogen, laminin 6 and the anchoring complex may
directly be adjoined to collagen IV, perlecan and indirectly be adjoined, through repeated
nidogen-ligand linkages, to laminin 10. Another mode of association between the
anchoring complex with the rest of the BMZ may occur through interaction of the heparin
binding domains of the laminin 3 chain G4 and G5 domains with perlecan, the high
molecular weight heparan sulfate basement membrane proteoglycan. Interaction of
perlecan with laminin 5 would theoretically occur only transiently, as the α3 chain G4 and
G5 domains are proteolytically removed (Marinkovich et al., 1992b) during assembly into
the basement membrane in vivo.
One of the properties of laminins is the ability to polymerize with the responsible
elements residing on the N terminal portions of the molecule (Yurchenco and Cheng,
1993). While it is clear that the truncations which are present on all three short arms of
the laminin 5 molecule prevent it from polymerizing, the ability of laminin 6 to
polymerize had remained in question. A study examining the polymerization of laminin 6
with laminins 1 and 2 showed that the truncations of laminin 6’s short arm prevents it
from self polymerizing or polymerizing with laminins 1 and 2 (Cheng et al., 1997). This
study suggested that domains present on laminin al and α2 chains required for self
polymerization are lacking on the laminin 6 α3 chain. Therefore, laminin polymerization
does not appear to be a mechanism of association of the anchoring complex with the rest
of the BMZ.
COLLAGEN VII
Collagen VII is the major constituent of anchoring fibrils (Sakai et al., 1986). The
importance of collagen VII towards the process of dermal-epidermal cohesion is well
demonstrated by its absence or by functional defects of this molecule due to underlying
gene mutations (Uitto et al., 1994) in the inherited blistering diseases known as dystrophic
epidermolysis bullosa. In these diseases, sublamina densa separation of the skin and
mucosa occurs due to a lack of intact anchoring fibrils. Like all collagens, collagen VII
assembles into a triple helix.
Analysis of the deduced amino acid sequence of collagen VII (Parente et al., 1991)
reveals the presence of a long central collagenous region characterized by repeating Gly-X-
Y sequences that contain a number of noncollagenous interruptions, including a 39 amino
acid noncollagenous segment in the center of the helix which corresponds to the “hinge
region” predicted by biochemical studies (Bachinger et al., 1990). These interruptions
account for the flexibility of the collagen VII molecule, and explain its ability to loop
around and entrap dermal matrix molecules (Keene et al., 1987), to provide its function of
stabilizing the BMZ to the underlying papillary dermis. The 145 kD N terminal end of
collagen VII contains the largest noncollagenous domain (Lunstrum et al., 1986). This
domain inserts onto the lamina densa and anchoring plaques.
Collagen VII triple helices are joined together at their processed NC-2 globular domains
to form antiparallel dimers (Morris et al., 1986). The C terminal noncollagenous globular
domain, termed NC-2, of the collagen VII molecule is processed prior to anti-parallel dimer
formation (Lunstrum et al., 1987). When this step is prevented, through mutations of this
region of the collagen VII gene (Bruckner-Tuderman et al., 1995), sub-lamina densa
blistering and dystrophic epidermolysis bullosa are the result. Anchoring fibrils probably
derive from lateral associations of the antiparallel dimers.
Collagen IV in the lamina densa and anchoring plaques specifically binds to collagen VII
NC1 domain (Burgeson et al., 1990). As anchoring fibrils extend as perpendicular
projections in areas directly underlying anchoring filaments, it was suspected that a direct
interaction between anchoring filaments and anchoring fibrils also existed. Recent studies
have confirmed a specific interaction between the anchoring filament component laminin
5 and collagen VII NC1 domain. Colocalization of laminin 5 and collagen VII NC1 domain
has been demonstrated both in the lamina densa and in anchoring plaques of the dermal-
epidermal BMZ. These studies provide the first evidence of direct linkage between
anchoring filaments and anchoring fibrils. A second component of anchoring fibrils has
been recently identified (Gayraud et al., 1997). This component which is recognized by the
mAb GDA-J/F3 appears to be a 50 kDa protein which localizes to the junction of
anchoring fibrils with the lamina densa.
SUMMARY
A model of the protein-protein interactions of the dermal-epidermal basement membrane
is presented in Figure 5.3. Intermediate filaments insert upon hemidesmosomes via
plectin and BPAG1. Plectin and BPAG1 in turn bind to β4 integrin and collagen XVII
endodomains respectively. The hemidesmosomal complex is further stabilized by co-
association of the β4 integrin and collagen XVII endodomains. Anchoring filaments arise
from the extracellular surface of hemidesmosomes through the exodomains of α6β4
integrin and collagen XVII. Further assembly of anchoring filaments takes place by
association of a6β4 integrin and possibly collagen XVII with laminin 5 or laminin 5/laminin
6 complex. The C terminal regions of laminin 5 and 6 face the basal cell while the N-terminal
domains are oriented towards the lamina densa. The entire anchoring complex is linked with
the lamina densa by the association of laminin 6 with nidogen, which in turn is capable of
binding collagen IV and perlecan. Through additional nidogen linkages, laminin 10 also
assembles with collagen IV and perlecan. Anchoring filaments are linked to anchoring
fibrils by a direct association between laminin 5 and the NC1 domain of collagen VII. In
addition, the NC1 domain of collagen VII binds to collagen IV in the lamina densa. finally,
due to the flexible nature of collagen VII, anchoring fibrils wind around and entrap
interstitial collagen and other molecules, which serves to fasten the lamina densa onto the
papillary dermis.
Figure 5.3 Overview of protein-protein interactions in the dermal-epidermal basement

membrane.
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6.
BIOLOGY AND PATHOLOGY OF
HEMIDESMOSOMES
LEENA PULKKINEN AND JOUNI UITTO
INTRODUCTION
Recent cutaneous biology research has successfully explored the complexity of the
cutaneous basement membrane zone (BMZ), which by ultrastructural, biochemical and
molecular means has been demonstrated to consist of a large number of adhesion
complexes. Several of these attachment complexes can be recognized by electron
microscopy (Figure 6.1). On the epidermal side, complex structures, known as
hemidesmosomes (HD), extend from the cytoplasmic milieu of basal keratinocytes to the
extracellular space (Borradori and Sonnenberg, 1996; Green and Jones, 1996). Within
the lamina lucida, thread-like structures, known as anchoring filaments, can be recognized
by transmission electron microscopy, and they are concentrated predominantly under the
HDs and form the HD-anchoring filament complex (Figure 6.1). On the dermal side,
anchoring fibrils, attachment structures with a wheat-stack appearance, extend from
lamina densa to the underlying dermis (Burgeson, 1993). It has been suggested that one
end of anchoring fibrils binds to lamina densa while the other end interacts with basement
membrane-like structures, known as anchoring plaques, within the upper papillary dermis
(Keene et al., 1987). Intertwining of the anchoring fibrils between the interstitial collagen
fibers, which consist primarily of type I, III and V collagens, provides stable association of
the lower portion of the dermal-epidermal basement membrane to the underlying dermis
(Figure 6.2).
Based on these and similar observations, a concept has been advanced which depicts the
dermal-epidermal BMZ as a continuum of adhesion molecules linked to a network
structure and, the integrity of this network is necessary for stable association of epidermis
to the underlying dermis (Uitto and Christiano, 1992; Christiano and Uitto, 1996a; Uitto
et al., 1997; Pulkkinen and Uitto, 1998). Consequently, structural weakness in this
network structure can manifest as fragility of skin and other epithelial basement
membranes, as depicted by a group of diseases, collectively known as epidermolysis
bullosa (EB).
BIOLOGY AND PATHOLOGY OF HEMIDESMOSOMES 107
Figure 6.1 Transmission electron microscopy of the cutaneous basement membrane zone
composed of lamina lucida (LL) and lamina densa (LD) separating epidermis (E) from dermis (D).
Several distinct attachment complexes, including hemidesmosomes (HD), anchoring filaments
(small arrows) and anchoring fibrils (arrow head) can be recognized. Hemidesmosomes consist of
intracellular inner (1) and outer plaques (2), and of extracellular electron dense structures known as
sub-basal dense plates (3). Original magnification x40,000 (The figure was kindly provided by Dr.
John A.McGrath, St. John’s Institute of Dermatology, London, UK).
ULTRASTRUCTURAL AND MOLECULAR FEATURES OF
HEMIDESMOSOMES
One of the critical attachment structures of the cutaneous BMZ is the hemidesmosome
(HD), which extends from the intracellular milieu of basal keratinocytes to the
extracellular matrix (Figures 6.1 and 6.2). Ultras true tur ally, HD can be recognized as
an electron dense organelle with distinct subcompartments, including an intracellular
inner plaque which is recognized in close association with keratin intermediate filaments,
and an intracellular outer plaque which is attached to the plasma membrane of the basal
keratinocyte on the cytoplasmic side (Borradori and Sonnenberg, 1996). The sub-basal
dense plate parallels the plasma membrane at the extracellular side and appears to serve as
an attachment site for the anchoring filaments.
At least four distinct proteins have been localized to the HDs by immunoelectron
microscopic means (Figure 6.2); these include plectin (Wiche et al., 1983; Wiche, 1989),
the 230-kD and the 180-kD bullous pemphigoid antigens (Ishiko et al., 1993; Guo et al.,
1995), and the α6β4 integrin (Stepp et al., 1990; Sonnenberg et al., 1991). These four
proteins and their subunit polypeptides are well characterized, their primary sequences
have been deduced through molecular cloning, and their chromosomal locations have
been determined (Table 6.1). In addition, several less well characterized proteins have
been suggested to be components of the hemidesmosomes at the cutaneous BMZ. These
include IFAP300, a ~300-kD polypeptide which has been suggested to be a member of
the plakin family of proteins (Ruhrberg and Watt, 1997; Skalli et al., 1994); and p200, a
200-kD molecule recognized by a monoclonal antibody 6A5 and deposited at the epithelial-
stromal interface (Kurpakus and Jones, 1991).
108 LEENA PULKKINEN AND JOUNI UITTO
Figure 6.2 Schematic representation of the components of the cutaneous basement membrane zone
and their molecular interactions. The individual components are identified by color code at the
bottom of the figure. (Reproduced with permission from Pulkkinen and Uitto, 1998).
In addition to the hemidesmosomal components referenced above, a number of lamina
lucida proteins have been identified, but their exact relationship to HDs is in most cases
unclear. One of the best characterized BMZ proteins is laminin 5 which consists of three
polypeptide subunits, the α3, β3 and γ2 chains, encoded by distinct genes on
chromosomes 18q 11.2, 1q32 and 1q25–31, respectively (Vailly et al., 1994; Ryan et al.,
1994). In addition to laminin 5, two other members of the laminin family, laminins 6 and
7, have been identified in the cutaneous BMZ covalently adducted to laminin 5 (Burgeson
Table 6.1 Hemidesmosomal proteins, their molecular features and associated genetic diseases
*EB-MD, EB with muscular dystrophy; GABEB, generalized atrophie benign EB; EB-PA, EB with pyloric atresia(for details, see Table II) .
et al., 1994). Ubiquitously expressed BMZ components, including nidogen and fibulins 1
and 2, as well as various proteoglycans, have also been identified in the cutaneous
basement membranes (Martin and Timpl, 1987; Miosge et al., 1996; Woods et al., 1996).
Finally, several additional, less well characterized proteins have been mapped to the
cutaneous BMZ. These include (a) another 200 kD protein recognized by sera of patients
with a novel autoimmune bullous disease (Zillikens et al., 1996) and localized by indirect
immunogold electron microscopy to the lower lamina lucida; (b) a 105-kD lower lamina
lucida antigen (p105), also recognized by sera from patients with an unusual blistering
skin disease and distinct from the γ2 chain of laminin 5 which is of the same size (Chan et
al., 1995); (c) uncein, a trimeric BMZ protein which is absent in the skin of patients
withjunctional forms of EB (Fine, 1990); and (d) a recently characterized 50-kD
component of epithelial basement membranes identified by a monoclonal antibody GDAJ/
F3 and localized by immunoelectron microscopy to lamina densa at the sites of insertion
of anchoring fibrils (Gayraud et al., 1997). The primary sequences and molecular features
of these four proteins are currently unknown. Finally , a novel, 45-kD BMZ protein,
designated as ladinin, has been recently identified (Motoki et al., 1997).
THE MOLECULAR COMPONENTS OF HEMIDESMOSOMES
The Plectin Family of Proteins

Plectin belongs to a family of widely expressed, cytoskeleton associated proteins versatile
in their binding activities (Wiche et al., 1981; Wiche, 1989). Plectin was initially isolated
as a high-molecular-weight protein which co-purified with type III intermediate filament
protein vimentin and was subsequently found to bind a number of intermediate filament
proteins, including keratins and nuclear lamins. Immunohistochemical analyses have
shown that plectin is widely expressed in a variety of epithelial as well as mesenchymal
tissues (Wiche et al., 1983). Within the cells, plectin is found in association with cell
membranes and junctional complexes, and it co-localizes with intermediate filaments, as
well as stress fibers and focal contacts.
Cloning of human plectin cDNA and genomic sequences (Liu et al., 1996; McLean et
al., 1996) has revealed that the primary polypeptide, approximately 518 kD in size, has
structural homologies with previously characterized plakin family of proteins, which in
addition to plectin, includes desmoplakin, the 230-kD bullous pemphigoid antigen,
envoplakin, and recently cloned periplakin (Ruhrberg et al., 1996, 1997; Ruhrberg and
Watt, 1997; Aho et al., 1998). All members of the plakin family of proteins have an
amino-terminal globular domain, central rod-like structure, and a carboxy-terminal tail
which contains homologous regions (see Figure 6.3). All these proteins have been
identified in association of desmosomes and/or hemidesmosomes within basal
keratinocytes, where their function appears to relate to binding of intermediate filaments
(Corden and McLean, 1996). Molecular analyses of plectin have also revealed
considerable heterogeneity due to alternative splicing or alternatively transcribed 5’ exon
of the gene (Elliott et al., 1997). Among the various forms of plectin, of particular interest
Figure 6.3 Demonstrations of the 180-kD bullous pemphigoid antigen (BP180) and β1 integrin
interactions, as probed by yeast two-hybrid system. A: The constructs (p376–p434) shown on the left,
corresponding to the intracellular domain of BP180, were used as bait in the GAL4 binding domain
in yeast two-hybrid system, while the sequence encoding intracellular domain of β4 integrin was
inserted with the GAL4 activation domain. Protein-protein interactions, determined by growth in -
his, -trp, -leu medium, is shown on the right. B: The amino-terminal end of BP180 responsible for
α1 integrin binding. The critical region spans amino acids E13-R26 which contains a predicted β-
sheet. (For details, see the original publication Aho and Uitto, 1998).
is the variant, designated as HD1, which is recognized by the monoclonal antibody

HD121 (Hieda et al., 1992). This monoclonal antibody identifies plectin epitopes within
the skin only in association with hemidesmosomes, suggesting that HD1 is a plectin
variant exclusively expressed in HDs, where it has been localized to the inner plaque
(Figure 6.2). Alternatively, this monoclonal antibody recognizes a unique epitope in
plectin expressed only in the context of hemidesmosomes. Immunohistochemical staining
of skin and muscle with the monoclonal antibody HD121 was instrumental in identifying
plectin defects underlying a variant of EB associated with late-onset muscular dystrophy
(EB-MD) (for review see Uitto et al., 1996).
The 230-kD Bullous Pemphigoid Antigen (BPAG1)

BPAG1, another member of the plakin family of proteins associated with
hemidesmosomes, was initially identified as an autoantigen in an acquired blistering skin
disease, bullous pemphigoid. BPAG1 has been localized, together with plectin, to the
inner plaque of HDs (Figure 6.2). Development of transgenic mice with ablated BPAG1
gene has provided insight into the function of this protein (Guo et al, 1995). Specifically,
the BPAG1 −/− mice demonstrated that this protein plays a critical role in binding of
intermediate keratin filaments to HDs. Most importantly, ultrastructural findings
demonstrated that HDs in these animals are otherwise normal, but they lack the inner
plaque, and the integrity of hemidesmosome/ cytoskeleton association is disturbed. The
keratin intermediate filaments were severed from the HDs and had retracted into
perinuclear locations. An interesting observation is that a neural-specific gene product,
known as dystonin, is essentially identical with BPAG1, with the exception of the most
amino-terminal end (Brown et al., 1995a). Specifically, sequencing of the dystonin gene,
in comparison with the BPAG1 gene, revealed that dystonin is a neural isoform of
BPAG1, and that the variation at the amino-terminus of the protein is due to the alternate
transcription of the 5’ exon. These two proteins, BPAG1 and dystonin, are clearly
products of the same gene, as transgenic mice lacking the BPAG1 gene locus also develop
characteristic neuromuscular abnormalities (Guo et al., 1995). Furthermore, a naturally
occurring mouse mutant, dt/dt, manifesting with neurologic disorders, is due to
mutations in the BPAG1 gene (Guo et al., 1995; Brown et al., 1995a,b). No human
heritable disease has been shown to be due to mutations in the BPAG1 gene as yet,
although the expression of this protein is markedly reduced in several patients with EB-
MD (see Uitto et al., 1996) which harbor the pathogenetic mutations in the plectin gene,
thus attesting to the close interdependence of BPAG1 and plectin gene expression.
The 180-kD Bullous Pemphigoid Antigen (BPAG2)—The

Type XVII Collagen
BPAG2 was initially characterized as an autoantigen in bullous pemphigoid and herpes
gestationis, two acquired blistering skin diseases (Morrison et al., 1988). Subsequent
molecular cloning of the BPAG2 gene revealed that the protein consists of an amino-
terminal globular domain and a carboxy-terminal segment, the latter one with
characteristic collagenous Gly-X-Y repeat sequences (Giudice et al., 1992; Li et al., 1993).
In fact, the carboxy-terminal two thirds of the protein were shown to contain 15
collagenous submodules separated by short non-collagenous segments (see Figures 6.2 and
Figure 6.7). Molecular cloning and cell biological studies also demonstrated that BPAG2
has a transmembrane domain, and that the amino-terminal domain resides in the
cytoplasmic side of basal keratinocytes. Thus, BPAG2 is a transmembrane collagen,
designated as type XVII collagen, in type II topography (Li et al., 1993). Recent
immunoelectron microscopic data have also supported the type II transmembrane
topography of BPAG2. Specifically, use of a monoclonal antibody anti-BV4 IgG, which
recognizes the carboxy-terminal end of BPAG2, revealed its association with anchoring
filaments in the extracellular milieu (Masunaga et al., 1997). Furthermore, these findings
suggested that the carboxyterminus extends all the way across the lamina lucida into the
lamina densa, implying that the carboxy-terminal segment of BPAG2 is in fact a
component of anchoring filaments which have been thought to consist primarily of laminin
5 (Figure 6.2).
The a6β4 Integrin Attachment Complex

Integrins, a family of dimeric transmembrane glycoproteins, consist of two subunit
polypeptides, the α and β chains, which are noncovalently associated on the cell surface.
The α6β4 integrin is characteristically found in a variety of epithelial tissues, including
human skin and the gastrointestinal tract, and it plays a major role as an attachment
molecule connecting basal keratinocytes to the underlying basement membrane (Stepp et
al., 1990; Sonnenberg et al., 1991). The two subunit components, α6 and β4, have been
extensively characterized by molecular cloning, and their intron-exon organizations and
chromosomal locations have been determined (see Table 6.1, and refs. Hogervorst et al.,
1990; Tamura et al., 1990; Pulkkinen et al., 1997a,b). The expression of a6 and β4
integrin polypeptides is variable in that α6 subunit expression is detected in a variety of
tissues and also during embryogenesis, while the expression of β4 subunit is more
restricted to epithelial tissues (Hogervorst et al., 1990; Tamura et al., 1990; Sonnenberg
et al., 1991; Thorsteinsdottir et al., 1995). The β4 integrin differs from other β subunits
in that it has an unusually long cytoplasmic domain encoding over 1000 amino acids. This
domain contains two pairs of fibronectin type III-like repeats (FNIII) separated from each
other by a connecting segment (Hogervorst et al., 1990). Furthermore, the β4 integrin
subunit has at least five different variants, primarily due to alternative splicing within the
region encoding the cytoplasmic domain, and the expression of such splice variants is also
tissue-specific (Hogervorst et al., 1990; Tamura et al., 1990; Van Leusden et al., 1997).
Two different splice variants of α6 integrin have been described, both of them being able
to form dimers with β1 and β4 integrins (Delwel et al., 1995).
HEMIDESMOSOMAL PROTEIN-PROTEIN INTERACTIONS

During the past few years, a significant amount of novel information on molecular
interactions between the BMZ attachment complexes has emanated from studies utilizing
a variety of cell and molecular biological technologies, including transient cell

transfections, yeast two-hybrid systems, development of transgenic “knock-out” mouse
models, and examination of mutation databases on human diseases affecting the BMZ. On
the basis of the currently available information, clearly defined interactions can be
recognized within this network.
The inner hemidesmosomal plaque consists of at least two proteins, BPAG1 and plectin
(Figure 6.2). BPAG1 “knock-out” mice revealed that this protein is responsible for
attachment of intermediate keratin filaments to the HDs, and this functional abnormality
is associated with ultrastructurally recognizable lack of the inner plaque of HDs (Guo et
al., 1995). In contrast, in “knock-out” mice deficient in plectin the formation of HDs in
basal keratinocytes is not noticeably impaired, although they are reduced in number
(Andrä et al., 1997). HDs in the latter mice are able to serve, to a certain extent, as
intermediate filament anchoring sites, as demonstrated by the presence of
ultrastructurally intact appearing filament structures radiating from the cytoplasmic side
of HDs. At the same time, a series of cell transfection studies utilizing plectin deletion
constructs and mutated cDNAs to disrupt the interactions of this protein with
intermediate filament network have identified a cluster of four basic amino acid residues
(Arg4277–Arg4280) within the carboxyl-terminal domain of plectin to be essential for
intermediate filament binding (Nikolic et al., 1996). The association of plectin with
intermediate filaments, and subsequent cross-linking, are mediated by mitosis-specific
phosphorylation involving p34cdc2 kinase (Foisner et al., 1996). Thus, the inner plaque
proteins, plectin and BPAG1, clearly participate in anchorage of intermediate filaments to
the HDs. Collectively, these data suggest that BPAG1 plays a critical role in intermediate
filament binding, while plectin, in addition to contributing to such binding, serves as a
stabilizer of the assembly of the inner plaque of HDs (Sanchez-Aparicio et al., 1997).
The outer plaque of HDs consists of at least three polypeptide components, the
cytoplasmic domains of the α6 and β4 integrins and the 180-kD bullous pemphigoid
antigen (Figure 6.2). The critical role of β4 integrin subunit in the assembly of HDs is
well established in studies which have revealed several interactions of this polypeptide
with other hemidesmosomal components, such as plectin (Niessen et al., 1997a,b). Using
yeast two-hybrid system, the cytoplasmic domains of BPAG2 and β4 integrin were
demonstrated to interact with each other (Aho and Uitto, 1998). Specifically, an
intracytoplasmic segment extending from amino acids 13 to 89 of BPAG2, including a
predicted β-sheet, and a region of β4 integrin spanning the connecting segment between
two pairs of FNIII repeats and the second pair of FNIII, as well as the carboxyl-terminal
intracellular tail, are required for this interaction (Figure 6.3). The same connecting
segment, together with the second FNIII of the first pair, both within the intracellular
domain of β4 integrin, is critical for interactions between this polypeptide and plectin, as
well as directing its hemidesmosomal localization (Borradori et al., 1997). At the same
time, interactions of the extracellular domain of the α6 integrin with BPAG2 have been
suggested to be mediated by discrete sequences within so-called NC16A domain in the
latter polypeptide (Hopkinson et al., 1995). The interdependency of the a6 and β4
integrin expression is also attested by the fact that 6 integrin expression is deficient in 4
“knock-out” mice (van der Neut et al., 1996). Similarly, in the skin of patients with a
variant of EB associated with pyloric atresia, EB-PA, and caused in most cases by
underlying mutations in the β4 integrin gene, α6 integrin expression is variably reduced
or undetectable (Shimizu et al., 1996; Pulkkinen et al., 1998a).
The interactions between extracellular components of HDs and the anchoring filament
proteins within lamina lucida are less well characterized. For example, the ligand for the
extracellular collagenous domain of BPAG2 has not been disclosed as yet. It is clear,
however, from several in vitro and in vivo studies that basal keratinocytes bind to laminin 5
via the extracellular domain of the α6β4 integrin, and the α3β1 integrin appears to
contribute to this association (Champliaud et al., 1996; Carter et al., 1997). Specifically,
the carboxy-terminal globular domain of laminin 5 has been shown to take part in this
binding, which is completely blocked by a monoclonal antibody (BM165) recognizing the
first G-domain of the α3 chain. This observation suggests that the G-domain is the binding
site for α6β4 integrin (Champliaud et al., 1996). Furthermore, the amino terminus of the
β3 chain of laminin 5 connects the anchoring filament-HD complex to the NC-1 domain
of type VII collagen (Rousselle et al., 1997).
GENETIC DISEASES OF HEMIDESMOSOMES

The genes encoding the components of hemidesmosomes could potentially serve as
candidates for genetic lesions in different variants of EB, and in fact, distinct mutations in
four genes expressed in HDs have been disclosed thus far (Table 6.2; Pulkkinen and
Uitto, 1998). These include EB variants with mutations in the genes encoding the α6 and
β4 integrin subunits (EB-PA), the 180-kD bullous pemphigoid antigen/type XVII collagen
(GABEB), or plectin (EB-MD). Clinically, these EB variants present with dermal-epidermal
blistering and characteristic extracutaneous manifestations.
Plectin Mutations in Patients with EB-MD

A clinically puzzling variant of EB is a recessively inherited skin blistering associated with
late-onset muscular dystrophy. Blistering of the skin is usually noted at birth, associated with
nail dystrophy (Figure 6.4). The level of blistering is intraepidermal within basal
keratinocytes at the basal cell/lamina lucida interface. Consequently, this variant of EB
has been traditionally classified as EB simplex (Niemi et al., 1988; Fine et al., 1989; Smith
et al., 1996; Gache et al., 1996). The skin manifestations are relatively mild, and the
blistering tendency usually improves with advancing age. Dramatically, however, these
patients develop late-onset muscle weakness, time of onset being highly variable. In some
cases, the signs of muscle involvement have been noted as early as two years of
age (McLean et al., 1996), while in some families, no muscle involvement has been
reported until in the middle of the fourth decade of life (Pulkkinen et al., 1996). In many
cases, however, the muscle involvement is progressive and can incapacitate the affected
individual.
Early clues to the gene/protein systems at fault in EB-MD came from
immunofluorescence studies which examined the affected individuals’ skin with an
antibody recognizing plectin/HD1 epitopes (Gache et al., 1996; Smith et al., 1996).
Specifically, staining of skin with an antibody HD-121, which recognizes plectin or its
variant associated with hemidesmosomes, was negative in the patients’ skin (Figure 6.5).
Although staining for BPAG1 was variably attenuated at the dermalepidermal junction of
the skin of the same individuals, staining for other BMZ components, including BPAG2
and type VII collagen, was normal (Figure 6.5). Furthermore, immunohistochemical
staining of muscle biopsies revealed absent plectin expression, which in normal muscle
can be found in association with sarcolemma and Z-lines (Gache et al., 1996; Smith et al.,
1996). Thus, the plectin gene (PLEC1) was considered as a candidate gene for mutations
in patients with EB-MD.
Mutation detection strategies, either based on amplification of genomic DNA
sequences followed by heteroduplex scanning with conformation-sensitive gel
electrophoresis and nucleotide sequencing (Pulkkinen et al., 1996), or utilizing protein
truncation tests (Dang et al., 1998) have been successful in identifying PLEC1 mutations
in families with EB-MD. Thus far, 21 distinct PLEC1 mutations in 14 different families
have been disclosed (McLean et al., 1996; Chavanas et al., 1996; Smith et al., 1996;
Pulkkinen et al., 1996; Mellerio et al., 1997; Dang et al., 1998; Rouan et al., 2000). The
majority of these mutations consist of insertions or deletions which result in premature
termination codons for translation (PTC) in this gene (Figure 6.7). The only exception is
a homozygous 9-bp deletion mutation, 2719de19 (see Figure 6.6), which results in in-
frame deletion of three amino acids, QAE. This particular mutation was considered to be
pathogenetic for the following reasons. First, this 9-bp deletion was not found in 95
unaffected control individuals, indicating that it was not a polymorphism in ethnically
matched (Japanese) population. Secondly, the precise amino acid sequence was conserved
between human, rat and mouse sequences, suggesting the functional importance of the
QAE tripeptide (Pulkkinen et al., 1996).
Most of the PTC-coding mutations discovered so far reside in exon 32 which encodes
the rod domain of plectin. The 9-bp deletion mutation resides in exon 22 which encodes a
portion of the amino-terminal globular domain. Collectively, the patients with EB-MD
have mutations in the plectin gene, and expression of this gene both in the skin at the
dermal/epidermal junction and in the muscle within the sarcolemmal adhesion zone, would
explain manifestations in these two organ system (Ditto et al., 1996).
Clinical Features and Molecular Basis of Generalized

Atrophie Benign Epidermolysis Bullosa (GABEB)
GABEB was initially described as a non-lethal variant of junctional EB (Hashimoto et al.,
1976; Hintner and Wolff, 1982). Clinically, the affected individuals demonstrate
protracted, life-long generalized blistering which results in cutaneous atrophy, associated
with diffuse scarring alopecia, characteristic pigmentary changes, as well as tooth and nail
abnormalities. It was subsequently shown that expression of the 180-kD bullous
pemphigoid antigen in the skin of these patients is reduced or absent, suggesting that the
corresponding gene, BPAG2/COL17A1, is the candidate gene for mutations (Jonkman et
al., 1995; Pohla-Gubo et al., 1995). Following cloning of BPAG2 genomic sequences and
Table 6.2 Hemidesmosomal variants of epidermolysis bullosa: diagnostic features and genetic basis
a)Abbreviations: BMZ, basement membrane zone; PTC, premature termination codon mutations.
b)For the specific mutations and their positions along the affected molecules, see Fig. 7.
Figure 6.4 Clinical features of a patient with EB-MD. Note superficial blistering and erosions, nail
dystrophy, and atrophy of intercostal and biceps muscles. (Published with permission from
Pulkkinen et al., 1996).
elucidation of its intron-exon organization (Gatalica et al., 1997), mutation detection
strategies were developed which were able to pinpoint specifie genetic lesions in this gene
in GABEB patients (Table 6.2). Again, the majority of the mutations cause PTCs either as
a result of nonsense mutations or small insertions or deletions in the BPAG2 gene
(Figure 6.7). In all these cases, immunofluorescence staining of the proband’s skin is
negative for the 180-kD bullous pemphigoid antigen expression. The only case with
missense mutations in both alleles (R1303Q/R1303Q), affecting the extracellular non-
collagenous segment NC4 of type XVII collagen, had a localized variant of EB with
predominantly acral blistering, however this patient had normal hair (Schumann et al.,
1997). Although the mechanistic consequences of this amino acid substitution are not
entirely clear, immunofluorescence of the proband’s skin was normal for type XVII
collagen, and his keratinocytes in culture synthesized full-length al (XVII) polypeptides.
Thus, the arginine substitution by glutamine at the amino acid position 1303 must alter a
critical function of type XVII collagen but allows its assembly into HDs, resulting in a
relatively mild phenotype.
A particularly interesting family with the combination of a PTC and a missense
mutation has been described by McGrath et al., (1996). The proband, a 50-year old
female, had extensive cutaneous blistering, as well as dental abnormalities characterized
by enamel pitting. She was shown to be a compound heterozygote for 3514ins25/G627V
Figure 6.5 Immunofluorescence of the skin in a control individual (panel A) and in a patient with
EB-MD (panels B-D). Note essentially negative staining for plectin (HD1) in the patient’s skin
(panel B) in comparison to the control skin (panel A). Staining of the patient’s skin with antibodies
recognizing the 180-kD bullous pemphigoid antigen (panel C) or type VII collagen (panel D)
epitopes was entirely normal. (Modified from Pulkkinen etal., 1996).
mutations in BPAG2/COL17A1. The patient also had two offspring, both of whom had
inherited the glycine substitution mutation G627V, whereas the paternal allele was normal.
In the latter individuals, there was no evidence of skin fragility, but they had dental
abnormalities with enamel hypoplasia and pitting, similar to those observed in the
mother. Thus, the proband in this family had a recessively inherited skin blistering,
characteristic of GABEB, while the dental abnormalities of her offspring appear to have
resulted from the glycine substitution in type XVII collagen alone, resulting in a
dominantly inherited clinical phenotype.
Another fascinating case with mutations in the BPAG2 gene was recently reported by
Jonkman et al., (1997). The proband, a 28-year old female, demonstrated characteristic
features of GABEB, including generalized blistering after minor trauma and resulting in
cutaneous atrophy, mild mucous membrane involvement, universal alopecia, pigmentary
changes, dental anomalies, and nail dystrophy. Careful physical examination disclosed
patches of clinically unaffected skin in a symmetrical leaf-like pattern over the extensor
surface of hands and upper arms, and these patches covered about 10% of the total body
surface area. Immunofluorescence staining using an anti-BPAG2/type XVII collagen
antibody was negative in clinically affected skin, while in clinically unaffected patches the
Figure 6.6 Illustration of the mutation detection strategy in a family with EB-MD. A: Genomic
sequences of the plectin gene (PLEC1) were PCR amplified from two patients with EB-MD (III-1
and III-3), their clinically unaffected mother and two younger sisters. CSGE revealed two bands
with mother’s PCR product (arrows) while the affected sisters showed the lower band and the
unaffected sisters showed the upper band only. B: Direct nucleotide sequencing of the PCR
products showed that the affected individuals were homozygous for a 9-bp deletion (2719de19) in
the plectin gene (upper panel), while the mother was heterozygous carrier (middle panel), in
comparison to normal sequence (lower panel). C: The mutation abolished a restriction enzyme site
for BglI, which was used to verify the mutation. (Published with permission from Pulkkinen et al.,
1996).
BPAG2 expression was present in approximately 50% of the basal cells, the positive cells
being in groups of about 10–50 adjacent cells (Jonkman et al., 1997). Staining for other
hemidesmosomal components, such as β4 integrin showed normal continuous pattern at
the dermal-epidermal junction. The proband’s skin in the affected area depicted
compound heterozygous mutations (1706delA/R1226X) in the BPAG2 gene, while the
clinically normal areas showed the presence of the maternal mutation (R1226X) only,
accompanied by loss of heterozygosity along a tract of at least 381 bp in keratinocytes
derived from clinically unaffected skin. This case represents revertant mosaicism of
the compound heterozygous proband with the autosomal recessive genodermatosis,
GABEB (Jonkman et al., 1997).
The a6β4 Integrin Mutations in Epidermolysis Bullosa with

Pyloric Atresia (EB-PA)
EB-PA is a distinct variant of epidermolysis bullosa associated with congenital intestinal
abnormalities, such as pyloric or duodenal atresia. Histology of the skin shows dermal-
Figure 6.7 Position of the mutations affecting 180-KD bullous pemphigoid antigen/type XVII collagen (upper panel), β4 integrin (middle panel) plectin
(lower panel).mutation above the schematic protein molecules are premature termination codon-causing mutations (PTC) while those below are missense
or in-frame deletion mutations The domain organaization of the polypeptides are color coded, as shown on the right (for original references,see text and
pulkkinen and uitto, 1998).
epidermal separation at the level of BMZ, and electron microscopy reveals that HDs are
frequently hypoplastic and reduced in number (Shimizu et al., 1996; Pulkkinen et al.,
1998a). In general, this condition is lethal during the postnatal period, and the affected
children often die from complications of skin involvement, in spite of surgical
intervention to correct the intestinal abnormalities. Nevertheless, non-lethal cases of EB-
PA have also been recognized (Fine et al., 1991; Pulkkinen et al., 1998b), and mutation
analyses in the a6β4 integrin genes have provided explanation for the milder phenotype
(see below).
Early immunocytochemical evidence suggested that the expression of a6β4 integrin is
reduced or absent in the skin of the affected individuals with EB-PA (Gil et al., 1994;
Brown et al., 1996), and subsequently, a number of mutations in the genes encoding
either one of the two subunits of the α6β4 integrin (ITGA6 and ITGB4) have been
demonstrated (Figure 6.7). In most lethal cases, the mutations consist of PTCs in both
alleles resulting in the absence of the corresponding protein due to accelerated mRNA
decay mechanism or the truncated polypeptides being nonfunctional and sensitive to
proteolytic degradation. Interestingly, our recent findings have revealed several missense
mutations within the β4 integrin gene in patients presenting with different degrees of
clinical severity varying from lethal phenotypes to very mild EB-PA (see Pulkkinen and
Uitto, 1998 and Figure 6.7). Three out of six missense mutations characterized thus far
are cysteine substitutions, which affect the extracellular domain of the β4 subunit. Two of
these cases were lethal, and one of them harbored a homozygous C61Y mutation while
the other one was a compound heterozygote for the missense mutation C245G combined
with a PTC mutation (120 delTG). A non-lethal case from consanguineous union with
homozygous C562R mutation in the cysteine-rich region had pyloric atresia at birth, and
developed localized, relatively mild skin blistering shortly after birth. Two other non-
lethal cases of EB-PA were compound heterozygotes, one for arginine substitution
mutations (R252C/R1281W) and the other one for a leucine-to-proline missense
mutation in combination with a nonsense mutation (L156P/R554X). The first of these two
cases was moderately affected at birth, but the condition improved with time. One of
these mutations (R1281W) affects the intracellular domain of β4 integrin polypeptide
within the putative region interacting with plectin. On the other hand, the other mutation
in this case, R252C, which creates a new cysteine residue in the extracellular domain of
β4 integrin, may participate in the formation of new intra- or inter-molecular disulfide
bonds, possibly disrupting ligand binding or affecting noncovalent association between the
a6 and β4 subunits. The second case had very mild blistering tendency and dystrophic
nails. It was demonstrated that the R554X mutation resulted in accelerated decay of the
corresponding mRNA transcript, and the phenotype was primarily determined by the
L156P mutation. The fourth non-lethal case was homozygous for R1281W, and as a
special clinical feature the patient presented with severe kidney failure. However, in all of
these cases the pylorus was similarly affected. It should be noted that all of these missense
mutations in the extracellular domain of ß4 integrin affect highly conserved amino acid
residues.
As indicated above, mutations in the α6 integrin subunit gene (ITGA6) have also been
reported in two cases with EB-PA (Pulkkinen et al., 1997b; Ruzzi et al., 1997). In both
cases, the clinical phenotype is indistinguishable from those caused by mutations in ITGB4,
with the exception that the patient described by Pulkkinen et al., (1997b) had also cleft lip
and cleft palate.
CLINICAL IMPLICATIONS OF BASIC RESEARCH ON

HERITABLE SKIN DISEASES
The progress made during the past decade in understanding the molecular basis of various
heritable skin diseases, as exemplified by EB, has been tremendous. For example in the
case of EB, distinct mutations in 10 different BMZ genes have been identified, and the
total number of allelic variants containing pathogenetic mutations in such genes is now in
excess of 400. Examination of the mutation database has resulted in better understanding
of how different variants with differential clinical severity reflect the underlying
mutations, although finer predictions of the genotype/phenotype correlations need to be
developed on the basis of extended database. From the practical point of view, this progress
raises the critical question: What are the benefits of the progress in basic research on
heritable blistering skin diseases to the patients and their families? Immediate benefits can
be seen through improved, molecularly based diagnosis with refined classification which
allows improved prognostication regarding the severity and the progress of the disease
(Christiano and Uitto, 1996b). Furthermore, knowledge of the underlying mutations in
different forms of EB has also provided the basis for development of DNA-based prenatal
testing in families at risk for severe forms of EB. Such testing can be performed from
chorionic villus samples as early as the 10th week of gestation or from amniocentesis
specimens at 12th week. In fact, such prenatal testing has already been performed in a
relatively large number of families at risk for extremely severe and life-threatening
variants of EB, including several cases with EB-PA (Pulkkinen et al., 1998a). A logic
extension of prenatal testing is the development of pre-implantation genetic diagnosis
(PGD), a technique that has been successfully applied to a variety of genetic diseases, such
as X-linked muscular dystrophy, cystic fibrosis and others (see McGrath and Handyside,
1998). Thus, couples with a previous child affected with EB can now initiate the next
pregnancy by knowing that there are ways to find out the state of the fetus in the early
stages of pregnancy, or even before the pregnancy is initiated.
Finally, one of the future prospects for the treatment of EB relates to development of
successful gene therapy approaches. This could involve ex vivo manipulation of cultured
cells in the manner that the mutation is corrected, with subsequent grafting of the cells to
the eroded areas of skin. Alternatively, direct application of DNA into the skin could be
used in attempts to elicit genetic reversal of the underlying mutation (Khavari and
Krulger, 1997). Although successful application of gene therapy for treatment of EB may
still be several years away, rapid development of new technologies, such as the use of
ribozymes or utilization of chimeric RNA/DNA nucleotides for correction of the
mutation by homologous recombination, hold promise for breakthroughs in the near
future.
ACKNOWLEDGMENTS
The authors thank numerous colleagues who contributed to the original studies cited in
this overview. Drs. Sirpa Aho and John A.McGrath kindly provided illustrations. The
original studies were supported by the United States Public Health Services, National
Institutes of Health grants PO1-AR38923 and T32-AR0781, and by Dermatology
Foundation and the D.E.B.R.A. of America.
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cytomatrix. CRC Grit Rev Biochem, 24:41–67.
Woods, A. and Couchman, J.R. (1996) Signaling from the matrix to the cytoskeleton: role of cell
surface proteoglycans in matrix assembly. J Invest Dermatol, 106:531–537.
Zillikens, D., Kawahara, Y., Ishiko, A., Shimizu, H., Mayer, J., Rank, C.V., Liu, Z., Giudice,
G.J., Tran, H.H., Marinkovich, M.P., Bröcker, E-B., and Hashimoto, T. (1996) A novel
subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement
membrane zone. J Invest Dermatol, 106:1333–1338.
7.
DERMAL-EPIDERMAL ADHESION
LEENA BRUCKNER-TUDERMAN
INTRODUCTION
The integrity of the skin and the strong resistance against external shearing forces are
provided by the epidermal basement membrane, a highly specialized structure which
separates and concomitantly attaches the epidermis and the dermis to each other.
Basement membranes are sheet-like, complex molecular networks that connect external
or internal epithelia with stromal mesenchyme but unique, highly specialised structures
within the basement membranes contribute to tissue-specific functions, such as the tight
dermal-epidermal adhesion in the skin.
The epidermal basement membrane which is known as the dermal-epidermal junction
zone (DEJZ) contains, beside the ubiqutous basement membrane molecules, several
unique components which form an auxiliary structure, the anchoring complex. This
connects the epidermal cells to the basement membrane and the basement membrane to
the dermal connective tissue through physical and chemical protein-protein interactions.
Both epithelial and mesenchymal cells contribute to the production of the DEJZ
macromolecules; the synthesis is regulated by paracrine signals from other cells and from
the extracellular matrix.
For studies on the structure and functions of the DEJZ, genetic and acquired diseases
leading to diminished dermal-epidermal adhesion are useful models. Examples of such
disorders are heritable epidermolysis bullosa (EB) and acquired bullous skin diseases with
autoantibodies targeting the DEJZ. A wealth of information on molecular mechanisms of
dermal-epidermal adhesion has been gained by studying mutations in the genes encoding
DEJZ proteins and their biological consequences, as well as by characterizing the
autoantigens in acquired blistering diseases. Basement membranes similar to the DEJZ
occur in the mucous membranes of the orifices, the eye, the gastrointestinal tract, and the
placental membranes, all tissues exposed to mechanical shearing forces. Therefore,
molecular studies on the DEJZ also provide novel information on the structure and
functions of basement membranes in other tissues and on epithelial-mesenchymal
interactions in general.
132 LEENA BRUCKNER-TUDERMAN
THE DERMAL-EPIDERMAL JUNCTION ZONE (DEJZ)
Morphology and Suprastructure of the DEJZ

The basement membrane at the DEJZ is unique in that it contains auxiliary structures,
anchoring complexes, which serve to strengthen the adhesion of the epithelial cells to the
extracellular matrix (Figure 7.1). Ultrastructural examination reveals the basement
membrane as a bilayer consisting of the electron lucid lamina lucida and the electron
dense lamina densa (Gedde-Dahl and Anton-Lamprecht, 1996, Keene et al., 1997). The
anchoring complexes consist of hemidesmosomes, anchoring filaments and the anchoring
fibrils and seem evenly distributed along the DEJZ (see Burgeson and Christiano, 1997).
Hemidesmosomes at the ventral plasma membrane of basal keratinocytes appear as small
electron dense complexes into which the cytoskeletal intermediate filaments insert
(Borradori and Sonnenberg, 1996). Outside the cell, thin threadlike anchoring filaments
traverse the lamina densa from the hemidesmosome into the lamina densa. Below, the
crossbanded anchoring fibrils extend from the lamina densa to the papillary connective
tissue where they either
Figure 7.1. A schematic representation of the anchoring complex. In the inner plaque of the
hemidesmosome, BP230 and plectin connect with the keratin intermediate filaments.
Transmembrane components of the hemidesmosome, collagen XVII and integrin α6β4, interact
with anchoring filament proteins in the lamina lucida, and laminin 5 binds to collagen VII in the
anchoring fibrils. The GDA-antigen is localized at the insertion points of the anchoring fibrils in the
lamina densa. Collagen IV is a component of the lamina densa and of the anchoring plaque.
insert in so called anchoring plaques or loop back to the lamina densa (Burgeson, 1993).
These distinct ultrastructural units result from highly specific ligand aggregation of the
structural macromolecules of the DEJZ. The individual molecules usually are large
DERMAL-EPIDERMAL ADHESION 133
Table 7.1 Molecular components of the DEJZ
K: keratinocytes; F: fibroblasts
oligomers composed of one or several polypeptides, and they polymerise into

suprastructures at several hierarchic levels, e.g. fibrils or filaments that are further
assembled into other suprastructures such as the anchoring complex.
Molecular Components of the DEJZ

The primary structure of many DEJZ components has been deduced from the cDNA
sequence, and studies on authentic and recombinant proteins or fragments have
contributed to understanding the molecular structures (for reviews, see Beck and Gruber,
1995, Timpl, 1996, Burgeson and Christiano, 1997). BP230, plectin, a6β4 integrin,
collagen XVII and laminin 5 are part of the anchoring complex; a3β1 integrin is localized
at the keratinocyte plasma membrane between the complexes. Collagen IV, nidogen and
perlecan contribute to the basic lamina densa network; and collagen VII in the anchoring
fibrils extends the anchoring complex into the dermis (Table 7.1). All these macromolecules
can be regarded as linear sequences of structural modules that are similar in a large variety
of proteins (Engel, 1991). While matrix suprastructures may be understood in terms of
molecular interactions, the specificity of cell-matrix interactions may depend on the
periodic occurrence of modules along the aggregated suprastructures. Experimental

evidence suggests following interactions: plectin β4 integrin (Niessen et al., 1997,
Rezniczek et al., 1998) α6 integrin—collagen XVII (Hopkinson et al., 1995), β4 integrin
—collagen XVII (Schaapveld et al., 1998, Aho et al., 1998), collagen XVII— BP230
(Borradori et al., 1998), collagen XVII—laminin 5 (Reddy et al., 1998), laminin 5—
laminin 6 (Champliaud et al., 1996), laminin 5—collagen VII (Rousselle et al., 1997,
Chen et al., 1997), laminin 6—nidogen, nidogen—collagen IV and nidogen—perlecan
(see Mayer et al., 1995, Timpl, 1996). In the following, the components of the different
suprastructures are commented.
Intracellular proteins
The 230 kD bullous pemphigoid antigen, BP230 or bullous pemphigoid antigen 1, is a
member of the plakin protein family (Stanley et al., 1988, Sawamura et al., 1991). Typical
of plakins, BP230 has a rod-like structure with an N-terminal globular domain and a C-
terminal tail which contains the homologous regions. Since it is located in the inner plaque
of the hemidesmosome, it is likely to bind intermediate filaments. This was confirmed by
generating mice with ablation of the BPAG1 gene encoding for BP230. Null mice
exhibited a phenotype with lack of the hemidesmosomal inner plaque, lack of
intermediate filament binding and cytolysis of basal cells. Surprisingly, the phenotype
included neuromuscular abnormalities (Guo et al., 1995). Molecular analysis of the mice
disclosed two isoforms of BP230, an epidermal specific isoform in the inner plaque of the
hemidesmosome which associates with the keratin intermediate filaments and a neuron
specific isoform that binds actin (Yang et al., 1996). The neural isoform is known as
dystonin; it differs from BP230 in ist most aminoterminal sequence which is created by
alternate transcription of a 5’exon.
Plectin is a 500 kD cytoskeleton-membrane anchorage protein widely distributed in
squamous epithelia and muscle (Wiche et al., 1983, 1991, Hieda et al., 1992).
Considerable heterogeneity exists as a result of alternative splicing or alternate
transcription, resulting in different tissue-specific isoforms. Like BP230, plectin belongs
to the plakin protein family and is localized to the inner plaque of the hemidesmosome.
Recent data provide evidence that both cytoskeletal components and β4 integrin serve as
plectin ligands (Niessen et al., 1997, Rezniczek et al., 1998). Ablation of the plectin gene
in mice resulted in skin blistering caused by degeneration of keratinocytes and demise 2–3
days after birth (Andra et al., 1997). Hemidesmosomes were found to be significantly
reduced in number, and apparently their mechanical stability was altered. The skin
phenotype of these mice was similar to that of patients suffering from epidermolysis
bullosa simplex with muscular dystrophy, caused by defects in the plectin gene (McLean
et al., 1996). In addition, plectin (−/−) mice revealed abnormalities reminiscent of
minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart.
These results suggest a general role of plectin in the reinforcement of mechanically
stressed cells.
Transmembrane proteins of basal keratinocytes

Collagen XVII is also known as the 180 kD bullous pemphigoid antigen or BP180. It is a
hemidesmosomal transmembrane protein (Nishizawa et al., 1993), with an intracellular
domain in the hemidesmosomal plaque and and a rod-like flexible ectodomain (Giudice et
al., 1992, Gatalica et al., 1997, Hirako et al., 1996, 1998, Schäcke et al., 1998). The
flexibility results from collagenous, triple-helical -Gly-X-Y- sequences interrupted with
non-collagenous sequences (Giudice et al., 1992 Hirako et al., 1996, 1998, Schäcke et al.,
1998). Being a transmembrane component, collagen XVII presumably plays a role in
maintaining the linkage and integrity between the intracellular and the extracellular
components of the anchoring complex and in mediating the epithelial cell adhesion to the
the basement membrane (Borradori et al., 1997). An intriguiging, recently discovered
regulatory feature is the shedding of the ectodomain of from the keratinocyte cell surface
by furin-mediated proteolytic processing (Schäcke et al., 1998). The function of the
processing is not fully understood yet, but it may serve as a mechanism to regulate cell
adhesion and differentiation. As intracellular ligands of collagen XVII, the intracellular
domains of both a6 and β4 integrin polypeptide chains have been implicated (Hopkinson
et al., 1995, Borradori et al., 1997, Aho et al., 1998).
Integrins are dimers of α and β transmembrane subunits involved both in organizing
the cytoskeleton and in binding to the extracellular matrix. The epithelial specific integrin
α6β4 in the hemidesmosome serves as a ligand for plectin and collagen XVII (Niessen et
al., 1997, Reznieczek et al., 1998, Hopkinson et al., 1995, Schaapveld et al., 1998, Aho et
al., 1998), and in the extracellular space as a laminin 5 receptor, thereby contributing to
the stability of the anchoring complex. Since α6 and β4 integrin chains form a dimeric
receptor, one might expect that defects of both integrin subunits will lead to diminished
dermal-epidermal adhesion. Consistent with this expectation, transgenic mice with
targeted elimination of either the α6 or the β4 integrin gene showed absence of
hemidesmosomes and dramatic skin blistering (Georges-Labouesse et al., 1996, Van der
Neut et al., 1996).
The distribution of the integrin α3β1 is wider than that of α6β4, indicating broader
ligand specificity. In the epidermis this integrin is mainly localized at the ventral
keratinocyte plasma membrane between the anchoring complexes, where it supposedly
interacts with laminin 5-laminin 6 complexes (Champliaud et al., 1996, Burgeson and
Christiano, 1997). In vitro, α3β1 localizes to the focal contacts suggesting interactions
with the actin cytoskeleton. Transgenic mice with ablation of the α3 integrin gene showed
disorganization of the basement membrane and mild skin blistering (DiPersio et al.,
1997). However, the frictional forces required to induce dermal-epidermal separation
were considerably stronger than those involved in blister formation in α6 or β4 integrin
deficient animals, implying that α3 integrin contributes to dermal-epidermal adhesion,
but in a less crucial manner than α6β4 integrin in the anchoring complex. Diverse studies
on determination of the extracellular ligands of α3β1 have not yielded unambigous results
(see Aumailley and Smyth, 1998).
Collagen XIII is a transmembrane protein with a wide tissue distribution (see Prockop
and Kivirikko, 1995). Remarkably, a strong epidermal expression, also on the ventral
keratinocyte plasma membrane facing the lamina lucida was observed. Similarly to
integrin α3β1, collagen XIII was found in focal adhesion plaques in cultured
keratinocytes. These plaques represent structures involved in the adhesion of cells to the
extracellular matrix. The α1 (XIII) collagen mRNA is characterized by complex
alternative splicing of both collagenous and noncollagenous sequences. Analysis of total of
17 splice combinations of nine exons suggest that the predicted length of the
corresponding polypeptides varies between 651 and 710 arnino acid residues (Peltonen et
al., 1997).
Syndecans are a family of transmembrane heparan sulphate proteoglycans. They bind
and modify the action of various growth factors/cytokines, proteases/ antiproteases, cell
adhesion molecules, and extracellular matrix components and thereby regulate cellular
growth, adhesion and movement. Syndecan 1 is located over the entire surface of
keratinocytes, whereas syndecan 4 mainly is found at the basal surface contacting matrix
(Gallo et al., 1996). Syndecans bind collagens and fibronectin and thus act as receptors for
the extracellular matrix. Similarly to collagen XVII, the syndecan ectodomain is shed
constitutively by cultured cells. Proteases and growth factors active during wound repair
can accelerate syndecan shedding from cell surfaces, suggesting physiological roles for the
soluble ectodomains (Fitzgerald et al., 2000).
The muscle and neural protein dystroglycan is a ubiquitous transmembrane receptor for
extracellular ligands, such as laminins and agrin, and it provides a linkage of the basement
membrane to the actin cytoskeleton (Henry and Campbell, 1996). Interestingly,
dystroglycan is also expressed by epithelial cells including keratinocytes. It is encoded by a
single gene and cleaved into two proteins, α- and β-dystroglycan, by posttranslational
processing. The functions of dystroglycan in the skin still remain elusive, but some
information was derived from targeted elimination of the dystroglycan gene in mice.
Homozygous embryos exhibited gross developmental abnormalities including an early
disruption of Reichert’s membrane, an extra-embryonic basement membrane. The
localization of two critical structural elements of Reichert’s membrane, laminin and
collagen IV, were specifically disrupted, suggesting that dystroglycan may be required for
basement membrane organization (Williamson et al., 1997).
Anchoring filament and lamina lucida proteins

Laminin 5 is a major consituent in anchoring filaments which extend from the
hemidesmosomes to the lamina densa. Laminin 5, the smallest member of the laminin
family, is a unique heterotrimer containing α3, β3 and γ2 subunit polypeptides, all of
which are proteolytically processed to truncated chains during biosynthesis and
supramolecular assembly (Marinkovich et al., 1992). Laminin 5 is the link between
anchoring filaments and anchoring fibrils. It is the major ligand of α6β4 integrin,
presumably binding to the extracellular domain of the integrin in the anchoring filaments.
It also binds strongly to the Nterminal globular domain of collagen VII (Chen et al., 1997,
Rousselle et al., 1997). Laminin 5 is essential for the dermal-epidermal adhesion, as
demonstrated by junctional epidermolysis bullosa Herlitz (see Pulkkinen and Uitto,
1998), a JEB subtype in which lack of laminin 5 leads to massive dermal-epidermal

separation and early demise.
The a3β3γ2 laminin 5 molecule is not the only molecular form of laminin α3 chain, it
can also associate with the β1γ1 dimer to form laminin 6. At the DEJZ, laminin 6 forms
disulphide-bonded complexes with laminin 5 (Champliaud et al., 1996). The complexes
interact with the hemidesmosomal α6β4 integrin, the interhemidesmosomal α3β1
integrin and with collagen VII, thereby strengthening the lamina lucida network of the
basement membrane.
The recently characterized laminin α5 chain is also found at the DEJZ (Miner et al.,
1997). Since it can associate with the β1γ1 dimer to form laminin 10, this laminin
presumably assists laminins 5 and 6 in forming stable complexes with other DEJZ
components. Likewise, laminin α2 chain which can form laminin 2 by association with the
β1γ1 dimer has been shown to be present at the DEJZ, but details of its distribution and
putative functions remain unclear (Burgeson and Christiano, 1997).
Full length collagen XVII is a homotrimeric transmembrane molecule of three 180 kD l
(XVII) chains, with the N-glycosylated 120 kD extracellular domain extending into the
lamina lucida. Based on immuno-EM studies (Masunaga et al., 1997), collagen XVII
ectodomain is likely to participate in the formation of the anchoring filaments together with
laminin 5 (Reddy et al., 1998). Similarly to syndecans, keratinocytes shed the ectodomain
of collagen XVII through furin mediated proteolytic processing, beside maintaining the
full length transmembrane protein (Hirako et al., 1998, Schäcke et al., 1998). It is enticing
to speculate about the potential functions of a soluble ectodomain of collagen XVII. The
cleavage of the ectodomain may be a process for rapidly downregulating the protein from
the cell surface. Alternatively, generation of a soluble form that has properties either
identical with, or subtly different from those of the membrane bound form may be a way
to fine-regulate signal transduction and/or cell attachment to the basement membrane
during proliferation and differentiation of the epidermis.
Using linear IgA dermatosis autoantisera and a monoclonal antibody, two forms of a
basement membrane protein, LAD-1, were identified as components of the lamina lucida
and the anchoring filaments (Marinkovich et al., 1996). In vitro, the 120 kD form secreted
by keratinocytes was processed to the 97 kD form during purification; this smaller form
was also isolated from epidermal extracts (Zone et al., 1990). Sequencing of the 97 kD
form revealed partial amino acid sequence identity with the ectodomain of collagen XVII
(Zone et al., 1998). These data, together with our current investigations with both linear
IgA dermatosis autoantisera and domain-specific antibodies against recombinant collagen
XVII fragments suggest that LAD-1 is identical with the soluble ectodomain of collagen
XVII.
Lamina densa proteins

The major lamina densa molecules discussed below are ubiquitous basement membrane
components. Through self-assembly and ligand interactions they build and stabilize the
major basement membrane network which serves as the basic scaffold for cell adhesion
and matrix attachment. At the DEJZ, this basic structure is strengthened through
interactions with the proteins in the anchoring complexes.
Collagen IV occurs as tissue specific isoforms, depending on the chain composition of the
heterotrimeric collagen molecules (see Beck and Gruber, 1995, Timpl, 1996). The DEJZ
contains three different α(IV) chains, each of about 100 kD. The ubiquitous α1(IV) and α2
(IV) chains form heterotrimeric α1(IV)2 α2(IV) 1 molecules that build the major basement
membrane network which probably exerts fundamental supportive functions. The
importance of these chains is alluded to by the fact that no mutations in the COL4A1 and
COL4A2 genes have been found, possibly because their defects are lethal during
embryonic development. The molecular structure of the third collagen IV polypeptide at
the DEJZ, the a5(IV) chain, is unknown at present. This chain does not seem to be crucial
for dermal-epidermal adhesion since patients with Alport syndrome, a hereditary kidney
disease, who lack the α5(IV) chain do not exhibit skin blistering (Antignac, 1995).
Collagen IV is a major adhesion molecule, it interacts at least with nidogen, BM-40/
SPARC and β1 integrin (see Timpl, 1996), thus mediating attachment of basal
keratinocytes to the basement membrane.
Nidogen is a ubiquitous 150 kD basement membrane glycoprotein which consists of
three globular domains and a central rod. It is a strongly interactive molecule with
multiple ligands, e.g. collagen IV, perlecan and fibulins, and it is believed to function as a
stabilizer of the basic protein scaffold of the basement membranes (Timpl, 1996). In
addition, the C-terminus of nidogen binds strongly to laminin γ1-chain (Mayer et al.,
1995), the interaction being calcium dependent. Therefore, at least laminins 2, 6 and 10
can act as nidogen ligands at the DEJZ.
Perlecan is the main proteoglycan of basement membranes and pericellular spaces (lozzo
et al., 1994). It has a large nearly 500 kD core protein with heparan sulphate attached to
the aminoterminal domain. In the skin, perlecan is manufactured by papillary fibroblasts,
but not by keratinocytes. It contributes to the stability of the basic protein scaffold of the
basement membrane by binding to nidogen and dystroglycan (Talts et al., 1999). Besides,
perlecan’s ability to capture growth factors and cytokines points to an important
regulatory function for basement membrane synthesis and turnover.
Anchoring fibril proteins

Anchoring fibrils represent polymers of collagen VII, a large homotrimeric protein with a
central triple helix, and flanking amino- and carboxyterminal globular domains. Collagen
VII consists of three identical α1(VII) chains of about 300 kD each (Burgeson, 1993,
Christiano et al., 1994). Keratinocytes synthesize and secrete collagen VII as a triplehelical
precursor—procollagen VII—into the extracellular matrix, where proteolytic removal of
the C-terminal propeptide is necessary for correct supramolecular assembly (Morris et al.,
1986, Bruckner-Tuderman et al., 1995). During fibrillogenesis, collagen VII molecules
form antiparallel tail-to-tail dimers with a central carboxy-terminal overlap and with the
ammo-termini pointing outwards (Burgeson, 1993). The dimers then aggregate laterally
in a nonstaggered manner into the anchoring fibrils which are further stabilised by
transglutaminase-2 cross-links (Raghunath et al., 1996). Anchoring fibrils are attached to
the basement membrane through binding to laminin 5 and collagen IV (Chen et al., 1997,
Rousselle et al., 1997).
A 50 kDa non-collagenous component of the DEJZ, coined GDA-J/F3 antigen:, was
identified as an epitope of a monoclonal antibody. The GDA-J/F3 antigen is a small
disulphide-bonded protein with a potential to interact with basement membrane
proteoglycans, however, its primary structure remains unknown at present. With
immunoelectron microscopy, it was localized to the insertion points of the anchoring
fibrils into the lamina densa. In vitro, GDA-J/F3 is synthesized and secreted by
keratinocytes, and to a lesser extent by normal human skin fibroblasts. Integration of the
GDA-J/F3 antigen into the histoarchitecture of the DEJZ is dependent on the presence of
collagen VII, since the GDA-J/F3 epitope was missing in several DEB patients with absent
or mutated collagen VII (Gayraud et al., 1997).
Biosynthesis, Processing and Regulation of DEJZ

Components
Both keratinocytes and fibroblasts contribute to the synthesis of the DEJZ. Ample
evidence points to active epithelial-mesenchymal crosstalk by the regulation of DEJZ
formation, the biosynthesis of molecules in one cell type is modulated byjuxtacrine or
paracrine signals from neighbouring cells or from the extracellular matrix. In vitro,
depending on the experimental conditions, the one or the other cell type plays a more
active role in the production of certain molecules (Marinkovich, 1992, König and
Bruckner-Tuderman, 1994, Fleischmajer et al., 1995). Epithelial cells manufacture
collagen VII and most laminin chains, whereas mesenchymal cells synthesize collagen IV,
nidogen, perlecan and the laminin α2 chain (Salmivirta et al, 1997). In skin equivalent
cultures (Fleischmajer et al., 1995) and in developing mouse organs (Ekblom et al., 1994)
fibroblasts are the only source of nidogen at the epithelial-mesenchymal interface.
An interesting, additional regulatory step of the supramolecular assembly of DEJZ is
provided by post-translational proteolytic processing of some components. The trimming
is thought to contribute to correct association and folding of the polypeptide subunits,
since propeptides can recognize binding sites for accurate alignment of the subunits or
prevent premature aggregation of the molecules. Two important components of the
anchoring complex are subject to such processing, laminin 5 and collagen VII. The 200 kD
α3 chain of laminin 5 is processed to a 145 kD mature chain by cleavage in the C-terminal
G-domain, and the 155 kD γ2 chain is cleaved within the N-terminal short arm to yield a
105 kD polypeptide (Marinkovich et al., 1992). The carboxyterminal NC-2 domain of
procollagen VII is removed prior to stabilization of the anchoring fibrils (Bruckner-
Tuderman et al., 1995). BMP-1 (bone morphogenetic protein 1), also known as
procollagen C-proteinase (Kessler et al., 1996), processes the γ2 chain of laminin 5
(Amano et al., 1997), and possibly procollagen VII, in the extracellular space.
Interestingly, BMP-1 is synthesized by fibroblasts, whereas both laminin 5 and collagen
VII are epithelial products suggesting that epithelial-mesenchymal interactions play an
important regulatory role at this step. The significance of the proteolytic processing for
assembly of the DEJZ was demonstrated in BMP-1 knock-out mice which showed a
disorganized basement membrane structure (Suzuki et al., 1996). This is further

corroborated by the finding that a deletion of the BMP-1 consensus sequence in human
procollagen VII was associated with dystrophic epidermolysis bullosa (Winberg et al.,
1997).
Yet another form of regulation may be proteolytic cleavage of the extracellular
domains of integral type 1 or type 2 transmembrane proteins. The ectodomains of
collagen XVII and syndecans are shed from the keratinocyte surface by selective post-
translational proteolysis (Subramanian et al., 1997, Hirako et al., 1998, Schäcke et al.,
1998). The process is catalyzed by a group of enzymes collectively referred to as secretases
or sheddases which have been only partially characterized, but many can be grouped as
metallo- and/or serine proteinases (Hooper et al., 1997). At least in some instances, e.g.
by processing of the collagen XVII ectodomain, proprotein convertases of the furin/
PACE-family are involved. Furin mediated proteolysis is also required to yield a heavy and
a light chain of the α6 integrin polypeptide, a necessary step for activation of the integrin
(Delwel et al., 1997). The proteolytic cleavage of the ectodomains of keratinocyte surface
proteins may provide means to regulate cell adhesion, differentiation and migration at the
DEJZ, however, the details of these events remain unclear.
The biology of the DEJZ is evidently also controlled by growth factors and cytokines.
Apart from proteoglycans in the dermis, perlecan and syndecans can trap various factors,
act as a reservoir for regulatory signals and release them during physiological and
pathological processes, such as development, repair or invasion. Factors relevant for
modulation of synthesis and maintenance of the DEJZ include TGF-β, CTGF, KGF, TNF-
α and interferon-γ (Eckes et al., 1997). Most potent stimulators of laminin 5, collagen
VII, collagen XVII, and perlecan expression are TGF-β1 and -β2 isoforms, both of which
bind to microfibrils and other extracellular matrix structures in the skin (Raghunath et al.,
1998).
DISORDERS OF DERMAL-EPIDERMAL ADHESION

Many DEJZ macromolecules are targeted in both hereditary and acquired blistering skin
diseases. In acquired autoimmune blistering disorders, tissue bound autoantibodies are
believed to perturb ligand interactions of the target proteins thereby contributing to
blister formation (for review, see Yancey, 1995, Pleyer et al., 1996). In hereditary
epidermolysis bullosa, mutated DEJZ components form instable anchoring complexes
resulting in detachment of the epidermis from the dermis after minor friction or trauma
(Gedde-Dahl and Anton-Lamprecht, 1996, Bruckner-Tuderman, 1993, Christiano and
Uitto, 1996).
Acquired Disorders of Dermal-Epidermal Adhesion:

Autoantibodies to DEJZ Molecules
Acquired disorders of dermal-epidermal adhesion are usually autoimmune diseases with
circulating and tissue-bound autoantibodies targeting DEJZ molecules. Molecular cloning,
protein isolation and generation of recombinant protein fragments have facilitated
Table 7.2 Autoimmunity to DEJZ components
characterization of the autoantigens and definition of the immunodominant epitopes

within the polypeptides (Table 7.2). BP230, plectin, collagen XVII, laminin 5 and
collagen VII serve as autoantigens in different disorders (Stanley et al., 1988, Woodley et
al., 1988, Domloge-Hultsch et al., 1992, Giudice et al., 1993, Iwasaki et al., 1995,
Fujiwara et al., 1996). In bullous pemphigoid, antibodies reactive with BP230 and/or
collagen XVII are common. More than 90% of the patients with collagen XVII reactivity
target the extracellular NC-16a domain (Giudice et al., 1993, Balding et al., 1996,
Zillikens et al., 1997). Two pemphigoid variants, pemphigoid gestationes and cicatricial
pemphigoid, show reactivity with the ectodomain of collagen XVII or/and with the
laminin a3 chain, respectively (Kawahara et al., 1998, Kirtschig et al., 1998). In linear IgA
dermatosis, the autoantibodies seem to target the 120 kD soluble ectodomain of collagen
XVII (Pas et al., 1997; Schumann et al., 2000). Previously, a 120 kD/97 kD linear IgA
dermatosis antigen, or LAD-1, was identified using patient sera and a monoclonal
antibody (Zone et al., 1990, Marinkovich et al., 1992). However, recent protein chemical
and sequence data support the assumption that LAD-1 does not represent a distinct gene
product, but is identical with the soluble ectodomain of collagen XVII. Antibodies to
collagen VII are associated with epidermolysis bullosa acquisita, bullous SLE and some
forms of inflammatory bowel disease (Chen et al., 1997). In most cases, the
immunodominant epitopes are within the globular NC-1 domain (Lapiere et al., 1993),
except in a novel ERA subgroup in children, in which the autoantibodies targeted only the
triple-helical central domain of collagen VII (Tanaka et al., 1997).
The molecular mechanisms involved in blister induction in these diseases are not fully
understood (Chan et al., 1998). Studies using newborn mice as an animal model indicated
pathogenicity of autoantibodies to collagen XVII and laminin 5; passive transfer of
antibodies into the animals induced binding of the antibodies at the DEJZ and dermal-
epidermal tissue separation (Liu et al., 1993, Lazarova et al., 1996). Besides, ample
evidence exists for involvement of proteolytic enzymes released from resident skin cells
or inflammatory cells in degradation of DEJZ components in autoimmune blistering skin

diseases (Liu et al., 1998). Somewhat puzzling is the role of autoantibodies to BP230 and
plectin (Stanley et al., 1988, Fujiwara et al., 1996), both intracellular proteins, in the
pathogenesis of skin blistering. Possibly, the antibody formation is secondary to blistering
and exposure of the intracellular epitopes and represents an epiphenomenon.
Heritable Disorders of Dermal-Epidermal Adhesion:

Epidermolysis Bullosa
Mutations in the genes for anchoring complex components impair the dermal-epidermal
adhesion and cause increased sensitivity to friction and skin blistering as a consequence of
minor trauma. The name epidermolysis bullosa (EB) which refers to the mechanically
induced detachment of the epidermis from the dermis, into the blister roof, was coined for
these conditions almost 80 years ago (Siemens, 1921). Despite modern knowledge about
the genetic and biological heterogeneity of the disease group, the name has stayed. Based
on ultrastructural criteria, three main categories of EB have been defined according to the
precise level of tissue separation: simplex, junctional and dystrophic subtypes. In EB
simplex (EBS) the separation occurs within the basal keratinocytes, as a consequence of
cytolysis of the cells; in junctional EB (JEB) the cleavage occurs along the lamina lucida;
and in dystrophic EB (DEB), below the basement membrane, within the uppermost dermis
(Figure 7.2). Predictably, abnormality of any DEJZ component can lead to impaired
interactions, to diminished adhesion of the skin layers and to blistering. The multitude of
pathologic alterations is alluded to by the extensive clinical heterogeneity of EB, more
than 20 genetic and clinical subtypes are known (Table 7.3; Fine et al., 1991). In
Table 7.4, the molecular defects in different EB subtypes are summarized.
EBS: a disorder affecting keratin intermediate filaments

The first indications for involvement of keratins in the etiopathogenesis of EBS came from
ultrastructural observations on clumping of the keratin filament bundles in basal
keratinocytes in EBS skin (Fuchs and Yang, 1999). The basal cell keratins 5 and 14, the
former a type II and the latter a type I keratin, form as a pair a heterodimer and
copolymerize into supramolecular intermediate filaments (Lane, 1993). Consistently,
transgenic mice with truncated keratin 14 exhibited a phenotype with tonofilament
clumping, basal cell cytolysis and skin blistering (Vassar et al., 1991), and subsequently a
large number of mutations in the genes for keratin 5 and 14 have been identified in EBS
families. Different mutations in the two genes underlie a variety of clinical EBS phenotypes,
i.e. the subtypes Köbner (Bonifas et al., 1991)) Dowling-Meara (Coulomb et al., 1991, Lane
et al., 1992), Weber-Cockayne (Chan et al., 1993) and EBS with mottled pigmentation
(Uttam et al., 1996). Filament assembly assays with mutated keratins from the patient
cells pointed to a tendency of mutations in the highly con server regions of the protein to
cause severe disruption of the filament network and more severe phenotypes. In contrast,
mutations in the less conserved domains had minor effects on the filament network and
were associated with a milder clinical presentation (Uttam et al., 1996).
Figure 7.2 Immunofluorescence staining of DEJZ components in spontaneously separated EB skin.

A: Laminin 5 on the floor of a junctional blister. B: Collagen XVII in the roof of a junctional blister.
C: Collagen VII on the floor of a junctional blister. D: Lack of collagen XVII in GABEB skin. E:
Collagen VII in the roof of a dystrophic blister. F: Intraepidermal accumulation of collagen VII in
TBDN.
A ligand of the intermediate filaments, the cytoskeleton-membrane linker protein
plectin is involved in the rare autosomal recessive EBS with muscular dystrophy (EBS-MD)
(McLean et al., 1996); nonsense mutations in the PLEC1 gene have been disclosed in about
a dozen EBS-MD families (Pulkkinen and Uitto, 1999). These lead to lack of plectin in the
hemidesmosomal plaque, to defective cytoskeleton anchorage and to cell fragility.
Clinically, the disease manifests with trauma-induced non-scarring skin blistering in the
neonatal period and late onset muscular dystrophy at the age of 2–30 years. The
expression of plectin isoforms in many tissues including epithelia and muscle explains the
combined EBS-MD phenotype (Wiche et al., 1983).
Mutations of the other intermediate filament connector, BP230, have not been found
yet. However, targeted elimination of the gene for BP230 in mice predictably produced a
cutaneous phenotype. Beside abnormal hemidesmosome structure, lack of anchorage of
Table 7.3 Subtypes of epidermolysis Bullosa
*AD=autosomal dominant; AR=autosomal recessive; X-=X-chromosomal; EBS-MD=EBS with

muscular dystrophy; GABEB=generalised atrophie benign EB; JEB-PA=JEB with pyloric atresia;
TBDN=transient bullous dermolysis of the newborn.
For more details, and for a revised classification the reader is referred to Fine et al., 2000 and
Gedde-Dahl and Anton-Lamprecht, 1996
the cytoskeleton and keratinocyte fragility, the structure, lack of anchorage of the
cytoskeleton and keratinocyte fragilityr, the mice unexpectedly developed sensory nerve
degeneration (Guo et al., 1995) and muscular dystrophy. This unusual phenotype led to
the discovery of a neural isoform of BP230 which also had been inactivated by the
targeting vector (Yang et al., 1996).
JEB: abnormalities of the anchoring filaments

Detachment of the basal keratinocytes from the lamina densa, rudimentary
hemidesmosomes and anchoring filaments are ultrastructural hallmarks in JEB (McGrath
and Eady, 1997, McMillan et al., 1998). Therefore, molecular components of the
anchoring filaments, laminin 5, α6β4 integrin and collagen XVII, were obvious candidates
Table 7.4 Human disease genes in heritable blistering disorders of the skin
Figure 7.3 A similar phenotype with skin blistering and parietal alopecia in two JEB patients with
mutations in different genes. On the left, an 18 year-old proband with the homozygous nonsense
mutation Q1016X in COL17A1 gene (Schumann et al., 1997). On the right, a 43 year-old patient with
the heterozygous LAMBS mutation R635X; the other mutation of this patient remains elusive.
for the different JEB subtypes. Indeed, mutations in the genes encoding these molecules
have been shown to cause clinically diverse JEB subtypes, ranging from the lethal Herlitz
JEB to mild localized blistering (Gedde-Dahl & Gedde-Dahl and Anton-Lamprecht, 1996,
Bruckner-Tuderman, 2000, Pulkkinen and Uitto, 1999).
Lack of laminin 5 results in extreme cutaneous and mucosal fragility in JEB Herlitz, the
most severe, often postnatally lethal JEB subtype. Homozygous or compound
heterozygous mutations in the LAMAS, LAMBS and LAMC2 genes encoding the α3, β3
and the γ2 chains of laminin 5, respectively, are associated with JEB Herlitz (see
Christiano and Uitto, 1996). Mutations causing premature termination codons often lead
to nonsense-mediated mRNA decay and to lack of synthesis of the corresponding
polypeptide chain, and absence of any one of the three subunit polypeptides prevents
assembly and secretion of the entire heterotrimeric laminin 5 molecule. Use of chain
specific laminin antibodies is therefore of limited value for determination of the defective
gene (McMillan et al., 1997). However, mutation screening is facilitated by the fact that
LAMBS mutations account for 80 % of all gene defects in JEB (Christiano and Uitto,
1996). In 50 % of the cases, one of the two common mutations R42X or R635X is seen
(Kivirikko et al., 1996). R635X and some other nonsense mutations in combination with
missense mutations has been found associated with milder, non-lethal JEB subtypes (
Table 7.4, Figure 7.3).
COL17A1 gene mutations underlie most cases of generalized atrophie benign EB,
GABEB (McGrath et al., 1995, 1996 a/b, Darling et al., 1997, 1998 a/b, Gatalica et al.,
1997, Schumann et al., 1997, Chavanas et al., 1997, Jonkman et al., 1997, Floeth et al.,
1998, 1999). This represents a JEB subtype with generalized blistering, skin atrophy,
extensive alopecia, scarce eyelashes, eyebrows and body hair, as well as nail dystrophy and
dental anomalies (Figure 7.3). Morphological hallmarks are rudimentary
hemidesmosomes and anchoring filaments (McGrath and Eady, 1997). Most of the
mutations known as far lead to a premature termination codon and absence of collagen
XVII in the skin. Missense mutations or missense/nonsense mutation combinations which
allowed synthesis of structurally abnormal collagen XVII caused milder JEB subtypes,
including the JEB localisata subtype with mostly acral blistering, but not with alopecia
(McGrath et al., 1996, Schumann et al., 1997, Floeth et al., 1998, Tasanen et al., 2000).
This raises a very intriguing biological quesr tion about the relation of collagen XVII
abnormalities with hair growth and loss.
Deficiency of α6β4 integrin is associated with abnormal hemidesmosomes, blistering of
the skin and pyloric atresia. This JEB subtype often has lethal outcome due to extensive skin
fragility (Vidai et al., 1995, Pulkkinen et al., 1997, Ruzzi et al., 1997). Consistently, α6
and β4 integrin knock-out mice exhibited severely attenuated epithelial adhesion in the
skin and other organs, and early demise (Van der Neut et al., 1996, Georges-Labouesse et
al., 1996). Recently, missense mutations in the gene for integrin β4, ITGB4, were found
to underlie pyloric atresia with extremely mild, late onset skin blistering (Pulkkinen et al.,
1998). The prognosis in these cases is very good after the pyloric atresia is surgically
corrected.
DEB: abnormalities of anchoring fibrils

In DEB, the cleavage plane is below lamina densa, at the level of the anchoring fibrils. In
contrast to normal skin with well-defined slender and centrosymmetrically cross-banded
anchoring fibrils (Keene et al., 1997), DEB skin presents with broad, blunt whispy fibrils
without cross-banding, or no fibrils at all (McGrath and Eady, 1994, Gedde-Dahl and
Anton-Lamprecht, 1996). Complete lack of anchoring fibrils and collagen VII are
characteristic signs of the most severe subtype, DEB mutilans (Bruckner-Tuderman et al.,
1989). In milder DEB forms mutated collagen VII polypeptides are synthesized, but the
morphology of the anchoring fibrils is abnormal. Over 100 mutations in COL7A1, the
gene for collagen VII, have been identified in different DEB subtypes (Christiano et al.,
1993, Bruckner-Tuderman et al., 1995, Gardella et al., 1996, Christiano and Uitto, 1996,
Dunnill et al., 1996, Hovnanian et al., 1997, Mellerio et al., 1997), but the complexity of
the mutation constellations and their biological consequences are only beginning to
emerge. Clinically, DEB presents with blistering of the skin followed by scarring. The
cleavage below the basement membrane explains the scar formation, since any wound
reaching into the dermis heals with a scar. DEB contains two dominantly and four
recessively inherited subtypes (Table 7.3). Apart from the inheritance pattern, the
subtypes differ in the distribution of the lesions. The spectrum varies from extensive
blistering and mutilating scarring to a mild acral affection, or in extreme cases, to mere
nail dystrophy. Despite this wide spectrum of phenotypes, all DEB forms are believed to
be allelic. COL7A1 mutations have been shown to underlie both recessive and dominant
DEB subtypes, and in some families combinations of recessive and dominant mutations
lead to unusual and variable phenotypes (Christiano et al., 1996, Winberg et al., 1997,
Hammami-Hauasli et al., 1998a). Similarly to the deficiencies of other anchoring complex
molecules, collagen VII nullizygotes present with the most severe DEB phenotypes (see
Christiano and Uitto, 1996). Milder phenotypes are due to missense mutations or
nonsense/ missense mutation combinations.
Molecular heterogeneity of DEB

Investigation of homozygous and heterozygous COL7A1 mutations and their biological
consequences have revealed extensive molecular heterogeneity of collagen VII defects.
From a protein chemical point of view, many pathogenetic mechanisms would seem
predictable. Dominant negative interference of amino acid substitution or deletion
mutations for formation of anchoring fibrils has been documented (Christiano et al., 1996,
Sakuntabhai et al., 1998). However, it has come as a surprise that the trimeric triple-
helical collagen VII molecule seems to tolerate structural aberrations without obvious
functional defects. This is best illustrated by the example of unaffected parents of patients
with recessive DEB. These individuals carry heterozygous missense mutations, express the
mutated polypeptides and have abnormal collagen VII molecules in the skin, however,
without obvious functional deficits. In this context, glycine substitutions are particularly
interesting. Usually a substitution of a small glycine residue within the triple-helix of a
collagen leads to dominant negative effects (Prockop and Kivirikko, 1995, Bruckner-
Tuderman and Bruckner, 1998, Bruckner-Tuderman et al., 1999). The mutated
polypeptides fold together with normal polypeptides into collagen triple-helices, and the
structurally aberrant trimeric molecules are incorporated into fibrils. The effects of the
initally rather small structural abnormality are thereby accentuated by the supramolecular
assembly, and the resulting collagen fibrils are rendered functionally inadequate. This is
not the case with collagen VII, as seen by the unaffected carriers of mutations. Recent
studies showed that glycine substitutions in critical positions within collagen VII molecule
interfered with the protein folding and suprastructure in a dominant negative manner
(Figure 7.4), whereas other glycine substitutions within the distal ends of the triple-helix
exhibited no adverse biological effects (Christiano et al., 1996, Hammami-Hauasli et al.,
1998 a,b, Terracina et al., 1998).
Figure 7.4 Immunofluorescence staining with antibodies to collagen VII of keratinocytes from a
patient with dominant DEB (A) and control (B). The dominant COL7A1 deletion mutation
6081del28 causes in-frame skipping of exon 73 (Sakuntabhai et al., 1998) and interferes with folding
and secretion of procollagen VII in a dominant negative manner. This results in intracellular
accumulation, partial degradation and delayed secretion of the protein.
Another example of unusual variable phenotypes is modulation of the phenotype by
second mutations. We have characterized two families with an exon skipping mutation
that prevented normal processing of procollagen VII to collagen VII, i.e. the
carboxyterminal propeptide was not removed. The deletion was combined with a
different glycine substitution mutation in each family resulting in distinct clinical
phenotypes (Winberg et al., 1996). Similarly, in a TBDN family, combination of
dominant and recessive glycine substitution mutations in COL7A1 resulted in modulation
of the phenotype. Two point mutations caused amino acid substitutions G1519D and
G2251E in the triple helical domain of collagen VII. In the heterozygous state the paternal
mutation G1519D was silent, and the maternal mutation G2251E led merely to nail
dystrophy, but not skin blistering (Figure 7.5). In the proband, compound heterozygosity
for the mutations caused massive, transitory retention of collagen VII in the epidermis, its
reduced deposition at the basement membrane zone and extensive dermo-epidermal

separation at birth (Hammami-Hauasli et al., 1998b).
Discovery of the extensive variability of COL7A1 mutation constellations has
consequences for genetic counseling of affected families. Detection of one or two
mutations in the index patient does not always allow unambiguous determination of the
inheritance pattern or prognostic predictions. Therefore, the counceling should be very
cautious.
Allelic diseases: heterogeneity of EB phenotypes

Molecular analysis of skin diseases has provided unambiguous evidence for the fact that
phenotypically different diseases can be allelic disorders. In EBS, keratin 5 and 14
abnormalities underlie at least four different clinical subtypes. DEB patients who are
nullizygotes for COL7A1 alleles suffer from a very severe mutilans variant of dystrophic
EB with extensive blistering and scarring, while patients with missense mutations in the
same gene exhibit the localisata EB dystrophica variant with milder skin affection.
Similarly, null alleles of COL17A1 gene underlie GABEB with loss of hair, but missense
mutations in the same gene cause junctional EB localisata with blistering at mechanically
exposed sites only. As further examples serve LAMBS or ITGB4 gene mutations with
lethal JEB phenotypes as a consequence of homozygous or compound heterozygous
nonsense mutations and milder clinical affections by missense mutations. The different
phenotypes probably reflect perturbation of particular functions exercised by the protein
domain affected by the mutation.
Recent investigations have revealed an unusual self-limiting postnatal blistering disease,
the transient bullous dermolysis of the newborn (TBDN), as allelic to DEB (Figure 7.5).
At birth, TBDN is characterized by extensive subepidermal blisters, reduced or abnormal
anchoring fibrils, and massive transitory retention of collagen VII in the epidermis.
However, within the first months and years of life, TBDN heals or improves dramatically.
In two families, compound heterozygosity for dominant and negative glycine substitution
mutations in COL7A1 (Hammami-Hauasli et al., 1998b; Shimizu et al., 1999) was found
to cause accumulation of collagen VII in keratinocytes, a situation that normalized within
approximately two years. The molecular mechanisms of the distinct accumulation of
collagen VII in the epidermis and in particular its disappearance from the epidermis and
deposition at the DEJZ after a certain time remain elusive at present.
Skin Blistering Phenotypes—Lessons for Normal Biology

Molecular studies on EB and the genes and proteins involved have produced a wealth of
information on the normal biology of the DEJZ. The function of keratins in maintaining
structural integrity of cells was established when keratin mutations were shown to lead to
skin blistering (Vassar et al., 1991). Since, several keratins and other epidermal proteins
have been implicated in heritable disorders of epidermal differentiation (Roop, 1995).
The investigations established plectin and BP230 as versatile cytoskeletal linker proteins in
the hemidesmosomes and showed that BP230 and plectin isoforms exist in the skin,
Figure 7.5 Modulation of DEB phenotype by a second mutation. A: A proband with the dominant
COL7A1 mutation G2251E has toe nail dystrophy, but no skin blistering. B: Her newborn child
who had inherited G2251E and another glycine substitution mutation, G1519D, from the father
presented with extensive skin blistering. G1519D was silent in heterozygous state, the father was
clinically unaffected.
muscle and neural tissues (see Fuchs and Yang, 1999). Disclosure of mutations in JEB has
shown that some proteins of the anchoring complex play a more pivotal role in epithelial
cell adhesion and epidermal resistance to friction than others. Lack of laminin 5 seems to
produce the most devastating, lethal JEB phenotype, whereas lack of another anchoring
filament component, collagen XVII, leads to generalized skin blistering with altogether
milder manifestations that are well compatible with life (Burgeson and Christiano, 1997,
Pulkkinen and Uitto, 1999). The ligand of laminin 5, integrin a6β4, likewise represents
an indispensable component of the anchoring complex, since homozygous null alleles of
either gene desmonstrated extensive skin blistering with lethal outcome (Georges-
Labouesse et al., 1996, Van der Neut et al., 1996, Pulkkinen et al., 1997, 1998). The
association of integrin 64 deficiency with congenital pyloric atresia (Vidai et al., 1995,
Ruzzi et al., 1997) suggests an important role for a6β4 integrin in the development of the
gastrointestinal tract. New information was also obtained about the polymerization of
collagen VII into anchoring fibrils. Patients with recessive DEB are compound
heterozygous or homozygous for COL7A1 gene mutations, and therefore, unaffected
parents are obligate carriers of heterozygous mutations (Christiano et al., 1996,
Hammami-Hauasli et al., 1998b). This is a situation not observed with other collagens.
Heterozygous mutations, typically glycine substitutions, usually cause dominant negative
effects which lead to more or less severe disturbances of molecular assembly or
supramolecular aggregation of collagens. Collagen VII therefore must use different
mechanisms for stabilisation of suprastructures than other collagens, perhaps binding of
other, yet unknown components of the basement membrane zone.
The functional significance of the anchoring complex proteins is further under-lined by
the fact that a quantitative reduction of a protein to 50% is not sufficient to impair dermal-
epidermal adhesion. This is illustrated by clinically unaffected heterozygous carriers of
nonsense mutations in the genes for plectin, a6β4 integrin, collagen XVII, laminin 5 or
collagen VII who only express one allelic product, and therefore theoretically synthesize
only one-half of the relevant protein. Indeed, keratinocytes from such individuals have
been shown to synthesize reduced amounts of protein in vitro (Hilal et al., 1993).
Remarkably, we have recently identified a clinically unaffected proband with a
heterozygous nonsense mutation in both LAMBS and COL17A1 genes (Floeth and
Bruckner-Tuderman, 1999). Therefore, it seems that a drastic reduction in the quantity
of the normal DEJZ molecules is required to cause symptoms and the molecular
constituents forming the biological suprastructures appear to be, at least in part, functionally
redundant.
Novel Candidate Genes and DEJZ Proteins

The DEJZ is known to contain a number of additional proteins which are candidates for
blistering skin disorders. The presence of these components at the DEJZ has been
demonstrated mostly with immunohistological techniques, however, their
suprastructures, ligands and roles in dermal-epidermal adhesion are not yet well denned.
Some of them may be expressed only under certain conditions, such as during
development or wound healing, others may have more permanent functions. Examples of
such components are are laminins 2 and 10, dystroglycan, syndecans, uncein, the GDA-J/
F3- antigen, fibulins, collagen XVIII, fibrillins, LTBPs and other components of the
microfibrils (for reviews, see Beck and Gruber, 1995, Ekblom and Timpl, 1996, Timpl,
1996, Burgeson and Christiano, 1997, Aumailley and Smyth, 1998, Bruckner-Tuderman
and Bruckner, 1998, Pulkkinen and Uitto, 1999).
Animal Models for Acquired and Heritable Blistering Skin

Diseases
Animals have been shown to suffer from autoimmune blistering disorders (Iwasaki et al.,
1995) similar to the human counter parts, and animal models have helped define some
aspects of dermal-epidermal adhesion. The role of autoantibodies in the pathogenesis of

bullous pemphigoid and cicatricial pemphigoid has been demonstrated in a mouse model,
using passive transfer of antibodies generated against collagen XVII (Liu et al., 1993) or
laminin 5 (Lazarova, 1995). Remarkably, neonatal mice injected with the antibodies
developed a blistering disorder that faithfully reproduced the key immunopathological
features of bullous pemphigoid or cicatricial pemphigoid, respectively, including
circulating autoantibodies, deposition of IgG and complement at the DEJZ, inflammatory
infiltration of the upper dermis, and subepidermal blistering in skin and/or mucous
membranes. These data suggest that the autoimmune response against the human collagen
XVII or laminin 5 is relevant in the pathogenesis of blister formation in patients.
Heritable mechanobullous disorders in animals are rare (see Bruckner-Tuderman et al.,
1991). Autosomal dominant EBS was reported in bulls, a severe JEB in a toy poodle, and
DEB in calves and dogs, but these forms have not been characterized in molecular terms.
Severe recessive DEB in inbred sheep was shown to result from lack of collagen VII and
anchoring fibrils in the skin, a phenotype similar to the mutilating DEB in humans
(Bruckner-Tuderman et al., 1991). More recently, gene ablation in mice has delivered
information of the functions of some novel DEJZ proteins. Studies on skin development in
integrin α3β1-deficient mice revealed regions of disorganized basement membrane in the
skin (DiPersio et al., 1997). In neonatal skin, matrix disorganization was frequently
accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix
proteins remained associated with both the dermal and epidermal sides of blisters,
suggesting rupture of the basement membrane itself, rather than detachment of the
epidermis from the basement membrane as occurs in some blistering disorders such as
epidermolysis bullosa. The findings support a novel role for α3β1 in establishment and/or
maintenance of basement membrane integrity. Ablation of the gene for laminin α5 chain
lead to abnormalities in many organs during embryogenesis and early lethality. Among
others, the basement membrane of skin and placenta was abnormal, indicating the
importance of this laminin chain for the development of the subepithelial basement
membranes (Miner et al., 1998). Targeted in activation of the collagen VII gene lead to
severe skin blistering and early demise of the animals (Heinonen et al., 1999).
Future Perspectives
Clarification of molecular pathomechanisms of blistering skin disorders not only extends
our knowledge on normal biology of the DEJZ but also forms a basis for novel therapeutic
approaches, such as somatic gene therapy. Optimal diseases to be treated with such
approaches are blistering skin disorders caused by null alleles, e.g. lack of collagen VII or
collagen XVII. The skin is an easily accessible organ for morphologic and biochemical
investigations, and keratinocyte culture and transplantation techniques have been well
developed for treatment of burns. Gene transfer into human keratinocytes in vitro has
been successfully performed in many laboratories, however stable transfection and
expression of correctly folded proteins still are problematic (see Khavari, 1998).
Future development of diverse successful therapies will also depend on the progress in
our understanding of suprastructure formation by structural DEJZ macromolecules. This
not only concerns the mechanisms of aggregate formation but also the structural
characteristics unique for each molecule. Further, functional redundancies of molecular
components within supramolecular aggregates will have to be defined in greater detail
than presently available. This endeavour will not only be assisted by the analysis of
aggregate formation by purified matrix macromolecules or their mixtures in vitro, but
also by the elucidation of further genetic defects and their consequences in animal or
human diseases as well as the generation of transgenic animals as models for human
diseases. This combined information will not only help to understand and treat heritable
skin diseases but also many common disorders currently considered as acquired.
ACKNOWLEDGMENTS
The author’s work was supported by grants SFB 492/A3 and SFB 293/B3 from the
Deutsche Forschungsgemeinschaft (DFG) and by the University of Münster IZKF 2/D5
from the Ministry for Education and Research. The expert help of Nadja Hammami-
Hauasli and Hauke Schumann with the illustrations is grate-fully acknowledged.
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LEUKOCYTE TRAFFICKING IN SKIN
DISEASES
8.
INTRODUCTION
JONATHAN N.W.N.BARKER
The skin, because of its privileged site, is continuously exposed to a number of potentially
injurious stimulae, not encountered in other organs. In much the same way that mucosal
structures, such as the gastro-intestinal tract, have developed specialised immunological
responses directed to its special needs, for example the production of secretory IgA and
mucosal homing T lymphocytes, so the skin has its own specialised structures. Critical
amongst these are epidermal antigen presenting Langerhans cells, which form a network
throughout the epidermis, and skin homing lymphocytes. Keratinocytes also critically
influence skin immune responses and should therefore be considered as specialised
immunologically active cells. To reflect the growing evidence for an active immunological
role for the skin, Bos coined the term Skin Immune System (SIS), building on skin
associated lymphoid tissue (SALT) defined by Streilein (1), to define the multiple cell
types and the complexity of immune responses within the skin (2).
Circulating blood leucocytes constantly scrutinize potential sites of pathogen entry,
rapidly moving from blood into tissue to mediate effective host defenses. The neutrophil
influx that characterises the immediate response is superseded by antigen-specific
mononuclear cell infiltration. In chronic inflammatory skin diseases such as psoriasis,
atopic dermatitis, lichen planus and lupus erythematosus and other T cell mediated
conditions including allergic contact dermatitis, this highly tuned surveillance system
appears awry, with inflammatory cells infiltrating skin in response to an unidentified
“pathogen” or “antigen”. Early vascular dilatation and perivascular accumulation of
lymphocytes, monocytes, and macrophages are followed by accumulation of T
lymphocytes within epidermis and at the tips of dermal papillae. Thus, mechanisms
responsible for leucocyte infiltration into skin are of great relevance to the pathogenesis of
the conditions mentioned above and may point to important targets for future treatment
strategies.
Cohnheim devised an elegant in vivo experimental system using intravital microscopy to
visualise the inflammatory response in rabbit ears following topical croton oil application
(3). He was the first to observe that passage of leucocytes from blood into tissue occurred
in sequential steps: initial vascular dilatation, followed by leucocytes rolling on
endothelia, stopping, and finally transendothelial migration towards the inflammatory
focus. Over the last 10 years, the molecular basis for this sequence of events has been
subject to intense research. Contrary to previous dogma, where postcapillary venules
JONATHAN N.W.N.BARKER 165
were thought to be passive bystanders, these vessels undergo profound morphological and
functional changes, actively seeking and filtering out leukocytes of appropriate phenotype
in a situation analogous to the role of high endothelial venules in peripheral lymph nodes.
Once through the vessel wall, further complex interactions between leucocytes and tissue
extracellular matrix determine the speed and direction of leucocyte movement and their
subsequent retention in skin or return to the circulation. These events are mediated by a
group of receptors/ ligands expressed on endothelial cells, leucocytes, and tissue
matrices, known collectively as adhesion molecules. Through multiple, sequential steps
these molecules organise and direct cell trafficking in skin in close collaboration with
cytokines, particularly chemoattractant chemokines.
Three main groups of adhesion molecules are recognised, based on structural and
functional characteristics: selectins, integrins and members of the immunoglobulin
supergene family (Figure 8.1).
SELECTINS
Selectins are a group of glycoproteins, characterised by an N-terminal lectin-like domain,
homologous to Ca2+ -dependent lectins and a variable number of repeated complement
regulatory protein-like residues. To date, three selectins have been identified: E-selectin
(endothelial leukocyte adhesion molecule-1), Pselectin (granule membrane protein Mr
140 kDa(GMP-140)), and L-selectin (LEC-CAM-1).
P-selectin and E-selectin act as ligands for neutrophils, eosinophils, monocytes, and
memory T lymphocytes. Both molecules require endothelial cell activation for
expression. P-selectin is present within resting vascular endothelial cells (together with
von Willebrand factor), in the form of granules known as Weibel-Palade bodies.
Following stimulation by acute inflammatory mediators such as histamine, thrombin or
components of complement (C5b-9) these cytoplasmic granules rapidly fuse with the cell
surface membrane to reach peak expression at 10 minutes. In contrast, E-selectin
expression on human umbilical vein endothelial cells, and in human skin in vivo, reaches
peak expression 4 hours after stimulation by interleukin (IL) -1, tumour necrosis factor-α
or bacterial lipopolysaccharide(LPS) and requires de novo mRNA and protein synthesis.
Selectins bind with low affinity to sialylated carbohydrate moities which are closely
related to sialyl lewis x (SLX) and its isomer sialyl lewis a (SLA). These carbohydrate
ligands are arranged in clusters, O-linked to much larger, serine-and threonine- rich
molecules, which have been likened to mucins due to their similarly extended, O-
glycosylated, rod-like, protein structure. P- and E-selectins mediate initial, potentially
reversible, leucocyte tethering to, and rolling along, the endothelial cell surface.
INTEGRINS
Integrins consist of non-covalently linked α and β subunits, with an extracellular ligand
binding site and an intracellular portion linked to the cell cytoskeleton. They are sub-
divided into various families according to the associated β subunit.
166 JONATHAN N.W.N.BARKER
Figure 8.1. Structure of selectins, immunoglobulins and integrins.

Figure 8.2. Molecular basis for leukocyte migration through vascular endothelium.
In addition to mediating leucocyte adhesion to endothelium, appropriate integrin

expression is also critical for many other biological processes including leucocyte adhesion
to extracellular matrix, antigen presentation, terminal epidermal keratinocyte
differentiation and anchoring of epidermis to the basement membrane. Leucocyte
function associated antigen-1 (LFA-1, CD 11a/ GD18), and MAC-1 (CD11b/
CD18;CR3) are both β2 integrins, but whereas LFA-1 is found on all leucocytes, MAC-1
is confined to monocytes, eosinophils and neutrophil cell surfaces. Both molecules bind
with high affinity to members of the immunoglobulin supergene family; LFA-1 to
intercellular adhesion molecule -1 (ICAM-1), ICAM-2 and ICAM-3; MAC-1 to ICAM-1
only. Very late activation antigen-4 (VLA-4, CD49d/CD29) is a β1 integrin, maximally
expressed on eosinophils, monocytes and memory type T cells, whose endothelial cell and
extracellular matrix ligands are vascular cell adhesion molecule-1 (VCAM-1) and
fibronectin, respectively. Firm integrin mediated binding to endothelia is dependent on
leucocyte activation. Functional upregulation of integrin “stickiness” is induced by locally
released chemokines. Different chemokines appear to act preferentially on different
leucocyte subpopulations, thereby conferring enormous flexibility to integrin-mediated
adhesion.
IMMUNOGLOBINS
Members of the immunoglobulin supergene family consist of single chain molecules with a
variable number of immunoglobulin-like, extracellular domains and include ICAM-1,
ICAM-2, ICAM-3 and VCAM-1. ICAM-1 is constitutively expressed on isolated human
umbilical vein endothelial cells (HUVEC) and on normal dermal endothelium in vivo and
possesses distinct binding sites for LFA-1 and MAC-1. IL-1, TNF-α and interferon-gamma
(IFN-γ) upregulate endothelial ICAM-1 expression in a process requiring gene
168 JONATHAN N.W.N.BARKER
transcription and protein synthesis. In vitro, mRNA is detectable at 2 hours following

cytokine stimulation, and persists for at least 72 hours. ICAM-2 is also strongly
constitutively expressed on isolated vascular endothelium, but does not appear to be
regulated by cytokines and may therefore have a role in leucocyte trafficking in
uninflamed tissue. ICAM-3 acts primarily as a T-cell accessory molecule. VCAM-1 is
absent from unstimulated vascular endothelium, but in common with ICAM-1 is
upregulated by a number of cytokines including IL-1 and TNF-α. Notably, IL-4 selectively
upregulates endothelial VCAM-1 expression, whilst having no demonstrable effect on that
of ICAM-1.
Further details of the structure and function of the selectins, integrins and
immunoglobulins together with detatils of the molecular basis of skin homing are
presented in Chapter 9.
Dynamic in vitro and in vivo models examining leucocyte adhesion to endothelia under
conditions of flow indicate that these different groups of adhesion molecules operate in a
sequential, interdependent manner (Figure 8.2). At sites of inflammation, local dilatation
of post capillary venules results in margination of leucocytes to the peripheral, slow
flowing, blood stream. Vascular P- and E-selectins capture these marginating, non-
activated leucocytes causing them to roll along the luminal surface of the vascular
endothelium. Provided the cell is activated, this weak, selectin-mediated adhesion, is
superceded by high avidity integrin/immunoglobulin binding at which point the leucocyte
becomes stationary (arrest). Diapedesis then occurs through the vessel wall followed by
directed migration to the inflammatory focus. Chemokines, released by cells at the
inflammatory focus and endothelial cells and bound by extracellular matrix to the luminal
surface of the vasculature, play a pivotal role in facilitating the switch between weak,
selectin-mediated adhesion, to firm, integrin-mediated adhesion.
There is clear evidence that these events are important in skin inflammation and
cutaneous chronic inflammatory diseases. E-selectin and ICAM-1 are upregulated on
vascular endothelium after epicutaneous application of antigen (4), intradermal injection
of tuberculin or exposure to UVB irradiation (5) in a time dependent fashion, parallelling
the influx of inflammatory cells. In chronic inflammatory conditions such as atopic
dermatitis and psoriasis there is strong and persistent upregulation of ICAM-1 and E-
selectin on dermal blood vessels which are themselves surrounded by skin homing
lymphocytes (see chapter 9). Furthermore frozen section adhesion studies demonstrate
that these vessels can support adhesion of activated T lymphocytes (6). Chemokines,
including IL-8 and MCP-1, which are also thought to play a key role in leukocyte
trafficking are also upregulated in these same cutaneous conditions (7).
In the ensuing chapters, the role of adhesion molecules and cytokines, including
chemokines, in the control of leukocyte activity, such as trafficking and activation, within
the skin are discussed in detail. The significance of the events detailed are highlighted by
the use of animal models to reveal their function in vivo and their importance to human skin
disease by the use of leukocyte adhesion and accessory molecules as important
immunotherapeutic targets for skin diseases.
REFERENCES:
1. Streilein, J.W. Lymphocyte traffic, T-cell malignancies and the skin. Journal of Investigative
Dermatology 71:167–171, 1978.
2. Bos, J.D., Das, P.K. and Kapsenberg, M.L. The skin immune system. In: Skin Immune System
(SIS), edited by Bos, J.D.Boca Raton: CRC, 1997, p. 9–16.
3. Cohnheim, J. The pathology of the circulation. New Sydenham Society 242–382, 1889.
4. Griffiths, C.E., Barker, J.N., Kunkel, S. and Nickoloff, B.J. Modulation of leucocyte
adhesion molecules, a T-cell chemotaxin (IL-8) and a regulatory cytokine (TNF-alpha) in
allergic contact dermatitis (rhus dermatitis). Brit JDermatol 124:519–526, 1991.
5. Norris, P., Poston, R.N., Thomas, D.S., Thornhill, M., Hawk, J. and Haskard, D.O. The
expression of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion
molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in experimental
cutaneous inflammation: a comparison of ultraviolet B erythema and delayed
hypersensitivity. Journal of Investigative Dermatology 96:763–770, 1991.
6. Barker JNWN, Groves RW, Allen MH and DM MacDonald DM. Adherence of T
lymphocytes and neutrophils to psoriatic epidermis. Brit.J. Dermatol.1992, 127:205–211.
7. Schroder, J.M. and Christophers, E. Identification of C5ades arg and an anionic neutrophil-
activating peptide (ANAP) in psoriatic scales. Journal of Investigative Dermatology 87:53–58,
1986.
9.
SKIN HOMING LYMPHOCYTES
CONRAD HAUSER AND RENÉ MOSER
INTRODUCTION
The migration of lymphocytes to nonlymphatic tissue is an essential step in the
homeostatic adaptation to injury and is involved in many reactive and neoplastic
disorders. Besides granulocytes and monocyte/macrophages, which are leukocytes of the
innate (non-adaptive) immune system, lymphocytes are cellular elements of the adaptive
immune system and play a very important role in defense and disease. The skin is one of
the principal organs that delimits the body from the outside world. As many injuries
which originate from outside can hit the skin, lymphocyte migration to this organ has to
be particularly well assured. Although B and natural killer lymphocytes can infiltrate the
skin in various pathologic conditions, little is known about the mechanisms that direct
them to the skin. The vast majority of lymphocytes which infiltrate the skin are T cells.
Therefore, this chapter deals with the migration of T cells to the skin.
LYMPHOCYTES IN NORMAL SKIN

Very few or no lymphocytes can be observed in histological sections of normal skin. It has
been estimated, however, that the entire skin can contain a large number of lymphocytes
—mainly T cells. It is currently unknown whether they migrate there because of
environmental stimulation (but without clinical signs) or whether some lymphocytes have
an intrinsic capacity to migrate to normal skin. Lymphocytes with intrinsic capacity to
migrate to skin are a subset of T cells in the mouse that express the γδ type T cell
receptor. This subset of T γδ cells can form a network of dendritically shaped cells,
similar to epidermal Langerhans cells. In humans, however, γδ T cells are rarely
encountered in normal skin. Extensive analysis of T cells in normal human epidermis has
shown that the majority lie within the most basal keratinocyte layer, often in close
apposition to Langerhans cells and comprise less than 1 % of all epidermal cells. Among
these CD3+ intraepidermal T cells, the majority express the αβ type T cell receptor
(Foster et al., 1990). Most of the CD3+ intraepidermal cells bear either CD4 or CD8 and
a minority are negative for these 2 markers. About 90% of intraepidermal T cells are
CD45RO+RA−, about 75% are CLA− (see below) and about half express the integrin aEb7
(Spetz et al., 1996). Although T cells located in normal dermis have been less
SKIN HOMING LYMPHOCYTES 171
characterized, they have been estimated to account for >95% of all lymphocytes in normal
skin. Most dermal T cells can be observed around postcapillary venules of the papillary
vascular plexus (Bos et al., 1987). The majority express the CD45RO+R− phenotype and
the CD4/CD8 ratio has been reported to be approximately 1.
LYMPHOCYTES IN INFLAMED SKIN
Molecules Involved in Lymphocyte Migration

During their ontogeny, and in order to exert effector function, lymphocytes migrate
between lymphoid organs and nonlymphoid tissues. Precursors from the bone marrow
migrate to the thymus and differentiate into mature T cells by negative and positive
selection. From there, they arrive via the blood and the high endothelial venules in lymph
nodes or spleen. Once activated by the appropriate antigen, they move into the
bloodstream and can enter into non-lymphoid tissue. Some of the lymphocytes in
peripheral nonlymphoid tissue may regain peripheral lymphoid tissue via the afferent
lymphatics. It is possible that an activated or memory cell can circulate between peripheral
lymphoid and nonlymphoid tissue more than one round. Postcapillary venules are the
principal blood vessel structures that control tissue entry of lymphocytes. Thus, much
effort has been invested to study the molecules and biology of lymphocyte— endothelium
interaction. Before focusing specifically on T cell migration to the skin, a brief overview
on the molecules involved in lymphocyte—endothelium adhesion and transmigration is
presented.
Selectins and their ligands

The selectin family of adhesion molecules is defined by their common protein structure
with an extracellular N-terminal C-type lee tin (sugar binding) domain, a single epidermal
growth factor -like domain, short consensus repeats, a transmembrane domain, and a
short C-terminal cytoplasmic domain. The known members are E-selectin (ELAM-1,
CD62E), L-selectin (LECAM-1, LAM-1, CD62L), and P-selectin (PADGEM, GMP-140,
CD62P). They bind with their lee tin domain to anionic oligosaccharides related to
sialylated Lewis x (sLex, CD15s) (Tedder et al., 1995). The binding is charactericed by
fast on- and fast offrates, considerable binding forces when exposed to fluid shear (so-
called tensile strength), and Ca++ dependence (for review see Bevilacqua and Nelson,
1993, Kansas, 1996).
(i)
L-selectin and its ligands
L-selectin (CD62L, LAM-1) is constitutively expressed at high levels by most leukocytes
except by the majority of activa ted/memory lymphocytes, and is intriguing in mediating
tethering and rolling adhesion of the different types of leukocytes to the activated
172 CONRAD HAUSER AND RENÉ MOSER
endothelium (Knol et al., 1994, Smith et al., 1991, Spertini et al., 1991, Spertini et al.,
1992, Tedder et al., 1995). The molecular cloning of L-selectin revealed a sequence
encoding a 37 kD core protein with eight possible sites for N-linked glycosylation and
contains a highly conserved lectin domain at the NH2-terminus, where the initial nine
amino acids are critical for ligand binding. The fact that L-selectin is rapidly shed from the
cell surface after leukocyte activation limits the capacity to provide tethering, rolling
(Kishimoto et al., 1989), and also arrested adhesion (Moser et al., 1993). The shedding of
L-selectin occurs by endoproteolytic cleavage in the membrane-proximal region of the
extracellular domain (Chen et al., 1995, Migaki et al., 1995).
Bonds between L-selectin and its ligand(s), once initiated, have considerable mechanical
strength, allowing initial tethering to the endothelium under significant fluid shear. In
addition, fast on and off rates seem to determine the rolling velocity in response to
hydrodynamic drag (finger et al., 1996). L-selectin binds in a Ca++ dependent manner to
sialylated derivatives of the Lewisx oligosaccharide on leukocytes (Tedder et al., 1995).
Sialylation, sulfation, and fucosylation of the oligosaccharides are necessary to bind to L-
selectin (Hemmerich et al., 1994, Hemmerich et al., 1994, Hemmerich et al., 1995,
Hemmerich and Rosen, 1994). There are different sialylated glycoproteins (SGP) which
fulfill these criteria. The most import SGP are the mucin-like SGP 50, GlyCAM-1 that is
restricted to high endothelial venules (Lasky et al., 1992), and the more broadly
distributed SGP 90, now identified as CD34 (Baumhueter et al., 1994, Baumhueter et al.,
1993). To date, the protein core ligand for L-selectin, which is induced after activation of
the endothelial cells by IL-1 or TNF (Spertini et al., 1991), and thought to be crucial in
recruiting leukocytes at the endothelial lining, is still unknown. One reason for the
difficulty in defining such membrane proteins is the relative promiscuity of carbohydrate
structures concerning their protein back bones. Recent research into the understanding of
the complex protein-oligosaccharide interactions indicates that different mucins, when
presented in unique spacing and/or clustered combinations, probably dictated by the
polypeptide backbone, may generate functional L-selectin ligands.
(ii)
E-selectin and its ligands
E-selectin (ELAM-1, CD62E) also provides rolling adhesion under conditions of
physiologic flow (Spertini et al., 1991) and is expressed on endothelial cells at sites of
inflammation, including inflamed skin. It is not expressed in normal tissue but induced by
proinflammatory cytokines such as interleukin (IL)-l, tumor necrosis factor-a (TNF-a),
lipopolysaccharide (LPS), IL-10 and lymphotoxin (Bevilacqua et al., 1987, Pober et al.,
1987, Vora et al., 1996). Recently, E-selectin induction has also been reported by CD40
ligand (Hollenbaugh et al., 1995, Karmann et al., 1995). The expression of E-selectin is
downregulated by transforming growth factor-β (TGF-β) (Gamble et al., 1993), IL-4 and
IL-13 (Etter et al., 1998). The expression of E-selectin on cultured human umbilical vein
endothelial cells (HUVEC) is known to be transient culminating at 4–6 hours, then
rapidly declining and disappearing after 24 hours. In vivo, E-selectin is continuously
expressed in different inflammatory skin diseases (Groves et al., 1991, Norris et al., 1991,
Groves et al., 1991, Norton et al., 1991, Brasch and Sterry, 1992, Rohde et al., 1992,
Norton et al., 1993, Norris et al., 1992, Bradley et al., 1994, Barlow et al., 1994, Wakita
and Takigawa, 1994, Menange et al., 1996, Jones et al., 1996, Sais et al., 1997) and
correlates with mixed leukocyte infiltrates (Barker et al., 1992). Endothelial E-selectin is
induced in experimental delayed hypersensitivity reaction and in late phase skin reactions
in atopies (Waldorf et al., 1991, Silber et al., 1994, Leung et al., 1991). Cutaneous graft-
versus-host disease is associated with expression of E-selectin (Norton et al., 1991,
Norton et al., 1993). It can also be induced by injection of TNF- or IL-1 into skin (Groves
et al, 1992, Groves et al., 1995). Prolonged expression of E-selectin was found when
human umbilical vein endothelial cells were cultured in human serum (Etter et al., 1998).
The detection of a factor in human serum providing sustained E-selectin expression
supports this observation (Sepp et al., 1994). In addition, monocytes may also provoke
late E-selectin expression by a yet unknown mechanism (Rainger et al., 1996).
Typically, E-selectin requires fucose for biologically relevant recognition. The efficient
binding to the E-selectin N-terminal lectin domain depends on fucosylated tetrasaccharide
sialyl LewisX (SLex; NeuAc, α2,3Galβ1,4(Fuc α1,3)GlcNAc; CD15s) or closely related
structures (Goelz et al., 1990, Phillips et al., 1990, Tiemeyer et al., 1991, Walz et al.,
1990). High levels of SLex and fucosylated lactosamins are constitutively expressed on
different leukocytes, including natural killer cells (Munro et al., 1992, Walz et al., 1990),
whereas peripheral T and B lymphocyte do not express SLex unless activated ex vivo
(Ohmori et al., 1993). CLA, an antibody defined carbohydrate related to sLex and bound
to PSGL-1 (CD162), is the major ligand for E-selectin in T cells (see also below). Due to
its eminent importance in certain inflammatory skin disorders, CLA is discussed
seperately (see below).
(iii)
P-selectin and its ligand
P-selectin (CD62P, PADGEM) GMP-140 (granule membrane protein-140) is also
expressed in endothelial cells and provides leukocyte rolling. It is constitutively expressed
in these cells and stored in Weibel-Palade bodies from where it can be rapidely
translocated to the cell membrane (Bonfanti et al., 1989, McEver et al., 1989). Thrombin
and histamine induce such fusions of granule- and cell membranes within seconds to
minutes, causing rapid redistribution of P-selectin to the cell surface (Hattori et al., 1989,
McEver et al., 1989). In addition, endothelial cells express C5a receptors, which induce P-
selectin expression when occupied (Foreman et al., 1994). Similar P-selectin expression is
induced by the C5b-9 membrane attack complex (Hattori et al., 1989), oxidized low-
density lipoprotein (Gebuhrer et al., 1995), and oxygen radicals (Patel et al., 1991). In
cultured endothelium, P-selectin induction is transient and downregulated by endocytosis
to basal levels within 1 hour (Hattori et al., 1989), whereas IL-4 or oncostatin M induce a
prolonged expression (Yao et al., 1996). Transcriptional upregulation of P-selectin by pro-
inflammatory cytokines and lipopolysaccharides points to an additional level of P-selectin
regulation in chronic inflammation.
The ligands of P-selectin are the Lewisx (Lex) trisaccharide (CD15) (Larsen et al.,
1990), Galβ1,4(Fuc α1,3)GlcNAc and with higher affinity the sialylated tetrasaccharide
SLex (Foxall et al., 1992, Handa et al., 1991, Policy et al., 1991, Zhou et al., 1991). Like E-
selectin, P-selectin binds also to SLea (Handa et al., 1991). In addition, sulfated glycolipids
and certain sulfated polysaccharides, like heparin, bind to the P-selectin lectin domain
(Aruffo et al., 1991, Handa et al., 1991, Needham and Schnaar, 1993, Skinner et al.,
1989). A major protein carrier of these ligands on T cells and granulocytes is also
PSGL-1, which requires O-linked sLex-like structures and sulfated N-terminal tyrosines
for optimal binding (Moore et al., 1995). Significant binding to P-selectin requires
coexpression of a fucosyl transferase (Sako et al., 1993). The binding of PSGL-1 to Pselectin
is calcium dependent and requires presentation of sialyl-Lewisx -type structures on the O-
linked glycans of PSGL-1 (Sako et al., 1993). PSGL-1 is widely distributed on different
leukocytes and is present on lymphocytes in a nonfunctional form. PSGL-1 can acquire
binding activity after cellular stimulation by post-translational modifications (Vachino et
al., 1995) such as glycosylation and sulfation (Li et al., 1996).
Integrins and their ligands

Integrins are adhesion molecules with diverse functions in processes such as
embryogenesis, maintenance of tissue integrity and leukocyte migration. They consist of a
large family of non-covalently associated α and β subunits generally of 150 and 100 kDa,
respectively (Hynes, 1992, Smyth et al., 1993). The β1, β2 and β7 subfamilies play a role
in lymphocyte migration. Whereas the role of the β1 and β2 integrins is well established in
migration of lymphocytes to skin, the β7 integrins are involved in lymphocyte migration
to gut-associated lymphoid tissue. The α4β7 integrin directs homing of lymphocytes to
Peyers patches. The αEβ7 intergin (αIELβ7, αHMLβ7)was first described to be involved in
interaction with intestinal epithelial cells. In the skin, this integrin is expressed on
intraepithelial lymphocytes in normal and inflamed skin as well as by intraepidermal
lymphocytes in cutaneous T cell lymphoma with epidermotropic T cell infiltration (Spetz
et al., 1996, Dietz et al., 1996). These cells may undergo adhesive interaction with
keratinocytes which express E-cadherin, a ligand for αEβ7 intergin (Cepek et al., 1994,
Simonitsch et al., 1994). Also dermal lymohocytes have been reported to express this
integrin to a significant percentage. An example with dermal αEβ7 integrin positive T cells
is atopic dermatitis (de Vries et al., 1997). Only 1–2% of peripheral blood lymphocytes
express this integrin which can be upregulated by mitogens or induced by TGF-β1.
Expression of integrins at the cell surface can be regulated either by biosynthesis of
these molecules or by their transport to the cell surface. In addition, once on the surface,
the affinity of integrins for their ligands can be regulated. Various signals can induce a
transient increase of affinity and thus permit firmer adhesion. Such signals include
activation by the T cell receptor, interaction with CD31, hepatocyte growth factor (HGF)
and most importantly chemokines which act via their numerous serpin (7 transmembrane
domain) receptors. Many of the previously known chemokines such as interleukin-8
(IL-8, NAP-1), γ-interferon-induced peptide (IP-10), macrophage inflammatory
protein-1α and-β (MIP-1α, MIP-1β), RANTES (regulated on activation, normal T-cell
expressed and secreted), and macrophage chemotactic protein-1, -2, -3 (MCP-1, -2, -3)
had activity on integrins expressed on T cells. Only very recently, several newly cloned
chemokines were shown to arrest T cells much faster (within minutes) on immobilized
ICAM-1 than the previously identified members of the chemokine family (Campbell et al.,
1998). These include stromal cell-derived factor-1 α (SDF-1α), 6-C-kine and macrophage
inflammatory protein-3β (MIP-3β). The latter, in contrast to the former two, was
selective for CD4+ activated/memory T cells.
(i)
b2 integrins and their ligands
The expression of β2-integrins is restricted to leukocytes. They consist of three
heterodimeric membrane glycoproteins, leukocyte function associated molecule-1
(LFA-1, CD11a/CD18, αLβ2 integrin), Mac-1 (CD11b/CD18, αMβ2 integrin) and p150,
95 (CD11c/CD18, αXβ2 integrin). The CD11 molecules and the common GDIS are the
products of different genes, exhibiting distinct though overlapping patterns of tissue- and
developmental stage-specific expression. The expression of CD11b and CD11c is almost
exclusively restricted to cells of the myeloid lineage, whereas that of CD11a and GD18 is
panleukocytic. The α subunits CD11a, CD11b and CD11c have different molecular
masses of 180 Kd, 155 Kd, and 150 Kd, respectively. The formation of α/β heterodimers
is stabilized by Ca2+ and Mg2+ (for review see Arnaout, 1990). Peripheral blood
lymphocytes express LFA-1, whereas neutrophils, monocytes and NK cells all express
Mac-1 (Arnaout, 1990). Dendritic cells of the myeloid lineage express CD11c. The
density of LFA-1 is increased on activated/memory T cells than on naïve T cells.
LFA-1 binds to the intercellular adhesion molecules-1, -2 and -3 (ICAM-1, -2, -3,
CD54, CD102, CD50). ICAM-1 belongs to the immunoglobulin (Ig) gene superfamily
and plays apivotal role in leukocyte adhesion. Endothelial cells constitutively express
ICAM-1 and continuously upregulate it within 24 hour in response to stimulation with
pro-inflammatory cytokines and lipopolysaccharides (Thornhill and Haskard, 1990).
ICAM-1 is a single copy gene located on chromosome 19, consisting of five Ig-like
domains (Katz et al., 1985, Simmons et al., 1988, Staunton et al., 1988). The first domain
binds to the A-domain of CD 11a (Staunton et al., 1990), whereas the third domain
confers binding to the A-domain of CD11b (Diamond et al., 1991). Only the third domain
of ICAM-1 requires N-linked glycosylation to confer binding (Diamond et al., 1991). The
cytoplasmic domain of ICAM-1, is connected to the cytoskeleton via α-actinin and thus
represents a fixed ligand on the endothelial surface (Simmons et al., 1988, Staunton et al.,
1988), whereas a glycophosphatidylinositol-linked variant can diffuse laterally within the
plasma membrane (Carpen et al., 1992). As a ligand of LFA-1 and Mac-1, the
transmembrane form of ICAM-1 is thought to mediate arrested adhesion and cell-attached
migration during the transendothelial migration of leukocytes. ICAM-1 is expressed on
both hemapoietic and non-hemapoietic cells, including T cells and endothelial cells.
Inflamed endothelium in skin expresses high levels of ICAM-1 and is important for
leukocyte recruitment to skin (Griffiths and Nickoloff, 1989, Griffiths et al., 1989, Lisby
et al., 1989, Lewis et al., 1989, Majewski et al., 1991, Norris et al., 1991, Norris et al,
1992, Brasch and Sterry, 1992, Das et al., 1994, Jung et al., 1996, Teina et al., 1996,
Jones et al., 1996, Dressler et al., 1997, Menage et al., 1996, Bennion et al., 1995,
Baranda et al., 1997, Gruschwitz and Vieth, 1997). ICAM-1 is upregulated in dermal
delayed hypersensitivity reactions, in human allergic contact dermatitis sites and in the
Arthus reaction (Silber et al., 1994, Brasch and Sterry, 1992, Norman et al., 1994). It is
also upregulated by injection of TNF-a into human skin (Groves et al., 1995). Its
expression can be further induced in cultured endothelial cells by pro-inflammatory
cytokines such as IL-1 and TNF-a. ICAM-1 is also expressed in keratinocytes in many
inflammatory skin disorders, but its role in skin inflammation has remained elusive
(Williams and Kupper, 1994). Resting skin endothelium expresses lower but detectable
levels of this adhesion molecules. ICAM-2 (CD102) is also involved in leukocyte
adhesion. It is constitutively expressed on endothelial cells at high levels and not
upregulated by TNF and IL-1. Human ICAM-2 is a single copy gene localized on
chromosome 17 and is composed of only two extracellular Ig-like domains (Hogg et al.,
1991, Staunton et al., 1989). ICAM-2 binds to LFA-1 (Staunton et al., 1989) and a peptide
derived from ICAM-2 can bind to purified CD11a/CD18 (Li et al., 1993). ICAM-2 also
binds to LFA-1 via its A domain (Xie et al., 1995). Like ICAM-1, the cytoplasmic domain
of ICAM-2 is linked to a-actinin of the cytoskeleton, suggesting anchored expression at
the surface of endothelial cells (Heiska et al., 1996). ICAM-3 (CD50) is constitutively
expressed on leukocytes, including resident epidermal Langerhans cells, but not on
endothelial cells in normal skin. ICAM-3 was found on skin endothelial cells by
immunohistology in only 5% of skin biopsies from various inflammatory and neoplastic
skin diseases (Montazeri et al., 1995).
(ii)
The β1 integrins and their ligands
The β1 integrins comprise different receptors for extracellular matrix proteins, including
fibronectin, collagen, laminin and vitronectin. CD29 is the common β subunit shared
among the different heterodimers. Only one member of the β1 integrin family, the α4β1
heterodimer also called very late antigen-4 (VLA-4, CD49d/CD29), has been shown to
confer binding of lymphocytes (Elices et al., 1990, Schwartz et al., 1990, van Kooyk et al.,
1993, Vennegoor, et al., 1992, Vonderheide and Springer, 1992), eosinophils (Bochner,
et al., 1991, Dobrina et al., 1991, Moser et al., 1992, Moser et al., 1992, Schleimer et al.,
1992) monocytes (Carlos et al., 1991, Jonjic et al., 1992), basophils (Bochner et al., 1991,
Schleimer et al., 1992) and NK cells (Allavena et al., 1991) to cytokine-activated
endothelial cells. Of importance, neutrophils do not express VLA-4 and therefore cannot
utilize vascular cell adhesion molecule-1 (VCAM-1), which is the most important ligand of
VLA-4 (Elices et al., 1990). Aside from providing firm adhesion to endothelium, α4
(CD49d) integrins, forming α4β1 and α4β7 heterodimers, have recently been shown to
initiate lymphocyte tethering under shear and in the absence of a selectin contribution
(Alon et al., 1995, Berlin et al., 1995). VLA-4 thus plays a crucial role in primary and
secondary adhesion of T cells to inflamed endothelium as well as in transmigration across
endothelium (Santamaria Babi et al., 1995a).
VCAM-1 (CD106, INCAM-110) belongs also to the Ig gene superfamily and primarily
mediates firm adhesion. In addition, VCAM-1 provides tethering and rolling of VLA-4-
expressing leukocytes under conditions of physiologic shear forces. It is important to
mention that the rolling adhesion on VCAM-1 does not require VLA-4 activation or the
presence of an α4 cytoplasmic domain (Alon et al., 1995). For peripheral T lymphocytes,
of which the majority lack both Lselectin and the ligand for E-selectin but express VLA-4,
rolling on VCAM-1 is thought to be important. In addition, VLA-4 has been shown to
participate in lymphocyte tethering to and rolling on VCAM-1 and mucosal addressin cell
adhesion molecule-1 (MAdCAM-1) in the absence of L-selectin (Berlin et al., 1995).
VCAM-1 can be found on inflamed cutaneous endothelium, but is not detectable in
resting skin (Das et al, 1994, Jung et al., 1996, Bradley et al., 1994, Barlow et al., 1994,
Jones et al., 1996, Petzelbauer et al., 1996, Wakita et al., 1994, Menage et al., 1996, Sais
et al., 1997, Groves et al., 1993, Norris et al., 1991, Norris et al., 1992, Brasch and
Sterry, 1992). VCAM-1 is upregulated in vitro by proinflammatory cytokines such as IL-1
and TNF. IL-4, a lymphokine with many anti-inflammatory actions, is also capable of
inducing VCAM-1 on endothelial cells (Moser et al., 1992, Schnyder et al., 1996). In
contrast to human umbilical vein endothelial cells, dermal microvascular endothelial cells
respond only to TNF-α but not IL-1 in induction of VCAM-1, showing that there is
heterogeneity among endothelial cells from different sources with regard to cytokine
induction of this adhesion molecule (Swerlick et al., 1992). This adhesion molecule can be
induced in vivo in human skin by IL-1 and TNF-α (Groves et al., 1992, Groves et al.,
1995). In baboons, the injection of TNF-α leads to sustained expression of VCAM-1 on
endothelial cells of the skin and correlated anatomically well with accumulation of T cells
(Briscoe et al., 1992). LPS injection was not capable of recruiting T cells to skin and this
correlated with the failure to induce VCAM-1 expression. It was also shown in this in vivo
model that ineffective doses of TNF-α together with IL-4 also induced VCAM-1 on
endothelial cells and T cell accumulation in skin (Briscoe et al., 1992). Similar observations
were made in pig skin where injection of TNF-α induced VCAM-1 better than IL-1.
Interestingly, injection of purified protein derivative (PPD) induced VCAM-1 only in
sensitized pigs but not naïve pigs whereas E-selectin was induced in both sensitized and
naïve pigs (Harrison, et al., 1997). Similarly, cutaneous delayed hypersensitivity reaction
to tuberculin in sensitized rhesus monkey was associated with VCAM-1 induction on
endothelial cells (Silber et al., 1994). Induction of VCAM-1 in skin endothelial cells was
often associated with induction of this molecule on other perivascular cells such as dendritic
cells.
MAdCAM-1, a further ligand of VLA-4, is an additional member of the Ig gene
superfamily. It was molecularly cloned in 1993 by screening a cDNA library derived from
a TNF-activated murine endothelioma cell line (Briskin et al., 1993), and in 1996 the human
counterpart was cloned based on sequence homology (Shyjan et al., 1996). MAdCAM-1
recognizes the VLA-4 and supports Lselectin-dependent rolling (Berg et al., 1993, Berlin
et al., 1993). MAdCAM-1 is known to be constitutively expressed but seems to depend on
TNF and lymphotoxin- (Eugster et al., 1996, Neumann et al., 1996). It participates in
migration of lymphocytes to the mucosa-associated lymphoid tissue of the gut (mesenteric
lymph nodes, Peyers patches, and intestinal lamina propria).
Other adhesion molecules

Another member of this family is PECAM-1 (CD31). It contributes to the control of the
leukocyte adhesion, and particularly to transmigration. In principle, PECAM-1 mediates
homophilic ligations but also reacts heterophilically with αvβ3, ADP-ribosyl cyclase
(CD38), glycosaminoglycans and permits binding of Plasmodium fadparum-infected
erythrocytes. It is expressed on most leukocytes, platelets and endothelial cells (Newman
et al., 1990, Simmons et al., 1990). On the latter, PECAM-1 is closely localized at
intercellular junctions while ICAM-1 is diffusely distributed on the apical cell surface
(Albelda et al., 1991, Muller et al, 1993, Simmons et al., 1990). CD31 has been claimed,
but not confirmed, to be a minor transplantation antigen in that a polymorphism in the
gene of this molecule determines different risks for graft-versus-host disease (Behar et
al., 1996, Nichols et al., 1996). Despite the fact that PECAM-1 is involved in
transendothelial migration of granulocytes it was not possible to demonstrate for
PECAM-1 a role in transendothelial migration of lymphocytes (Bird et al., 1993). In
contrast, binding of CD31 mAbs to CD4+ and CD8+ T-lymphocytes increased the
adhesive function of VLA-4 to VCAM-1 and fibronectin (Tanaka et al., 1992). Thus,
PECAM-1 is another adhesion molecule providing inside-out signalling. PECAM-1 is
involved in transendothelial migration of leukocytes.
CD44 is a cell surface glycoprotein with many isoforms generated by alternative
splicing from a single gene. It binds to hyaluronic acid but also interacts with collagen,
laminin and fibronectin. CD44 can bind chemokines as demonstrated for MIP-1β through
its heparan or chondroitin sulfate side chains and may thus participate in lymphocyte
migration via intergins. It plays a crucial role in the extravasation of activated T cells into
the peritoneal cavity and most likely also to skin (DeGrendele et al., 1997). Skin
infiltrating lymphocytes express CD44 in a variety of inflammatory skin conditions
(Jalkanen et al., 1990).
Vascular adhesion molecule-1 (VAP-1) is an endothelial cell adhesion molecule for
lymphocytes existing in two forms, a 90 kD and a 170 kD molecule. VAP-1 is present on
mucosal, peripheral and synovial high endothelial venules but is not expressed on
endothelium of large vessels, including cultured HUVEC. Only the heavily sialylated 170
kD form of VAP-1 confers lymphocyte binding (Salmi and Jalkanen, 1996). At
inflammatory sites, such as in inflammatory bowel diseases and chronic dermatoses,
expression of VAP-1 is clearly increased (Salmi et al., 1993). VAP-1 is functional in inflamed
skin because lymphocytes adhering to inflamed skin can be inhibited with antibodies to
this adhesion molecule (Arvilommi et al., 1996a). L-VAP-2 (CD73) expressed on
lymphocytes has been shown to confer adhesion to vascular endothelium in inflamed skin
(Arvilommi et al., 1996b). In addition, L-VAP+ T cells appear to accumulate in skin in
several inflammatory conditions.
The Dynamic Model of Lymphocyte Endothelium

Interaction
In principle, leukocyte extravasation, consists of a sequential order of molecular
interactions involving different classes of endothelial and leukocyte adhesion molecules
(Butcher, 1991). A model of multistep binding of leukocytes to endothelium was
postulated by Butcher. It was mostly elaborated with neutrophils but is also generally valid
for lymphocytes. Before focusing specifically on T cell migration to the skin, a brief
introduction on the different steps guiding the circulating leukocytes to penetrate the
endothelium at sites of inflammatory or immune reactions is presented.
As for neutrophils, the first adhesive interaction of lymphocytes with endothelium
(primary adhesion) is located at the level of postcapillary venules. It is transient, reversible
and of low affinity/avidity. Morphologically, it may impress as continuous tethering and
rolling of the lymphocytes along the endothelium. Blood vessel dilatation which occurs in
many inflammatory conditions may favor this process by lowering the blood flow rate.
This first step is mediated by lectin— carbohydrate interactions involving principally the
selectin family of adhesion molecules (for review see Tedder et al., 1995). Later, the α4β1
and the a4β7 integrins and CD44 were shown to be involved in rolling adhesion (Berlin et
al., 1995). Indeed, lymphocytes roll on E- and P-selectins in vitro under conditions of
capillary blood flow (Diacovo et al., 1996, Luscinskas et al., 1995). In this context it
appears logical that L-selectin, the a4 integrins and probably PSGL-1 are concentrated on
the tips of lymphocyte microvilli (Berlin et al., 1995, Erlandsen et al., 1993, Moore et al.,
1995). Reversibility is the major characteristic of rolling unless accompanied by a second
event, due to the continuous activation of the leukocyte by focal adhesive contacts to the
activated endothelium, local stimuli like chemokines, and platelet-activating factor
(Kuijpers et al., 1991, Kuijpers et al., 1990, Lo et al., 1991, Zimmerman et al., 1996).
This second event is also referred to as triggering which leads to activation of integrins
(see also above). Chemokines may participate in this step not only by being released in
soluble form but also by immobilization on carbohydrates such as heparan sulfate (Tanaka
et al., 1993). Induction of avidity changes in LFA-1 have been reported by ligation of CD2,
CD3, CD43, and CD44 (Shimizu et al., 1992). In addition, binding of T cells to
endothelial CD31 (PECAM-1) increased the function of β1 integrins (Tanaka et al.,
1992). Further co-stimulatory signals derive from binding to ICAM-1, ICAM-2 and
VCAM-1 generating signals for T-cell proliferation and IL-2 secretion (Burkly et al.,
1991, Damle and Aruffo, 1991, Damle et al., 1992, Damle et al., 1992, van Seven ter et
al., 1991). Neutrophils, as a best characterized example, continuously shed L-selectin,
permitting the reversion of primary adhesion, and upregulate Mac-1 during rolling.
Rolling-dependent activation triggers a conformational change in the integrin
heterodimers which increases the avidity of binding to the endothelial ligands (Ginsberg et
al., 1992, Hogg et al., 1993, Hynes, 1992, Sastry and Horwitz, 1993, Schwartz, 1992,
Simon et al., 1995, Smyth et al., 1993). Such “inside-out” signalling allows quick
transformation of the integrins from low-avidity to a high-avidity state. The increasing
formation of integrin-dependent ligations strengthens the binding forces, slowing and,
eventually, terminating rolling adhesion. As a result, leukocytes firmly adhere to the
activated endothelium. This third step of firm irreversible adhesion (secondary adhesion)
is resistant to shear forces of the bloodflow due to higher affinity/avidity interactions.
Integrins, including the α4β1 integrin, the α4β7 integrin, LFA-1 and MAC-1, have been
implicated in this step (Arnaout, 1990, Elices et al., 1990, Berlin et al., 1993). As
mentioned before, in their activated state, they have higher affinity/avidity for their
ligands. The final step, diapedesis or transmigrations across the endothelial lining, begins
immediately after firm adhesion. This process, in itself a multistep sequence of events,
involves integrins, CD44 and CD31. Induction of a 72 kDa gelatinase via ligation of the
α4β1 integrin may help in digesting basement membranes underlying endothelial cells and
may facilitate migration into the surrounding extravascular tissue (Romanic and Madti,
1994).
It is thought that the combined and possible, sequential action of adhesion molecules on
lymphocytes and their corresponding receptors on endothelial cells can confer tissue-
specific homing of lymphocytes. Some adhesion molecules such as LFA-1 may have a role
in homing to most tissues and thus have a more general function in migration. Others may
serve exclusively for migration to one particular organ system. Therefore, a combination
of general and tissue selective adhesion receptors should determine the migratory pattern
of lymphocytes, especially T cells. Springer has made an analogy of tissue-specific homing
of lymphocytes based on a combination of adhesion molecules this with telephone
numbers which consist of general codes such as country and area code and specific codes
such as the local number. The best characterized homing pathways of lymphocytes include
migration of naïve T cells to peripheral lymph nodes. It is directed by L-selectin but
LFA-1 and G protein-linked receptors are also involved. The leading molecule for
migration to the gut-associated lymphoid tissue is the α4β7 integrin. Migration of T cells
to the skin appears to be principally directed by CLA but adhesion molecules with a more
general function may also be needed for migration to the skin.
Molecules Required for T Lymphocyte Migration to the

Skin
Before mentioning the adhesion molecules required in vivo for lymphocyte migration to
skin it should be mentioned that the vast majority of skin infiltrating T cells in disease and
immunopathology are activated/memory cells with an CD45RA−CD45RO+ surface
phenotype (Bos et al., 1989, Markey et al., 1990, Sterry and Hauschild, 1991, Frew and Kay,
1991). Numerous studies have directly addressed the question of which adhesion
molecules are required for T cell migration to the skin. Most of them investigated the role
of adhesion molecules in skin inflammation induced by T cell-dependent skin reactions
such as contact hypersensitivity and dermal delayed hypersensitivity reactions. There is
good evidence that E- and P-selectin, a4ß1 (integrin/VCAM-1 and LFA-1/ICAM-1 as well
as CD44 play an important role in the generation of cutaneous lymphocyte infiltration
induced by antigen or hapten challenge in sensitized animals such as mice, pigs and non-
human primates (Ferguson et al., 1991, Ferguson and Kupper, 1993, 1994, Scheynius et
al., 1993, Chisholm et al., 1993, Elices et al., 1993, Issekutz, 1993, Camp et al., 1993,
Labwohl et al., 1994, Silber et al., 1994, Kondo et al., 1994, Tipping et al., 1996, Staite et
al., 1996, Binns et al., 1996a, 1996b). In mice, both P- and E-selectin play a role for
migration of T cells to skin in delayed hypersensitivity reactions but it is not clear whether
the contribution of P-selectin stems from endothelial cells or platelets (Austrup et al.,
1997, Borges et al., 1997). In any case, rolling of leukocytes in non-inflamed skin depends
in vivo on P-selectin (Nolte et al., 1994). As cutaneous delayed type hypersensitivity
reactions can be mediated by CD4+ T cells with a type 1 lymphokine pattern (IL-2+IFN-β
+IL- 4-IL-5-, Th1 cells) the migration of Th1 cells was confirmed to depend on both Pand
E-selectin. In contrast, CD4+T cells with type 2 lymphokine pattern (IL- 2-IFN-γ-IL-4
+IL-5+, Th2 cells) were unable to migrate to skin and to induce delayed hypersensitivity
reaction. The absence of E-selectin and P-selectin ligands on Th2 cells, in contrast to Thl
cells which expressed ligands for both selectins, may explain their deficiency to migrate to
skin in this murine model (Austrup et al., 1997, Borges et al., 1997). Among the
chemokines that may participate in the triggering of integrins, IL-8 and MCP-1 have been
shown in vivo to contribute to the migration of T cells to skin (Larsen et al., 1995, Rand et
al., 1996).
The Cutaneous Lymphocyte-Associated Antigen (CLA)

Because CLA is selectively associated with skin-infiltrating T cells in humans and as it
represents a ligand for E-selectin, this determinant has stimulated intense research. The
results are summarized in a separate paragraph. The mouse homologue of CLA has not yet
been identified, although E-selectin ligands on a subpopulation of T cells that migrate to
skin and mediate delayed hypersensitivity responses have been demonstrated (Austrup et al.,
1997, Borges et al., 1997).
Cellular distribution of CLA

The term Cutaneous Lymphocyte-associated Antigen (CLA) was coined when Picker et al.
found that the monoclonal rat IgM antibody HECA-452 stained the majority of skin-
infiltrating lymphocytes in a variety of reactive inflammatory skin disorders but only in a
subpopulation of circulating blood T cells and in a minority of lymphocytes infiltrating
other organs than skin (Picker et al., 1990a). Studies from other labs confirmed the
selective association of CLA+ T cells and skin. For example, only GvH of the skin (but not
of gut) is associated with infiltration of CLA+ T cells (Davis and Smoller, 1992). Skin of
psoriatic patients contained significantly more CLA+ T cells than synovium from patients
with psoriatic arthritis or rheumatoid arthritis, and skin DTH reactions from patients with
rheumatoid arthritis accumulated more CLA+ T cells than their synovial membranes
(Pitzalis et al. 1996, Jones et al., 1997).
Positive staining with HECA-452 is also found on circulating monocytes and
polymorphonuclear leukocytes. In contrast to these cells, CLA expression in T cells was
not uniform. CLA was expressed in about 16% of T cells from the blood, in about 10% of
tonsil T cells but was virtually absent in thymocytes. CD45RO- T cells did not express
CLA and only a subpopulation of CD45RO+ activated/ memory T cells expressed the
determinant, suggesting CLA induction as a consequence of T cell activation (Picker et al.,
1990a). CLA did not appear to be a classical activation marker because activation of
peripheral blood mononuclear cells in vitro with phytohemagglutinin and concanavalin-A
downregulated CLA expression (Picker et al., 1993b).
Further study of CLA expression revealed that CLA+ T cells and those labeling with a
mAb to the mucosal homing receptor integrin a4b7 were mutually exclusive in the blood
but that the majority of CLA+ cells expressed L-selectin, a homing receptor for lymph
nodes (Picker et al., 1990b). Both CD4+ and CD8+ T cells as well as T cells with αβ and
γδ T cell receptors could be identified in CLA+ circulating T cells. To obtain more
information from T cells infiltrating the skin, suction blister were raised, overlying
delayed type hypersensitivity reactions in the skin. The blisters were tapped at different
time points and the cellular content analyzed. With this technique, it was confirmed that
the majority of skin infiltrating T cells expressed high levels of CLA and L-selectin. In
addition, HLA-DR, an activation marker of T cells was mainly expressed on CLAhigh
expressing cells, suggesting that activation of T cells in the skin leads to further
upregulation of CLA (Picker et al., 1994). In contrast to CLA, the integrins aeb7 and a4b7
in skin blister T cells had a distribution that resembles T cells in the blood. Comparison
with lung lavage T cells from normal and diseased respiratory systems confirmed the
selective association of CLA with skin (Picker et al., 1994). CLA expression was also
compared in T cell clones derived from the skin and the blood. Clones from the skin had a
much higher density of CLA and adhered better to E-selectin than those from the blood.
In contrast, binding to P-selectin was equal among skin and blood derived clones (Rossiter
et al., 1994).
Molecular aspects of CLA

Initial immunochemical analysis of CLA revealed molecular masses of 200 and 125 Kd.
Periodate treatment abrogated immunoreactivity, suggesting that CLA was a
carbohydrate-dependent epitope (Picker et al., 1990a). The hypothesis of expression of
organ selective adhesion molecules in T cells gained support when Picker showed that
CLA+ T cells selectively adhered to E-selectin transfected COS cells (Picker et al., 1991).
E-selectin is expressed on endothelial cells in inflamed skin. It was postulated that
circulating CLA+ T cells are directed by CLA to migrate and accumulate in the skin.
However, many authors could not confirm that endothelial cells of the skin preferentially
expressed E-selectin when compared to endothelial cells in other inflamed organs. Thus,
the differential expression of CLA alone but not its receptor on endothelial cells could not
fully explain the apparent selective migration of CLA+ T cells to the skin. In subsequent
studies it was confirmed that the HECA-452 reactive material extracted and affinity
purified from tonsils was a ligand for E-selection. It was further shown that the binding of
E-selection to HECA-452 antigen was neuraminidasc and Ca- sensitive (Berg et al., 1991).
Biochemical and antibody inhibition studies subsequently established the close relation
between CLA and sialyl Lewis x (sLex, CD15s), the carbohydrate blood group antigen
which is the major E-selectin ligand on neutrophils. E-, P- and L-selectin ligands are complex
carbohydrates, glycans which are under the control of an ordered series of glycosylation
reactions whereby the terminal steps are controlled by specific fucosyltransferases. The
leukocytes of mice who are deficient in fucosylransferase VII have no ligands for E- and P-
selectin, demonstrating that this enzyme was necessary for the build up of ligands for both
E-and P-selectin (Mali et al., 1996). On the other hand, the transfection of
fucosyltransferase IV and VII into E-selectin negative hemopoiectic cell lines renders them
reactive with E-selectin. But only the transfection of fucosyltransferase VII reconstituted
the staining with HECA-452, indicating that fucosyltransferase VII was specifically
involved in the synthesis of the CLA epitope. Further, fucosyltransferase VII generated E-
selectin ligands with higher affinity than fucosyltransferase IV (Wagers et al., 1997).
Finally, the protein carrying the CLA carbohydrate epitope was identified as PSGL-1,
previously known to bear determinants responsible for binding to P-selectin (Fulbrigge et
al., 1997). It thus appears that PSGL-1 can be glycosylated to bear ligands for either P-
selectin or both P- and E-selectin. Differential glycosylation of PSGL-1 may thus regulate
lymphocyte migration to skin.
CLA+ T cells in the circulation mirror their counterparts in the skin

The association of CLA and skin was tested in blood T cells. If circulating CLA+ T cells
are predestined to migrate to the skin then they should also contain cells that respond to
cutaneous antigens. This was tested with nickel, an exclusive skin antigen (hapten) and
tetanus toxoid which is not expected to exclusively induce T cells that migrate to skin.
CLA+ and CLA− CD45RA− T cells from the blood were incubated with these antigens.
The proliferative response to nickel, but not tetanus toxoid, was confined to the CLA+
subset (Santamaria Babi et al., 1995b). On the other hand, the response to the same
antigen in diseases with different organ localization was investigated. Asthma and atopic
dermatitis are both atopyassociated diseases and affected individuals are often sensitized to
house dust mite. The CLA+ blood T cells of patients with atopic dermatitis proliferated more
vigorously than CLA- cells in contrast to asthmatics in which CLA- cells responded better
than CLA− cells. In addition, expression of HLA-DR, an activation marker in human T
cells, and release of IL-4, a Th2 marker lymphokine associated with atopy, were
selectively increased in CLA+ cells from patients with atopic dermatitis. Together, these
results supported the contention that CLA+ T cells migrate to the skin. Further support was
provided when it was shown that the increased HLA-DR expression in circulating CLA+ T
cells from patients with atopic dermatitis was reduced after combined UVA and B
treatment, as was the extent and severity of this condition (Piletta et al., 1996).
Analoguous findings were made for IL-2 receptor and CD30 expression in CLA1 blood T
cells with this treatment. When the CLA+ circulating T cells of these patients were
further analyzed, they were shown to proliferate spontaneously, to release IL-13
spontaneously, and to stimulate IgE synthesis in autologous B cells (Akdis et al., 1997).
Because the cytokine release occured early in culture and was not inhibited by cycloheximide
it can be assumed that they have been turned on in vivo. A selective increase of HLA-DR in
CLA+ circulating T cells was also found in delayed cutaneous drug reactions (Gonzalez et
al., 1997).
The regulation of CLA expression in T cells

As previous observations suggested that CLA may be induced in response to activation,
the regulation of CLA expression was further studies in vitro. With peripheral blood
mononuclear cells. Naïve T cells, which express CD45RA but no CD45RO, go from
CD45RA+RO− to a CD45RA+RO+ intermediate to become finally CD45RA-RO+ when
activcated (Picker et al., 1993b). Some T cells in the double positive intermediate fraction
were CLA+, suggesting that CLA is induced in the conversion from the naïve to activated
state (Picker et al., 1993a). This CLA+ intermediate was also identified in lymph node
cells. The percentage of CLA+ cells in this fraction was correlated with their anatomic
origin: lymph nodes from sites close to skin contained a higher percentage of CLA+
intermediates than lymph nodes from sites remote from the skin (e.g. lymph nodes from
the appendix), further supporting the association of CLA and the skin (Picker et al.,
1993a). In addition, these data suggested that the conditions for CLA induction were
more favorable in skin draining lymph nodes than those draining the gut. Most likely, the
microenvironment in the lymph node regulates the induction of CLA. For this reason, the
response of naïve T cells to activation using a panel of cytokines was investigated in vitro.
TGF-β, and to a lesser extent IL-6, were capable of upregulating CLA on T cells. This
was the case for naïve T cells from adults and neonatal chord blood T cells but CLA could
also be further upregulated on activated T cells when restimulated and exposed to these
cytokines (Picker et al., 1993a). Later it was shown that IL-12, a cytokine that plays an
important role in the initiation of immune T cell responses, also upregulated the density of
CLA. Inducers of CLA via IL-12 include SEE, TSST-1 and streptococcal pyrogenic
exotoxins A and C (Leung et al., 1995) . Recently, it was shown that T cells activated and
cultured in fetal calf serum-free media and IL-2 express high levels of CLA on virtually all
T cells (Fuhlbrigge et al., 1997). Fetal calf serum therefore appears to have a suppressive
effect on CLA expression on T cells. But this suppression seems to be overcome to some
extent by IL-6, TGF-β and IL-12. Furthermore, activation of T cells via CD2 induces
strong CLA expression in T cells, even in the presence of fetal calf serum. IL-4 inhibited
CLA induction under these conditions. In contrast, activation by either CD3 or CD3 plus
CD28 or mitogens induces CLA weakly and only transiently. Antigens could also induce
CLA in culture (Liu et al., 1996) . When peripheral blood mononuclear cells were
cultured with casein or Candida, CLA (but not L-selectin) increased in the T cells exposed
to casein. This phenomenon was selective for patients with milk-induced atopic dermatitis
but not milk-induced enterocolitis, allergic eosinophilic gastroenteritis or normal controls
(Abernathy-carver et al., 1995).
CLA in the interaction of T cells with endothelium

In vitro studies attempted to clarify the role of CLA in the interaction with endothelial
cells. In a static migration assay across endothelial cells, it was demonstrated that CLA+ T
cells migrated in greater numbers across IL-1β and TNF-α activated (but not non-
activated) endothelial cell layers than the corresponding CLA− T cells. CLA appeared to
be directly involved in enhanced transendothelial migration because HECA-452 blocked
Figure 9.1 A combination of adhesion molecules and their receptors direct T lymphocytes to skin.
Circulating memory T lymphocytes undergo adhesive interaction with activated endothelium on the level of postcapillary skin venules. The initial adhesive
interaction is reversible and impresses as rolling. P- and E-selectin appear primarily to be involved in this step. Subsequent firm adhesive interaction
terminates rolling. CLA, an E-selectin ligand, directs T lymphocytes selectively to skin. Activated integrins (LFA-1 and VLA-4) and their respective
receptors (ICAM-1 and VCAM-1) appear primarily to be implicated in this step. firm adhesive interaction is triggered by chemokines and other factors via
activation of integrins. Once firm adhesion is initiated migration across the inflamed endothelium starts. After completion of this step, T lymphocytes are
located in interstitial skin tissue where they can interact with antigen presenting cells such as Langerhans cells. This interaction leads to T cell activation,
secretion of lymphokines, and induction of cytotoxicity.
to levels of CLA− CD45RA– T cells (Santamaria Babi et al., 1995a). As integrins are
involved in adhesion to endothelial cells, the influence of these molecules on endothelial
transmigration of CLA+ and CLA− T cells was investigated. Blocking of the VLA-4 and its
endothelial receptor VCAM-1 selectively blocked enhanced transmigration of CLA+ T
cells across activated endothelial cells. Because CLA+ and CLA− T cells expressed the same
density of VLA-4 integrin these results indicated that this integrin was selectively involved
in transmigration of CLA+ (but not CLA− T cells) . In contrast, blocking of LFA-1 and its
receptor ICAM-1 on endothelial cells inhibited transmigration, regardless of whether CLA
+ or CLA− T cells and activated or nonactivated endothelial cells were used,
demonstrating that these adhesion molecules are not selective for the transmigration of
CLA+ T cells (Santamaria Babi, et al., 1995a). As chemokines and their receptors are
involved in lymphocyte homing and integrin activation their effect on transmigration of
CLA+ and CLA+ T cells and CLA+ and CLA- subclones of the cutaneous T cell lymphoma
cell line HUT-78 was studied in this system. Pertussis toxin and antibodies to IL-8 and
antibodies to its receptor B (CXCR-1) blocked the enhanced transmigration of CLA+ (but
not CLA−) T cells across activated umbilical and skin microvascular endothelial cells, again
showing the selectivity of this chemokine and the CXCR1 for CLA+ T cells (Santamaria
Babi et al., 1996). Collectively, these transmigration studies under static conditions clearly
demonstrate that adhesion molecules and chemokine receptors have a distinctly different
role in CLA+ than in CLA− activated/memory T cells. The interaction of CLA expressing
T cells and adhesion molecules was also investigated under conditions of flow. CLA+ T
cells adhered much better to
E-selectin transfectants than CLA− memory T cells, supporting a role for CLA in
rolling, the first adhesive step in the interaction with endothelium (Jones et al., 1994,
Ronen et al., 1994).
To have an in vivo demonstration for the role of CLA in the migration of T cells to the
skin, attempts were made with SCID mice transplanted with human skin and the CLA+
and CLA− subclones of the mentioned T cell line. When the grafts were stimulated with
TNF-a, the frequency of positive signals for the T cell receptor of the HUT-78 cell line
was somewhat higher after intraperitoneal injection of the CLA+ subclone than the CLA−
subclone. But the overall positive signal frequency was low in the skin and therefore the
difference not statistically significant (Rosenblatt-Velin et al., 1997). Another approach
with SCID mice transplanted with human skin and i.p. injection of lymphocytes was more
successful. Transplants of skin containing superficial, but not deep, vascular plexus
accumulated CLA+ T cells (Kunstfeld et al., 1997).
SUMMARY AND CONCLUSION

The skin accumulates activa ted/memory effector T lymphocytes in a variety of
inflammatory skin diseases. In the last few years, some insight into the mechanisms of T
cell migration to skin has been gained. General and tissue-selective adhesion molecules in
T lymphocytes and their counter-receptors expressed on inflamed microvascular
endothelium in skin determine the migratory capacity of T lymphocytes. These adhesion
molecules, together with soluble factors such as chemokines, participate in a complex
process of interaction between T lymphocytes and endothelium. The interaction includes

primary adhesion (rolling) of T lymphocytes along endothelium followed by firm adhesion
(secondary adhesion), arrest and diapedesis across the endothelial barrier. There is good
evidence that on the inflamed endothelium, the classic adhesion principles mediated by E-
and P-selectin (primary adhesion), ICAM-1, and VCAM-1 (secondary adhesion) with
their respective leukocyte integrins (LFA-1 and VLA-4) drive T cell migration to the skin.
On the other hand, circulating T cells appear to be selectively directed to skin by the E-
selectin ligand CLA, which is a carbohydrate determinant on PSGL-1 and dependent on
fucosyltransferase VII (Figure 9.1). It is possible that the understanding of CLA expression
or interference with its binding may provide new strategies for the treatment of T
lymphocyte-dependent inflammatory skin diseases.
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10.
T-CELL ACCESSORY MOLECULES
RALF W.DENFELD AND JAN C.SIMON
INTRODUCTION
In many inflammatory skin diseases, T cells accumulate around the dermal perivascular
plexus and some subsequently migrate into the epidermis. These T cells are thought to
make a major contribution to inflammation and tissue damage in these skin diseases. On
the other hand, T cells residing in skin are supposed to play an important role in
protecting the epidermis from potentially dangerous immune responses. In mice, a unique
T-cell subset, namely dendritic epidermal γδ-TCR positive T cells (DETC), has been
characterized as being responsible for the maintenance of immunologie homeostasis within
skin, however, a human epidermotrophic T-cell analogue has not been identified as of yet
(Shiohara and Moriya, 1997). Specialized antigen-presenting cells (APC) resident in skin,
namely Langerhans cells, are considered to play a central role in the induction of T cell-
mediated cutaneous immune responses. Upon antigen-capture, i.e. by epicutaneously
applied haptens or invading pathogens, these sentinel cutaneous APC migrate via dermal
lymphatics into the T-cell areas of skin-draining lymphoid tissues where T cells are
activated. Subsequently, these antigen-specific T cells travel via the blood stream to the
original site of antigen-deposition. Also, cutaneous APC, i.e. Langerhans cells and dermal
dendritic cells, are well equipped to stimulate resident T cells intracutaneously
(Banchereau and Steinman, 1998). However, it must be noted that the integrated immune
system within skin is not only comprised of a distinctive population of resident and
recirculating T cells and cutaneous APC. Additionally, neighboring cells like
keratinocytes, fibroblasts, and mast cells regulate T-cell responses via cytokine-mediated
intercellular communication and cell-cell interactions (Takashima and Bergstresser,
1996). Thus, mechanisms of intracutaneous as well as extracutaneous T-cell activation are
of central importance for the pathophysiology of inflammatory and some malignant skin
diseases.
Today it is generally accepted that activation of T cells requires two signals from APC
(Bretscher, 1992). The first signal provides specificity via binding of the T-cell receptor
(TCR)-CD3 complex to its antigen-MHC ligand. The second signal is mediated by
additional costimulatory signals delivered to the T cell via a number of accessory
molecules which serve as receptors for specific antigen-independent ligands expressed on
APC. Over the years, CD28 and its homologue cytotoxic T lymphocyte antigen-4
T-CELL ACCESSORY MOLECULES 203
(CTLA-4, CD 152) have emerged as key regulators of T-cell responses. CD28 is the
primary T-cell costimulatory receptor, and upon interaction with its ligands, B7–1
(CD80) and B7–2 (CD86), it enhances T-cell proliferation and cytokine synthesis,
induces T-cell differentiation and the expression of anti-apoptotic proteins (Lenschow et
al., 1996a). By contrast, CTLA-4 functions to inhibit T-cell responses, which is not
simply due to competition with CD28 for costimulation (Thompson and Allison, 1997).
Furthermore, another key player in T-cell activation is the ligand for CD40 (CD40L,
CD154). CD40–CD40L interactions play a pivotal role in the development of T cell-
dependent immune responses by upregulating APC costimulatory molecule expression
and sustaining T-cell clonal expansion (Grewal and Flavell, 1998). How the T cell
integrates signals through the TCR-CD3 complex, CD28, CTLA-4 and CD40L to
initiate, maintain and/or terminate antigen-specific immune responses is currently a
central issue in understanding T-cell activation. Furthermore, the knowledge of the
mechanisms involved in T-cell activation in inflammatory and malignant skin diseases will
have implications for future dermatologic therapy. For example, allergic and autoimmune
skin diseases are characterized by imbalanced T-cell differentiation (Th1 vs. Th2) as a result
of differential costimulation, offering a potential target for interfering with these
pathways. Also, introduction of costimulatory molecules into melanoma cells augmented
anti-tumor immunity in mice in vivo.
CD28, A POSITIVE REGULATOR OF T-CELL ACTIVATION

The CD28 receptor is a member of the immunoglobulin supergene family and exists as a
disulfide-linked homodimeric glycoprotein in situ. CD28 is constitutively expressed at the
cell surface on the majority of both resting and activated human T cells. The CD28
ligands, B7–1 (CD80) and B7–2 (CD86), are constitutively expressed on resting APC at
low levels. Both B7 molecules are upregulated upon activation but with different kinetics.
Following APC activation first B7–2 and then B7–1 expression is increased (Lenschow et
al., 1996a). T-cell responses require a nonantigen-driven costimulatory signal provided by
CD28 (and possibly other accessory molecules), in addition to ligation of the TCR-CD3
complex with CD4 or CD8. Indeed, in vitro interruption of the B7-CD28 pathway
combined with TCR occupancy leads to failure of proliferation in human T cells upon
restimulation with normal APC plus antigen, a state termed anergy. However, the
induction of anergy can be reversed by the addition of exogenous IL-2 or, more
importantly, by ligation of CD28 during the initial activation event (Lenschow et al.,
1996a). Several other costimulatory molecules have been found to regulate T-cell
activation, i.e. integrins (Dubey et al., 1995), CD43 (Sperling et al., 1995), CD44 (Guo et
al., 1996), CD47 (Waclavicek et al., 1997), heat-stable antigen (CD24, Wu et al., 1998)
and 4–1BB (CDw 137, Kim et al., 1998), but none of these molecules can block the
induction of T-cell anergy or regulate T-cell activation as profoundly as CD28. In most T
cells, CD28 lowers the threshold needed for activation and increases response longevity,
effects linked to enhanced stability and transcription of lymphokine mRNA, in particular
those encoding IL-2 and IL-4 (Lenschow et al., 1996a). Analysis of CD28-deficient mice
indicated many T cell responses to be impaired, i.e. T-cell responses to superantigens
204 RALF W.DENFELD AND JAN C.SIMON
(Saha et al., 1996), however, some responses proceed normally (Lenschow et al., 1996a).
In addition, it has been shown that CD28-deficient T cells can initiate, but,
more importantly, cannot sustain, proliferative responses (Kündig et al., 1996).
Furthermore, CD28 costimulation may amplify an immune response by recruitment of T
cells to inflammatory sites via the production of chemokines (Herold et al., 1997a).
Together, these results suggest that CD28 is critically important for initiating and
sustaining T-cell responses.
The B7-CD28 pathway also amplifies cytolytic responses in tumor models. The first
reports to document the importance of CD28 costimulation in tumor rejection in vivo
demonstrated that the murine melanoma K1735 transfected with B7–1 was rejected by
syngeneic hosts. This resulted in immunity to rechallenge with the parental, B7–1
negative, tumor cells. Subsequent work demonstrated B7–1 transfected tumor cells to
induce regression of established mouse melanoma requiring both CD4+ and CD8+ T
cells (Allison et al., 1995), and the effect of B7–2 transfected tumor cells in elicitating
antitumor immunity (Allison et al., 1995, Martin-Foncheta et al., 1996). It is well
documented that multiple antigenic peptides or tumor antigens presented by the MHC on
tumor cells are recognized by T cells. Nevertheless, tumor-infiltrating T cells against
most of these epitopes of autologous cancer cells are usually not active in vivo, a
phenomenon called immunological ignorance of silent tumor antigens, possibly resulting
in tumor escape (Chen, 1998). Recently, it has been shown that, as a result of B7-CD28
costimulation, T-cell responses could be spread to normally silent tumor antigens
(Johnston et al., 1996). Despite these successes, early reports showed clearly that the
expression of a CD28 ligand on poorly immunogenic tumor cells is not sufficient to
induce regression in all tumor models (Allison et al., 1995), suggesting that increased B7-
CD28 costimulation may not be suitable for conversion of all silent tumor antigens.
Instead, tumor immunogenicity, that is threshold of TCR molecules to be engaged with
the peptide-MHC-complex, determines the effect of CD28 costimulation via B7 on tumor
immunity (Chen, 1998). However, in some models the coexpression of B7 plus other
costimulators, i.e. CD48 (Li et al., 1996) and 4–1BB (Melero et al., 1998), induced a
vigorous T-cell response to silent epitopes. Taken together, B7-CD28 costimulation
provides an important mechanism to enhance immune responses against (silent) tumor
antigens for immunotherapy of cancer.
Moreover, the relevance of B7-CD28 interactions in vivo in transplantation models and
autoimmune diseases is well documented. The first observation was made in mice
transplanted with xenogeneic human pancreatic islet cells. Inhibition of B7-CD28
interactions by treatment with CTLA-4 fusion protein (CTLA-4-Ig) resulted in prevention
of pancreatic islet rejection and, additionally, led to long-term donor-specific tolerance,
which was later confirmed using antibodies (mAb) to B7–1 and B7–2 (Lenschow et al.,
1996a). Other investigators have demonstrated that CTLA-4-Ig treatment in cardiac and
renal allogeneic transplant models can result in indefinitive graft survival (Sayegh et al.,
1995, Sayegh et al., 1997). Similar findings were made in murine graft-versus-host disease
(Via et al., 1996, Blazar et al., 1996). Most recently, Olthoff et al. (1998) elegantly
demonstrated that following gene transfer of sequences encoding CTLA-4-Ig allogeneic
liver transplants produced the recombinant protein shortly after revascularization,
resulting in intact liver function, indefinite allograft survival, and the development of
donor-specific unresponsiveness. Furthermore, at least three rodent autoimmune models
corroborate the importance of B7-CD28 costimulation in vivo. First, in the spontaneous
non-obese diabetic mouse model, blocking the B7-CD28 pathway by CTLA-4-Ig or anti-
B7–2 mAb greatly reduced the incidence of diabetes when administered before or even
after the onset of insulitis (Lenschow et al., 1995), which seems to be IL-4 dependent
(Arreaza et al., 1997). Also in streptozotocin-induced diabetes CD28 expression is
necessary since CD28-deficient animals and wildtype animals treated with anti-B7– 2
mAb develop neither hyperglycemia nor insulitis (Herold et al., 1997b). Second, in
experimental allergic encephalomyelitis (EAE), a rodent model of multiple sclerosis,
CTLA-4-Ig protected against EAE induced by either active immunization or adoptive
transfer of activated peptide-specific T cells (Cross et al, 1995, Racke et al., 1995, Gallon
et al., 1997). The profound inhibition of the clinical and histological manifestations of EAE
continued after cessation of CTLA-4-Ig treatment (Cross et al., 1995). Treatment with
anti-B7-l mAb ameliorated EAE and resulted in the predominant generation of Th2 clones
whose transfer both prevented induction of EAE and abrogated established disease
(Kuchroo et al., 1995). Even epitope spreading and clinical relapses in EAE were
prevented following treatment with non-cross-linking Fab fragments of anti-B7–1 mAb
(Miller et al., 1995). Third, in lupus-prone (NZB/ W Fl) mice, a murine model
resembling human systemic lupus erythematosus, CTLA-4-Ig treatment suppressed the
lupus-like illness and prolonged life even when treatment was administered late in disease
(Finck et al., 1994). However, both anti-B7–1 and anti-B7–2 mAb were needed to
prevent the development and progression of lupus, with B7–2 playing a more critical role
and contributing to Th2-mediated cytokine production (Nakajima et al., 1995). In
summary, the manipulation of the B7-CD28 pathway in vivo can prevent transplant
rejection, the initiation of autoimmune responses, as well as suppress ongoing autoimmune
processes.
Furthermore, costimulatory signals delivered through the CD28 molecule have been
shown to have profound effects on the differentiation of Th1 (i.e. IL-2, IFNγ) and Th2
(i.e. IL-4, IL-5) subsets (Mosmann and Sad, 1996). Kucheroo and collegues first
suggested that interactions of CD28 with B7–2 but not B7–1 preferentially induce a Th2
response (Kuchroo et al., 1995). Additional in vitro (Freeman et al., 1995) and in vivo
(Subramanian et al., 1997, Keane-Myers et al., 1998) studies supported a selective role of
B7–2 in Th2 development, indicating B7–1 and B7–2 to deliver qualitatively distinct
signals via CD28. However, other groups have not observed significant differences in the
induction of Th1/Th2 cytokines when using B7–1 and B7–2 transfectants (Lanier et al.,
1995) or APC lacking B7–1 and B7–2 (Schweitzer et al., 1997). In vivo, in the non-obese
diabetic mouse strain, early disruption of the B7-CD28 pathway using CD28-deficient
mice promoted the development and progression of spontaneous autoimmune diabetes
correlating with an enhanced Th1 response (Lenschow et al., 1996b), indicating that the
early differentiation of naive diabetogenic T cells into the Th2 subset is dependent upon
CD28 signaling. Subsequently, using antigen-specific TCR-transgenic T cells, Rulifson et
al. (1997) demonstrated that Th2 but not Th1 cytokine production was highly dependent
on CD28 ligation, suggesting that CD28-mediated costimulation can drive the
differentiation of T cells towards a Th2 phenotype. Further evidence for the necessity of
CD28 costimulation for Th2 immune responses was obtained in vivo in a Th2-mediated
murine model of allergic airway hyperresponsiveness. Blocking the B7-CD28 pathway
ablated allergen-induced airway dysfunction concomitant with a significant reduction in
the Th2 response (Keane-Myers et al., 1998). However, in other in vivo Th2-mediated
immune responses to a nematode parasite no CD28 dependency on T-cell differentiation
was found (Cause et al., 1997), implying that the specific kind of immune response
determines whether Th2 differentiation is CD28 dependent. Also, the point in time
during an ongoing immune response at which CD28 signaling is provided or blocked
(Arreaza et al., 1997) and the strength of the TCR signal (Tao et al., 1997) seem to be
crucial for the requirement of CD28 costimulation in Th1/Th2 differentiation.
Finally, recent reports have suggested a role for CD28 costimulation in regulating T-
cell survival (Boise et al., 1995). It has been shown that following a TCR signal, CD28
costimulation dramatically upregulates expression of Bcl-xL on T cells. Indeed, following
the initiation of T-cell activation, the amount of cl-xL protein is associated with resistance
to Fas- and TNF receptor 2-mediated apoptosis (Boise et al., 1995, Radvanyi et al., 1996,
Noel et al., 1996, Lin et al., 1997). Since both, the Fas/Fas ligand system and TNF/TNF
receptor system, are the major pathways mediating activation induced T-cell death in the
periphery, CD28 costimulation can maintain adequate numbers of functional T cells to
ensure the successful outcome of a productive immune response (Boise et al., 1995,
Lenardo, 1997). Although CD28 costimulation does not have a significant effect on Bcl-2
expression, it results in significant IL-2 accumulation, which can subsequently upregulate
Bcl-2 expression providing a possible additional mechanism for clonal expansion (Mueller
et al., 1996). In conclusion, CD28 is a positive regulator of T-cell activation.
CTLA-4, A NEGATIVE REGULATOR OF T-CELL ACTIVATION

CD28 and CTLA-4 are related glycoproteins of the immunoglobulin supergene family,
with both molecules existing as disulfide-linked homodimers. The variable domains of
CD28 and CTLA-4 contain conserved sequences necessary for binding their ligands B7–1
and B7–2. However, CTLA-4 has a much higher affinity and avidity for both B7
molecules compared to CD28. Although CTLA-4 and CD28 share a number of common
structural properties and have the same ligands, their patterns of expression are quite
distinct. Conversly to CD28, CTLA-4 protein levels are low or undetectable on resting T
cells, but following activation CTLA-4 expression is maximal at 48 hours at the peak of
the T-cell response (Thompson and Allison, 1997). CTLA-4 protein is primarily localized
and stored intracellularly in the perinuclear Golgi vesicles, and upon activation of resting
T cells, CTLA-4 transits from the internal stores to the cell surface and becomes directed
towards the site of TCR engagement (Linsley et al., 1996). These differences in CD28 and
CTLA-4 expression patterns have important functional implications.
As mentioned earlier, many T-cell responses are impaired in CD28-deficient mice.
Similarly, initial experiments demonstrating antibodies to CTLA-4 to enhance T-cell
proliferation were interpreted to mean that CTLA-4 synergized with CD28 in enhancing
T-cell costimulation (Lenschow et al., 1996a, Thompson and Allison, 1997). This result,
however, may be a consequence of the removal of an inhibitory signal by interrupting B7-

CTLA-4 interactions. In support of this idea, it was demonstrated that the inhibition of
the B7-CTLA-4 pathway with anti-CTLA-4 mAb augmented alloreactive and peptide-
specific T-cell responses in vitro (Wahmas et al., 1996). Furthermore, T-cell proliferation
induced by CD3 and CD28 cross-linking was inhibited by simultaneous CTLA-4 cross-
linking (Krummel and Allison, 1996). These effects were due to the inhibition of cell
cycle progression (Krummel and Allison, 1996, Walunas et al., 1996). This was supported
by the finding that CTLA-4 ligation diminished IL-2 production and permitted induction
and expression of the anti-apoptotic gene Bcl-xL (Blair et al., 1998). Very recently,
Walunas and Bluestone (1998) reported that CTLA-4 blockade inhibits tolerance
induction, possibly by skewing T-cell differentitation towards a Th2 response. These
results indicate that CTLA-4 transduces an inhibitory signal modulating the outcome of
CD3 and/or CD28 signalling.
The first demonstration that CTLA-4 blockade can augment antigen-specific T-cell
responses in vivo was reported by Kearney et al. (1995). Here, adoptively transferred
TCR-transgenic T cells expanded in response to immunization with OVA-peptide, a
response which was markedly augmented by treatment with antiCTLA-4 mAb. Similarly,
CTLA-4 blockade increased T-cell responses to superantigen in vivo (Krummel et al.,
1996). Using CTLA-4-deficient mice the importance of CTLA-4 in regulating T-cell
activation was shown dramatically. The absence of the CTLA-4 molecule led to a massive
T-cell lymphoproliferative disease, i.e. lymphadenopathy and splenomegaly, with early
lethality at 3–4 weeks of age (Thompson and Allison, 1997, Chambers et al., 1997).
These results demonstrated the essential functional role of CTLA-4 in vivo for maintaining
peripheral T-cell homeostasis. Furthermore, the phenotype of CTLA-4-deficient mice is
remarkably different from that of CD28-deficient mice (Lenschow et al., 1996a), again
indicating that CTLA-4 and CD28 have distinct functions.
In addition, the role of the B7-CTLA-4 pathway in autoimmune disease and tumor
models has recently been addressed. In EAE, treatment with antibodies to CTLA-4
resulted in exacerbation and, if treatment was initiated during disease remission, relapses
of disease, which was associated with enhanced production of the encephalitogenic
cytokines TNF-α, IFN-γ, and IL-2 (Thompson and Allison, 1997). In an elegant study,
Leach et al. (1996) examined an alternative strategy to augment anti-tumor T-cell
responses in vivo by blocking B7-CTLA-4 interactions. Treatment with anti-CTLA-4 mAb
enhanced the rejection of B7-transfected tumor cells in vivo. Most strikingly, animals
completly rejected the B7 negative parental tumor when treated with anti-CTLA-4 mAb,
even when treatment was delayed until a palpable tumor was established (Leach et al.,
1996, Yang et al., 1997). Although the exact mechanism for this rejection is not yet
known, these results suggest that CTLA-4 blockade can augment T-cell responses to
tumor antigens presented by host APC in vivo. In summary, the most compelling
interpretation of all this data is that CTLA-4 is a negative regulator of T-cell activation.
THE ROLE OF CD40L IN COSTIMULATION AND T-CELL

ACTIVATION
CD40 ligand (CD40L, CD154) is a member of the tumor necrosis factor (TNF) cytokine
superfamily, including TNF, Lymphotoxin α/β, Fas ligand, as well as the ligands for
CD27, CD30, 4–1BB, and OX40. Like other members of this superfamily, CD40L is
expressed as a homotrimeric complex of type II integral membrane glycoproteins. In
humans, naturally occuring soluble forms of CD40L have also been described. Although
the distribution of CD40L is not yet completly denned, it is preferentially expressed on
activated CD4+ T cells, but also on mast cells, eosinophils, B cells and dendritic cells. For
example, activated CD4+ T cells rapidly express CD40L in response to TCR ligation.
The counter-receptor for CD40L is CD40, a member of the TNF receptor superfamily. The
type I integral membrane glycoprotein CD40 is found on APC, i.e. B cells, dendritic cells
and macrophages, some T cells, but also on non-hematopoetic cells, including endothelial
cells, fibroblasts, and epithelial cells, i.e. keratinocytes (Grewal and Flavell, 1998).
The importance of the CD40L in humans was revealed in patients suffering from
Hyper-IgM syndrome, an immunodeficiency characterized by mutations in the CD40L
gene locus, leading to high levels of IgM but severly reduced levels of circulating IgG and
IgA, and the absence of germinal centers. Although patients show abnormal antibody
responses, i.e. their B cells are unable to switch to produce other Ig-classes and to
establish B-cell memory, their B cells are capable of producing normal antibodies in vitro
when co-cultured with normal T-helper cells, indicating the deficient ability of T cells to
activate B cells. Furthermore, the lack of functional expression of CD40L on activated T
cells in Hyper-IgM syndrome patients makes them susceptible to recurrent upper-
respiratory-tract and opportunistic infections, i.e. with Pneumocystis carinii,
Cryptosporidium, and cytomegalovirus, pathogens usually cleared by T cell-dependent
mechanisms. Similarly, CD40L-deficient mice are severely impaired in primary T-cell
responses to protein antigens and clonal expansion of CD4+ T cells. Moreover, it is well
documented that CD40 signals are required for germinal center formation and antibody
isotype class switching (Grewal and Flavell, 1998). Furthermore, CD40– CD40L
interactions affect APC function via upregulation of costimulatory and/or adhesion
molecule expression, i.e. B7–1, B7–2, ICAM-1, LFA-3, CD44 (Caux et al,, 1994, Kiener
et al., 1995, Guo et al., 1996), but also cytokine secretion, i.e. TNF-a, IL-12 (Kiener et
al., 1995, Kato et al., 1997). Another important effect of CD40L signaling is its ability to
enhance APC longevity by inhibiting either spontaneous or activation-induced cell death.
It was shown that CD40 ligation via CD40L promoted sustained viability of dendritic cells
and monocytes in culture (Peguet-Navarro et al., 1995, Kiener et al., 1995) and allowed
naive and memory B cells as well as dendritic cells to resist T cell-induced Fas-mediated
apoptosis (Lagresle et al., 1996, Bjorck et al., 1997), thereby possibly sustaining and
amplifying APC-driven T-cell responses. The CD40–CD40L pathway is also critical in T
cell-dependent macrophage activation by stimulating the release of proinflammatory
cytokines, nitric oxide, and metalloproteinases (Stout and Suttles, 1996), the latter of
which are thought to facilitate the penetration of cells into inflammatory sites. There is
also evidence for direct effects of the CD40– CD40L pathway on T-cell function. For
example, Blotta et al (1996) showed that crosslinking CD40L on T cells can enhance T-
cell proliferation and cytokine production.
CD40L-deficient mice have been tested for their ability to resist Leishmania infection,
which is believed to be contained by T cell-activated macrophages. Soong et al (1996)
found CD40L-deficient mice to have an enhanced susceptibility to Leishmaniasis with
higher parasite load associated with low levels of proinflammatory cytokine and nitric
oxide production compared to wild type animals. Similar results were obtained by other
investigators (Campbell et al., 1996), additionally demonstrating a failure to mount a Th1
type immune response. The impairment of the Th1 response was dependent on the
inability of macrophages to produce IL-12, and, thus, could be overcome by
administration of exogenous IL-12 to infected mice. Also in hapten-induced colitis, an
experimental model for Thl-mediated disease, the early blockade of CD40–CD40L
interactions by antibody to CD40L prevented the detrimental Thl response, while IL-12
injections caused an exacerbation of disease (Stüber et al., 1996).
The impact of a lack of CD40–CD40L interactions in vivo has also been studied in
models of T cell-mediated autoimmune diseases. In type II collagen-induced arthritis the
in vivo administration of antibody to CD40L prevented autoimmune disease (Durie et al.,
1993). In EAE, a T-cell priming deficiency became apparent, when the CD40L mutation
was bred onto encephalitogenic peptide-specific TCR transgenic mice (Grewal et al.,
1996): EAE could not be provoked in these mice by immunization with the specific
peptide, whereas the same peptide induced EAE in CD40L+ TCR transgenic mice. Also,
lupus-prone mice did not develop renal disease when treated with anti-CD40L early in
life (Early et al., 1996) or, when treated at the time of established nephritis, showed a
decrease in severitiy of renal disease (Railed et al., 1998).
Finally, the importance of the CD40–CD40L pathway was demonstrated in several
transplantation models. Inhibition of this pathway with antibodies to CD40L dramatically
prolonged murine islet and cardiac allograft survival (Parker et al., 1995, Larsen et al.,
1996). These results were consistent with findings of other investigators in graft-versus-
host disease models in which disease was inhibited by antiCD40L mAb or by CD40L gene
disruption (Durie et al., 1994, Blazar et al., 1997). More recently, Rirk and collegues
(1997) have demonstrated the remarkable potency of blocking CD40–CD40L
interactions in a Rhesus renal allograft model. Overall, the data suggest that CD40L is a
master regulator of the immune system, with strong influence on T-cell activation, as
well as on macrophage, dendritic cell, B-cell and T-cell effector functions.
THE COMPLEXITIES OF T-CELL ACTIVATION AND

COSTIMULATION
It is now more than 25 years since Bretscher and Cohn first proposed that the generation
of an antigen-specific T-cell response requires at least two distinct signals from the APC
(Bretscher, 1992). We now know that the first signal occurs upon engagement of the
antigen-specific TCR-CD3 complex with antigenic peptide bound to major
histocompatibility complex (MHC) antigens, while another set of non-cognate cell-cell
interactions provides the second, so called costimulatory signal to the T cells via the B7-
CD28 pathway (Lenschow et al., 1996a). Over the years, numerous studies have
supported the two-signal hypothesis. Interactions between T cells and APC must be
carefully regulated such that activation of T cells is sufficient to generate a necessary
immune response, but also that activation of quiescent self-reactive or bystander T cells is
avoided. As described earlier in this review, the presence of MHC and CD40 molecules
on resting APC, in conjunction with the TCR-CD3 complex and CD28 on resting T cells,
is sufficient to trigger an immune response, while the lack of CD40 and B7 costimulation
leads to T-cell anergy or tolerance (Lenschow et al., 1996a, Grewal and Flavell, 1998).
Cognate interaction followed by CD40–CD40L interaction is necessary for the
activation of costimulatory activity on APC, which has been shown for several APC,
including B cells, macrophages, and dendritic cells. However, it is the dendritic cell which
is implicated in the initiation of an immune response (Banchereau and Steinmann, 1998).
And, indeed, DC, generated from human cord blood CD34+ progenitor cells, upregulate
B7–1 and B7–2 dramatically, upon activation by CD40L (Caux et al., 1994). But the
question remains whether a T cell can activate its APC before the T cell itself is activated.
Addressing this issue, it was shown that the upregulation of CD40L on resting T cells
requires stimulation via the TCR (signal 1) whereas costimulation (signal 2) is not
required (Jaiswal and Croft, 1997). Therefore, a two-step model for the activation of T
cells in the initiation of an immune response can be proposed (Grewal and Flavell, 1998).
First, dendritic cells take up antigen at the site of injury or infection and migrate to the
lymph node, where the antigen is presented to naive T cells. The T cell receives the
antigenic signal 1, causing the rapid upregulation of CD40L (step 1). This in turn activates
the expression of costimulatory molecules/activity on the APC. In the second phase, the
costimulatory signal 2 is received by the T cell via CD28 which drives the T cell into cell
cycle and full activation (step 2). These activated T cells can now enter into secondary
cognate, antigen-specific CD40–CD40L-dependent and/or B7-CD28-dependent T-cell
helper or T-cell effector functions in the effector phase of the immune response. Hence, a
reciprocal dialogue between APC and T cell can only follow if either APC or T cell is
activated.
This two-step model of T-cell activation was confirmed in vivo in the already mentioned
EAE model using CD40L-deficient, peptide-specific TCR transgenic mice (Grewal et al.,
1996). Here, T cells could be primed to produce IFN-γ and mediate EAE only when B7–
1+ APC were provided by adoptive transfer. These findings suggest that CD40L is
required for the induction of in vivo costimulatory activity on APC, which in turn is
required for activation and priming of T cells evoking EAE. However, in a transplant
model the simultaneous blockade of both the CD40–CD40L and the B7-CD28 pathways
was required to permit engraftment of highly immunogenic allografts (Larsen et al., 1996,
Konieczny et al., 1998), indicating that B7–1/2 induction and T-cell activation can occur
in the absence of CD40–CD40L interactions. Thus, although they are interrelated, both
pathways are independent regulators of T-cell-mediated immune responses. Similar
observations were made in autoimmune/transplant models where the synergistic effects of
a brief simultaneous blockade of CD40–CD40L and B7-CD28 interactions led to long-
term inhibition of murine lupus (Daikh et al., 1997), and renal allograft rejection (Kirk et
al., 1997), whereas blocking either the CD40– CD40L or B7-CD28 pathway alone was
not sufficient to inhibit disease progression. Taken together, these data suggest that there
are both CD40-CD40L-dependent and -independent mechanisms for B7–1 and B7–2
induction in vivo.
As described earlier, full T-cell activation occurs in the presence of MHC-peptide
ligand complexes and TCR occupancy sufficient for adequate TCR-CD3 complex
signalling (signal 1) and increased expression of B7–1 and B7–2, initially on dendritic cells
and subsequently on activated B cells, resulting in CD28 costimulation (signal 2). This
response is characterized by cell cycle progression, induction of anti-apoptotic proteins, T-
cell clonal expansion, T-cell differentiation, and the generation of effector functions, such
as cytolytic responses or directed T-cell help by polarized cytokine production. In this
setting, CD28-mediated positive costimulation is not only important for initiating but also
for sustaining T-cell responses (Kündig et al., 1996). However, longevity and strength of a
T-cell response need to be controlled. Here, CTLA-4, the negative regulator of T-cell
activation, comes to play its role. Optimal T-cell activation induces the synthesis and peak
cell surface expression of CTLA-4. The increased localized density of B7–1, B7–2 and
CTLA-4 molecules (Linsley et al., 1996) would facilitate high avidity B7-CTLA-4
interactions. The resultant inhibitory signals could limit T-cell clonal expansion, decrease
the duration of the response, and/or influence the effector function, all of which would
ultimately lead to downregulation of the ongoing T-cell response necessary for
maintaining peripheral T-cell homeostasis. Indeed, in a very recent study by Saito and
coworkers (1998) acute graft-versus-host disease (GVHD) was induced by allogeneic
CD28-deficient T cells, which was inhibited by treatment with antibody to CD40L, but was
exacerbated by treatment with antibodies to B7–1 and B7–2, thereby interrupting B7-
CTLA-4 interactions. These findings indicate the B7-CTLA-4 pathway to act protectively
in the development of GVHD by downregulating the allogeneic T-cell response.
Overall, the complex array of cognate and non-cognate interactions, i.e. between the
CD40–CD40L, B7-CD28 and B7-CTLA-4 pathways, and the reciprocal dialogue between
APC and T cells allow for a well balanced T cell-mediated immune response.
Nevertheless, how the T cell integrates these signals to initiate, maintain and/or
terminate antigen-specific immune responses still remains one of the central issues in the
understanding of T-cell activation.
T-CELL ACCESSORY MOLECULE EXPRESSION IN

INFLAMMATORY AND MALIGNANT SKIN DISEASES
The in situ expression of the receptor/ligand pairs B7-CD28 and CD40-CD40L has been
examined in different T-cell-mediated inflammatory skin diseases. In allergic contact
dermatitis and lichen planus, we found CD28 to be expressed on the majority of dermal
and epidermal T cells. B7–1/2 and CD40 expression was clearly detectable on
Langerhans cells, dermal dendritic cells (DC)/ macrophages, and occasionally on dermal
T cells. Moreover, strong CD40 staining was observed on endothelial cells (EC) in
diseased skin. In normal skin, however, CD28 was observed only occasionally on
perivascular T cells, whereas B7–1/2 could not be detected in situ.
Only a faint CD40 staining was found on cutaneous DC, EC and keratinocytes (KG) of
the basal layer (Simon et al., 1994, Hollenbaugh et al., 1995 and unpublished results). By
contrast, no significant expression of CTLA-4 and CD40L could be detected, most likely
due to their limited and transient expression patterns. In experimental murine contact
hypersensitivity (CHS), a T-cell-mediated response to epicutaneous hapten sensitization
and challange, the in vivo relevance of both pathways has recently been highlighted.
Disruption of B7-CD28 interactions in vivo by CTLA-4-Ig or mAb to B7–1 and B7–2
during hapten sensitization inhibits CHS with distinct roles for B7–1 and B7–2 during the
priming of CD8+ and CD4+ T cells (Tang et al., 1996, Xu et al., 1997). Furthermore,
the additional inhibition of the CD40-CD40L pathway induced tolerance in CHS, but had
no effect on the elicitation phase of the CHS response (Tang et al., 1996, Tang et al., 1997),
indicating both pathways to be essential for complete sensitization to haptens.
In psoriasis, CD28 and B7–1 were found to be expressed by virtually all T cells in the
epidermis and dermis of psoriatic lesions (Nickoloff et al., 1994). This expression pattern
may permit self-costimulation and thereby contribute to the ongoing T-cell proliferation.
Additionally, dermal DC, being more prevalent in psoriatic plaques than in normal skin,
have been shown to express high levels of B7–2 and intermediate levels of B7–1 (Mitra et
al., 1995). Moreover, in psoriatic skin we have demonstrated a markedly enhanced
expression of CD40 on KC in the suprabasal layers of the epidermis as well as on the
dermal mononuclear infiltrate and on EC. The CD40 expression co-localized with the
enhanced expression of the adhesion molecule ICAM-1 and the anti-apoptotic protein Bcl-
x on KC and an influx of juxtaposed T cells (Denfeld et al., 1996). These findings suggest
that direct interactions between infiltrating T cells and KC via the CD40–CD40L pathway
might contribute to the induction of CD40, ICAM-1 and Bcl-x on KC and, therefore,
could have implications for the pathogenesis of psoriasis.
In diseased skin of patients with systemic, subacute cutaneous and chronic discoid lupus
erythematosus (SLE, SCLE, CDLE), we detected CD28 expression on most T cells
infiltrating the dermis and epidermis. B7–1 and B7–2 were expressed on dermal and
epidermal DC, particularly when in apposition to CD28+ T cells, but not on KC. By
contrast, in uninvolved skin of SLE, SCLE, and CDLE patients, CD28, B7–1, and B7–2
expression was negligible. Treatment of SLE patients with systemic corticosteroids
revealed a correlation between clinical improvement, the resolution of the infiltrate and
the reduced expression of B7–1, B7–2, and CD28 in formerly lesional skin. These findings
suggest that in LE, B7–1+ and B7–2+ DC could contribute to the intracutaneous
activation of CD28+ T cells, thus making an inhibition of B7-CD28 interactions worth
considering in the therapy of LE (Denfeld et al., 1997). Indeed, in a murine model
resembling SLE, disruption of the B7-CD28 pathway suppressed the lupus-like illness and
prolonged life (Finck et al., 1994, Nakajima et al., 1995). Furthermore, it has been shown
that T cells of patients suffering of SLE have an increased and prolonged expression of
CD40L, documenting an impaired regulation of CD40L expression on T cells in SLE,
which might result in abnormal costimulation for autoantibody production (Datta and
Kalled, 1997). Specific immunotherapy that blocks CD40-CD40L interactions may thus
be of value for treatment of this (and other) autoimmune disease, which has already been
demonstrated in lupus-prone mice (Early et al., 1996, Daikh et al., 1997, Kalled et al.,
1998).
In addition, EC play an important role in the pathogenesis of T-cell-mediated skin
diseases. Although T cells reside within skin under physiological conditions, at sites of
cutaneous inflammation effector T cells need to be recruited and activated. Evidence is
emerging that CD40 may play an important role in the regulation of leukocyte
recruitment into peripheral tissues like the skin. The low level of CD40 expression by EC
in situ is enhanced by proinflammatory cytokines, i.e. TNF-α, IL-1β, IFN-γ. Ligation of
CD40 on isolated EC induces the expression of the adhesion molecules ICAM-1,
VCAM-1 and E-selectin suggesting that signalling through CD40 during T cell-EC
interactions may be an important step in leukocyte recruitment (Hollenbaugh et al., 1995,
Dechanet et al., 1997). In vivo, we have found a marked upregulation of CD40 expression
on EC in T-cell-mediated inflammatory skin disease, such as psoriasis and allergic contact
dermatitis, compared to normal skin (Hollenbaugh et al., 1995). Nevertheless, it is not
known whether CD40 is actually required for T-cell-dependent EC activation in vivo. For
example, although inhibition of CD40–CD40L interactions with antibody against CD40L
prolonged allograft survival in several transplantation models including murine skin
allografts (Durie et al., 1994, Parker et al., 1995, Larsen et al., 1996, Blazar et al., 1997, Kirk
et al., 1997), it is unknown whether such a blockage impedes T-cell recruitment into the
graft. In fact, Larsen and coworkers (1996) have demonstrated the expression of B7–1,
B7–2 and T-cell cytokine transcripts in anti-CD40L mAb treated allografts suggesting that
T cells may still enter the graft in the first days after transplantation. Hence, CD40
expression on EC may not be pivotal for early T-cell recruitment, but the CD40L on T cells
may become an important factor in sustaining adhesion molecule expression and
leukocyte recruitment during the effector phase of T-cell-mediated inflammatory skin
disease.
Finally, the expression of costimulatory molecules, especially regarding both, the B7-
CD28 and the CD40-CD40L pathway, has been studied in skin tumors. In human
malignant melanoma, we and others failed to detect B7–1 and B7–2 surface expression on
melanoma cells of primary and metastatic malignant melanoma in situ. However, B7–1
and B7–2 expression was found on tumorinfiltrating APC in apposition to CD28+ T
cells. Additionally, B7–1 and B7–2 protein expression was at low or undetectable levels
in the majority of cell lines derived from cultured primary and metastatic melanomas,
while B7–2 RNA was detectable in some cell lines. The important exceptions were
primary melanomas with partial spontaneous regression, in which a focal expression of
B7–1 and B7–2 was detectable on melanoma cells in situ, suggesting that the absence of
B7–1 and B7–2 favors the escape of malignant melanoma from immunosurveillance
(Denfeld et al., 1995). In contrast to molecules of the B7 family, CD40 is expressed on
melanomas in situ and in vitro following tissue culture (Thomas et al., 1996, van den Oord
et al., 1996). Recently, we found that CD40 ligation on human melanoma cells in vitro led
to secretion of proinflammatory cytokines, an increased expression of adhesion and MHC
molecules, and augmented tumorspecific CTL-mediated lysis and apoptosis of melanoma
cells (von Leoprechting et al. submitted). In vivo, CD40-CD40L interactions also
contribute to the development of protective immunity against melanoma, since anti-
CD40L mAb treatment inhibited the generation of anti-tumor immune responses in a

murine melanoma model (Mackey et al., 1997). Furthermore, skin cancers of KC origin,
namely basal cell carcinoma and squamous cell carcinoma, do not express B7 in situ
(Simon et al., 1994, Nestle et al., 1997) and exhibit a down-regulation of CD40 compared
to the proliferative basal layers of normal skin or benign viral induced skin lesions (Viac et
al., 1997). Moreover, only a minority of tumor-associated APC express functional B7–1
and B7–2, whereas CD40 expression was unaffected compared to inflammatory skin
disease (Nestle et al., 1997, Viac et al., 1997). Hence, both, the paucity of B7-molecule
expression and function of tumor-infiltrating APC as well as the lack of B7 and CD40
expression on epidermal tumor cells, might account for the failure of tumor-infiltrating T
cells to become fully activated, thus allowing tumor escape from immunosurveillance.
In conclusion, over the years significant evidence has accumulated that both, B7-CD28
and CD40-CD40L interactions play a central role in neoplastic and inflammatory skin
diseases. This may bear direct clinical implications since the manipulation of one or both
pathways may offer novel and powerful immunotherapeutic strategies by disrupting
costimulation in inflammatory skin diseases and enhancing costimulation in malignant skin
diseases.
ACNOWLEDGEMENTS
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Si 397/7–
1, 8–1)
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11.
ANIMAL MODELS OF SKIN
INFLAMMATION
BENJAMIN E.RICH AND THOMAS S.KUPPER
INTRODUCTION
As the major interface with the environment, the skin encounters a steady array of injuries
and incursions of foreign materials and pathogens. In response to these continuous
challenges, an effective system of cutaneous immunity has evolved to defend the skin
against pathogens, to expel foreign materials and to facilitate the repair process. The
detection of foreign antigen within tissue initiates a series of reactions that result in a
concerted set of physiological changes. Inflammation is the most outwardly evident of
these changes. In the normal course of events inflammation serves to protect the organism
and initiate the repair of damaged tissue; however, in numerous different circumstances
inappropriate inflammation can itself be pathological. The imperative to understand and
develop means to control pathological inflammation of skin has led to the study of animals
with cutaneous inflammatory disorders. Furthermore, the development of genetic
engineering methods has led to the creation of a number of strains of mice in which
improper expression of certain genes causes inflammatory disorders. In this chapter we
will review the use of such animals to study the process of inflammation in vivo.
NORMAL CUTANEOUS INFLAMMATION

The normal cutaneous immune response involves at least two qualitatively distinct
components. While these components have distinct mechanisms with different triggering
events and ultimate outcomes, they each contribute to the successful defense of the
cutaneous barrier. One component, called immediate hypersensitivity, can be a nearly
instantaneously response to the presence of antigens within the tissue. Immediate
hypersensitivity ensues when these antigens are engaged by antigen-specific IgE bound to
mast cells. This triggers those mast cells to activate and release factors including
vasoactive amines which permeablize cutaneous vessels thereby allowing blood cells to
enter the tissue. This initial inflammation subsides within an hour or so, however several
hours later a second cellular infiltrate arises at the site of antigen exposure. In this
secondary inflammation effector cells, including basophils, neutrophils and eosinophils,
seek out and destroy pathogens such as parasites. The immediate hypersensitive response
is wholly dependent upon pre-existing antigen-specific IgE molecules. The rapid kinetics
222 BENJAMIN E.RICH AND THOMAS S.KUPPER
of the reaction results from the direct activation of mast cells via the IgE molecules.
Therefore previous exposure to the antigen is a prerequisite for the response. Indeed,
adoptive transfer of antigen-specific IgE molecules from an immunized animal to a naive
recipient conveys the ability to mount an immediate hypersensitive response.
Unlike the immediate hypersensitive response, the delayed hypersensitive response has
no immediate inflammatory phase. Rather, a cellular infiltration and inflammation in
response to the detection of foreign antigens within the tissue occurs only after several
hours. While this reaction is also dependent upon previous immunization with the antigen,
it is unlike the immediate hypersensitive response in that adoptive transfer of cells from an
immunized animal can confer reactivity to a naive recipient but serum (cell free
immunoglobulins) cannot. The delay in the response is due to the time required for
antigen presenting cells (APCs) to take up antigenic molecules, process them internally
and display peptides on their major histocompatability complex (MHC) molecules for
presentation to T cells. At the same time the APCs migrate to lymphoid tissue where they
encounter and activate T cells which facilitate the inflammatory response.
SECONDARY EFFECTS OF INFLAMMATION: PERTURBATION

OF SKIN ARCHITECTURE
In addition to the infiltration of effector cells, the normal process of inflammation also causes
secondary effects on the structure of the skin. One of the most prominent effects of
inflammation on the structure of the skin involves alterations of the growth and
differentiation behavior of epidermal keratinocytes. Epidermal keratinocytes ordinarily
divide in the basal layer and undergo a process of differentiation while they move towards
the surface as a result of proliferation below and ablation above. Therefore the structure of
the epidermis is determined by the balance among the rates at which the keratinocytes
divide, differentiate to cornified squamous cells, migrate to the surface and slough off.
This epidermal homeostasis is altered when the rate of cell division of basal keratinocytes
increases as a result of signals associated with the inflammation, such as cytokines released
by infiltrating cells. This phenomenon seems to make sense in the context of a wound or
pathogenic intrusion because more keratinocytes may be needed to repair the lesion.
However in prolonged pathological inflammations, such as psoriasis, the
hyperproliferation of keratinocytes can significantly alter the architecture and functional
properties of the skin and become a significant aspect of the pathology.
The most prominent perturbation of the epidermis seen in pathological inflammations
is an increase in the number of keratinocytes and the thickness of the epidermal layer of
living cells. This phenomenon, termed acanthosis, is often accompanied by elongation of
the ridges at the dermal/epidermal junction increasing the convolutions and hence the area
of the dermal/epidermal interface. This can in turn, provide a greater surface area for the
interactions between the dermis and the epidermis.
A second alteration of the epidermal structure that is often associated with acanthosis is
hyperkeratosis, a thickening of the layer of cornified cells called the stratum corneum.
Hyperkeratosis generally appears to be due to an increased rate of production of cornified
keratinocytes (corneocytes) however it may also be due to changes in the integrity of the
ANIMAL MODELS OF SKIN INFLAMMATION 223
stratum corneum and a reduced rate of sloughing of the outer cells. This feature is
generally the most evident at the macroscopic level.
A third morphological feature that is associated with disturbances in epidermal
homeostasis is parakeratosis or the presence of imperfections and gaps between cells in the
stratum corneum. These perturbations are generally associated with the appearance of
incompletely differentiated cells with nuclei in the stratum corneum. This can arise as a
result of the program of differentiation of keratinocytes being delayed relative to the
proliferation of the basal layer. Parakeratosis may also caused by certain types of trauma
which cause cellular injury such as “tape stripping”. Regardless of the etiology,
parakeratosis can be associated with degradation of the barrier properties of the skin.
Cellular infiltrates are often seen in the dermis and occasionally in the epidermis of
inflamed skin. These may consist of myeloid and/or lymphoid cells and are often seen
clustered around dermal vessels. Dermal vasculature may be dilated or otherwise
compromised.
Another mechanism by which inflammation can disrupt the skin architecture is
hydrostatic pressure. Perhaps the most clear example of this is in the autoimmune disease
bullous pemphigoid in which autoantibodies reactive with the basal layer provoke
extensive release of fluid from vessels near the junction between the epidermis and the
dermis. This fluid accumulates and forces the epidermis away from the dermis forming
blisters. Another example of the effects of hydrostatic pressure is spongiosis where fluid is
forced between keratinocytes leaving only desmosomal contacts between the cells. This
leads to a characteristic spongy appearance of the tissue in which keratinocytes assume a
star-shaped morphology as a result of the tension on the desmosomes. This is distinct from
acantholysis in which cell to cell contacts are lost and keratinocytes become round. This
can be caused by extreme fluid infiltration, in which case it is called secondary
acantholysis. In contrast, primary acantholysis is caused by impairment of the function of
cell to cell adhesion molecules by autoantibodies or genetic defects.
CONTACT HYPERSENSITIVITY: A MODEL OF SKIN

INFLAMMATION
Contact hypersensitivity (CHS) is an immunological reaction in which epicutaneous
administration of various compounds elicits a T cell mediated inflammatory response in
the skin. A standardized experimental method for the generation and measurement of CHS
has been developed and utilized extensively. Experimental CHS has proven to be a very
useful model for the study of delayed-type hypersensitivity as well as cutaneous immune
responses. Detailed methodology of CHS reactions are described elsewhere (1). While
some materials that provoke contact hypersensitivity are inherently antigenic, many are
chemically reactive compounds, called haptens, that modify skin cell proteins which then
become antigenic. Compounds that have been used to provoke CHS include picryl
chloride (2,4,6-trinitrochlorobenzene), fluorescein isothiocyanate, oxazalone and
dinitrofluorobenzene. In addition to antigenicity, the general irritant properties of the
haptenizing compound are important since it has been shown that the simultaneous
application of an unrelated irritant compound with the antigen greatly enhances the
inflammatory response to low levels of the antigen (2).
Experimental CHS is a cutaneous form of a delayed-type hypersensitive response in
that the eliciting exposure to the sensitizing agent does not provoke an immediate
response. It requires at least two exposures to the sensitizing agent to cause the reaction
and these exposures must be separated by several days to a week. Furthermore, the
second, eliciting exposure can be at a site distal to the site of initial sensitization.
Therefore it relies upon both afferent and efferent arms of the immune system and is a
sensitive measure of their combined action.
The inflammatory lesions that arise in response to the second exposure to the
sensitizing agent are characterized by edema and perivascular cellular infiltrates. If the
second application of the sensitizing agent is applied to an ear, the resulting edema and
thickening of the ear provides a convenient index of the extent of the inflammation. The
quantitative measurement of the CHS reaction in mouse ears was first described in 1968
(3). Since then it has been exploited extensively as a simple and reliable measure of
immunological function. Much of our current understanding of cellular immune
responses is derived from experiments using this model.
IMMEDIATE HYPERSENSITIVITY: A MODEL OF ATOPIC

DERMATITIS
Recently there has been increased interest in immediate hypersensitivity provoked by
model antigens such as chicken ovalbumin (OVA). This research has focused on airway
and cutaneous inflammatory responses after sensitization by direct application of the
antigen to the skin. In this method, a concentrated aqueous solution of OVA (1 mg/ml) is
absorbed in cotton gauze and held against the skin by an adhesive patch dressing for one
week. This sort of topical immunization appears to favor Th2 type responses and
generates the requisite antigen specific IgE titers for a constrictive airway response upon
subsequent challenge in the lungs. In addition to airway hypersensitivity, Balb/c mice
repeatedly challenged by epicutaneous application of OVA develop lesions exhibiting
acanthosis and dermal infiltrates of eosinophils, neutrophils and lymphocytes that are very
similar to human atopic dermatitis (4).
REGULATORY CIRCUITS IN INFLAMMATION

In order to maintain healthy skin, cutaneous inflammation must be rapidly inducible to
respond effectively to environmental and infectious challenges, but the effects of
inflammation can be deleterious, therefore the inflammation must also be controllable.
While the large number of cell types and gene products involved in inflammation clearly
make the control of inflammation an enormously complex phenomenon, some simple
regulatory mechanisms in the skin have been identified.
One of these is the autocrine and paracrine induction of proinflammatory genes by
interleukin 1α (IL-1α), IL-1β and tumor necrosis factor (TNFα) (5). IL-1α and TNFα
stimulate activation of the transcription factor NF-κB in target cells. NF-κB acts directly
to promote transcription of the IL-1α , IL-1β and TNFα genes, causing more release of
these factors. Thus a small induction of either IL-1 or TNFα can be amplified and
perpetuated within one cell and this induction can be communicated to other adjacent
cells by diffusion of the cytokines. This inherently unstable control circuit is modulated by
the action of I-κB, an efficient intracellular inhibitor of NF-κB. As discussed below, in the
description of motheaten mice, the absence or impaired function of I-κB leads to runaway
signaling and profound pathological inflammatory processes.
A second regulatory circuit in cutaneous inflammation involves a reciprocal paracrine
dialog between keratinocytes and T cells (6, 7). Activated T cells can release interferon γ
(IFN-γ) among other products. Keratinocytes exposed to IFN-γ increase transcription of
the IL-7 gene and secrete more of this factor. IL-7 is absorbed into extracellular matrix
and thus does not diffuse rapidly, though it remains fully active. As a potent mitogen for
activated T cells, IL-7 augments their IL-2-mediated autocrine growth stimulation,
upregulates expression of IL-2 receptor (IL-2R) and acts to block activation induced
programmed cell death. Therefore keratinocytes in the vicinity of activated T cells are
stimulated to enhance the activity of those T cells by release of IL-7. Conversely, the
expanded population of T cells will release more IFN-γ, perpetuating the cycle. Since this
control circuit involves two cell types it is likely to be slower and perhaps less unstable
than the IL-1/TNF-α circuit. Nonetheless, this reciprocal positive regulatory loop is likely
to have a role in normal inflammation. As described below, deregulated expression of
either IL-7 or IFN-γ provokes chronic inflammatory disorders. It remains to be
determined to what extent this sort of runaway reciprocal intercellular signaling may
contribute to human disease.
SPONTANEOUS GENETIC MODELS

A number of mutant strains of mice with aberrant cutaneous inflammation have been
identified (8). Some of these are similar to human diseases while others are distinct. We
present here a brief discussion of a few of the more prominent of these.
The Flaky Skin Mouse

The flaky skin mouse, which arose spontaneously at The Jackson Laboratory in Bar
Harbor, Maine, USA, carries an autosomal recessive mutation, fsn, that causes a cutaneous
disorder that resembles human psoriasis. The fsn gene has been mapped to a locus at 56
centimorgans of mouse chromosome 17 but has not been molecularly characterized, flaky
skin mice have sparse fur, and a scaly and thickened epidermis. A number of
ultrastructural features of the disorder are also found in psoriasis vulgaris (9). The flaky
skin phenotype is transferred to SCID mice by transplant of fsn/fsn bone marrow
indicating that the defect is primarily in bone marrow derived cells. Like human psoriasis,
the lesions of the flaky skin mouse are significantly diminished by administration of
cyclosporine. While a direct effect of cyclosporine on keratinocytes has not been ruled
out, it appears likely that the interference with the activation of T cells and their
production of inflammatory cytokines is the basis of this phenomenon. Therefore, in
addition to the morphological resemblance of the disorder of the flaky skin mouse to
human psoriasis, it is likely that both diseases involve some sort of reactive T cells.
However, recent reports of extensive systemic autoimmunity in the flaky skin mouse may
point to an etiology that is distinct from that of most human psoriasis (10, 11).
The Scurfy Mouse

A recessive mutation in the murine X-linked scurfy gene, sfy, causes an immunodeficiency
with extensive leukocyte infiltration of cutaneous tissues. The scurfy disorder appears to
involve improper maturation of T cells since the disease is suppressed in nude mice, can
be transmitted to immunodeficient mice by T cell transplants and fails to develop in wild-
type mice that have been engrafted with scurfy bone marrow (12). The disorder has been
shown to be caused by a class of CD4+ T cells and is associated with increased expression
of certain cytokines including IL-4, IL-6, IL-7 and TNF-α (13). The scurfy disease has
similarities with the X-linked recessive human immunodeficiency disease Wiskott-Aldrich
Syndrome (WAS) and interestingly the scurfy mutation maps very close to the murine
homolog of the WAS gene. This has lead to the suggestion that the two disorders may be
mechanistically related (14). While this has not been absolutely ruled out, the recent
observation that mice with an engineered null mutation of the WAS gene have T cell
defects but do not have the characteristic extreme cutaneous inflammation of the scurfy
disorder (15) makes it seem likely that these two genes are distinct.
The NC/Nga Mouse

The inbred NC/Nga strain of mice exhibits spontaneous skin lesions that appear similar to
human atopic dermatitis (16). Lesions fail to arise in NC/Nga mice housed under
pathogen-free conditions implying that development of these lesions is dependent upon
exposure to certain pathogens. The disorder seems mechanistically related to atopic
dermatitis in that the levels of circulating IgE in the NC/Nga mice are elevated and
individuals with higher IgE levels tend to have more severe dermatitis. This syndrome has
been shown to be a multigenic trait in that the dermatitis segregates from the elevated IgE
when the NC/Nga mice are crossed with Balb/c mice. The dermatitis appears to be
caused by a single recessive gene while the elevated IgE is caused by cooperation of two
recessive genes (17). Thus it remains somewhat unclear to what extent the elevated level
of IgE in the NC/Nga mice contributes to their disorder. Nonetheless, the recent
demonstration that topical application of the immunosuppressive agent FK506 can
mitigate the disease of NC/Nga mice lends further support to the role of IL-4 production
by Th2 cells in atopy and may lead to the use of this compound to treat human atopic
dermatitis (18).
The Chronic Proliferative Dermatitis Mouse

Chronic proliferative dermatitis (cpdm) is an autosomal recessive mutation that arose
spontaneously in C57BL/Ka mice (19). The phenotype associated with this gene is first
evident as inflammatory skin lesions that appear spontaneously on the back and belly at 5
to 6 weeks of age. While the lesions spread progressively over the trunk, the ears, tail and
footpads are spared. The epidermis within the lesion is hyperproliferative and
parakeratosis and hyperkeratosis are evident. The dermal vasculature in the lesions is
enlarged and cellular infiltrates are found in the dermis and epidermis that consist
primarily of myeloid cells such as granulocytes, eosinophils and macrophages. Mast cells
are found to accumulate in the dermis of lesional skin. Increased numbers of IgE+ mast
cells are found in tissues of cpdm/ cpdm mice prior to the development of gross symptoms
while the mast cells in wild type mice are IgE− (20). Treatment of the mice with
cyclosporin had little effect on the cutaneous symptoms, however corticosteroid treatment
was effective in reducing the lesions (21).
Transplantation of spleen or bone marrow to syngeneic mice failed to confer the
disease on the recipients. Skin grafted from cpdm/ cpdm mice onto unaffected syngeneic or
nude mice retained the inflammatory phenotype but did not confer the disorder to host
tissue. Conversely, skin from wild type mice grafted onto cpdm/ cpdm mice remained
healthy (22). It is clear that the genotype of resident skin cells is critical for the disease to
develop and the genotype of the hematopoietic cells is not. The binding of neutrophils to
frozen sections of affected skin is elevated while lymphocyte binding to the sections is not
(23). This reflects increased levels of expression of ICAM-1 and L-selectin and low
expression of E-selectin in the skin. Thus it appears that the defect caused by the cpdm
mutation is in resident cells of the skin. This alteration seems to provoke inflammatory
behavior in myeloid cells which may, in turn, elicit further abnormalities in the resident
skin cells.
The Motheaten Mouse

Motheaten mice have an autosomal recessive disorder in which cutaneous inflammation is
one aspect of a complex immunodeficiency syndrome (24, 25). Their skin abnormality is
evident soon after birth as inflammatory cutaneous infiltrates involving granulocytes.
These inflammations lead to alopecia and give rise to the characteristic “motheaten”
appearance of the mice. Adoptive transfer of splenocytes conveys the disease to syngeneic
recipients indicating that the defect is in hematopoietic cells (26). There are two variants
of motheaten mice that differ by the severity of their diseases. The original motheaten
mice live an average of 22 days while “viable motheaten” mice live an average of 61 days.
Both of these were discovered to be caused by mutations in the protein-tyrosine
phosphatase gene Hcph (27). The product of this gene appears to act in removing
phosphate groups from molecules involved in signal transduction from cytokine
receptors, thereby limiting the duration and extent of the signals conveyed. In its absence
these signals persist, leading to pathological responses. This is demonstrated by the response
of motheaten macrophages to GM-CSF which has been shown to be significantly
exaggerated (28). Perhaps the most significant substrate of the phosphatase is IκB, the
inhibitor of the transcription factor NF-κB. When IκB is phosphorylated it becomes
targeted for proteolytic destruction by ubiquitination. In the absence of IκB, NF-κB is
able to translocate to the nucleus and initiate transcription of a number of genes involved
in immune responses, such as cytokines, chemokines, adhesion molecules and enzymes

involved in effector functions. Among these are the genes encoding IL-1β and TNF-α. As
mentioned above, these two genes have been implicated in the perpetuation of
inflammatory reactions since their products activate the NF-κB pathway, creating a
positive regulatory loop (5). The activity of this loop is enhanced by the rapid destruction
of IκB leading to activation of NF-κB. This model is further supported by the observation
of an even more severe, neonatal-lethal inflammatory disorder that arises in gene-targeted
mice lacking the IκBα gene (29). Furthermore, transgenic mice expressing TNF-α under
the control of the K14 promoter have cutaneous lesions as well as systemic cachexia (30).
Recently motheaten mice were used to test the ability of soluble TNFR-1 to block the
action of TNF-α in vivo (31). Motheaten mice treated with this compound had twice the
lifespan and significant reductions in arthritis, pneumonitis and skin lesions as compared to
motheaten mice receiving control treatment. The success of this study confirms the
critical role of TNF-α in this inflammatory disorder and illustrates the value of motheaten
mice in evaluating candidate therapeutics.
ENGINEERED GENETIC MODELS: CYTOKINES EXPRESSED IN

SKIN
In efforts to elucidate the roles of various biologically active molecules, a number of
investigators have created transgenic mice that express these molecules inappropriately.
Among the different phenotypes exhibited by these transgenic mice, several of them
develop inflammatory disorders of the skin. Foremost among these are transgenic mice
expressing cytokines.
One of the earliest examples of cutaneous inflammation caused by deregulated cytokine
expression is a strain of transgenic mice in which IL-2 was expressed under the control of
the MHC class I promoter (H2Kd) in order to test the possibility that broad expression of
IL-2 might support indiscriminate maturation of T cells and lead to autoimmunity (32).
While no specific autoimmunity was detected in these mice, they were found to have
spontaneous alopecia that develops at about 8 weeks of age as well as pneumonia and
splenic hypercellularity. The affected skin exhibited thickened epidermis, absent or
disfigured hair follicles and sporadic lymphocytic infiltrates. H2Kd-IL2 mice examined at 5
weeks, before the alopecia developed, had increased numbers of Thy-1+ dendritic
epidermal T cells (DETC) but very little signs of cutaneous inflammation or any
proliferative disorder.
The mechanism behind the profound cutaneous inflammation in older H2Kd-IL2 mice
is not entirely clear. Since IL-2 acts principally on lymphocytes and is not known to exert
any direct proliferative effects on keratinocytes it is likely that the effect on epidermis is
indirect and mediated by lymphocytes. When skin grafts were performed between wild
type and transgenic mice, only the trans-genic skin became affected; in each configuration
the wild-type skin was spared. This demonstrated that the skin disorder is caused by
expression of the transgene in resident skin cells and that the effects of the IL-2 expression
on circulating cells do not persist when those cells migrate to non-transgenic tissue. The
proinflammatory behavior of the circulating cells must be sustained by localized exposure

to IL-2.
The specific lymphocytes involved in the infiltrates are also unclear. The authors
postulated involvement of Thy-1+ DETC because they are ubiquitous in the epidermis and
their numbers are expanded prior to the development of alopecia. On the other hand, the
subcutaneous lymphocytic infiltrates were seen only in association with the alopecia and
acanthosis. While IL-2 is not directly chemotactic for T cells, it can have an indirect effect
in that it blocks their chemotactic responses to certain chemokines (33). Thus T cells
passing through tissue in response to relatively long range chemokine gradients may stop
when they encounter high levels of IL-2. This might cause T cells to linger and accumulate
in the skin of these mice.
MHC class I genes are active in a broad spectrum of cells including keratinocytes, and
their expression is enhanced by exposure to cytokines such as interferons (IFN), TNF-α
and lymphotoxin (LT) that can be produced by activated T cells (34, 35). The IL-2
released by keratinocytes will enhance the proliferation and viability of activated T cells.
Those activated T cells can, in turn, release factors that are mitogenic for keratinocytes
and also induce higher expression of endogenous MHC genes and the transgenic IL-2.
Therefore these reciprocal agonistic signals between lymphocytes and the transgenic
keratinocytes may constitute a dialog involving mutual activation of lymphocytes and
keratinocytes that leads to inflammation.
While exogenous IL-2 will clearly enhance the response of activated T cells, their
activation will necessarily depend upon T cell receptor (TCR) mediated signals. No
autoreactive antibodies were detected in these mice and adoptive transfer of splenocytes
failed to convey the disease to recipients. Thus the role of any autoantigens in this disease
is uncertain.
IL-7 AND SKIN

The IL-7 signaling pathway overlaps the IL-2 signaling pathway in that they both utilize
the common gamma chain (γc) which transmits signals via the JAK-3 kinase (36).
However, unlike IL-2 which is principally expressed by activated lymphocytes, IL-7 is
normally a product of epithelial cells. IL-7 was first identified as a growth factor for
immature B lymphocytes that is produced by bone marrow stromal cells (37).
Subsequently it was found to stimulate proliferation of thymocytes (38–42) and to be
expressed at high levels in thymic epithelial cells (37, 43). Mature B cells generally lose their
responsiveness to IL-7 however most T cells remain sensitive to IL-7 (44). The most clearly
understood role of IL-7 is in the development of lymphocytes and the maintenance of
lymphocyte homeostasis. IL-7 plays a central role in the expansion of early lymphocyte
populations and the rearrangement of antigen receptor genes that is essential for
generation of their diversity. This is demonstrated by the severely reduced numbers of T
and B lymphocytes found in IL-7 deficient mice (45, 46). The IL-7 mediated signaling that
controls lymphoid development and homeostasis appears to originate from the thymus
and bone marrow. However, in addition to bone marrow and thymic stromal cells, IL-7
has also been shown to be expressed by keratinocytes (47) as well as certain epithelial
cells of the intestine (48). The expression of IL-7 in other epithelial tissues may be more
important for regulating the behavior of intraepithelial lymphocytes. γδ T cells, including
DETC, are particularly responsive and dependent upon IL-7 (6, 49–51).
Much less is known about IL-7 mediated signaling in the immune response. A number
of effects of IL-7 signaling on T cell responses have been detected in vitro and in vivo,
however the role of IL-7 in reactive immunity has not been well denned. IL-7 stimulates
proliferation and enhances the viability of many mature T cells, particularly after TCR
stimulation (52). The expression of the IL-2R in T cells is induced by exposure to IL-7
thereby increasing the sensitivity of these cells to IL-2 (53). The responsiveness of T cells
to IL-7 is enhanced by their activation which has been shown to bring the IL-7 receptor
into association with the γc chain (54). IL-7 also specifically promotes the development
and function of lymphokine activated killer (LAK) cells (55–57) and cytotoxic T
lymphocytes (CTL) (57, 58).
Several observations point to a significant role for IL-7 in skin biology. As discussed
above, IL-7 and IFN-γ expression constitute a regulatory circuit involving T cells and
keratinocytes. While epidermal keratinocytes appear to express IL-7 constitutively, they
are induced to produce higher levels by exposure to IFN-γ (6, 47). The secreted IL-7
supports the viability and prevents apoptosis of DETC and other T cells in the skin. These
T cells may indeed be the source of the IFN- γ. Therefore, intraepidermal T cells that
become activated and release IFN-γ are likely to encounter reciprocally expressed IL-7
that will in turn enhance their viability. This reciprocal dialog between cutaneous T cells
and keratinocytes may be a mechanism that maintains homeostasis of the cutaneous
immune system. It may also be a means by which the active participation of keratinocytes
enhances the response of intraepithelial T cells (6, 7).
Human cutaneous T cell lymphomas (CTCL) are distinguished by their propensity to
migrate into the dermis and epidermis and cause pathological inflammatory lesions. CTCL
cells have been found to proliferate in response to IL-7 (59–61) and many have detectable
autocrine expression of IL-7 (59). However, the expression of the IL-7R by individual
tumors does not appear to correlate with their epidermotropism (62).
IL-7 is unusual among cytokines in that it can be absorbed into extracellular matrix
(ECM) (63–65). Moreover, T cells that encounter IL-7 (either free or matrix-bound) are
induced to bind to ECM components such as fibronectin as well as to the adhesion molecule
VCAM-1, that is expressed by stimulated endothelial cells (66). This mechanistic link
between the IL-7 signaling pathway and T cell extravasation may provide insight into the
apparently unique role of IL-7 in the skin.
Three different strains of transgenic mice have been generated in which deregulated
expression of IL-7 leads to cutaneous infiltrates of lymphocytes and profound cutaneous
inflammatory disorders. One of these expresses IL-7 in lymphocytes that are responsive to
the IL-7 in an autocrine fashion. A second strain expresses IL-7 under the control of viral
sequences that appear to be active in skin. The transgene in third strain directs expression
of IL-7 to keratinocytes and therefore delivers IL-7 to responsive cells in a paracrine
fashion.
Transgenic mice carrying an IL-7 cDNA under the control of immunoglobulin heavy
chain promoter and enhancer sequences (EµPµ-IL7) develop a progressive cutaneous
Figure 11.1 Progression of inflammatory disorder in EµPµ-IL7 transgenic mice. Three

representative EµPµ-IL7 transgenic mice illustrate the progressive nature of the spontaneous lesions.
A 57 day old mouse exhibits minimal skin disorders. Prominent lesions are seen on a 115 day old
mouse and a 235 day old mouse is nearly fully denuded with extensive alopecia, inflammation and
hyperkeratinization.
disorder involving inflammatory infiltrates leading to alopecia (67). In addition to the
cutaneous disorder these mice have expanded populations of lymphocytes and develop
sporadic lymphomas that both express and respond to IL-7. The Igµ promoter is active in
both T and B cells and therefore both populations are affected. In contrast to the
stochastic kinetics of the lymphomas, the cutaneous lesions develop in adult mice in a very
uniform fashion. They are first evident at about 12 weeks of age as a thinning of fur on the
belly and then progress until the trunk is wholly denuded (see Figure 11.1). Histological
examination of the lesions reveals lymphoid infiltrates in the dermis and a
hyperproliferative epidermis. Occasional foci of lymphocytes in the epidermis are seen.
Acanthosis, hyperkeratosis and parakeratosis are evident and hair follicles appear to be lost
in the hyperproliferation (see Figure 11.2). The infiltrating cells are predominately T cells
that lack expression of the co-receptors CD4 or CD8. Both αβ and γδ T cells are present
in the infiltrates, however αβ cells appear to predominate. When affected skin or
dissociated lymphoid tissues were transplanted to syngeneic mice the characteristic skin
lesions arose in the recipients. While no clonal TCR gene rearrangements were detected
in the affected skin of transgenic mice, TCR-β gene rearrangements were found in the
affected skin of the graft recipients. Therefore it appears that the skin infiltrating
lymphocytes of the µEµP-IL7 transgenic mice are initially polyclonal, however clonal
variants arise and are enriched by selection in transplant experiments. It is clear that the
autocrine expression of IL-7 in these mice leads to the development of a class of T
lymphocytes that are predisposed to migrate into and accumulate in the dermis. These
Figure 11.2 Histological micrographs of skin from wild type and µEµP -IL7 transgenic mice.
Standard paraffin preparations of wild type (left) and µEµP-IL7 transgenic (right) skin were stained
with hematoxylin and eosin. The epidermis of wild type skin is only a few cells thick and sections of
two hair follicles are present in the field. The epidermis of the µEµP-IL7 transgenic skin has
markedly thickened epidermis (acanthosis), parakeratosis and hyperkeratosis. Lymphocytic
infiltrates are seen in the dermis and hair follicles are absent.
cells then convey proliferative signals to epidermal cells which respond and thereby
disrupt the normal architecture of the skin.
Viral transcriptional control elements were used to direct the expression of IL7 to
lymphocytes in transgenic mice that develop a similar disorder involving cutaneous
lymphoid infiltrates (51). The cellular infiltrates found in the skin lesions of these mice are
predominately composed of γδ T cells, however they are polyclonal and are not
predominated by the Vγ5 TCR that is found on all DETC. Recently these cells have been
shown to express IL-4 (68). Consistent with this is the finding that the serum levels of IgE
are elevated in these mice, possibly indicating a Th2 phenotype for the expanded
population of lymphocytes in the skin.
The effects of deregulated paracrine expression of IL-7 on lymphocytes in the skin was
investigated by creating a strain of transgenic mice in which an IL-7 cDNA was placed
under the control of the human keratin 14 promoter (K14-IL7) leading to strong
expression in basal keratinocytes (69). Much like the autocrine IL-7 transgenic mice, these
mice develop inflammatory infiltrates of T lymphocytes that lead to alopecia. However,
unlike the autocrine IL-7 transgenic mice, the cutaneous alterations are evident in young
animals and appear most severe at about 3 weeks of age. Thereafter they become less
intense although a characteristic inflammation of the eyelids remains prominent
throughout life. Many of the lymphocytes that accumulate in the dermis of each of these
IL-7 transgenic mice are T cells that express intermediate levels of the TCR complex in
addition to the signaling natural killer (NK) cell receptor NK1.1. Thus they appear related
to the T cells of similar phenotype termed intermediate T cells that mature without
passing through the thymus (70). Indeed, the characteristic skin lesions arise in nude mice
bearing the µEµP-IL7 transgene demonstrating that the pathogenic cells are not
dependent upon maturation in the thymus (67). However, the autocrine expression of
IL-7 in T cells appears to relieve them of the need to mature in the thymus (71). The fact
that these cells have the NK1.1 receptor raises the possibility that they may be able to
perpetuate inflammatory reactions in the absence of foreign antigens since the NK1.1
receptor is able to convey activation signals when it engages cognate receptor (72).
IFN-γ AND SKIN

IFN-γ is a key component to the model of keratinocyte—T cell dialog discussed above.
To evaluate the effects of constitutive signaling by IFN-γ, transgenic mice were generated
in which IFN-γ is expressed under the control of the involucrin promoter which is active
in suprabasal keratinocytes (73). These mice have variable skin abnormalities ranging from
simple hypopigmentation to gross scaling, flaking and hair loss. The more severely
affected skin is characterized by cellular infiltrates in the dermis, dilated dermal capillaries,
acanthosis, parakeratosis and hyperkeratosis. The keratinocytes are hyperproliferative and
express a proliferation-associated antigen Ki-67 as well as ICAM-1 and MHC class II
molecules. The response of these mice to CHS was exaggerated and prolonged. Since IL-7
expression of keratinocytes is stimulated by IFN-γ, it is likely that a significant component
of the inflammatory disorder seen in these mice is related to the release of IL-7 by
keratinocytes and thus similar to the K14-IL7 transgenic mice discussed above. The
phenotypes of the two strains are largely similar, however, hypopigmentation was not
seen in the few pigmented K14-IL7 mice that were examined.
IL-1 AND SKIN

As discussed briefly above, IL-1 is a central signaling molecule of the immune system in that
it induces several types of cells to synthesize and release other cytokines and chemokines
that recruit effector cells and coordinate inflammation. The high levels of expression of
IL-1 and the IL-1R by keratinocytes in inflamed skin led to the speculation that autocrine
stimulation of keratinocytes helps to enhance and perpetuate inflammatory reactions in
the skin. Strains of transgenic mice expressing IL-1α or the IL-1R in the skin were
generated to test the hypothesis that IL-1 signaling can initiate and maintain inflammation.
Transgenic mice expressing IL-1α in basal keratinocytes under the control of the human
K14 promoter develop a spontaneous skin disorder that is marked by hair loss and
hyperkeratosis (74). Foci of inflammatory lesions arise in these mice that have cellular
infiltrates and hyperproliferative epidermis leading to acanthosis and parakeratosis.
Transgenic mice constitutively expressing the type-1 IL-1R (K14-IL1R1) on their
keratinocytes have exaggerated responses to IL-1. When the K14-IL1R1 and K14-IL1α
transgenic lines were crossed together, the resulting mice developed spontaneous
inflammation that was more severe than either parental strain and appeared similar to the
response to PMA, a potent inducer of protein kinases that stimulates expression of IL-1 in
keratinocytes (75). The type 2 IL-1R (IL-1R2) binds IL-1 with high affinity but does not
appear to transduce any signals. Moreover, the extracellular domain of the molecule can
be shed by cells in a soluble form. Therefore it has been considered an inhibitor of the
action of IL-1. The IL-1R2 gene appears to be regulated in parallel with the IL-1 gene in
keratinocytes and has also been found to be overexpressed in psoriasis. To test the
hypothesis that IL-1R2 modulates the activity if IL-1 in vivo, IL-1R2 was expressed under
the control of the K-14 promoter in transgenic mice. Basal keratinocytes of these mice
express IL-1R2 constitutively and consequently have a blunted responds to induction of
IL-1 by PMA while contact hypersensitivity is not affected (76).
CHEMOKINE EXPRESSION IN SKIN

Chemokines are a large family of structurally related factors that are principally involved
in chemotactic migration of leukocytes (77). The ability of the expression of the
chemokine JE in epidermal cells to recruit neutrophils was demonstrated by the
characterization of transgenic mice expressing this molecule in basal keratinocytes (78).
These K14-JE mice have dramatic increases of neutrophils in their dermis and epidermis
but do not exhibit any abnormal inflammation. The chemokines MCP-1 and IP-10 are
induced in skin during contact hypersensitive reactions (79) and treatment of mice with
anti-MCP-1 antibodies inhibits cutaneous DTH reactions by blocking the recruitment of T
cells (80). Tissue specific transgenic expression of MCP-1 recruits macrophages and
monocytes to those tissues (81, 82) but these cells cause little inflammation in the absence
of antigenic challenge. However, when mice expressing MCP-1 in their basal
keratinocytes were challenged by CHS their responses were greatly enhanced (81). Thus
the expression of MCP-1 appears to induce leukocytes to travel towards the site of
expression without promoting effector function by those cells. However, their presence
in greater numbers within the skin provides for a greater inflammatory response. By
contrast, expression of IP-10 in keratinocytes of transgenic mice does not affect
inflammation spontaneously but it does inhibit wound healing and interfere with repair of
vasculature (83).
KERATINOCYTE EXPRESSION OF INTEGRINS

Integrin molecules expressed on the surface of keratinocytes and other cells interact with
extracellular matrix. The β1 integrin dimerizes with the α2 integrin to bind collagen, with
the α3 integrin to bind laminin and with the α5 integrin to bind fibronectin. The α2β1 and
α3βl integrins are present on normal epidermal keratinocytes while the α5βl complex is
expressed at higher levels during wound healing in normal skin and constitutively on
keratinocytes in psoriatic skin. To investigate the effects of deregulated expression of
these integrin molecules, lines of transgenic mice were created that express α2, α5 or β1
integrins under the control of the involucrin promoter (84). Constitutive expression of
the βl integrin in combination with α2 or α5 or by itself, causes a disorder of the epidermis

that involves hyperproliferation, altered keratinocyte differentiation and inflammatory
lymphocytic infiltrates. Like the flaky skin mouse, the involucrin- βl transgenic mice have
a syndrome that shares features of human psoriasis.
CONCLUSIONS
As the most studied mammalian model of human biology the mouse is close to an ideal
system in which to study cutaneous inflammation. While there are significant differences
between human and murine skin, the cellular and molecular principles governing the
behavior of the skin and its interactions with the immune system are largely conserved.
The development of techniques for genetic manipulation of mice has led to the creation of
a large number of different mice in which to study various aspects of cutaneous
inflammation. While a number of strains exhibit features of human diseases, few have
absolutely identical pathology. Ultimately this may reflect a limitation in the mouse as a
model because of inherent differences between the species, however it is clear that the
underlying similarities make murine models ideal for investigating cutaneous biology and
testing candidate therapeutic regimens.
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12.
LANGERHANS CELL MIGRATION
GEORG STINGL AND DIETER MAURER
DEVELOPMENT OF LANGERHANS CELLS FROM

HEMOPOIETIC PRECURSORS (HPC) (FIGURE)
Both in humans and the mouse, Langerhans cells (LC) can already be identified in the
fetus. Unlike the fetal murine epidermis, which contains phenotypically immature Ia− LC
until the end of gestation (1), HLA-DR+/ATPase+ dendritic cells (DC) can be identified
in the human epidermis by 6 to 7 weeks of estimated gestational age (2). These cells must
originate from HPC in the yolk sac or fetal liver, the primary sites of hemopoiesis during
the embryonic period. Until the twelfth week of pregnancy, these cells are CD1a− and
lack Birbeck granules (BG). Thereafter, there occurs a dramatic increase in LC CD1a
expression, an event which coincides with the initiation of bone marrow function (2).
A major breakthrough in the understanding of LC development came from the
observation that the exposure of CD34+ HPC to granulocyte/macrophage colony-
stimulating factor (GM-CSF) and tumor necrosis factor α (TNF-α) gives rise to a progeny
of CD1a+, E-cadherin+, BG-containing cells with immunostimulatory properties
strikingly resembling those of LC isolated from human skin (3,4). Subsequent studies have
tried to delineate the phenotype of LC progenitors at their various states of maturation/
differentiation. It is now quite clear that, already at the CD34+ HPC stage, cells exist
which are committed to the LC lineage. An apparently useful marker to identify these
cells is the cutaneous lymphocyte-associated antigen (CLA) which is detectable not only
on skin-homing T cells but also on LC in situ. CLA is abundantly expressed by LC
precursors rather than by cells giving rise to non-LC DC (5). It has yet to be determined
whether this molecule, similar to its function on skin-seeking T cells, helps to direct LC/
LC progenitors to the skin. Around day 4–6 of in vitro culture in GM-CSF- and TNF-α
supplemented medium, LC precursors become CD1a+ and, upon prolongation of the
culture until day 12–14, preferably on fibronectin-coated tissue culture plates (6),
develop into typical DC displaying all the features found in and on epidermal LC. Besides
the CD1a+ LC precursor, CD14+ CD1a− cells emerge early during the culture. The
lineage commitment of these cells is apparently less restricted as they can give rise to a
monocyte/macrophage phenotype when exposed to M-CSF while differentiating into non-
LC DC in the presence of GM-CSF and TNF-α(7). Phenotypically, these non-LC DC are
characterized by the abundant expression of factor XIIIa, CD1a, CD68, CD11b, CD36
242 GEORG STINGL AND DIETER MAURER
and the virtual absence of E-cadherin and BG and, thus, resemble dermal dendritic cells
(DDC).
Additional factors governing the development of LC/DC from CD34+ progenitors are
stem cell factor (SCF) and flt3-ligand. These cytokines amplify the DC differentiation
pathways initiated by GM-CSF and TNF-α without any apparent selectivity for LC or non-
LC DC development (8,9). In contrast, TGF-β1 seems to be of unique importance in LC
ontogeny. This is evidenced by the lack of LC in TGF-β1−/− mice (10), i.e., mice whose
TGF-β1-encoding genes have been deleted by homologous recombination, and by the
preferential development of CD1a+, BG+ cells in GM-CSF- and TNF-α-containing, TGF-
β1-supplemented serum-free stem cell cultures (11). It is not yet entirely clear which cell
types serve as the biologically relevant source of TGF-β1 in LC differentiation. Cell
transfer studies in mice suggest that radiation-resistant host cells other than KG are
important in this regard (12). Recent studies show that TGF-β1 may have a LC-
promoting effect at the CD14+ DC precursor stage (12) and, perhaps even, at the level of
peripheral blood monocytes (13). While these cells transform into non-LC DC under the
influence of GM-CSF and IL-4 (7,14,15) they upregulate E-cadherin as well as CD la and
display BG-like structures when additionally stimulated by TGF-β1 (13).
LANGERHANS CELL HOMING TO THE SKIN (FIG.)

The exact maturational stage at which LC precursors enter the skin/epidermis is still
unknown as are the mechanisms operative in this process. Some evidence exists that
various members of the chemokine (CK) system are of importance in this regard. CK
constitute a multipartite superfamily of chemoattractant cytokines which, upon binding to
G protein-coupled receptor proteins, induce the directional as well as the non-directed
migration of leukocytes and other cells (16–18). Currently, chemokines are subdivided
into 4 groups according to the position of the first cysteine pair (CXC, CC), the lack of
two of the four cysteines (C), or the presence of three spacing amino acids in the first
cysteine tandem (CX3C). Up to now, several receptors for CXC (CXCR1 to 5) and CC
chemokines (CCR1 to 9) have been identified (18).
While experimental data on the chemokine responsiveness of skin-derived LC are still
sparse, a considerable amount of information exists concerning the chemokine responses
of other DC, e.g., of those generated from monocytes by GM-CSF plus IL-4 (14, 15).
These monocyte-derived (md)DC display migratory responsiveness to a broad repertoire
of inflammatory CC chemokines, including macrophage chemotactic protein (MCP)-1,
MCP-2, MCP-4, regulated upon activation, normal T cell expressed and secreted
(RANTES), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-5/HCC2, the
CXC chemokines IL-8 and stromal cell-derived factor (SDF)-1 (19–23), and macrophage-
derived chemokine (MDC) (24). In correlation to this response profile, mdDC express
receptors for inflammatory chemokines such as CCR1, CCR2, CCR3, CCR5, CXCR1
and constitutively expressed chemokines (i.e., CXCR4 (19, 21, 23, 25)). Interestingly,
LC/DC generated from CD34+ stem cells but not mdDC express CCR6 and respond to
MIP-3/liver and activation regulated chemokine (LARC) (26,27). In a recent study, we
obtained convincing evidence that MIP-3 is indeed a prime candidate involved in the
LANGERHANS CELL MIGRATION 243
recruitment of LC/LC precursors to the skin epidermis (27a). This argument is supported
by (i) the sensitivity of CD1a+ LC precursors for MIP-3α, (ii) the expression of CCR6,
the specific receptor for MIP-3α, both by LC in vivo and LC generated in vitro, (iii) the
lack of CCR6 expression by non-LC DC which are not present in nonperturbed
epidermis, (iv) the loss of CCR6 expression by cytokine-matured epidermal LC, thought
to be correlates of lymph node-bound LC, and, importantly, by (v) the constitutive
expression of MIP-3α in keratinocytes and skin endothelial cells. One should not forget
that MIP-3α, although absent from spleen and bone marrow, is also expressed in fetal
liver and lung, appendix, thymus, and tonsils (28–31). However, in tonsils (27), the skin,
and, perhaps, in the other organs containing epithelial cells (e.g., liver, lung, gut,
thymus), MIP-3α is constitutively expressed by ectodermal rather than mesenchymal/
bone marrow-derived cells. It also appears that mechanisms other than MIP-3α:
expression determine whether LC precursors appear and populate a given organ. These may
include the organ-specific molecular composition of the vascular barrier and the tissue-
specific cytokine milieu.
LANGERHANS CELL EMIGRATION AND HOMING TO

SECONDARY LYMPHOID ORGANS (FIG.)
Finally, there remains the question about the ultimate fate of the epidermal LC
population. Despite the shedding of ATPase+ dendritic cells into the para-keratotic horny
layer after epidermal injury (32), it is unclear whether elimination through the stratum
corneum occurs under physiologic conditions. Instead, evidence exists that perturbation of
the cutaneous microenvironment leads to major phenotypic changes in the LC population
allowing them to leave their cutaneous residence and to travel to the regional lymphoid
organs. The phenotypic and functional metamorphosis of LC has first been observed in single
epidermal cell suspension cultures. Over the course of only few days, molecules/
structures linked to or responsible for antigen uptake and processing (e.g., FcεRI, FcγRII,
Birbeck granules) as well as for the attachment to their symbionts (e.g., E-cadherin)
progressively decrease and often disappear. In contrast, their dendricity as well as their
expression of surface moieties needed for T cell priming (e.g., MHC class I and class II;
costimulatory molecules CD40, CD54, CD58, CD80, CD86) increase sharply (33,34).
Thus, LC recovered from epidermal cell cultures are essentially indistinguishable from
MHC class II-bearing DC of lymphoid organs known to be potent stimulators of primary
and secondary T cell responses (35,36).
It is clear that the behavior of LC in epidermal cell cultures is mediated by cytokines
provided by non-LC. Whereas LC highly enriched or even purified from epidermal cell
suspensions enjoy only a short survival in normal culture medium, the addition of the
cytokines TNF-α and GM-CSF prolongs their viable state. In contrast, the induction of
phenotypic changes is mediated mainly by GM-CSF and IL-1 but not by TNF-α (37,38).
From a (patho)physiologic view-point, it is important to note that these cytokines can be
produced by KG either constitutively (IL-1α) or upon stimulation (GM-CSF, TNF-α).
In and ex vivo studies in both the murine and human system have convincingly
corroborated the biological relevance of the observations made in single epidermal cell
cultures. In skin transplants (39) as well as in skin expiant cultures (39,40), LC begin to
enlarge, exhibit increased amounts of surface-bound MHC class II molecules,
spontaneously emigrate from the epidermis and, together with DDC, assemble in dermal
afferent lymphatics to begin their journey out of the skin. In similar fashion, application of
contact sensitizers to the skin results in the consecutive appearance of strongly anti-MHC
class II-reactive cells in the dermis (41), of veiled cells in the lymphatics (42) and, finally,
of antigen-bearing LC/DC in the draining nodes (43,44).
In the past few years, we have learned a great deal concerning the factors governing LC
migration from the skin into draining lymph nodes. Antibodies to TNF-α and IL-1β
prevent the early migration of LC from the epidermis, the accumulation of DC in lymph
nodes, and the development of optimal contact sensitization (45–47). In keeping with
these observations is the finding that mice deficient in IL-1β manifest impaired contact
hypersensitivity to haptens (48). Conversely, the intradermal injection of TNF-α or IL-1β
stimulates the migration of LC out of the epidermis and the accumulation of DC in
draining lymph nodes (49). The effect of TNF-α is apparently mediated by the 75 kD
TNF-RII as LC migration in TNF-RI gene-targeted knock out mice is unchanged but still
sensitive to TNF-α neutralization (50). The further sequence of events occurring in LC
emigration include the loosening of the E-cadherin-dependent attachment of LC to
neighboring KC which can be, at least partly, explained by a LC maturation-related
downregulation of this molecule (51). Recently, two additional LC surface receptors have
been implicated as being essential for LC emigration to occur. One is CD44, a hyaluronic
acid receptor putatively involved in the tissue homing of leukocytes and certain cancer
cells. Antibody blocking studies suggest that an N-terminal epitope of CD44 is involved in
LC emigration while the differentiation-related expression of the CD44 splice variant v6
allows for LC binding to T cell rather than to B cell areas of lymph nodes (52). The other
LC-bound receptor structure involved in tissue emigration is a heterodimer built up by
the integrin chains α6/β1 or α6/β4 (53). Importantly, the prototype cell expressing the
latter moiety is the KC. These cells express this receptor in the hemidesmosome where it
mediates KC attachment to laminin, a major constituent of the basement membrane. It is
conceivable that in vivo-stimulated LC loosen their KC-binding sites, and use 6-containing
integrin receptors to specifically recognize basement membrane components. The antigenic
stimulus itself, stimulation-induced KC products, or the receptor-mediated interaction
with extracellular matrix proteins may then induce LC to secrete proteolytic activity,
e.g., type IV collagenase (MMP-9) (54), allowing them to penetrate the basement
membrane and to pave their route through the dense dermal network into the lymphatic
system.
At least upon in vitro maturation, CD34+ HPC-derived LC/DC acquire
responsiveness to MIP-3β/Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC)
due to the de novo expression of CCR7, a specific receptor for the factors MIP-3β and
secondary lymphoid tissue chemokine (SLC; refs. 27, 55, 56). In keeping with an
important role of CCR7 signaling for the proper function of mature DC in vivo is the
observation that pit mice, which lack expression of SLC, have reduced numbers of mature
DC in T cell areas of lymph node but, importantly, display an epidermal LC population
that is normal in terms of cell numbers and distribution (57).
Figure 12.1Ontogenetic, migration and maturation pathways of Langerhans cells and dermal dendritic cells.
E, epidermis; B, basement membrance; HPC, hemopoietic percursor cell; CLA, cutaneous leukocyte antigen; DC, dendritic cell; LC Langerhans cell;
DDC, dermal dendritic cell; BM, bone marrow; PB, peripheral blood; LN, lymph node; IL-1β, interleukin-1β; IL-4, interleukin-4; SCF, stem cell factor;
GM-CSF, granulocyte/macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; TNF-α, tumor necrosis factor-α; flt-3-L,
flt-3 ligand; TGF-β, transforming growth factor-β; CKs, cemokines; MIP-3α, macrophage inflammatory protein 3α; MIP-3β, macrophage inflammatory
protein 3β; SLC, secondary lymphoid tissue chemokine; α6/β1, 4, α6/β1 and α6/β4 intergrins
Where and how does the life cycle of a LC end? This question has not yet been
completely resolved but some evidence exists that DC upon cognate interaction with T cells
physically disappear from the lymph nodes (58). It remains to be seen whether this event
is due to their emigration from the nodes or, alternatively, due to their elimination by
cytolysis or apoptosis induced by responder T cells.
ACKNOWLEDGEMENT
This work was supported, in part, by grants from Novartis Pharma, Basel, Switzerland,
and the Austrian Science Foundation, Vienna, Austria.
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13.
LEUKOCYTE ADHESION AND ACCESSORY
MOLECULES AS THERAPEUTIC TARGETS
FOR INFLAMMATORY SKIN DISEASES
KIMBERLY E.FOREMAN AND BRIAN J.NICKOLOFF
INTRODUCTION
Given the visibility and accessibility of human skin to investigators interested in
inflammatory and immunologically-mediated disorders, it should not be too surprising
that dermatological disease-related studies are continually being reported in the world-
wide literature. As such, it is difficult to keep pace with developments regarding the
pathophysiology and treatment of various skin disorders. Even though many advances have
been made regarding; identification of a new virus that may cause skin lesions (i.e.
HHV-8) (Chang et al., 1994), characterization of proteins that inhibit bacterial invasion of
the skin (Frohm et al., 1997), and gene mutations responsible for causing cutaneous
neoplasms such as basal cell carcinoma (Xie et al., 1998; Dahmane et al., 1997; Oro et al.,
1997; Fan et al, 1997), this chapter will be devoted exclusively to the molecular and
cellular basis for leukocyte (i.e. neutrophil) and immunocyte (i.e. T-cell) adhesion in the
skin. Indeed, the past decade has been characterized by rapid progress in elucidating key
adhesion molecules, cytokines, and chemotactic polypeptides which govern the non-
random trafficking of circulating cells into the dermis and epidermis.
Figure 9.1 Optimal stimulation of T-cells by antigen presenting cells requires two signals.
252 KIMBERLY E.FOREMAN AND BRIAN J.NICKOLOFF
By taking a rather broad overview of the entire field of investigation, it is possible to

sub-divide the topic of cell adhesion and migration involved in skin diseases into two
aspects. These components can perhaps best be appreciated in a temporal fashion, and
include the initial involvement and highlighting the role of vascular endothelial cells,
followed secondarily by the participation of epidermal keratinocytes. Thus, for the sake of
brevity this chapter will primarily focus on the indigenous cells of the dermis-endothelium
and epidermis-keratinocyte, and will not cover the other constituents that compose the
confederacy of cell types that normally reside in human skin such as: pericytes, mast cells,
dermal dendritic cells, fibroblasts, adnexal structures, melanocytes or Langerhans cells.
For those interested in the role of these other cell types in dermatological disorders, the
dermal immune system and skin immune system have been extensively reviewed
elsewhere. (Luger et al., 1996; Williams and Kupper, 1996; Santamaria et al., 1995;
Nestle and Nickoloff, 1995) Returning to the two principle cell types of interest, the basis
for cell adhesion can be broken down into specific steps. The interaction involving
emigration of leukocytes out of the blood vessels includes four different steps (i.e. rolling,
activation, firm adhesion, and extravasation), whereas the movement of immunocytes into
the epidermis includes three different steps (i.e. recruitment, retention, and return to
circulation), as described further below. (Nickoloff, 1988; Butcher, 1991; Shimizu et al.,
1992; Springer, 1994)
Before reviewing more detailed aspects of the adhesive cascade, it is worth emphasizing
that the adhesion molecules themselves are more than elegant forms of velcro—simply
holding cells in contact with each other. Cell surface molecules which mediate cell-cell
adhesion are clearly very important as signaling molecules transducing important
activation messages from the cell surface into the cytoplasm and nucleus of the cell.
(Resales et al., 1995) It is also evident that one cannot solely rely on the mere presence or
absence of an adhesion molecule, but the number of adhesion molecules, their affinity
state (i.e. low affinity/high affinity), and presence of soluble forms, all contribute in both
positive and negative ways to cell adhesion and migration and activation in the skin. Also
in this introduction, we would like to emphasize the point that it is highly likely that many
of the principles established for the role of adhesion molecules in the skin will be relevant
to other organ systems such as: synovial tissue, gastrointestinal tract, pulmonary system,
etc.
As mentioned above, adhesion molecules promote critically important activation
signals to the cells. With respect to a resting T-cell, at least two distinct signals are
required for full activation including proliferation and cytokine production. Signal #1 is
the antigen-specific signal delivered via the CD3-T-cell receptor complex in which an
antigen presenting cell (APC) displays appropriately processed antigen or superantigen in
the context of class I or class II major histocompatibility complex (MHC) molecules.
However, this signal #1 by itself is generally insufficient to fully trigger T-cell activation
and a signal #2 is required which is not directly related to the antigen (see Figure 13.1).
This socalled second signal can involve a variety of molecules. Perhaps the best
characterized accessory signaling molecules include expression of CD28 and CTLA-4 by
the T-cell interacting with B7–1 (CD80), B7–2 (CD86) or B7–3 (BB1) on the APC.
There is evidence that adhesion molecules such as LFA-1/ICAM-1 can subserve this
LEUKOCYTE ADHESION AND ACCESSORY MOLECULES 253
accessory signaling function, as well as certain cytokines—although this latter point is

somewhat controversial. Nonetheless, with respect to dermatology, the importance of
focusing on signal #2 is very relevant because in many (if not most) inflammatory/
immunological skin diseases, the nature of the inciting agent/antigen is unknown. From a
therapeutic perspective, it is thus possible to envision treating these disorders by targeting
the second signal aspect of the T-cell activation pathway. Another caveat for this approach
is that a drug that can block signal #2 effectively, may be efficacious in a wide variety of
skin diseases.
In this chapter, adhesion molecules that govern cell: cell interaction and movement of
cells into/out of the skin will be examined for their role in pathophysiology and as
molecular targets for therapeutic intervention. In addition, the role of accessory
molecules that regulate the activation state of the immunocytes once they have entered
the skin will be covered in a secondary but complementary fashion. Since adhesion
molecules may also mediate cell activation, it is probably that certain drugs may have a
dual role of not only blocking adhesion, but also to interrupt the signal #2 component for
T-cell activation. It is hoped that after reading this chapter a clear understanding of the
importance of adhesion molecules in various skin diseases will be achieved, as well as
being integrated with the concept that accessory signaling molecules are excellent targets
for therapeutic intervention in dermatology.
BASIC REVIEW OF THE ADHESION CASCADE IN THE SKIN
Endothelial Cells
Trafficking of leukocytes into an area of inflammation is a multi-step sequence of cellular
and molecular events that are necessary for an effective inflammatory and/or
immunological response to occur. At least four distinct steps have been defined for
endothelial cell:leukocyte interactions including (1) attachment and rolling, (2) adhesion
triggering, (3) firm adhesion, and (4) transendothelial migration. At sites of cutaneous
inflammation, the release of pro-inflammatory mediators, including cytokines, induce
changes in the adhesive properties of endothelial cells lining the surrounding vasculature.
In the first step of the adhesion cascade, leukocytes attach and begin to roll along the
surface of the endothelial cells. This process is mediated by the selectin family of adhesion
molecules (see Table 13.1) consisting of L-selectin (constitutively expressed on
leukocytes), P-selectin and E-selectin (expressed on activated endothelial cells).
Table 13.1
The selectins interact with the carbohydrate ligands CLA (cutaneous lymphoid antigen)
and sialyated Lewis X (sLeX) expressed on leukocytes and activated endothelial cells.
(Fuhlbrigge et al., 1997) If the stimulus persists, the rolling leukocytes become firmly
bound to the activated endothelial cell surface through interactions occurring between
members of the immunoglobulin gene superfamily and integrin adhesion molecules
(Table 13.1) expressed on leukocytes. Interestingly, there is direct evidence that integrins
such as LFA-1 (CD11a/ GDIS) exist in two different affinity states. (Lollo et al., 1993;
Carlos and Harlan, 1990). An increase in cell surface expression of these molecules is
neither necessary nor sufficient for increased adhesiveness; instead, they must undergo a
conformational change to become fully activated. Such activation has been referred to as
“inside-out” signaling. Data indicate that integrins also act as classic receptors, and
engagement of these molecules results in intracellular signaling or “outside-in signaling”.
(Petty and Todd, 1996; Resales et al., 1995). Finally, the leukocytes migrate into the
epidermal compartment by moving between the endothelial cells towards the area of
inflammation where they can interact with keratinocytes.
Keratinocytes
In addition to mediating the binding of leukocytes to endothelial cells, adhesive
interactions play an important role in the interaction of keratinocytes with T-cells.
Previously, epidermal keratinocytes were thought of as merely passive targets in
immunologie reactions; however, it has since become clear that keratinocytes can be
stimulated by a variety of mediators resulting in upregulation of adhesion molecules, such
as ICAM-1, and binding of T-cells. Such interactions facilitate functional participation and
an active role for keratinocytes in inflammation, and are important to understand how
keratinocytes play a key role in the pathogenesis of many cutaneous diseases. Both in vitro
and in vivo studies have indicated that a variety of substances can induce keratinocytes to
express ICAM-1 including cytokines (such as TNF-a and IFN-γ), phorbol esters, poison
ivy antigen (urushiol), nickel ion, ultraviolet light, irritants, and bacterial derived
superantigens. Not only can stimuli induce keratinocyte ICAM-1 expression, but certain
stimuli can also suppress keratinocyte ICAM-1 expression in vitro. (Norris et al., 1990)
ROLE OF ADHESION MOLECULES IN PSORIASIS

Psoriasis is a common, chronic inflammatory skin disease which is characterized by
epidermal hyperproliferation, parakeratosis, and the presence of an inflammatory
infiltrate consisting of neutrophils and activated T-cells in the dermis. While the precise
mechanisms by which these cells are recruited selectively into the dermis is unknown;
studies have demonstrated altered expression of adhesion molecules on keratinocytes and
endothelial cells in this disease. ICAM-1 is constitutively expressed on endothelial cells in
normal human skin and uninvolved skin in psoriatic patients; however, both keratinocytes
and endothelial cells upregulate ICAM-1 expression in involved psoriatic lesions. (Barker
et al., 1992; Veale et al., 1995) In contrast, E-selectin expression on endothelial cells is
minimal in normal skin, and is widely expressed in psoriatic lesions. Similarly, VCAM-1 is
expressed on the endothelium and some dendritic cells in psoriatic lesions, but little to no
VCAM-1 is not found in normal or uninvolved psoriatic skin. (Veale et al., 1995) It is
thought that the increased expression of E-selectin on endothelial cells in psoriasis may be
responsible for preferential recruitment of T-cells into the area.
In addition to expression of membrane bound adhesion molecules, soluble forms of
ICAM-1, ICAM-3, and E-selectin have been demonstrated in serum from patients with
psoriasis at significantly increased levels. (Griffiths et al., 1996; Bonifati et al., 1995;
Schopf et al., 1993) These soluble forms of adhesion molecules may serve as functional
decoys that can interfere with the binding of ligand mediated interactions with membrane
bound adhesion molecules. Of interest, these elevated levels correlate with clinical
severity of the disease as measured by the psoriasis area and severity index (PASI), and it
has been postulated that the increased serum levels of these soluble adhesion molecules
may represent a protective mechanism.
ADHESION MOLECULES AND TREATMENT OF PSORIASIS
Ultraviolet Light
Ultraviolet B (UVB) phototherapy, in combination with coal-tar preparations, is a highly
effective treatment for moderate to severe psoriasis resulting in a reduction in T-cell
infiltrate and disease severity within a few weeks. (Lauharanta, 1997; Lowe et al., 1997)
Recent studies indicate that the reduced inflammatory infiltrate observed following
treatment may be the result of decreased adhesion molecule expression on endothelial
cells in psoriatic plaques. (Cai et al., 1996) Using immunohistochemical staining, Cai et al.
(1996) demonstrated significant reductions in ICAM-1 and E-selectin expression on
endothelial cells in treated psoriatic plaques compared with untreated controls. In
contrast, VCAM-1 expression was induced following UVB treatment. (Cai et al., 1996)
The decrease in ICAM-1 and E-selectin expression following UVB treatment inhibited the
binding of psoriatic peripheral blood mononuclear cells (PBMC), but not normal PBMC,
to psoriatic lesions in cell adhesion assays, indicating that these molecules play an
important role in the influx of inflammatory cells into the lesion. Additional functional
studies in vitro demonstrated that antibodies to E-selectin, but not ICAM-1 or VCAM-1,
significantly inhibited the ability of PBMC to bind to psoriatic tissue providing direct
evidence for the involvement of E-selectin in the adhesion of circulating lymphocytes to
psoriatic endothelial cells. In addition to alterations in endothelial cell adhesion molecule
expression, UVB therapy inhibits ICAM-1 expression on keratinocytes exposed to IFN-γ
(Krutmann et al., 1990; Krutmann and Trefzer, 1992; Norris et al., 1990) UVB,
however, is not associated with decreased levels of soluble ICAM-1 or soluble E-selectin
in psoriasis. (Petersen et al., 1997; Kowalzick et al., 1993; Kowalzick et al., 1994) UVB
therapy also induces keratinocytes to express Fas ligand (CD95L) which can
mediate induction of apoptosis on intraepidermal T-cells bearing the Fas antigen (CD95).
(Gutierrez-Steil et al. 1998)
Photochemotherapy, known as PUVA therapy, combines the use of ultraviolet A (UVA)

radiation with a skin sensitizer (psoralen) and is a well-established and effective treatment
for severe psoriasis. (Lauharanta, 1997; Lowe et al., 1997) While the exact mechanism of
action of PUVA is unknown, it is thought that alterations in adhesion molecule
expression, in addition to anti-proliferative effects, may be important. Studies have shown
that PUVA treatment significantly reduced ICAM-1 expression on keratinocytes in
psoriasis patients resulting in a concurrent reduction in epidermal and dermal T-cell
infiltration and disease severity. (Lisby et al., 1989) Similarly, PUVA therapy has been
shown to reduce ICAM-1, E-selectin and VCAM-1 expression on human umbilical vein
endothelial cells in vitro and suppressed LFA-1 and VLA-4 expression on PBMC in
response to stimulation by concanavalin A and phytohemagglutinin. (Laing et al., 1995;
dL, 1995)
Cyclosporin
Cyclosporin A is a fungal metabolite with potent immunosuppressive activity. This
compound is an effective treatment for psoriasis with an overall reduction in epidermal
hyperplasia, inflammatory infiltrate and severity of the disease as measured by PASI;
however, the mechanism of action has been controversial. (Baker et al., 1989; Ho et al.,
1990b) It has been hypothesized that the drug acts through inhibition of cytokine release
from activated T-cells, an anti-proliferative effect on keratinocytes or inhibition of
adhesion molecule expression on keratinocytes and/or endothelial cells. (Buurman et al.,
1986; Elder et al., 1993; Urabe et al., 1989; Ho et al., 1990a; Petzelbauer et al., 1991;
Petzelbauer and Wolff, 1992; Edwards et al., 1993; Horrocks et al., 1991)
Recently, it has been shown that following Cyclosporin therapy, endothelial cells in
psoriasis showed a slight reduction of ICAM-1 expression while keratinocyte expression
was significantly reduced compared to pre-treatment samples. (Servitje et al., 1996) This
data confirms earlier studies which also showed a reduction of ICAM-1 on keratinocytes.
(Ho et al., 1990a; Petzelbauer and Wolff, 1992; Horrocks et al., 1991) It has been
hypothesized that Cyclosporin acts by interfering with the production of IFN-γ which in
turn reduces adhesion molecule expression and the accompanying inflammatory infiltrate.
The efficacy of Cyclosporin may indeed reflect multiple cell targets including activated T-
cells, keratinocytes and endothelial cells which are present on psoriatic plaques.
Cetirizine
Cetirizine dihydrochloride is a third generation HI antihistamine which has been shown to
inhibit expression of ICAM-1 on epithelial cells and eosinophils. (Caproni et al., 1995) A
recent clinical trial by Caproni et al., (1995) evaluated adhesion molecule expression in
psoriasis patients following a 15 day course of oral Cetirizine. Following treatment, a
significant decrease in the number of infiltrating CD3, CD4 and CD8-positive T-cells was
noted in the epidermis and dermis, and there was improvement in epidermal thickening
as well as scaling and erythema in 80% of the patients. The results correlated with a
significant reduction in ICAM-1 and HLA-DR expression on the keratinocytes and dermal
endothelial cells. A dose-dependent reduction of VCAM-1 expression on human

endothelial cells in vitro following Cetrizine treatment has also been reported. (Jean-Louis
et al., 1998)
Anti-LFA-1 Antibodies
Given the importance of adhesive interactions between endothelial cells, keratinocytes,
and T-cells that involve LFA-1/ICAM-1, it should not be surprising that anti-LFA-1
monoclonal antibody has been given to psoriasis patients. Previous investigators have
observed that the use of monoclonal antibodies against the T-cell surface antigen CD3 or
CD4 were associated with improvement of psoriasis skin lesions. (Morel et al., 1992)
With the increasing advances made in the production of fully or partially humanized
antibodies, more recent clinical trials have replaced mouse monoclonal antibodies
directed against various human cell surface adhesion molecules with humanized versions
of such blocking antibodies. (Neuberger and Bruggemann, 1997) Preliminary results in 31
patients with moderate to severe psoriasis who received a single intravenous dose of
humanized monoclonal antibody (hu 1124) against LFA-1 (CD11a) produced a mean
decrease in PASI scores of 33%. (Gottlieb et al., 1998) Biopsies of treated patients
revealed decreased numbers of LFA-1 positive T-cells in the epidermis with diminished
keratinocytes and endothelial cell ICAM-1 expression.
Vitamin D
The most active metabolite of vitamin D used to treat psoriasis patients is 1,25-
dihydroxyvitamin D3. This form of vitamin D is very effective in the treatment of
psoriasis, although its precise mechanism of action has yet to be fully characterized. (Smith
et al., 1988; Kragballe et al., 1988) Using an in vitro tissue culture system, 1,25-
dihydroxyvitamin D3 was capable of decreasing IFN-γ induced HLA-DR, but ICAM-1
expression was not significantly reduced on keratinocytes. (Tamaki et al., 1990; Gerritsen
et al., 1993) It is possible that this vitamin D derivative improves psoriasis by direct
interaction on keratinocytes and T-cells that does not involve modulation of adhesion
molecules.
Nonsteroidal Anti-inflammatory Drugs (NSAIDs)

NSAIDs have been used successfully for years in the treatment of many acute and chronic
inflammatory diseases. While it is known that these compounds block cyclooxygenase and
inhibit prostaglandin synthesis, this may not be the sole mechanism of action of NSAIDs.
(Vane, 1971) Recent studies have indicated that several NSAIDs including indomethacin,
diclofenac, aspirin, aceclofenac, mefenamic acid and flufenamic acid strongly inhibit
neutrophil adhesion to endothelial cells in vitro. (Diaz-Gonzalez and Sanchez-Madrid,
1998; DiazGonzalez et al., 1995; Gonzalez-Alvaro et al., 1996) This effect appears to
result from a decrease in the expression of neutrophil L-selectin, an adhesion molecule
involved in the initial (rolling) interaction between these two cell types. (DiazGonzalez
and Sanchez-Madrid, 1998) Studies indicate that the mechanism of action of these NSAIDs
is through metalloproteinases, which cleave L-selectin resulting in shedding of this
molecule from the neutrophil surface. However, not all NSAIDs function the same way.
Piroxicam and meloxicam do not reduce L-selectin expression and do not inhibit initial
adhesion of neutrophils to endothelial cells. However, these NSAIDs are able to prevent
the activation of integrins and thereby decrease cell adhesiveness. (Garcia-Vicuna et al.,
1997; Abramson et al., 1994)
CO-STIMULATORY MOLECULES
As mentioned earlier, besides an antigen-specific signal (i.e. signal #1), the full activation
of T-cells requires a second non-antigen specific signal generally mediated by co-
stimulatory molecules. Three different co-stimulatory molecules will be mentioned which
include (1) CD28/B7; (2) LFA-2/LFA-3; and (3) CD44 variant V7. For skin diseases such
as psoriasis, the nature of the antigen responsible for inducing T-cell activation/
proliferation is unknown. However, it has been established that the disease is mediated by
T-cells since either treatment with a highly specific reagent for activated T-cells
(DAB369IL-2 fusion protein that attach to IL-2 receptor bearing T-cells) improved
psoriasis, or introduction of pathogenic T-cells into symptomless skin engrafted onto
SCID mice produced psoriasis. (Gottlieb et al., 1995; Wrone-Smith and Nickoloff, 1996)
The role for co-stimulatory molecules in psoriasis was initially supported by the ability of
a fusion protein (i.e. CTLA4Ig) designed to interrupt CD28-B7 interactions to block the
ability of psoriatic plaque derived dendritic cells to activate autologous resting T-cell
proliferation and cytokine release. (Nestle et al., 1994b; Nestle et al., 1994a) Clinical
trials using CTLA4Ig in psoriasis have yielded promising early results.
The T-cell surface protein LFA-2 (CD2) and its ligand LFA-3 (CD58) are also involved
in co-stimulation of T-cells resulting in activation. (Selvaraj et al., 1987) Recent clinical
studies by Magilavy et al. (1998) used a human fusion protein designed to bind to LFA-2
and thereby inhibit T-cell activation via the costimulatory pathway mediated by CD2/
LFA-3 interactions. The results demonstrated a decrease in peripheral lymphocytes in
normal volunteers as well as patients with chronic psoriasis with few side effects indicating
potential as a treatment for psoriasis.
The CD44 gene codes for a family of alternatively spliced, multifunctional adhesion
molecules that participate in lymphocyte activation and cell migration. Increased
expression of CD44 standard has been shown on the inflammatory and endothelial cells in
psoriasis lesions making this molecule a potential target for therapy. (Reichrath et al.,
1997) Similarly, recent studies using a murine model for inflammatory bowel disease
(remember some patients with psoriasis also suffer from Crohn’s Disease) found that
antibody against the CD44 variant V7 was highly efficient at preventing or reversing
chronic colitis. (Wittig et al., 1998)
SUMMARY
Adhesion molecules provide attractive targets for immimo-interaction strategies for
several skin diseases including psoriasis. Indeed, many investigators and pharmaceutical
companies interested in adhesion molecules are beginning to use inflammatory skin
disease for their phase I and phase II clinical trials given the accessibility of skin and early
promising results with several anti-adhesion molecule therapies. Thus, even though skin
diseases may not be their ultimate disease target, a demonstration of efficacy in a disorder
such as psoriasis can provide compelling proof of concept evidence for novel treatments.
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INDEX
acanthosis 58, 222, 232 betal integrin 177, 179–180

accessory plaque protein 74–76 beta2 integrin 177, 178–179
adherens junction 1 beta4 integrin 108, 110, 115, 146
adhesion cascade in the skin 253–254 beta4 knock out mice 116
adhesion molecule 168 beta7 integrin 177, 251
allelic diseases 150–152 Birbeck granules 241
allergic contact dermatitis 167, 178, 212 blistering skin diseases, animal models 153–154
alopecia 231 BP230 135, 143, 146
alpha3beta1 integrin 116, 135, 137 BPAG1 10, 91, 101, 102, 108, 110, 113, 115,
alpha4 integrin 179 136
alpha4betal integrin 179 BPAG1 knockout mice 115
alpha6 integrin 108, 110, 146, 146 BPAG2 10, 108, 110, 113–114, 115
alpha6beta4 integrin 10, 93, 94–95, 101, 102, bullous congenital ichthyosiform erythroderma
109, 110, 114, 134, 135, 137, 245 (BCIE) 2, 36, 37, 39–40, 47
mutations 124–125 bullous pemphigoid 5, 113, 142, 143, 223
alphaEbeta7 integrin 177 bullous SLE 143
anchoring complex 133
BMZ associations 99–100 cadherin 57
anchoring fibril 107, 108 CD28 203, 204–207, 251
proteins 140–141 CD28-B7 pathway 205, 258
anchoring filament 107, 108 CD29 179
and lamina lucida proteins 138–139 CD31 177
anchoring plaques 90 CD34 253
antigen presenting cell (APC) 203, 222, 251, CD40 204
252 CD40L 204, 208–210
anti-LFA-1 antibodies 257 CD43 204
Arthus reaction 178 CD44 181, 204, 258
atopic dermatitis 167, 177, 224 CD47 204
autoimmune bullous disease 89 CD50 178
CD54 178
bacterial lipopolysaccharide 168 CD80 203, 204
basal cell carcinoma 251 CD86 203, 204
basement membrane 89–106, 107, 245 CD102 178
ultrastructure 89–90 cell adhesion recognition (CAR) 61–62
basophils 221 cell-cell attachment 7–85
INDEX 265
cell-matrix attachment 87–163 dermal connective tissue 1

cetirizine 256–257 dermal dendritic cell (DDC) 212, 241, 245
chemoattractant cytokine 168 dermal elastin/collagen 102
chemokine 177, 188, 242 dermal T cell 212
expression in skin 234 dermal-epidermal adhesion 133–163
chronic discoid lupus erythematosus (CDLE) disorders 142–154
213 aquired 142–143
chronic proliferative dermatitis mouse 227 dermal-epidermal basement membrane zone
cicatricial pemphigoid 96, 142, 143 (BMZ) 89–106
CLA 176, 188 laminins 97–99
collagen 179 ultrastructure 89–90
fibril 108 dermal-epidermal junction (DEJ) 133–142
collagen IV 99, 100–101, 102, 103, 134, 135, anchoring fibril 140–141
139 anchoring filament 138–139
collagen VII 134, 135, 140, 143, 144, 146 biosynthesis, processing and regulation 141–
collagen XIII 137 142
collagen XVII 93, 95–97, 101, 102, 103, 134, intracellular proteins 136
135, 136, 143, 144, 146 lamina densa protein 139–140
collagen XVII ectodomain 139 molecular components 135–141
contact hypersensitivity (CHS) 212, 223–224 morphology and suprastructure 133–135
corneocytes 9, 222 transmembrane proteins 136–138
corneodesmosin (CDE) 11, 16–17 dermis 107
cornified cell envelope (CE) 5, 9–25 desmin 27
co-stimulatory molecules 258 desmocalmin 76
CR3 253 desmocollin (Dscs) 4, 10, 57, 58
CTLA-4 207–208 desmocollin 1 59
fusion protein 205 desmocollin 2 59
cutaneous lymphocyte-associated antigen (CLA) desmocollin 3 10, 59
183–189, 241, 254 desmoglea 57
cellular distribution 184–185 desmoglein (Dsgs) 4, 10, 57, 58
in the circulation 185–186 desmoglein 1 59
molecular aspects 185 desmoglein 2 10, 59
regulation 186–187 desmoglein 3 10, 59
and T cell-endothelium interaction 187– desmoplakin (Dp) 4, 31, 57, 58, 72–73, 91
189 desmoplakin I 59
cutaneous T cell lymphoma (CTCL) 230 desmoplakin II 59
cyclosporin 256 desmosomal glycoprotein 58–67
cystatin A (CYA) 11, 114 and cell adhesion 58–63
cytokeratin expression pattern 33 and cytoplasmic interactions 63–64
cytotoxic T lymphocyte antigen-4 (CTLA-4) in epidermis 64–67
203, 204 desmosomal plaque 67–76
desmosome 1, 4, 5, 29, 31, 57–85
Darier’s disease 10 dystroglycan 138
dendritic cell 241, 245
dendritic epidermal gammadelta-TCR positive EHK 10
T cell (DETC) 203 elafin (ELA) 10, 13–14
dermal adhesion 5 endothelial cell (EC) 212, 253–254
266 INDEX
envoplakin (EPL) 11, 15–16, 57, 73–74 E-selectin (ELAM-1) 168, 169, 171, 174, 175–
eosinophils 221 176, 188, 253
epidermal differentiation complex (EDC) 10 extracellular matrix 230
epidermal keratinocyte 9, 222, 252
epidermis 1, 9, 107, 245 fibronectin 170, 179
epidermolysis bullosa (EB) 35, 89, 109, 134, fibrosarcoma 60
143–152 filaggrin 14–15
acquisita 143 flaky skin mouse 225–226
dystrophic (DEB) 10, 35, 101, 144, 145, focal contact junction 1
148–149, 148–150 focal non-epidermolytic palmoplantar
generalisata 145 keratoderma (FNEPPK) 42
inversa 145
localisata 145, 150 gamma delta T cell 173
mutilans 145, 148, 150 gamma-interferon-induced peptide (IP-10) 177
junctional 10, 35, 121, 144, 145, 147–148, gap junction I
147–148 GDA-J/F3 antigen 135, 140
cicatricans 145 gene therapy 19–21, 45–47
generalisata mitis 145 generalized atrophie benign epidermolysis
Herlitz 145, 147 bullosa (GABEB) 95, 117, 121–124, 144
inversa 145 glial fibrillary acidic protein (GFAP) 27
localisata 145, 147, 148 gly-CAM-1 253
progressiva 145 GM-CSF 245
pyloric atresia 145, 148 graft-versus-host disease 212
recessive forms 38–39 granulocyte/mactophage colony stimulating
epidermolysis bullosa simplex (EBS) 2, 10, 35– factor (GM-CSF) 241
38, 45, 116, 144, 145–146
Dowling-Meara (EBS-DM) 2, 36, 47, 145, Hailey-Hailey 10
146 haptens 223
Kallin 145 heat-stable antigen 204
Kobner (EBS-K) 36, 39, 145, 146 hemidesmosome 1, 5, 90, 101
Mendes da Costa 145 biology and pathology 107–131
mottled pigmentation (EBS-MP) 38, 45, genetic diseases 116–125
146 inner plaque 108
muscular dystrophy (EBS-MD) 116–121, outer plaque 108
145, 146 protein-protein interaction 115–116
Ogna 145 ultrastructural and molecular features 109–
Weber-Cockayne (EBS-WC) 36, 38, 145, 111
146 hemopoietic precursor cell 245
epidermolysis bullosa-pyloric atresia (EB-PA) hepatocyte growth factor (HGF) 177
118, 124–125 heritable skin diseases, clinical implications 125–
epidermolytic hyperkeratosis (EH) 126
see bullous congenital Herlitz junctional epidermolysis bullosa 5
ichthyosiform erythroderma herpes gestationis 113
epidermolytic palmoplantar keratoderma HHV-8 251
(EPPK) 2, 41 histamine 176
Epstein-Barr virus induced molecule 1 ligand human umbilical vein endothelial cell (HUVEC)
chemokine (ELC) 244 170, 175, 180
erythrokeratoderma (EK) 2, 10, 18–19
INDEX 267
hyperkeratosis 222, 232 bundles 29

gene mutation 34
ICAM-1 169, 170, 177, 178, 188, 251, 253 genetics research 45–47
ICAM-2 169, 170, 171, 178, 179, 253 intermediate filament 5
ICAM-3 170, 171, 178, 179, 253 medical aspects 34–45
ichthyosis bullosa of Siemens (IBS) 2, 36, 41 molecular structure 27–34
IFAP300 108, 109 protein domain structure 30
IL-1 178 trichocyte 27
IL-10 175 type I intermediate filament 31–34
IL-13 175 type II intermediate filament 31–34
IL-lbeta 245 keratinocyte 1, 89, 254
IL-2 receptor (IL-2R) 225 differentiation 9
IL-4 171, 175, 180, 245 expression of integrins 235
IL-7 and skin 229–233
IL-8 171 LAD-1 96
immunoglobulin 170–171 lamellar ichthyosis 10, 17–18
immunoglobulin supergene family 168 lamina densa 90, 107, 108, 134
inflammatory and malignant skin disease 212– proteins 139–140
215 lamina lucida 90, 107, 108, 134
inflammatory bowel disease 143, 181 laminin 179
integrin 168–170, 177–180 laminin 1 99
interferon gamma (IFN-gamma) 171, 225 laminin 2 135, 139
and skin 233 laminin 5 93, 97–99, 102, 103, 108, 109, 116,
interleukin 1 alpha (IL-1 alpha) 224 10, 135, 138, 143, 144, 146
interleukin 1beta (IL-lbeta) 224 laminin 6 93, 97–99, 102, 103, 135, 143
interleukin-1 (IL-1) 168, 171, 175 laminin 10 99, 102, 103, 135, 138
and skin 233–234 Langerhans cell (LC) 167, 173, 188, 203, 212,
interleukin-8 (IL-8) 177 245
intermediate filament 27, 89, development from hemopoietic precursors
attachment 74–76 (HPC) 241–242
hemidesmosome interaction 90–91 emigration to secondary lymphoid organs
molecular structure 27–34 243–246
intracellular protein 136 homing to the skin 242–243
involucrin 10, 12–13 migration 241–249
leukocyte adhesion and accessory molecules
keratin 1 10 251–262
keratin 10 10 leukocyte function associated molecule-1
keratin 14 2, 110, 90, 102, 134, 135, 146 (LFA-1) 169, 170, 178, 188, 251, 253
keratin 2e 10 leukocyte function associated molecule-2
keratin 5 2, 10, 90–91, 102, 134, 135, 146 (LFA-2) 258
keratin 9 10 leukocyte function associated molecule-3
keratin (LFA-3) 258
acidic 27 leukocyte trafficking in skin diseases 165–262
basic 27 lichen planus 167, 212
disorders 27–55 lipopolysaccharide (LPS) 175
dominant-negative mutation clusters 46 liver and activation regulated chemokine
filament 64 (LARC) 242
268 INDEX
liver defects 44 NC/Nga mouse 226

loricrin 2, 10, 12 nestin 27
mutation 18 neurofilament 27
L-selectin (LAM-1) 168, 169, 174–175, 253 neutrophils 221
lupus erythematosus 167 nidogen 99, 102, 103, 135, 140
lymphocyte non-epidermolytic palmoplantar keratoderma
endothelium interaction 181–183 (NEPPK) 2, 40–41, 45
in inflamed skin 174–189 non-Herlitz junctional epidermolysis bullosa 5
migration 174–181 nonsteroidal anti-inflammatory drugs (NSAIDs)
in normal skin 173–174 257–258
lymphotoxin 175 novel candidate genes 153
MAC-1 170, 178 p200, 108, 109

macrophage 212 pachyonychia congenita (PC) 2, 41–42
macrophage chemotactic protein-1 (MCP-1) Jackson-Lawler (PC-2) 42
177, 242 Jadassohn-Lewandowsky (PC-1) 42, 43
macrophage chemotactic protein-2 (MCP-2) papillary dermis 90, 108
177, 242 parakeratosis 223, 232
macrophage chemotactic protein-3 (MCP-3) PECAM-1 180–181, 253
177 pemphigoid gestationis 142, 143
macrophage chemotactic protein-4 (MCP-4) pemphigus 4, 58, 66
177, 242 foliaceous 4, 67
macrophage inflammatory protein-1alpha vulgaris 4, 67
(MIP-1 alpha) 177, 242 antigen 10, 60
macrophage inflammatory protein-1beta (MIP- periphenin 27
lbeta) 177, 242 periplakin (PPL) 11, 16, 57, 73–74
macrophage inflammatory protein-3alpha perlecan 99, 102, 103, 135, 140
(MIP-3alpha) 242–243 photochemotherapy (PUVA therapy) 256
macrophage inflammatory protein-3beta plakoglobin (Pg) 4, 57, 58, 59, 68–72, 91
(MIP-3beta) 177 plakophilin (Pp) 4, 47, 57, 58, 68–72
macrophage inflammatory protein-5 (MIP-5) plakophilin 1 59, 69, 91
242 plakophilin 2 59, 69
macrophage-derived chemokine (MDC) 242 plasma membrane 108
MAdCAM-1 169, 180, 253 plectin 10, 47, 73–74, 91–94, 101, 108, 109,
major histocompatibility complex (MHC) 210, 110, 111–113, 115, 134, 135, 136, 143, 146
222, 251, 252 PPK 10
malignant melanoma 214 pre-implantation genetic diagnosis (PGD) 125
M-CSF 245 prenatal testing 47, 125
Meesmann’s corneal dystrophy (MCD) 43, 44– profilaggrin (PFN) 10, 11, 14–15
45, 47 progressive symmetric erythrokeratoderma
MIP-3alpha 245 (PSEK) 18–19
MIP-3beta 245 protein-protein interaction 5
monilethrix 2 , 44 P-selectin (PADGEM) 168, 169, 171, 174, 176–
motheaten mouse 227–228 177, 253
MPC-1 171 PSGL-1 176–177
muscular dystrophy 116 psoralen 256
psoriasis 167, 213, 254–255
INDEX 269
and anti-LFA-1 antibodies 257 stromal cell-derived factor-1 alpha

and cetirizine 256–257 (SDF-1alpha) 177
and cyclosporin 256 subacute cutaneous lupus erythematosus (SCLE)
and nonsteroidal anti-inflammatory drugs 213
(NSAIDs) 257–258 sub-basal dense plate 107
and ultraviolet light 255–256 subcutaneous fat 1
and vitamin D syndecans 1 and 4 135, 138
purified protein derivative (PPD) 180 systemic lupus erythematosus (SLE) 213
RANTES 177, 242 T cell 251

accessory molecule 203–220
S100 protein family 11 14 expression 212–215
S100A 10 activation and costimulation 210–212
scurfy mouse 226 mediated skin disease 213
secondary lymphoid tissue chemokine 245 receptor (TCR) 251
selectin 168, 174 CD3 complex 203
sialylated Lewis X (sLeX) 168, 174, 254 T lymphocyte migration to the skin 183
skin TGF-beta 245
architecture perturbation 222–223 TGF-betal 177
associated lymphoid tissue (SALT) 167 thrombin 176
blistering phenotypes 152–153 tight junction 1
disease, inflammatory and TNF-alpha 245
malignant 212–215 transcription factor NF-kappaB, 225
homing lymphocyte 167, 173–201 transforming growth factor-beta (TGF-beta)
immune system (SIS) 167 175
animal models 221–240 transforming growth factor-betal (TGF-betal)
contact hypersensitivity 223–224 175, 242
delayed hypersensitive response 222 transglutaminase (TG) 10, 11–12
engineered genetic models 228–229 mutation 17–18
immediate hypersensitivity 221, 224 transient bullous dermolysis of the newborn
normal cutaneous inflammation 221–222 (TBDN) 151
regulatory circuits 224–225 transmembrane proteins of basal keratinocytes
secondary effects 222–223 136–138
spontaneous genetic models 225–228 trichohyalin (THH) 10, 11, 15
tumour 214 tumor necrosis factor alpha (TNF-alpha) 168,
small proline-rich protein (SPRP) 10, 13 171, 175, 178, 180, 224, 241
spongiosis 223 type VII collagen 10, 108
SPRR 10 tyrosine activation motif (TAM) 94
stem cell factor (SCF) 242, 245
stratum basalis 9 ultraviolet light 255–256
stratum corneum 1, 9, 222–223
stratum granulosum 9 vascular adhesion molecule-1 (VAP-1) 181
stratum lucidum 9 vascular cell adhesion molecule-1 (VCAM-1)
stratum spinosum 9 169, 170, 171, 179, 188, 253
striate palmoplantar keratoderma 4 vimentin 27, 31, 32
stromal cell-derived factor-1 (SDF-1) 242 vitamin D 257
vitronectin 179
270 INDEX
VLA-4 (alpha4betal) 169, 170, 179, 188

Vohwinkel’s syndrome 2, 10, 17, 18
Weibel-Palade bodies 168, 176

white sponge nevus of Cannon (WSN) 42–44,
47
x-linked ichthyosis (XLI) 20–21

Cell Adhesion and Migration in Skin Disease (J Barker)

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CELL ADHESION AND MIGRATION IN SKIN

harwood academic publishers

ISBN 0-203-30459-4 Master e-book ISBN

ISBN 0-203-34336-0 (Adobe eReader Format)

Preface to the Series vii

LEUKOCYTE TRAFFICKING IN SKIN DISEASES

10. T-cell Accessory Molecules 202

The development and normal functioning of all multicellular organisms is governed to a

Jonathan N.W.N. Barker

Dundee DD1 9SY

Jefferson Medical College

Subsequently, pathogenetic missense mutations were demonstrated in the corresponding

MAJOR COMPONENTS OF THE CORNIFIED CELL ENVELOPE

Small Proline-rich Proteins

S100 Protein Family

terminus of profilaggrin reveals a high degree of homology to the small calcium-binding

(52). Envoplakin shows homology to the intermediate filament-associated proteins

MOLECULAR BASIS OF DISEASES AFFECTING THE CE

Lamellar Ichthyosis and Transglutaminase Mutations

Vohwinkel’s Syndrome and Loricrin Mutations

Figure 2.3 Clinical presentation of Vohwinkel’s syndrome, (a) Characteristic honeycomb

Progressive Symmetric Erythrokeratoderma and Loricrin

POSSIBILITIES OF FUTURE GENE THERAPY FOR DISORDERS

(“epidermal differentiation complex”) on human chromosome Iq21. J Invest Dermatol 106:

MOLECULAR ASPECTS OF KERATIN INTERMEDIATE

The Intermediate Filament Family of Cytoskeletal Proteins

THE MOLECULAR STRUCTURE OF INTERMEDIATE

Table 3.1 Types of intermediate filament

keratins: Protein Domain Structure

Keratins: the Type I and Type II Intermediate Filaments

Table 3.2 Cytokeratin expression patterns

MEDICAL ASPECTS OF KERATIN INTERMEDIATE FILAMENTS

Mutations in Keratin Genes Cause a Wide Range of Human

Epidermolysis Bullosa Simplex

Recessive Forms of EBS—Nature’s Gene Knockout

Mutations in K1 and K10 Cause The Skin Thickening

Alternate Phenotypes Caused by K1 Mutation

Ichthyosis Bullosa of Siemens and K2e Mutations

K9 Mutations Cause EPPK—Fragility of Palm and Sole Skin

Pachyonychia—Thick Nails due to Mutations in K6a, K6b,

K4 and K13 Mutations Cause Fragility of Mucosal Tissues

K18 Mutation May Cause Liver Disease Predisposition

Monilethrix—Beaded Hair Due to Mutations in Hair

Focusing on K3 and K12 Mutations in Meesmann’s Corneal

THE FUTURE OF KERATIN GENETICS RESEARCH

More Keratins, More Associated Molecules, More Diseases

Prevention and Therapy of Keratin Diseases

deactivation rather than replacement therapy must be developed. Despite these

Figure 4.1 Cartoon showing major components of desmosomes. ML=mid-line, PM=plasma

Desmosomal Glycoproteins and Cell Adhesion

For further details, other components and references, see text.

Cytoplasmic Interactions of Desmosomal Glycoproteins

Desmosomal Glycoproteins in Epidermis

important in regulating epidermal morphology and differentiation. Secondly, expression

desmosomal glycoproteins have distinct distribution patterns leading to the conclusion

ORGANISATION OF THE DESMOSOMAL PLAQUE

Plakoglobin and Plakophilin

Desmoplakin Homologues: Envoplakin, Periplakin and

Intermediate Filament Attachment and Accessory Plaque

Garrod, D., Chidgey, M. & North, A. (1996). Desmosomes; differentiation, development,

ULTRASTRUCTURAL ORGANIZATION OF THE DERMAL-

Figure 5.1 Ultrastructural appearance of the dermal-epidermal basement membrane. Human

muscle leads to muscular dystrophy remains to be determined. Plectin knockout mice