Histology and Cell Biology: An

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Abraham L Kierszenbaum M.D. Ph.D.
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HISTOLOGY AND CELL BIOLOGY
An Introduction to Pathology
Thispageintentionallyleftblank
HISTOLOGY AND CELL BIOLOGY
An Introduction to Pathology

Fifth Edition

Abraham L. Kierszenbaum, M.D., Ph.D.

Medical (Clinical) Professor Emeritus
Former Chair of the Department of Cell Biology and Anatomy
The Sophie Davis School of Biomedical Education*
The City University of New York
New York, New York USA

Laura L. Tres, M.D., Ph.D.

Medical (Clinical) Professor Emerita
The Sophie Davis School of Biomedical Education*
The City University of New York
New York, New York USA

*Now CUNY School of Medicine/ Sophie Davis Biomedical Education Program

Elsevier
1600 John F. Kennedy Blvd.
Ste 1600
Philadelphia, PA 19103-1899

HISTOLOGY AND CELL BIOLOGY: ISBN: 978-0-323-67321-1

AN INTRODUCTION TO PATHOLOGY, FIFTH EDITION

Copyright © 2020 by Elsevier, Inc. All rights reserved

No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying, recording, or any information storage
and retrieval system, without permission in writing from the publisher. Details on how to
seek permission, further information about the Publisher’s permissions policies and our
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the Publisher (other than as may be noted herein).

Notice
Practitioners and researchers must always rely on their own experience and knowledge
in evaluating and using any information, methods, compounds or experiments described
herein. Because of rapid advances in the medical sciences, in particular, independent
verification of diagnoses and drug dosages should be made. To the fullest extent of the
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jury and/or damage to persons or property as a matter of products liability, negligence or
otherwise, or from any use or operation of any methods, products, instructions, or ideas
contained in the material herein.

Previous editions copyrighted 2016, 2012, 2007, and 2002.

Library of Congress Control Number: 2019940508

Content Strategist: Alexandra Mortimer

Senior Content Development Specialist: Ann Ruzycka Anderson
Publishing Services Manager: Catherine Jackson
Senior Project Manager: Daniel Fitzgerald

Printed in Canada.
Last digit is the print number: 9 8 7 6 5 4 3 2 1
 To our daughters, Adriana and Silvia
To our grandchildren, Ryan, Trevor, Kyle and Marielle
To the beloved memory of our parents

This book is also dedicated to you, the teacher,

who transmits enthusiastically the significance of
knowledge in a way that goes beyond what is being
taught; and to you, the student, who transforms the
act of learning into the passion of learning.
Thispageintentionallyleftblank
 PREFACE
The fifth edition of Histology and Cell Biology: An Introduction to Pathology contains
substantial revisions and additions. They strengthen the visual approach to learning histology
within the context of cell biology and pathology introduced in the previous editions. New in
the fifth edition are a number of Primers included in most chapters. Each Primer transmits
information about a specific topic in a concise and visual format to transcend concepts and
stimulate further exploration. The convergence of histology–cell biology–pathology intends
to prepare medical students for the forthcoming learning of pathophysiology and clinical
medicine. The practice of medicine changes relentlessly as new knowledge emerges. Future
physicians can find in this book the basis for continuing education to better help their
patients by constantly integrating basic and clinical sciences.

New in the fifth edition are Primers
Each Primer transmits information
about a specific topic in a concise
and visual format to transcend
concepts and stimulate further
exploration.
The visual approach presented in this book emerged from many years of practicing pathol-
ogy and teaching cell biology, histology and pathology to medical students. The conver-
gence of histology, cell biology and pathology promotes unity in diversity. Diversity leads to
the transforming power of new knowledge. The cell biology and pathology components,
although not complete, provide the necessary foundation for further learning and integra-
tion with medical sciences. Pathology students and residents may find this book useful for
refreshing basic concepts of histology and cell biology. Histology and pathology are visually
oriented sciences, and the visual cues included in this book can facilitate interpretation op-
portunities in clinical practice.

Similar to the previous editions, the fifth edition consists of six parts, bringing together
histology, cell biology and general pathology within the context of the basic tissues and
organ systems. Chapter 3, Cell Signaling | Cell Biology | Pathology, is an uncommon section
in a histology book. It serves to unify the concept that the study of tissues and organs cannot
be separated from the increasing impact of molecular biology in the practice of medicine.

New in this edition is the use of a light green background to identify in each chapter sec-
tions presenting essential concepts of histology. A number of teachers may find this offering
Each
NewConcept Mapping
in the fifth editionprovides a
is the use useful as a starting point for further learning. The expectation is that the additional mate-
basic
offramework
a light green of background
interconnected
concepts arranged
to identify sectionsin presenting
a hierarchi- rial could stir curiosity to unfold the indispensable complement of fragmented knowledge.
cal form leading concepts
essential to integration
of and All the information is presented in a clear, concise and student-friendly manner using color
critical
histology andthinking.
cell biology, graphics and photographs that are meant to be studied. In some cases the graphics reiterate
a starting point for further learning. the succinct text; in others they add new information complementing or extending the text.
Several boxes dispersed in all chapters introduce students to clinical and pathological condi-
tions and to recent and evolving molecular and biochemical knowledge.

Each Concept Mapping provides a
basic framework of interconnected
concepts arranged in a hierarchical
form, leading to integration and
critical thinking.
Students may find the online
animation version of Concept
Mappings convenient for group
interaction, transforming the pas-
sivity of learning into a dynamic
and collective activity.
Most chapters include one or more Concept Mappings. Each Concept Mapping provides
a basic framework of interconnected concepts arranged in a hierarchical form, leading to
integration and critical thinking. Concept Mapping and Essential Concepts highlight key
issues to review and integrate when the time of in-course and board examinations arrives.
Students may find the online animation version of Concept Mappings convenient for group
interaction, transforming the passivity of learning into a dynamic and collective activity.
Activity inspired by a new mode of communication, depending not only on content but also
the integrative vision and shared values of the information.
There are many people to be acknowledged and thanked. We are grateful for the
numerous suggestions, comments and encouragements from faculty and students. We thank
publishers who made available to faculty and students the Chinese, French, Greek, Japanese,
Portuguese, Spanish and Turkish editions. We thank the British Medical Association for
awarding the First Prize in Basic and Medical Sciences to the second edition. Our special
appreciation goes to Alexandra Mortimer, Ann Ruzycka Anderson and Daniel Fitzgerald
in London, New York and St. Louis offices for their magnificent effort in making sure that
the fifth edition met high publishing standards.

Abraham L. Kierszenbaum | Laura L. Tres

vii
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 HISTOLOGY AND CELL BIOLOGY | An Introduction to Pathology | CONTENTS

Protein nuclear import and export, 44

BASIC TISSUES | CELL BIOLOGY X chromosome inactivation, 44
Cellular localization of nucleic acids, 44
Chapter 1 | EPITHELIUM | CELL BIOLOGY Primer 1-H | Ran GTPase directs nucleocytoplasmic
Classification of epithelia, 2 bidirectional transport, 45
Epithelial cell polarity, 2 Cell cycle, 46
Box 1-A | General characteristics of epithelia, 2 Autoradiography and FACS, 47
Concept Mapping | Epithelium, 3 Box 1-I | PAS and Feulgen reactions, 47
Apical domain, 3 Box 1-J | Basophilia and acidophilia, 47
Cilia, 3 Disassembly of the nuclear envelope, 47
Multiple motile cilia, 3 The mitotic cycle, 47
Single or primary non-motile cilium, 3 Karyotyping (chromosome analysis), 47
Microvilli, 7 Box 1-K | Cytochemistry and histochemistry methods used in
Stereocilia (stereovilli), 7 Histology and Pathology, 49
Cell adhesion molecules and cell junctions, 7 Retinoblastoma (Rb) protein, 50
Ca2+-dependent molecules, 7 Retinoblastoma tumors, 51
Cadherins, 7 Primer 1-I | Disassembly and reassembly of the nuclear
Selectins (C-type lectins), 8 envelope, 52
Primer 1-A | Ciliogenesis. Primary cilium and Hedgehog Box 1-L | Summary of cell division, 54
signaling, 9 p53 protein, a transcription regulator, 55
Ca2+-independent molecules, 9 Box 1-M | Li-Fraumeni syndrome, 56
Superfamily of immunoglobulin-like cell adhesion Telomerase: Aging, senescence and cancer, 56
molecules (Ig-CAMs), 9 Basic concepts of Medical Genetics, 58
Integrins,11 Chromosomal disorders, 58
Primer 1-B | Transmigration of leukocytes through the Concept Mapping | A glossary of Human Genetics, 59
endothelial barrier, 12-13 Mendelian inheritance, 60
Cell junctions, 12 Box 1-N | Pedigree analysis, 60
Tight junctions, 13 Non-mendelian inheritance, 61
Adhering junctions, 14 Concept Mapping | Epithelial Differentiations, 62
Primer 1-C | ADAM, a member of the family of sheddase Essential Concepts | Epithelium, 62
proteins, 15
Hemidesmosomes, 17 Chapter 2 | EPITHELIAL GLANDS | CELL BIOLOGY
Box 1-B | Tight junctions and disease, 18 Epithelial glands, 68
Gap junctions or communicating junctions, 19 Types of epithelial glands, 68
Connexin mutations, 20 Components of exocrine glands, 68
Basement membrane, 21 Composition of secretion of an exocrine gland, 69
Box 1-C | Periodic acid–Schiff (PAS) reaction, 21 Components of a branched (compound) gland, 72
Primer 1-D | Cell adhesion molecules, cell junction and Mechanisms of secretion of an exocrine gland, 72
basement membrane, 22 Plasma membrane and cytomembranes, 73
Cytoskeleton, 23 Plasma membrane, 73
Microfilaments, 23 Types of lipids and lipid domains, 73
Primer 1-E | Actin microfilaments: Assembly and Box 2-A | Lipid rafts, 73
disassembly, 25 Plasma membrane proteins, 74
Microtubules, 26 Transporter and channel proteins, 75
Box 1-D | Wiskott-Aldrich syndrome, 26 Box 2-B | Glycocalyx, 75
Centrosomes, 27 Endoplasmic reticulum, 75
Axoneme, 29 Primer 2-A | Freeze-fracture, 76
Microtubule-targeting agents, 29 Freeze-fracture technique, 77
Box 1-E | Centrosomes, centromeres and kinetochores, 29 Protein synthesis and sorting, 77
Ciliopathies, 30 Golgi apparatus, 79
Box 1-F | Bardet-Biedl syndrome, 30 Functions of the Golgi apparatus, 79
Cargo transport and motor proteins, 30 Primer 2-B | Protein synthesis, 80
Primer 1-F | Intraciliary and axonal (neuronal) transport, 31 Vesicle transport, 81
Intraciliary transport, 31 Lysosomal sorting pathway, 82
Axonal (neuronal) transport, 32 Cholesterol uptake by receptor-mediated endocytosis, 82
Myosin motor proteins, 32 Sorting of clathrin- and COP-coated vesicles, 83
Primer 1-G | Myosin motor proteins, 33 Box 2-C | Familial hypercholesterolemia, 83
Smooth muscle and myosin light-chain kinase, 34 Primer 2-C | Clathrin- and COP-mediated vesicle transport
Intermediate filaments, 35 and targeting of transporting vesicles, 84-85
Box 1-G | Summary: Intermediate filament proteins, 36 Lysosomes, 84
Hemidesmosomes and skin blistering diseases, 37 Box 2-D | Macroautophagy and autophagy, 85
Cell nucleus, 38 Primer 2-D | Lysosomes, 86
Nuclear lamina, 39 Phagocytosis, endocytosis and macroautophagy, 87
Laminopathies, 40 Lysosome storage disorders, 87
Box 1-H | Clinical aspects of several laminopathies, 40 Box 2-E | Lysosome hydrolytic enzymes can be secreted, 87
Chromatin, 40 Primer 2-E | Lysosome storage disorders: Tay-Sachs disease
Chromatin condensation and transcription, 42 and Gaucher’s disease, 88
Nucleolus, 43 Mitochondria, 89

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CONTENTS | HISTOLOGY AND CELL BIOLOGY | An Introduction to Pathology
Mitochondria participate in apoptosis, steroidogenesis
and thermogenesis, 91
Mitochondria maternal inheritance, 92
Mitochondria replacement therapies, 93
Peroxisomes, 93
Peroxisome biogenesis, 94
Peroxisome biogenesis disorders (PBDs), 95
Concept Mapping | Epithelial Glands, 96
Essential Concepts | Epithelial Glands, 96
Chapter 3 | CELL SIGNALING | CELL BIOLOGY | PATHOLOGY
Cell signaling mechanisms, 100
Cell signaling and feedback action, 100
Types of signaling molecules and their ligands, 100
Steroid hormones, 100
Box 3-A | Steroid hormones, 101
Peptide hormones and growth factors, 102
Nitric oxide, 102
Box 3-B | Peptide hormones, 102
Receptor and non-receptor tyrosine kinases, 103
Neurotransmitters, 104
Eicosanoids, 104
Box 3-C | Eicosanoids, 104
Cell surface receptors, 104
G protein–coupled receptors, 104
Cytokine receptors, 105
Receptors with tyrosine phosphatases activity, 106
Major signal transduction pathways, 106
The cAMP pathway, 106
The cGMP pathway, 107
The phospholipid–calcium pathway, 107
The calcium–calmodulin pathway, 108
The Ras–Raf / MAP kinase (MEK–ERK) pathway, 108
The JAK-STAT pathway, 109
NF-gB transcription factor pathway, 109
The integrin-actin pathway, 109
Specific signaling pathways, 110
Stem cell niches and stemness, 110
Primer 3-A | Specific cell signaling pathways, 111
Primer 3-B | Specific cell signaling pathways, 112
Regenerative medicine and cell plasticity, 112
Box 3-D | Epithelial-mesenchymal transition (EMT), 113
Cell culture, 115
Cell and tissue injury, 115
Concept Mapping | Cell death, necrosis and apoptosis, 116
Necrosis, 116
Apoptosis, 117
What a nematode worm told us about apoptosis, 117
Extrinsic and intrinsic signaling of apoptosis, 118
Caspases: Initiators and executioners of cell death, 119
Intrinsic pathway: Mitochondria cytochrome c, 120
Apoptosis and the immune system, 120
Apoptosis and neurodegenerative diseases, 120
Necroptosis, 121
Primer 3-C | Necroptosis, 122
Mitochondria permeability transition, 123
Intracellular degradation, 123
Autophagy pathway, 123
Ubiquitin–proteasome pathway, 124
Mitophagy signaling pathway, 125
Neoplasia, 125
Proto-oncogenes, oncogenes and tumor suppressor
genes, 126
Concept Mapping | Neoplasia, 127
Box 3-E | Proto-oncogenes and tumor suppressor
proteins, 128
Concept Mapping | Oncogenes and tumor suppressor
genes, 129
x
Identification of oncogenes in retroviruses, 130
Box 3-F | Proto-oncogenes and oncogenes, 130
Concept Mapping | Cell Signaling, 131
Essential Concepts | Cell Signaling, 131
Chapter 4 | CONNECTIVE TISSUE
Classification, 136
Box 4-A | Types of collagens, 136
Components of connective tissue, 137
Fibroblast, 137
Box 4-B | Diverse cell sources of collagen, 138
Synthesis, secretion and assembly of collagen, 139
Ehlers-Danlos syndrome, 140
Elastic fibers, 140
Box 4-C | Characteristics of collagens, 142
Marfan’s syndrome, 143
Macrophages, 145
Box 4-D | Metachromasia, 145
Mast cells, 146
Plasma cells, 146
Box 4-E | Allergic hypersensitivity reactions, 146
Extracellular matrix (ECM), 147
Degradation of the ECM, 148
Molecular biology of tumor invasion, 149
Primer 4-A | Tumor invasion and metastasis, 150-151
Adipose tissue or fat, 151
Adipogenesis, 152
Lipid storage and breakdown (lipolysis), 154
Box 4-F | Visualization of fat in histology sections, 154
Leptin and obesity, 154
Cartilage, 155
Box 4-G | Survival of chondrocytes, 155
Box 4-H | Cartilage repair after injury, 155
Chondrogenesis, 155
Types of cartilage, 156
Box 4-I | Cartilage of the joints, 160
Box 4-J | Sox9 transcription factor, 160
Bone, 160
Macroscopic structure of mature bone, 160
Microscopic structure of mature bone, 161
Periosteum and endosteum, 161
Bone matrix, 162
Cellular components of bone, 163
The osteoblast, 163
Primer 4-B | Genes involved in osteoblast differentiation, 166
Differentiation of preosteoblasts to osteoblasts to
osteocytes, 166
The osteoclast, 167
Osteoclastogenesis, 169
Osteoporosis, 170
Osteopetrosis and osteomalacia, 171
Concept Mapping | Connective Tissue, 172
Essential Concepts | Connective Tissue, 172
Chapter 5 | OSTEOGENESIS
Osteogenesis (Bone development or ossification), 178
Intramembranous ossification, 178
Endochondral ossification, 178
Secondary centers of ossification, 180
Zones of endochondral ossification, 181
Growth in length of the diaphysis, 181
Hedgehog signaling: The epiphyseal growth plate
and dwarfism, 182
Conversion of trabecular bone into osteons, 184
Box 5-A | Metaphyseal chondrodysplasia, 184
Bone remodeling, 188
Bone fracture and healing, 189
Primer 5-A | Bone fracture and healing, 190
 HISTOLOGY AND CELL BIOLOGY | An Introduction to Pathology | CONTENTS

Bone disorders, 191 Box 7-A | Myasthenia gravis, 244

Concept Mapping | Metabolic and hereditary bone T tubules, calcium ions and muscle contraction, 245
disorders, 192 Box 7-B | Functional types of muscle fibers, 245
Joints, 192 Muscular dystrophies, 246
Rheumatoid arthritis, 192 Satellite cells and muscle repair, 249
Concept Mapping | Osteogenesis, 195 Neuromuscular spindle and Golgi tendon organ, 249
Essential Concepts | Osteogenesis, 195 Primer 7-A | Satellite cells and muscle repair, 250
Cardiac muscle, 253
Chapter 6 | BLOOD AND HEMATOPOIESIS Transport proteins and the sarcolemma, 253
Blood, 200 Myocardial infarction, 253
Plasma, 200 Smooth muscle, 255
Red blood cells (RBC; erythrocytes), 200 Mechanism of smooth muscle contraction, 256
Cytoskeletal and hemoglobin abnormalities of red blood Concept Mapping | Muscle Tissue, 258
cells, 200 Essential Concepts | Muscle Tissue, 258
Hemoglobin A1c (glycated hemoglobin) and diabetes
mellitus, 201 Chapter 8 | NERVOUS TISSUE
Erythroblastosis fetalis, 201 Development of the nervous system, 262
Box 6-A | Hemolysis in erythroblastosis fetalis, 202 Box 8-A | Ectoderm germ cell layer, 263
Leukocytes, 202 Cell types: Neurons, 263
Granulocytes, 202 Box 8-B | Brain development, 264
Neutrophils, 202 Types of neurons, 264
Eosinophils, 203 Designation of neurons and axons, 264
Box 6-B | Blood cells /+L or mm3, 203 Synaptic terminals and synapses, 265
Box 6-C | Primary and specific granules, 204 Axonal transport, 265
Basophils, 204 Box 8-C | Neural tube defects, 265
Box 6-D | Eosinophilic esophagitis, 205 Box 8-D | Neuronal migration, 265
Agranulocytes, 205 Box 8-E | Cerebral cortex, 267
Lymphocytes, 205 Glial cells, 270
Monocytes, 206 Astrocytes, 271
Leukocyte recruitment and inflammation, 207 Box 8-F | Neurotransmitters: Mechanisms of action, 271
Box 6-E | Leukocyte adhesion deficiency (LAD), 208 Oligodendrocytes, 272
Mast cell, eosinophil and asthma, 208 Myelinization, 272
Platelets, 208 Myelin, 274
Coagulation disorders, 211 Box 8-G | Charcot-Marie-Tooth disease, 275
Hemostasis and blood clotting, 211 Unmyelinated nerve fibers, 275
Box 6-F | Hemophilia, 211 Demyelinating diseases, 277
Primer 6-A | Blood clotting pathways, 212 Neurodegenerative diseases, 254
Hematopoiesis, 213 Box 8-H | Amyloid deposits, 281
The vascular niche, 213 Microglia, 281
The endosteal niche, 213 Function of microglia, 281
Hematopoietic cell populations, 216 Primer 8-A | Microglia, 282
Hematopoietic growth factors, 216 Ependyma, 283
Erythroid lineage, 216 Choroid plexus, 283
Leukopoiesis, 218 Cerebrospinal fluid, 283
Granulocytes, 219 Brain permeability barriers, 285
Box 6-G | Anemia, 219 Peripheral nervous system, 287
Agranulocytes: Lymphocytes, 221 Structure of a peripheral nerve, 287
Monocytes, 224 Segmental demyelination and axonal degeneration, 287
Colony-stimulating factors and interleukins, 225 Autonomic nervous system, 291
c-kit receptor and its ligand stem cell factor, 225 Enteric nervous system, 291
Leukemias, 227 Sympathetic and parasympathetic nervous divisions, 291
Megakaryocytes and platelets, 229 Box 8-I | Neurotrophins, 291
Iron-overload disorders, 229 Box 8-J | Schwannomas, 291
Primer 6-B | Uptake of iron by internalization of Autonomic (sympathetic) ganglia, 293
transferrin and iron-linked disorders, 230 Sensory (spinal) ganglia, 293
Megaloblastic anemia, 232 Neurohistochemistry, 293
Concept Mapping | Blood and Hematopoiesis, 233 Box 8-K | Neurotransmitters: Classification, 295
Essential Concepts | Blood and Hematopoiesis, 233 Concept Mapping | Nervous Tissue, 295
Essential Concepts | Nervous Tissue, 296
Chapter 7 | MUSCLE TISSUE
Skeletal muscle, 238 Chapter 9 | SENSORY ORGANS: VISION AND HEARING
Skeletal muscle cell or fiber, 238 Eye, 300
The sarcomere, 240 Development of the eye, 300
Components of the sarcomere, 241 Outer tunic: Sclera and cornea, 301
Sarcomere changes during muscle contraction, 243 Cornea, 301
Creatine phosphate, 244 Box 9-A | Development of the cornea, 301
Neuromuscular junction: Motor end plate, 244 Middle tunic: Uvea, 302
Disorders of neuromuscular synaptic transmission, 244 Box 9-B | Cornea transplantation, 302

xi
 CONTENTS | HISTOLOGY AND CELL BIOLOGY | An Introduction to Pathology
Box 9-C | Uvea, 303
Three chambers of the eye, 305
Lens, 309
Box 9-D | Cataracts, 312
Accommodation, 312
Inner layer: Retina, 312
Cell layers of the retina, 313
Box 9-E | Detachment of the retina, 313
Photoreceptor neurons: Rods and cones, 313
Box 9-F | The retina, 314
Conducting neurons: Bipolar and ganglion cells, 314
Association neurons: Horizontal and amacrine
cells, 317
Supporting glial cells: Müller cells, 317
Fovea centralis and optic disk, 317
Box 9-G | The synaptic ribbon, 321
The eyelids, conjunctiva and the lacrimal gland, 321
Box 9-H | Retinitis pigmentosa, 322
Box 9-I | Red eye and conjunctivitis, 323
Ear, 325
External ear, 326
Middle ear, 326
Inner ear: Development of the inner ear, 327
General structure of the inner ear, 328
Vestibular system, 328
Semicircular canals, 328
Otolithic organs: Utricle and saccule, 331
Box 9-J | Ménière’s disease, 332
Cochlea, 332
Organ of Corti, 334
Molecular and mechanical aspects of the hearing
process, 334
Deafness and balance, 338
Concept Mapping | Sensory Organs: Eye, 338
Essential Concepts | Sensory Organs: Vision and
Hearing, 339
Concept Mapping | Sensory Organs:
Ear, 341
ORGAN SYSTEMS | PROTECTION OF THE BODY
Chapter 10 | IMMUNE-LYMPHATIC SYSTEM
Components of the immune–lymphatic system, 344
Types of immunity, 345
Box 10-A | Toll-like receptors, 345
Properties of adaptive or acquired immunity, 346
Development and maturation of B cells in bone
marrow, 346
Major histocompatibility complex (MHC) and the human–
equivalent leukocyte antigens (HLA), 347
Box 10-B | CD antigens, 347
T-cell receptor, 348
LCK and CD4 and CD8 coreceptors, 348
Box 10-C | The immune synapse, 348
Thymocyte maturation in the thymus: Positive and negative
selection, 348
Primer 10-A | Structure of the T cell receptor and class I
and II major histocompatibility complex (MHC), 349
Box 10-D | Immunoglobulins, 351
CD4+ T-cell subsets: TH1, TH2, TH17 and TFH cells, 351
How do CD4+ helper T cells help?, 352
Box 10-E | Multiple myeloma, 352
How do CD8+ cytolytic T cells kill?, 353
Natural killer cells, 353
Acquired immunodeficiency syndrome (AIDS), 354
Box 10-F | HIV reproductive cycle, 355
Hypersensitivity reactions, 355
xii
Primer 10-B | Immune system and HIV infection, 356
Complement system, 358
Primer 10-C | Complement system, 359
Inflammation, 360
Acute inflammation, 360
Concept Mapping | Acute inflammation, 360
Concept Mapping | Acute and chronic inflammation
compared, 362
Resolution of acute inflammation, 363
Types of acute inflammation, 363
Chronic inflammation, 363
Lymphoid organs, 365
Lymph nodes, 365
Lymphadenitis and lymphomas, 368
Box 10-G | Lymph flow and dendritic cell migration, 368
Thymus, 368
Development of the thymus, 368
Development of thymic epithelial cells, 369
Box 10-H | Aire gene and autoimmunity, 369
Structure of the thymus, 370
Box 10-I | DiGeorge syndrome, 370
Spleen, 375
Vascularization of the spleen, 377
White pulp, 379
Red pulp, 379
Sickle cell anemia, 379
Asplenia, 380
Cancer immunotherapy, 380
Tumor cells secrete exosomes carrying PDL1, 382
Concept Mapping | Immune-Lymphatic System, 383
Essential Concepts | Immune-Lymphatic System, 383
Chapter 11 | INTEGUMENTARY SYSTEM
Organization and types of skin, 390
Epidermis, 390
Differentiation of keratinocytes, 391
Melanocytes, 392
Melanin production by melanocytes, 394
Box 11-A | Cornified cell envelope disorders, 394
Box 11-B | Disorders of keratinization, 397
Box 11-C | Differentiation of melanocytes, 398
Langerhans cells (dendritic cells), 399
Merkel cell, 400
Dermis, 400
Box 11-D | Leprosy, 400
Wound healing, 401
Concept Mapping | Wound healing, 401
Psoriasis, 402
Tumors of the epidermis, 404
Box 11-E | Tumors of the epidermis, 404
Epithelial antimicrobial proteins, 405
Skin: Blood and lymphatic supply, 405
Sensory receptors of the skin, 406
Box 11-F | Vascular disorders of the skin, 407
Hypodermis (superficial fascia), 409
Development of the hair follicle, 409
Structure of the hair follicle, 409
Lgr5+ stem cell pathways, 410
Glands of the skin. Sebaceous glands, 412
Sweat glands, 414
Cystic fibrosis, 415
Fingernails, 415
Concept Mapping | Integumentary System, 417
Essential Concepts | Integumentary System, 417
 HISTOLOGY AND CELL BIOLOGY | An Introduction to Pathology | CONTENTS
ORGAN SYSTEMS | BLOOD CIRCULATORY SYSTEMS
Chapter 12 | CARDIOVASCULAR SYSTEM
The cardiovascular system, 422
Heart, 422
Conductive system of the heart, 422
Purkinje fibers, 422
Blood vessels, 423
Arteries, 423
Large elastic arteries (conducting vessels), 424
Aortic aneurysms, 424
Muscular arteries (distributing vessels), 424
Arterioles (resistance vessels), 425
Capillaries (exchange vessels), 425
Types of capillaries, 426
Arterial and venous portal systems, 427
Veins (capacitance, or reservoir, vessels), 428
Vasculitis, 430
Box 12-A | Capillary endothelial barriers, 430
Lymphatic vessels, 432
Primer 12-A | Vasculitis, 433
Box 12-B | Lymphatic vascular disorders, 433
Edema, 434
Hemorrhage, 435
Atherosclerosis, 435
Functions of the endothelium, 435
Primer 12-B | Atherosclerosis, 436
Primer 12-C | Vasculogenesis and angiogenesis, 438-439
Vasculogenesis and angiogenesis, 439
Angiogenesis and tumor progression, 440
Box 12-C | Kaposi’s sarcoma, 440
Concept Mapping | Cardiovascular pathogenesis, 441
Thrombosis, embolism and infarction, 441
Hypertension, 442
Concept Mapping | Hypertension, 442
Concept Mapping | Cardiovascular System, 443
Essential Concepts | Cardiovascular System, 443
Chapter 13 | RESPIRATORY SYSTEM
The respiratory system, 448
Nasal cavities and paranasal sinuses, 448
Nasopharynx, 448
Olfactory epithelium, 449
Box 13-A | Olfactory epithelium, 449
Larynx, 451
Box 13-B | True vocal cords or folds, 451
Trachea and primary bronchi, 453
Box 13-C | Airway mucus, 453
Cystic fibrosis, 453
Box 13-D | Cystic fibrosis gene, 455
Segmentation of the bronchial tree, 455
Pulmonary lobule and pulmonary acinus, 458
Club cells (formerly Clara cells), 460
Respiratory portion of the lung, 460
The alveolus, 460
Alveolar type 2 (AT2) cells, 464
Bronchopulmonary diseases, 467
Asthma, 467
Chronic obstructive pulmonary disease, 467
Acute respiratory distress syndrome, 468
Primer 13-A | Asthma, 468-469
Lung cancer, 472
Box 13-E | Lung cancer immunotherapy, 473
Pleura, 473
Disorders of the pleura, 473
Essential Concepts | Respiratory System, 475
Concept Mapping | Respiratory System, 476
Chapter 14 | URINARY SYSTEM
The kidneys, 480
The renal vascular system, 480
Vasa recta, 480
Renal medullary pyramid, renal lobe and renal lobule, 482
The uriniferous tubule, 482
The renal corpuscle, 483
Glomerular filtration barrier, 484
Pathology of the GBM, 485
Box 14-A | Acute kidney injury, 485
Mesangium, 489
Podocyte injury, 490
Primer 14-A | Pathology of the renal corpuscle:
Glomerulonephritis, 492
Juxtaglomerular apparatus, 493
Proximal convoluted tubule, 493
Loop of Henle, 495
Box 14-B | Fibroblast growth factor (FGF) 23, the kidneys
and phosphate metabolism, 495
Distal convoluted tubule, 495
Collecting tubule/duct, 497
Renal interstitium, 497
Excretory passages of urine, 501
Box 14-C | Osmoregulation, 501
Regulation of water and NaCl absorption, 501
Primer 14-B | Renin-angiotensin system (RAS), 502
Renin-angiotensin system (RAS), 503
Countercurrent multiplier and exchanger, 504
Mechanism of action of diuretics, 506
Essential Concepts | Urinary System, 507
Concept Mapping | Urinary System, 507
ORGAN SYSTEMS | THE ALIMENTARY SYSTEM
Chapter 15 | UPPER DIGESTIVE SEGMENT
Mouth (oral cavity), 512
Lips, 512
Gingiva, hard and soft palate, 513
Tongue, 513
Types and function of taste receptor cells, 513
Tooth, 516
Tooth development, 517
Dental pulp, 517
Periodontium, 517
Odontoblasts, 519
Ameloblasts, 519
Non-neoplastic and neoplastic lesions of the oral
mucosa, 520
General organization of the digestive tube, 521
Box 15-A | Peptic ulcer disease (PUD), 522
Microvasculature of the stomach, 522
Enteric nervous system (ENS), 523
Esophagus, 524
Box 15-B | Gastroesophageal reflux disease (GERD), 526
Stomach, 526
Cardia region of the stomach, 528
Fundus-body region of the stomach, 529
Chief cells and parietal cells, 531
Secretion of hydrochloric acid, 531
Box 15-C | Ménétrier’s disease, 531
Box 15-D | Autoimmune gastritis, 531
Secretion of hydrochloric acid, 531
Infection with Helicobacter pylori, 533
Primer 15-A | Helicobacter pylori, chronic gastric
inflammation and ulcers, 534
Gastroenteroendocrine cells, 535
Box 15-E | Zollinger-Ellison syndrome, 537
xiii
 CONTENTS | HISTOLOGY AND CELL BIOLOGY | An Introduction to Pathology
Pyloric region of the stomach, 537
Concept Mapping | Upper Digestive Segment, 538
Essential Concepts | Upper Digestive Segment, 538
Chapter 16 | LOWER DIGESTIVE SEGMENT
Small intestine, 544
The peritoneum, 544
Intestinal wall, 544
Microcirculation of the small intestine, 545
Innervation and motility of the small intestine, 546
Histologic differences between the duodenum,
jejunum and ileum, 547
Villi and crypts of Lieberkühn, 547
Enterocytes: Absorptive cells, 547
Trafficking of peptides and sugars, 549
Trafficking of lipids and cholesterol, 551
Goblet cells, 553
Enteroendocrine cells, 553
Tuft cells, 553
Intestinal stem cells (ISCs), 553
Protection of the small intestine, 554
Intestinal tight junction barrier, 554
Peyer’s patches, 555
Follicle-associated epithelium (FAE), 556
Box 16-A | Development of Peyer’s patches, 558
Polymeric IgA, 559
Paneth cells, 560
Intestinal antimicrobial proteins (AMPs), 561
Box 16-B | Lgr5+-intestinal stem cells are regulated by
FoxL1+-telocytes located in the lamina propria, 561
Inflammatory bowel diseases, 562
Malabsorption syndromes, 563
Large intestine, 563
The appendix, 566
The rectum, 566
Hirschsprung’s disease, 568
Colorectal tumorigenesis, 568
Primer 16-A | APC (adenomatous polyposis coli) and
cancer of the colon, 569
Box 16-C | Lynch syndrome, 570
Concept Mapping | Lower Digestive Segment, 571
Essential Concepts | Lower Digestive Segment, 571
Chapter 17 | DIGESTIVE GLANDS
Structure of a salivary gland, 576
Box 17-A | Classification of exocrine glands, 576
Saliva, 576
Parotid gland, 576
Submandibular (submaxillary) gland, 577
Sublingual gland, 577
Box 17-B | Parotid gland: Mumps, rabies, autoimmunity
and tumors, 579
Exocrine pancreas, 579
Pancreatic tumors, 582
Functions of the pancreatic acinus, 585
Pancreatitis and cystic fibrosis, 586
Liver, 587
Organization of the hepatic lobule, 587
Concepts of the hepatic lobule, 589
Hepatocyte, 590
Peroxisomes, 594
Perisinusoidal cells, 594
Perisinusoidal cells and chronic liver disease, 594
Box 17-C | Liver iron-overload disorders, 594
Alcoholism and fatty liver (alcoholic steatohepatitis), 596
Chronic hepatitis and cirrhosis, 596
Box 17-D | Liver regeneration, 596
Primer 17-A | Metabolism of bilirubin, 598
xiv
Metabolism of bilirubin, 598
Gallbladder, 599
Hyperbilirubinemia, 599
Mechanism of bile secretion, 599
Composition of the bile, 601
Conditions affecting bile secretion, 602
Concept Mapping | Digestive Glands, 602
Essential Concepts | Digestive Glands, 602
ORGAN SYSTEMS | THE ENDOCRINE SYSTEM
Chapter 18 | NEUROENDOCRINE SYSTEM
Hypophysis, 608
Embryologic origin of the hypophysis, 608
Hypothalamohypophyseal portal circulation, 608
Histology of the pars distalis (anterior lobe), 611
Hormones secreted by acidophils: Growth hormone and
prolactin, 612
Growth hormone, 612
Gigantism (in children) and acromegaly (in adults), 613
Prolactin, 613
Hyperprolactinemia, 615
Hormones secreted by basophils: Gonadotropins,
TSH and ACTH, 615
Gonadotropins: Follicle-stimulating hormone and
luteinizing hormone, 615
Infertility, 616
Thyroid-stimulating hormone (thyrotropin), 616
Hypothyroidism, 617
Adrenocorticotropic hormone, 617
Cushing’s disease, 618
Neurohypophysis, 618
Histology of the neurohypohysis, 618
Function of VP/ADH and oxytocin, 619
Hypothalamic diabetes insipidus, 621
Pineal gland, 622
Development of the pineal gland, 622
Histology of the pineal gland, 625
Pinealocytes secrete melatonin, 625
Light is a regulator of circadian rhythms, 625
Pinealomas, 627
Concept Mapping | Neuroendocrine System, 628
Essential Concepts | Neuroendocrine System, 628
Chapter 19 | ENDOCRINE SYSTEM
Thyroid gland, 632
Development of the thyroid gland, 632
Histologic organization of the thyroid gland, 632
Function of the thyroid gland, 632
Graves’ disease and hypothyroidism, 636
Box 19-A | Pathology of the thyroid gland, 636
Calcium regulation, 638
Parathyroid glands, 639
Development of the parathyroid glands, 639
Histology of the parathyroid glands, 639
Signal transduction mediated by CaSR, 639
Functions of the parathyroid hormone, 639
Dysfunction of the parathyroid glands, 641
CaSR and Gq/11 mutations, 641
Box 19-B | Rickets and osteomalacia, 641
C cells (thyroid follicle), 641
Vitamin D (calcitriol), 643
Adrenal (suprarenal) glands, 644
Development of the adrenal gland, 644
Functions of the fetal adrenal cortex, 644
Histology of the adrenal cortex, 644
Zona glomerulosa, 648
 HISTOLOGY AND CELL BIOLOGY | An Introduction to Pathology | CONTENTS
Zona fasciculata, 648
Zona reticularis, 650
Dysfunction of the adrenal cortex, 650
Box 19-C | Congenital adrenal hyperplasia, 650
Adrenal medulla, 650
Blood supply to the adrenal gland, 653
Box 19-D | Pheochromocytoma, 653
Endocrine pancreas, 653
Development of the pancreas, 653
Islets of Langerhans, 653
Peptides produced by cells of the islets of Langerhans, 656
Cell entry and fate of insulin, 656
Cell entry and fate of glucose, 656
Diabetes mellitus, 658
Box 19-E | The Nrf2–Keap1 pathway and diabetes, 659
Concept Mapping | Endocrine System, 660
Essential Concepts | Endocrine System, 660
ORGAN SYSTEMS | THE REPRODUCTIVE SYSTEM
Chapter 20 | SPERMATOGENESIS
The testes, 664
Seminiferous epithelium, 664
Basal and adluminal compartments, 666
The spermatogenic cell progeny, 666
Primer 20-A | The spermatogenic cell progeny, 667
Sertoli cells, 670
Box 20-A | Androgens and spermatogenesis, 671
Spermatogonia, 671
Box 20-B | Sertoli cell–only syndrome (SCOS), 672
Regulation of spermatogonia cell function, 672
Spermatocytes, 673
Meiosis, 673
Primer 20-B | Meiosis I: Prophase I (from leptotene to
pachytene), 675
Primer 20-C | Meiosis I: Prophase I (from diplotene to
diakinesis), 676
Primer 20-D | Molecular structure of the synaptonemal
complex, 677
Spermatids, 678
Box 20-C | Acroplaxome, 678
Primer 20-E | Manchette and acroplaxome, 682
Shaping spermatids into fertilizing sperm, 683
Box 20-D | Intramanchette transport (IMT), 683
Completion of spermiogenesis, 683
Structure of the sperm, 683
Conditions affecting male fertility, 685
Box 20-E | Semen analysis, 685
Temperature, 685
Cryptorchidism, 685
Inguinal hernia, cysts and hydrocele, 685
Cancer chemotherapy, 685
Viral orchitis, 686
Spermatic cord torsion, 687
Varicocele, 687
Leydig cells, 687
Steroidogenic acute regulatory protein (StAR), 687
Bioregulation of spermatogenesis, 688
Box 20-F | Actions of testosterone in the male
reproductive system, 688
The spermatogenic cycle, 689
Epigenetics reprogramming, 692
Testicular tumors, 695
Essential Concepts | Spermatogenesis, 696
Concept Mapping | Spermatogenesis, 697
Chapter 21 | SPERM TRANSPORT AND MATURATION
Development of the gonads, 702
Development of the testes, 702
Primer 21-A | Migration of primordial germinal cells from
the yolk sac to the gonadal ridges, 703
Development of internal genitalia, 704
Box 21-A | Development of internal genitalia, 704
Testicular descent, 704
Androgen insensitivity syndrome (AIS), 704
Steroid 5_-reductase 2 deficiency, 705
Sperm maturation pathway, 705
The epididymal ducts, 707
Box 21-B | Epididymal duct, 709
Vas deferens, spermatic cord and ejaculatory duct, 709
The azoospermia factor (AZF), 710
Box 21-C | Klinefelter’s syndrome, 710
Accessory genital glands, 710
Seminal vesicles, 711
Box 21-D | Seminal fluid (semen), 711
Prostate gland, 711
Benign prostatic hyperplasia, 714
The androgen receptor, 715
Prostate cancer and tumor suppressor genes, 716
Male and female urethra, 716
Penis, 717
Box 21-E | Erectile dysfunction, 718
Bulbourethral glands, 719
Concept Mapping | Sperm Transport and
Maturation, 719
Essential Concepts | Sperm Transport and
Maturation, 719
Chapter 22 | FOLLICULOGENESIS AND MENSTRUAL CYCLE
Development of the female reproductive tract, 724
Development of the ovary, 724
Development of the female genital ducts, 724
Box 22-A | Turner’s syndrome, 724
Box 22-B | Müllerian duct development. The role of anti-
müllerian hormone in folliculogenesis, 724
Primer 22-A | From the indifferent gonad to the ovary
and testis, 725
Development of the external genitalia, 726
The ovaries, 726
Box 22-C | Lgr5+ stem cells in the ovarian surface
epithelium, 726
The ovarian cycle, 726
Granulosa cell–primary oocyte interaction, 729
Primer 22-B | Granulosa cell–primary oocyte interaction, 730
Box 22-D | Polycystic ovary syndrome, 731
Box 22-E | Ovarian hormones, 731
Theca interna–granulosa cell interaction, 731
Follicular atresia or degeneration, 732
Ovulatory phase, 732
Box 22-F | Follicular atresia, 733
Luteal phase: Luteinization and luteolysis, 733
Hormonal regulation of the menstrual cycle, 736
Oviduct, fallopian or uterine tube, 737
The uterus, 737
Box 22-G | Decidualization, 743
Hypogonadotropic hypogonadism and GnRH, 743
Endometriosis, 744
Cervix and vagina, 744
Cervical cancer and high-risk human papillomavirus
infection, 746
Diagnostic cytopathology, 748
Mons pubis, labia majora and labia minora, 748
Female urethra and glands (paraurethral glands and
Bartholin’s glands), 748
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 CONTENTS | HISTOLOGY AND CELL BIOLOGY | An Introduction to Pathology
Concept Mapping | Folliculogenesis and
Menstrual Cycle, 749
Essential concepts | Folliculogenesis and
Menstrual Cycle, 749
Chapter 23 | FERTILIZATION, PLACENTATION AND LACTATION
Fertilization, 756
Sperm capacitation, 756
Acrosome reaction and sperm-egg fusion, 756
Box 23-A | Tetraspanins, 757
Box 23-B | Oocyte activation, 757
Conditions leading to fertilization, 759
Box 23-C | Fertilization in vitro, 759
Implantation of the blastocyst, 760
Box 23-D | Timetable of implantation, 760
Differentiation of the trophoblast, 760
Immunoprotective decidua during implantation, 761
Primary, secondary and tertiary villi, 762
Box 23-E | Trophoblast cells, 763
Structure of the placenta, 763
Decidua basalis and chorion, 764
Placental blood circulation, 764
Structure of the chorionic villus, 767
Functions of the placenta, 769
Exchange of gases, 769
Transfer of maternal immunoglobulins, 769
Rh (D antigen) isoimmunization, 769
The fetoplacental unit, 769
The luteal-placental shift, 769
xvi
Active transport of ions and glucose, 770
Fetal alcohol syndrome, 770
Infectious agents, 770
Placenta and fetal tissues and the maternal
immune system, 770
Abnormal placentation, 771
Box 23-F | Ectopic pregnancy, 771
Box 23-G | Hydramnios, 771
Gestational trophoblastic diseases, 772
Box 23-H | Placenta previa, 772
Lactation, 773
The mammary glands, 773
Morphogenesis of the mammary glands, 774
Mammary gland development, 774
Mammary glands during puberty and pregnancy, 776
Histology of the mammary glands, 776
Suckling during lactation, 776
Mammary cell lineages and the branched epithelial
ductal tree, 778
Primer 23-A | Distinct cell lineages form the branched
epithelial ductal tree of the mammary glands, 779
Box 23-I | Lactation, 780
Benign breast diseases and breast cancer, 781
Concept Mapping | Fertilization, Placentation and
Lactation, 783
Essential Concepts | Fertilization, Placentation
and Lactation, 783
INDEX, 787
HISTOLOGY AND CELL BIOLOGY
An Introduction to Pathology
Thispageintentionallyleftblank
BASIC TISSUES | CELL BIOLOGY 1 | EPITHELIUM |
CELL BIOLOGY
Epithelia separate the internal environment from
the external environment by forming sheets of po-
larized cells held together by specialized junctional
complexes and cell adhesion molecules. Epithe-
lial cells participate in embryo morphogenesis and
organ development in response to intrinsic and
extrinsic signaling by tailoring cell proliferation, dif-
ferentiation and cell death. We address the structural
characteristics of epithelial cells within a biochemi-
cal and molecular framework as an introduction to
the transition from a normal to a pathologic status.

1
1 | EPITHELIUM | CELL BIOLOGY
CLASSIFICATION OF EPITHELIA (1-1 to 1-4)
The epithelium is a tightly cohesive sheet of cells
that covers or lines body surfaces and forms the
functional units of secretory glands. The main
characteristics of epithelia are summarized in Box
1-A.
The traditional classification and nomenclature
of different types of epithelia are based on two
parameters:
1. The shapes of individual cells.
2. The arrangement of the cells in one or more
layers.
Individual epithelial cells can be flattened (squa-
mous cells), have equal dimensions (cuboidal cells)
and be taller than wider (columnar cells).
According to the number of cell layers, an epi-
thelium consisting of a single cell layer is classified
as simple epithelium.
Simple epithelia, in turn, are subdivided into
simple squamous epithelium, simple cuboidal
epithelium and simple columnar epithelium, ac-
cording to the shape of their cell components. The
specific name endothelium is used for the simple
squamous epithelium lining the blood and lym-
phatic vessels. Mesothelium is the simple squamous
epithelium lining all body cavities (peritoneum,
pericardium and pleura).
Stratified epithelia are composed of more than
one cell layer. Stratified epithelia are subclassified
according to the shapes of the cells at the superficial
or outer layer into stratified squamous epithelium,
stratified cuboidal epithelium and stratified co-
lumnar epithelium.
Stratified squamous is the epithelium most fre-
quently found and can be subdivided into moder-
ately keratinized (also known as non-keratinizing)
or highly keratinized types. The cells of the outer
Box 1-A | General characteristics of epithelia
• Epithelia derive from the ectoderm, mesoderm and endoderm.
• Epithelia line and cover all body surfaces except the articular cartilage, the enamel
of the tooth and the anterior surface of the iris.
• The basic functions of epithelia are protection (skin), absorption (small and
large intestine), transport of material at the surface (mediated by cilia), secretion
(glands), excretion (tubules of the kidneys), gas exchange (lung alveolus) and
gliding between surfaces (mesothelium).
• Most epithelial cells renew continuously by mitosis.
• Epithelia lack a direct blood and lymphatic supply. Nutrients are delivered by
diffusion.
• Epithelial cells have almost no free intercellular substances (in contrast to
connective tissue).
• The cohesive nature of an epithelium is maintained by cell adhesion molecules
and junctional complexes.
• Epithelia are anchored to a basal lamina. The basal lamina and connective tissue
components cooperate to form the basement membrane.
• Epithelia have structural and functional polarity.
2
layer of a non-keratinizing squamous epithelium
retain nuclei (for example, esophagus and vagina).
Nuclei are absent in the outer layer of the highly
keratinized stratified squamous epithelium (for
example, the epidermis of the skin).
Stratified epithelia have basal cells aligned along
the basal lamina. Basal cells are mitotically active
and continuously replace the differentiating cells
of the upper layers.
Although rare, there are also stratified cuboidal
epithelia (for example, in the ovarian follicles and
lining the intralobular ducts of salivary glands).
Two special categories are the pseudostratified
epithelium and the urothelium. The pseudostrati-
fied epithelium consists of basal and columnar cells
resting on the basal lamina. Only the columnar cells
reach the luminal surface. Because the nuclei of the
basal and columnar cells are seen at different levels,
one has the impression of a stratified epithelial
organization.
Within this category are the pseudostratified
columnar ciliated epithelium of the trachea and
the pseudostratified columnar epithelium with
stereocilia of the epididymis.
The epithelium of the human urinary passages,
also referred to as urothelium, has the characteristics
of a pseudostratified epithelium. It consists of basal
cells, intermediate cells and columnar dome-shaped
cells, each extending thin cytoplasmic processes
reaching the basal lamina. An important feature of
this epithelium is its transitional height that varies
with distention and contraction of the organ.
Epithelial cell polarity (1-5)
An important aspect of an epithelium is its polarity.
Polarity is essential to carry out specific functions of
the various organ systems. Polarity is determined by
the distribution of plasma membrane proteins and
lipids and the rearrangement of the cytoskeleton.
Most epithelial cells lining surfaces and cavities
have three geometric domains:
1. The apical (uppermost) domain is exposed
to the lumen or external environment and displays
apical differentiations.
2. The lateral domain faces neighboring epi-
thelial cells linked to each other by cell adhesion
molecules and junctional complexes.
3. The basal domain is associated with a basal
lamina that separates the epithelium from underly-
ing connective tissue, representing the internal and
nutritional supporting environment.
The basal lamina, of epithelial cell origin, is
reinforced by components of the connective tis-
sue. The basal lamina–connective tissue complex
is designated the basement membrane.
 1 | EPITHELIUM | CELL BIOLOGY

1-1 | Epithelium: Concept Mapping

Epithelia

Shapes of individual Arrangement of the cells

Special categories
cells (side view) (one or more layers)

Cuboidal
Squamous Columnar cell
cell
cell (taller than it Pseudostratified
(about equal Urothelium
(flattened) is wide) epithelium
dimensions)
One layer More than one layer

Simple epithelia Stratified epithelia

Endothelium
Simple Simple Simple Stratified Stratified Stratified
squamous cuboidal columnar squamous cuboidal columnar
epithelium epithelium epithelium epithelium epithelium epithelium
Mesothelium
Named according to the shapes
of the surface layer cells

From the functional perspective, sealing junc-
tions segregate the plasma membrane of an epithe-
lial cell into an apical domain and a basolateral
domain.
This segregation is supported by the asymmetric
distribution of transporting molecules, ensuring
polarized secretory and absorptive functions of
an epithelium. For example, the apical domain
has structures important for the protection of the
epithelial surface (such as cilia in the respiratory
tract) or for the absorption of substances (such as
microvilli in the intestinal epithelium). In con-
trast, the basolateral domain facilitates directional
or vectorial transport functions prevented from
trespassing the sealing junctions.
Apical domain
The apical domain of some epithelial cells can
display three types of differentiation:
1. Cilia.
2. Microvilli.
3. Stereocilia.
Cilia (1-5)
There are two types of cilia (singular, cilium):
multiple motile cilia and a single or a primary
non-motile cilium.
Ciliogenesis, the assembly process of both types
of cilia, is initiated by the basal body, a structure
originated from a basal body precursor located in
the centrosome.
Basal bodies migrate to the apical plasma mem-
brane and extend into the extracellular space.
Multiple motile cilia (1-6)
Multiple motile cilia function to coordinate fluid
or cargo flow on the surface of an epithelium.
They are cell projections originating from basal
bodies anchored by rootlets to the apical portion
of the cytoplasm.
A basal body contains nine triplet microtubules
in a helicoid array without a central microtubular
component. By contrast, a cilium consists of an
axoneme formed by a central pair of microtubules
surrounded by nine concentrically arranged micro-
tubular pairs. This assembly is known as the 9 + 2
microtubular doublet arrangement. The axoneme
is also a component of the sperm tail, or flagellum.
The trachea and the oviduct are lined by ciliated
epithelial cells. In these epithelia, ciliary activity is
important for the local defense of the respiratory
system and for the transport of the fertilized egg to
the uterine cavity.
Ciliary motility is characterized by wide asym-
metric bending motions. In contrast, flagellar
motility is defined by a symmetric sine-wave
bending pattern.
Single or primary non-motile cilium (Primer 1-A)
Some cells have a single or primary non-motile
cilium. The importance of a single cilium emerges
from rare recessive human disorders known as
ciliopathies, caused by structural or functional
abnormalities of cilia.
The significant aspects of a primary cilium are:
1. It functions as a sensor, providing the cell with
information about the surrounding environment.

3
1 | EPITHELIUM | CELL BIOLOGY

1-2 | Types of epithelium: Simple epithelia

Simple squamous epithelium (endothelium)

Red blood cells

in the lumen
Basal lamina
Flat nucleus of
an endothelial Lumen
cell supported
by the basal
lamina

Simple squamous
epithelium Red blood cell

The inner lining of all blood vessels consists of a single layer of squamous endothelial cells. The thinness of the simple squamous epithelial
cells reflects their primary function in rapid exchange of substances between blood and tissue. A similar epithelium (called mesothelium)
covers the peritoneum, pleura and pericardium.

Simple cuboidal epithelium (collecting tubule, kidneys)

Basal lamina
Lumen

Lumen Lumen

Simple cuboidal
epithelium

The inner lining of kidney tubules and thyroid follicles consists of a single layer of cuboidal cells. Cuboidal cells are highly polarized and
participate in absorption, secretion (thyroid gland) and active ion transport (kidneys). Similar to the endothelium, a basal lamina attaches the
cell to the subjacent connective tissue.

Simple columnar epithelium (small intestine)

Brush border
Goblet cell Basal lamina

Lumen

Lamina propria

Simple columnar epithelium

The small intestine is lined by columnar epithelial cells with the nucleus in the medial portion of the cell. The apical domain contains finger-like
projections called microvilli forming a brush border. Microvilli participate in the absorption of proteins, sugar and lipids, which are released at
the basolateral domain into the blood circulation for transport to the liver. Goblet cells, present among the columnar epithelial cells, have a
dilated, goblet-like apical cytoplasm containing a light-stained mucus. Mucus, released into the lumen, coats and protects the epithelial cell
surface. The lamina propria consists of loose connective tissue located beneath the epithelium and the supporting basal lamina.

4
 1 | EPITHELIUM | CELL BIOLOGY

1-3 | Types of epithelium: Stratified epithelia

Stratified squamous epithelium with moderate keratin (esophagus)

Nuclei are seen in the

outermost cells
Nucleated superficial squamous cells

Mitotic basal
cell

Basement
membrane

Basement membrane

This epithelium consists of basal cells specialized for mitotic division. Stratified cells covering the basal layer are differentiating cells. Cells of
the outer layer are highly differentiated: they increase their keratin content to protect the tissue from the mechanical action of ingested food.
The outermost cells retain their nuclei. This epithelium is also known as non-keratinizing.

Stratified squamous epithelium with abundant keratin (epidermis)

Nuclei are not seen in

the outermost cells
Highly keratinized keratinocytes of
the superficial layer lack nuclei

Basal cell
Basement
membrane

Melanocyte
Basement membrane

This highly keratinized epithelium also consists of basal cells specialized for mitotic division. Melanocytes are present in the basal layer.
Stratified cells above the basal layer are differentiating keratinocytes. Keratinocytes of the outer layer contain abundant keratin to prevent water
loss and penetration of chemical and physical insults. The outermost cells lack nuclei. This epithelium is also known as keratinizing.

5
1 | EPITHELIUM | CELL BIOLOGY

1-4 | Types of epithelia: Pseudostratified epithelia

Pseudostratified columnar ciliated epithelium (trachea)

Goblet cell Columnar ciliated cell

Cilia

Basal bodies alignment

Columnar ciliated cell

Basal cell

Basement membrane Basal lamina

This epithelium consists of three major cell types: (1) columnar cells with cilia on their apical domain, (2) basal cells anchored to the basal
lamina, part of the basement membrane and (3) goblet cells, mucus-secreting epithelial cells with basally located nuclei. Columnar ciliated and
goblet cells attach to the basal lamina and reach the lumen. Basal cells do not reach the lumen.

Pseudostratified columnar epithelium (epididymis)

Principal cell with
Clump of stereocilia/ Sperm
stereocilia/stereovilli
stereovilli
Basal cell Golgi
region
Principal cell
with stereocilia/
stereovilli
Basal cell
Sperm

The epididymal epithelium contains two major cell types: (1) columnar cells (called principal cells) with stereocilia and highly developed Golgi
apparatus and (2) basal cells attached to the basal lamina. Basal and principal cells are associated with the basal lamina. Only principal cells reach
the lumen. Sperm can be visualized in the lumen. “Stereocilia” is an early misnomer as they lack microtubules. An appropriate name is stereovilli.

Urothelium (urinary bladder)

Dome-shaped superficial cell
Plaques
Intermediate cell Dome-shaped superficial cell
Plaques Superficial
Basal cell
cell
Urothelium of an empty
Intermediate
urinary bladder.
cell
Basal cell

Urothelium of a urinary Plaques

bladder filled with urine.

The epithelium lining the urinary passages (also called urothelium), consists of three cell types: (1) dome-shaped superficial cells (often
binucleated); (2) pyriform-shaped intermediate cells and (3) polyhedral-shaped basal cells, all of them extending cytoplasmic processes
anchored to the basal lamina. In humans, the urothelium is a pseudostratified epithelium. A characteristic of the urothelium is its transitional
configuration in response to distention and contraction tensional forces caused by urine. Plaques of aggregated proteins (uroplakins) are found
on the apical plasma membrane of the dome-shaped superficial cells.

6

1-5 | Cell polarity
Differentiations of the apical domain (cilia,
Lumen microvilli and stereocilia/stereovilli)
Apical domain
Microvilli Lumen
Tight junction
Basolateral
domain
Basement membrane
Nucleus
2. It participates in the early stages of embryonic
patterning, leading to organogenesis.
3. Many components of the Hedgehog signaling
pathway, essential at least in early development, are
present in a single cilium.
4. The position of the single cilium, called ki-
nocilium, of the hair cell of the organ of Corti in
the inner ear determines the correct polarity of the
adjacent actin-containing stereocilia, essential for
maintaining body balance and for hearing.
Microvilli (1-7)
Microvilli (singular, microvillus) are finger-like
cell projections of the apical epithelial cell surface
containing a core of cross-linked microfilaments
(a polymer of G-actin monomers).
At the cytoplasmic end of the microvillus,
bundles of actin and other proteins extend into the
terminal web, a filamentous network of cytoskeletal
proteins running parallel to the apical domain of
the epithelial cell.
The intestinal epithelium and portions of the
nephron in the kidney are lined by epithelial cells
with microvilli forming a brush border. In general,
a brush border indicates the absorptive function
of the cell.
Stereocilia (stereovilli) (1-7)
Stereocilia (singular, stereocilium) are long and
branching finger-like projections of the apical
epithelial cell surface. Similar to microvilli, ste-
reocilia contain a core of cross-linked actin with
other proteins.
Stereocilia (or stereovilli) do not have axonemes.
Stereocilia/stereovilli are typical of the epithelial lin-
ing of the epididymis and contribute to the process
of sperm maturation occurring in this organ.
1 | EPITHELIUM | CELL BIOLOGY
Cell adhesion molecules and cell junctions (1-8)
A sheet of epithelial cells results from the tight
attachment of similar cells to each other and to
the basal lamina, a component of the extracellular
matrix. Cell adhesion molecules enable interepi-
thelial cell contact and this contact is stabilized by
specialized cell junctions. A consequence of this
arrangement is the apical and basolateral domain
polarity of an epithelial sheet.
There are two major groups of cell adhesion
molecules:
1. Ca2+-dependent molecules, including cadher-
ins and selectins.
2. Ca2+-independent molecules, including the
immunoglobulin-like cell adhesion molecules
superfamily and integrins.
Many cells can use different cell adhesion
molecules to mediate cell-cell attachment. Integrins
are mainly involved in cell–extracellular matrix in-
teractions. Cadherins and integrins establish a link
between the internal cytoskeleton of a cell and the
exterior of another cell (cadherins) or the extracel-
lular matrix (integrins).
Ca2+-dependent molecules
Cadherins (1-8)
Cadherins are a family of Ca2+-dependent mol-
ecules with a major role in cell adhesion and
morphogenesis.
A loss of E-cadherins is associated with the acqui-
sition of invasive behavior by tumor cells (metasta-
sis), as we discuss in Chapter 4, Connective Tissue
and Chapter 17, Digestive Glands.
There are more than 80 different cadherins,
including desmogleins and desmocollins. Classical
cadherins were originally named for the tissue in
which they were particularly expressed–for example,
epithelial cadherin (E-cadherin) in epithelial cells;
neural cadherin (N-cadherin) in the nervous sys-
tem; vascular endothelial cadherin (V-cadherin) in
endothelia; and placenta cadherin (P-cadherin).
E-cadherin is found along the lateral cell surfaces
and is responsible for the maintenance of most
epithelial layers. The removal of calcium or the use
of a blocking antibody to E-cadherin in epithelial
cell cultures breaks down cell-cell attachment and
the formation of stabilizing junctions is disrupted.
E-cadherin molecules form cis-homophilic di-
mers (“like-to-like”), which bind to dimers of the
same or different class of cadherins in the opposite
cell membrane (trans-homophilic or heterophilic
[“like-to-unlike”] interaction). These forms of bind-
ing require the presence of calcium and result in a
specialized zipper-like cell-cell adhesion pattern.
The cytoplasmic domain of cadherins is linked
to actin through intermediate proteins known
7
1 | EPITHELIUM | CELL BIOLOGY

1-6 | Cilia and ciliogenesis 3. Catenins control the adhesive state of the
Cilium: a core of microtubule doublets Cilium
extracellular domain of cadherins.
in a 9 + 2 concentric arrangement The association of actin to the cadherin-catenin
surrounded by plasma membrane complex is essential for cell morphogenesis, changes
in cell shape and the establishment of cell polarity.
Basal body anchored 0.25 +m Members of the cadherin family also are present
to the cytoplasm by
between cytoplasmic plaques of the zonula and the
striated rootlets Basal body
macula adherens. `-catenin plays a significant role
Multiplication of the in colorectal carcinogenesis (see Chapter 16, Lower
basal body precursor 0.2 +m Digestive Segment).
Basal body precursor Selectins (C-type lectins) (1-8; Primer 1-B)
Centrosome Selectins, similar to cadherins, are Ca2+-dependent
(centriolar pair surrounded Striated rootlets cell adhesion molecules. In contrast to cadherins,
by a microtubular-organizing selectins bind to carbohydrates and belong to the
Basal lamina center) family of C-type lectins (Latin lectum, to select).
Components of a cilium Each C-type selectin (for calcium requiring) has
Cilia develop from basal bodies located in the apical domain of the cytoplasm.
a carbohydrate-recognition domain (CRD) of 120
Basal body precursors derive from the centrosome, multiply, mature and dock to amino acids with binding affinity to a specific oli-
the apical plasma membrane of the cell. gosaccharide attached to a protein (glycoprotein) or
A basal body, consisting of nine peripheral microtubule triplets (93 [triplets] + a lipid (glycolipid). The molecular configuration of
0) in a helicoidal arrangement, extends into the extracellular space as an the CRD is controlled by calcium. Calcium acts as
axoneme, a microtubular structure surrounded by the plasma membrane. a linker between the CRD and the hydroxyl groups
Rootlets anchor the basal body to the cytoplasm. Central microtubules are not of the sugar target.
present in basal bodies and centrioles. There are three major classes of cell surface C-
The cilium consists of a concentric array of nine microtubule doublets type lectins:
surrounding a central pair of microtubules (92 [doublets] + 2).
1. P-selectin, found in activated platelets and
activated endothelial cells lining blood vessels.
2. E-selectin, found on activated endothelial cells.
Plasma 3. L-selectin, found in leukocytes.
membrane C-type lectins are transmembrane receptors
(CLRs) involved in antimicrobial immunity and
Cilium
autoimmunity but can also be found as soluble
molecules (growth factors, antimicrobial proteins
and components of the extracellular matrix).
Cilium 92 + 2 CLRs can activate signaling pathways resulting
in the activation or inhibition of cellular functions.
For example, a signalling pathway can induce
Basal body primarily nuclear factor-gB (NF-gB)-dependent
proinflammatory responses. Some others can pro-
mote and suppress antitumour immune responses,
Rootlet
such as in natural killer (NK) cells, where C-type
lectins facilitate the recognition of cancer cells and
prevent the attack of healthy cells by inducing the
cytotoxic activities of NK cells. Yet, metastasis of
Oviduct Basal body 93 + 0
cancer cells is increased by expression of C-type
collectively as the catenin (Latin catena, chain) lectins, such as L-selectin, which facilitates cancer
complex. The complex includes catenins _, ` and cell adherence to the endothelium.
a and actin-binding proteins _-actinin, vinculin Selectins participate in the movement of leuko-
and formin-1, among others). cytes (Greek leukos, white, kytos, cell) circulating
The catenin complex has at least three distinct in blood (neutrophils, monocytes, B and T cells)
roles in the function of cadherins: toward tissues by extravasation.
1. The protein _-catenin mediates a direct link Extravasation is the essence of homing, a mecha-
to filamentous actin. nism that enables leukocytes to escape from blood
2. Catenins interact with regulatory molecules circulation and reach the sites of inflammation.
of the actin cytoskeleton. Homing also permits thymus-derived T cells to

8
 1 | EPITHELIUM | CELL BIOLOGY

Primer 1-A | Ciliogenesis. Primary cilium and Hedgehog signaling

Assembly of the cilium Cytoplasmic dynein-2

The cilium is formed and maintained by the transport of motor
tubulins along the axoneme mediated by proteins of the IFT proteins
intraflagellar transport (IFT) system. IFT trafficking from the
base of the cilium to the tip (anterograde transport; to the
microtubule plus end) is mediated by kinesin-2 motor
proteins mobilizing IFT protein complexes. Cytoplasmic Anterograde transport Retrograde transport
dynein-2 motor participates in retrograde transport (to the
microtubule minus end; base of the cilium). IFT proteins form Kinesin-2 motor
a platform for transporting cargo between the base and tip of
the cilium. IFT proteins Cilium
Disruption of the kinesin-2 motor or IFT proteins blocks cilia
Ciliary membrane
formation. Basal body proteins influence ciliary trafficking.

YY
YY
Among these are components of the BBSome, which are Distal appendage
named after their association with Bardet-Biedl syndrome
(BBS). BBSome proteins, bound to ciliary proteins,
facilitate their crossing through the distal appendage. Basal body
Proteins of the Hedgehog signaling pathway participate in
intraciliary and intraflagellar transport.
Subdistal appendage
All motile and non-motile cilia extend from a basal body that Protein components of the BBSome (for
consists of a microtubule triplet and distal and subdistal Bardet-Biedl Syndrome) bind to retrograde IFT
appendages. Distal appendages (also known as transition and cargoes to facilitate their lateral transport
fibers) and Y-shaped structures anchor and connect the basal through the porous barrier of the distal appendage.
body to the base of the ciliary membrane.
6
Primary cilium and Hedgehog signaling Gli Gli A
Hedgehog (Hh) signaling requires primary cilia for
4 Sufu
activation. The trafficking of Hh pathway proteins along
primary cilia is a key factor in epithelial cell differentiation. Tip of cilium
1 In the absence of Hh secretory protein , Ptc ( for 7
patched; the receptor of Hh), the transmembrane protein Gli
Gli
Smo ( for Smoothened) is blocked from entering the A
cilium. Smo is stored in vesicles near the basal body. 5 IFT proteins
2 Upon Hh binding, Ptc is internalized and Smo, free from
Dynein-2 motor
blocking, moves to the plasma membrane 3 and activates Ptc
the Hh pathway by antagonizing the function of Sufu Gli Primary cilium
(suppressor of fused) 4 .
Hh Gli
5 The motor kinesin KIF7 transports Gli (for glioma) 3 A
transcription factors to the tip of the cilium. If Smo is not
available to inactivate Sufu (because of the absence of Hh), Basal body
Gli is degraded or processed to become a repressor. If the 2
suppressive function of Sufu is antagonized, Gli Gli is
processed to an activator form ( Gli A GliA) 6 . 1 KIF7 To the nucleus
7 Activated GliA is then transported out of the cilium into
the nucleus (by dynein motor and IFT proteins ) to
activate epithelial differentiation genes. Smo Vesicle

home in peripheral lymph nodes.
P-selectin is stored in cytoplasmic vesicles in en-
dothelial cells. When endothelial cells are activated
by inflammatory signaling, P-selectin appears on
the cell surface.
On their surface, leukocytes contain sialyl Lewis-
x antigen, a specific oligosaccharide ligand for
P-selectin. P-selectin binding to the antigen slows
down streaming leukocytes in blood and they begin
to roll along the endothelial cell surfaces. P-selectins
get additional help from members of the superfam-
ily of immunoglobulin-like cell adhesion molecules
and integrins to stabilize leukocyte attachment,
leading to extravasation.
Ca2+-independent molecules
Superfamily of immunoglobulin-like cell adhesion
molecules (Ig-CAMs) (1-9)
In contrast to cadherins and selectins, members of
the Ig-CAMs superfamily are Ca2+-independent
cell adhesion molecules and are encoded by a single

9
1 | EPITHELIUM | CELL BIOLOGY

1-7 | Microvilli and stereocilia (stereovilli)

Microvillus
Microvillus: A core of Glycocalyx
actin-containing microfilaments Cap
Microvillus

Tight junction Actin filament core

and belt 0.08 +m
desmosome,
end points of
the actin
terminal web Terminal web
region

Basal lamina
Oviduct | Microvilli and cilia (cross section)
Glycocalyx

Microvilli Actin

Microtubule

Cilia
Small intestine | Microvilli (longitudinal section)

Stereocilium (stereovillus)
Sperm tail
Stereocilia/stereovilli
contain a core of actin
microfilaments No glycocalyx

Branching
stereocilium/stereovillus

Endocytotic vesicles

Basal lamina
Microvilli and stereocilia (stereovilli) have the same substructure:
A core of actin microfilaments and actin-associated proteins.
In the intestinal epithelium, actin extends into the terminal web, a
network of cytoskeletal proteins in a collar-like arrangement at the apical
Epididymis
domain of the cytoplasm of the intestinal cell. Although microvilli have
comparable length, stereocilia/stereovilli are longer and branch and the
apical domain of the cell contains endocytotic vesicles. The bridges
connecting adjacent stereocilia (blue arrows) are indicators of their branching.

gene. Members of the Ig superfamily are generated
by the alternative messenger RNA (mRNA) splic-
ing and have differences in glycosylation.
A conserved feature shared by all members of
the Ig superfamily is an extracellular segment
with one or more folded loops characteristic of
immunoglobulins.
Similar to cadherins, cell–cell adhesion takes
place by homophilic interactions between Ig-
CAMs, although for Ig-CAMs the binding is Ca2+-
independent. The cytoplasmic tail of the Ig-CAMs
is associated with components of the cytoskeleton,
such as actin, ankyrins and spectrin.
In addition to their cell adhesion properties, Ig-
CAMs mediate adhesion-independent signaling by
interacting with growth factor receptors, transcrip-
tion cofactors and other signaling proteins.
Ig-CAMs are dysfunctional in a broad range of
diseases such as cancer, vasculopathies, epithelial
and neurological disorders.
Of particular interest is CD4, a member of the
Ig-CAM superfamily and the receptor for the

10
 1 | EPITHELIUM | CELL BIOLOGY

1-8 | Ca2+-dependent cell adhesion molecules on endothelial cell surfaces. ICAM-1 is expressed
when an inflammation is in progress to facilitate
Cadherin
the transendothelial migration of leukocytes (see
Chapter 6, Blood and Hematopoiesis).
Domains in the extracellular portion cis-
Ca2+
of cadherin bind to calcium Members of the ADAM family (for an extracel-
homophilic
dimer
lular disintegrin and metalloprotease) are sheddases
Ca2+ Cadherin cis-homophilic dimers of that can cleave and release the soluble ectodomain
opposite cell membranes establish of various Ig-CAMs. The released, or shedded,
Ca2+
trans-homophilic interaction
protein fragments are involved in biological activi-
Ca2+ ties such the activation of growth factor receptors.
Plasma membrane
trans-homophilic Integrins (1-9; Primers 1-B and 1-C)
a Cytoplasm interaction Integrins differ from cadherins, selectins and
` members of the Ig superfamily in that integrins are
_ heterodimers formed by two associated _ and `
subunits encoded by different genes (see 1-9). There
Vinculin are about 22 integrin heterodimers consisting of 17
Actin-binding
Actin Formin-1 forms of _ subunits and 8 forms of ` subunits.
_-Actinin proteins
Almost every cell expresses one or several integ-
`-catenin binds to the intracellular domain of cadherin. The `-catenin/cadherin rins. Similar to cadherins, the cytoplasmic domain
complex recruits _-catenin, an adaptor protein that binds directly to actin. of ` integrin subunit is linked to actin filaments
a-catenin (also called plakoglobin) is a regulator of cadherin function. through connecting proteins (see 1-9).
The extracellular domain of ` integrin subunit
binds to the tripeptide RGD (Arg-Gly-Asp) se-
Selectin (C-type lectins) quence present in laminin and fibronectin, two
Transmembrane receptor Soluble molecules major components of the basement membrane, a
specific type of extracellular matrix. Laminin and
Carbohydrate- fibronectin interact with various collagen types (in-
recognition cluding type IV collagen), heparan sulfate proteo-
domain (CRD) Tetranectin
Ca2+ Ca2+
glycan perlecan and entactin (also called nidogen).
The integrin–extracellular matrix relationship
Calcium acts as a linker Eosinophil is critical for cell migration to precise sites during
between CRD and major basic embryogenesis and can be regulated when cell
hydroxyl groups of the protein (MBP) motility is required. In addition to their role in
sugar target
cell-matrix interactions, integrins also mediate
cell-cell interaction.
Plasma membrane Tetranectin, present in
serum, increases
Integrins, containing `2 subunits, are expressed
Cytoplasmic tail plasminogen activation, on the surface of leukocytes and mediate cell-cell
can bind fibrin and binding in preparation for extravasation. An exam-
C-type lectins have a carbohydrate-recognition heparin and participates ple is _1`2integrin on non-adherent leukocytes that
domain (CRD) with conserved residues that confer in wound healing. bind to ligands on endothelial cell surfaces following
binding specificity for a particular sugar: EPN motif MBP is involved in activation by extracellular stimulation, resulting in
(Glu–Pro–Asn) for mannose-type carbohydrates antiparasitic defense and leukocyte extravasation during the recruitment of
and QPD motif (Gln–Pro–Asp) for galactose-type immune hypersensitivity leukocytes to extravascular spaces (see Primer 1-B).
carbohydrates. reactions.
Integrins are bidirectional signaling receptors.
Integrins can be activated by proteins binding to
human immunodeficiency virus type 1 (HIV-1) their extracellular and intracellular domains. When
in a subclass of lymphocytes known as T cells or integrins bind to extracellular matrix molecules,
helper cells. a protein complex binds to the cytoskeleton and
We discuss the significance of several members several signaling pathways are activated.
of the Ig superfamily in Chapter 10, Immune- Genetic mutations of integrins or integrin regula-
Lymphatic System. tors have been associated with Glanzmann’s throm-
Other members of the Ig-CAM superfamily boasthenia (mutations in `3 integrin subunit),
play important roles in the homing process during leukocyte adhesion deficiency (type I, caused by
inflammation. Examples include intercellular adhe- mutations in `2 integrin subunit; type II, resulting
sion molecules 1 and 2 (ICAM-1 and ICAM-2) from the absence of fucosyl-containing ligands for

11
1 | EPITHELIUM | CELL BIOLOGY

Primer 1-B | Transmigration of leukocytes through the endothelial barrier

Rolling

Endothelium

Extravascular
space

1 Leukocytes
(neutrophils) in circulation Endothelium
resist shear forces to slow
down along the vascular
endothelium. Selectin phase Integrin phase

Rolling Integrins `1 and `2

Neutrophil Transendothelial
VCAM-1 migration
ICAM-1
1
Shear forces Adhesion
Carbohydrate-containing ligand
2 3
4

Selectins Extravascular space

2 Loose adhesion to the endothelium 3 Integrin receptors for endothelial

4 Transendothelial
under conditions of slow flow causes the ICAM-1 and VCAM-1 ligands are
leukocytes to roll. Selectins present on the rapidly activated on the leukocyte migration is mediated
endothelial cell surface bind to surface during rolling. Chemical by integrins interacting
carbohydrate ligands on the leukocyte mediators in the sites of inflammation with ligands on
surface. stimulate the activation of integrins `1 endothelial cell
and `2. Integrins strengthen leukocyte surfaces. F-actin
binding to endothelial cell surfaces. dynamics participate in
this process.

selectins due to a hereditary defect of endogenous CELL JUNCTIONS (1-10)

fucose metabolism; and type III, determined by
mutations in kindlin) and skin diseases (mutations
in kindlin, _2, _6 and `4 integrin subunits).
Integrin-mediated cell binding to extracellular
ligands can be disrupted by ADAM sheddases (see
Primer 1-C). ADAMs have roles in fertilization,
angiogenesis, neurogenesis, heart development,
cancer and Alzheimer’s disease.
Although cell adhesion molecules are responsible
for cell-cell adhesion, cell junctions are necessary
for providing stronger stability to epithelia and also
for separating different tissue compartments from
the external environment.
The movement of solutes, ions and water
through an epithelial layer occurs across and be-
tween individual cell components. The transcel-
lular pathway is controlled by numerous channels
and transporters whereas the paracellular pathway

12

Most leukocytes circulate in blood without interacting with other blood cells or
endothelial cells lining the blood vessels. However, a subset of lymphocytes
participates in a continuous recirculation process through lymphoid tissues. This
homing process involves many diverse adhesion molecules that help lymphocytes
to “home” to various lymphoid compartments of the body.
The lymphocyte–endothelial cell interaction requires two types of cell adhesion
proteins: selectins and integrins.
Neutrophils use a similar mechanism to escape from blood vessels, primarily
postcapillary venules, into inflammatory sites.
A series of events enable circulating leukocytes to identify the vascular
endothelium in an inflammatory site and to interact with the blood vessel wall
through a series of steps known as:
(1) Leukocyte capturing.
(2) Leukocyte rolling.
(3) Leukocyte arrest.
(4) Leukocyte crawling to sites of exit.
(5) Leukocyte transmigration through the barriers of endothelial cells, the
supporting basement membrane, and pericytes or smooth muscle cells of the
vascular wall.
The expression of endothelial selectins, E-selectin and P-selectin, are induced
by chemoattractants produced by the endothelial cells or released by
inflammatory cells and appear on the surface of endothelial cells in inflamed
tissues. The up-regulation of selectins on the surface of endothelial cells
represents an essential molecular step for the capture of leukocytes from the
rapidly flowing blood.
The leukocyte capturing event is transient. Then, the captured leukocytes,
propelled by the blood flow, start rolling on the endothelial cell surface and remain
stationary or arrested.
At this point, leukocytes start to contribute to their crawling and transmigration
process by the activation of leukocyte integrins `1 and `2. Integrins bind to
members of the immunoglobulin superfamily, such as intercellular adhesion
molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), whose
expression is induced on endothelial cells by pro-inflammatory cytokines.
Cytokines represent the guidance cues that mediate the activation of leukocyte
integrins, the initial indication of imminent cellular migration. If integrins are not
activated, the arrested leukocyte would be rapidly dislocated from the endothelial
attachment site by shear forces of the blood flow.
The strong leukocyte integrin–endothelial cell intercellular adhesion enables the
attachment and spreading of the leukocytes on the endothelial cell surface,
followed by crawling and transmigration across the endothelial barrier at
transiently opened endothelial cell tight junctions or through the endothelial cell
cytoplasm. A number of endothelial cell adhesion molecules and receptors
participate in the transmigration process.
Transendothelial migration, also called diapedesis, takes about 2-3 minutes. In
about 15-20 minutes, leukocytes permeate the endothelial basement membrane,
pericytes and smooth muscle cells of the vascular wall.
is regulated by continuous intercellular contacts or
cell junctions.
Cell junctions are symmetrical structures formed
between two adjacent epithelial cells. An exception
is the hemodesmosome, an asymmetrical structure
anchoring the basal domain of an epithelial cell to
the basal lamina.
There are four major classes of cell junctions:
1. Tight junction.
2. Adherens junctions, including zonula adher-
ens (belt desmosome) and macula adherens (spot
desmosome).
1 | EPITHELIUM | CELL BIOLOGY
3. Hemidesmosome.
4. Gap or communicating junction.
Tight junctions (1-10)
Tight junctions (also called occluding junctions)
have three major functions:
1. They determine epithelial cell polarity by
separating the apical domain from the basolateral
domain and preventing the free diffusion of lipids
and proteins between them.
2. They prevent the free passage of substances
across an epithelial cell layer, creating gates that
control the paracellular diffusion of ions and solutes.
3. Apart from serving as permeability barriers,
tight junctions are connected to signaling networks
that regulate proliferation and cell differentiation
and transmit information to and from the cyto-
skeleton, the nucleus and different cell adhesion
complexes.
Cell membranes of two adjacent cells come
together at regular intervals to seal the apical inter-
cellular space. These areas of close contact continue
around the entire surface of the cell like a belt,
forming anastomosing strips of the transmembrane
proteins occludin and claudin. Occludin and clau-
din belong to the family of tetraspanins with four
transmembrane domains, two outer loops and two
short cytoplasmic tails.
Occludin interacts with four major zonula occlu-
din (ZO) proteins: ZO-1, ZO-2, ZO-3 and afadin.
Claudin (Latin claudere, to close), a family of
16 proteins forming linear fibrils in the tight junc-
tions, confers barrier properties on the paracellular
pathway.
A mutation in the gene encoding claudin 16 is
the cause of a rare human renal magnesium wasting
syndrome characterized by hypomagnesemia and
seizures (see Box 1-B).
Two members of the Ig superfamily, nectins
and junctional adhesion molecules (JAMs), are
present in tight junctions. Both form homodimers
(cis-homodimers) and trans-homodimers across the
intercellular space.
Nectins are connected to actin filaments through
the protein afadin. The targeted deletion of the
afadin gene in mice results in embryonic lethality. A
mutation in the nectin-1 gene is responsible for cleft
lip/palate and ectodermal dysplasia (CLEPD1) of
skin, hairs, nails and teeth in humans. Nectin-2–
deficient male mice are infertile.
Freeze fracture electron microscopy, a technique
that enables the visualization of the hydrophobic
interior of a cell membrane, reveals tight junctions as
a meshwork of anastomosing sealing strands formed
by rows of transmembrane particles regarded as
representing the diffusion barriers.
13
1 | EPITHELIUM | CELL BIOLOGY

1-9 | Ca2+-independent cell adhesion molecules

Immunoglobulin (Ig) -like cell adhesion molecule (CAM) superfamily

VCAM-1
Ig-CAM
Immunoglobulin-like (vascular cell adhesion molecule 1)
The extracellular segment of an Ig-CAM, is domains
folded into two to six immunoglobulin-like NCAM-1
domains. (neural cell adhesion
Ig-CAMs on one cell can bind to identical molecule 1)
molecules on another cell (trans-homophilic
binding) or to other members of the family
(trans-heterophilic binding) to produce
zipper-like structures that stabilize cell–cell
contacts. Ig-CAMs are further stabilized by Extracellular space
anchoring their cytoplasmic tail to actin-binding Plasma membrane
proteins such as spectrin.
ICAM and VCAM molecules play important
roles in T cell interactions and binding of ICAM-2
ICAM-1
leukocytes to activated or resting endothelial Spectrin Actin (intercellular cell (intercellular cell
cells. adhesion molecule 1) adhesion molecule 2)
Cytoplasm

Integrin

Integrins differ from the other cell adhesion proteins: Actin Cytoplasm
(1) They consist of two subunits.
(2) They have a dual function: they bind to the _-actinin
extracellular matrix and the internal cytoskeleton. Vinculin
IPP complex
The _ subunit of an integrin has two chains linked
by a disulfide linkage and a globular head with
Talin
binding sites for divalent cations. Kindlin Paxillin
Focal adhesion
The ` subunit has two significant characteristics:
kinase
(1) The extracellular chain contains repeating
cysteine-rich regions. (2) The intracellular portion Plasma membrane
interacts with actin filaments through connecting
proteins: talin and focal adhesion kinase, the first Extracellular space
Only the `subunit binds S
proteins to be recruited. S
to the cytoskeleton
In its active conformation, talin binds the `-integrin Disulfide bridge
cytoplasmic domain. Kindlin, an integrin Cysteine-rich domains
_ subunit
coactivator, binds to the `-integrin cytoplasmic
domain and increases talin-induced integrin Divalent cation
activation. The IPP complex (consisting of ` subunit binding sites
intergrin-linked kinase, PINCH [for Particularly RGD
Interesting New Cysteine-Histidine-rich protein] and (arginine-glycine-
parvin) recruits _-actinin and paxillin to the cell aspartic acid) RGD
adhesion site.
In its active conformation state, `-integrin connects S S
S S
actin to the extracellular matrix proteins fibronectin
and laminin, through their RGD-binding sites. Fibronectin Laminin

Adhering junctions (1-11 and 1-12)
Adherens (Latin adhaereo, sticking to) junctions
include zonula adherens junctions and macula
adherens junctions, both found below the tight or
occluding junctions, usually near the apical surface
of an epithelium (see 1-11).
Similar to the tight junctions, zonula adherens
junctions form a circumferential belt (belt des-
mosome) around the apical domain of epithelial
cells. In contrast, macula adherens junctions in the
epidermis are small, spot-like and provide strong
adhesion.
Zonula adherens junctions are associated with
actin microfilaments. This association is mediated
by the catenin complex: _-catenin, `-catenin
and a-catenin (also called plakoglobin). `-catenin
and a-catenin bind to the cytoplasmic domains of
desmocollins and desmogleins, two families of des-
mosomal cadherins. _-catenin, bound to `-catenin,
is an adapter protein responsible for connecting the

14
 1 | EPITHELIUM | CELL BIOLOGY

Primer 1-C | ADAM, a member of the family of sheddase proteins

Intracellular domain ADAM (a disintegrin and metalloprotease) proteins are

(phosphorylation sites or membrane-anchored metalloproteases. The proteolytic activity of
proline-rich sites that bind metalloproteases depends on a metal ion (Zn2+) for their activity.
Src-homology [SH3] domains) An ADAM protein has an extracellular domain that contains an
N-terminal metalloprotease domain and a disintegrin domain.
Transmembrane domain
Glycoprotein Platelet
Extracellular domain gpIIb/IIIa
Shedding The disintegrin domain has
EGF-like domain
high sequence similarity to
Cysteine-rich domain snake-venom disintegrins. On
Disintegrin domain
shedding, the disintegrin
Metalloprotease
domain, which contains the
domain
Pro-domain (an RGD tripeptide (Arg-Gly-Asp),
intramolecular chaperone) binds to the platelet

RG
Shedding glycoprotein gpIIb/IIIa,

D
preventing platelet aggregation.
Metalloprotease Disintegrin
Ectodomain shedding
Ectodomain Soluble ectodomain
shedding Ectodomain shedding allows membrane-
The metalloprotease degradesr anchored growth factors or cytokines to
e
components of the extracellular participate in paracrine signaling (at a
matrix during cell migration.
n It also distance from the site of cleavage) or to
participates in the cleavagee of a enter the bloodstream. Shedding also can
membrane protein at the plasma generate a soluble decoy receptor that
membrane level, which results
s in could sequester a ligand.
the release of its soluble
ectodomain. This process is Plasma membrane
known as ectodomain shedding.
Transmembrane protein
The reversal of integrin-mediated cell binding to the extracellular protein cleaved adjacent to the plasma membrane. ADAMs are
matrix can be disrupted by proteins called ADAM (for A Disintegrin members of the family of sheddases.
And Metalloprotease). ADAMs have pivotal roles in fertilization, Ectodomain shedding targets for cleavage the proinflammatory
angiogenesis, neurogenesis, heart development, cancer and cytokine tumor necrosis factor ligand (TNFL) and all ligands of the
Alzheimer’s disease. epidermal growth factor receptor.
A typical ADAM protein contains an extracellular domain and an A released soluble ectodomain of a cytokine or growth factor can
intracellular domain. The extracellular domain consists of several function at a distance from the site of cleavage (paracrine signaling).
portions including a disintegrin domain and a metalloprotease domain. Ectodomain shedding of a receptor can inactivate the receptor by
(1) A disintegrin domain binds to integrins and competitively functioning as a decoy sequestering soluble ligands away from the
prevents integrin-mediated binding of cells to laminin, fibronectin and plasma membrane-bound unoccupied receptor.
other extracellular matrix proteins. A defect in TNF receptor 1 (TNFR1) shedding, determined by a
(2) A metalloprotease domain degrades matrix components and mutation in the receptor cleavage site, causes a periodic febrile
enables cell migration. condition because of continuous availability of TNFR1 for TNFL
A significant function of ADAMs is protein ectodomain shedding, binding. Consequently, recurring fever occurs by increased
consisting of the proteolytic release of the ectodomain of a membrane inflammatory responses.

intracellular domain of cadherins to actin filaments.
Macula adherens junctions, also called spot
desmosomes, are punctate-like junctions associated
with keratin intermediate filaments (also known as
tonofilaments) extending like ropes from one spot
to another on the lateral cell surfaces of epithelial
cells (see 1-12). Spot desmosomes provide strength,
rigidity and also flexibility to an epithelial cell layer.
Desmoglein 1 and desmoglein 3 are found in
the macula adherens junctions of the epidermis.
The intracellular domains of desmocollins and
desmogleins are associated with plakoglobin. Plako-
globin is an adapter protein that interacts with the
N-terminus globular domain of desmoplakin; the
opposite C-terminus globular domain is connected
to intermediate filaments (keratin, vimentin or
desmin).
Plakoglobin, desmoplakin and plakophilin are
components of the cytoplasmic plaques.
Note that actin microfilaments are members of
the zonula adherens, whereas intermediate filaments
are components of the macula adherens.

15
1 | EPITHELIUM | CELL BIOLOGY

1-10 | Epithelial cell junctions

Tight junctions Note that gap junctions are not

They define cell polarity and control the associated with cytoskeletal
passage of substances between adjacent components.
cells. Tight junctions have a beltlike
distribution like a ribbon internally bracing Tight
the cells and are associated with actin junction
filaments.
Zonula adherens or belt desmosome
Zonula
This anchorage junction has a beltlike adherens
distribution and is associated with actin
filaments.
Macula
Macula adherens or spot desmosome adherens
This anchorage junction has a spotlike
distribution and is associated with
intermediate filaments. Basal lamina

Hemidesmosome Gap or communicating junctions

Hemidesmosomes link the basal domain of They connect functionally two adjacent cells.
an epithelial cell to the basal lamina. A gap junction is formed by connexons,
Intermediate filaments are associated with channel-like structures that enable the passage
a plaque. of small molecules (~1.2 kd) between cells.

Tight junctions

Tight junctions are circumferential belts at junctions seal, like kissing points, the between adjacent epithelial cells
the apical domain of epithelial cells and space between epithelial cells and regulate (paracellular pathway). Molecules across
linking adjacent endothelial cells. Tight the passage of water and flux of ions the cell follow a transcellular pathway.

Afadin-nectin complex is anchored to ZO-1. Nectins F-actin JAMs

form cis-homodimers, which interact with each other Afadin Nectin
(trans-homo interaction) through the extracellular region.
Junctional adhesion molecules (JAMs) are Occludin
associated to afadin and ZO-1. JAMs cis-homodimers Claudin
interact with each other (trans-homo interaction) and
determine the formation of cell polarity. ZO-1
ZO-2
Occludin and claudins are the molecular ZO-3
basis for the formation of tight junction strands seen in
freeze-fracture preparations.

Kissing point Paracellular Transcellular

Zonula occludens proteins (ZO-1, ZO-2 and
pathway pathway
ZO-3) facilitate the reciprocal interaction of occludin,
claudins and JAMs with F-actin.

In contrast to occluding junctions, adjacent cell
membranes linked by zonula and macula adherens
junctions are separated by a relatively wide inter-
cellular space. This space is occupied by the gly-
cosylated extracellular portion of desmogleins and
desmocollins, anchored to cytoplasmic plaques.
As we have already seen, the interlocking of simi-
lar cadherins connects two adjacent cells by Ca2+-
dependent homophilic or heterophilic interaction.
It is important to emphasize that, in the stratified
squamous epithelium of the epidermis, the C-termi-
nus of desmoplakin interacts with the intermediate
filaments and that desmoglein 1 and desmoglein 3
maintain the cohesiveness of the epithelium.
Autoantibodies to desmoglein-1 cause a blis-
tering disease, called pemphigus foliaceus, by
disrupting cell-cell adhesion of the upper layers of
the epidermis. Autoantibodies to desmoglein-3 pro-
duce also a blistering disease, called pemphigus vul-
garis, localized in the basal layers of the epidermis.

16
 1 | EPITHELIUM | CELL BIOLOGY

1-11 | Epithelial cell junctions: Zonula adherens (belt desmosome)

Zonula adherens (belt desmosome)

Lumen

Actin filaments

Tight junction

Zonula adherens

Plaque

Afadin Afadin-nectin
Actin filament complex
anchored to afadin Nectin
and _-catenin
Adapter protein
_-catenin, bound to
Catenin complex
a-catenin, connects F-actin
to cadherins
Cadherins
Plaque: Desmoplakin, (desmocollins and Plasma
plakoglobin and plakophilin desmogleins) membrane
Desmoglein 1 predominates
Pemphigus foliaceus is an autoantibody-mediated blistering disease
above the stratum spinosum
in which antibodies against desmoglein 1 cause a loss of adhesion
Desmoglein 3 predominates in of keratinocytes in the superficial layers of the epidermis
the strata basale and spinosum
Layers of the epidermis
Anti-desmoglein 1
Stratum corneum immunoglobulin
Stratum granulosum Blister

Stratum spinosum

Stratum basale

Basal lamina Dermis Dermis

Hemidesmosomes (1-13) mosome is found in hemidesmosomes.

Hemidesmosomes are asymmetrical structures
anchoring the basal domain of an epithelial cell
to the underlying basal lamina. Hemidesmosomes
increase the overall stability of epithelial tissues by
linking intermediate filaments of the cytoskeleton
with components of the basal lamina.
Hemidesmosomes have a different organization
compared with adherens junctions. Although
hemidesmosomes look like half-desmosomes, none
of the biochemical components present in the des-
A hemidesmosome consists of these components:
1. An inner cytoplasmic plate associated with
keratin intermediate filaments (or tonofilaments).
2. An outer membrane plaque linking the
hemidesmosome to the basal lamina by anchoring
filaments (composed of laminin 5) and integrin
_` and proteins of the plakin family, including
plectin and BPAG1 (bullous pemphigoid antigen
1). We come back to the hemidesmosome in Chap-
ter 11, Integumentary System.

17
1 | EPITHELIUM | CELL BIOLOGY

1-12 | Epithelial cell junctions: Macula adherens (spot desmosome)

Macula adherens (spot desmosome)

Keratin intermediate filaments

(tonofilaments)

Plasma membrane
Outer dense plaque

Macula adherens

Inner dense plaque

Dense middle line

Dense
middle line

Intermediate filament
anchored to the C-terminus
of desmoplakin

Cadherins
Plaque: Desmoplakin, Plasma
(desmocollins and
plakoglobin and plakophilin desmogleins) membrane

Plakoglobin interacts directly with the

intracellular region of cadherins and also binds to Keratin, vimentin
desmoplakin and plakophilins. Outer dense plaque Inner dense plaque or desmin
Plakophilins (types1-4) participate in the Cadherin (intermediate
recruitment of proteins to the plasma membrane. filaments)
Desmoplakins (types I-II) bridge plakoglobin
to intermediate filaments keratin, vimentin or
desmin (bound to the C-terminus of desmoplakins).
N C

Inherited disorders affecting the skin and heart

Plakoglobin gene mutation: Naxos disease
(arrhythmogenic right ventricular cardiomyopathy Plakophilin Desmoplakin
[ARVC], woolly hair and palmoplantar
Plakoglobin Adapter protein plakoglobin links
keratoderma). Fat cells replace cardiocytes.
(also called plakophilin and desmoplakin to
Desmoplakin gene mutation: ARVC. Woolly hair.
a-catenin) intermediate filaments

Box-1-B | Tight junctions and disease

• Several diseases affecting tissues and organs have been related Claudin 16: Familial hypomagnesemia.
to tight junctions. Some diseases are caused by mutations in tight Claudin 19: Familial hypomagnesemia, hypercalciuria and
junction-associated proteins or by viral and bacterial infections targeting nephrocalcinosis.
tight junctions. • Junctional protein targeted by a virus/bacterium
• A number of diseases, such as chronic inflammation and cancer, are Claudin 1, claudin 6, claudin 9 and occludin: Hepatitis C virus
associated with dysfunctional tight junctions. infection.
• Junctional protein–associated genetic diseases JAMs: Infection caused by retroviruses.
Claudin 1: Neonatal ichthyosis; sclerosing cholangitis. Occludin: Vibrio cholera infection.
Claudin 5: Velo-cardial-facial syndrome. ZO1, ZO2: Dengue virus infection.

18
 1 | EPITHELIUM | CELL BIOLOGY

1-13 | Hemidesmosomes and gap junctions

Hemidesmosome

Keratin filaments Epidermis Keratin intermediate filaments

(tonofilaments)

Plate
Plate
Plasma
membrane Plaque

Plaque

Anchoring filaments Integrin _6`4 Anchoring filament

(laminin 5) (laminin 5)
Basal lamina
Basal lamina

Gap or communicating junction

The intercellular channel is an axial Clusters of intercellular Large patch of uniformly dense and closely packed
channel that allows the direct channels are known as particles corresponding to connexons seen on the PF
passage of small signaling gap junctions because (protoplasmic face) fracture face of a gap junction
molecules between adjacent cells of the narrow

Freeze-fracture electron micrograph from Robert RL, Kessel RG, Tung H-N: Freeze
to coordinate cell responses. extracellular gap that

Fracture Images of Cells And Tissues. New York, Oxford University Press, 1991.
separates the apposed
plasma membranes.
cAMP
Plasma
Ca2+ membrane 1

Ca2+
Connexon
cAMP
Plasma
Gap (2–4 nm) Connexin membrane 2

Six connexin monomers assemble to form a hexameric connexon, a Closely spaced pits on the EF (extracellular face) fracture
cylinder with a central open channel. Connexons in the plasma membrane face complementary to the particles on the PF fracture
of one cell align with connexons of an adjacent cell, forming a hydrophilic face. The EF and PF are artificially produced by splitting
intercellular channel connecting the cytoplasm of the apposed cells. the membrane bilayer along its hydrophobic core.

Gap junctions or communicating junctions (1-13)
Gap junctions are symmetrical communicating
junctions formed by integral membrane proteins
called connexins.
Six connexin monomers associate to form a con-
nexon, a hollow cylindrical structure that spans the
plasma membrane.
The end-to-end alignment of connexons in
adjacent cells provides a direct channel of com-
munication (1.5 to 2 nm in diameter) between the
cytoplasm of two adjacent cells. Connexons have
a clustering tendency and can form patches about
0.3 mm in diameter.
Gap junctions facilitate the movement of mol-
ecules 1.2 nm in diameter between cells (for ex-
ample, Ca2+ and cyclic adenosine monophosphate,
cAMP). The connexon axial channels close when
the concentration of Ca2+ is high.
Gap junctions enable the chemical and electrical
“coupling” between adjacent cells. For example,
cardiac muscle cells are connected by gap junctions
for the transmission of electrical signals.

19
1 | EPITHELIUM | CELL BIOLOGY

1-14 | Basement membrane. Laminin and fibronectin

Epithelial cell Under light microscopy, basal

Tight junction and recticular laminae are
resolved as a single basement
membrane after staining with the
PAS technique (kidney; cortex)

Nucleus
Basolateral
domain
Hemidesmosome
Basal domain

Basal lamina
Reticular lamina
The basement membrane, an extracellular component in direct contact with the basal
domain of epithelial cells, is visible under the light microscope after staining with the
periodic acid–Schiff (PAS) reagent technique.
At the electron microscopic level, the basement membrane is defined by two
layers or laminae:
(1) A basal lamina, which contains laminin, fibronectin, type IV collagen, heparan
sulfate proteoglycans and nidogen (also called entactin).
(2) A reticular lamina, which contains type III collagen (reticular fibers)
The components of these two laminae are glycoproteins. They are PAS positive.

Basement membrane: Electron microscopy can resolve each lamina as a separate entity
Epithelial cell

Nucleus of a fibroblast producing

Basal lamina Reticular lamina components of the reticular lamina

CONNEXIN MUTATIONS Connexin 32 (Cx32) protein is expressed in

Several diseases occur when genes encoding con-
nexins are mutated. Mutations in the connexin 26
(Cx26) gene, highly expressed in cells of the cochlea,
are associated with deafness. Mutations in the con-
nexin 32 (Cx32) gene are found in X-linked Charcot-
Marie-Tooth demyelinating neuropathy, resulting
in progressive degeneration of peripheral nerves,
characterized by distal muscle weakness and atrophy
and impairment of deep tendon reflexes.
Schwann cells, which are involved in the produc-
tion of rolled myelin tubes around the axons in the
peripheral nervous system (see Chapter 8, Nervous
Tissue). Gap junctions couple different parts of the
rolled myelin tubes of the same Schwann cell, rather
than different cells. A loss of the functional axial chan-
nels in myelin leads to the demyelinating disorder.
Mutations in the connexin 50 (Cx50) gene are asso-
ciated with congenital cataracts, leading to blindness.

20
 1 | EPITHELIUM | CELL BIOLOGY

Bone cells (osteoblasts/osteocytes) are connected

by gap junctions and express connexin 43 (Cx43)
Laminin and connexin 45 (Cx45) proteins. A deletion of the
Cx43 gene determines skeletal defects and delays in
_ chain mineralization.
` chain a chain
Basement membrane (1-14; Primer 1-D)
Collagen binding site The basement membrane consists of two compo-
Nidogen (entactin)
nents:
Collagen Cell binding site (RGD) 1. The basal lamina, a sheet-like extracellular
Integrin _6`1 for integrins matrix in direct contact with epithelial cell surfaces.
The basal lamina results from the self-assembly of
laminin molecules with type IV collagen, entactin
Binding site for heparin/
Proteoglycan and proteoglycans.
heparan sulfate and
_-dystroglycan
2. A reticular lamina, formed by type III collagen
fibers, supports the basal lamina and is continuous
with the connective tissue.
Laminin is the major component of the basal lamina. It consists of three
The basal and reticular laminae can be distin-
disulfide-linked polypeptide chains designated _, ` and a chains. Variants for
each chain give rise to several laminin isoforms with different structure and guished by electron microscopy. Under the light
function. microscope, the combined basal and reticular
Laminins have binding sites for cell surface receptors (integrins), type IV laminae receive the name of basement membrane,
collagen and other adhesion proteins (for example, nidogen, also known as which can be recognized by the periodic acid–Schiff
entactin). (PAS) stain (see Box 1-C). The PAS stain enables
Laminin monomers self-associate to form a network that is part of the basal the pathologist to determine whether an epithelial
lamina. malignant tumor has invaded the subjacent connec-
tive tissue by cancerous cells breaking through the
Fibronectin basement membrane.
Fibrin The basal lamina has specific functions in dif-
Collagen ferent tissues. The double basal lamina of the renal
C-terminal corpuscle constitutes the most important element
S S
S S
of the glomerular filtration barrier during the
initial step in the formation of urine (see Chapter
14, Urinary System). In skeletal muscle, the basal
Proteoglycans lamina maintains the integrity of the tissue and its
(heparan sulfate) Integrin (_5`1) binding sites disruption gives rise to muscular dystrophies (see
Chapter 7, Muscle Tissue).
Fibronectin, another component of the basal lamina, is a glycoprotein formed by Laminin is a cross-shaped protein consisting of
two identical chains joined by disulfide linkages close to the C-terminal. There three chains: the _ chain, the ` chain and the a
are two forms of fibronectin: chain. Laminin molecules can associate with each
(1) Plasma fibronectin, produced by hepatocytes and secreted into the other to form a mesh-like polymer.
bloodstream.
Laminin and type IV collagen are the major com-
(2) Cellular fibronectin, produced by fibroblasts, is a component of the
extracellular matrix, including the basal lamina.
ponents of the basal lamina, and both are synthesized
Fibronectin has binding sites for integrins, collagen, heparan sulfate and by epithelial cells resting on the lamina.
fibrin. Laminin has binding sites for nidogen (also called
entactin), proteoglycans (in particular, heparan
sulfate perlecan), _-dystroglycan (see Chapter 7,
Box 1-C | Periodic acid–Schiff (PAS) reaction Muscle Tissue) and integrins.
• PAS is a widely used histochemical technique to show 1,2-glycol or 1,2-aminoal- Fibronectin consists of two protein chains cross-
cohol groups, such as those present in glycogen, mucus and glycoproteins. linked by disulfide bonds. Fibronectin is the main
adhesion molecule of the extracellular matrix of the
• Periodic acid, an oxidant, converts these groups to aldehydes. The Schiff connective tissue and is produced by fibroblasts.
reagent, a colorless fuchsin, reacts with the aldehydes to form a characteristic red- Fibronectin has binding sites for heparin present
purple (magenta) product. in proteoglycans, several types of collagens (types I,
II, III and V) and fibrin (derived from fibrinogen
• Some important PAS-positive structures are the basement membrane, glycoca-
lyx, mucus produced by goblet cells, stored glycoprotein hormones in cells of the during blood coagulation or hemostasis; see Chapter
pituitary gland and collagens. 6, Blood and Hematopoiesis).

21
1 | EPITHELIUM | CELL BIOLOGY

Primer 1-D | Cell adhesion molecules, cell junctions and basement membrane

Zonula adherens junction (belt desmosome)

It consists of a dense plaque associated with the catenin complex (_-catenin, `-catenin and
a-catenin), _-actinin, vinculin and formin-1. Actin filaments are attached to _-catenin. The
intercellular space is bridged by cadherins (desmocollins and desmogleins) and the
afadin-nectin complex connecting the opposite dense plaques.
Immunoglobulin (Ig)
superfamily cell adhesion Tight junction (occluding junction)
molecule (Ig-CAM) Consists of the transmembrane proteins Macula adherens
Ig-CAMs belong to the occludin and claudins, associated with junction (spot
immunoglobulin superfamily ZO-1, ZO-2, ZO-3, junctional adhesion desmosome)
because they contain domains molecules (JAMs) and the afadin-nectin Desmosomes are
similar to immunoglobulins. complex. symmetrical structures
Ig-CAMs do not require Ca2+ to Catenin consisting of: (1) plaques
Claudin
maintain homophilic adhesive complex Occludin containing desmoplakin,
interactions. Actin Junctional adhesion molecule (JAM) plakoglobin and
Afadin-nectin complex
ZO-1, ZO-2, and ZO-3
plakophilin; (2) linking
cadherins (mainly
desmocollins and
Afadin-nectin complex desmogleins); and (3)
Selectin Cadherins keratin filaments attached
Selectins are to desmoplakin.
Plaque
Ca2+-dependent molecules
with binding affinity for Integrin
sugars. Selectins have an On the extracellular
important role in the homing side, integrins interact
process directly with fibronectin
and laminin. On the
Gap junction intracellular side, the `
Aggregate of connexons subunits of integrin
interact with actin through
intermediate proteins
(including _-actinin,
S S vinculin and talin).
Basement membrane S S Perlecan

Hemidesmosome
Hemidesmosomes consist of
an inner plate, the anchoring
site of the intermediate
filament keratin and an outer
plaque, attached to the basal
lamina by two major
components: anchoring
filaments (laminin 5) and
integrin _6`4.
Fibronectin
Collagens
Type IV collagen
Laminin
Laminin consists of
Nidogen
(entactin) three polypeptide
Proteoglycan chains (_, ` and a) with
Proteoglycans (mainly binding sites for type IV
heparan sulfate perlecan) collagen, proteoglycan
interact directly with perlecan, integrin and
fibronectin and laminin. nidogen.

Fibronectin circulating in blood is synthesized mobilized fibronectin binds to integrin expressed

in the liver by hepatocytes. It differs from fibro-
nectin produced by fibroblasts in that it lacks one
or two repeats (designated EDA and EDB for
extra domain A and extra domain B) as a result of
alternative mRNA splicing.
Circulating fibronectin binds to fibrin, a com-
ponent of the blood clot formed at the site of
blood vessel damage. The RGD domain of im-
on the surface of activated platelets and the blood
clot enlarges. We return to the topic of blood co-
agulation or hemostasis in Chapter 6, Blood and
Hematopoiesis.
Primer 1-D integrates most of the basic informa-
tion that you should remember about cell adhe-
sion molecules, cell junctions and the basement
membrane.

22

1-15 | Immunocytochemistry
Direct immunofluorescence
The immunoglobulin molecule
After detergent treatment, the
immunoglobulin molecule enters
cannot enter into an intact cell
the cell and binds to the antigen
Antigen
Direct immunocytochemistry involves a specific antibody or some agent with
specific binding affinity to an antigen tagged with a visible marker. Visible markers
attached to the immunoglobulin molecule can be a fluorescent dye such as
fluorescein (green fluorescence) or rhodamine (red fluorescence). When
examined with a fluorescence microscope, only labeled components are visible as
bright, fluorescent structures. Direct immunofluorescence involves a single incubation
step and provides a simple detection method.
Gold particles (electron-dense) attached to an immunoglobulin are convenient
markers for immunocytochemistry at the electron microscopic level.
Indirect immunofluorescence
Antigen Second immunoglobulin Antigen
with a fluorescent tag
First immunoglobulin Sperm
without a nucleus Nuclei
fluorescent tag
bound to the antigen
Indirect immunocytochemistry involves a second antibody tagged with a
fluorescent marker. This second antibody binds to an already antigen-bound
nontagged first antibody specific for an antigen.
The indirect method requires two separate incubations (one each for the first and
second antibodies) and is regarded as more specific than direct
immunofluorescence for the identification and localization of antigens (see image of
a testicular section. The nuclear red staining corresponds to propidium iodide; the
green staining identifies a fluorescein-labeled antigen).
CYTOSKELETON (1-15)
Cytoskeleton is a three-dimensional network of
proteins distributed throughout the cytoplasm
of eukaryotic cells. The cytoskeleton has roles in:
1. Cell movement (crawling of blood cells along
blood-vessel walls, migration of fibroblasts during
wound healing and movement of cells during em-
bryonic development).
2. Support and strength for the cell.
3. Phagocytosis.
4. Cytokinesis.
5. Cell-cell and cell–extracellular matrix adher-
ence.
6. Changes in cell shape.
1 | EPITHELIUM | CELL BIOLOGY
The components of the cytoskeleton were origi-
nally identified by electron microscopy. These early
studies described a system of cytoplasmic “cables”
that fell into three size groups:
1. Microfilaments (7 nm thick)
2. Intermediate filaments (10 nm thick)
3. Microtubules (25 nm in diameter)
Biochemical studies, involving the extraction
of cytoskeletal proteins from cells with detergents
and salts and in vitro translation of specific mRNA,
showed that each class of filaments has a unique
protein organization.
When cytoskeletal proteins were purified, they
were used as antigens for the production of antibod-
ies. Antibodies are used as tools for the localization
of the various cytoskeletal proteins in the cell.
The immunocytochemical localization of cyto-
skeletal proteins and cell treatment with various
chemical agents disrupting the normal organiza-
tion of the cytoskeleton have been instrumental
in understanding the organization and function of
the cytoskeleton.
Microfilaments (1-16; Primer 1-E)
F-actin is a versatile and abundant cytoskeletal
Testis component forming static and contractile bundles
and filamentous networks specified by actin-binding
proteins and their distinctive location and function
in a cell.
F-actin anchors to the plasma membrane and, by
this mechanism, contributes to cell shape changes.
Actin filaments can branch in the leading edge
(lamellipodia) of cells involved in either motility
or interaction with other cell types.
F-actin bundles are present in the microvilli
of the intestinal and renal epithelial cells (brush
border) and the stereocilia from the hair cells of
the inner ear.
Microvilli and stereocilia are comparable struc-
tures, although they differ in length and the number
of actin filaments:
1. Intestinal microvilli are 1 to 2 +m long, 0.1
+m wide and consist of 20 to 30 bundled actin
filaments.
2. Stereocilia in hair cells of the inner ear have a
tapered shape at their base, the length range is 1.5
to 5.5 +m and each actin bundle contains up to
900 actin filaments.
Hair cells are extremely sensitive to mechanical
displacement and a slight movement of the stereo-
cilium is amplified into changes in electric potential
transmitted to the brain.
The main component of microfilaments is actin.
Actin filaments are composed of globular monomers
(G-actin, 42 kd), which polymerize to form long
helical filaments intertwined in a helix (F-actin).
23
1 | EPITHELIUM | CELL BIOLOGY

1-16 | Intestinal microvilli (brush border) We have already seen that the intracellular por-
tion of the cell adhesion molecules cadherins and
Small intestine epithelium: Microvilli
integrin `1 interacts with F-actin through adapter
proteins. As discussed in Chapter 6, Blood and
Hematopoiesis, actin, together with spectrin, forms
a filamentous network on the inner face of the red
blood cell membrane that is essential for main-
taining the shape and integrity of red blood cells.
Spectrin is a tetramer consisting of two distinct
polypeptide chains (_ and `).
Actin filaments are polar. Growth of actin fila-
ments may occur at both ends; however, one end
(the “barbed end” or plus end) grows faster than the
other end (the “pointed end” or minus end). The
names correspond to the asymmetric arrowhead
appearance when myosin binds at an angle along
Brush border, formed by a closely Goblet cell the length of actin filaments.
packed layer of microvilli, at the apical Density Actin monomers have a binding site for adenos-
domain of the intestinal columnar corresponding to ine triphosphate (ATP), which is hydrolyzed to
epithelial cells. The brush border is the terminal web adenosine diphosphate (ADP) as polymerization
also seen in cuboidal epithelial cells of
proceeds. Actin polymerization is ATP-dependent.
the proximal convoluted tubule
(nephron). The kinetics of actin polymerization involves
Formin, a protein of the cap and
interacting with the fast-growing
a mechanism known as treadmilling: G-actin
Intestinal Formin barbed ends of F-actin, promotes the monomers assembled at one end of the filament
microvillus cap elongation of unbranched F-actin. concurrently disassemble at the other end.
Four types of actin-binding proteins control
Glycocalyx treadmilling by binding to actin monomers, sever-
ing F-actin, capping F-actin ends, nucleating and
cross-linking F-actin, stabilizing F-actin or moving
F-actin along F-actin:
Membrane- F-actin 1. `-Thymosin sequesters pools of G-actin
linking cross-linking monomers within cells, thus inhibiting their addi-
proteins proteins tion on the barbed end of F-actin.
Myosin I 2. Profilins, bound to G-actin, suppress nucle-
Villin
Calmodulin ation of G-actin. The main pool of actin avail-
Fimbrin
able for polymerization is represented by G-actin
bound to profilin. Profilin can favor the assembly
of monomeric G-actin into F-actin by facilitating
the exchange of bound ADP for ATP. Only ATP-
actin monomers can be assembled into filaments.
3. Cofilin, also known as actin depolymer-
izing factor (ADF), triggers depolymerization of
Actin filament Terminal Spectrin isoform Intermediate filaments ADP-bound actin at the pointed end. The F-actin
rootlets web connecting fibrils (keratins) severing property of cofilin/ADF triggers the rapid
disassembly of F-actin networks, a process that
replenishes the pool of G-actin for subsequent
In the core of the intestinal microvilli, the as- network assembly.
sembly of G-actin monomers into filaments and the 4. Gelsolin has a dual role; it is a capping protein
organization of these filaments into thick bundles that prevents the loss and addition of actin mono-
are controlled by various types of actin-binding mers and it is a severing protein. In the presence of
or actin-related proteins. A bundle of parallel Ca2+, gelsolin fragments F-actin and remains bound
non-branching actin filaments, forming the core to the barbed end, forming a cap that prevents
of the microvillus, is held together by actin-linking further filament growth.
proteins, villin and fimbrin. Side arms of myosin-I F-actin can branch or grow in length. F-actin
and the Ca2+-binding protein calmodulin anchor branching is initiated from the side of pre-existing
the bundle to the plasma membrane. F-actin by Arp2/3 (for actin-related protein 2/3),

24
 1 | EPITHELIUM | CELL BIOLOGY

Primer 1-E | Actin microfilaments: Assembly and disassembly

F-actin polymerization and depolymerization Arp2/3-mediated branched F-actin network

`4-thymosin sequesters G-actin into a reserve pool.

Profilin binds to ATP G-actin and regulates filament assembly.

G-actin carries an
ATP molecule (one
`4-thymosin per monomer)

Profilin
Barbed end
Polymerizing end of Old filament
a growing actin
filament Barbed end
An ATP G-actin nucleation cap
is formed to promote further
addition of G-actin. New filament

ATP is hydrolyzed. Arp2/3 Pointed end

Polymerized G-actin complex
contains bound
ADP. Arp2/3, a complex of seven proteins, initiates
the growth of F-actin from the sides of a pre-existing
Gelsolin severs actin filament. Arp2/3 assembles branches growing at
filaments and binds to the 7 nm thick
their free barbed ends. Arp2/3 attaches to the side
newly formed barbed end or of the older filament.
to the sides, blocking further
Severed actin filament
polymerization or Formin-mediated linear F-actin network
dissociation (capping).
Capped
Profilin regulates filament barbed end Profilin
assembly by catalyzing the Microvilli
exchange of G-actin bound ADP for
Formin G-actin
ATP and promotes the transfer of
actin monomers to the `4-thymosin
reserve pool and from there to the Barbed end
barbed end of F-actin. Pointed end
Depolymerizing F-actin
G-actin carrying end of a grown
bound ATP G-actin carrying actin filament
bound ADP
Profilin Cofilin, an actin
depolymerizing factor,
stimulates the
dissociation of Pointed end
ADP-bound G-actin
Treadmilling is the dynamic balance between the polymerizing and Formins have a common FH2 domain. Two FH2
depolymerizing ends to maintain the length of an actin filament. A treadmilling domains form a ring at the barbed end of an actin
actin filament contains ATP-bound G-actin monomers at the barbed end, filament that grows linearly by adding profilin-targeting
whereas the ones at the pointed end are ADP-bound. ATP-bound G-actin monomers to the barbed end.

Cytochalasins bind to the
fast-growing end (barbed end),
preventing further addition of G-actin.
A cytochalasin cap is formed.
Cytochalasins are alkaloids produced
by fungi.
F-actin inhibitors
Phalloidin binds to F-actin
preventing depolymerization.
Fluorescent-labeled phalloidin is used to
stain F-actin in cells. Phalloidin is an
alkaloid produced by the mushroom
Amanita phalloides.
Latrunculins disrupt F-actin by
binding to G-actin and inducing directly
F-actin depolymerization. Latrunculins
derive from the Red Sea sponge
Latrunculla magnifica.

Barbed end Pointed end

25
Another random document with
no related content on Scribd:
 Settlements were closed, and orders were sent to all Ningpo
men—and they form 50 per cent. of the population—to go out on
strike.

"Some of the principal Ningpo merchants came forward in the

afternoon, and through the good offices of a peacemaker came
to an understanding with the French Consul, under which it was
agreed that the French should postpone taking possession of the
cemetery for one month, during which time the Ningpo Guild
trust to come to some amicable arrangement. The French Consul
has given them to understand that he will not recede from his
position; the cemetery must be given up, but he is willing
that this should be done in any way that will be most pleasing
to the Ningpo residents."

On the 23d of August it was announced by telegram from

Shanghai that "the dispute arising out of the Ningpo Josshouse
is about to be settled by French withdrawing their claims to
remove the buildings in consideration for an extension of
their concession as far as Si-ca Wei, an addition of 20 square
miles." This raised protests from Great Britain and the United
States, many of whose citizens owned property within the area
thus proposed to be placed under French jurisdiction; and the
distracted Tsung-li Yamên was threatened and pulled about
between the contending parties for months. The final outcome
was an extension of the general Foreign Settlement at Shanghai
(principally British and American) and a limited extension of
that especially controlled by the French. The adjustment of
the question was not reached until near the end of 1899.

Great Britain, Papers by Command:

China, Number 1, 1899. and Number 1, 1900.

CHINA: A. D. 1898-1899 (June-January).

Anti-missionary insurrection at Shun-ch'ing,
in Central Szechuan.
The following is from a communication addressed by the British
Minister to the Tsung-li Yamên, August 2, 1898:

"At Shun-ch'ing Fu the local officials, with a few of the

gentry, have for years shown themselves determinedly hostile
to foreigners, and have refused to allow houses to be let to
missionaries. British missionaries have in consequence been
forced to quarter themselves in an inn for the last six years,
but even that was apparently objected to; in 1893 there was a
riot, and in 1895 another, in which two missionaries were
treated with brutal violence. In May of this year a house was
finally rented; the District Magistrate was notified, but
neither he nor the Pao-ning Taotai issued proclamations or
gave protection, though requested by the missionaries to do
so. The Shun-ch'ing Prefect, instead of giving protection,
connived at the local opposition, and with the usual results.
On the 15th June three missionaries were attacked and stoned,
and one severely wounded, while passing through the city, and
though protection was asked of the Prefect, he gave none, and
later he and the other officials repeatedly refused to issue a
Proclamation or to take any measures whatever to avert
disturbances. On the 20th the Prefect feigned sickness, and
could not be appealed to, and on the 27th the house leased by
the missionaries was destroyed, with all the property it
contained. The Roman Catholic establishment was also gutted.

"As usual the disturbance did not end at Shun-ch'ing. Later on

a Roman Catholic Chapel at Yung ch'ang Hsien was attacked and
looted, two native Christians killed, 10,000 taels of silver
stolen, and a French priest [Father Fleury] seized and held to
ransom by a band of rioters. I am also informed that other
acts of brigandage have occurred, and that the Protestant
missions at Pao-ning and Shê-Hung are in grave danger. The
Provincial Government appears to absolutely ignore the recent
Imperial Decrees for the prevention of missionary troubles.
All the conditions point to this, or to an utter incapacity on
the part of those officers to exercise satisfactory control. Her
 Majesty's Consul, indeed, informs me that there is one band of
brigands, led by an outlaw known as Yü Mau Tzu, which is able
to terrorise two important districts in the centre of Szechuan
and even to overawe the Chêngtu authorities. In connection
with the Shun-ch'ing affair Her Majesty's Consul has made the
following demands:

1. Immediate restoration of their house to the missionaries,

the officers to pay the whole cost of repairs;

2. Punishment of the ring-leaders;

3. The local headmen to give security for future good conduct;

4. Compensation for all property destroyed;

5. Punishment of officials in fault.

I shall look to the Yamên and see that these demands are fully
satisfied and with the least possible delay."

{98}

But urgency from both England and France failed to stimulate

action on the part of the Chinese government, energetic enough
to stop this anti-missionary movement in Central Szechuan. What
seems to have been a riot at first became a formidable revolt.
"The action of the Provincial Government was paralysed by the
fact that Father Fleury was still in Yü's hands, and would be
killed if a move was made against the brigands. On the other
side the Viceroy was informed by M. Haas, the French Consul,
that if anything happened to the Father the consequences to
China would be disagreeable. The Taotai was in consequence
making efforts to secure the release of the prisoners by
paying blackmail." In September it was reported that "Yü's
power was increasing, his emissaries were scattered about in
places beyond his immediate sphere of influence, and were
attempting to stir up Secret Societies, and he had issued a
manifesto. A riot took place at Ho Chou, 60 miles north of
Chungking, on the 14th September, the American mission
hospital being partially looted and a Roman Catholic
establishment destroyed by fire. The Provincial Government was
acting weakly and unprofitably."

Towards the end of September, "Yü marched with about 2,000

uniformed men and took up a position on the Ch'êng-tu road;
thence he moved east to Tung-Liang, pillaging and burning the
houses of Christians, and levying contributions on the rich.
The Viceroy, at the request of the Consuls, was said to have
sent 4,000 troops from Ch'êng-tu, Lu-chou, and Ho-chiang to
converge on Chungking. Twelve of the rebels, who had been
seized at a place only 30 miles south of the port, were
publicly executed with torture in Chungking on the 30th
September." In October the report was that "fresh troops were
arriving, and were taking up positions along the Ch'êng-tu
road, and the passes north of Chungking. Further executions
had taken place. … The Procureur of the French Mission
estimated that up to date the total damage done by Yü Mau-tzu
was twenty persons killed, the houses of 6,000 Christians
burned and their property stolen, and twelve Missions
destroyed."

Great Britain, Papers by Command: China,

Number 1, 1899, page 249, and Number 1, 1900, page 152.

On the 12th of October the United States Minister, Mr. Conger,

sent to the State Department at Washington the following
translation of a decree issued by the Empress Dowager on the
6th:

"From the opening of ports to foreign trade to the present

time, foreigners and Chinese have been as one family, with
undivided interests, and since missionaries from foreign
countries are living in the interior, we have decreed, not
three or four times, but many times, that the local officials
must protect them; that the gentry and people of all the
provinces must sympathize with our desire for mutual
benevolence; that they must treat them truthfully and
honestly, without dislike or suspicion, with the hope of
lasting peaceful relations.

"Recently, there have been disturbances in the provinces which

it has been impossible to avoid. There have been several cases
of riot in Szechuan, which have not been settled. The stupid
and ignorant people who circulate rumors and stir up strife,
proceeding from light to grave differences, are most truly to
be detested. On the other hand, the officials, who have not
been able at convenient seasons to properly instruct the
people and prevent disturbances, can not be excused from
censure.

"We now especially decree again that all high provincial

officials, wherever there are churches, shall distinctly
instruct the local officials to most respectfully obey our
several decrees, to recognize and protect the foreign
missionaries as they go to and fro, and to treat them with all
courtesy. If lawsuits arise between Chinese and native
Christians, they must be conducted with justice and speedily
concluded. Moreover, they must command and instruct the gentry
and people to fulfill their duties, that there may be no
quarrels or disagreements. Wherever there are foreigners
traveling from place to place, they must surely be protected
and the extreme limit of our hospitality extended. After the
issue of this decree, if there is any lack of preparation and
disturbances should arise, the officials of that locality will
be severely dealt with; whether they be viceroys or governors
or others they shall be punished, and it will not avail to say
we have not informed you."

United States Consular Reports,

February, 1899, page 299.
On the 1st of November the British Consul at Chungking
announced "an alarming extension of the rebellion. Flourishing
communities of Christians in four districts were destroyed,
and heavy contributions were laid on non-Christians. The
continued inactivity of the Government troops was chiefly
attributable to orders from the Yamên to the effect that the
first and foremost consideration was the rescue of Père
Fleury. Negotiations were being carried on by the Chungking
Taotai and the Chinese Generals for the Father's release; a
ransom of 100,000 taels was offered, presents were sent to Yü
and his mother, and he and his lieutenants were given buttons
of the third rank." The next month, however, brought on the
scene a new Viceroy who really wished to suppress the
insurrection, though he "complained that his hands were tied
by the Yamên's instructions, which urged him to come to terms
with Yü." At last this singularly energetic Viceroy got
permission to fight the rebels. On arriving in front of the
terrible Yü he "found that the troops who had been stationed
there previously were quite untrustworthy, and that the
Generals and local officials were all more or less in league
with the rebels. However, as soon as it was learnt that the
Treasurer meant business, a number of the rebels dispersed.
The main band, under Yü Mau-tzu, about 6,000 in number, was
then surrounded in Ta Tsu Hsien, after a preliminary encounter
in which the rebels lost some 100 men. By the 19th January, a
Maxim was brought to bear on Yü's camp, and the rebels fled
like rabbits. Yü begged Père Fleury to save his life, and next
day released the Father, who found his way to the Treasurer,
after some narrow escapes. Yü then surrendered."

It was said at the time that the French government was

demanding an indemnity in this case to the amount of £150,000.
Later it was understood that the French Minister at Peking
"had taken advantage of the pending missionary ease to revive
an old request for a Mining Concession."
 Great Britain, Papers by Command:
China, Number 1, 1900.

{99}

CHINA: A. D. 1899.
Anti-missionary outbreaks, increasing piracy, and other
signs of growing disorder in the country.

During 1899 there was a notable relaxation of the hard and

ceaseless pressure upon China which governments, capitalists
and speculators had been keeping up of late, in demands more
or less peremptory for harbor leases, settlement grounds,
railway franchises, mining privileges, and naval, military and
commercial advantages of every possible sort. But the irritation
of the country under the bullying and "nagging" of the
treatment it had received from the European nations revealed
itself in increasing outbreaks of popular hostility to
foreigners; and these called out threats and demands, for
indemnity and punishment, which were made, as a rule, in the
truculent tone that had become habitual to western diplomacy
in dealing with the people of the East. It was a tone which
the Chinese provoked, by the childish evasions and treacherous
deceptions with which their officials tried to baffle the
demands made on them; but it gave no less offense, and is no
less plainly to be counted among the causes of what afterwards
occurred.

Throughout most of the year, the British Legation at Peking

and the Consulate at Chungking were busied in obtaining
satisfaction for the murder of Mr. Fleming, a missionary in
the China Inland Mission, stationed at Pang Hai, near
Kwei-Yang. He had been killed and the mission looted in
November, 1898. The Chinese authorities claimed that he had
been killed by a band of rebels. The British Consul
investigated and became convinced that the missionary had been
the victim of a deliberate plot, directed by the headman of the
village of Chung An Chiang, where the murder occurred, and
connived at by the military official Liu. The Chinese
government, yielding to this conviction, caused two of the
murderers to be executed, degraded and exiled all of the local
officials who were involved in the crime, except the headman,
and paid an indemnity of $30,000. But the headman escaped, and
it was claimed by the governor of the province that he could
not be found. The British authorities found evidence that he
was being shielded by the governor, and demanded the dismissal
of the governor, which was persistently refused. Finally, in
October, 1899, the guilty headman was hunted down.

On the 18th of February, the British Minister complained

energetically to the Tsung-li Yamên of the rapid increase of
piracy on the Canton River. "Since November, 1898," he wrote,
"that is in three months, no less than forty-seven cases of
piracy in the Canton waters have been reported in the papers.
In several of these cases life was taken, and it may almost be
said that a reign of terror exists on the waterways of the Two
Kuang. Cargo boats are afraid to travel at night, or to move
about except in company, and trade is becoming to a certain
extent paralysed. The Viceroy is always ailing, and it is
difficult to obtain an interview with him. Her Majesty's
Consul has repeatedly addressed him on the subject of these
piracies in the strongest terms, but can only obtain the
stereotyped reply that stringent instructions have been sent
to the officials concerned. Admiral Ho, the
Commander-in-chief, who should properly be the officer to
inaugurate a vigorous campaign against the pirates, appears
absolutely supine and incapable of dealing with the evil. The
complaints of the Hong Kong Government and Her Majesty's
Consuls show a state of affairs in Canton waters which is
quite intolerable. There is no security for life or property,
and as British subjects are closely concerned, it is my duty
to inform your Highness and your Excellencies that unless
measures are immediately taken to prevent such outrages, I
shall have to report, for the consideration of Her Majesty's
Government, the advisability of taking steps to protect
British lives and property, either by patrolling the waterways
or by placing guards on the steamers, the expense of which
would be the subject of a claim on the Chinese Government."

On the 28th of February, Herr von Billow, the German Imperial

Secretary of State for Foreign Affairs, announced in the
Reichstag, at Berlin, the reception of a telegram from
Tien-tsin, reporting that several Germans had been attacked
and insulted in that town on the 24th, and had been compelled
to take refuge in the side streets and narrow alleys. The
Imperial Government, he said, had been already aware for some
weeks past that a considerable feeling of irritation had
manifested itself against foreigners in China, especially in
the southern portion of the Province of Shantung. The Chinese
Government were thereupon warned of the necessity of
maintaining order and securing public safety, and, upon the
receipt of the telegram above referred to, the German Minister
at Peking was instructed to impress upon the Chinese
Government that, if such incidents were permitted to recur, or
the perpetrators allowed to escape unpunished, the
consequences for China would be very serious. "We have," Herr
von Bülow declared, "neither the occasion nor the desire to
interfere in the internal affairs of China. But it is our duty
to watch lest the life and property of our fellow subjects,
whether missionaries or traders, should be made to suffer
through the internal complications in China."

In March, however, the state of things at and near Peking was

so far improved that the Legation guards were being withdrawn.
Sir Claude MacDonald obtained leave of absence and left Peking
on the 23d for a visit to England, and the business of the
Legation was conducted for a time by Mr. Bax-Ironside, the
Charge d'Affaires.

In June there was an anti-missionary riot at Kienning, in the

neighborhood of Foochow, excited by the murder of a boy,
 popular rumor ascribing the murder to foreigners. "In all
directions," wrote one of the threatened missionaries, who
gave an account of the occurrence, "murders were said to be
taking place, though no bodies were ever found; that seemed of
no consequence to the people, and the story was current, and
was apparently generally believed, that these murders were
done by men in our employ, and that we used the eyes and legs
to make medicine. There were endless stories of people being
kidnapped, chloroformed in the road by a bottle being held out
to their nose, and the day I arrived it was said that eyes had
been found at Tai-lui, a suburb of Kienning, and as I passed a
crowd was actually on the spot seeking for the said eyes." The
missionaries succeeded in escaping from the mob; but one
native convert was killed, and the mission hospital and other
premises were looted.

The ideas and the state of feeling out of which this attack on
the missionaries and their converts grew are revealed in the
following translation of a placard that was posted in Kienning
in June:

"We of this region have hitherto led a worthy life. All the
four castes (scholars, agriculturists, artizans, traders) have
kept the laws and done their duty. Of late foreigners have
suddenly come among us in a disorderly march and preaching
heretical doctrines. They have had from us indulgent
treatment, but they have repaid us by endangering our lives.
This year, in town and country, people have been hewn in two,
men and women in numbers have fallen upon evil days.
{100}
Everywhere the perpetrators have been seized, and everyone of
them has confessed that it was by the missionary chapels they
were ordered to go forth and slay men and women; to cut out
their brains and marrow to make into medicine. The officials
deliberately refrained from interfering. They garbled the
evidence and screened the malefactors. The whole country side
is filled with wrath; the officials then posted Proclamations,
and arrested spreaders of false reports. The hewing down of
men is hateful; but they issued no Proclamations forbidding
that. Now fortunately the people is of one mind in its wrath.
They have destroyed two chapels. The Ou-ning ruffian has
issued another Proclamation, holding this to be the work of
local rowdies. He little knows that our indignation is
righteous, and that it is a unanimous expression of feeling.
If the officials authorize the police to effect unjust
arrests, the people will unite in a body, in every street
business will be stopped, and the Wu-li missionary chapel will
be destroyed, while the officials themselves will be turned
out of the city, and the converts will be slain and
overthrown. When cutting grass destroy the roots at the same
time. Do not let dead ashes spring again into flame."

A settlement of the Kienning case was arranged locally between

the British Consul at Foochow, Mr. Playfair, and the Viceroy
of the province. The views of the Consul were expressed in a
communication to Mr. Bax-Ironside as follows:

"Since the missionaries established themselves there it is the

sixth attempt made to drive them out of the region. The common
people (from what I can gather) have no animus against the
foreign preacher of the Gospel, and show none. On the
contrary, whether moved to accept Christianity or not, they
appear to recognize that missionaries are in any case there
for benevolent and beneficial objects. Schools and hospitals
are independent of proselytizing, and, even if the
missionaries were never to make another convert, they would be
doing good and useful work in spreading Western knowledge and
healing the sick. In addition to this, the presence of
missionaries in out-of-the-way places in China has one
unquestionable advantage. To use the phrase of Sir Thomas
Wade, they 'multiply the points of contact,' and familiarize
the Chinese with the sight of the European. To the missionary,
either as a preacher of the Gospel or as a dispenser of benefits,
the populace at Kienning does not seem to have shown any
aversion; yet six times this populace has risen and tried to
drive the missionaries from the place. The logical inference
is that the Kienning peasant, though tolerant by nature, is
subject to some outside influence. He is moved, not by what he
sees, but by what he is told exists beyond his range of
vision; and these things are pointed out to him by such as he
believes to be his intellectual superiors, and as have,
therefore, the faculty of perceiving what is hid from himself.
The history of almost every anti-missionary movement in China
points to the same process. Why the educated classes of this
land should be so inveterately hostile to the foreigner is a
difficult question to answer. It has been suggested that the
Chinese of this type have an ineradicable conviction that
every European is at heart a 'land-grabber'; that missionaries
are the advance agents of their Governments; that the Bible is
the certain forerunner of the gun-boat; and that where the
missionary comes as a sojourner he means to stay as a
proprietor; consequently, that the only hope of integrity for
China is that her loyal sons should on every occasion destroy
the baneful germ. Extravagant and (in the instance of
Kienning) far-fetched as these notions may seem, I am
convinced that the literati and gentry have been at the root
not only of the present outbreak, but of the others which have
preceded it. While, therefore, I have insisted throughout that
Kienning must be made a place of safe residence for the
British missionary, I have considered that the only way to
attain that result will be to shackle the hands of the gentry
by making any further breach of the peace a sure precursor of
punishment."

To this end, Mr. Playfair's exactions included, not only the

trial and punishment of those guilty of the riot and the
murder, but the signing of a bond by twenty-four leading
notables of Kienning, binding them, with penalties, to protect
the missionaries. His demand was complied with, and, on the
29th of September, he reported that the required bond had been
given. He added: "Before accepting it, however, I required the
authorities to inform me officially that the terms, which to me
seemed somewhat vague, were understood by the authorities to
extend the responsibility of the gentry to any outbreak of the
populace. I received this intimation and I consider that it
supplements the original wording effectually."

In November the German government made public the substance of

an official telegram received from Peking, reporting a serious
state of disturbance in the German missionary districts of
Shantung: "It appears from this communication that the
followers of the sects of the 'Red Fist' and the 'Great Knife'
are in a state of revolt against the Administration and the
people in that province, and are engaged in plunder and rapine
in many places. The native Christians suffer no less than the
rest of the population by this revolt. Money was usually
extorted from them, and their dwellings were pillaged or
destroyed. The Italian Mission, situated in the adjoining
district, were faring no better, and their chapel had just
been burned down. Owing, however, to the unremitting
representations of the German Minister, the Chinese Government
have caused several of the agitators to be arrested by the local
authorities, and they are taking further steps in this
direction, with the result that order is gradually being
restored. At several places the native Christians, with their
non-Christian fellow-countrymen, repulsed the rebels by force
of arms. The Provincial Governor has promised the authorities
of the Mission a full indemnity for the losses suffered by
them and by the other Christians, and several payments have
already been made."

On the 4th of December, the following despatch was sent to

London from the British Legation at Peking:

"During the delimitation of French leased territory at

Kwang-chou-wan on the 13th November, Chinese villagers seized
two French officers and decapitated them. The execution of a
dignitary—the Prefect concerned in the murder—has been
 demanded by the French Minister, as well as the dismissal of
the Canton Viceroy, who is also implicated. The Chinese
Commissioner engaged in the delimitation and the gunboat in
which he travelled are held by the French as hostages."

{101}

CHINA: A. D. 1899 (March).

The Tsung-li Yamên.
Its character and position.
The power of the Empress Dowager.

The Tsung-li Yamên is a small body of Councillors who form a

species of Cabinet, with a special obligation to advise the
Emperor on foreign affairs. "They have no constitutional
position whatever, they have no powers except those derived
from the Emperor, and they are very much afraid for
themselves. He may by mere fiat deprive them of their rank,
which is high; he may 'squeeze' them of their wealth, which is
often great; he may banish them from the delights of Pekin to
very unpleasant places; or he may order them to be quietly
decapitated or cut slowly into little pieces. At such times
their preoccupation is neither their country nor their
immediate business, nor even their own advancement, but to
avoid offending the irritable earthly deity who holds their
lives and fortunes in his hands. Such a time it is just now.
The Empress-Dowager is Emperor in all but name, she has ideas
and a will, and she is suspicious to the last degree. There is
no possibility of opposing her, for she has drawn together
eighty thousand troops round Pekin, who while she pays their
Generals will execute anybody she pleases; there is no
possibility of appeal from her, for she represents a
theocracy; and there is no possibility of overpowering her
mind, for she is that dreadful phenomenon four or five times
revealed in history, an Asiatic woman possessed of absolute
power, and determined to sweep away all who oppose, or whom
she suspects of opposition, from her path. Under her regime
 the members of the Tsung-li Yamên are powerless nonentities,
trembling with fear lest, if they make a blunder, they may
awaken the anger of their all-powerful and implacable
Sovereign, whose motives they themselves often fail to
fathom."

The Spectator (London),

March 18, 1899.

CHINA: A. D. 1899 (March-April).

Agreement between England and Russia concerning
their railway interests in China.

On the 28th of April, 1899, the governments of Great Britain

and Russia exchanged notes, embodying an agreement
(practically arrived at in the previous month) concerning
their respective railway interests in China, in the following
terms:

"Russia and Great Britain, animated by the sincere desire to

avoid in China all cause of conflict on questions where their
interests meet, and taking into consideration the economic and
geographical gravitation of certain parts of that Empire, have
agreed as follows:

1. Russia engages not to seek for her own account, or on

behalf of Russian subjects or of others, any railway
concessions in the basin of the Yang-tsze, and not to
obstruct, directly or indirectly, applications for railway
concessions in that region supported by the British
Government.

2. Great Britain, on her part, engages not to seek for her own
account, or on behalf of British subjects or of others, any
railway concessions to the north of the Great Wall of China,
and not to obstruct, directly or indirectly, applications for
railway concessions in that region supported by the Russian
Government. The two contracting parties, having nowise in view
to infringe in any way the sovereign rights of China or of
existing treaties, will not fail to communicate to the Chinese
Government the present arrangement, which, by averting all
cause of complication between them, is of a nature to
consolidate peace in the far East, and to serve the primordial
interests of China herself."

Second Note. "In order to complete the notes exchanged this

day respecting the partition of spheres for concessions for
the construction and working of railways in China, it has been
agreed to record in the present additional note the
arrangement arrived at with regard to the line
Shanghaikuan-Newchwang, for the construction of which a loan
has been already contracted by the Chinese Government with the
Shanghai-Hongkong Bank, acting on behalf of the British and
Chinese corporation. The general arrangement established by
the above-mentioned notes is not to infringe in any way the
rights acquired under the said loan contract, and the Chinese
Government may appoint both an English engineer and a European
accountant to supervise the construction of the line in
question, and the expenditure of the money appropriated to it.
But it remains understood that this fact can not be taken as
constituting a right of property or foreign control, and that
the line in question is to remain a Chinese line under the
control of the Chinese Government, and can not be mortgaged or
alienated to a non-Chinese Company. As regards the branch line
from Siaoheichan to Sinminting, in addition to the aforesaid
restrictions, it has been agreed that it is to be constructed
by China herself, which may permit European—not necessarily
British—engineers to periodically inspect it, and to verify
and certify that the work is being properly executed. The
present special agreement is naturally not to interfere in any
way with the right of the Russian Government to support, if it
thinks fit, applications of Russian subjects or establishments
for concessions for railways, which, starting from the main
Manchurian line in a southwesterly direction, would traverse
 the region in which the Chinese line terminating at Sinminting
and Newchwang is to be constructed."

Great Britain, Papers by Command,

Treaty Series, Number 11, 1899.

CHINA: A. D. 1899 (April).

Increasing ascendancy of Manchus in the government.

On the 17th of April, Mr. Bax-Ironside reported to Lord

Salisbury: "There has been no change of importance to note in
the political situation. The tendency to replace Chinese by
Manchus in the important political posts of the Empire is
increasing. There are sixty-two Viceroys, Governors,
Treasurers, and Judges of the eighteen provinces and the New
Dominion. Twenty-four of these posts are now held by Manchus,
whereas before the coup d'etat only thirteen of them were so
occupied. So large a percentage of Manchus in the highest
positions tends to indicate a retrograde administration, as
the Manchus are, as a race, very inferior to the Chinese in
intelligence and capacity, and their appointment to important
positions is viewed with disfavour by the Chinese themselves.
The Dowager-Empress has sent special instructions both to
Moukden and Kirin to raise the present standard of the Manchu
schools in those towns to that existing in the ordinary
schools in Peking."-

Great Britain, Papers by Command,

China, Number 1, 1900, page 129.

{102}

CHINA: A. D. 1899 (May-July).

Representation in the Peace Conference at The Hague.

See (in this volume)

PEACE CONFERENCE.
CHINA: A. D. 1899 (August).
Talienwan declared a free port.

"The Emperor of Russia in a quaintly worded Imperial Order

issued on Sunday last [August 13] and addressed to the
Minister of Finance, has declared that after the completion of
the railway Talienwan shall be a free port during the whole
duration of the lease from China. In the course of the Order
the Emperor says: 'Thanks to the wise decision of the Chinese
Government, we shall through the railway lines in course of
construction be united with China,—a result which gives to all
nations the immeasurable gain of easy communication and
lightens the operations of the world's trade.' The Emperor
also speaks of 'a rapprochement between the peoples of the
West and East' (brought about apparently by obtaining an
outlet for the great Siberian rail way) as 'our historic
aim.'"

Spectator (London),
August 19, 1899.

CHINA: A. D. 1899 (December).

Li Hung-chang appointed Acting Viceroy at Canton.

On the 20th of December it was announced that the Viceroy at

Canton had been ordered to Peking and that Li Hung-chang had
been appointed Acting Viceroy of Kwangtung and Kwangsi—the
provinces of which Canton is the Viceroyal seat.

CHINA: A. D. 1899-1900 (September-February).

Pledges of an "open-door" commercial policy in China obtained
by the government of the United States from the governments of
Great Britain, Russia, France, Germany, Italy and Japan.

On the 6th of September, 1899, the American Secretary of

State, Mr. John Hay, despatched to the United States
Ambassador at Berlin the following instructions, copies of
which were forwarded at the same time to the Ambassadors at
London and St. Petersburg, to be communicated to the British
and Russian governments:

"At the time when the Government of the United States was
informed by that of Germany that it had leased from His
Majesty the Emperor of China the port of Kiao-chao and the
adjacent territory in the province of Shantung, assurances
were given to the ambassador of the United States at Berlin by
the Imperial German minister for foreign affairs that the
rights and privileges insured by treaties with China to
citizens of the United States would not thereby suffer or be
in anywise impaired within the area over which Germany had
thus obtained control. More recently, however, the British
Government recognized by a formal agreement with Germany the
exclusive right of the latter country to enjoy in said leased
area and the contiguous 'sphere of influence or interest'
certain privileges, more especially those relating to
railroads and mining enterprises: but, as the exact nature and
extent of the rights thus recognized have not been clearly
defined, it is possible that serious conflicts of interest may
at any time arise, not only between British and German
subjects within said area, but that the interests of our
citizens may also be jeopardized thereby. Earnestly desirous
to remove any cause of irritation and to insure at the same
time to the commerce of all nations in China the undoubted
benefits which should accrue from a formal recognition by the
various powers claiming 'spheres of interest,' that they shall
enjoy perfect equality of treatment for their commerce and
navigation within such 'spheres,' the Government of the United
States would be pleased to see His German Majesty's Government
give formal assurances and lend its cooperation in securing
like assurances from the other interested powers that each
within its respective sphere of whatever influence—

"First. Will in no way interfere with any treaty port or any

vested interest within any so-called 'sphere of interest' or
leased territory it may have in China.

"Second. That the Chinese treaty tariff of the time being

shall apply to all merchandise landed or shipped to all such
ports as are within said 'sphere of interest' (unless they be
'free ports'), no matter to what nationality it may belong,
and that duties so leviable shall be collected by the Chinese
Government.

"Third. That it will levy no higher harbor duties on vessels

of another nationality frequenting any port in such 'sphere'
than shall be levied on vessels of its own nationality' and no
higher railroad charges over lines built, controlled, or
operated within its 'sphere' on merchandise belonging to
citizens or subjects of other nationalities transported
through such 'sphere' than shall be levied on similar
merchandise belonging to its own nationals transported over
equal distances.

"The liberal policy pursued by His Imperial German Majesty in

declaring Kiao-chao a free port and in aiding the Chinese
Government in the establishment there of a custom-house are so
clearly in line with the proposition which this Government is
anxious to see recognized that it entertains the strongest
hope that Germany will give its acceptance and hearty support.
The recent ukase of His Majesty the Emperor of Russia declaring
the port of Ta-lien-wan open during the whole of the lease
under which it is held from China, to the merchant ships of
all nations, coupled with the categorical assurances made to
this Government by His Imperial Majesty's representative at
this capital at the time, and since repeated to me by the
present Russian ambassador, seem to insure the support of the
Emperor to the proposed measure. Our ambassador at the Court
of St. Petersburg has, in consequence, been instructed to
submit it to the Russian Government and to request their early
consideration of it. A copy of my instruction on the subject to