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Collectins and Cationic Antimicrobial Peptides of the Respiratory Epithelia


B. Grubor, D. K. Meyerholz and M. R. Ackermann
Vet Pathol 2006 43: 595
DOI: 10.1354/vp.43-5-595

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Vet Pathol 43:595–612 (2006)

REVIEW ARTICLE

Collectins and Cationic Antimicrobial Peptides of the


Respiratory Epithelia
B. GRUBOR, D. K. MEYERHOLZ, AND M. R. ACKERMANN
Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University,
Ames, IA

Abstract. The respiratory epithelium is a primary site for the deposition of microorganisms that are
acquired during inspiration. The innate immune system of the respiratory tract eliminates many of these
potentially harmful agents preventing their colonization. Collectins and cationic antimicrobial peptides
are antimicrobial components of the pulmonary innate immune system produced by respiratory
epithelia, which have integral roles in host defense and inflammation in the lung. Synthesis and secretion
of these molecules are regulated by the developmental stage, hormones, as well as many growth and
immunoregulatory factors. The purpose of this review is to discuss antimicrobial innate immune
elements within the respiratory tract of healthy and pneumonic lung with emphasis on hydrophilic
surfactant proteins and b-defensins.

Key words: Defensins; innate immunity; lung; surfactant proteins.

Introduction and brush cells.7 The number of basal cells


decreases with the decreased diameter of airway
The respiratory epithelium is a mucosal surface
branches, and the epithelium becomes simple
that routinely comes in contact with environmental
cuboidal. At the bronchiolo-alveolar junction,
substances including particulate matter, vapors,
nonciliated (Clara) cells are abundant. The alveolar
aerosols, and microbial pathogens.32 The innate
epithelium consists of a continuous layer of 2
immune components of the respiratory tract
principal cell types, thin, elongate type I pneumo-
eliminate many of these potentially harmful sub-
cytes (ATI cells), which cover 95% of the alveolar
stances. These components include coughing, the
surface, and cuboidal to rounded type II pneumo-
mucociliary escalator, phagocytosis by leukocytes
cytes (ATII cells).80 In addition, there are also
such as pulmonary alveolar macrophages and
mucous and serous epithelial cells of the tracheal
pulmonary intravascular macrophages,3,91 the com-
and bronchial submucosal glands, which together
plement cascade,66 interferons,50,106 neuroki-
with the pseudostratified epithelium and alveolar
nins,11,112 and antioxidants.91 More recently, addi-
epithelial cells serve as an important physical
tional components of the mucosal secretions such
barrier, and also produce and modify airway
as defensin peptides, anionic peptide (AP), and
surface liquid (ASL).131
surfactant proteins are increasingly becoming
appreciated for their role in preventing microbial Function of the Respiratory Epithelium
colonization/infection and also for triggering the
adaptive immune response.17,146 Ciliated epithelial cells participate in mucocili-
ary clearance, form a physical barrier, and secrete
a broad spectrum of antimicrobial factors.32,131
Cellular Components of the Respiratory Epithelium
Clara cells are the major progenitors of ciliated
The upper respiratory tract (nasal cavity, sinuses, cells after bronchiolar injury,28,64 and are involved
and trachea) and larger airways of the lower in the detoxification of xenobiotic compounds
respiratory tract are lined by pseudostratified, through cytochrome P-450 monooxygenase system
ciliated epithelium and submucosal glands activity.91 Clara cells produce surfactant proteins
(Fig. 1).32 Other epithelial cells include mucus- and Clara cell secretory 10-kd protein (CCSP/
secreting goblet cells, small mucous granule cells, CC10), which are natural regulators of airway
595
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596 Grubor, Meyerholz, Ackermann Vet Pathol 43:5, 2006

Fig. 1. Schematic presentation of the respiratory epithelium and its production of antimicrobial peptides
and proteins.

inflammation and cytodifferentiation mar- cells may play a role in the maintenance of normal
kers.28,107 ATII cells are responsible for pulmonary alveolar homeostasis and protection from injury,
surfactant synthesis, storage in lamellar bodies, lung development and remodeling, host defense,
secretion, and recycling.80,124 They also act as stem tumor/growth suppression, surfactant metabolism,
cells by repopulating the lung with ATI and ATII and other activities.30
cells during normal tissue turnover and during the
repair of alveolar epithelium after injury. Recently, Airway Surface Liquid
it has been shown that the dendritic cell-lysosomal The airway surface liquid represents a first line of
associated membrane protein (DC-LAMP/CD208) defense against inhaled pathogens.94 It consists of
is constitutively expressed by mouse, sheep, and the superficial gel or mucus layer and the layer of
human ATII cells and can be used as a marker for periciliary fluid, which are separated by a thin layer
normal as well as transformed (adenocarcinoma) of surfactant (Fig. 1).119 The mucus layer is 93–95%
ATII cells.124 ATII cells also express cathepsins, water and 5–7% solid material principally com-
major histocompatibility complex class II mole- posed of mucins (large glycoproteins) admixed with
cules, intercellular adhesion molecule-1 (CD54), phospholipids, proteoglycans, cellular debris, and
and costimulatory molecules such as CD80 and various proteins some of which possess antimicro-
CD86.124 For ATI (also known as membranous bial, antiprotease, and antioxidant activity.85 The
pneumocytes), one of the most useful differentia- mucus traps inhaled particles and microbial patho-
tion markers is T1a because it is not expressed by gens, neutralizes soluble gases, and is cleared by an
ATII cells or endothelial cells.145 The main interaction between airflow and cilia activity.91 The
function of ATI cells is to facilitate gaseous periciliary fluid layer is a watery layer that
exchange. There is limited data on ATI cells’ surrounds the cilia on the apical surface of the
metabolic and immune functions; however, these ciliated cells.94 This layer is necessary for efficient

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Vet Pathol 43:5, 2006 Antimicrobial Substances of the Respiratory Epithelia 597

Table 1. Antimicrobial factors or substances in airway surface liquid.

Factors Cell and Tissue Distribution in Lung


Lysozyme34,43,131 Epithelia—serous cells of tracheobronchial submucosal glands, tracheal
and bronchial surface epithelium; neutrophils; alveolar macrophages;
monocytes
Lactoferrin34,43 Epithelia—serous cells of tracheobronchial submucosal glands, tracheal
surface epithelium; neutrophils
Secretory leukoprotease inhibitor43,104,125 Epithelia—serous cells of respiratory submucosal glands and Clara cells
(in vivo); monocytes, alveolar macrophages, neutrophils, and
tracheal, bronchial, bronchiolar, and ATII cells (in vitro)
Elafin104,125 Epithelia—tracheal, Clara and ATII cells; alveolar macrophages
Phospholipase A25,47,88 Epithelia; neutrophils
Bactericidal/permeability-increasing Neutrophils
protein22
IgA secretory component36 Epithelia
Surfactant proteins SP-A and SP- Epithelia—ATII and Clara cells
D27,28,52,62,87,96
Anionic peptides17,18,40,41 Epithelia—trachea, bronchioles, alveoli
a- and b- Defensins17,32,43,97,131 Neutrophils; epithelia—conducting airways (and alveoli?)
h-Defensins142 Neutrophils
Cathelicidins9,17,154 Neutrophils; epithelia—conducting airways, serous and mucous cells of
respiratory submucosal glands
Peroxidases32 Neutrophils; epithelia
Clara cell secretory 10-kd protein Clara cells
(CC10)71,107,150,152
Nitric oxide32 Endothelial cells, macrophages
Uric acid32 Epithelia—respiratory submucosal glands
Aminopeptidase75 Neutrophils; alveolar macrophages; epithelia—bronchial and alveolar
Proline-rich proteins*123 Epithelia—bronchus
Statherin*123 Epithelia—serous cells of respiratory submucosal glands
Cystatin*123 Epithelia—bronchus
Plunc13,143 Epithelia—trachea and bronchus
* Note: These proteins have highest production levels in salivary glands. SP-A 5 surfactant protein A; SP-D 5 surfactant
protein D.

ciliary beating and mucin hydration. Surfactant is strength of the ASL, the antimicrobial activity of
80% phospholipids (phosphatidylcholine being the ASL components decreases dramatically.
most abundant), 12% neutral lipids (cholesterol Lysozyme and lactoferrin are the most abundant
being the most abundant), and 8% surfactant proteins in ASL and are present at 0.1–1 mg/ml
proteins (SP) including hydrophilic SP-A and SP- concentration.43 Lysozyme was first recognized by
D, and hydrophobic SP-B and SP-C.108 The Alexander Fleming in 1922. It is a 14-kd enzyme
primary function of surfactant is to regulate the produced by neutrophils, monocytes, macro-
surface tension at the air–liquid interface, prevent- phages, and epithelial cells.43,131 It enzymatically
ing alveolar collapse at low lung volumes and cleaves glycosidic bonds of the bacterial membrane
overinflation at high lung volumes. Surfactant also peptidoglycans or kills bacteria by a nonenzymatic
facilitates effective spreading of mucus, preserva- mechanism. Lactoferrin is an 80-kd cationic iron-
tion of gel and periciliary fluid integrity, and binding protein and is also produced by neutro-
beating of cilia without entanglement. phils and epithelial cells. This protein inhibits
growth of iron-requiring bacteria and can also be
Antimicrobial Factors of Airway Surface Liquid directly microbicidal through its N-terminal cat-
Airway surface liquid contains several proteins ionic fragment.
and peptides with antimicrobial activity (Ta- Antiproteinases are also important constituents
ble 1).22,32,34,75,123,125,126,131,143,144 Most of these fac- of ASL that are involved in the acute phase of
tors act cooperatively in their microbicidal activity inflammation.104,125 Their molecular characteristics
and the degree of their activity depends on the ionic include low-molecular weight, positive charge, and
strength of the solution.131 With increased ionic numerous disulfide bonds. They are present at the

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598 Grubor, Meyerholz, Ackermann Vet Pathol 43:5, 2006

mucosal surfaces and protect against noxious lectin domain, known as a carbohydrate recogni-
effects of proteolytic enzymes released by phago- tion domain (CRD), which is attached to a col-
cytic cells thereby controlling excessive inflamma- lagen-like region via an alpha-helical coiled-coil
tion and septic shock. An important member of neck region. To distinguish between self and non-
antiproteinases is secretory leukoprotease inhibitor self, collectins need to recognize and bind complex
(SLPI), a low-molecular-weight cationic proteinase glycoconjugates on microorganisms. Collectin
inhibitor produced by epithelial cells and macro- binding takes place via the CRD, which preferen-
phages. This factor acts as an effective inhibitor of tially binds mannose-like ligands such as maltose,
neutrophil elastase via the C-terminal domain. which is favored by SP-D, N-acetyl-D-glucosea-
Secretory type II phospholipase A2 (PLA2) is mine, which is favored by conglutinin, and CL-46,
another antiproteinase that belongs to a growing or D-mannose, which is favored by CL-43. This
family of enzymes produced by epithelia and leads directly to agglutination or neutralization of
blood-derived cells. Such enzymes are involved in the microorganism. The binding recruits inflam-
cytokine-mediated inflammation, surfactant degra- matory cells and causes opsonization of the
dation, and bactericidal activity especially against microorganisms for phagocytosis. Although some
Gram-positive bacteria.5,47,88 Other antiproteinases collectins such as MBL and conglutinin have an
include locally synthesized and secreted elastase- important function in systemic response to micro-
specific inhibitor (elafin), and systemic antiprotei- bial challenge, the principal area of interest for this
nases produced in the liver and secreted in the review include hydrophilic surfactant proteins SP-
circulation such as a1-antichymotrypsin and a1- A and SP-D, also known as lung collectins, which
proteinase inhibitor. are a part of more localized response of the lung to
Antimicrobial peptides and proteins from the injury.27
ASL that have received the least attention are those
produced by respiratory epithelia, including CC10, Lung Collectins
APs, cationic antimicrobial peptides (AMP), and Functional Roles
collectins. CC10 protein is induced by corticoster- A large body of evidence has been collected for
oids and is a potent anti-inflammatory cytokine at least four potential functional roles of lung
produced by Clara cells. Although tumor necrosis surfactant proteins based on in vitro and in vivo
factor- (TNF-a) released during lung inflammation studies.29 These roles include antimicrobial, anti-
increases the expression of both CC10 and PLA2, inflammatory, immunomodulatory, and surfactant
CC10 inhibits the activity of PLA2.71,88,152 Addi- regulatory activities.
tional features of CC10 include a dose-dependent Lung collectins bind, aggregate, and opsonize
suppressive effect on TH2 cytokine expression and different microorganisms including Gram-positive
a potential protective role in lung tumorigene- and Gram-negative bacteria, enveloped (influenza
sis.71,150 A virus [IAV], respiratory syncytial virus [RSV])
Anionic peptides are small, hydrophilic peptides and nonenveloped viruses (Rotavirus), and fungi to
that contain homopolymeric regions (e.g., 5–7 enhance phagocytosis, killing, and clearance of
residues) of aspartic acid, which are responsible microorganisms from the lung (Table 2).68 Initially,
for their negative charge.16,41 They have been SP-A and SP-D have an antimicrobial role that is
detected in the lungs, bronchoalveolar lavage often followed by a proinflammatory response to
(BAL) fluid, and pulmonary surfactant of humans, destroy the pathogen and prevent further spread.
sheep, and cattle. For maximal bactericidal activ- In vitro, SP-D enhances phagocytosis of RSV by
ity, APs require zinc as a cofactor, which may allow alveolar macrophages and neutrophils, and sup-
the peptide to overcome the net negative charge presses the inhibitory effect of RSV on oxygen
once on the microbial surface.17 radical production by these cells.82 SP-D also
inhibits the infectivity and hemagglutination activ-
Collectins
ity of IAV in vitro by blocking pathogen-target cell
Collectins produced by vertebrates are a family attachment sites.29 Next, SP-D binds, agglutinates,
of proteins that include mannan-binding lectin and/or kills pathogens, resulting in enhanced
(MBL/mannose-binding protein), collectin liver 1 physical or cellular clearance.25 SP-D agglutinates
(CL-L1), collectin placenta 1 (CL-P1), SP-A, SP-D, Mycobacterium tuberculosis but inhibits its inter-
conglutinin, 43-kd collectin (CL-43), and 46-kd nalization;29 a more recent study shows that both
collectin (CL-46), the last three being found only in SP-A and SP-D may enhance mannose receptor-
the bovidae.57,62,68 These proteins share a common mediated phagocytosis of Mycobacterium avium by
structure made of a C-terminal located C-type macrophages.79 SP-A can also cause direct killing

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Vet Pathol 43:5, 2006 Antimicrobial Substances of the Respiratory Epithelia 599

Table 2. Interaction of surfactant protein A (SP-A) and surfactant protein D (SP-D) with pathogens.

SP-A SP-D
Bacteria Dimannose repeating unit associated with Rough LPS29
capsular polysaccharides of some capsular LTA and peptidoglycan of Gram-positive
serotypes27 bacteria68
Rough LPS Lipid component of the cell membrane of
Outer membrane protein of Haemophilus Mycoplasma pneumoniae68
influenzae type A Mycobacterium tuberculosis68
Lipid A domain of Escherichia coli Enhances mannose receptor-mediated
Gram-positive bacteria68 phagocytosis of Mycobacterium avium by
Mycobacterium tuberculosis68 macrophages79
Enhances mannose receptor-mediated
phagocytosis of Mycobacterium avium by
macrophages79
Viruses Inhibits influenza virus hemagglutination and Inhibits influenza virus hemagglutination and
infectivity; neutralizes influenza A virus (IAV) infectivity; binds high-mannose
by directly occupying and likely blocking the oligosaccharides present on HA of IAV60,61
coat hemagglutinin (HA) cell attachment site60,61 Binds to G and F proteins of RSV82
Binds to F proteins of respiratory syncytial Hemagglutination inhibition and neutralization
virus (RSV)45,128 of bovine rotavirus116
Fungi Binds glycoprotein gpA of cyst and trophozoite Binds glycoprotein gpA of cyst and trophozoite
forms of Pneumocystic carinii, which enhances forms of Pneumocystic carinii, which enhances
binding of organism to alveolar binding of organism to alveolar
macrophages27,68 macrophages27,68
Binds to uncapsulated forms of Cryptococcus Binds to uncapsulated forms of Cryptococcus
neoformans neoformans leading to agglutination
Binds to conidia and specific cell wall Binds to conidia and specific cell wall
glycoproteins of Aspergillus fumigatus glycoproteins of Aspergillus fumigatus

of microorganisms by inducing the formation of cus and H.influenzae.29 In rats, after the intratra-
reactive oxygen species (ROS).53 In addition, both cheal inoculation of lipopolysaccharide (LPS),
SP-A and SP-D can inhibit the proliferation of message levels and BAL fluid contents of both
pathogens such as Gram-negative bacteria by SP-A and SP-D significantly increase at 24 hours
increasing the permeability and disrupting the and 72 hours postinoculation, respectively, under-
microbial cell membrane.147 scoring the role of these 2 proteins in the acute
According to in vivo studies, the antimicrobial phase of lung inflammation.98 In lambs, during the
role of SP-A and SP-D appears to depend on the progression of Mannheimia haemolytica-induced
type of pathogen and may be species-specific. In bronchopneumonia into chronicity, the SP-D pro-
wild-type mice, SP-D levels increase markedly after tein is reduced to near absence in hyperplastic
IAV infection,117 whereas decreased viral clearance bronchiolar epithelial cells.52 indicating that the
is observed after challenge of SP-A knockout (SP- state of epithelial differentiation may influence the
A 2/2) mice with RSV96 or SP-D knockout (SP-D SP-D expression. Our recent studies indicate that
2/2) mice with RSV and IAV.29,82 In neonatal elevated levels of SP-A and SP-D (as well as sheep
lambs infected intratracheally with parainfluenza b-defensin-1) mRNA in the lungs of neonatal
type 3 (PI-3) virus, SP- A and SP-D mRNA levels lambs infected with PI-3 virus are associated with
are significantly elevated in the lung.51 Regarding simultaneous decrease in PI-3 virus replication.51
bacterial infection, SP-A 2/2 mice exhibit delayed These findings may suggest that lung collectins
microbial clearance of group B streptococcus, (and defensins) are synthesized to bind to PI-3
Haemophilus influenzae, and Pseudomonas aerugi- virus, as collectins bind to other viruses, and to
nosa.96 SP-D 2/2 mice clear tested bacteria as neutralize it directly or indirectly.
efficiently as wild-type mice;57 however, there is The second role of lung surfactant proteins is
increased inflammation and oxidant production, anti-inflammatory. This role can be explored by
and reduced macrophage phagocytosis in response using SP-A 2/2 and SP-D 2/2 mice. The
to intratracheal instillation of group B streptococ- phenotypic features of SP-D 2/2 mice include

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600 Grubor, Meyerholz, Ackermann Vet Pathol 43:5, 2006

chronic low-grade pulmonary inflammation, ATII properties, and altered macrophage production of
cell hyperplasia with giant lamellar bodies, excess MMPs.25 One study even suggests that exogenous
alveolar surfactant phospholipid, and emphysema- SP-A can significantly protect lung from injury
tous changes with dilated airspaces and pulmonary induced by ischemia-reperfusion of isolated rat
fibrosis.25 The inflammation is characterized by lungs by reducing the SP-A loss and by activating
peribronchial monocytic infiltrate with an excessive the upregulation of endogenous NO synthetase
number of alveolar macrophages that produce (eNOS).99
increased amounts of ROS and matrix metallopro- The fourth role, which was the first discovered
teinases (MMP). Inflammation in this model can be functional role of lung surfactant proteins, is
explained by increased lung expression of mono- nonimmunologic and is related to the function of
cyte chemoattractant protein-1 (MCP-1) mRNA, surfactant.96 SP-A is more important than SP-D in
which can be corrected by the treatment of SP-D this regard, and has been reported to regulate
2/2 mice with recombinant SP-D.25 Another surfactant homeostasis by controlling the secretion
mechanism of lung inflammation would be in- and uptake of surfactant by ATII cells, to protect
creased numbers of apoptotic macrophages present the surface activity properties of surfactant from
in SP-D 2/2 mice, because failure in clearing functional inhibitors, to inhibit phospholipase A1,
apoptotic cells can lead to triggering of inflamma- and to be required for the formation of tubular
tion.25 SP-A 2/2 mice do not show detectable myelin. In addition, it has been suggested that an
abnormalities in histology, lung wet weight, lung appropriate SP-A level in fetal lung may determine
volume, compliance, or surfactant lipid metabo- the time of labor.26 According to this theory, when
lism, but clearly demonstrate increased susceptibil- SP-A reaches a yet undefined critical threshold in
ity to infection by respiratory pathogens, enhanced the ASL of fetal lung, it stimulates macrophages
lung inflammation, and increased incidence of via TLRs causing proinflammatory cytokine re-
splenic dissemination after intratracheal instillation lease and macrophage migration to the uterus,
of bacteria.53,62 In addition, surfactant isolated which in turn leads to increased uterine contractil-
from these mice lacks tubular myelin, but surpris- ity and parturition.
ingly these mice have normal postnatal survival
with no detectable alteration in pulmonary func- Modulation of SP-A and SP-D function
tion.62 Certain ligands and inflammatory products can
SP-D is capable of regulating an inflammatory serve as competing ligands for collectins that
response to microorganisms and microbial prod- reduce their activity, or can cause their partial
ucts.29 After bacterial challenge, SP-D inhibits cleavage or complete degradation.29,65,120 Glucose
pulmonary secretion of the proinflammatory cyto- concentrations encountered in diabetes mellitus can
kines such as IL-1, IL-6, and TNF-a.57 SP-A 2/2 interfere with the ability of SP-D to interact with
mice show increased production of TNF-a and specific strains of IAV in vitro and cause increased
nitric oxide (NO) upon intratracheal challenge with susceptibility of mice to certain influenza viruses in
P. aeruginosa or LPS.53 In addition, peptidoglycan- vivo.115 Both SP-A and SP-D bind free DNA and
and zymosan-induced secretion of TNF-a can be the DNA present on apoptotic cells via their
inhibited by direct interaction of SP-A with Toll- CRD’s and collagen-like domains, although SP-D
like receptor (TLR)-2 on macrophages.79 binds nucleic acids more effectively at physiological
The third role of lung collectins is immunomo- salt conditions.111 This feature may allow lung
dulation. SP-D mediates inhibition of IL-2 secre- collectins to play a role in decreasing the in-
tion by monocytes, and leads to reduced T- flammation caused by DNA in lung, which at the
lymphocyte proliferation.57 It can bind to oligo- same time may inhibit lung collectin carbohydrate
saccharides associated with dust mite allergens and binding ability. Interaction of SP-A with other
inhibit the binding of specific IgE molecules to proteins such as fibrinogen can induce inactivation
those allergens.29 Glycosylation of viral coat of SP-A.54 Phosphatidylinositol (PhI) from surfac-
proteins such as those of IAV, an adaptation that tant may serve as a ligand for SP-D and suppress
is believed to help viruses evade antibody-mediated interaction between SP-D and microorganisms in
neutralization, is associated with enhanced reactiv- diseases in which PhI levels are elevated such as in
ity of SP-D.117 SP-A can also inhibit proliferation acute respiratory distress syndrome (ARDS).105
of stimulated lymphocytes and affect IL-2 pro- Furthermore, neutrophil serine proteases including
duction.53 Other functions revealed by in vitro cathepsin G, elastase, and proteinase-3 contribute
studies include chemotaxis of alveolar macro- to degradation of SP-A; this may be the reason
phages, neutrophils, and monocytes, antioxidant for low levels of SP-A in patients with cystic

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Vet Pathol 43:5, 2006 Antimicrobial Substances of the Respiratory Epithelia 601

fibrosis.120 These proteases also cleave highly alveoli (hypophase), which leads to formation of
conserved CRD subregions of SP-D markedly tubular myelin.62,108 Regulated exocytosis of the
reducing the ability of SP-D to promote bacterial lamellar body content involves about 10% of the
aggregation and binding to yeast mannan in intracellular surfactant pool per hour, but the
vitro.65 In addition, bacterial products such as P. availability of SP-A that is free to interact with
aeruginosa elastase, a zinc-metalloprotease, also ATII cell receptors in the intact lung is unknown.62
cause formation of nonfunctional SP-D degrada- The secretory granules of Clara cells, on the other
tion products4,146 Finally, it has been shown that hand, are primarily responsible for storing SP-D
the function of SP-A depends on cation concen- and it seems likely that SP-D secretion is regulated
tration, especially calcium, in the environment, via granule exocytosis at this level of the re-
which affects the quaternary organization of the spiratory tract.29 Although both SP-A and SP-D
SP-A protein.118 are expressed in highest amounts in the distal lung,
expression at various extrapulmonary sites has
Regulation of SP-A and SP-D expression
been reported and it appears to be species
Synthesis and secretion of surfactant compo- specific.62,87 The extrapulmonary sites of SP-A
nents are developmentally regulated. Surfactant expression include porcine eustachian tube epithe-
starts to be synthesized in progressively increasing lium,110 rabbit sinus and middle ear mucosa,38
amounts during the last stage of lung development, human small and large intestine,86 vaginal muco-
the alveolar stage.77,100 In humans, SP-A is not sa,92 trachea, thymus, pancreas, and prostate.93 The
detectable in fetal lung until the 30th week, extrapulmonary sites for SP-D include porcine
whereas SP-D is first detected in the second eustachian tube epithelium,110 human salivary
trimester of gestation (40 weeks being the full gland, kidney, heart, small intestine, prostate
term).35,77,108 In sheep, mRNA content of SP-A is gland, and pancreas, rat gastric mucosa, and many
low to nondetectable at 99–100 days of gestation other sites.62
(145–150 days being the full term), and it pro-
gressively increases starting at about 120 days of Protein structure
gestation to term and into the postnatal peri- Pulmonary collectins are organized as oligomers
od.100,139 Because pre- and full-term infants and assembled from multiple copies of a single poly-
animals lack fully effective adaptive immune peptide chain consisting of 4 structural do-
response and the maternal immunoglobulin levels mains.27,62 The first domain is a short amino-
can vary from very high to low or absent,37 terminal disulfide-rich domain of 7 (SP-A) to 25
regulated expression of surfactant is especially (SP-D) amino acids which is involved in the
important in this age group that largely depends interchain interactions. The second domain is
on its innate immune response. a collagen-like domain that forms an extended
In addition to being dependent on the develop- fibrillar triple helix 20 (SP-A) to 46 (SP-D) nm
mental stage of lung maturation, lung surfactant long. The third domain forms a short trimeric
proteins are also regulated by hormones, growth coiled-coil domain, and links the collagen-like
factors, and other regulatory substances (Ta-
sequence to the fourth domain, globular CRD.
ble 3).44 In Table 3, discrepancies in the effect of
SP-A is the most abundant surfactant protein in the
different factors on SP-A and SP-D expression can
lung (5.3% of total surfactant weight) and its
be observed, suggesting that these 2 proteins are
monomer measures 28–36 kd.53,63 This protein is
not coordinately regulated.35
usually assembled into octadecamers that consist of
Tissue distribution 6 trimeric subunits that form a ‘‘bunch-of-tulips’’
SP-A and SP-D are synthesized by ATII and shape.27,63 SP-D is predominantly assembled as
Clara cells.28 Alveolar macrophages show strong dodecamers that contain 4 homotrimeric subunits
cytoplasmic and/or membrane labeling with anti- arranged in a cruciform shape. Each SP-D mono-
body against SP-D; however, because macrophages mer weighs 43 kd.
express no detectable SP-D mRNA, it is likely that Receptors
intracellular SP-D protein is caused by phagocyto-
sis in conjunction with surfactant metabolism.29 The best characterized host defense receptor for
The lamellar bodies of ATII cells serve as storage SP-A is a 210-kd cell surface protein (SP-R210)
organelles for surfactant phospholipids and the present on rat ATII cells and alveolar macro-
content of these organelles is rapidly associated phages.68,69 The interaction of SP-A and SP-R210
with SP-A upon its secretion into the liquid lining occurs through the collagen-like domain of SP-A,

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602 Grubor, Meyerholz, Ackermann Vet Pathol 43:5, 2006

Table 3. Regulation of SP-A and SP-D expression by hormones, growth factors, and other regulatory
substances.

SP-A SP-D
Insulin Decreases SP-A (and SP-B) mRNA in
human fetal lung explants (HFLE);
may account for increased incidence
of respiratory distress syndrome in
infants of diabetic mothers31
Glucose Ligand for SP-D; may account for
increased susceptibility of diabetic
mice to Influenza virus infection115
Glucocorticoids Biphasic response in HFL—increases Increases SP-D mRNA in HFL122
SP-A mRNA at low concentration
and with short exposure,
but decreases with higher
concentration;35 in immature lambs,
increases SP-A protein6,139
Vascular endothelial growth Increases SP-A protein and mRNA
factor in HFLE; possible autocrine and
paracrine regulatory role in
pulmonary epithelial cell growth
and differentiation20
Keratinocyte growth factor Increases SP-A mRNA in adult rat Increases SP-D mRNA in adult
lung151 rat lung151
Transforming growth factor b1 Reduces SP-A mRNA in HFLE
and arrests lung morphogenesis
in pseudoglandular stage of
development;156 potential role
of TGF-b1 in pathogenesis of
bronchopulmonary dysplasia12
cAMP, IFN-c Increase SP-A mRNA in HFLE35
Lipoteichoic acid, tumor Decrease SP-A mRNA in HFLE35,103
necrosis factor-a, phorbol
myristate acetate
IL-4 Increases SP-D in rat ATII cells; may
provide a link between innate and
adaptive immunity during allergic
inflammation21
85–95% O2 Increases SP-A in rat lung98 Increases SP-D in rat lung29
Nitric oxide Increases SP-A protein and mRNA
in lamb lung138
Retinoic acid Increases SP-D mRNA in ovine ATII
cells (unpublished data, Grubor B,
Ackermann MR)

and leads to binding of SP-A to ATII cells and resulting in reduced TLR2 binding to peptidogly-
alveolar macrophages, SP-A-mediated uptake and cans.87 Other proposed receptors for collectins
killing of Mycobacterium bovis, and inhibition of include cC1qR (calreticulin), CD14, and 30-kd
phospholipid secretion.68,96 Both SP-A and SP-D alveolar cell membrane protein.57,63
bind to glycoprotein gp-340, a member of the
macrophage scavenger receptor cysteine-rich pro- Lung collectin genes
tein family that has both soluble and alveolar In humans and nonhuman primates, SP-A is
macrophage membrane-bound components.96 The encoded by 2 genes, SP-A1 and SP-A2, with several
interaction of lung collectins with gp-340 leads to alleles at each locus.76,83 In other species such as
binding and aggregation of some bacteria, such as rabbits, rats, mice, and dogs SP-A is encoded by
Streptococcus mutans.113 SP-A also binds to TLR2 a single-copy gene.83 Human SP-D is encoded by

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Vet Pathol 43:5, 2006 Antimicrobial Substances of the Respiratory Epithelia 603

a single gene, which has also been confirmed in kidney,8 whereas MBD-3 exhibits homology at the
rodents and ruminants.27,48 Both human SP-A and gene level to HBD-2, and is detected in lung,
SP-D genes are found on the long arm of salivary gland, and reproductive organs.8 Bovine b-
chromosome 10 at q22.2–23.1 region, near the defensin family members include neutrophil defen-
MBL gene.27 Bovine SP-D gene is found at the sins (BNBD) 1–13 that are expressed in bovine
same locus as the conglutinin gene on Bos taurus neutrophils and macrophages; tracheal antimicro-
chromosome 28 (BTA 28) at position q1.8, bial peptide (TAP) expressed in nasal epithelium,
and comprises 9 exons spanning approximately trachea, bronchioles, and alveolar macrophages;
10.5 kb.49 The bovine gene encoding SP-A is and lingual antimicrobial peptide (LAP) expressed
located proximal to the bovine SP-D gene at in the epithelial cells of tongue and alveolar
BTA 28 at position q1.8–1.9.48 The order of bovine macrophages.17,43 Two ovine b-defensins have been
genes for SP-A, MBL-A, and SP-D is homologous described, sheep b-defensin (SBD)-1 and -2. SBD-1
with the order of the analogous genes found in expression has been detected from the tongue to
humans and mice. Regulation of lung surfactant colon with the exception of distal ileum where
protein gene expression is multifactorial and SBD-2 predominates.72,101 SBD-1 is also expressed
controlled by transcriptional and/or post-transcrip- in the trachea,72 whereas both SBD-1 and SBD-2
tional (mRNA stability) mechanisms.14 The func- are expressed in the lung.2,51
tion of surfactant protein promoter depends on the Cathelicidins, another important family of cat-
combined activity of many transcription factors, ionic AMP, have been found in cows, pigs, sheep,
one of them being thyroid transcription factor 1 goats, horses, mice, guinea pigs, rabbits, and
(TTF-1/Nkx2.1), a common positive regulator of humans.17,132 Their main structural feature is a high-
surfactant protein promoter activity.14 level sequence identity in the 59 region, named the
N-terminal cathelin domain because it is also found
Cationic Antimicrobial Peptides in cathelin, a cysteine protease inhibitor. The C-
Two major families of cationic AMP are terminal domain of cathelicidins provides them
defensins and cathelicidins.78 Defensins can be with antimicrobial activity. In most species, cathe-
further divided into 3 classes, a-, b-, and h- licidins are expressed in myeloid precursor cells.
defensins based on pairing of their 6 cysteine Other cells and tissues known to express cathe-
residues in the disulfide bridges.32,140,149 Human a- licidins include mature peripheral porcine neutro-
defensins are 29–35 amino acids in length and phils, human airway epithelium, and the testis.9,114
contain 3 disulfide bridges in a 1–6, 2–4, 3–5 Some examples of cathelicidins include bovine
arrangement.78 These peptides were first described myeloid antimicrobial peptide (BMAP)-34,
in human tissues and include 6 members.140 Four BMAP28, Bac5, Bac7 in bovine; OaBac11, sheep
members, named human neutrophil peptides myeloid antimicrobial peptide (SMAP)-29,
(HNPs) 1–4, have been found in the azurophilic SMAP34 in sheep; porcine myeloid antimicrobial
granules of neutrophils. The other 2, human peptide (PMAP)-23, cecropin P1, PR-39, protegrin-
defensin (HD)-5 and HD-6, are located in secretory 1 in pigs; equine cathelicidins eCATH-1, -2, and -3
granules of intestinal Paneth’s cells and in female in horses; and LL-37 (also known as hCAP-18) in
genital epithelial cells. In the early 1990s, b- humans.17,154
defensins were identified.155 b-defensins are 36–42
Functional roles
amino acids in length and have 3 disulfide bridges
arranged in a 1–5, 2–4, 3–6 pattern.78 More than 28 Cationic AMPs have multiple roles and the best-
human b-defensins have been identified7 among studied ones are included in Table 4. These
which the best studied include human b-defensin peptides have the potential to kill or neutralize
(HBD)-1 expressed in the epithelial cells of re- bacteria (Gram-positive and Gram-negative), fungi
spiratory tract including lung and trachea, pancre- (including yeasts), viruses (even enveloped, like
as, kidney, gastrointestinal epithelium,72,97 urogen- human immunodeficiency virus [HIV]), and para-
ital tract,157 and reproductive tract;84 HBD-2 sites (including planaria, nematodes, trypano-
expressed in skin, trachea, and lung;130 and HBD- somes, and plasmodia).132 The peptides are selec-
3 expressed predominantly in the skin but is also tive in that normal host cells are relatively resistant
expressed in the respiratory tract.74 In mice, over 40 to their action. This is based on a few important
b-defensins have been identified including mouse b- differences between cytoplasmic membranes of
defensin (MBD)-1, -2, -3, and -4.7 MBD-1 is prokaryotic and eukaryotic cells.95 The most
homologous to HBD-1 at the gene level, and is striking difference is the composition and topologi-
found in multiple epithelia with highest levels in cal arrangement of lipids. The outer surface of

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604 Grubor, Meyerholz, Ackermann Vet Pathol 43:5, 2006

Table 4. Multifunctional roles of cationic antimicrobial peptides (AMP).

Activities Examples
1. Antimicrobial Minimal inhibitory concentration of synthesized peptides used in vitro
experiments is 2–16 mM17
Addition of pig protegrin-1 to methicilin resistant Staphylococcus aureus
infected mice improved survival from 0 to 100%137
Decreased production of antifungal peptide drosomycin in knockout
Drosophila made animals more susceptible to Aspergillus fumigatus129
Over-expression of human LL-37 in mouse airways improved survival from
10 to 75% in Escherichia coli infected animals10
2. Synergistic action Act in synergy with other AMP, host molecules (e.g., lactoferrin, lysozyme),
or conventional antibiotics46,56,135
3. Anti-inflammatory a-defensins regulate complement activation and secretion of protease
inhibitors67,141
Indolicidin and to a lesser extent bactenecin show selective cytotoxicity to T
lymphocytes132
PR-39 peptide inhibits leukocyte (particularly neutrophil) recruitment into
inflamed tissue and attenuate myocardial reperfusion injury in a murine
model67
Defensins can inhibit serine protease (e.g., elastase)132
Defensins can inhibit fibrinolysis43
Cationic AMP can downregulate neutrophils’ ability to generate superoxide
(by affecting NADPH oxidase)
4. Proinflammatory a-defensins stimulate production of IL-8140
HBD-1 and -2 are chemotactic for immature dendritic cells and memory T
lymphocytes1
HBD-6 causes neutrophil recruitment and mild aggravation of concurrent
PI-3 virus infection in lambs102
CAP-11, magainin-2, and HBD-2 can stimulate activation and
degranulation of mast cells131,140
5. Wound healing Promotion of re-epithelialization of damaged surfaces55
6. Increased bacterial colonization Observed in patients with chronic obstructive pulmonary disease; possibly,
defensins stimulate adherence of Haemophilus influenzae to expose it to
high concentrations of epithelial-cell derived AMP140
7. Inhibition of adrenocorticotroph
hormone (ACTH)-stimulated
cortisol production43
8. Bridge between innate and Recruitment of immature dendritic cells and memory T lymphocytes likely
adaptive immune system through interaction with chemokine receptor CCR6148
Mice challenged with ovoalbumin antigen produce higher levels of antigen-
specific serum IgG when treated with HNP-1 and HBD-219

mammalian cell membranes is composed of elec- cholesterol, which also contributes to prokaryotic
trically neutral (zwitterionic phospholipids), mainly cells being a better target. The last striking
phosphatidylcholine (PC) and sphingomyelin. Bac- differentiating factor of prokaryotic cells is that
terial membranes, on the other hand, contain large they possess a large transmembrane potential of
amounts of negatively charged phospholipids, 2140 mV, compared to the low membrane poten-
phosphatidylglycerole (PG) and cardiolipin (CL). tial of eukaryotic cells (15 mV). Unfortunately,
In addition, the outer membranes of Gram- there are some exceptions to these rules and certain
negative bacteria are covered with polyanionic cationic AMP peptides, like melittin from bees,
LPS. Therefore, cationic peptides prefer binding mastoparan from wasps, or charybdotoxin from
to bacteria because of electrostatic interactions scorpions, are potent toxins.132
between positive charges of the peptides and Given the diversity of AMP and of target
negative charges of the lipids. The second impor- microorganisms, it is likely that there is not one
tant difference is that unlike eukaryotic cell unique mechanism for their mode of action.39 The
membranes, prokaryotic cell membranes lack only constant is that these peptides do not need

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Vet Pathol 43:5, 2006 Antimicrobial Substances of the Respiratory Epithelia 605

a metabolically active cell to exert their effect. cathelicidin SMAP29) are showing promising
Based on studies done with magainins, cationic results in enhancing AMP activity.134 Another
AMP isolated from frog skin, a general mecha- factor influencing AMP activity is pH, which can
nism of action on Gram-negative (2-cell mem- affect the electrical charge of peptides.136 This may
brane) bacteria has been proposed.95 It includes 2 lead to changes in peptide conformation and
steps, with a note that the second step could also activity. The ability of some peptides to be active
be applied to Gram-positive (1-cell membrane) at low pH conditions can be used for drug
bacteria. In the first step, positively charged AMP generation, because this feature would allow the
interacts with polyanionic LPS at sites of LPS peptides to act in phagolysosomes. Furthermore,
where divalent cations usually bind to cross because of their cationic properties, cationic AMP
bridge adjacent LPS molecules and stabilize the easily forms complexes with various serum proteins
outer membrane. The competitive displacement of like serum albumin and a2-macroglobulin, or
these divalent cations (Ca2+, Mg2+) by the bulkier bacterial degradation products of extracellular
peptides leads to lesions in the outer bacterial matrix, which may abolish their activity.1,70
membrane visualized as surface blebbing. This
model has been described by Robert Hancock, Regulation of expression
and is called ‘‘self-promoted uptake.’’56 In the All cationic AMPs can have constitutive (nor-
second step, AMP associates with the negatively mally present in animals) and/or inducible (upon
charged phospholipid membrane and form holes stimulation) expression.78 Two examples of pep-
(‘‘aggregate channels’’) in the membrane when tides with constitutive properties are HBD-1 and
certain, critical AMP concentrations have been MBD-1. The expression of inducible peptides can
reached. Examples of peptides that cause forma- be stimulated during infection or inflammation
tion of membrane channels are cecropins and with LPS, lipoteichoic acid (LTA), TNF, IL-1b,
defensins.17 Other cationic AMP like Bac5 and IFN-c, and other mediators of inflammation.55,58
Bac7 alter transport and energy metabolism in the These AMP include human HBD-2, -3, -4, and LL-
cytoplasmic membrane of pathogens. Once the 37,74,78,89 bovine LAP and TAP,33,121 mouse MBD-2
peptides are within the cells, they can bind to and -3,78 and others. In addition, like with
cytoplasmic polyanions such as DNA and in- collectins, the expression of pulmonary defensins
terfere with DNA/protein synthesis (e.g., PR-39), seems to be developmentally regulated in late
and then stimulate autolytic enzymes.17,39 It has gestation and through the neonatal period,101 which
also been demonstrated that AMP can inactivate leaves a window of immature peptide expression in
bacterial heat-shock proteins (DnaK) without the infant that may provide an environment with
binding to human analogous heat shock protein increased susceptibility to respiratory tract infec-
Hsp70 and affecting its normal function.109 The tion.
mechanism of antiviral action is less understood, The mechanism of AMP gene induction is poorly
but studies show that b-defensins can cause understood. One of the proposed induction path-
disruption of the viral envelope, whereas a- ways is LPS-mediated upregulation of AMP genes,
defensins may also interfere with intracellular cell which has been directly shown for the TAP gene in
signaling inhibiting viral replication.23,102 bovine and HBD-2 gene in human airway epithelial
cells.133 Cationic AMP also bind LTA, although
Modulation of AMP function with a lower affinity compared to LPS, leading in
In vitro experiments have shown that defensins some cases to production of TNF-a and IL-6 by
are microbicidal at mM concentrations against murine macrophages.132 In addition, recent studies
many Gram-positive and Gram-negative bacteria, indicate that retinoic acid (RA) may have an
yeast, fungi, and some enveloped viruses if there is important effect on AMP gene expression.59 In
low-salt concentration (e.g., 10 mM sodium phos- porcine bone marrow cells, RA induced expression
phate) in the environment.43 As the salt concentra- of cathelicidin PR-39; however, in human kerati-
tion increases, the activity of lung innate antibac- nocytes, induction of HBD-2, -3, and -4 mediated
terial factors such as HBD-1 and -2, as well as by high Ca2+ concentration, proinflammatory
lactoferrin, lysozyme, histatin, and cathelicidin can cytokines, phorbol myristate acetate (PMA), and
be completely inhibited, which likely occurs in the bacteria, was abolished when keratinocytes were
lungs of patients with cystic fibrosis.127 Using the preincubated with RA. The biological effect of RA
osmolyte xylitol to reduce airway surface liquid salt is achieved by its binding to and activation of the
concentration,153 and/or exogenously introducing nuclear retinoic acid receptors (RAR) that belongs
cationic AMP that are not salt-sensitive (e.g., sheep to the steroid/thyroid receptor family.42,59 RAR, in

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606 Grubor, Meyerholz, Ackermann Vet Pathol 43:5, 2006

turn, form heterodimers with the retinoid x with regulatory elements in their promoter regions.
receptors, which is followed by its direct binding The primary translational product of exons is
to specific cis-acting retinoic acid response elements a prepropeptide, which consists of an N-terminal
or by its antagonistic effect on AP-1 (c-Jun/c-Fos)- signal sequence for targeting of the endoplasmic
mediated gene expression. Because the promoter reticulum, a pro segment for correct folding of the
regions of all inducible HBD genes contain putative mature peptide, intracellular trafficking, and/or
binding sites for AP-1, it is possible that AP-1 plays inhibition of activity of the mature peptide, and
an important role in the signal transduction during a C-terminal cationic peptide that has antimicro-
induction and downregulation of defensin gene bial activity after cleavage.78,81 Cleavage of the
expression.59 propeptide occurs during later stages of intracellu-
lar processing or after secretion. AMP can be
Peptide characteristics
stored in cells as propeptides or as mature C-
Cationic AMP are 12–50 amino acids in length, terminal peptides.
and have a net positive charge (+2 to +7) owing to
an excess of basic amino acids (arginine, lysine, and Conclusions
histidine).55 Different AMP are needed to deal with
different microorganisms and because of this, A significant amount of evidence from both in
single animal species can have more than 24 vitro and in vivo studies suggests that SP-A and
different AMPs.17 AMPs fall into 4 principal SP-D serve numerous functions in innate immune
categories based on their size, conformational processes. The use of recombinant lung surfactant
structure, or predominant amino acid structure.17,78 proteins in the treatment of diseases such as
These comprise group I: linear, a-helical peptides respiratory distress syndrome in neonates, emphy-
without cysteines (e.g., SMAP and porcine cecro- sema and chronic obstructive pulmonary disease,
pin P1); group II: b-sheet structures stabilized by 2– cystic fibrosis, and asthma has already shown
3 disulfide bridges (e.g., a- and b-defensins, rhesus promising results.24,25 Development of collectin-
theta defensin-1, and protegrins); group III: linear based therapies for various pulmonary diseases
peptides rich in 1 or more amino acids (e.g., PR-39, warrants further investigation.
Bac5, Bac7, indolicidin, and prophenin); and group With increasing development of antibiotic re-
IV: peptides with loop structures (e.g., bactenecin sistance, there is a need to develop new classes of
and ranalexin). In general, cationic AMPs with best antibiotics. Cationic AMPs have many of the
antimicrobial activity are molecules that have the desirable features of such substances, which include
charged and hydrophilic portions separated from a broad spectrum activity, rapid killing of bacteria,
the hydrophobic areas. This means that either activity against classic antibiotic-resistant variants,
amphipathic or cationic double-wing structures synergy with antibiotics, neutralization of endotox-
with a hydrophobic core separating 2 charged ins, and activity in vivo.132 However, despite all
segments are preferred, which is determined by these positive features, there are still many issues
their tertiary structure.132 There is also a group of that need to be addressed in order for these
cationic AMP, which are formed by proteolytic molecules to be used as systemic therapeutic agents.
digestion of larger peptides, such as lactoferricins These include production costs as a result of their
that are derived from lactoferrin.15 high molecular weight, potential toxicity, and
peptide liability to proteases in the body.55
Cationic AMP genes
AMPs of vertebrates are ribosomally synthe- Acknowledgements
sized, gene-encoded peptides, meaning that 1 gene
codes for 1 peptide.78 In humans, mice, and rats, This work was supported in part by the National
both a- and b-defensins colocalize on the chromo- Institutes of Health NIAID Awards 5R01AI062787-02
some region that is on human chromosome 8p21– and 5K08AI055499-03, USDA/CSREES/NRI-CGP
23, mouse chromosome 8, and rat chromosome 2003-35204-13492, and the JG Salsbury Endowment.
Special thanks to Jack M. Gallup for his contributions
16.55,90,131 In sheep, b-defensin genes that comprise to our projects.
two exons (e.g. SBD-1 and -2) have been mapped
to sheep chromosome 26, whereas the 4-exon
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