Cell Adhesion and Migration in Skin Disease (Cell Adhesion and Communication)

CELL ADHESION AND MIGRATION IN SKIN DISEASE Cell Adhesion and Communication A series of books encompassing monographs...

Author: Jonathan Barker | John McGrath

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CELL ADHESION AND MIGRATION IN SKIN DISEASE

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

CELL ADHESION AND MIGRATION IN SKIN DISEASE Edited by

Jonathan Barker and John McGrath St.Thomas’ Hospital, London, UK

harwood academic publishers Australia • Canada • France • Germany • India • Japan Luxembourg • Malaysia • The Netherlands • Russia • Singapore Switzerland

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Copyright © 2001 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage or retrieval system, without permission in writing from the publisher. Printed in Singapore. Amsteldijk 166 1st Floor 1079 LH Amsterdam The Netherlands British Library Cataloguing in Publication Data Cell adhesion and migration in skin disease.—(Cell adhesion and commnunication; v. 8) 1. Skin—Diseases—Cytopathology 2. Cell adhesion 3. Cell adhesion— Molecular aspects I.Barker, J. (Jonathan) II. McGrath, John 616.5′07 ISBN 0-203-30459-4 Master e-book ISBN

ISBN 0-203-34336-0 (Adobe eReader Format) ISBN: 90-5823-067-8 (Print Edition) ISSN: 1023–7046

CONTENTS

1.

Preface to the Series

vii

Preface

ix

Contributors

x

Introduction John A.McGrath

1

CELL-CELL ATTACHMENT 2.

The Cornified Cell Envelope Jorge Frank and Angela M.Christiano

8

3.

Keratin and Keratin Disorders Laura D.Corden and W.H.Irwin McLean

26

4.

Desmosomes David R.Garrod, Martyn A.J.Chidgey, Alison J.North, Sarah K.Runswick and Chris Tselepis

56

CELL-MATRIX ATTACHMENT 5.

Protein-Protein Interactions at the Dermal-Epidermal BMZ M.Peter Marinkovich

88

6.

Biology and Pathology of Hemidesmosomes Leena Pulkkinen and jouni Uitto

106

7.

Dermal-Epidermal Adhesion Leena Bruckner-Tuderman

131

LEUKOCYTE TRAFFICKING IN SKIN DISEASES 8.

Introduction Jonathan N.W.N.Barker

164

9.

Skin Homing Lymphocytes Conrad Hauser and René Moser

170

vi

10.

T-cell Accessory Molecules Ralf W.Denfeld and Jan C.Simon

202

11.

Animal Models of Skin Inflammation Benjamin E.Rich and Thomas S.Kupper

221

12.

Langerhans Cell Migration Georg Stingl and Dieter Maurer

241

13.

Leukocyte Adhesion and Accessory Molecules as Therapeutic Targets for Inflammatory Skin Diseases Kimberly E.Foreman and Brian J.Nickoloff

251

Index

264

PREFACE TO THE SERIES

The development and normal functioning of all multicellular organisms is governed to a large part by the interactions cells undergo with neighbouring cells-and with their acellular environment. Many of these interactions are mediated by cell-cell adhesion molecules and by extracellular matrix components and their cellular receptors, that is by molecules which establish direct cell-cell and cell-matrix contacts. These molecules are particularly important for determining whether a cell remains where it is or moves elsewhere and, if a cell moves, where it goes and when it stops migrating. These ness and metastasis. Moreover, recent advances in the field show that most, if are of course key events during normal development, but they play equally crucial roles in adult physiology and pathology, such as the extravasation of white blood cells, inflammatory processes and wound healing, tumour invasive-not all, cell adhesion molecules are capable of triggering intracellular events, in the same way as diffusible growth and differentiation factors and their cellular receptors do. It is thus hardly surprising that clinicians are devoting increasing attention to the molecular mechanisms underlying cell adhesion, and that cell adhesion molecules are not being considered as suitable targets for drug development. This book series is aimed at scientists, both in academia and in industry, at graduate students planning to move into the area, at the clinician, who wants to become familiar with a field with many clinical implications, and at scientists already working in the field, who want to keep abreast with the recent developments outside their own speciality. Hence, each volume of the series reviews a particular segment of the field and provides a critical assessment of recent discoveries and future developments. Each volume has a volume editor, who is an expert in the field and invites contributors to cover the different aspects of the topic. By keeping the number of contributors to each volume small, we hope to avoid overlaps and redundancies, common pitfalls of multi-author volumes. By looking at the previous volumes, I have the impression that we have been successful. Previous volumes of the series addressed the role of cell adhesion in selected physiological and pathological phenomena or concentrated on important structural families. The volumes on Cell Adhesion Molecules in Cancer and Inflammation and on Adhesion Molecules and Chemokines in Lymphocyte Trafficking are examples of the first, those on The Laminins and on the CEA Family are examples of the second kind. Whilst the previous volume, Epithelial Morphogenesis in Development and Disease, focused on the epithelia, whose development and integrity relies heavily on cell adhesion phenomena, this volume addresses this crucial phenomena in an especially important

viii

epithelium, the epidermis of the skin. Together the two books will constitute an invaluable source of information for scientists and clinicians interested in skin biology and diseases.

PREFACE

Cell adhesion and migration play a critical role in many cutaneous diseases. These include blistering disorders both inherited and acquired, and inflammatory conditions, such as eczema and psoriasis. In recent years, considerable advances have been made in our understanding of the molecular basis of cell adhesion and migration in the skin and the precise role of many adhesive proteins have been defined. In this book, we bring together a series of articles written by international experts, describing cell-cell, and cell-matrix adhesion and the biological processes involved in maintaining integrity of the skin and in the orchestration of inflammatory events. In the first part of the book, the molecular mechanisms involved in epidermal cell-cell attachment and keratinocyte structural integrity are discussed in detail. Emphasis is placed on the cornified cell envelope, intermediate filaments and desmosomes. Abnormalities of these structures, for example as a result of genetic mutation or disruption by autoantibodies, lead to specific blistering skin diseases such as epidermolysis bullosa simplex or pemphigus. In the second part of the book, the molecular basis for securing adhesion between the epidermis and the dermis via the intricate network of macromolecules that comprise the cutaneous basement membrane zone is discussed. The importance of perturbations in these adhesion complexes is highlighted by diseases such as junctional epidermolysis bullosa, resulting from inherited abnormalities in genes encoding structural components of hemidesmosomes and anchoring filaments and bullous pemphigoid resulting from autoimmune assault against a hemidesmosomal collagen. In the third part of the book, the molecular events responsible for accumulation of inflammatory cells into skin are discussed. Leukocyte adhesion molecules play a critical role in the control of lymphocyte recruitment into skin and as such are attractive targets for therapeutic intervention in designing new strategies to combat common inflammatory skin diseases including psoriasis and eczema. Indeed, clinical trials are presently under way using biotechnological approaches to anti-leukocyte adhesion molecule therapy and the rationale for such studies is discussed. It is hoped that the high quality chapters from international experts and the layout of this book will appeal to basic scientists and clinicians working in skin biology. Further we believe the book will also be of invaluable assistance to industry by providing information on important molecular targets in bullous and inflammatory skin diseases.

CONTRIBUTORS

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

xi

Dundee DD1 9SY UK Ralf W. Denfeld Department of Dermatology Albert-Ludwigs-Universität Freiburg Germany Kimberly E.Foreman Department of Pathology Skin Cancer Research Laboratories Cardinal Bernardin Cancer Center Loyola University Medical Centre Maywood, IL USA Jorge Frank Department of Dermatology College of Physicians and Surgeons Columbia University 630 West 168th Street, VC-1526 New York, NY 10032 USA David R.Garrod School of Biological Sciences 3.239 Stopford Building University of Manchester Oxford Road Manchester M13 9PT UK Conrad Hauser Allergy Unit Division of Immunology and Allergy, and Department of Dermatology (DHURDV) University Hospitals 1211 Geneva 14 Switzerland Thomas S.Kupper Division of Dermatology Harvard Skin Disease Research Center Brigham and Women’s Hospital Boston, MA 02115 USA M.Peter Marinkovich

xii

MSLS Building Rm. p208 Stanford University School of Medicine Stanford, CA 94305 USA Dieter Maurer Division of Immunology, Allergy and Infectious Diseases (DIAID) Department of Dermatology University of Vienna Medical School Vienna Austria John A.McGrath St. John’s Institute of Dermatology St. Thomas’ Hospital Lambeth Palace Road London, SE1 7EH UK W.H.Irwin McLean Epithelial Genetics Group Department of Molecular and Cellular Pathology Ninewells Medical School University of Dundee Dundee DDI 9SY UK René Moser Institute for Biopharmaceutical Research Inc. 9545 Waengi Switzerland Brian J.Nickoloff Department of Pathology Skin Cancer Research Laboratories Cardinal Bernardin Cancer Center Loyola University Medical Centre Maywood, IL USA Alison J.North School of Biological Sciences 3.239 Stopford Building University of Manchester Oxford Road Manchester M13 9PT

xiii

UK Leena Pulkkinen Department of Biochemistry and Molecular Pharmacology Thomas Jefferson University Philadelphia, PA 19107 USA Benjamin E.Rich Division of Dermatology Harvard Skin Disease Research Center Brigham and Women’s Hospital Boston, MA 02115 USA Sarah K.Runswick School of Biological Sciences 3.239 Stopford Building University of Manchester Oxford Road Manchester M13 9PT UK Jan C.Simon Department of Dermatology Albert-Ludwigs-Universität Freiburg Germany Georg Stingl Division of Immunology, Allergy and Infectious Diseases (DIAID) Department of Dermatology University of Vienna Medical School Vienna Austria Chris Tselepis School of Biological Sciences 3.239 Stopford Building University of Manchester Oxford Road Manchester M13 9PT UK Jouni Uitto Department of Dermatology and Cutaneous Biology

xiv

Jefferson Medical College Thomas Jefferson University Philadelphia, PA 19107 USA

1. INTRODUCTION JOHN A.McGRATH

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

2 JOHN A.MCGRATH

Figure 1.1 Ultrastructural appearances of cell adhesion junctions in human epidermis. (a): hemidesmosomes (arrow) at the dermal-epidermal junction (×40,000); (b): a desmosome (arrow) between adjacent keratinocytes (×70,000).

Subsequently, pathogenetic missense mutations were demonstrated in the corresponding genes, KRT14 and KRT5, in DNA from patients with this autosomal dominant skinfragility syndrome. Thus, epidermolysis bullosa simplex was established as the first keratin gene disorder and the pathological importance of cytoskeletal integrity to cell adhesion was established. In subsequent years, keratin gene defects have been delineated in a large number of other skin diseases, many involving abnormalities of cell adhesion (Irvine and McLean, 1999). To date, mutations have been described in 15 different epithelial keratins as well as two hair keratins. The associated diseases include bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), ichthyosis bullosa of Siemens, epidermolytic palmoplantar keratoderma, diffuse and focal non-epidermolytic palmoplantar keratoderma, pachyonychia congenita and monilethrix. Further insights into cell adhesion have also been established from studies of diseases affecting keratinocyte terminal differentiation. Specifically, some cases of the autosomal dominant mutilating keratoderma, Vohwinkel’s syndrome, and the disorder, progressive symmetric erythrokeratoderma, have been shown to result from mutations in the gene encoding loricrin, the major component of the cornified cell envelope (Maestrini et al., 1996; Ishida-Yamamoto et al., 1998). Immunoelectron microscopy studies have revealed

INTRODUCTION 3

that the mutant loricrin fails to be incorporated into the cell membrane/envelope but instead has an intranuclear localisation. Desquamation normally involves alterations in cell adhesion through degradation of lamellated lipid and loss of residual desmosomes but this is perturbed by the presence of a dominant-negative loricrin mutation. This process may also be disrupted by abnormalities in keratinocyte transglutaminase, mutations in which may lead to the autosomal recessive disorder, lamellar ichthyosis. Studies of skin diseases involving pathological changes in desmosomes have also provided significant information about keratinocyte adhesion. Desmosomes comprise a collection of transmembranous glycoproteins (desmogleins and desmocollins) and several intracellular proteins that link the cell membrane to the cytoskeleton, including plakoglobin, desmoplakin and plakophilin 1 (Smith and Fuchs, 1998). The acquired blistering skin disease, pemphigus, involves pathogenic autoantibodies directed against conformational epitopes on some of the desmosomal cadherins leading to a loss of cellcell adhesion and acantholysis. Studies of the different subtypes of pemphigus and the distribution of target epitopes in skin and mucosal epithelium have provided further new insights into cell adhesion (Amagai, 1999). For example, patient sera containing antidesmoglein 3 antibodies alone lead to a predominantly mucosal form of pemphigus vulgaris with limited skin involvement. Sera containing both anti-desmoglein 3 and antidesmoglein 1 antibodies may lead to pemphigus vulgaris, with disrupted desmosomal adhesion in both skin and mucosa. In contrast, the presence of desmoglein 1 antibodies alone results in pemphigus foliaceous with skin blisters but no mucosal involvement. Thus, targeting of particular desmosomal cadherins by autoantibodies compromises cell adhesion with different clinical consequences. Naturally occurring mutations in the desmoglein 1 gene have also been described. Specifically, an amino-terminal deletion in desmoglein 1 has been shown to underlie some cases of striate palmoplantar keratoderma, an autosomal dominant genodermatosis (Rickman et al., 1999). This condition does not lead to blistering, but keratinocyte terminal differentiation is affected through perturbed desmosome physiology leading to hyperkeratosis of the palms and soles. This genodermatosis may also result from haploinsufficiency in the desmosomal plaque protein, desmoplakin (Armstrong et al., 1999). In such patients, cell adhesion is disrupted in that electron microscopy reveals that some of the desmosomes are small with widening of intercellular spaces between keratinocytes and disruption of the keratin filament network which is compacted in a perinuclear distribution. Similar ultrastructural features are also seen in patients who have complete ablation of plakophilin 1 (McGrath et al., 1997). In addition to skin fragility with loss of cell-cell adhesion, these patients show features of a hypohidrotic ectodermal dysplasia with abnormalities of hair, nails, teeth and sweating. Such changes arise because plakophilin 1 not only has a role as a structural component of desmosomes but, in addition, as a member of the armadillo gene/protein family, it also contributes to epidermal development and morphogenesis. Components of other epithelial junctions may also have broad biological significance. For example, in gap junctions, mutations in some of the connexin genes (connexins 26 and 31) may either affect keratinocyte terminal differentiation and lead to keratoderma or the mutations may result in non-syndromic deafness, or even clinical overlap between the two depending on the type or combination of connexin mutations (Kelsell et al., 1997; Richard et al., 1998).

4 JOHN A.MCGRATH

Figure 1.2 Inherited and acquired blistering skin diseases provide insights into the mechanisms of epidermal cell adhesion, (a): blisters and erosions in a 40-year-old male with inherited mutations in the type XVII collagen gene (COL17A1) resulting in complete ablation of the protein; (b): extensive conjunctival scarring in a 65-year-old male with acquired autoantibodies to the α3 chain of laminin 5; (c) erosions and scarring on the dorsum of the hand of a 48-year-old male resulting from autoantibodies against the NC-1 domain of type VII collagen; (d) prominent sacral erosions in a 72year-old female with an inherited nonsense/missense combination of mutations in the LAMB3 gene of laminin 5; (e) blisters, erosions, scars and milia on the knees of a 14-year-old girl with an inherited homozygous nonsense mutation in the type VII collagen gene, COL7A1; (f) tense blisters and urticated erythema on the lower leg of a 62-year-old female with acquired autoantibodies against the NC16A domain of type XVII collagen.

INTRODUCTION 5

Cell adhesion between basal keratinocytes and the underlying dermis also involves interactions between multiple structural macromolecules, many of which may be targets for autoantibodies in acquired blistering skin diseases or subjects of inherited mutations in the genes encoding these proteins in genetic blistering skin disorders. Both types of abnormality have offered fresh insights into the structural dynamics of cell adhesion as well as the pathology of certain skin diseases (Borradori and Sonnenberg, 1999). For example, autoantibodies against type XVII collagen (also known as the 180-kDa bullous pemphigoid antigen) lead to blister formation through the lamina lucida and the clinical disorder, bullous pemphigoid. Mutations in the corresponding gene, COL17A1, result in the autosomal recessive genodermatosis, non-Herlitz junctional epidermolysis bullosa. Similar paradigms for acquired-inherited disorders of cell adhesion exist for other hemidesmosome-associated components, including laminin 5 and type VII collagen and, to a lesser extent, plectin and α6β4 integrin (McGrath and Eady, 1998). These diseases provide information about the particular roles of individual structural proteins in cell adhesion. In addition, they offer clues to the relative contributions of specific proteins involved in cell adhesion. For example, total ablation of laminin 5, resulting from inherited nonsense mutations on both alleles of LAMA3, LAMB3 or LAMC2 (the three genes encoding laminin 5), results in the lethal, Herlitz form of junctional epidermolysis bullosa, in contrast to the milder, non-Herlitz phenotype resulting from complete ablation of type XVII collagen (Jonkman, 1999). Just as some components of desmosomes have roles beyond simple mechanical adhesion, the same is true for hemidesmosomes. For example, the α6β4 integrin not only helps secure adhesion between the hemidesmosome and epidermal basement membrane, but it is also able to transduce signals from the extracellular matrix to the cell interior and thereby modulate keratinocyte processes such as differentiation, proliferation and cytoskeletal organisation. The aim of this first section of the book (chapters 2–8) is to examine the mechanics of keratinocyte cell adhesion in more detail. Specifically, the chapters cover the topics of the cornified cell envelope, the keratin intermediate filaments, desmosomes, hemidesmosomes, dermal adhesion, and protein-protein interactions. The goal is to outline the complexity and intricacy of the biology of keratinocyte cell adhesion as well as to demonstrate the rapidly evolving state of knowledge in this field and its relevance to skin scientists and dermatologists. REFERENCES Amagai M. Autoimmunity against desmosomal cadherins in pemphigus. J Dermatol Sci 20: 92–102, 1999. Armstrong DKB, McKenna KE, Purkis PE et al.Haploinsufficency of desmoplakin causes a striate type of palmoplantar keratoderma. Hum Mol Genet 8:143–148, 1999. Borradori L, Sonnenberg A. Structure and function of hemidesmosomes: more than simple adhesion complexes. J Invest Dermatol 112:411–418, 1999. Irvine AD. McLean WHI. Human keratin diseases: the increasing spectrum of disease and subtlety of the phenotype-genotype correlation. Br J Dermatol 140:815–828, 1999.

6 JOHN A.MCGRATH

Ishida-Yamamoto A, McGrath JA, Chapman SJ, Leigh IM, Lane EB, Eady RAJ. Epidermolysis bullosa simplex (Dowling-Meara) is a genetic disease characterized by an abnormal keratin filament network involving keratins K5and K14. J Invest Dermatol 97:959–968, 1991. Ishida-Yamamoto A, Takahashi H, lizuka H. Loricrin and human skin diseases: molecular basis of loricrin keratodermas. Histol Histopathol 13:819–826, 1998. Jonkman MF. Hereditary diseases of hemidesmosomes. J Dermatol Sci 20:103–121, 1999. Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387:80–83, 1997. Maestrini E, Monaco AP, McGrath JA, Ishida-Yamamoto A, Camisa C, Hovnanian A, Weeks DE, Lathrop M, Uitto J, Christiano AM. A molecular defect in loricrin, the major component of the cornified cell envelope, underlies Vohwinkel’s syndrome. Nat Genet 13: 70–77, 1996. McGrath JA, McMillan JR, Shemanko CS, Runswick SK, Leigh IM, Lane EB, Garrod DR, Eady RAJ. Mutations in plakophilin 1 result in ectodermal dysplasia-skin fragility syndrome. Nat Genet 17:240–244, 1997. McGrath JA, Eady RAJ. Molecular basis of blistering skin diseases. Hosp Med 59:28–32, 1998. Richard G, Smith LE, Bailey RA, Itin P, Hohl D, Epstein EH Jr, DiGiovanna J.J, Compton JG, Bale SJ. Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis. Nat Genet 20:366–369, 1998. Rickman L, Simrak D, Stevens HP, Hunt DM, King IA, Bryant SP, Eady RAJ, Leigh IM, Arnemann J, Magee AI, Kelsell DP, Buxton RS. Amino-terminal deletion in a desmosomal cadherin causes the skin disease striate palmoplantar keratoderma. Hum Mol Genet 8:971–976, 1999. Smith EA, Fuchs E. Defining the interactions between intermediate filaments and desmosomes. J Cell Biol 141:1229–1241, 1998.

CELL—CELL ATTACHMENT

2. THE CORNIFIED CELL ENVELOPE JORGE FRANK AND ANGELA M.CHRISTIANO

SUMMARY The skin is the largest organ of the human body, and is the home of constant cycles of exquisitely regulated cell migration, differentiation, and regeneration. In stratifying squamous epithelia, such as the skin, the cornified cell envelope (CE), a highly insoluble peripheral layer of crosslinked proteins, replaces the plasma membrane in a complex and sequential fashion from precursor proteins initially dispersed in the cytoplasm. The insolubility of the CE is based on the presence of N-ε(γ-glutamyl) lysine isodipeptide cross-links formed by epidermal transglutaminases. Because of its mechanical resilience and impenetrability, the CE provides the human skin with a protective barrier against the environment. Here, we provide an overview of the different components of the CE and their properties and function within this complex scaffolding of cross-linked proteins. INTRODUCTION The human body is continuously exposed to a variety of external environmental aggressors. The first and most important barrier of defense and protection against these hazards is provided by the skin, in particular by the epidermis, the outermost skin layer. The epidermis consists of four different cell layers, the basal layer (stratum basalis), the spinous layer (stratum spinosum), the granular layer (stratum granulosum), and the cornified layer (stratum corneum). Additionally, a transitional cell layer (stratum lucidum) can be differentiated. This stratum lucidum is located between the stratum granulosum and the stratum corneum and, thus, separates the “living” parts of the epidermis from the “dead” epidermal layers. The epidermal network consists of a variety of different cell types, the most important of which is the epidermal keratinocyte, and in recent years, a large number of inherited disorders involving the keratinocyte have been described (Figure 2.1). Through a complex and highly structured cycle of differentiation, keratinocytes are responsible for constructing a protective barrier against the environment. The last step in keratinocyte differentiation is characterized by the formation of cornified cells, so called corneocytes. These corneocytes are enucleated,

THE CORNIFIED CELL ENVELOPE 9

Figure 2.1 A summary of recent progress in the molecular basis of genodermatoses. The inherited skin disorders involving specific skin layers are indicated on the left, and the genes proteins are listed on the right (BPAG1, bullous pemphigoid antigen 1; BPAG2, bullous pemphigoid antigen 2; EB, epidermolysis bullosa; EHK, epidermolytic hyperkeratosis; HD1, plectin; PPK, palmoplantar keratodermas; SPRRs, small proline-rich proteins; VS, Vohwinkel’s syndrome). Reproduced with permission from Ref. 79.

flattened polyhedrons, which consist of a stabilized array of keratin filaments encased within the cornified cell envelope (CE) (1). The CE represents the most insoluble component of stratified squamous epithelial cells and appears as an electron dense and homogeneous band, initially about 15 nm in thickness, replacing the plasma membrane in the uppermost cell layers of squamous epithelia in vertebrates (2, 3). Later, it gradually increases its thickness and rigidity, and in cornified cells, the CE is about 20 nm thick (3). CE formation occurs as a complex but highly structured sequence of events, involving the sequential deposition of distinct proteins (2). These proteins are cross-linked by the formation of Nε-(γ-glutamyl) lysine isodipeptide and disulfide bonds. Nε-(γ-glutamyl) lysine cross-linkage is catalyzed by distinct isoforms of transglutaminases (TG), TG1 and TG 3, and the formation of disulfide bonds is catalyzed by sulfhydryl oxidase (2, 3). Apart from the TGs, the best characterized

10 JORGE FRANK AND ANGELA M.CHRISTIANO

proteins of the CE are loricrin, involucrin and the small proline-rich proteins (SPRRs), which are all encoded by genes localized within a 2 megabase region on human chromosome 1q21, known as the epidermal differentiation complex (EDC) (Figure 2.2) (4, 5). Further components of the CE include elafin (ELA; synonym cysteine-rich protein or skin-derived antileukoproteinase, SKALP), profilaggrin (PFN), trichohyalin (THH), cystatin A (CYA; synonym keratolinin), the S100 protein family (S100A1–S100A11), envoplakin (EPL), periplakin (PPL), and corneodesmosin (CDE) (1–3, 5–9). Here, we discuss the major components of the CE and their properties and function within this complex scaffolding of cross-linked proteins. MAJOR COMPONENTS OF THE CORNIFIED CELL ENVELOPE Transglutaminases The TGs constitute a family of calcium- and thiol-dependent enzymes, catalyzing the formation of Nε-(γ-glutamyl) lysine isodipeptide bonds and N1,N8-bis(γ-glutamyl) spermidine bonds (3). In humans, the TG family consists of six known members, and at least two of these, TG1 and TG3, are believed to play an important role in the formation and maintenance of the CE (10). TG1 is an approximately 92 kDa protein, consisting of 817 amino acids. It is thought to function in a plasma membrane-bound form via its amino terminal region, and also as a soluble form in the cytoplasm (3). The major expression sites are in the upper spinous and granular layers (11), although expression of TG1 can also be detected in undifferentiated epidermal basal cells (12). The gene encoding human TG1 is located on human chromosome 14q 11.2 (13). TG3 is a 77 kDa soluble pro-enzyme, consisting of 692 amino acids. The intact TG3 protein can be subdivided into two globular domains, a 50 kDa amino-terminus containing catalytic regions, and a 27 kDa carboxy-terminus. These two domains are separated by a flexible hinge, corresponding to the cleavage site for conversion to the active protein (14, 15). TG3 is exclusively expressed in the stratum granulosum and the expression is regulated by calcium and the Sp1 and ets transcription factors (16). The gene coding for TG3 has been mapped to human chromosome 20q 11.2 (13, 17). Loricrin Loricrin is a 26 kDa protein that is located in the stratum granulosum of the epidermis and on the inner surface of purified/sonicated CEs (18, 19). The protein consists of 315 amino acids, is rich in glycine (55%), serine (22%), and cysteine (7%), and is highly insoluble. The insolubility is attributed to the high content of glycine residues and the formation of intra- and intermolecular disulfide bonds (1, 3). Loricrin contains repeats of a unique, highly flexible glycine-rich sequence, held in place by aromatic amino acid interactions, and referred to as a “glycine-loop” (1, 3). The amino- and carboxy-terminal ends of loricrin are rich in glutamine and lysine residues (3). Loricrin is the major


Figure 2.2 The epidermal differentiation complex (EDC). The three gene families residing in the EDC region on human chromosome 1q21 are indicated to the right of the diagram. The loricrin, involucrin and SPRR gene families are shown as purple rectangles, the trichohyalin and filaggrin gene families are shown in green, and the S100 gene family is shown in black. Genomic distances are represented in kilobases (kb). Reproduced with permission from Ref. 79.

component of the CE, comprising about 75% of the total CE mass, or 85–95% of the cytoplasmic two-thirds of the CE (20). Using recombinant loricrin molecules, it was demonstrated that TG1 is involved in establishing inter-molecular cross-links resulting in very large loricrin oligomers, whereas most of the cross-links formed by TG3 are intramolecular bridges (20). Loricrin is mainly expressed in the granular layers of the epidermis, and its expression is upregulated by calcium, cell density, and phorbol ester, and downregulated by retinoids (3). The loricrin gene is a single copy gene and resides within the EDC on human chromosome 1q21 (4,21).


Involucrin Involucrin is an acidic, water-soluble, rod-shaped protein of 68 kDa and consists of 585 amino acids, rich in glutamine and glutamic acid (3). The protein can be subdivided into amino- and carboxy-terminal flanking domains, showing homology to those of loricrin, and a central domain containing 39 repeats of a consensus decapeptide (22). All 39 repeats contain, on average, 3 glutamine residues, each of them representing a potential cross-linking site (1). Involucrin is ubiquitously synthesized by all stratified squamous epithelia and is expressed in the spinous layer of normal epidermis (3) and in cells of the inner root sheath of the hair follicle (23). Recent studies in transgenic mice demonstrated that over-expression of involucrin in mouse epidermis is associated with a delay in hair ingrowth, alterations in hair appearance, and changes in the appearance of the epidermis (24). Involucrin is believed to act as a major initial component of the CE, functioning as a scaffold onto which other proteins are incorporated (3). However, not all involucrin protein is incorporated into the CE, and it is conceivable that it might exhibit other specific biological functions in the cytoplasm (3). Involucrin expression is positively regulated by phorbol ester, calcium, vitamin D, and hydrocortisone, and negatively regulated by retinoids (3). Involucrin is encoded by a unique gene on human chromosome 1q21 within theEDC (4,21). Small Proline-rich Proteins The SPRRs are small (about 10 to 30 kDa), basic, soluble, proline-rich proteins (3). Members of this family of proteins have also been named pancornulins and cornifin (25, 26). To date, three classes of SPRRs have been distinguished, SPRR1, SPRR2, and SPRR3 (2). They contain a variable number of repeating elements in their central portions with a common sequence motif (XKXPEPXX) and end domains revealing high homology with the amino- and carboxy-terminal domains of loricrin and involucrin (2). Cross-linking occurs at these amino- and carboxy-terminal regions, suggesting that SPRRs may function as molecular cross-bridges connecting two proteins (6, 27). Expression of SPRRs is distinctively regulated in various stratified epithelia (2, 3). In normal human epidermis, SPRR1 is mainly expressed in the skin appendages and SPRR2 is expressed in the stratum granulosum, whereas SPRR3 is absent (3). The expression of SPRR2 and SPRR3 is downregulated by retinoic acid (2). Calcium, interleukin-1 and interleukin-3 upregulate the expression SPRR1, and whereas TGF-β results in downregulation. Human SPRR genes constitute a multigene family clustered within a 300 kb DNA region in the EDC on chromosome 1q21, close to the genes encoding for loricrin and involucrin (4, 28). This gene cluster contains two known SPRR1 genes, eight SPRR2 genes, and a single SPRR3 gene (28). Elafin Elafin is a basic, 6 kDa protein derived from the 12 kDa precursor preproelafin (1,3, 29, 30). Preproelafin consists of 117 amino acids; the initiation methionine, a putative 25-amino


acid signal peptide (“pre” sequence), a 35-amino acid “pro” sequence, and the 57 amino acids that constitute the mature elafin protein (31). Elafin is a potent inhibitor of elastase and proteinase-3, both derived from polymorphonuclear leukocytes (3). It has a high content of cysteine (14%), glycine (13%), and proline (12%), and its structure suggests the potential to form disulfide bonds (1). Elafin contains an internal segment that is a highly reactive TG substrate. This sequence consists of a series of six amino acids repeated five times (VKGQDP) (32). In normal adult epidermis, there is only little, if any, expression of elafin, but it is highly expressed in differentiated keratinocytes in various pathological conditions, including psoriatic epidermis (3). Further, it is transiently expressed during fetal and neonatal development of the epidermis. The expression of elafin is induced by interleukin Iβ and TNFα (33). The gene encoding elafin has been mapped to chromosome 20q12–q13 (34). In contrast to other CE precursors, the elafin gene contains a signal sequence and proelafin is stored in secretory vesicles and secreted extracellularly (35). Cystatin A Cystatin A is a 12 kDa, lysine-rich protein, belonging to the evolutionarily related family of cystatins which inhibit cysteine proteinases (3, 36). Phosphorylated cystatin A is a natural substrate of epidermal TG and a constituent of the CE (6, 37). It functions as a bacteriostatic barrier (38) and is expressed in the cells of the epidermal spinous layer (39). Cystatin A is upregulated by calcium and forskolin, a cAMP elevating agent (40). The human cystatin A gene is localized on human chromosome 3cen-q21 (41). S100 Protein Family The S100 proteins are a group of small (10–12 kDa), acidic proteins that form homo- and heterodimers and bind calcium via two EF hand motifs (27, 42). They share a common sequence and structure and are expressed in a tissue- and differentiation-specific manner (42). Some of the S100 proteins are expressed in the skin, and S100A10 (calpactin light chain) as well as S100A11 (S100C, calgizzarin) are precursors of the CE (27). Since S100 proteins have no known enzymatic activity, they are thought to function in a calmodulinlike manner to regulate calcium-dependent cell signaling, proliferation, and morphology (27, 42). Recent studies revealed that S100A10 binds to annexin II to form a tetramer, called calpactin 1 (27). S100A11 interacts with annexin I in a calcium-dependent manner, an interaction that requires the 12 first amino acids of the annexin I amino terminus (27). The genes encoding the proteins of the S100 family reside in the EDC on chromosome 1q21 (21). Profilaggrin/Filaggrin Profilaggrin is a highly phosphorylated protein component of the keratohyalin granules of mammalian epidermis in the stratum granulosum (43). It consists of 10 or more tandemly repeated filaggrin units plus an amino- and a carboxy-domain (43, 44). The amino


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


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


fact, Corneodesmosin (58) and that the protein contains a high percentage of serine, glycine, and proline residues (60). Although there are significant similarities with the proteins encoded by loricrin, keratin 1, and keratin 10, all major components of the stratum granulosum, the S gene is structurally unrelated to other known genes of the HLA complex (60), and, in a study investigating the potential role of the S gene in psoriasis vulgaris, several genetic intragenic polymorphisms have been identified (61). MOLECULAR BASIS OF DISEASES AFFECTING THE CE Lamellar Ichthyosis and Transglutaminase Mutations Lamellar ichthyosis (LI) (OMIM accession numbers 242300 and 601277) is a genetic skin disorder, inherited in an autosomal recessive fashion (62). Clinically, ichthyotic lesions are present at birth and almost always involve the entire skin surface. The children are usually born encased in a collodion membrane that desquamates during the first 10 to 14 days of life. The skin is erythematous and covered with large scales, fissures of the hands and feet, scarring alopecia, dystrophic nails, and ectropion are common symptoms. Because of an obstruction of the eccrine glands, the patients do not sweat normally, which can lead to hyperpyrexia (63). Recently, it was demonstrated that there exist at least three genetically distinct forms of recessively inherited LI characterized by typical pathology of the skin, with direct sequelae of primary cutaneous lesions (for example ectropion, joint contractures, and digital necrosis) and without in volvement of other organ systems (64, 65). The form that maps to chromosome 14q 11 has been designated LI Type 1. This form of LI is characterized by mutations in TG1, one of the major components of the CE (64, 66–74). A second form, LI Type 2, maps to chromosome 2q33–q35 (65); however, no candidate gene for LI Type 2 has been identified so far in this chromosomal region. A third form of LI, LI Type 3, has been mapped to chromosome 20q l l.2 in the region of the TG3 gene (13, 17). A large number of mutations in the TG1 gene in LI have enabled genotype/ phenotype correlation in families with lamellar ichthyosis (73). Linkage analyses using microsatellite markers spanning the region of the TG1 gene confirmed genetic heterogeneity. In patients not linked to the TG1 locus, the second region identified on chromosome 2q33–q35 and the third candidate region on chromosome 20 were excluded, suggesting the existence of at least four loci for lamellar ichthyosis (73). Vohwinkel’s Syndrome and Loricrin Mutations Vohwinkel’s syndrome (OMIM number 124500), first reported in 1929, is an autosomal dominant palmoplantar is keratoderma with pseudoainhum, and sometimes associated with deafness (75, 76) (figure 3). In an extended Vohwinkel’s syndrome family, Maestrini et al. demonstrated linkage to the EDC on chromosome Iq21, the calculated maximum multipoint lod score being 14.3 (75), and a mutation consisting of a 1 bp insertion after nucleotide 730 (730insG) was detected in the loricrin gene (77). This additional G nucleotide was found in an area of six subsequent wild-type G nucleotides (codons 230


Figure 2.3 Clinical presentation of Vohwinkel’s syndrome, (a) Characteristic honeycomb keratoderma on the palm, (b) Pseudoainhum is present on the third, fourth and fifth digits, (c) Bilateral autoamputation of the fifth toes in an affected individual. Reproduced with permission from Ref. 79.

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


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


POSSIBILITIES OF FUTURE GENE THERAPY FOR DISORDERS OF THE CE The skin represents an accessible somatic tissue for gene transfer and, depending on therapeutic goals, a variety of cutaneous gene delivery approaches are currently available (84–86). Recently, corrective gene transfer was demonstrated in LI, a disfiguring skin disease characterized by abnormal epidermal differentiation and defective cutaneous barrier function (84). LI is associated with a decreased activity of keratinocyte TG1, the enzyme necessary for normal formation of the cornified epidermal barrier. Using LI as a prototype for therapeutic cutaneous gene delivery, Choate et al. developed a human skin/ immunodeficient mouse xenograft model to correct the molecular, histological and functional abnormalities of skin derived from LI patients in vivo. After retroviral transduction of TG1 into TG1-deficient primary keratinocytes, they regenerated engineered human LI epidermis on immunodeficient mice. The engineered LI epidermis displayed normal TG1 expression in vivo, in contrast to unengineered LI epidermis where TG1 was absent. Epidermal architecture was also normalized by TG1 restoration, as was expression of the epidermal differentiation marker filaggrin. Engineered LI skin demonstrated restoration of cutaneous barrier function to levels seen in epidermis regenerated by keratinocytes from patients with normal skin, indicating functional correction of the proposed primary pathophysiologic defect in LI. These results confirm a major role for TG1 in epidermal differentiation, and demonstrate a potential future approach to therapeutic gene delivery in human skin (84). In 1997, the same group of researchers examined the regeneration of skin from TG1-deficient patients with LI on nude mice by direct injection of naked plasmid DNA (85). Regenerated LI patient skin receiving repeated in vivo injections with a TG1 expression plasmid displayed restoration of TG1 expression in the correct tissue location in the suprabasal epidermis. However, unlike LI skin regenerated from keratinocytes first transduced with a retroviral expression vector for TG1 prior to grafting, directly injected LI skin displayed a non-uniform TG1 gene expression pattern (85). Further, direct injection failed to correct the central histologie and functional abnormalities of the disease. These data demonstrate that partial restoration of gene expression can be achieved via direct injection of naked DNA in human skin, but underscore the need for new advances to achieve efficient and sustained plasmid-based gene delivery to the skin. Recently, another group of researchers has also demonstrated successful corrective gene transfer in the human skin disorder X-linked ichthyosis (XLI) (OMIM number 308100) (86). XLI is characterized by loss of function of the steroid sulfatase arylsulfatase C (STS). Freiberg et al. developed a model of corrective gene delivery to human skin in vivo (86). A retroviral expression vector was produced and utilized for STS gene transfer into primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with


defective skin barrier function, effectively recapitulating the human disease. Transduced XLI keratinocytes from the same patients, however, regenerated an epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin (86). STS expression in transduced XLI epidermis was demonstrated in vivo by immunostaining as well as a normalization of histologie appearance at 5 weeks postgrafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. Thus, these findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy (86). Collectively, these data support the notion that the epidermis is an attractive site for therapeutic gene delivery due to its accessibility and the potential for delivering polypeptides to the systemic circulation. A number of obstacles, however, have emerged in attempts at cutaneous gene delivery, and central among these is the inability to sustain therapeutic gene production. The challenges for the future of gene therapy in the skin have been identified in these early studies, and provide a foundation of knowledge for the growth of this exciting and important field. CONCLUSIONS In recent years, chromosomal mapping and cloning of the genes encoding the different protein components of the CE, as well as the identification of TG1 and loricrin mutations underlying diseases affecting the CE, has greatly enhanced our understanding of the structure and organization of this important structure of the skin. However, more discoveries will be made before the mysteries of this complex protein cross-linked treasury are solved. The number of newly identified precursors of the CE continues to grow, and elucidation of their relevance to the maintenance of skin integrity, inherited skin disorders and approaches to gene therapy is our challenge for the future. REFERENCES 1. 2.

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3. KERATINS AND KERATIN DISORDERS LAURA D.CORDEN AND W.H.IRWIN MCLEAN

MOLECULAR ASPECTS OF KERATIN INTERMEDIATE FILAMENTS The Intermediate Filament Family of Cytoskeletal Proteins In addition to the actin microfilament and microtuble cytoskeletal systems found in all mammalian cells, there is a third cytoskeletal network comprised of filaments which are intermediate in size between the other two systems and which are therefore termed intermediate filaments (Figure 3.1 & 3.2). Lazarides described five classes of intermediate filament within higher eukaryotic cells, distinguishable biochemically and immunologically: keratin, desmin, vimentin, neurofilaments and glial filaments (Lazarides, 1980). The current classification of the different types of intermediate filament is shown in Table 3.1, below. THE MOLECULAR STRUCTURE OF INTERMEDIATE FILAMENTS All intermediate filaments have a diameter of approximately lOnm (Lazarides, 1980) and share a common structure composed of a central a-helical rod domain of approximately 310 amino acids (Geisler and Weber, 1982; Hanukoglu and Fuchs, 1982; Hanukoglu and Fuchs, 1983; Lewis et al., 1984; Quax et al., 1983; Steinert et al., 1983; Tyner et al., 1985; Weber and Geisler, 1982), corresponding to 46nm in length (reviewed in (Steinert and Bale, 1993; Steinert and Parry, 1985)). The domain structures of type I and II keratins, typical of all intermediate filament types, are shown in Figure 3.3. The rod domain is interspersed by three non α-helical regions which are predicted to take the form of β-turns (Hanukoglu and Fuchs, 1983; Steinert et al., 1983), and divide the rod into four sub-domains termed 1A, 1B, 2A and 2B (Steinert and Parry, 1985) (Figure 3.3). The four helical domains are reasonably constant in size between all intermediate filament types, approximately 30–50, 95, 35 and 95 amino acid residues in length respectively (Hanukoglu and Fuchs, 1983).

KERATINS AND KERATIN DISORDERS 27

Table 3.1 Types of intermediate filament

Figure 3.1 Epithelial cell line PtK2 transfected with a human K17 cDNA clone in eukaryotic expression plasmid pCR3.1, stained with monoclonal antibody F3 against K17 and FITC secondary antibody. This confocal scanning laser microgaph shows the typical dense cytoplasmic meshwork of keratin filaments seen in mammalian epithelial cells. Photograph provided by Drs Frances J.D.Smith and Seana P.Covello, Epithelial Genetics Group, Thomas Jefferson University, Philadelphia.

The non-helical linker regions possess different net charges, implying that ionic interactions between these regions on opposite molecules might play a part in stabilizing the filament (North et al., 1994). The neutral/basic LI linker separates the 1A and 1B subdomains of the rod (Figure 3.3) and consists of between 8–12 residues dependent upon filament type. The acidic linker LI2 separates the rod domain between the IB region and 2A (Figure 3.3). This linker is variable in length (16–22 residues) but shows some degree of sequence conservation between the intermediate filament types. The acidic linker L2, separating the 2A and 2B subdomains of the rod, is the shortest of the three linkers. It is uniform in length, consisting of 8 residues and shows sequence conservation

28 LAURA D. CORDEN AND W.H. IRWIN MCLEAN

Figure 3.2 Transmission electron micrograph of human epidermis, showing keratin filament bundles (black arrows) in the cytoplasm of epidermal keratinocytes. Also seen are desmosomes (white arrow), transmembrane complexes which connect the keratin networks of neighbouring cells. Magnification 35,000× approx. figure kindly provided by Dr John A.McGrath, St John’s Institute of Dermatology, St Thomas’s Hospital, London.

(North et al., 1994). The linker regions are thought to impart flexibility on the rod domain (Steinert, 1993). The 2B domain contains a “stutter”, which is a highly conserved discontinuity in the regularity of heptad periodicity in the coiled-coil, of unknown function (North et al., 1994; Steinert and Parry, 1985). The a-helical conformation is slightly distorted in the region of the “stutter”, which could represent a point of weakness within the coiled-coil, enabling it to bend (North et al., 1994). Alternatively, this helix inversion could be necessary to allow filament packing. These hypotheses cannot be formally proven without X-ray crystallographic data. At the amino terminal end of the central rod domain is a region termed the helix initiation motif and at the carboxy terminal end of the rod, the helix termination motif (Steinert, 1993) (red areas, Figure 3.3). These regions show striking sequence conservation among and between intermediate filament types (Conway and Parry, 1988). Conservation of these sequences indicates that they must play an important role in filament assembly (Steinert et al., 1993b; Wilson et al., 1992) (Figure 3.3). This is supported by the identification of mutations in these regions which have dramatic effects on filament assembly and function, as discussed below. The central a-helical region is flanked by a variable domain V1 at the amino-terminus and V2 domain at the carboxy-terminus (Hanukoglu and Fuchs, 1982; Weber and Geisler, 1982) (Figure 3.3). These domains are highly variable both in length and


sequence, accounting for size differences specific for each keratin polypeptide. These domains usually contain tandem peptide repeats and are rich in serines and glycines (Korge et al., 1992; Steinert et al., 1985a). The type II keratins also possess short, complex sequences, H1 and H2 (homology) subdomains, which lie between the rod domain and the V1 and V2 domains, respectively (Steinert et al., 1985a). Mutations in the H1 domain of type II keratins have been shown to cause skin disease, as discussed below. keratins: Protein Domain Structure

Figure 3.3 Schematic diagram showing the protein domain organisation of the type I and type II keratins, a structure which is similar for all intermediate filament types. The a-helical coiled-coil rod domain, which is responsible for heteropolymerisation, consists of 4 subdomains, 1A, 1B, 2A and 2B, separated by non-helical linkers L1, L12 and L2. In helix 2B, is a stutter or discontinuity in the coiled-coil heptad repeats (S). The rod domain is flanked by globular V1 and V2 domains, which vary greatly in size and amino acid composition between keratins compared to the rod domains, which are well conserved in size and sequence. The most highly conserved sequences are the helix boundary motifs (shaded red), at the beginning and end of the rod domain and which are thought to be involved in molecular overlap interactions in filament assembly. Type II keratins have additional conserved features within their variable domains: the homology subdomains H1 and H2, which probably play a role in polymerisation; and the ISIS box, which is thought to be a site of interaction of keratins with other protein complexes.

In contrast to the rod domain, the head and tail regions show great variability not only between individual keratins, but also differ between the intermediate filament types (Geisler and Weber, 1982; Hanukoglu and Fuchs, 1983), reviewed in (Steinert and Parry, 1985; Steinert et al., 1985b). It has been proposed that these differences specify the function of the intermediate filament in which they are found (Steinert et al., 1985b). Experiments carried out to examine filament formation in vitro using the rod domain of desmin, have shown that this domain alone is incapable of forming lOnm filaments, indicating that the head and tail domains are required for polymerisation into mature filaments (Geisler et al., 1982). Studies involving expression of deleted keratin proteins, where either the head or tail domain had been deleted from one or both keratins, have revealed that one member of the heterotypic keratin pair requires an intact head and tail domain for normal filament assembly and elongation to occur (Lu and Lane, 1990). These studies showed that homotypic head/tail interactions of like polypeptides played an


important role in filament elongation. More recent investigations have pointed to a role for the type II head domain in elongation and lateral alignments/registration of neighbouring molecules, possibly mediated by HI, whereas the tail domains of type I and II keratins have been implicated in filament stabilization (Hatzfeld and Burba, 1994; Steinert and Parry, 1993; Wilson et al., 1992). An evolutionarily conserved nonapeptide motif within the head domain of vimentin (also found in desmin and peripherin) has been shown to be necessary in assembly of soluble tetramers into filaments (Herrmann et al., 1992). The amino acid sequence of the rod domain of an intermediate filament shows over most of its length, a heptad periodicity of hydrophobic residues at positions a and d of every seven (abcdefg). This periodicity is indicative of a protein that forms coiled-coil polymers (Crick, 1953); reviewed in (Cohen and Parry, 1986). A single polypeptide chain, which is predicted to assume a-helical structure is generally unstable in an aqueous environment due to the presence of hydrophobic or apolar residues within the molecule, as reviewed in (Cohen and Parry, 1986; Conway and Parry, 1991). Dimerisation of two a-helical molecules involves twisting of the two polypeptide chains together, placing the hydrophobic residues in a position protected from the aqueous environment, thus forming a stable coiled-coil molecule. Residues in positions e and g are charged and thought to be involved in interchain ionic interactions which stabilize the arrangement of two a-helices within the coiled-coil (Cohen and Parry, 1986). The polar hydrophilic residues b, c and f are located at the periphery of the coiled-coil, in contact with the aqueous environment. The importance of the stability of interations within the coiled-coil has been demonstrated in experiments where head and tail deleted keratin polypeptides were expressed within cultured fibroblasts. Interactions within the heterotypic coiled-coil protected the individual type I or type II keratin from proteolysis which occurs when a single type keratin is expressed alone. Keratins deleted in the head and/or tail regions were capable of resisting proteolysis in combination with a keratin of the opposite type (Lu and Lane, 1990). Keratins: the Type I and Type II Intermediate Filaments Studies on sheep wool proteins suggested that there were two groups of keratins (Parry et al., 1977). Fuchs and colleagues described the existence of two different classes of keratin genes (Fuchs et al., 1981), the type I and II intermediate filaments which constitute the largest fraction of the intermediate filament protein family, with >30 different human keratins described to date (Quinlan et al., 1994). Keratins contribute to the intermediate filament cytoskeleton within epithelial cells, forming a basket-like network around the nucleus in eukaryotic cells extending to the cell periphery, where they contact the plasma membrane through interaction with specific protein junctions, the hemidesmosomes (on the basal cell surface) and the desmosomes (at intercellular membrane sites). It is through interaction with the desmosomes that the intermediate filaments link the cells into a three dimensional structure, increasing mechanical integrity of individual cells and the tissue as a whole.


The desmosomal component desmoplakin has been shown to associate directly through its carboxy terminal tail with the amino terminal head domain of several type II epidermal keratins (Kouklis et al., 1994). This association was not found with the simple epithelial type II keratins, the type I keratins or vimentin (Kouklis et al., 1994). It is the amino terminus of desmoplakin that targets the protein to the desmosome and the carboxy terminus which attaches intermediate filaments to the desmosomal plaque (Stappenbeck et al., 1993; Stappenbeck et al., 1994). Phosphorylation of a serine residue within the carboxy terminus of desmoplakin has been found to be likely to negatively regulate its interaction with keratin intermediate filament networks (Stappenbeck et al., 1994). Keratins are obligate heteropolymers (Hatzfeld and Franke, 1985; Lee and Baden, 1976; Steinert et al., 1976) and the first step in keratin filament assembly has been shown to be the formation of a coiled-coil type I/II heterodimer (Figure 3.4) (Hatzfeld and Weber, 1990; Steinert, 1990). Thus the obligatory heteropolymer step in keratin intermediate filament assembly occurs at the dimer stage and not during tetramer formation (Hatzfeld and Weber, 1990). This heterotypic association appears to be mediated by the α-helical rod domains, as deletions of the head and tail domains do not prevent this association (Lu and Lane, 1990). Within the coiled-coil dimer, the two molecules are parallel and in axial register, as reviewed in (Cohen and Parry, 1990; Steinert, 1993). Chemical cross-linking studies using the rod domain of desmin, capable of homopolymerisation, indicated that intermediate filament structure is based on a tetramer, composed of a dimer of two double stranded coiled-coils (Geisler and Weber, 1982). The tetramer is considered the most stable intermediate filament oligomeric species in vitro (Quinlan et al., 1984). The precise manner in which tetramers associate to form mature 1Onm filaments is not known with certainty, however the free tetramer is composed of two coiledcoil dimers which are thought to exist in an anti-parallel and staggered arrangement (Geisler et al., 1985; Meng et al., 1996; Steinert, 1993; Steinert et al., 1993a; Steinert et al., 1994; Stewart et al., 1989) (see Figures 3.3 & 3.4). This arrangement within the filament raises the possibility of a large number of interactions between adjacent molecules (Figure 3.4). Elongation involves end to end interactions of additional tetramers, with an overlap between the first 10–11 residues of the 1A region of one dimer and the last 10–11 residues of the 2B region of the other (emphasising the importance of the helix initiation and termination motifs) (Steinert, 1993; Steinert et al., 1993a; Steinert et al., 1994) (Figure 3.4). Higher orders of filament assembly have not been conclusively deduced, although protofilaments (2–3nm) and protofibrils are thought to assemble by longitudinal extension of laterally associated tetramers and “half-filaments” by longitudinal extension of oligomers containing 12–20 laterally associated dimers, as reviewed by (Lane, 1993; Quinlan et al., 1994; Steinert, 1993). Accuracy of the type I/II keratin pairing is thought to be important in terms of threedimensional quality of the keratin filament network within the cell (Lu and Lane, 1990). Type I keratins are acidic (K9–K20) and their genes have been found to map to chromosome 17q (Milisavljevic et al., 1996; Rosenberg et al., 1988), except for K18 which maps to the type II (K1–K8) keratin gene cluster (Waseem et al., 1990; Yoon et al., 1994) on chromosome 12q (Rosenberg et al., 1991). The keratins are designated as type I


Figure 3.4 Potential domain alignments within keratin intermediate filaments, adapted from Steinert, 1993a; Steinert et al., 1993b; see also Meng et al., 1996. Cross-linking experiments (Steinert et al., 1993) have established four modes of alignment of two nearest neighbour molecules in the keratin intermediate filament. (A12) the two molecules are aligned antiparallel and in almost axial register. (A11) the two molecules are arranged anti-parallel to each other and staggered, bringing 1B sub-domains into approximate alignment. (A22) the two molecules are again anti-parallel and staggered to bring the 2B sub-domains into close alignment. Alignments A11 and A22 reveal the fourth mode (ACN), here two similarly directed molecules overlap so that the first 10 amino acid residues of 1A overlap with the last 10 amino acid residues of sub-domain 2B on the other molecule (revealing the importance of these conserved regions). Intermediate filament protein domain interactions (vimentin) were studied using the two-hybrid system (Meng et al., 1996). These studies indicated that the 1B sub-domains interact strongly with one another and that the 2B domains also interact strongly with each other. The A11 and A22 modes of alignment were considered to reveal true domain interactions, whereas the A12 and ACN modes may describe adjacent molecules within the filament, but not interacting molecules (as no interaction was found between different constructs containing the 1A or 2B domains).

or II according to their apparent molecular weight and isoelectric point in 2D gel electrophoresis (Moll et al., 1982). The differences in size between the type I and II keratins result from differing lengths of the amino and carboxy terminal regions of the proteins, rather than from significant insertions or deletions within the central rod domain (Hanukoglu and Fuchs, 1983). Keratins are generally expressed in specific pairs depending upon cell type, developmental stage, growth environment and differentiation or disease state of the cell (Lazarides, 1980; Sun et al., 1983; Sun et al., 1985). The tissue-specific expression


Table 3.2 Cytokeratin expression patterns

*indicates that mutations in this keratin have been found to cause human disease.

patterns of human keratins are shown in Table 3.2. For example, keratins K5 and K14 are expressed in the basal cells of the epidermis (Moll et al., 1982; Purkis et al., 1990), although K15 may also be expressed to a lesser extent in these cells (Moll et al., 1982). As cells leave this layer and undergo differentiation, expression of K5 and K14 is downregulated, with increased expression of the keratin pair K1 and K10. In conjunction with K1/K10 expression, K2e (the epidermal form of K2) is expressed in the more superficial suprabasal cells (Collin et al., 1992). The amount of K1 and K10 within the cell increases as it becomes more suprabasal (Coulombe, 1993). In addition to the keratins discussed above, other keratins are also expressed in the epidermis, with the particular keratin expressed being dependent upon tissue location. K9 is a major keratin expressed suprabasally within palmar and plantar skin (Langbein et al., 1993). Keratins K6 and K16 are also found in sole and palm skin and are expressed in regions of the skin which are actively proliferating, for example during wound healing or in psoriasis (Weiss et al., 1984). The anterior corneal epithelium, a non-cornified stratified squamous epithelium, expresses K3 and K12 predominantly, with low levels of K5 and K14 expression within the basal cells (Sun et al., 1984). The non-stratified oesophageal mucosa specifically expresses K4 and K13 (Moll et al., 1982). The expression of two major epidermal keratins in human skin is shown in Figure 3.5.


MEDICAL ASPECTS OF KERATIN INTERMEDIATE FILAMENTS Mutations in Keratin Genes Cause a Wide Range of Human Diseases Since 1991, mutations have been found in 15 different epithelial keratin genes and two hair keratin genes causing a wide range of human diseases affecting the epidermis and various other epithelia. In each case the phenotype observed has been dependent upon the expression pattern of the defective keratin. All these disorders display keratinocyte fragility, demonstrating that a major function of keratin filaments is to enable epithelial cells to resist physical damage.

Figure 3.5 Indirect immunoperoxidase staining of human epidermis using antibodies against (a) keratin 1 (LH1) and (b) keratin 14 (RCK107). K14 expression is confined to the basal cell compartment (b), whereas K1 expression is found in all other (suprabasal) cell layers of the epidermis. The precise reasons for these striking comparmentalised keratin expression patterns in differentiated epithelia still remain a mystery but are presumed to indicate tissue-specific and/or differentiationspecific functions of the keratin cytoskeleton. Photographs courtesy of Declan P.Lunny, CRC Cell Structure Research Group, Department of Anatomy and Physiology, University of Dundee.


Epidermolysis Bullosa Simplex Epidermolysis bullosa (EB) is a clinically and genetically heterogeneous group of inherited disorders characterised by skin blistering. There are three main forms of EB: junctional, dystrophic and simplex which were originally classified according to the level within the skin where the blister occurs (Fine et al., 1991). These can now be better classified according to which of the 10 known EB genes are involved, as reviewed by (Uitto et al., 1997). In EB simplex (EBS), the split is intraepidermal and cytolysis occurs within the basal keratinocyte, usually midway between the nucleus and the hemidesmosomes. Mutations in the K5 and K14 genes have been shown to underlie this form of EB. The identification of these mutations confirmed that the primary function of keratin filaments is structural, since these mutations lead to fragility of the tissue in which the mutant keratin is expressed.

Figure 3.6 Clinical appearance of the phenotypes arising from mutations in three major epidermal keratins, (a) The Dowling-Meara form of epidermolysis bullosa simplex (EBS-DM)—widespread epidermal blistering caused by mutations in or near the helix boundary motifs of basal cell keratins K5 or K14. (b) The thickened, hyperkeratotic skin of a patient with bullous congenital ichthyosiform erythroderma (BCIE), which can be caused by mutations in K1 or K10, the keratins expressed in all suprabasal layers of the epidermis. (c) Ichthyosis bullosa of Siemens (IBS), a mild epidermolytic ichthyosis caused by mutations in K2e, a keratin expressed only in high suprabasal layers of the epidermis. Thanks to Dr Alan D.Irvine, Department of Dermatology, Royal Victoria Hospital, Belfast; Dr Marion White, Department of Dermatology, Aberdeen Royal Infirmary, Aberdeen; and Prof Irene M.Leigh, Academic Department of Dermatology, The Royal London School of Medicine and Dentistry, London, for providing these figures.

There are three main forms of EBS (Fine et al., 1991) which generally exhibit autosomal dominant inheritance. The Dowling-Meara form (EBS-DM) is considered to be the most severe, with blisters clustered at any body site as a result of mild physical trauma (Fine et al., 1991). The clinical appearance of EBS-DM is shown in Figure 3.6. A widespread distribution but less severe blistering is associated with the Köbner form of EBS (EBS-K). The mildest variant, Weber-Cockayne EBS (EBS-WC), causes blistering localized to the hands and feet, the major trauma sites of the epidermis (Fine et al., 1991). Electron


Figure 3.7 Transmission electron micrograph of epidermis from a patient with bullous congenital ichthyosiform erythroderma (BCIE), the phenotype due to K1 or K10 mutations shown in Fig. 3.6b. The basal cells at the bottom of the figure, in contact with the basement membrane (arrowheads), do not express K1 or K10 and therefore have normal appearing keratin filaments (compare Fig. 3.2). In contrast, all the upper suprabasal cells can be seen to contain dense filament aggregates due to the mutant keratin expression (arrows) and consequently are undergoing cytolysis. The scale bar represents 5µm. Thanks to Prof Robin A.J.Eady and Dr Akemi IshidaYamamoto, St John’s Institute of Dermatology, St Thomas’s Hospital, London for providing this illustration.

microscopy of biopsy material from EBS-DM patients revealed keratin filament collapse within basal keratinocytes, leading to the formation of electron-dense aggregates within the cytoplasm. Immuno-electron microscopy revealed that these aggregates contained K5 and K14 (Ishida-Yamamoto et al., 1991), suggesting these keratins as candidate genes for EBS. Expression of mutant keratins in cultured cells had been shown to produce similar keratin aggregates (Albers and Fuchs, 1987; Albers and Fuchs, 1989). Filament aggregation can also be seen by EM in many other keratin disorders. Typical tonofilament aggregates are shown in Figure 3.7, in the skin of a patient carrying a K1 mutation. Expression of a truncated K14 gene in the skin of transgenic mice where more than 30% of the rod domain and all of the V2 domain had been replaced by a lacZ fusion protein, led to a phenotype resembling EBS (Vassar et al., 1991). Like EBS-DM, electron


microscopy showed the presence of electron dense aggregates within basal keratinocyte cytoplasm (Vassar et al., 1991). Mice in which only 50 amino acids had been removed from the carboxy terminus of the protein displayed no skin blistering or basal cell cytolysis, although keratin aggregates were seen in some basal cells. This showed that phenotypic severity can depend on the particular mutation involved (Vassar et al., 1991). Further transgenic experiments revealed that different K14 mutations could generate phenotypes of mild and severe blistering in mice (Coulombe et al., 1991b). Linkage analysis provided further support to K5 and K14 as candidate genes for EBS. Genetic linkage mapped the EBS-K gene to the type I keratin gene cluster on chromosome 17q and later, the causative mutation in this family (L384P) was discovered within the 2B segment of the K14 rod domain (Bonifas et al., 1991a; Bonifas et al., 1991b). Mutations encoding different amino acid substitutions within the same codon in K14 (R125H/ R125C) were also identified in EBS-DM cases (Coulombe et al., 1991a). These mutations involved the 125th amino acid, located within the highly conserved helix initiation motif of the K14 polypeptide (Coulombe et al., 1991a). Arginine 125 is the 10th codon within the 1A domain and contains a CpG sequence which is conserved in most type I keratins (McLean and Lane, 1995). Many mutations have now been found to affect analogous residues in different keratins, showing this to be a mutation hot spot. The first mutation in K5 was revealed in a large family with EBS-DM. This mutation was found within the helix termination motif of the K5 helix 2B domain (Lane et al., 1992). Mutations in the K5 and K14 genes associated with EBS-WC soon followed (Chan et al., 1993; Humphries et al., 1993; Rugg et al., 1993). Mutations in the milder forms of EBS have been found to occur outside the highly conserved helix boundary motifs and filaments examined by electron microscopy appear normal (McLean and Lane, 1995). To date, there have been numerous reports of mutations in the K5 and K14 genes in dominant EBS families, reviewed (Corden and McLean, 1996). Pre-natal diagnosis has now been carried out for the Dowling-Meara form of EBS, using PCR amplification and sequencing of genomic DNA (Rugg et al., 1997). More recently a heterozygous point mutation, P25L, was found in the non-helical V1 domain of K5 in individuals from a number of unrelated families with EBS with mottled pigmentation (EBS-MP) (Irvine et al., 1997b; Uttam et al., 1996). Although keratin aggregates are not characteristic of this disorder, organelle distribution was reportedly aberrant in the basal keratinocytes from affected individuals (Uttam et al., 1996). Filament assembly studies carried out in vitro with the mutant K5 revealed subtle effects on the length of the 10nm keratin filaments. Expression of the mutant K5 within transfected PtK2 cells which express K8 and K18, produced keratin filament networks indistinguishable from wild-type (Uttam et al., 1996). How a mutation within this domain acts to weaken the keratin filaments is unknown but it indicates a subtle, previously undetected role for the V1 domain in filament dynamics. It is also unknown how this mutation leads to pigmentary changes, however the slight change in the keratin network within keratinocytes may influence longevity of the melanin granules, or possibly efficiency of melanin transfer from the melanocytes (Uttam et al., 1996).


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


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


K14 network is a pre-requisite for the formation of a normal K1/K10 network, which might explain how mutations in K5/K14 could have a detrimental effect in suprabasal cells (Kartasova et al., 1993). Alternate Phenotypes Caused by K1 Mutation One family with diffuse non-epidermolytic palmoplantar keratoderma (NEPPK) has been reported with a missense mutation outside the K1 rod domain (Kimonis et al., 1994). This mutation occurs within the ISIS box, a 22 amino acid motif conserved within the V1 domain of several type II keratins (Kimonis et al., 1994). The lysine to isoleucine substitution generated by this mutation, occurs in an invariant lysine residue within the ISIS box consensus sequence. This lysine residue in the type II keratins has been found to be frequently involved in the formation of intermolecular cross-links during cornified cell envelope formation (Marekov and Steinert, 1995). The ISIS box sequence within the amino terminal head of type II keratins is thought to be involved in binding of the desmoplakin tail (Kouklis et al., 1994), and so the K73I mutation may act by interfering with the interaction of K1 with other molecules. Ichthyosis Bullosa of Siemens and K2e Mutations Ichthyosis bullosa of Siemens (IBS) is a type of mild epidermolytic ichthyosis localised mainly on the flexures (Siemens, 1937). The outer layers of the epidermis undergo a characteristic moulting/shedding, not seen in patients with EH/BCIE (Siemens, 1937). In a number of kindreds, IBS was found to be linked to the type II keratin gene cluster on chromosome 12q (Steijlen et al., 1994). Tonofilament aggregation and cytolysis are both characteristic of this disorder, but these phenomena are restricted to the upper spinous and granular layers of the epidermis, unlike BCIE (McLean et al., 1994). K2e expression was found to closely mirror the distribution of affected cells in IBS (Collin et al., 1992), which suggested that this was a candidate gene for this disease. Mutations were initially found in the K2e gene in six families affected with IBS, although two of the families were mis-diagnosed as having EH (Rothnagel et al., 1994b). These mutations affected codon 493 within the helix termination motif of K2e (Rothnagel et al., 1994b). Five of the mutations were identical (E493K), and are likely to represent a hot spot for mutations within this gene, involving a CpG dinucleotide. Further cases of this E493K mutation have been found, as well as other mutations in both helix boundary motifs, reviewed by (Corden and McLean, 1996). K9 Mutations Cause EPPK—Fragility of Palm and Sole Skin Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant skin disorder characterised by diffuse thickening of the epidermis on the entire palmar and plantar surfaces, bounded by erythematous margins (Vörner, 1901). The expression of K9 specifically within the suprabasal keratinocytes of the palm and sole led to this gene becoming the candidate for EPPK (Langbein et al., 1993). The gene for EPPK was initially mapped


to the type I keratin gene cluster on 17q12–21 in a large German kindred (Reis et al., 1992). Reis and colleagues then went on to discover three different mutations within the K9 gene, all in the helix initiation motif (Reis et al., 1994). One mutation was found in five unrelated kindreds (R162W), the type I keratin CpG hotspot mutation in the helix initiation motif (McLean and Lane, 1995). Further mutations have been found within the K9 gene, all involving the 1A rod domain, as reviewed (Corden and McLean, 1996). Pachyonychia—Thick Nails due to Mutations in K6a, K6b, K16 & K17 Pachyonychia congenita (PC) is a group of autosomal dominant ectodermal dysplasias characterised by hypertrophie nail dystrophy with various constellations of other ectodermal abnormalitites. There are two main sub-types of PC (McKusick, 1998). The Jadassohn-Lewandowsky variant or type 1 (PC-1) involves pachyonychia in conjunction with severe non-epidermolytic palmoplantar hyperkeratosis, hyperhidrosis, infrequent blistering, oral leucokeratosis and follicular keratoses (Jadassohn and Lewandowsky, 1906). The Jackson-Lawler form or PC-2, has minor oral involvement but displays multiple pilosebaceous cysts (Jackson & Lawler, 1951). This form of the disease may also be associated with pili torti (twisted hairs), natal teeth (some teeth prematurely erupted at birth) and other ectodermal abnormalities (McLean et al., 1995). The fingernails of a patient with PC-1, carrying a mutation in K6a, are shown in Figure 3.8. Genetic linkage to the type I keratin gene cluster was obtained in a large Scottish kindred with PC-2 (Munro et al., 1994). The mutation in this family was found to be a heterozygous missense mutation N92D in the K17 gene (McLean et al., 1995). A missense mutation L130P was simultaneously reported in the K16 gene, in an individual with sporadic PC-1 (McLean et al., 1995). Both of these mutations occur within the highly conserved helix initiation motif of the specific type I keratin. Further K17 mutations have been reported in PC-2 kindreds and also in families diagnosed with steatocystoma multiplex (Covello et al., 1998; Smith et al., 1997). Kl6 mutations have been found in families suffering from focal non-epidermolytic palmoplantar keratoderma (FNEPPK) and extremely subtle nail changes were found in the affected individuals (Shamsher et al., 1995). Mutations were subsequently reported in PC-1 cases in K6a, the type II keratin partner of K16 (Bowden et al., 1995; Smith et al., 1998b). One surprising result has emerged from the study of PC-2. K17 was not known to be specifically expressed in conjunction with any other keratin and was thought to polymerise with K5 in basal cells and K6 or another type II protein, when expressed suprabasally (Lane, 1993). Thus, there was no type II keratin in which mutations could be predicted to produce a PC-2 phenotype and many PC-2 families were shown to have K17 defects, further implying genetic homogeneity. However, a PC-2 family was recently identified where no K17 mutation could be found, but genetic linkage was obtained to markers in the type II keratin cluster and subsequently, a mutation was identified in K6b (Smith et al., 1998a). The PC-2 phenotype in the K6b family was very similar to individuals carrying K17 mutations. K17 and K6b were shown to be co-expressed in many epidermal appendages, confirming that these proteins are expression partners in general,


although they differed in a few places (Smith et al., 1998a). Thus, the study of this keratin disorder revealed the previously undiscovered role of K6b in epithelial biology. There are at least six highly homologous copies of the K6 gene (K6a–f), at least four of which are expressed (Takahashi et al., 1995). To date, mutations are known in only two of these genes, K6a and K6b. It is not yet known what phenotypes might arise from mutations within the remaining expressed K6 isogenes. K4 and K13 Mutations Cause Fragility of Mucosal Tissues White sponge nevus of Cannon (WSN) is an autosomal dominant disorder which affects non-cornifying stratified squamous epithelia (McKusick, 1998). White “spongy” plaques are found in the mouth, as seen in Figure 3.8, and to a lesser extent in the oesophagus and anogenital mucosa (Jorgenson and Levin, 1981). As K4 and K13 are specifically expressed within these tissues, they became the candidate genes for this disorder (Moll et al., 1982). Two Scottish families were found to carry a mutation in K4 (Rugg et al., 1995) and the complementary mutation in K13 was published simultaneously (Richard et al., 1995). Mutations in these two genes were the first found to affect a tissue other than the epidermis and its appendages. K18 Mutation May Cause Liver Disease Predisposition Expression of K18 carrying a missense mutation in the 1A domain was found to produce filament aggregation (Ku et al., 1995). Transgenic mice expressing mutant K18 developed chronic hepatitis and hepatocyte fragility (Ku et al., 1995). These experiments indicated that liver defects within humans might also be caused by mutations in either of the simple epithelial keratins, K8 or K18. One heterozygous K18 mutation has been published, H127L, which affects the last amino acid of the L1 domain and is believed to predispose towards or be responsible for the development of cryptogenic cirrhosis within the patient (Ku et al., 1997). Mutations in this domain, which is less well conserved at the sequence level and whose function is currently unclear, have not been reported in any other keratin disorder. However, in vitro assembly studies with this K18 mutant showed defective filaments by electron microscopy, giving some indication that this mutation may be pathogenic (Ku et al., 1997). Monilethrix—Beaded Hair Due to Mutations in Hair Keratins Monilethrix is an autosomal dominant hair disorder showing variable phenotype (Winter et al., 1997a; Winter et al., 1997b). Hairs from affected individuals have a characteristic beaded appearance and so trichocyte hair keratins were good candidate genes. The first monilethrix mutations were found in the type II hair cortex keratin, hHb6. Again, these mutations involved amino acid substitution in the helix termination motif (Winter et al., 1997b). A mutation in another type II hair keratin, hHbl, also in the helix termination


Figure 3.8 Clinical appearance of three diverse disorders of differentiation-specific keratins, (a) The hypertrophie nail dystrophy which is a major characteristic of pachyonychia congenita type PC-1 and PC-2, seen here in a PC-1 patient with a K6a mutation. Mutations in four keratins, K6a, K6b, K16 and K17 produce variants of this disease, (b) The buccal hyperkeratosis seen in white sponge nevus syndrome (WSN), due to mutations in mucosal keratins K4 or K13. (c) Retroillumination slit lamp photo-graph of microcysts within the corneal epithelium (arrows) in a patient with Meesmann’s corneal dystrophy (MCD), caused by mutations in keratins K3 or K12. Thanks to Dr Kevin McKenna, Department of Dermatology, Craigavon Area Hospital, Northern Ireland; Dr Colin Munro, Department of Dermatology, Southern General Hospital, Glasgow and Drs Ole and Beate Swensson, Departments of Dermatology and Ophthalmology, Christian-AlbrechtsUniversität, Kiel, Germany for kindly providing these illustrations.


motif, has been described in a family with monilethrix (Winter et al., 1997a), as well as further mutations in hHb6 (Korge et al., 1998). Focusing on K3 and K12 Mutations in Meesmann’s Corneal Dystrophy The anterior epithelium of the cornea is the outermost protective layer of the eye and consists of about four layers of stratified, non-cornified keratinocytes which predominantly express cornea-specific keratins K3 and K12 (Schermer et al., 1986). Given that mutations in epidermal keratins produce skin fragility disorders, it was logical to think that mutations in K3 or K12 would produce a corneal fragility disease. At the end of 1996, our group considered the corneal dystrophies, of which there are many types (Grayson, 1983), as candidates for K3 and K12 mutations. However, Meesmann’s corneal dystrophy (MCD), a clinically mild disorder characterised by fragility of the anterior corneal epithelium (Meesmann and Wilke, 1939), seemed to be the best candidate for a variety of reasons. Firstly, it is autosomal dominant, like most of the keratin diseases. Secondly, MCD affects only the anterior corneal epithelium, the single tissue where K3 and K12 are specifically expressed. Thirdly, there had been a number of ultrastructural reports of intracellular aggregates of unknown composition in MCD corneocytes (Tremblay and Dube, 1982). Although there had been no suggestion in the literature that these were keratin aggregates, this was nevertheless a valuable clue. The clinical appearance of an MCD cornea, myriads of microcysts seen by slit-lamp examination, is shown in Figure 3.8. Analysis of two Northern Irish families and a large pedigree representing the descendants of Meesmann’s original German family gave positive genetic linkage to the keratin loci on chromosomes 12 and 17 (Irvine et al., 1997a). Mutations were found in the helix boundary motifs of both K3 and K12 in these initial families (Irvine et al., 1997a). Further K12 mutations have also been reported in Japanese MCD kindreds (Nishida et al., 1997). Overall, these results revealed the role of K3 and K12 in maintaining the structural integrity of corneal keratinocytes as well as the underlying molecular pathology in MCD. THE FUTURE OF KERATIN GENETICS RESEARCH More Keratins, More Associated Molecules, More Diseases Since the discovery of the first human keratin mutations at the end of 1991, a large part of the mutation-based research has been focused on the identification of further keratin diseases and in determining the complete range of mutations which are pathogenic in keratins. There are two main outcomes from this research. Firstly, there are now 17 keratins known in human disease and this list will probably continue to grow until there is a genetic disease association for all keratins. Secondly, certain molecular features of keratins have come to light as being structurally important due to the emergence of mutation clusters in these regions, as shown in Figure 3.9. This has been particularly


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

Prevention and Therapy of Keratin Diseases Although the study of keratin diseases has been valuable in understanding the basic biology of these protein systems, this work has also enhanced patient care for these disorders through the application of molecular diagnostic techniques. Some keratin diseases, such as MCD or WSN are so mild in their effects that demand for prenatal testing is low. In other disorders with more devastating phenotypes, such as EBS-DM and BCIE, affected families do request prenatal testing. In these cases, knowledge of the mutation in a particular family allows prenatal diagnosis by chorionic villus biopsy to be done at a very early stage of pregnancy. These preventative measures for severe keratin disorders are of no use in prevention of the sporadic mutations which account for many cases of keratin disease and so there will always be a need for improved treatment. Gene therapy is made doubly difficult in the case of keratins since most mutations are dominant-acting, so that gene

Figure 3.9 Clustring of dominant-negative mutations in all keratin diseases within the type I and II protain domain structures. actual numbers of mutations per domain are shown, as of mid-1998. Red bar indicate mutations affecting the helix boundery motif sequences, which can be seen to be mutation hotspots. Mutations outside these highly conserved regions have been shown to result in milder desease phenotypes.

Keratin Dominant-Negative Mutation Clusters



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4. DESMOSOMES DAVID R.GARROD, MARTYN A.J.CHIDGEY, ALISON J.NORTH, SARAH K.RUNSWICK AND CHRIS TSELEPIS

INTRODUCTION Desmosomes are punctate, adhesive intercellular junctions that bind cells together and provide membrane anchoring points for the intermediate filament cytoskeleton. They are circular membrane domains of up to 0.5 µm in diameter. The intercellular material or desmoglea occupies a region of about 30 nm wide between the apposed plasma membranes. It has a highly organised ultrastructure consisting of an electron-dense mid line from which cross-bridges extend to the plasma membranes. Close to the cytoplasmic faces of the plasma membranes are dense outer plaques. These are approximately 20 nm in thickness and are the most consistent and easily-recognisable feature of these junctions. At about 50 nm from the plasma membranes are less dense inner plaques that appear to associate with the intermediate filaments. Thus the total thickness of a desmosome from one inner plaque to the other is approximately 130 nm. Desmosomes may be thought of as the “scaffold couplings” that link the intermediate filament cytoskeleton throughout a tissue. Widely distributed, desmosomes are especially abundant in stratified epithelia where strong intercellular adhesion is required to resist external friction. They are present in almost all epithelia and also in cardiac muscle, the arachnoid and pia of the meninges, and the follicular dendritic cells of the lymphoid system. Desmosomes are macromolecular complexes consisting of at least six interacting proteins (Figure 4.1, Table 4.1). Their adhesion molecules are the desmocollins (Dscs) and desmogleins (Dsgs), members of the cadherin family of calcium-dependent adhesion molecules. These transmembrane proteins have their extracellular domains in the desmoglea and their cytoplasmic domains in the outer dense plaque. The outer plaque contains two proteins that are members of the armadillo family named after the Drosophila segment polarity signalling gene. These are plakoglobin (Pg), also known as γ catenin and present in other intercellular junctions, and plakophilin (Pp). The outer and inner plaques are bridged by the plakin family member desmoplakin (Dp), which mediates interaction between the adhesive domain and the intermediate filaments (reviewed by Schwartz et al., 1990; Buxton and Magee, 1992; Legan et al., 1992; Garrod, 1993; Garrod et al., 1996; Green and Jones, 1996; Chidgey, 1997; Garrod et al., 1998; Burdett, 1998).

D.R.GARROD ET AL. 57

Figure 4.1 Cartoon showing major components of desmosomes. ML=mid-line, PM=plasma membranes OP=outer plaque, IP=inner plaque, IF=intermediate filaments. The numbers at the bottom of the picture indicate distances in nanometres from the plasma membranes of the various plaque components as determined by quantitative measurements from immunogold labelling with domain-specific antibodies. Dsc=desmocollin, a=“a” form, b=“b” form; Dsg=desmoglein; DP=desmoplakin, PG=plakoglobin; PP=plakophilin. N=N-terminus; C=C-terminus. Only one half of the desmosome is shown: it is symmetrical about ML. The intercellular space is not drawn to scale as it would be almost 50% wider if shown at the same magnification as the plaque region.

The most recent additions to the tally of desmosomal components are the two plakin family members, envoplakin and periplakin (Ruhrberg et al., 1996; . Ruhrberg et al., 1997; Ruhrberg and Watt, 1997), which although not exclusively epidermal, appear to be of considerable importance in cornified envelope formation. DESMOSOMAL GLYCOPROTEINS Desmosomal Glycoproteins and Cell Adhesion Desmosomes have long been regarded as points of strong adhesion between cells. Apart from their very “solid” appearance by electron microscopy, this view has been based on their particular abundance in stratified epithelia, such as epidermis, which are subject to substantial frictional forces, and to the loss of adhesion (acantholysis) that occurs in the group of autoimmune epidermal blistering diseases known as pemphigus, where loss of adhesion is caused by antibodies against desmosomal glycoproteins (Stanley, 1993; Stanley and Kárpáti, 1994). Recent experimental demonstrations of the importance of desmosomal adhesion in the epidermis and the heart have been achieved, respectively, by dominant negative and null mutations of the pemphigus vulgaris antigen Dsg3 (Allen et

58 D.R.GARROD ET AL.

al., 1996; Koch et al., 1997) and plakoglobin (Ruiz et al., 1996; Bierkamp et al., 1996). Furthermore, the first human desmosomal mutation, effectively a Pp1 knockout, gives rise to loss of adhesion within the epidermis (McGrath et al., 1997). It has always been assumed that desmosomal adhesion is mediated by the major desmosomal glycoproteins, Dsc and Dsg. Two early pieces of evidence supporting this view were, firstly, that the glycoproteins were greatly enriched in isolates of desmosomal cores (Gorbsky and Steinberg, 1981) and, secondly, that inhibition of desmosome assembly could be obtained in MDBK cells using high concentrations of Fab’ fragments of anti-Dsc antibodies (Cowin et al., 1984). The adhesive roles of these glycoproteins seemed even more likely when it was shown that both were members of the cadherin family of calcium-dependent adhesion molecules (Holton et al., 1990; Koch et al., 1990). Despite all these indications, it has proved extremely difficult to provide experimental proof of the adhesive roles of Dsc and Dsg. Classical cadherins generally participate in homophilic, calcium-dependent cell-cell adhesion (Nose et al., 1990; Shapiro et al., 1996; Nagar et al., 1996). Such homophilic adhesion by Dsc or Dsg is, however, negligible (Amagai et al., 1994a; Chidgey et al., 1996; Kowalcyzk et al., 1996). Recent work by three groups has now provided definitive evidence for mediation of cell-cell adhesion by desmosomal glycoproteins and some indication of how it may operate (Chitaev and Troyanovsky, 1997; Marcozzi et al., 1998; Tselepis et al., 1998). The human fibrosarcoma cell line HT1080 adheres by means of adherens junctions mediated by N-cadherin, but also synthesises Dsg2 (Sacco et al., 1995). Chitaev and Troyanovsky (1997) utilized transfection of these cells with bovine Dscl and human Pg to study desmosomal adhesion. Marcozzi et al., (1998) and Tselepis et al., (1998) used multiple transfection of the non-adhesive L929 mouse fibroblast line to generate adhesion. These studies suggest a number of novel conclusions about desmosomal adhesion, as follows:(i) Both Dsc and Dsg are required for intercellular adhesion. Previous work had shown that expression of single desmosomal glycoproteins in L929 cells did not generate adhesion (Amagai et al., 1994b; Chidgey et al., 1996; Kowalcyzk et al., 1996). Both Marcozzi et al., (1998) and Tselepis et al., (1998) were able to generate extensive aggregation of these cells by simultaneous expression of Dsc, Dsg and Pg. These results suggest that both Dsc and Dsg are required in order to generate adhesion. (ii) Dsc and Dsg can interact heterophilically. Chitaev and Troyanovsky (1997) elegantly demonstrated heterophilic interaction between Dsc and Dsg. They transfected one population of Dsg-expressing HT1080 cells with Dsc which was truncated cytoplasmically to remove the Pg binding domain. Another population of the same cells was transfected with myc-tagged Pg. The two cell populations were co-cultured, then solubilized and immimoprecipitated with anti-Dsc

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

Table 4.1 The Principal Components of Desmosomes



antibody. It was found that myc-Pg co-precipitated with Dsc, a result explicable only in terms of interaction between Dsc and Dsg on adjacent cells. By contrast, Marcozzi et al. (1998) were unable to precipitate Dsc-Dsg complexes, and Tselepis et al. (1998) found no convincing evidence for adhesive interaction between L cells expressing the different individual glycoproteins. (iii) Cytoplasmic interaction with Pg is required for adhesion Marcozzi et al. (1998) demonstrated that adhesion between L929 cells expressing both desmosomal glycoproteins took place when the cells were further transfected with Pg. Tselepis et al. (1998) demonstrated co-localization of Dsc, Dsg and Pg at the cell surface in transfected L929 cells, but detected no evidence of junction assembly by electron microscopy. (iv) Different glycoprotein isoforms and desmosomal glycoproteins from different species interact Dsc and Dsg each occur as three different isoforms which are the products of different genes. These isoforms are called Dsc1, 2 and 3, and Dsgl, 2 and 3. Dsc2 and Dsg2 are ubiquitous and occur in all desmosome-bearing tissues, but Dscl and 3 and Dsgl and 3 occur principally in stratified epithelia (Legan et al., 1994; Nuber et al., 1995; Schäfer et al., 1994). Marcozzi et al. (1998) used the human Dsgl and Dsc2 isoforms in their transfection experiments showing that these can participate in mutual interaction. Chitaev and Troyanovsky (1997) demonstrated interaction between human Dsg2 and bovine Dscl indicating that not only different isoforms but also proteins from different species can interact. The latter result is consistent with previous studies showing desmosome formation between cells of a variety of different species and phyla including anuran amphibians, birds, and mammals (Overton, 1977; Mattey and Garrod, 1985). North et al. (1996) showed that where different desmocollin isoforms are expressed in the same cells in epidermis, they occur in the same individual desmosomes. It is likely that desmoglein isoforms are similarly distributed (Shimizu et al. 1995). In view of the above experiments, it is probable that different glycoprotein isoforms interact in vivo. (v) Cell adhesion recognition (CAR) sites function in desmosomal glycoprotein interaction E- and N-cadherin possess tripeptide sequences (the CAR sites) in their N-termi nal regions that have been shown to be involved in adhesive binding (Blaschuk et al., 1990). In these molecules the tripeptide sequence is -histidine-alanine-valine- (HAV). The desmosomal cadherins have similarly located, related sequences, which have previously been referred to as putative CAR sites. In bovine Dscl and Dsgl these sites are YAT and


RAL, respectively. Tselepis et al. (1998) showed that 10mer peptides centred around these putative CAR sites blocked desmosomal adhesion. The peptides were effective at concentration of 0.5 mM. Furthermore, the Dsc peptide and the Dsg peptide were equally effective in inhibiting adhesion, and either peptide alone was as effective as the two together. This demonstrates that the CAR sites are functionally important in adhesion mediated by desmosomal glycoproteins and supports the involvement of heterophilic DscDsg interaction. (vi) The “b” form of Dsc is not essential for adhesion All Dscs occur as two alternatively spliced variants with different sized cytoplasmic domains, that of the “a” form being longer than the “b”. The “a” and “b” forms occur with approximately equal stoichiometry in all desmosome-containing cells and tissue so far examined. Troyanovsky et al. (1993) provided evidence that the cytoplasmic domain of the “a” form contains the signals necessary for plaque assembly. Smith and Fuchs (1998) showed that the three major plaque constituents Pg, Pp and Dp can all interact with “a” form cytoplasmic domain. To obtain adhesion of L cells, Tselepis et al., (1998) expressed both the “a” and “b” forms of bovine Dscl together with Pg and Dsg1. However, Marcozzi et al. (1998) were able to obtain apparently similar adhesion using only the “a” form of Dscs. It therefore appears that the “b” form is not essential for development of adhesion. However, the “b” form is present in all desmosome-containing cells and therefore may have an essential function, possibly in regulating the binding affinity and organisation of desmosomes. (vii) Adhesion between desmosomal glycoproteins is at least partially calcium dependent Both Chitaev and Troyanovsky (1997) and Tselepis et al. (1998) showed that the adhesion generated in their systems was partially abolished by removal of calcium, so that some residual adhesion occurred even in the absence of calcium. Although desmosomal assembly is calcium dependent (Hennings and Holbrook 1983, Watt et al., 1984; Mattey and Garrod, 1986a), desmosomes can also exist in the calcium independent state (Mattey and Garrod, 1986b). It is thus not entirely surprising to find that interaction between desmosomal glycoproteins was not completely abolished by calcium removal. (viii) Clustering of Dsc and Dsg is dependent on the extracellular domains Utilizing Dsc mutants where either the cytoplasmic domain or the N-terminal region of the extracellular domain were deleted, Chitaev and Troyanovsky (1997) showed that mutual clustering of Dsc and Dsg was dependent on the extracellular domain. Thus


clustering took place after deletion of the entire cytoplasmic domain but was abolished by deletion of the N-terminal region of the extracellular domain. (ix) Classical cadherin-mediated adhesion is not required to initiate adhesive interactions of desmosomal glycoproteins The HT1080 cells used by Chitaev and Troyanovsky (1997) form adherens junctions via Ncadherin. Their results are consistent with previous studies suggesting that cellular interaction via cadherin is essential to general desmosomal adhesion and that there is cross talk between adherens junctions and desmosomes (Wheelock and Jensen, 1992; Amagai et al., 1995a; Lewis et al., 1997). It was possible therefore that adhesive interaction generated by desmosomal glycoproteins in L929 cells was dependent on up-regulation of a cadherin (Tselepis et al., 1998). Two lines of evidence suggest that this is not the case. Firstly, adhesion was blocked by desmosomal glycoprotein CAR pep tides, but not by corresponding conventional cadherin peptides. Furthermore, no up-regulation of expression of cadherin or β-catenin could be detected by immunoblotting in transfected L929 cells exhibiting desmosomal adhesion. Thus, adhesion between desmosomal glycoproteins does not require prior interaction by conventional cadherins or adherens junctions. The results of these studies represent a substantial advance in our understanding of the mechanism of desmosomal adhesion. However, many issues remain to be resolved. Like conventional cadherins, the extracellular domains of the desmosomal cadherins consist of five subdomains (EC1 to 5) each of approximately 100 amino acids. On the basis of binding studies between recombinant proteins of different lengths, Chitaev and Troyanovsky (1997), proposed a model for interaction in which the extracellular domains of the molecules on apposing cells overlap by 3 EC domains. This is different from the results of structural studies of E- and N-cadherins (Shapiro et al., 1996; Naga et al., 1996) where interaction was shown to be between the N-terminal EC1 domains. These are the domains that contain the HAV sites which are located on the faces of the molecules that interact in adhesion (Shapiro et al., 1996). The demonstration that the desmocollin and desmoglein CAR sites are involved in adhesion (Tselepis et al., 1998) suggests that interaction between desmosomal glycoproteins may resemble that seen between conventional cadherins. In addition, the distance between the desmosomal plasma membranes (25 to 35 nm) appears to correspond well with an interaction between the apposed EC1 domains. Furthermore, although our pep tide inhibition studies on transfected cells showed that both Dsc and Dsg peptides were equally effective in inhibiting adhesion, and also as effective as the two peptides combined (Tselepis et al., 1998), provisional experiments with desmosome-forming cells indicate that both peptides are required to block desmosome assembly (Runswick, O’Hare, Streuli and Garrod, unpublished). These results are not entirely consistent with the involvement of heterophilic interaction between Dsc and Dsg in desmosome formation. It may be that the interactions seen in transfected cells, which do not form desmosomes even though they


adhere to each other (Tselepis et al., 1998), do not mimic precisely the interaction seen in whole desmosomes. Cells in stratified squamous epithelia, transitional epithelia and squamous cells carcinomas possess an additional desmosomal glycoprotein, the E-48 antigen. This 20- to 22 kDa, glycophospatidyl inositol anchored protein has been shown to function in cell-cell adhesion by monoclonal antibody inhibition and transfection studies (Schrijvers et al., 1991; Brakenhoff et al., 1995). Cytoplasmic Interactions of Desmosomal Glycoproteins In in vitro overlay assays Dsc la has been shown to bind to Pp1, Pg and the Dp N-terminus, , while Dsgl binds Pp1 and Pg, the former more weakly and the latter more strongly than Dscla (Smith and Fuchs, 1998). The interactions of Pg with the cytoplasmic tails of the desmosomal glycoproteins have been studied in detail by Troyanovsky and colleagues, using expression studies in epithelial cells (principally A431) with chimeric proteins consisting of the gap junction protein connexin 32 and the cytoplasmic domain of either Dsc1a or Dsg1. These constructs target the desmosomal glycoprotein C-termini to the plasma membrane as part of a gap junction-like structure, and the association of cytoplasmic proteins Dp, Pg and keratin filaments (KF) can be studied by immunofluorescence, immunoprecipitation and electron microscopy. The first use of this technique showed that the Dscla cytoplasmic domain would support Pg, Dp and KF association and plaque formation, and that Dsgl had a dominant negative effect leading to disruption of endogenous desmosomes (Troyanovsky et al., 1993). Deletion mutagenesis of the Dscla tail localised the site for Pg interaction to 37 amino acids at the extreme C-terminus, and this deletion also abolished KF association with the plaque (Troyanovsky et al., 1994a). Dp association was localised to a 10 amino acid region of the Dscla cytoplasmic tail close to the plasma membrane. The dominant negative effect of the Dsgl cytoplasmic domain was not abolished by deletion of 262 amino acids from the extreme C-terminus, but further deletion of 41 amino acids abolished both the dominant negative effect and Pg binding, thus localising the Pg binding domain to this 41 amino acid stretch (Troyanovsky et al., 1994b). This Pg binding region of Dsgl corresponds to a region that is conserved in other cadherins, including Dscla, where it located at the extreme C-terminus (Chitaev et al., 1996). More detailed analysis of Dsg-Pg binding by alanine scanning mutagenesis showed that a series of large hydrophobic amino acids in the conserved region are involved in Pg interaction (Chitaev et al., 1998). Similarly it was shown that nine hydrophobic amino acids within the arm repeats 1–3 of Pg are involved in Dsg-Pg interaction (Chitaev et al., 1998). Chitaev et al. (1998) found a 1:1 stoichiometry of Pg-Dsg binding, in contrast to the suggestion from immunoprecipitation data of a 6:1 Pg:Dsg binding ratio by Kowalczyk et al. (1996). If correct, the latter suggestion might provide an explanation for the dominant negative effect of the Dsgl cytoplasmic domain, since overexpression would be expected to sequester substantial amounts of Pg making insufficient availability for junctional assembly. A further possibility for the dominant negative effect of the Dsgl tail arose from the demonstration that expression in A431 cells of chimeras of E-cadherin extracellular domain and Dsg1 tail resulted in a 3-fold decrease


inE-cadherin bound to plakoglobin and a 5- to 10-fold reduction in the steady state levels of endogenous desmosomal cadherins, Dsg2 and Dsc2 (Norvell and Green, 1998). Binding of Pp to desmosomal glycoproteins (Smith and Fuchs, 1998) is clearly of functional importance since the human mutations described by McGrath et al. (1997) result in weakened desmosomal adhesion as well as detachment of Dp and KF from the desmosomal plaque. Desmosomal Glycoproteins in Epidermis The different isoforms of the desmosomal glycoproteins have distinct patterns of expression in epidermis and other stratified epithelia (Arnemann et al., 1993; Legan et al., 1994; King et al., 1995; Yue et al., 1995; North et al., 1996). In bovine epidermis, Dsc1 is associated with the upper, terminally-differentiating layers but is absent from the basal layer and the bases of the deep rete ridges. Dsc2 is most strongly expressed in cells immediately above the basal layer that have begun terminal differentiation. This region of greatest Dsc2 expression corresponds with the location in rete ridges of transit amplifying cells, the most rapidly proliferating keratinocyte population (Lavkar and Sun, 1983). Dsc3 is strongly present in the basal layer and gradually fades in the suprabasal layers (Legan et al., 1994; Yue et al., 1995; North et al., 1996). In human epidermis the expression patterns of Dscs are similar (Arnemann et al., 1993; King et al., 1995; 1996). The desmoglein patterns in epidermis are similarly differentiation-related with Dsg3 being most strongly expressed in the basal regions and Dsg1 increasing in expression suprabasally (Arnemann et al., 1993; Shimizu et al., 1995; Amagai et al., 1996). Curiously, Dsg2 appears to be expressed predominantly in the basal layer (Theis et al., 1993). An intriguing pattern of desmosomal glycoprotein expression is also found in mammary epithelium. Here Dsc1 and Dsg1 are not expressed. Dsc2 and Dsg2 are present in both lumenal and myoepithelial cells but Dsc3 and Dsg3 are confined to the latter (Runswick et al., 1996), thus again occupying a basal location. Preliminary studies also reveal interesting Dsc distributions in corneal epithelium. Dscl is absent from this nonkeratinizing epithelium, Dsc2 is expressed throughout, and Dsc3 is confined to the basal and suprabasal limbal region where the corneal stem cells are believed to reside (Messent et al., 2000). Quantification of the distribution of Dscl and Dsc3 isoforms by immunogold labelling of desmosomes at different levels in the epidermis, revealed that they are expressed in a reciprocally graded manner, with Dsc3 decreasing suprabasally from the basal layer and Dsc1 increasing (North et al., 1996). An inversely graded pattern of Dsg3 and Dsg1 expression in human epidermis has also been observed (Shimizu et al., 1995). It may be inferred that different Dsg isoforms are also present in the same junctions, since at intermediate levels of epidermis all desmosomes can be labelled using sera specific for either Dsg1 or Dsg3. These reciprocally-graded expression patterns suggest two conclusions. Firstly, since their molecular composition changes, desmosomes in epidermis are presumably continually turning over as the cells ascend through the layers. At present the functional significance of this is not clear. It may be that changes in cell-cell adhesive properties are


important in regulating epidermal morphology and differentiation. Secondly, expression of the different glycoprotein isoforms appears to be linked. All six human Dsc and Dsg genes are located at chromosome 18q12, the mouse desmoglein genes are closely linked in the proximal region of chromosome 18 and the bovine desmocollin genes cluster on chromosome 24q21/q22 (King et al., 1995; Arnemann et al., 1992; Wang et al., 1994; Amagai et al., 1995b; Simrak et al., 1995; Buxton et al., 1994; Solinas-Toldo et al., 1995). This clustering may be required for the regulation of desmosomal glycoprotein gene expression. The functional significance of these remarkable desmosomal glycoprotein distributions in epidermis is an important area for future investigation. During development of the mouse embryo three of the six desmosomal cadherin isoforms (Dsc1, Dsc3 and Dsg1) have so far been shown to be first expressed in the developing epidermis. In the mouse embryo, desmosomes first form at the 32-cell stage in a trophectoderm-specific location coincident with the onset of cavitation and blastocoel formation (Fleming et al., 1991). The only Dsc isoform present at this stage is Dsc2. Embryonic Dsc2 gene expression first occurs at the 16-cell stage and is specific to the trophectoderm lineage (Collins et al., 1995). Dsc2 proteins appear immediately after detection of its mRNA and coincides with initial desmosome assembly (Fleming et al., 1991; Collins et al., 1995). Presumably the Dsg isoform present is the ubiquitous Dsg2, though this has not been directly demonstrated. Desmosome formation between trophectoderm cells may be regulated by transcription of the glycoprotein genes although studies on the Dsg genes will be required to confirm this view. The desmocollin isoforms Dsc1 and Dsc3 are not expressed in the early mouse embryo. Dscl is first detected in the epidermis of the external nares at E13.5 (King et al., 1996; Chidgey et al. 1997). Dsc3 is initially expressed 12 hours earlier in nasal epidermis, whisker and the most mature vibrissa follicles (Chidgey et al., 1997). Both genes are upregulated in the general body epidermis at E14.5. At the same time both isoforms are expressed in the suprabasal layers of the newly stratified epidermis. However, by day 18.5 the region of maximum Dsc3 expression becomes basal whilst that of Dscl remains suprabasal (King et al., 1996; Chidgey et al., 1997). Thus the adult pattern of Dsc expression (see North et al., 1996) is established, simultaneously with the onset of the adult pattern of differentiation including the basal location of cell proliferation and the formation of stratum corneum. We have suggested the ratio of Dsc1 to Dsc3 expression at different levels in the epidermis is fundamental to establishing this pattern of differentiation (Chidgey et al., 1997). However, Dsg isoforms are also likely to play a role in this process. It has recently been shown that the Dsgl gene is first expressed at day 13.5 and shows a regional pattern of induction similar to that of Dsc1, being present in the outer cell layers of stratifying epidermis (King et al., 1996). An in situ hybridisation study of desmosomal glycoprotein gene expression during mouse development concluded that the DSC and DSCG genes are transcribed in hierarchical, overlapping temporal and spatial patterns, and that the spatial order in which they are expressed correlates with the physical order of the genes at the desmosomal cadherin locus (King et al., 1997) Denning et al., (1998) investigated the regulation of Dsg isoform expression in cultured human keratinocytes, HaCaT cells and squamous carcinoma cell lines. In particular they showed that down-regulation of PKC by long term treatment with phorbol ester or


bryostatin 1 led to decreased expression of Dsgl and Dsg3 but not Dsg2. These results are consistent with a role for PKC activation in regulation of the expression of Dsgl and Dsg3 in substantial cells. Adams et al. (1998) expressed a 4.2kb region of the human DSG1 promotor linked to the lacZ reporter gene in transgenic mice. They found that this region does not contain all the regulatory elements necessary for correct expression of the gene but might have elements that regulate activity during hair growth. Desmosomal glycoproteins in the epidermis are the target antigens in the autoimmune blistering disease pemphigus. There are two main forms of the disease, pemphigus foliaceous (PF) and pemphigus vulgaris (PV). Both are associated with formation of epidermal blisters: in PV these tend to be flaccid and fluid-filled, whereas PF the blisters burst readily and become encrusted. PV, but not PF, is also characterised by mucous membrane lesions, particularly of the oral mucosa. Histologically PV blisters show loss of adhesion between keratinocytes or acantholysis immediately above the basal layer while in PF this occurs in the granular layer or directly below it. Thus the blister roof is much thicker in PV than in PF which accounts for the different characteristics of the blisters. Pemphigus is characterised by the presence of circulating autoantibodies to the desmosomal glycoproteins (Dsg1 in PF and Dsg3 in PV) (reviewed by Stanley, 1993; Stanley and Kárpáti, 1994). These autoantibodies, which become bound in the epidermis, have been shown to be pathogenic by a number of criteria: they induce acantholysis of cultured human keratinocytes (Schiltz and Michel, 1976; Hashimoto et al., 1983); they cause epidermal acantholysis when injected into new born mice (Anhalt et al., 1982; Roscoe et al., 1985), even in the absence of complement (Anhalt, et al., 1986); their activity in the new born mouse assay is abrogated by prior incubation with recombinant Dsgl (Amagai et al., 1994a; 1995c). The binding of PV and PF antisera to the extracellular regions of epidermal desmosomes appears to occur in a reciprocally graded fashion; in PV autoantibodies bind more extensively to basal cells whereas in PF autoantibodies bind more extensively to the granular layer (Shumizu et al., 1995). Thus the location of greatest binding corresponds approximately with the level of acantholysis. Because autoantibody binding is clearly not precisely localized to the level of acantholysis we have suggested that acantholysis occurs at the weakest points in the system, where keratinocyte adhesion is most reduced (Domchowski et al., 1995). The mechanism by which acantholysis occurs is not clear. Targeted disruption of the Dsg3 gene in mice produces epidermal and mucosal lesions closely resembling those seen in PV blisters (Koch et al., 1997). This would be consistent with a direct effect on desmosomal adhesion by PV antibodies. However, activation of proteases that cleave adhesion molecules is also a possibility (Hashimoto et al., 1983). It has also been shown that pemphigus sera cause transient changes in cytoplasmic calcium concentration, protein kinase C relocation and activation, and phospholipase C activation in keratinocytes (Esaki et al., 1995; Seishima et al., 1995; Osada et al., 1997). These changes may generate an adhesion-disrupting signal. Further examination of the Dsg3 −/− mouse indicates a role for Dsg3 in anchoring hairs into follicles at the telogen phase of the hair cycle, since young mice lose hair during this phase (Koch et al., 1998). A histochemical study of human hair folicles shows that the


desmosomal glycoproteins have distinct distribution patterns leading to the conclusion that compositionally different desmosomes are present in various compartments of the follicle (Kruzen et al., 1998). ORGANISATION OF THE DESMOSOMAL PLAQUE The cytoplasmic domains of the Dscs and Dsgs are located in the outer dense cytoplasmic plaque of the desmosome (Figure 4.1, OP) (North et al., 1999). Here they associate with at least three other proteins, Pg, Pp and Dp. Pg and Pp are members of the armadillo family of junctional/signalling molecules, which both participate in the organisation of intercellular junctions and, as complexes with transcription factors, enter the nucleus to regulate gene activity. Pg contributes to the structure of both desmosomes and adherens junctions (Cowin et al., 1986), and has been shown to cause axis duplication in Xenopus development (Karnovsky and Klymkowsky, 1995), while Pp has both junctional and nuclear location (Mertens et al., 1996; Schmidt et al., 1997). The possibility that desmosomal components can also regulate gene expression and cellular differentiation is an important area for current and future investigation. Dp appears to provide a molecular link between the adhesive components of the desmosome and the intermediate filament cytoskeleton (Stappenbeck and Green, 1992; Bornslaegar et al., 1996). Its C-terminus is located in the desmosomal inner plaque (IP, Figure 4.1), into which KF insert (Miller et al., 1987; North et al., 1999). Plakoglobin and Plakophilin Pg and Pp belong to the armadillo family which includes the adherens junction-associated proteins β-catenin and p120cas, and the tumour supressor protein APC (for reviews see Peifer, 1995; Gumbiner, 1995; Klymkowsky and Parr, 1995; Barth et al., 1997; Cowin and Burke, 1996; Huber et al., 1996). Pg occurs as a single isoform, which is a universal component of the cytoplasmic plaques of desmosomes, whereas the two isoforms of Pp have restricted tissue distributions. Members of the armadillo family interact with several different proteins. These interactions appear to be mediated by overlapping regions of a centrally located group of so-called armadillo repeats (arm motifs) (Figure 4.2), each consisting of 42 amino acids (Peifer, 1995; Klymkowsky and Parr, 1995). Pg contains 13 consecutive arm repeats flanked by distinct N-terminal and C-terminal domains (Riggleman et al., 1989). It shares 60–70% sequence identity with armadillo and β-catenin. The Pps consist of 9 arm repeats followed by a C-terminal extension of 13 (Pp 1) or 11 (Pp 2) amino acids and no Nterminal flanking domain (Hatzfeld et al., 1994; Heid et al., 1994; Mertens et al., 1996). The Pps show amino acid identity of 33% to p120 with which they form a sub-group, but less than 25% to Pg, β-catenin and armadillo (Cowin and Burke, 1996). Pg interacts with the cytoplasm domains of Dsc and Dsg, and the classical cadherins (Korman et al., 1989; Kowalczyk et al., 1994; Mathur et al., 1994; Peifer et al., 1992; Roh and Stanley, 1995; Troyanovsky et al., 1994a,b; Butz and Kemler, 1994; Jou et al., 1995; Knudsen and Wheelock, 1992; Piepenhagen and Nelson, 1993). It binds with high affinity


to Dsg, with lower affinity to Dsc “a” and only weakly to E-cadherin (Chitaev et al., 1996). It also forms cytoplasmic complexes with APC and β-catenin, the latter being a component of the adherens junction (Rubinfeld et al., 1995). Deletion analyses show that the central arm repeats mediate association with classical cadherins, whereas the flanking repeats are essential for desmosomal cadherin binding (Mathur et al., 1994; Troyanovsky et al., 1994a, b; Roh and Stanley, 1995; Chitaev et al., 1996; Troyanovsky et al., 1996; Wahl et al., 1996; Witcher et al., 1996). Competition for binding domains on Pg may determine which complexes it forms. For example, the Dsg binding domain in the first three arm repeats overlaps the β-catenin binding domain (Sacco et al., 1995; Witcher et al., 1996; Chitaev et al., 1996; Wahl et al., 1996; for review see Cowin and Burke, 1996), and may regulate whether Pg associates with desmosomes or with classical cadherins in adherens junctions. Chitaev et al. (1998) have identified, by alanine scanning mutagenesis, nine hydrophobic amino acids in arm repeats 1–3 of Pg that are required for binding to Dsg and Dsc. Human, bovine, murine and rat Pg sequences are available (Franke et al., 1989, Cowin et al., 1986; Butz et al., 1992; Hiipakka, 1996). A variant of Pg from a human transitional carcinoma cell line contains a 120bp deletion corresponding to fourth arm repeat, believed to result from alternative splicing (Ozawa et al., 1995a). This variant binds with lower affinity to E-cadherin, Dsg2 and APC than full-length Pg, but both forms bind acatenin equally (Ozawa et al., 1995b). Where the alternative form is expressed in vivo is not clear. Since Pg is common to both adherens junctions and desmosomes it may have a regulatory function injunction assembly. One such role may be segregating components between these junctions. The defective heart muscle of Pg −/− mice (Ruiz et al., 1996; Bierkamp et al., 1996) has no desmosomes and Dp becomes merged with the components of adherens junctions into extended junctions, while Dsg2 is scattered over the surface of the cells. The force exerted by contraction of heart muscle may cause intermixing of junctional components. Discrete but abnormal desmosomes are present in epithelia of Pg −/− mice. Thus in null mice surviving until birth, abnormal structure of epidermal desmosomes was accompanied by blistering and subcorneal acantholysis (Bierkamp et al., 1996) These desmosomes lacked either the inner or outer plaque, or both, and intermediate filament attachment was impaired. Deletion of Pg and yeast two hybrid analysis suggests that the N-terminus of Dp binds to Pg at a site located in the central arm repeats (Palka and Green, 1997; Kowalczyk et al., 1997). Co-expression of a chimera of the E-cadherin extracellular domain and the Dsgl cytoplasmic domain with Pg and truncated Dp (containing the N-terminus) in L929 and COS cells produced plaque-like junctional structures at points of cell-cell contact (Kowalczyk et al., 1997). Pg, therefore, acts as a link between the cytoplasmic domain of Dsg and Dp. Whether it also links Dsc and Dp is not yet clear. Pg, β-catenin and armadillo share substantial sequence homology in the arm repeats but the N- and C-termini are divergent, possibly conferring specific functions on the different molecules. To address this, mutant Pgs lacking either or both N- and C-termini were expressed in A-431 cells (Palka and Green, 1997). N-terminal deletion did not affect desmosome assembly or morphology but Pg lacking the C-terminus was

Figure 4.2 Comparsion of the domain structures of human plakglobin (adapted from Wahl et al., 1996) and human plakophilins 1a, 1b, 2a & 2b. The imperfect armadilo repeats are shown as nymbered boxes. The domains of plakoglobin necessary for association with desmosomal cadherins, classical cadherins, α-catenin, APC and desmoplakin as determined by deletion analyses are indicated. The fourth armadillo repeat which is lacking in the alternative form of plakoglobin due to a 120bp deletion is shown (hatched box). The plakophilin la mutations (Q304X in arm repeat 1 and 1132ins28 in arm repeat 3) which result in ectodermal dysplasia/skin fragility syndrome are shown. The additional 21 amino acids and 44 amino acids inserts found in the alternative splice forms, plakophilin 1b and 2b, are indicated (solid box). Incomplete carboxyl-terminal armadillo repeats are shown (patterned boxes). Numbers represent amino acid residues. Diagram is not to scale.



incorporated into abnormally long desmosomes, possibly indicating that the C-terminus is involved in limiting desmosome length. The C-terminus may mask sites which bind other desmosomal components. Consistent with this a sequence of amino acids in arm repeat 13, which interacts with and masks cadherin binding sites located further upstream (Troyanovsky et al., 1996). The C-terminus may mask cryptic binding sites for Dp, so limiting over-recruitment of components and desmosome length (Palka and Green, 1997). It should be remembered, however, that normal desmosomes appear to fuse when brought together by low calcium medium-induced contraction of MDCK cells (Mattey and Garrod,1986b). Such large desmosomes do not disperse when normal extracellular calcium concentration and cell morphology are restored (unpublished observations). Expression of N-terminally-truncated Pg in both Xenopus and cultured epithelial cells increased the cytosolic levels of endogenous and ectopic Pg (Rubenstein et al., 1997; Palka and Green, 1997). This may reflect enhanced stability of Pg in the non-cadherin associated pool. Stabilisation of β-catenin depends upon dephosphorylation of a serine/threonine glycogen synthase kinase 3β (GSK3β) consensus site in the N-terminus (Peifer et al., 1994b; Rubenstein et al., 1997). GSK3β is the vertebrate homologue of Drosophila Zestewhite-3, a segment polarity gene that is negatively regulated by wingless leading to dephosphorylation, stabilisation and increased cytoplasmic levels of armadillo. Pg also contains a GSK3β consensus site in its N-terminus, deletion of which generates increased cytosolic levels in Xenopus embryos (Rubenstein et al., 1997). Overexpression of β-catenin or Pg suggests that these molecules exert signalling effects. Micro-injection of β-catenin or Pg mRNA into Xenopus embryos causes duplication of the body axis, replicating the effect of Wnt-1 overexpression (Funayama et al., 1995; Karnovsky and Klymkowsky, 1995). The balance between cadherin-bound and unbound pools of β-catenin and Pg may be crucial for signalling during development. The architectural HMG box transcription factor, LEF-1/XTCF-3 interacts with β-catenin (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996) and Pg (Huber et al., 1996). In mammalian cells, β-catenin/LEF-1 or Pg/LEF-1 complexes translocate to the nucleus. The former complex binds to the Ecadherin promoter and may regulate E-cadherin transcription thus affecting assembly of intercellular junctions. Pg also forms complexes with the tumour suppresser protein APC (Rubinfeld et al., 1995). This complex targets proteins for ubiquitination and degradation, thus reducing their levels. APC competes with cadherins for the association with Pg and β-catenin (for review see Polakis, 1995) and may thus regulate the levels of cytosolic Pg. Mutant forms of APC, unable to down-regulate β-catenin and Pg, are associated with colonie hyperplasia. Hence Pg may be a key regulator in cell adhesion, differentiation and proliferation. It is not clear how the diverse functions of Pg are regulated, but modulation of tyrosine phosphorylation may be important. Growth factor-dependent tyrosine phosphorylation of Pg correlates with a more invasive cell state (Shibamoto et al., 1994) and may regulate junction assembly. Additionally, it has been reported that the assembly and formation of adherens junctions precedes desmosome assembly (O’Keefe et al., 1987; Wheelock and Jensen, 1992; Lewis et al., 1997). Studies of the role of Pg injunction assembly suggest that association of Pg with a classical cadherin is necessary before desmosomes can assemble (Lewis et al.,


1997). However, it has also been shown that Pg preferentially assembles into desmosomes rather than adherens junctions (Näthke et al., 1994; Adams et al., 1996). Furthermore, desmosomal adhesion can form in the absence of classical cadherin-mediated cell interactions (Tselepis et al., 1998). The events leading to junction formation are thus complex with Pg playing an important modulatory role. Steric hindrance between binding partners, molecular conformation, levels of expression and variation in binding kinetics between cadherin cytoplasmic domains may be mechanisms for controlling Pg complex formation. Pp, formerly designated “band 6” in enriched desmosome preparations from bovine nasal epidermis (Skerrow and Matoltsy, 1974), occurs as two isoforms, Pp1 and Pp2. Sequences are available for human and bovine Pp1 (Heid et al., 1994; Hatzfeld et al., 1994). Alternative splicing of exon 7 which encodes 21 amino acids at the beginning of arm repeat 4 of the human Ppl gene results in two variants, 1a (726 amino acids) and 1b (747 amino acids) (Schmidt et al., 1997). Pp2 occurs as two variants, 2a (837 amino acids) and 2b (881 amino acids), through alternative splicing of an exon between the second and third arm repeats. Pp1 and 2 are widely expressed proteins that occur in the nuclei of both epithelial and non-epithelial cells (Mertens et al., 1996; Schmidt et al., 1997). The nuclear function of Pps, if any, is presently unknown. The desmosomal complement of Pps is cell-type specific. Thus, in desmosomes of the suprabasal layers of stratified epithelia, only Pp1 is present, whereas in simple epithelia or myocardial cells only Pp2 can be detected. In other epithelia, both Pp1 and 2 can coexist in desmosomes (Heid et al., 1994; Mertens et al., 1996; Schmidt et al., 1997). Only Pp 1a localises to desmosomes, whereas the 1b variant is restricted to nuclei, probably because the additional sequence in the 1b variant somehow restricts its distribution (Schmidt et al., 1997). Pp1 has a major role in maintaining desmosome function in epidermis. The firstdescribed human desmosomal mutations result in functional knockout of Pp1 (McGrath et al., 1997). In the affected individual, absence of Pp1 results in skin fragility and ectodermal dysplasia, affecting skin, hair and nails. No abnormalities in other organs have been detected. Mutational analysis revealed different nonsense mutations in both of the Pp1 alleles, each causing premature termination of translation. These mutations would result in severely truncated forms of Pp1 and immunostaining was indeed absent from epidermis. Desmosomes in the affected skin are small and greatly reduced in number, lacking inner plaques and IF attachment. Large intercellular spaces indicate weakened desmosomal adhesion. Dp immunostaining was diffuse rather than cell-peripheral, whereas staining for other desmosomal components appeared normal. This suggests that Pp1 links Dp to the desmosomal cadherins and is important for desmosomal adhesion. Desmoplakin DpI and II are major components of the desmosomal plaque, extending across both outer and inner plaque domains (North et al., 1999) (Figure 4.1). Dp is also present in complexus adherentes junctions in vascular endothelial cells (Schmelz and Franke, 1993; Schmeltz et al., 1994; Valiron et al. 1996). Dps are constitutive desmosomal components


except that DpII is absent from cardiac muscle (Angst et al., 1990). Derived by alternative splicing from a single gene, DpI and DpII have predicted MWs of 332 and 260 kDa, respectively (see Bornslaeger et al., 1994 for review). DpI is a homodimer with a central α-helical coiled-coil rod domain flanked by globular end domains (Bornslaeger et al., 1994). By rotary shadowing, this rod domain is ~130 nm in length, while that of DpII is ~ 43 nm (O’Keefe et al., 1989). However, simultaneous immunogold labelling of both globular end domains in desmosomes of bovine nasal epi dermis showed the length of Dp, measured perpendicular to the plasma membrane, to be only ~40 nm (North et al., 1999). Thus DpI may be folded or coiled in tissue. The rod domain of DpI is characterized by periodic distribution of charged residues indicating that it may aggregate with itself or with similar proteins to form higher order filamentous structures (Stappenbeck and Green, 1992). The Dp C-terminal domain consists of three 176 amino acid subdomains, A, B and C, plus a 68 residue “tail” at the C-terminus. Each subdomain is composed of a 38 residue repeat motif with the same periodicity of acidic and basic residues as the 1B rod domain of IF proteins, suggesting that DP could interact with the latter (Bornslaeger et al., 1994). The N-terminal domain consists of a series of shorter heptad repeats which are predicted to form compact bundles (Virata etal, 1992). By transfection of specific Dp domains into cultured epithelial cells, Green and colleagues have demonstrated that the C-terminus mediates interactions with IF (Stappenbeck and Green, 1992; Stappenbeck et al., 1993; see also below). At least two Cterminal subdomains are involved in this binding. An additional 48–68 residue region at the C-terminus is critical for keratin, but not vimentin, association (Stappenbeck et al., 1993). The rod domain apparently mediates aggregation of desmoplakin molecules, thus contributing to desmosomal plaque architecture (Stappenbeck and Green, 1992; Bornslaeger et al., 1996): yeast two-hybrid studies indicate that the carboxyl portion of the rod contains a site essential for dimerization (Meng et al., 1997). The N-terminal domain targets Dp to the outer plaque (Stappenbeck et al., 1993; Bornslaeger et al., 1996) and clusters plakoglobin-desmosomal cadherin complexes into discrete plasma membrane domains (Kowalczyk et al., 1997). Consistent with this, immunogold labelling shows the Dp N-terminus to lie in the outer plaque and the C-terminus in the zone of IF attachment (North et al., 1999) (Figure 4.1). Smith and Fuchs (1998) have shown that targeting of Dp to desmosomes is disrupted by deletion of only about 10% of its N-terminal sequence, and that only 86 residues of the N-terminal residues are sufficient for desmosomal targeting. Transfection of cells with Dp lacking the C-terminus (Bornslaeger et al., 1996), causes formation of abnormal junctions in which desmosomal (Dp, desmosomal cadherins) and adherens junction (E-cadherin, α- and β-catenin) components merge, suggesting that the C-terminal domain may be involved in segregating desmosomes from other junctions. In endothelial cells, molecular interactions and junctional associations of Dp differ from those in epithelial cells. Kowalczyk et al. (1998) have shown that a Dp amino terminal peptide is recruited into cell junctions in association with VE-cadherin, plakoglobin and βcatenin, suggesting the formation of junctional complexes which associate with both the actin and intermediate filament cytoskeletons.


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


Intermediate Filament Attachment and Accessory Plaque Proteins The principal desmosomal component involved in mediating interactions between IFs and the desmosomal plaque is Dp. Cell transfection experiments using mutant desmoplakin constructs have shown that the C-terminus of desmoplakin can interact with both keratin and vimentin IFs (Stappenbeck and Green, 1992; Stappenbeck et al., 1993) in a phosphorylation-dependent manner (Stappenbeck et al., 1994). Direct interactions between Dp and IF proteins have also been demonstrated in vitro (Kouklis et al., 1994; Meng et al., 1997). Specific IF types interact with DP via distinct sequences in different IF domains (Meng et al., 1997). Thus type II epidermal keratins bind to the Dp C-terminus via sequences in the N-terminal head domain of a single keratin polypeptide chain, while the interaction of Dp with simple epithelial keratins requires the presence of both type I and type II partner proteins, indicating the importance of the tertiary structure of the αhelical coiled coil (Meng et al., 1997). The interactions of the Dp C-terminus with type III IF proteins (e.g. vimentin) is weaker than with epidermal keratins (Meng et al., 1997), but may be strengthened in vivo by the formation of coiled-coil Dp dimers, since strong alignment of Dp with vimentin IF in transient transfections has been shown to require the Dp rod domain (Stappenbeck and Green, 1992; Stappenbeck et al., 1993). The physiological relevance of Dp-IF binding has been confirmed by the expression of Dp Nterminal polypeptides in stably transfected cell lines: these polypeptides act in a dominant negative manner to produce disrupted Junetional structures which lack associated keratin filaments (Bornslaeger et al., 1996). Further members of the plakin family are strong candidates for a role in IF-desmosome attachment, both by their localisation to the region where IF insert into the plaque and by their homology to Dp. Plectin has been reported to bind both Dp and IF and by immunoelectron microscopy lies further from the desmosomal membrane than Dp: it could thus function as a linker between them (Eger et al., 1997). Intermediate filament Associated Protein (IFAP) 300, a vimentin IF-binding protein, localises to desmosomes and hemidesmosomes at the region where IF attach to the inner plaque and has been demonstrated to bind keratins in vitro (Skalli et al., 1994): the relationship of this protein to plectin remains unclear. The sequence similarity of envoplakin and periplakin to Dp, together with their localisation to desmosomes and along keratin IF, may also indicate an involvement in anchoring filaments to desmosomes (Ruhrberg et al., 1997). Alternatively these proteins may mediate cross-linking and stabilisation of desmosome-associated IF, rather than themselves anchoring the filaments. A further component which may be particularly important in this latter role, namely the organisation and/or stabilisation of a mature desmosome-IF network is pinin (originally known as the O8L antigen; Ouyang and Sugrue, 1992). This 140kDa desmosomal accessory protein is located at the periphery of the plaque of only mature desmosomes of epithelia, its temporal appearance here apparently correlating with the establishment of a highly-organised desmosome- IF complex (Ouyang and Sugrue, 1996). However, reservations about a desmosomal role for pinin have recently been expressed (Brandner et al., 1997).


A number of other desmosomal components located nearer to the plasma membrane may also be involved in IF attachment. Visualisation of isolated Dsg1 tails by rotary shadowing has revealed a head portion of ~ 4 nm diameter, presumed to reside near to the membrane, and a thin tail some 19 nm long (Rutman et al., 1994). It has been proposed that this long tail portion, which contains basic residues, could stretch across the plaque and thus be favourably located to bind IF (Nilles et al., 1991). Such binding has not been demonstrated. Pp1 has been demonstrated to bind cytokeratins in vitro (Kapprell et al., 1988; Hatzfeld et al, 1994; Smith and Fuchs, 1998) yet by immunogold labelling lies within the outer, rather than inner, plaque domain (North et al., 1999, Figure 4.1). Desmocalmin (bovine form; the human form is known as keratocalmin (Fairley et al., 1991) is a 240 kDa calmodulin-binding plaque protein which is expressed only in stratified epithelia (Tsukita and Tsukita, 1985). Like Pp1, desmocalmin is localised immediately subjacent to the plasma membrane yet binds to polymerised keratin IF in vitro (Tsukita and Tsukita, 1985). How can the in vitro binding results be reconciled with apparent physical separation of Pp1 and desmocalmin from the inserting IF? Two possible explanations are either that the in vitro binding to keratin IF of some of the above proteins is not physiologically relevant, or that the keratins may actually approach nearer to the membrane than previously supposed. Leloup et al. (1979) suggested that the traversing filaments were unraveled protofilaments from terminating IF. Recent expression studies have shown that DP proteins containing both the rod and C-terminal domains aggregate with IF proteins to form structures that resemble the 4–5 nm traversing filaments of the inner plaque (Stappenbeck and Green, 1992). This meshwork is not seen with Dp alone, suggesting that IF may be present in the plaque as an anastomosing network of fine filaments associated with Dp (Bornslaeger et al., 1994). CONCLUSION Experimental confirmation of the adhesive role of desmosomal glycoproteins and recent discoveries on the interactions between molecules in the desmosomal plaque have greatly increased our understanding of these junctions. However, much more detailed knowledge is required, as well as a clearer insight into how they relate to desmosome structure. Further important issues are the process of desmosome assembly and breakdown, and the dynamic regulation of desmosomal adhesion. These topics have been reviewed recently (Garrod et al., 1996; 1998; Burdett, 1998). These reviews indicate the likelihood that both “inside-out” and “outside-in” signalling are involved in the regulation of desmosomal adhesion. A much greater understanding of the function of desmosomes in the epidermis is required. An important question relates to the function of the differential expression of desmosomal glycoproteins and plakophilins in epidermis. Do the patterns represent differential adhesion in different epidermal layers and, if so, how is this important in relation to epidermal structure and morphogenesis? Alternatively, could these patterns somehow generate signals that provide positional information to regulate epidermal differentiation?


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CELL—MATRIX ATTACHMENT

5. PROTEIN-PROTEIN INTERACTIONS AT THE DERMAL-EPIDERMAL BMZ M.PETER MARINKOVICH

Specific protein-protein interactions are required for the assembly and continued integrity of all basement membranes. In the highly specialized dermal-epidermal basement membrane zone (BMZ) an intricate and precise organization of multiple components is required to maintain dermal-epidermal cohesion. Disruption of this organized array of components, either by inherited (epidermolysis bullosa) (Marinkovich et al., 1999) or acquired (autoimmune bullous) disease (Lin et al., 1997), can result in extensive skin and mucosal blistering. ULTRASTRUCTURAL ORGANIZATION OF THE DERMALEPIDERMAL BMZ BMZ ultrastructure has traditionally been visualized by standard transmission electron microscopy (Figure 5.1a). Through this technique, it can be appreciated that at the superior end of the dermal-epidermal BMZ, intermediate filaments insert upon electron dense condensations of the keratinocyte plasma membrane termed hemidesmosomes. Anchoring filaments appear as thin threadlike structures (Ellison and Garrod, 1984) which extend from the extracellular surface of hemidesmosomes, span the electron lucent space in the BMZ termed the lamina lucida, and appear to connect hemidesmosomes to the electron dense portion of the extracellular BMZ termed the lamina densa. Anchoring fibrils are banded structures which extend perpendicularly from the lamina densa into the adjacent superficial papillary dermis. Anchoring fibrils either extend back to reinsert onto the lamina densa or connect with electron dense structures of the papillary dermis termed anchoring plaques (Keene et al., 1987). In connecting to these structures, anchoring fibrils entrap interstitial collagen and other dermal molecules and help to firmly attach the lamina densa onto the papillary dermis. Newer techniques in the analysis of tissues by electron microscopy combine high pressure, cryopreservation and freeze substitution to yield improved preservation of tissue architecture (Hippe-Sanwald, 1993). During the initial cryopreservation step, water in tissues is converted to noncrystaline ice, through the combined application of very low temperature and very high pressure. Subsequently, absolute acetone or methanol with aldehyde or osmium is substituted for the ice, and crosslinking occurs at very low

PROTEIN-PROTEIN INTERACTIONS AT THE DERMAL-EPIDERMAL BMZ 89

Figure 5.1 Ultrastructural appearance of the dermal-epidermal basement membrane. Human neonatal foreskin was examined by transmission electron microscopy using standard fixation (A), or high pressure freeze substitution (B). Note absence of anchoring fibrils and lamina lucida structures in B. Magnification: A, 72, 660; B, 55,650. Micrographs provided by Douglas Keene, Shriners Hospital, Portland Oregon.

temperatures. This technique greatly reduces the loss of soluble substances such as proteoglycans. Recent studies with high-pressure freeze substitution electron microscopy have demonstrated that dermal-epidermal BMZ ultrastructure seen with conventional fixation may be artifactual (Keene and McDonald, 1993) (Keene et al., 1997). While hemidesmosomes and intermediate filaments are clearly visible with these techniques, anchoring fibrils and the lamina lucida appear absent (Figure 5.1b). Instead, a continuous felt-like matrix extends from the basal keratinocyte plasma membrane into the papillary dermis. These studies suggest that the lamina lucida seen after conventional fixation may arise as a dehydration artifact caused by a shrinking away of the basal cell plasma membrane from the underlying dermis after water removal. Likewise, the lamina densa, anchoring filaments and anchoring fibrils may appear due to artifactual precipitation resulting from removal of proteoglycans and other soluble substances during conventional fixation methods. These studies provide additional insight into BMZ organization. INTERMEDIATE FILAMENT-HEMIDESMOSOME INTERACTIONS Basal keratinocyte intermediate filaments contain the acidic keratin 14 and the basic keratin 5. Initially these keratins combine together as polar coiled-coil dimers. The dimers go on to form staggered anti-parallel tetramers and then undergo additional lateral and end to end associations to form 2 to 3 nm diameter apolar protofilaments and 4 to 5 nm

90 M.PETER MARINKOVICH

diameter protofibrils. finally two to four protofilaments associate into a coiled coil lattice to form 10 nm diameter intermediate filaments (Fuchs and Cleveland, 1998). Recent studies have suggested that the nonhelical keratin 5 head domain may be important in the anchoring of intermediate filaments to other structures (Smith and Fuchs, 1998). The desmosomal components desmoplakin, plakophilin 1 and plakoglobin, all appear to interact with this domain. Intermediate filaments through the keratin 5 head domain may also associate with hemidesmosomal components although this remains to be proven. The keratin 5 head domain is also suspected to be involved with the binding of intermediate filaments to melanosomes (Uttam et al., 1996) as mutations which affect this domain are associated with epidermolysis bullosa simplex with mottled pigmentation. BPAG1 Hemidesmosomes contain two plaque proteins, including BPAG1 (BP230) and plectin (HD1). BPAG1 is a 230 kD protein which consists of a central rod flanked by carboxyl (C) and amino (N) terminal globular domains (Tanaka et al., 1991) (Sawamura et al., 1991) (Tamai et al., 1995) (Tang et al., 1996). BPAG1 has significant homology both to desmoplakin as well as to plectin in its two C terminal repeating globular domains. This homologous region contains the keratin binding region. BPAG1 localizes to the inner plaque on the cytoplasmic surface of the hemidesmosome and functions in the connection between hemidesmosomes and intermediate filaments. BPAG1 negative transgenic mice lack a hemidesmosomal inner plaque and the connection between hemidesmosomes and intermediate filaments is severed, creating a cytoplasmic zone of mechanical fragility just above the hemidesmosomes, which results in intraepidermal blistering (Guo et al., 1995). In these mice neither hemidesmosome stability or cell substratum adhesion appear to be weakened. Thus BPAG1 does not appear vital for hemidesmosome or BMZ assembly. BPAG1 also exists as an alternatively spliced neural isoform termed BPAG1n (dystonin). As a result of the ablation of the domains common for both BPAG1 and BPAGln, BPAG1 knockout mice also develop severe dystonia and sensory nerve degeneration typical of dystonia musculorum (dt/dt) mice (Bowling et al., 1997). BPAG1 is also a major autoantigen targeted by sera of most patients with the blistering disease, bullous pemphigoid (Stanley, 1991). PLECTIN Plectin is a 500 kD cytoskeletal linker protein (Liu et al., 1996). Plectin associates with a variety of types of cytoskeletal structures including keratin, vimentin, neurofilaments, microfilaments, and microtubules. It shows a very widespread expression in the majority of tissues and cell types studied. In stratified epithelial cells, plectin promotes cell-cell adhesion by linking keratin containing intermediate filaments and desmosomes, while plectin’s role in promoting dermal-epidermal cohesion lies in its ability to link keratin into the hemidesmosomal plaque.


Figure 5.2 Anchoring filaments: central organizers of specialized basement membrane assembly. Four anchoring filament proteins, α6β4 integrin, collagen XVII, laminin 5 and laminin 6 are depicted schematically on the left. Important structural domains and proposed functions are listed to the right.


Rotary shadowing electron microscopic studies have shown that plectin, like many BMZ proteins, assumes a dumbbell shape and contains a central 200 nm helical coiled-coil rod, which is flanked at either end by large globular domains (Wiche, 1998). Gel permeation studies which predicts plectin’s molecular weight to be 1100 kD, suggest that plectin exists as a dimer in solution. Immuno-gold electron microscopy studies have demonstrated localization of plectin with intermediate filaments (Foisner et al., 1995). The C terminal globular domain of plectin contains 6 repeating domains containing approximately 300 amino acids each. Each domain contain multiple copies of a tandemly repeated 19 amino acid motif. A 50 amino acid segment bridging repeat domains 5 and 6 contains a basic amino acid residue cluster located in a bipartite nuclear localization sequence that functions as the intermediate filament binding site (Nikolic et al., 1996). Near this site of domain 6 is a phophorylation site for p34cdc2 protein kinase that may be involved with dissociation of plectin from intermediate filaments (Malecz et al., 1996). Plectin’s interaction with hemidesmosomes studied by immuno-gold electron microscopy with domain specific antibodies (Foisner et al., 1994) reveal a lack of orientation of plectin’s association between intermediate filaments and hemidesmosomal plaque. In this study, the same domains were seen to insert both into keratin filaments as well as hemidesmosomal plaque. These studies suggest that two binding sites of plectin exist which each are capable of interacting with other hemidesmosomal elements. These findings are consistent with direct ligand binding studies (Rezniczek et al., 1998) which demonstrate that both the N and C terminal globular domains of plectin are capable of binding to β4 integrin. Furthermore, plectin is capable of associating with two different regions of the β4 integrin cytoplasmic domain. The significance of these multiple interactions remains to be elucidated. Plectin has been recently shown to exist in multiple isoforms generated by alternative splicing of the amino termini (Elliott et al., 1997). At least eight isoforms have been detected thus far. Isoform expression levels as shown by RNAase protection assays, demonstrate a diversity of different isoform expression in individual tissues. The splicing diversity affects both coding and noncoding regions and may affect plectin function and/ or expression. Other molecules, including HD1 (Heida et al., 1992) and IFAP 300 (Skalli et al., 1994), which show similarities as well as differences with plectin in terms of tissue distribution as well as other parameters may represent some of these alternatively spliced plectin isoforms, however this remains to be determined. Plectin deficient patients show a blistering phenotype with separation of the skin occurring above the level of the hemidesmosome (Smith et al., 1996) (Gache et al., 1996) (McLean et al., 1996) (Chavanas et al., 1996). Hemidesmosomes are intac suggesting that plectin is not essential for hemidesmosome formation. Plectin deficient patients have been shown to have an associated muscular dystrophy, which accounts for the name, epidermolysis bullosa simplex with muscular dystrophy. Premature stop codon mutations of the plectin gene were found to be associated with the majority of these patients, although in one case, a three amino acid deletion was found in the N terminal globular region. This region, which is involved with the interaction of plectin with β4 integrin, is likely to be important in plectin function. The mechanism as to how a lack of plectin in


muscle leads to muscular dystrophy remains to be determined. Plectin knockout mice (Andrä et al., 1997) also demonstrate a blistering phenotype similar to epidermolysis bullosa patients described above, but show more extensive muscle pathology.

α6β4 INTEGRIN Hemidesmosomes also contain the transmembrane proteins collagen XVII and α6β4 integrin (Figure 5.2). The cytoplasmic portions of these molecules make up part of the hemidesmosome dense plaque whereas the extracellular portions of these molecules make up portions of the anchoring filament and may contribute to the structure known as the subbasal dense plate which underlies hemidesmosomes in the lamina lucida region. α6β4 integrin is an essential and central component of the hemidesmosome (Sonnenberg et al., 1991). Like all of the members of the family of molecules known as integrins (Hynes, 1992), it consists of two transmembrane subunits assembled together in a noncovalent fashion (Tamura et al., 1990) (Sonnenberg et al., 1990). The extracellular domains of these subunits combine together to form a ligand binding site, whereas the intracellular domains interact with other hemidesmosomal components. β4 integrin is only known to combine with the α6 subunit, whereas the α6 subunit can combine with β4 or β1 subunits. The α6β1 or α6β4 integrin combinations have been shown to act as receptors for laminin, and α6β4 integrin has been shown to act as a receptor for laminin 5. β4 integrin contains an especially large approximately 1000 amino acid cytoplasmic domain, which consists of two pairs of fibronectin repeats separated by a connecting segment. Replacement of β4 integrin deletion constructs in β4 integrin negative EB-PA keratinocytes revealed a 27 amino acid region of the connecting segment was required for hemidesmosome assembly (Niessen et al., 1997). This region of β4 integrin is critical in localizing plectin to hemidesmosomes, and a direct interaction between β4 integrin cytoplasmic domain and plectin has been demonstrated (Rezniczek et al, 1998). β4 integrin directly interacts with collagen XVII (Schaapveld et al., 1998) (Borradori et al., 1997) and influences its localization to hemidesmosomes. The sequences on β4 integrin which appear to mediate this interaction lie on the C terminal section of the connecting segment and the third fibronectin repeat. In addition to binding collagen XVII and plectin, β4 integrin cytoplasmic domain, under some circumstances may fold over and bind to itself. Ligand binding studies show that the C terminal 85 amino acids of β4 integrin can interact with and bind the connecting segment and regions N terminal to it. This folding appears to influence the affinity of β4 integrin for plectin and may be involved in hemidesmosome assembly/disassembly. β4 integrin contains a tyrosine activation motif (TAM) in its connecting segment. Mutations involving the tyrosines in this motif have been shown to have variable effects. In one study, these mutations appeared to prevent the incorporation of β4 integrin into hemidesmosomes (Mainiero et al., 1995).In another study, TAM mutations appeared to have only a minor effect on the localization of collagen XVII to hemidesmosomes (Borradori et al., 1997). As mentioned above, α6β4 integrin appears to play a central role in hemidesmosome formation in that transgenic mice lacking the β4 integrin show skin devoid of


hemidesmosomes as well as blistering and severe deficits in cell adhesion (Bowling et al., 1996). Patients with absence of the β4 (Vidal et al., 1995) or 6 integrin (Ruzzi et al., 1997) due to underlying gene mutations demonstrate very rudimentary hemidesmosome formation at best, widespread blistering and pyloric atresia (epidermolysis bullosa with pyloric atresia, EB-PA). COLLAGEN XVII Collagen XVII (BP180/BPAG2) is another transmembrane component of hemidesmosomes. It is collagenous protein with a type II transmembrane orientation (Giudice et al., 1992). Based on electron microscopy and crosslinking studies, collagen XVII appears to assemble into a homotrimer (Schumann and Bruckner-Tuderman, 1996) and contains three main regions; an intracellular N terminal globular head, a central rod and an extracellular flexible tail (Hirako et al., 1996). The C terminal portion of the molecule appears to be involved with polarization of the collagen XVII molecule to the basal surface of keratinocytes. As mentioned above the cytoplasmic domain of collagen XVII colocalizes and binds with the β4 integrin in the hemidesmosome (Borradori et al., 1998) (Aho and Uitto, 1998). A subset of epidermolysis bullosa patients who lack collagen XVII expression also show a lack of localization of BPAG1 into hemidesmosomes. Analysis of deleted collagen XVII cDNA constructs suggests that the C terminal half of the collagen XVII cytoplasmic domain is required for BPAG1 localization into hemidesmosomes. Thus the collagen XVII endodomain has two important functions: ligation of BPAG1 (which links intermediate filaments to the hemidesmosome) and ligation of the β4 integrin endodomain (which serves to correctly localize collagen XVII to the hemidesmosome). Collagen XVII assists in promoting epidermal adhesion, as evidenced by the widespread blistering that occurs in patients with generalized atrophie benign epidermolysis bullosa (GABEB) who lack this molecule. Interestingly, retrovirally mediated collagen XVII gene replacement in primary GABEB keratinocytes corrected adhesion defects in vitro and corrected blistering defects in vivo after grafting of these cells onto immunodeficient mice (Seitz et al., 1999). The large non-collagenous region of collagen XVII near the transmembrane region, termed the NC16A domain may be involved with association of collagen XVII with 6 integrin in keratinocytes in vitro, indicating that this portion of the molecule may be necessary for binding to α6β4 integrin (Hopkinson et al., 1995). This portion of the molecule also contains the epitopes recognized by bullous pemphigoid autoantibodies (Liu et al., 1995). The central rod and flexible tail region of collagen XVII contains a number of interrupted collagenous domains which assemble into a triple helix. The most distal extracellular portion of this molecule contains epitopes recognized by autoantibodies from some patients with cicatricial pemphigoid (Balding et al., 1996). Based on rotary shadowed electron microscopic measurements, the extracellular portions of collagen XVII may extend up to 193 nm. It is therefore likely that collagen XVII extends the full length of the lamina lucida and inserts into the lamina densa. This is consistent with the


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


LAMININS OF THE DERMAL-EPIDERMAL BMZ In addition to α6β4 integrin and collagen XVII, anchoring filaments contain the molecules laminin 5 (Verrando et al., 1988) (Rousselle et al., 1991) (Carter et al., 1991) and laminin 6 (Marinkovich et al., 1992a) (Figure 5.2). Laminins are family of hetero trimeric BMZ proteins which contain a, β and γ chains (Burgeson et al., 1994). Laminin 5 contains α3, β3, and γ2 chains. All previously described laminins were shown to have three short arms and one long arm, forming a cross shape as shown by rotary shadowing analysis. In contrast, laminin 5 appears as a dumbbell shape which is the result of severe truncations of the short arms. Each of these short arm reductions occur primarily as a result of truncations of the genes coding for these subunits, however some of this reduction occurs post-translationally as extracellular processing events on the α3 and γ2 short arms (Marinkovich et al., 1992b). Because of these short arm truncations, laminin 5 cannot polymerize or bind to nidogen. Instead laminin 5 forms a disulfide bonded complex with laminin 6 (Champliaud et al., 1996). The importance of laminin 5 in mediating epithelial adhesion is best illustrated by the widespread blistering, mucosal erosions and extracutaneous epithelial adhesion defects that occur in patients with Herlitz junctional epidermolysis bullosa, where laminin 5 expression is absent (Meneguzzi et al., 1992) (Marinkovich et al., 1993) due to underlying gene mutations (Uitto et al., 1995). Laminin 5 is initially synthesized and secreted by keratinocytes in an unprocessed form with an α3 chain of 200 kD, a β3 chain of 140 kD, and the γ2 chain of 155 kD (Marinkovich et al., 1992b). The domains I and II in all three chains are believed to adopt a helical coil conformation, account for the rod-like central portion of the laminin molecule, and are essential for laminin assembly, βγ dimers accumulate intracellularly and assembly of these dimers with the α chain appears to be the rate limiting step in laminin assembly (Matsui et al., 1995). Shortly after secretion, the α3 chain becomes processed from 200 to 165 kD. In addition, the γ2 chain is processed from 155 to 105 kD by the enzyme BMP-1 (Lee et al., 1997). These processing steps occur both in vitro in cultured keratinocytes, as well in vivo in human tissues. An additional processing step predominantly occurs in vivo, which results in the conversion of the a three chain from 165 to 145 kD (Marinkovich et al., 1992b). Analysis of laminin 5 extracted from human tissue shows that the former two steps occur to completion while the latter processing step occurs in approximately half of the laminin 5 molecules. In addition, further processing via MMP-2, of the γ2 chain from 105 kD to 80 kD, at the boundary of domains III and II; occurs in certain proliferative states, such as squamous cell carcinoma and the developing mammary gland (Giannelli et al., 1997). This processing which has only been demonstrated in rat tissues, appears to facilitate a promigratory epithelial phenotype. It remains to be shown whether this processing step occurs in human tissues. The laminin 3 chain contains an especially large C terminal globular domain which consists of five repeating EGF-like segments. The G domain of laminin 5 α3 chain contains the major cell binding activity, which provides laminin 5 the ability to serve as the major attachment ligand for a number of epithelial cell types including keratinocytes and squamous carcinoma cells. Processing of the α3 chain from 200 to 165 kD appears to


occur near the junction of the G3 and G4 domains, and laminin 5 containing processed 165 kD α3 chain supports cell attachment (Rousselle and Aumailley, 1994) (Rousselle et al., 1995). These observations make it clear that the cell binding activity and the binding sites for integrins α3β1 and α6β4 are located somewhere in the G1 to G3 domains. Further confirming this, an antibody, termed BM165, which recognizes the G1 domain, has been shown to inhibit the attachment of keratinocytes to underlying culture substrate and to interfere with the attachment of epidermis to dermis (Rousselle et al., 1991). Laminin 5 additionally has heparin binding properties contained within the G4 and 5 domains (Sung et al., 1997). The significance of the heparin binding is as yet unclear. It is possible that, prior to processing and insertion into the BMZ, laminin 5 may bind to BMZ heparan sulfate proteoglycan, also known as perlecan, or alternatively cell surface heparan sulfate proteoglycan, syndecans, may bind to laminin 5 and laminin 6 to influence BMZ formation and cell migration. The G5 domain has been shown to have a promigratory activity (Sung et al., 1997) and removal of this domain through processing appears to decrease the ability of laminin 5 to support cell migration and increase the ability of laminin 5 to support hemidesmosome formation (Goldfinger et al., 1998). Laminin 6, the other known anchoring filament laminin, contains α3, β1 and γ1 chains (Marinkovich et al., 1992a), and based on sequence analysis, it has been hypothesized that one of the unpaired cysteine residues in the laminin 6 α3 chain domain 3 EGF1 region binds to an unpaired cysteine residue in the laminin 5 β3 domain 6 region (Champliaud et al., 1996). The α3 and γ2 chains of laminin 5 are consistently and completely processed when laminin 5 is complexed with laminin 6, and these processing steps are probably necessary in the covalent association of laminin 5 and laminin 6. Laminin 5 has also been shown to complex with another component, laminin 7, which contains the β2 laminin chain, however laminin 7 does not appear to be present in skin to any significant extent. Additionally, the N terminal portion of the α3 chain exists in two spliced variant forms (Ryan et al., 1994). The functional significance of this alternative splicing is not yet clear. As laminin 6 also contains the α3 G domain, it can also theoretically combine with basal cell surface receptors. This suggests that the laminin 5, laminin 6 heterodimer contains two cell binding regions per dimeric complex. Laminins-5 and 6 function as autoantigens in a subset of patients with the blistering disease cicatricial pemphigoid (Domloge-Hultsch et al., 1992). The autoantibodies recognize the processed laminin 5 α3 chain (Kirtschig et al., 1995) and thus the autoantibody resides on this chain must reside somewhere between the N terminus and G domain 3 of this chain. As the autoantibodies disrupt skin cohesion, it is possible that autoepitope site(s) are located in G domains 1–3 near the cell binding region. The current data suggest that laminin 5 is oriented in the BMZ with its C terminal G domain located adjacent to the basal cell surface and the N terminal short arms facing the lamina densa complexing with laminin 6. Laminin 6, located in the lower lamina lucida and lamina densa forms interactions with other BMZ components through linkage of its γ1 chain with nidogen (Mayer et al., 1993). Through this interaction, the divalent nidogen can link laminin-6 and the anchoring complex with its other known BMZ ligands, collagen IV and perlecan (Dziadek et al., 1985). In addition to its interaction with laminin 6, laminin 5 also is capable of binding with the NC1 domain of collagen VII (Chen et al.,


1997) (Rousselle et al., 1997) determined by solid phase binding assays and rotary shadowing analysis. It is not entirely clear whether laminin 5 can simultaneously associate with both collagen VII and laminin 6. Even if laminin 5 in incapable of such a dual association, only approximately half of the laminin 5 purified from tissue is complexed with laminin 6. This would leave a significant portion of laminin 5 available to bind to collagen VII. The above model predicts that the N terminal globular short arm domains are probably the regions of laminin 5 that come in contact with collagen VII and thus these regions probably contain the binding sites. Preliminary studies suggest that collagen XVII exodomain binds to laminin 5, (D. Reddy, manuscript in preparation) indicating another possible important interaction of laminin 5 with the extracellular BMZ. Due to its multiple interactions, laminin 5 is a molecule of central importance in the assembly of extracellular components of the dermal-epidermal BMZ. The dermal-epidermal BMZ contains at least one other large laminin in addition to laminin 6 which contains β1 and γ1 chains as well as an a chain recognized by mAb 4C7 (Engvall et al., 1986). While it was originally believed that mAb 4C7 recognized laminin a1 chain, recent evidence has shown that this mAb instead recognizes laminin a5 chain (Tiger et al., 1997). Therefore, it is likely that the dermal-epidermal BMZ contains the laminin combination, a5β1γ1, which has been termed laminin 10. Laminin 1 (a1β1γ1), in contrast, does not appear to be a significant constituent of the dermal-epidermal BMZ. ANCHORING COMPLEX—BMZ ASSOCIATIONS All BMZs contain some type of laminin, collagen IV, nidogen and perlecan, a large heparan sulfate proteoglycan (Martin and Timpl, 1987). A number of studies have demonstrated that these purified components are capable of assembling into structures resembling the lamina densa of the basement membrane (Yurchenco and O’Rear, 1994). This occurs through a number of interactions, which have been extensively studied. Collagen IV, through a quaternary association via its 7-A regions, and a binary association through its NC-1 domain, has the ability to self-polymerize into an extended network which probably represents the central feature of the lamina densa (Yurchenco, 1994). Laminins, as discussed below, also are capable of self-polymerizing and also contribute to the self assembly of the BMZ. Linkages of collagen IV, laminins and perlecan with the 150 kD linker protein nidogen (Dziadek et al., 1985) are additional mechanisms which underly the self assembly of the lamina densa/ lamina lucida in all BMZs. In specialized BMZs, such as the one at the dermal-epidermal junction, the assembly of the ubiquitous BMZ structures into a lamina densa/lamina lucida, and the assembly of anchoring complex structures may occur independently. Impairment of anchoring complex, but not lamina densa/lamina lucida formation is seen in knockout murine or EBPA patient skin which lacks a6β4 integrin (Bowling et al., 1996) (Smith, 1993). In contrast, impairment of lamina densa/lamina lucida formation, but not anchoring complex formation is seen in the skin of α3 integrin knockout mice which lacks the a3β1 integrin (Taverna et al., 1998). These studies suggest that both α6β4 and α3β1 integrin perform important complementary functions in the development of the dermal-epidermal


BMZ and that development of the ubiquitous and specialized portions of the dermalepidermal BMZ may occur via separate mechanisms. A primary mode of linkage of the specialized components of the anchoring complex with the rest of the ubiquitous BMZ structures probably occurs through association between the 4th EGF repeat on domain III of the γ1 chain (Mayer et al., 1993) of laminin 6 with nidogen. Through this linkage with nidogen, laminin 6 and the anchoring complex may directly be adjoined to collagen IV, perlecan and indirectly be adjoined, through repeated nidogen-ligand linkages, to laminin 10. Another mode of association between the anchoring complex with the rest of the BMZ may occur through interaction of the heparin binding domains of the laminin 3 chain G4 and G5 domains with perlecan, the high molecular weight heparan sulfate basement membrane proteoglycan. Interaction of perlecan with laminin 5 would theoretically occur only transiently, as the α3 chain G4 and G5 domains are proteolytically removed (Marinkovich et al., 1992b) during assembly into the basement membrane in vivo. One of the properties of laminins is the ability to polymerize with the responsible elements residing on the N terminal portions of the molecule (Yurchenco and Cheng, 1993). While it is clear that the truncations which are present on all three short arms of the laminin 5 molecule prevent it from polymerizing, the ability of laminin 6 to polymerize had remained in question. A study examining the polymerization of laminin 6 with laminins 1 and 2 showed that the truncations of laminin 6’s short arm prevents it from self polymerizing or polymerizing with laminins 1 and 2 (Cheng et al., 1997). This study suggested that domains present on laminin al and α2 chains required for self polymerization are lacking on the laminin 6 α3 chain. Therefore, laminin polymerization does not appear to be a mechanism of association of the anchoring complex with the rest of the BMZ. COLLAGEN VII Collagen VII is the major constituent of anchoring fibrils (Sakai et al., 1986). The importance of collagen VII towards the process of dermal-epidermal cohesion is well demonstrated by its absence or by functional defects of this molecule due to underlying gene mutations (Uitto et al., 1994) in the inherited blistering diseases known as dystrophic epidermolysis bullosa. In these diseases, sublamina densa separation of the skin and mucosa occurs due to a lack of intact anchoring fibrils. Like all collagens, collagen VII assembles into a triple helix. Analysis of the deduced amino acid sequence of collagen VII (Parente et al., 1991) reveals the presence of a long central collagenous region characterized by repeating Gly-XY sequences that contain a number of noncollagenous interruptions, including a 39 amino acid noncollagenous segment in the center of the helix which corresponds to the “hinge region” predicted by biochemical studies (Bachinger et al., 1990). These interruptions account for the flexibility of the collagen VII molecule, and explain its ability to loop around and entrap dermal matrix molecules (Keene et al., 1987), to provide its function of stabilizing the BMZ to the underlying papillary dermis. The 145 kD N terminal end of


collagen VII contains the largest noncollagenous domain (Lunstrum et al., 1986). This domain inserts onto the lamina densa and anchoring plaques. Collagen VII triple helices are joined together at their processed NC-2 globular domains to form antiparallel dimers (Morris et al., 1986). The C terminal noncollagenous globular domain, termed NC-2, of the collagen VII molecule is processed prior to anti-parallel dimer formation (Lunstrum et al., 1987). When this step is prevented, through mutations of this region of the collagen VII gene (Bruckner-Tuderman et al., 1995), sub-lamina densa blistering and dystrophic epidermolysis bullosa are the result. Anchoring fibrils probably derive from lateral associations of the antiparallel dimers. Collagen IV in the lamina densa and anchoring plaques specifically binds to collagen VII NC1 domain (Burgeson et al., 1990). As anchoring fibrils extend as perpendicular projections in areas directly underlying anchoring filaments, it was suspected that a direct interaction between anchoring filaments and anchoring fibrils also existed. Recent studies have confirmed a specific interaction between the anchoring filament component laminin 5 and collagen VII NC1 domain. Colocalization of laminin 5 and collagen VII NC1 domain has been demonstrated both in the lamina densa and in anchoring plaques of the dermalepidermal BMZ. These studies provide the first evidence of direct linkage between anchoring filaments and anchoring fibrils. A second component of anchoring fibrils has been recently identified (Gayraud et al., 1997). This component which is recognized by the mAb GDA-J/F3 appears to be a 50 kDa protein which localizes to the junction of anchoring fibrils with the lamina densa. SUMMARY A model of the protein-protein interactions of the dermal-epidermal basement membrane is presented in Figure 5.3. Intermediate filaments insert upon hemidesmosomes via plectin and BPAG1. Plectin and BPAG1 in turn bind to β4 integrin and collagen XVII endodomains respectively. The hemidesmosomal complex is further stabilized by coassociation of the β4 integrin and collagen XVII endodomains. Anchoring filaments arise from the extracellular surface of hemidesmosomes through the exodomains of α6β4 integrin and collagen XVII. Further assembly of anchoring filaments takes place by association of a6β4 integrin and possibly collagen XVII with laminin 5 or laminin 5/laminin 6 complex. The C terminal regions of laminin 5 and 6 face the basal cell while the N-terminal domains are oriented towards the lamina densa. The entire anchoring complex is linked with the lamina densa by the association of laminin 6 with nidogen, which in turn is capable of binding collagen IV and perlecan. Through additional nidogen linkages, laminin 10 also assembles with collagen IV and perlecan. Anchoring filaments are linked to anchoring fibrils by a direct association between laminin 5 and the NC1 domain of collagen VII. In addition, the NC1 domain of collagen VII binds to collagen IV in the lamina densa. finally, due to the flexible nature of collagen VII, anchoring fibrils wind around and entrap interstitial collagen and other molecules, which serves to fasten the lamina densa onto the papillary dermis.


Figure 5.3 Overview of protein-protein interactions in the dermal-epidermal basement membrane.


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6. BIOLOGY AND PATHOLOGY OF HEMIDESMOSOMES LEENA PULKKINEN AND JOUNI UITTO

INTRODUCTION Recent cutaneous biology research has successfully explored the complexity of the cutaneous basement membrane zone (BMZ), which by ultrastructural, biochemical and molecular means has been demonstrated to consist of a large number of adhesion complexes. Several of these attachment complexes can be recognized by electron microscopy (Figure 6.1). On the epidermal side, complex structures, known as hemidesmosomes (HD), extend from the cytoplasmic milieu of basal keratinocytes to the extracellular space (Borradori and Sonnenberg, 1996; Green and Jones, 1996). Within the lamina lucida, thread-like structures, known as anchoring filaments, can be recognized by transmission electron microscopy, and they are concentrated predominantly under the HDs and form the HD-anchoring filament complex (Figure 6.1). On the dermal side, anchoring fibrils, attachment structures with a wheat-stack appearance, extend from lamina densa to the underlying dermis (Burgeson, 1993). It has been suggested that one end of anchoring fibrils binds to lamina densa while the other end interacts with basement membrane-like structures, known as anchoring plaques, within the upper papillary dermis (Keene et al., 1987). Intertwining of the anchoring fibrils between the interstitial collagen fibers, which consist primarily of type I, III and V collagens, provides stable association of the lower portion of the dermal-epidermal basement membrane to the underlying dermis (Figure 6.2). Based on these and similar observations, a concept has been advanced which depicts the dermal-epidermal BMZ as a continuum of adhesion molecules linked to a network structure and, the integrity of this network is necessary for stable association of epidermis to the underlying dermis (Uitto and Christiano, 1992; Christiano and Uitto, 1996a; Uitto et al., 1997; Pulkkinen and Uitto, 1998). Consequently, structural weakness in this network structure can manifest as fragility of skin and other epithelial basement membranes, as depicted by a group of diseases, collectively known as epidermolysis bullosa (EB).

BIOLOGY AND PATHOLOGY OF HEMIDESMOSOMES 107

Figure 6.1 Transmission electron microscopy of the cutaneous basement membrane zone composed of lamina lucida (LL) and lamina densa (LD) separating epidermis (E) from dermis (D). Several distinct attachment complexes, including hemidesmosomes (HD), anchoring filaments (small arrows) and anchoring fibrils (arrow head) can be recognized. Hemidesmosomes consist of intracellular inner (1) and outer plaques (2), and of extracellular electron dense structures known as sub-basal dense plates (3). Original magnification x40,000 (The figure was kindly provided by Dr. John A.McGrath, St. John’s Institute of Dermatology, London, UK).

ULTRASTRUCTURAL AND MOLECULAR FEATURES OF HEMIDESMOSOMES One of the critical attachment structures of the cutaneous BMZ is the hemidesmosome (HD), which extends from the intracellular milieu of basal keratinocytes to the extracellular matrix (Figures 6.1 and 6.2). Ultras true tur ally, HD can be recognized as an electron dense organelle with distinct subcompartments, including an intracellular inner plaque which is recognized in close association with keratin intermediate filaments, and an intracellular outer plaque which is attached to the plasma membrane of the basal keratinocyte on the cytoplasmic side (Borradori and Sonnenberg, 1996). The sub-basal dense plate parallels the plasma membrane at the extracellular side and appears to serve as an attachment site for the anchoring filaments. At least four distinct proteins have been localized to the HDs by immunoelectron microscopic means (Figure 6.2); these include plectin (Wiche et al., 1983; Wiche, 1989), the 230-kD and the 180-kD bullous pemphigoid antigens (Ishiko et al., 1993; Guo et al., 1995), and the α6β4 integrin (Stepp et al., 1990; Sonnenberg et al., 1991). These four proteins and their subunit polypeptides are well characterized, their primary sequences have been deduced through molecular cloning, and their chromosomal locations have been determined (Table 6.1). In addition, several less well characterized proteins have been suggested to be components of the hemidesmosomes at the cutaneous BMZ. These include IFAP300, a ~300-kD polypeptide which has been suggested to be a member of the plakin family of proteins (Ruhrberg and Watt, 1997; Skalli et al., 1994); and p200, a 200-kD molecule recognized by a monoclonal antibody 6A5 and deposited at the epithelialstromal interface (Kurpakus and Jones, 1991).

108 LEENA PULKKINEN AND JOUNI UITTO

Figure 6.2 Schematic representation of the components of the cutaneous basement membrane zone and their molecular interactions. The individual components are identified by color code at the bottom of the figure. (Reproduced with permission from Pulkkinen and Uitto, 1998).

In addition to the hemidesmosomal components referenced above, a number of lamina lucida proteins have been identified, but their exact relationship to HDs is in most cases unclear. One of the best characterized BMZ proteins is laminin 5 which consists of three polypeptide subunits, the α3, β3 and γ2 chains, encoded by distinct genes on chromosomes 18q 11.2, 1q32 and 1q25–31, respectively (Vailly et al., 1994; Ryan et al., 1994). In addition to laminin 5, two other members of the laminin family, laminins 6 and 7, have been identified in the cutaneous BMZ covalently adducted to laminin 5 (Burgeson

*EB-MD, EB with muscular dystrophy; GABEB, generalized atrophie benign EB; EB-PA, EB with pyloric atresia(for details, see Table II) .

Table 6.1 Hemidesmosomal proteins, their molecular features and associated genetic diseases



et al., 1994). Ubiquitously expressed BMZ components, including nidogen and fibulins 1 and 2, as well as various proteoglycans, have also been identified in the cutaneous basement membranes (Martin and Timpl, 1987; Miosge et al., 1996; Woods et al., 1996). Finally, several additional, less well characterized proteins have been mapped to the cutaneous BMZ. These include (a) another 200 kD protein recognized by sera of patients with a novel autoimmune bullous disease (Zillikens et al., 1996) and localized by indirect immunogold electron microscopy to the lower lamina lucida; (b) a 105-kD lower lamina lucida antigen (p105), also recognized by sera from patients with an unusual blistering skin disease and distinct from the γ2 chain of laminin 5 which is of the same size (Chan et al., 1995); (c) uncein, a trimeric BMZ protein which is absent in the skin of patients withjunctional forms of EB (Fine, 1990); and (d) a recently characterized 50-kD component of epithelial basement membranes identified by a monoclonal antibody GDAJ/ F3 and localized by immunoelectron microscopy to lamina densa at the sites of insertion of anchoring fibrils (Gayraud et al., 1997). The primary sequences and molecular features of these four proteins are currently unknown. Finally , a novel, 45-kD BMZ protein, designated as ladinin, has been recently identified (Motoki et al., 1997). THE MOLECULAR COMPONENTS OF HEMIDESMOSOMES The Plectin Family of Proteins Plectin belongs to a family of widely expressed, cytoskeleton associated proteins versatile in their binding activities (Wiche et al., 1981; Wiche, 1989). Plectin was initially isolated as a high-molecular-weight protein which co-purified with type III intermediate filament protein vimentin and was subsequently found to bind a number of intermediate filament proteins, including keratins and nuclear lamins. Immunohistochemical analyses have shown that plectin is widely expressed in a variety of epithelial as well as mesenchymal tissues (Wiche et al., 1983). Within the cells, plectin is found in association with cell membranes and junctional complexes, and it co-localizes with intermediate filaments, as well as stress fibers and focal contacts. Cloning of human plectin cDNA and genomic sequences (Liu et al., 1996; McLean et al., 1996) has revealed that the primary polypeptide, approximately 518 kD in size, has structural homologies with previously characterized plakin family of proteins, which in addition to plectin, includes desmoplakin, the 230-kD bullous pemphigoid antigen, envoplakin, and recently cloned periplakin (Ruhrberg et al., 1996, 1997; Ruhrberg and Watt, 1997; Aho et al., 1998). All members of the plakin family of proteins have an amino-terminal globular domain, central rod-like structure, and a carboxy-terminal tail which contains homologous regions (see Figure 6.3). All these proteins have been identified in association of desmosomes and/or hemidesmosomes within basal keratinocytes, where their function appears to relate to binding of intermediate filaments (Corden and McLean, 1996). Molecular analyses of plectin have also revealed considerable heterogeneity due to alternative splicing or alternatively transcribed 5’ exon of the gene (Elliott et al., 1997). Among the various forms of plectin, of particular interest


Figure 6.3 Demonstrations of the 180-kD bullous pemphigoid antigen (BP180) and β1 integrin interactions, as probed by yeast two-hybrid system. A: The constructs (p376–p434) shown on the left, corresponding to the intracellular domain of BP180, were used as bait in the GAL4 binding domain in yeast two-hybrid system, while the sequence encoding intracellular domain of β4 integrin was inserted with the GAL4 activation domain. Protein-protein interactions, determined by growth in his, -trp, -leu medium, is shown on the right. B: The amino-terminal end of BP180 responsible for α1 integrin binding. The critical region spans amino acids E13-R26 which contains a predicted βsheet. (For details, see the original publication Aho and Uitto, 1998).


is the variant, designated as HD1, which is recognized by the monoclonal antibody HD121 (Hieda et al., 1992). This monoclonal antibody identifies plectin epitopes within the skin only in association with hemidesmosomes, suggesting that HD1 is a plectin variant exclusively expressed in HDs, where it has been localized to the inner plaque (Figure 6.2). Alternatively, this monoclonal antibody recognizes a unique epitope in plectin expressed only in the context of hemidesmosomes. Immunohistochemical staining of skin and muscle with the monoclonal antibody HD121 was instrumental in identifying plectin defects underlying a variant of EB associated with late-onset muscular dystrophy (EB-MD) (for review see Uitto et al., 1996). The 230-kD Bullous Pemphigoid Antigen (BPAG1) BPAG1, another member of the plakin family of proteins associated with hemidesmosomes, was initially identified as an autoantigen in an acquired blistering skin disease, bullous pemphigoid. BPAG1 has been localized, together with plectin, to the inner plaque of HDs (Figure 6.2). Development of transgenic mice with ablated BPAG1 gene has provided insight into the function of this protein (Guo et al, 1995). Specifically, the BPAG1 −/− mice demonstrated that this protein plays a critical role in binding of intermediate keratin filaments to HDs. Most importantly, ultrastructural findings demonstrated that HDs in these animals are otherwise normal, but they lack the inner plaque, and the integrity of hemidesmosome/ cytoskeleton association is disturbed. The keratin intermediate filaments were severed from the HDs and had retracted into perinuclear locations. An interesting observation is that a neural-specific gene product, known as dystonin, is essentially identical with BPAG1, with the exception of the most amino-terminal end (Brown et al., 1995a). Specifically, sequencing of the dystonin gene, in comparison with the BPAG1 gene, revealed that dystonin is a neural isoform of BPAG1, and that the variation at the amino-terminus of the protein is due to the alternate transcription of the 5’ exon. These two proteins, BPAG1 and dystonin, are clearly products of the same gene, as transgenic mice lacking the BPAG1 gene locus also develop characteristic neuromuscular abnormalities (Guo et al., 1995). Furthermore, a naturally occurring mouse mutant, dt/dt, manifesting with neurologic disorders, is due to mutations in the BPAG1 gene (Guo et al., 1995; Brown et al., 1995a,b). No human heritable disease has been shown to be due to mutations in the BPAG1 gene as yet, although the expression of this protein is markedly reduced in several patients with EBMD (see Uitto et al., 1996) which harbor the pathogenetic mutations in the plectin gene, thus attesting to the close interdependence of BPAG1 and plectin gene expression. The 180-kD Bullous Pemphigoid Antigen (BPAG2)—The Type XVII Collagen BPAG2 was initially characterized as an autoantigen in bullous pemphigoid and herpes gestationis, two acquired blistering skin diseases (Morrison et al., 1988). Subsequent molecular cloning of the BPAG2 gene revealed that the protein consists of an aminoterminal globular domain and a carboxy-terminal segment, the latter one with


characteristic collagenous Gly-X-Y repeat sequences (Giudice et al., 1992; Li et al., 1993). In fact, the carboxy-terminal two thirds of the protein were shown to contain 15 collagenous submodules separated by short non-collagenous segments (see Figures 6.2 and Figure 6.7). Molecular cloning and cell biological studies also demonstrated that BPAG2 has a transmembrane domain, and that the amino-terminal domain resides in the cytoplasmic side of basal keratinocytes. Thus, BPAG2 is a transmembrane collagen, designated as type XVII collagen, in type II topography (Li et al., 1993). Recent immunoelectron microscopic data have also supported the type II transmembrane topography of BPAG2. Specifically, use of a monoclonal antibody anti-BV4 IgG, which recognizes the carboxy-terminal end of BPAG2, revealed its association with anchoring filaments in the extracellular milieu (Masunaga et al., 1997). Furthermore, these findings suggested that the carboxyterminus extends all the way across the lamina lucida into the lamina densa, implying that the carboxy-terminal segment of BPAG2 is in fact a component of anchoring filaments which have been thought to consist primarily of laminin 5 (Figure 6.2). The a6β4 Integrin Attachment Complex Integrins, a family of dimeric transmembrane glycoproteins, consist of two subunit polypeptides, the α and β chains, which are noncovalently associated on the cell surface. The α6β4 integrin is characteristically found in a variety of epithelial tissues, including human skin and the gastrointestinal tract, and it plays a major role as an attachment molecule connecting basal keratinocytes to the underlying basement membrane (Stepp et al., 1990; Sonnenberg et al., 1991). The two subunit components, α6 and β4, have been extensively characterized by molecular cloning, and their intron-exon organizations and chromosomal locations have been determined (see Table 6.1, and refs. Hogervorst et al., 1990; Tamura et al., 1990; Pulkkinen et al., 1997a,b). The expression of a6 and β4 integrin polypeptides is variable in that α6 subunit expression is detected in a variety of tissues and also during embryogenesis, while the expression of β4 subunit is more restricted to epithelial tissues (Hogervorst et al., 1990; Tamura et al., 1990; Sonnenberg et al., 1991; Thorsteinsdottir et al., 1995). The β4 integrin differs from other β subunits in that it has an unusually long cytoplasmic domain encoding over 1000 amino acids. This domain contains two pairs of fibronectin type III-like repeats (FNIII) separated from each other by a connecting segment (Hogervorst et al., 1990). Furthermore, the β4 integrin subunit has at least five different variants, primarily due to alternative splicing within the region encoding the cytoplasmic domain, and the expression of such splice variants is also tissue-specific (Hogervorst et al., 1990; Tamura et al., 1990; Van Leusden et al., 1997). Two different splice variants of α6 integrin have been described, both of them being able to form dimers with β1 and β4 integrins (Delwel et al., 1995). HEMIDESMOSOMAL PROTEIN-PROTEIN INTERACTIONS During the past few years, a significant amount of novel information on molecular interactions between the BMZ attachment complexes has emanated from studies utilizing


a variety of cell and molecular biological technologies, including transient cell transfections, yeast two-hybrid systems, development of transgenic “knock-out” mouse models, and examination of mutation databases on human diseases affecting the BMZ. On the basis of the currently available information, clearly defined interactions can be recognized within this network. The inner hemidesmosomal plaque consists of at least two proteins, BPAG1 and plectin (Figure 6.2). BPAG1 “knock-out” mice revealed that this protein is responsible for attachment of intermediate keratin filaments to the HDs, and this functional abnormality is associated with ultrastructurally recognizable lack of the inner plaque of HDs (Guo et al., 1995). In contrast, in “knock-out” mice deficient in plectin the formation of HDs in basal keratinocytes is not noticeably impaired, although they are reduced in number (Andrä et al., 1997). HDs in the latter mice are able to serve, to a certain extent, as intermediate filament anchoring sites, as demonstrated by the presence of ultrastructurally intact appearing filament structures radiating from the cytoplasmic side of HDs. At the same time, a series of cell transfection studies utilizing plectin deletion constructs and mutated cDNAs to disrupt the interactions of this protein with intermediate filament network have identified a cluster of four basic amino acid residues (Arg4277–Arg4280) within the carboxyl-terminal domain of plectin to be essential for intermediate filament binding (Nikolic et al., 1996). The association of plectin with intermediate filaments, and subsequent cross-linking, are mediated by mitosis-specific phosphorylation involving p34cdc2 kinase (Foisner et al., 1996). Thus, the inner plaque proteins, plectin and BPAG1, clearly participate in anchorage of intermediate filaments to the HDs. Collectively, these data suggest that BPAG1 plays a critical role in intermediate filament binding, while plectin, in addition to contributing to such binding, serves as a stabilizer of the assembly of the inner plaque of HDs (Sanchez-Aparicio et al., 1997). The outer plaque of HDs consists of at least three polypeptide components, the cytoplasmic domains of the α6 and β4 integrins and the 180-kD bullous pemphigoid antigen (Figure 6.2). The critical role of β4 integrin subunit in the assembly of HDs is well established in studies which have revealed several interactions of this polypeptide with other hemidesmosomal components, such as plectin (Niessen et al., 1997a,b). Using yeast two-hybrid system, the cytoplasmic domains of BPAG2 and β4 integrin were demonstrated to interact with each other (Aho and Uitto, 1998). Specifically, an intracytoplasmic segment extending from amino acids 13 to 89 of BPAG2, including a predicted β-sheet, and a region of β4 integrin spanning the connecting segment between two pairs of FNIII repeats and the second pair of FNIII, as well as the carboxyl-terminal intracellular tail, are required for this interaction (Figure 6.3). The same connecting segment, together with the second FNIII of the first pair, both within the intracellular domain of β4 integrin, is critical for interactions between this polypeptide and plectin, as well as directing its hemidesmosomal localization (Borradori et al., 1997). At the same time, interactions of the extracellular domain of the α6 integrin with BPAG2 have been suggested to be mediated by discrete sequences within so-called NC16A domain in the latter polypeptide (Hopkinson et al., 1995). The interdependency of the a6 and β4 integrin expression is also attested by the fact that 6 integrin expression is deficient in 4 “knock-out” mice (van der Neut et al., 1996). Similarly, in the skin of patients with a


variant of EB associated with pyloric atresia, EB-PA, and caused in most cases by underlying mutations in the β4 integrin gene, α6 integrin expression is variably reduced or undetectable (Shimizu et al., 1996; Pulkkinen et al., 1998a). The interactions between extracellular components of HDs and the anchoring filament proteins within lamina lucida are less well characterized. For example, the ligand for the extracellular collagenous domain of BPAG2 has not been disclosed as yet. It is clear, however, from several in vitro and in vivo studies that basal keratinocytes bind to laminin 5 via the extracellular domain of the α6β4 integrin, and the α3β1 integrin appears to contribute to this association (Champliaud et al., 1996; Carter et al., 1997). Specifically, the carboxy-terminal globular domain of laminin 5 has been shown to take part in this binding, which is completely blocked by a monoclonal antibody (BM165) recognizing the first G-domain of the α3 chain. This observation suggests that the G-domain is the binding site for α6β4 integrin (Champliaud et al., 1996). Furthermore, the amino terminus of the β3 chain of laminin 5 connects the anchoring filament-HD complex to the NC-1 domain of type VII collagen (Rousselle et al., 1997). GENETIC DISEASES OF HEMIDESMOSOMES The genes encoding the components of hemidesmosomes could potentially serve as candidates for genetic lesions in different variants of EB, and in fact, distinct mutations in four genes expressed in HDs have been disclosed thus far (Table 6.2; Pulkkinen and Uitto, 1998). These include EB variants with mutations in the genes encoding the α6 and β4 integrin subunits (EB-PA), the 180-kD bullous pemphigoid antigen/type XVII collagen (GABEB), or plectin (EB-MD). Clinically, these EB variants present with dermal-epidermal blistering and characteristic extracutaneous manifestations. Plectin Mutations in Patients with EB-MD A clinically puzzling variant of EB is a recessively inherited skin blistering associated with late-onset muscular dystrophy. Blistering of the skin is usually noted at birth, associated with nail dystrophy (Figure 6.4). The level of blistering is intraepidermal within basal keratinocytes at the basal cell/lamina lucida interface. Consequently, this variant of EB has been traditionally classified as EB simplex (Niemi et al., 1988; Fine et al., 1989; Smith et al., 1996; Gache et al., 1996). The skin manifestations are relatively mild, and the blistering tendency usually improves with advancing age. Dramatically, however, these patients develop late-onset muscle weakness, time of onset being highly variable. In some cases, the signs of muscle involvement have been noted as early as two years of age (McLean et al., 1996), while in some families, no muscle involvement has been reported until in the middle of the fourth decade of life (Pulkkinen et al., 1996). In many cases, however, the muscle involvement is progressive and can incapacitate the affected individual. Early clues to the gene/protein systems at fault in EB-MD came from immunofluorescence studies which examined the affected individuals’ skin with an antibody recognizing plectin/HD1 epitopes (Gache et al., 1996; Smith et al., 1996).


Specifically, staining of skin with an antibody HD-121, which recognizes plectin or its variant associated with hemidesmosomes, was negative in the patients’ skin (Figure 6.5). Although staining for BPAG1 was variably attenuated at the dermalepidermal junction of the skin of the same individuals, staining for other BMZ components, including BPAG2 and type VII collagen, was normal (Figure 6.5). Furthermore, immunohistochemical staining of muscle biopsies revealed absent plectin expression, which in normal muscle can be found in association with sarcolemma and Z-lines (Gache et al., 1996; Smith et al., 1996). Thus, the plectin gene (PLEC1) was considered as a candidate gene for mutations in patients with EB-MD. Mutation detection strategies, either based on amplification of genomic DNA sequences followed by heteroduplex scanning with conformation-sensitive gel electrophoresis and nucleotide sequencing (Pulkkinen et al., 1996), or utilizing protein truncation tests (Dang et al., 1998) have been successful in identifying PLEC1 mutations in families with EB-MD. Thus far, 21 distinct PLEC1 mutations in 14 different families have been disclosed (McLean et al., 1996; Chavanas et al., 1996; Smith et al., 1996; Pulkkinen et al., 1996; Mellerio et al., 1997; Dang et al., 1998; Rouan et al., 2000). The majority of these mutations consist of insertions or deletions which result in premature termination codons for translation (PTC) in this gene (Figure 6.7). The only exception is a homozygous 9-bp deletion mutation, 2719de19 (see Figure 6.6), which results in inframe deletion of three amino acids, QAE. This particular mutation was considered to be pathogenetic for the following reasons. First, this 9-bp deletion was not found in 95 unaffected control individuals, indicating that it was not a polymorphism in ethnically matched (Japanese) population. Secondly, the precise amino acid sequence was conserved between human, rat and mouse sequences, suggesting the functional importance of the QAE tripeptide (Pulkkinen et al., 1996). Most of the PTC-coding mutations discovered so far reside in exon 32 which encodes the rod domain of plectin. The 9-bp deletion mutation resides in exon 22 which encodes a portion of the amino-terminal globular domain. Collectively, the patients with EB-MD have mutations in the plectin gene, and expression of this gene both in the skin at the dermal/epidermal junction and in the muscle within the sarcolemmal adhesion zone, would explain manifestations in these two organ system (Ditto et al., 1996). Clinical Features and Molecular Basis of Generalized Atrophie Benign Epidermolysis Bullosa (GABEB) GABEB was initially described as a non-lethal variant of junctional EB (Hashimoto et al., 1976; Hintner and Wolff, 1982). Clinically, the affected individuals demonstrate protracted, life-long generalized blistering which results in cutaneous atrophy, associated with diffuse scarring alopecia, characteristic pigmentary changes, as well as tooth and nail abnormalities. It was subsequently shown that expression of the 180-kD bullous pemphigoid antigen in the skin of these patients is reduced or absent, suggesting that the corresponding gene, BPAG2/COL17A1, is the candidate gene for mutations (Jonkman et al., 1995; Pohla-Gubo et al., 1995). Following cloning of BPAG2 genomic sequences and

Table 6.2 Hemidesmosomal variants of epidermolysis bullosa: diagnostic features and genetic basis


b)For

BMZ, basement membrane zone; PTC, premature termination codon mutations. the specific mutations and their positions along the affected molecules, see Fig. 7.

a)Abbreviations:



Figure 6.4 Clinical features of a patient with EB-MD. Note superficial blistering and erosions, nail dystrophy, and atrophy of intercostal and biceps muscles. (Published with permission from Pulkkinen et al., 1996).

elucidation of its intron-exon organization (Gatalica et al., 1997), mutation detection strategies were developed which were able to pinpoint specifie genetic lesions in this gene in GABEB patients (Table 6.2). Again, the majority of the mutations cause PTCs either as a result of nonsense mutations or small insertions or deletions in the BPAG2 gene (Figure 6.7). In all these cases, immunofluorescence staining of the proband’s skin is negative for the 180-kD bullous pemphigoid antigen expression. The only case with missense mutations in both alleles (R1303Q/R1303Q), affecting the extracellular noncollagenous segment NC4 of type XVII collagen, had a localized variant of EB with predominantly acral blistering, however this patient had normal hair (Schumann et al., 1997). Although the mechanistic consequences of this amino acid substitution are not entirely clear, immunofluorescence of the proband’s skin was normal for type XVII collagen, and his keratinocytes in culture synthesized full-length al (XVII) polypeptides. Thus, the arginine substitution by glutamine at the amino acid position 1303 must alter a critical function of type XVII collagen but allows its assembly into HDs, resulting in a relatively mild phenotype. A particularly interesting family with the combination of a PTC and a missense mutation has been described by McGrath et al., (1996). The proband, a 50-year old female, had extensive cutaneous blistering, as well as dental abnormalities characterized by enamel pitting. She was shown to be a compound heterozygote for 3514ins25/G627V


Figure 6.5 Immunofluorescence of the skin in a control individual (panel A) and in a patient with EB-MD (panels B-D). Note essentially negative staining for plectin (HD1) in the patient’s skin (panel B) in comparison to the control skin (panel A). Staining of the patient’s skin with antibodies recognizing the 180-kD bullous pemphigoid antigen (panel C) or type VII collagen (panel D) epitopes was entirely normal. (Modified from Pulkkinen etal., 1996).

mutations in BPAG2/COL17A1. The patient also had two offspring, both of whom had inherited the glycine substitution mutation G627V, whereas the paternal allele was normal. In the latter individuals, there was no evidence of skin fragility, but they had dental abnormalities with enamel hypoplasia and pitting, similar to those observed in the mother. Thus, the proband in this family had a recessively inherited skin blistering, characteristic of GABEB, while the dental abnormalities of her offspring appear to have resulted from the glycine substitution in type XVII collagen alone, resulting in a dominantly inherited clinical phenotype. Another fascinating case with mutations in the BPAG2 gene was recently reported by Jonkman et al., (1997). The proband, a 28-year old female, demonstrated characteristic features of GABEB, including generalized blistering after minor trauma and resulting in cutaneous atrophy, mild mucous membrane involvement, universal alopecia, pigmentary changes, dental anomalies, and nail dystrophy. Careful physical examination disclosed patches of clinically unaffected skin in a symmetrical leaf-like pattern over the extensor surface of hands and upper arms, and these patches covered about 10% of the total body surface area. Immunofluorescence staining using an anti-BPAG2/type XVII collagen antibody was negative in clinically affected skin, while in clinically unaffected patches the


Figure 6.6 Illustration of the mutation detection strategy in a family with EB-MD. A: Genomic sequences of the plectin gene (PLEC1) were PCR amplified from two patients with EB-MD (III-1 and III-3), their clinically unaffected mother and two younger sisters. CSGE revealed two bands with mother’s PCR product (arrows) while the affected sisters showed the lower band and the unaffected sisters showed the upper band only. B: Direct nucleotide sequencing of the PCR products showed that the affected individuals were homozygous for a 9-bp deletion (2719de19) in the plectin gene (upper panel), while the mother was heterozygous carrier (middle panel), in comparison to normal sequence (lower panel). C: The mutation abolished a restriction enzyme site for BglI, which was used to verify the mutation. (Published with permission from Pulkkinen et al., 1996).

BPAG2 expression was present in approximately 50% of the basal cells, the positive cells being in groups of about 10–50 adjacent cells (Jonkman et al., 1997). Staining for other hemidesmosomal components, such as β4 integrin showed normal continuous pattern at the dermal-epidermal junction. The proband’s skin in the affected area depicted compound heterozygous mutations (1706delA/R1226X) in the BPAG2 gene, while the clinically normal areas showed the presence of the maternal mutation (R1226X) only, accompanied by loss of heterozygosity along a tract of at least 381 bp in keratinocytes derived from clinically unaffected skin. This case represents revertant mosaicism of the compound heterozygous proband with the autosomal recessive genodermatosis, GABEB (Jonkman et al., 1997). The a6β4 Integrin Mutations in Epidermolysis Bullosa with Pyloric Atresia (EB-PA) EB-PA is a distinct variant of epidermolysis bullosa associated with congenital intestinal abnormalities, such as pyloric or duodenal atresia. Histology of the skin shows dermal-

Figure 6.7 Position of the mutations affecting 180-KD bullous pemphigoid antigen/type XVII collagen (upper panel), β4 integrin (middle panel) plectin (lower panel).mutation above the schematic protein molecules are premature termination codon-causing mutations (PTC) while those below are missense or in-frame deletion mutations The domain organaization of the polypeptides are color coded, as shown on the right (for original references,see text and pulkkinen and uitto, 1998).



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


cases, the clinical phenotype is indistinguishable from those caused by mutations in ITGB4, with the exception that the patient described by Pulkkinen et al., (1997b) had also cleft lip and cleft palate. CLINICAL IMPLICATIONS OF BASIC RESEARCH ON HERITABLE SKIN DISEASES The progress made during the past decade in understanding the molecular basis of various heritable skin diseases, as exemplified by EB, has been tremendous. For example in the case of EB, distinct mutations in 10 different BMZ genes have been identified, and the total number of allelic variants containing pathogenetic mutations in such genes is now in excess of 400. Examination of the mutation database has resulted in better understanding of how different variants with differential clinical severity reflect the underlying mutations, although finer predictions of the genotype/phenotype correlations need to be developed on the basis of extended database. From the practical point of view, this progress raises the critical question: What are the benefits of the progress in basic research on heritable blistering skin diseases to the patients and their families? Immediate benefits can be seen through improved, molecularly based diagnosis with refined classification which allows improved prognostication regarding the severity and the progress of the disease (Christiano and Uitto, 1996b). Furthermore, knowledge of the underlying mutations in different forms of EB has also provided the basis for development of DNA-based prenatal testing in families at risk for severe forms of EB. Such testing can be performed from chorionic villus samples as early as the 10th week of gestation or from amniocentesis specimens at 12th week. In fact, such prenatal testing has already been performed in a relatively large number of families at risk for extremely severe and life-threatening variants of EB, including several cases with EB-PA (Pulkkinen et al., 1998a). A logic extension of prenatal testing is the development of pre-implantation genetic diagnosis (PGD), a technique that has been successfully applied to a variety of genetic diseases, such as X-linked muscular dystrophy, cystic fibrosis and others (see McGrath and Handyside, 1998). Thus, couples with a previous child affected with EB can now initiate the next pregnancy by knowing that there are ways to find out the state of the fetus in the early stages of pregnancy, or even before the pregnancy is initiated. Finally, one of the future prospects for the treatment of EB relates to development of successful gene therapy approaches. This could involve ex vivo manipulation of cultured cells in the manner that the mutation is corrected, with subsequent grafting of the cells to the eroded areas of skin. Alternatively, direct application of DNA into the skin could be used in attempts to elicit genetic reversal of the underlying mutation (Khavari and Krulger, 1997). Although successful application of gene therapy for treatment of EB may still be several years away, rapid development of new technologies, such as the use of ribozymes or utilization of chimeric RNA/DNA nucleotides for correction of the mutation by homologous recombination, hold promise for breakthroughs in the near future.


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Sonnenberg, A., Calafat, J., Janssen, H., Daams, H., van der Raaij-Helmer, L.M., Falcioni, R., Kennel, S.J., Aplin, J.D., Baker, J., Loizidou, M., and Garrod, D. (1991) Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol, 113:907–917. Stepp, MA, Spurr-Michaud, S., Tisdale, A., Elwell, J., and Gipson. I.K. (1990) 64 integrin heterodimer is a component of hemidesmosomes. Proc Natl Acad Sci USA, 87:8970–8974. Tamura, R.N., Rozzo, C., Starr, L., Chambers, J., Reichardt, L.F., Cooper, H.M., and Quaranta, V. (1990) Epithelial integrin 64: complete primary structure of 6 and variant forms of 4. J Cell Biol, 111:1593–1604. Thorsteinsdottir, S., Roelen, B.A.J., Freund, E., Gaspar, A.C., Sonnenberg, A., and Mummery, C.L. (1995) Expression patterns of laminin receptor splice variants 61 and 6B1 suggest different roles in mouse development. Develop Dyn, 204:240–258. Uitto, J. and Christiano, A.M. (1992) Molecular genetics of the cutaneous basement membrane zone. Perspectives on epidermolysis bullosa and other blistering skin diseases. J Clin Invest, 90: 687–692. Uitto, J., Pulkkinen, L., Smith, F.J.D., and McLean, W.H.I. (1996) Plectin and human genetic disorders of the skin and muscle. The paradigm of epidermolysis bullosa with muscular dystrophy. Exp Dermatol, 5:237–246. Uitto, J., Pulkkinen, L., and McLean, W.H.I. (1997) Epidermolysis bullosa: a spectrum of clinical phenotypes explained by molecular heterogeneity. Molec Med Today, 3:457–465. Vailly, J., Szepetowski, P., and Mattei, M.G. (1994) The genes for nicein/kalinin 125- and 100kDa subunits, candidates for junctional epidermolysis bullosa, map to chromosomes Iq32 and Iq25-q31. Genomics, 21:286–288. van der Neut, R., Krimpenfort, P., Calafat, J., Niessen, C.M., and Sonnenberg, A. (1996) Epithelial detachment due to absence of hemidesmosomes in integrin 4 null mice. Nat Genet, 13:366–369. Van Leusden, M.R., Kuikman, I., and Sonnenberg, A.(1997) The unique cytoplasmic domain of the human integrin variant 4E is produced by partial retention of intronic sequences. Biochem Biophys Res Commun, 235:826–830. Wiche, G., Herrmann, H., Leichtfried, F., and Pytela, T. (1981) Plectin—a high-molecularweight cytoskeletal polypeptide component that copurifies with intermediate filaments of the vimentin type. Cold Spring Harbor Symposia on Quantitative Biology, 46:475–482. Wiche, G., Krepler, R., Artlieb, U., Pytela, R., and Denk, H. (1983) Occurrence and immunolocalization of plectin in tissues. J Cell Biol, 97:887–901. Wiche, G. (1989) Plectin: general overview and appraisal of its potential role as a subunit of the cytomatrix. CRC Grit Rev Biochem, 24:41–67. Woods, A. and Couchman, J.R. (1996) Signaling from the matrix to the cytoskeleton: role of cell surface proteoglycans in matrix assembly. J Invest Dermatol, 106:531–537. Zillikens, D., Kawahara, Y., Ishiko, A., Shimizu, H., Mayer, J., Rank, C.V., Liu, Z., Giudice, G.J., Tran, H.H., Marinkovich, M.P., Bröcker, E-B., and Hashimoto, T. (1996) A novel subepidermal blistering disease with autoantibodies to a 200-kDa antigen of the basement membrane zone. J Invest Dermatol, 106:1333–1338.

7. DERMAL-EPIDERMAL ADHESION LEENA BRUCKNER-TUDERMAN

INTRODUCTION The integrity of the skin and the strong resistance against external shearing forces are provided by the epidermal basement membrane, a highly specialized structure which separates and concomitantly attaches the epidermis and the dermis to each other. Basement membranes are sheet-like, complex molecular networks that connect external or internal epithelia with stromal mesenchyme but unique, highly specialised structures within the basement membranes contribute to tissue-specific functions, such as the tight dermal-epidermal adhesion in the skin. The epidermal basement membrane which is known as the dermal-epidermal junction zone (DEJZ) contains, beside the ubiqutous basement membrane molecules, several unique components which form an auxiliary structure, the anchoring complex. This connects the epidermal cells to the basement membrane and the basement membrane to the dermal connective tissue through physical and chemical protein-protein interactions. Both epithelial and mesenchymal cells contribute to the production of the DEJZ macromolecules; the synthesis is regulated by paracrine signals from other cells and from the extracellular matrix. For studies on the structure and functions of the DEJZ, genetic and acquired diseases leading to diminished dermal-epidermal adhesion are useful models. Examples of such disorders are heritable epidermolysis bullosa (EB) and acquired bullous skin diseases with autoantibodies targeting the DEJZ. A wealth of information on molecular mechanisms of dermal-epidermal adhesion has been gained by studying mutations in the genes encoding DEJZ proteins and their biological consequences, as well as by characterizing the autoantigens in acquired blistering diseases. Basement membranes similar to the DEJZ occur in the mucous membranes of the orifices, the eye, the gastrointestinal tract, and the placental membranes, all tissues exposed to mechanical shearing forces. Therefore, molecular studies on the DEJZ also provide novel information on the structure and functions of basement membranes in other tissues and on epithelial-mesenchymal interactions in general.

132 LEENA BRUCKNER-TUDERMAN

THE DERMAL-EPIDERMAL JUNCTION ZONE (DEJZ) Morphology and Suprastructure of the DEJZ The basement membrane at the DEJZ is unique in that it contains auxiliary structures, anchoring complexes, which serve to strengthen the adhesion of the epithelial cells to the extracellular matrix (Figure 7.1). Ultrastructural examination reveals the basement membrane as a bilayer consisting of the electron lucid lamina lucida and the electron dense lamina densa (Gedde-Dahl and Anton-Lamprecht, 1996, Keene et al., 1997). The anchoring complexes consist of hemidesmosomes, anchoring filaments and the anchoring fibrils and seem evenly distributed along the DEJZ (see Burgeson and Christiano, 1997). Hemidesmosomes at the ventral plasma membrane of basal keratinocytes appear as small electron dense complexes into which the cytoskeletal intermediate filaments insert (Borradori and Sonnenberg, 1996). Outside the cell, thin threadlike anchoring filaments traverse the lamina densa from the hemidesmosome into the lamina densa. Below, the crossbanded anchoring fibrils extend from the lamina densa to the papillary connective tissue where they either

Figure 7.1. A schematic representation of the anchoring complex. In the inner plaque of the hemidesmosome, BP230 and plectin connect with the keratin intermediate filaments. Transmembrane components of the hemidesmosome, collagen XVII and integrin α6β4, interact with anchoring filament proteins in the lamina lucida, and laminin 5 binds to collagen VII in the anchoring fibrils. The GDA-antigen is localized at the insertion points of the anchoring fibrils in the lamina densa. Collagen IV is a component of the lamina densa and of the anchoring plaque.

insert in so called anchoring plaques or loop back to the lamina densa (Burgeson, 1993). These distinct ultrastructural units result from highly specific ligand aggregation of the structural macromolecules of the DEJZ. The individual molecules usually are large

DERMAL-EPIDERMAL ADHESION 133

Table 7.1 Molecular components of the DEJZ

K: keratinocytes; F: fibroblasts

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


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


Transmembrane proteins of basal keratinocytes Collagen XVII is also known as the 180 kD bullous pemphigoid antigen or BP180. It is a hemidesmosomal transmembrane protein (Nishizawa et al., 1993), with an intracellular domain in the hemidesmosomal plaque and and a rod-like flexible ectodomain (Giudice et al., 1992, Gatalica et al., 1997, Hirako et al., 1996, 1998, Schäcke et al., 1998). The flexibility results from collagenous, triple-helical -Gly-X-Y- sequences interrupted with non-collagenous sequences (Giudice et al., 1992 Hirako et al., 1996, 1998, Schäcke et al., 1998). Being a transmembrane component, collagen XVII presumably plays a role in maintaining the linkage and integrity between the intracellular and the extracellular components of the anchoring complex and in mediating the epithelial cell adhesion to the the basement membrane (Borradori et al., 1997). An intriguiging, recently discovered regulatory feature is the shedding of the ectodomain of from the keratinocyte cell surface by furin-mediated proteolytic processing (Schäcke et al., 1998). The function of the processing is not fully understood yet, but it may serve as a mechanism to regulate cell adhesion and differentiation. As intracellular ligands of collagen XVII, the intracellular domains of both a6 and β4 integrin polypeptide chains have been implicated (Hopkinson et al., 1995, Borradori et al., 1997, Aho et al., 1998). Integrins are dimers of α and β transmembrane subunits involved both in organizing the cytoskeleton and in binding to the extracellular matrix. The epithelial specific integrin α6β4 in the hemidesmosome serves as a ligand for plectin and collagen XVII (Niessen et al., 1997, Reznieczek et al., 1998, Hopkinson et al., 1995, Schaapveld et al., 1998, Aho et al., 1998), and in the extracellular space as a laminin 5 receptor, thereby contributing to the stability of the anchoring complex. Since α6 and β4 integrin chains form a dimeric receptor, one might expect that defects of both integrin subunits will lead to diminished dermal-epidermal adhesion. Consistent with this expectation, transgenic mice with targeted elimination of either the α6 or the β4 integrin gene showed absence of hemidesmosomes and dramatic skin blistering (Georges-Labouesse et al., 1996, Van der Neut et al., 1996). The distribution of the integrin α3β1 is wider than that of α6β4, indicating broader ligand specificity. In the epidermis this integrin is mainly localized at the ventral keratinocyte plasma membrane between the anchoring complexes, where it supposedly interacts with laminin 5-laminin 6 complexes (Champliaud et al., 1996, Burgeson and Christiano, 1997). In vitro, α3β1 localizes to the focal contacts suggesting interactions with the actin cytoskeleton. Transgenic mice with ablation of the α3 integrin gene showed disorganization of the basement membrane and mild skin blistering (DiPersio et al., 1997). However, the frictional forces required to induce dermal-epidermal separation were considerably stronger than those involved in blister formation in α6 or β4 integrin deficient animals, implying that α3 integrin contributes to dermal-epidermal adhesion, but in a less crucial manner than α6β4 integrin in the anchoring complex. Diverse studies on determination of the extracellular ligands of α3β1 have not yielded unambigous results (see Aumailley and Smyth, 1998). Collagen XIII is a transmembrane protein with a wide tissue distribution (see Prockop and Kivirikko, 1995). Remarkably, a strong epidermal expression, also on the ventral


keratinocyte plasma membrane facing the lamina lucida was observed. Similarly to integrin α3β1, collagen XIII was found in focal adhesion plaques in cultured keratinocytes. These plaques represent structures involved in the adhesion of cells to the extracellular matrix. The α1 (XIII) collagen mRNA is characterized by complex alternative splicing of both collagenous and noncollagenous sequences. Analysis of total of 17 splice combinations of nine exons suggest that the predicted length of the corresponding polypeptides varies between 651 and 710 arnino acid residues (Peltonen et al., 1997). Syndecans are a family of transmembrane heparan sulphate proteoglycans. They bind and modify the action of various growth factors/cytokines, proteases/ antiproteases, cell adhesion molecules, and extracellular matrix components and thereby regulate cellular growth, adhesion and movement. Syndecan 1 is located over the entire surface of keratinocytes, whereas syndecan 4 mainly is found at the basal surface contacting matrix (Gallo et al., 1996). Syndecans bind collagens and fibronectin and thus act as receptors for the extracellular matrix. Similarly to collagen XVII, the syndecan ectodomain is shed constitutively by cultured cells. Proteases and growth factors active during wound repair can accelerate syndecan shedding from cell surfaces, suggesting physiological roles for the soluble ectodomains (Fitzgerald et al., 2000). The muscle and neural protein dystroglycan is a ubiquitous transmembrane receptor for extracellular ligands, such as laminins and agrin, and it provides a linkage of the basement membrane to the actin cytoskeleton (Henry and Campbell, 1996). Interestingly, dystroglycan is also expressed by epithelial cells including keratinocytes. It is encoded by a single gene and cleaved into two proteins, α- and β-dystroglycan, by posttranslational processing. The functions of dystroglycan in the skin still remain elusive, but some information was derived from targeted elimination of the dystroglycan gene in mice. Homozygous embryos exhibited gross developmental abnormalities including an early disruption of Reichert’s membrane, an extra-embryonic basement membrane. The localization of two critical structural elements of Reichert’s membrane, laminin and collagen IV, were specifically disrupted, suggesting that dystroglycan may be required for basement membrane organization (Williamson et al., 1997). Anchoring filament and lamina lucida proteins Laminin 5 is a major consituent in anchoring filaments which extend from the hemidesmosomes to the lamina densa. Laminin 5, the smallest member of the laminin family, is a unique heterotrimer containing α3, β3 and γ2 subunit polypeptides, all of which are proteolytically processed to truncated chains during biosynthesis and supramolecular assembly (Marinkovich et al., 1992). Laminin 5 is the link between anchoring filaments and anchoring fibrils. It is the major ligand of α6β4 integrin, presumably binding to the extracellular domain of the integrin in the anchoring filaments. It also binds strongly to the Nterminal globular domain of collagen VII (Chen et al., 1997, Rousselle et al., 1997). Laminin 5 is essential for the dermal-epidermal adhesion, as demonstrated by junctional epidermolysis bullosa Herlitz (see Pulkkinen and Uitto,


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


and matrix attachment. At the DEJZ, this basic structure is strengthened through interactions with the proteins in the anchoring complexes. Collagen IV occurs as tissue specific isoforms, depending on the chain composition of the heterotrimeric collagen molecules (see Beck and Gruber, 1995, Timpl, 1996). The DEJZ contains three different α(IV) chains, each of about 100 kD. The ubiquitous α1(IV) and α2 (IV) chains form heterotrimeric α1(IV)2 α2(IV) 1 molecules that build the major basement membrane network which probably exerts fundamental supportive functions. The importance of these chains is alluded to by the fact that no mutations in the COL4A1 and COL4A2 genes have been found, possibly because their defects are lethal during embryonic development. The molecular structure of the third collagen IV polypeptide at the DEJZ, the a5(IV) chain, is unknown at present. This chain does not seem to be crucial for dermal-epidermal adhesion since patients with Alport syndrome, a hereditary kidney disease, who lack the α5(IV) chain do not exhibit skin blistering (Antignac, 1995). Collagen IV is a major adhesion molecule, it interacts at least with nidogen, BM-40/ SPARC and β1 integrin (see Timpl, 1996), thus mediating attachment of basal keratinocytes to the basement membrane. Nidogen is a ubiquitous 150 kD basement membrane glycoprotein which consists of three globular domains and a central rod. It is a strongly interactive molecule with multiple ligands, e.g. collagen IV, perlecan and fibulins, and it is believed to function as a stabilizer of the basic protein scaffold of the basement membranes (Timpl, 1996). In addition, the C-terminus of nidogen binds strongly to laminin γ1-chain (Mayer et al., 1995), the interaction being calcium dependent. Therefore, at least laminins 2, 6 and 10 can act as nidogen ligands at the DEJZ. Perlecan is the main proteoglycan of basement membranes and pericellular spaces (lozzo et al., 1994). It has a large nearly 500 kD core protein with heparan sulphate attached to the aminoterminal domain. In the skin, perlecan is manufactured by papillary fibroblasts, but not by keratinocytes. It contributes to the stability of the basic protein scaffold of the basement membrane by binding to nidogen and dystroglycan (Talts et al., 1999). Besides, perlecan’s ability to capture growth factors and cytokines points to an important regulatory function for basement membrane synthesis and turnover. Anchoring fibril proteins Anchoring fibrils represent polymers of collagen VII, a large homotrimeric protein with a central triple helix, and flanking amino- and carboxyterminal globular domains. Collagen VII consists of three identical α1(VII) chains of about 300 kD each (Burgeson, 1993, Christiano et al., 1994). Keratinocytes synthesize and secrete collagen VII as a triplehelical precursor—procollagen VII—into the extracellular matrix, where proteolytic removal of the C-terminal propeptide is necessary for correct supramolecular assembly (Morris et al., 1986, Bruckner-Tuderman et al., 1995). During fibrillogenesis, collagen VII molecules form antiparallel tail-to-tail dimers with a central carboxy-terminal overlap and with the ammo-termini pointing outwards (Burgeson, 1993). The dimers then aggregate laterally in a nonstaggered manner into the anchoring fibrils which are further stabilised by transglutaminase-2 cross-links (Raghunath et al., 1996). Anchoring fibrils are attached to


the basement membrane through binding to laminin 5 and collagen IV (Chen et al., 1997, Rousselle et al., 1997). A 50 kDa non-collagenous component of the DEJZ, coined GDA-J/F3 antigen:, was identified as an epitope of a monoclonal antibody. The GDA-J/F3 antigen is a small disulphide-bonded protein with a potential to interact with basement membrane proteoglycans, however, its primary structure remains unknown at present. With immunoelectron microscopy, it was localized to the insertion points of the anchoring fibrils into the lamina densa. In vitro, GDA-J/F3 is synthesized and secreted by keratinocytes, and to a lesser extent by normal human skin fibroblasts. Integration of the GDA-J/F3 antigen into the histoarchitecture of the DEJZ is dependent on the presence of collagen VII, since the GDA-J/F3 epitope was missing in several DEB patients with absent or mutated collagen VII (Gayraud et al., 1997). Biosynthesis, Processing and Regulation of DEJZ Components Both keratinocytes and fibroblasts contribute to the synthesis of the DEJZ. Ample evidence points to active epithelial-mesenchymal crosstalk by the regulation of DEJZ formation, the biosynthesis of molecules in one cell type is modulated byjuxtacrine or paracrine signals from neighbouring cells or from the extracellular matrix. In vitro, depending on the experimental conditions, the one or the other cell type plays a more active role in the production of certain molecules (Marinkovich, 1992, König and Bruckner-Tuderman, 1994, Fleischmajer et al., 1995). Epithelial cells manufacture collagen VII and most laminin chains, whereas mesenchymal cells synthesize collagen IV, nidogen, perlecan and the laminin α2 chain (Salmivirta et al, 1997). In skin equivalent cultures (Fleischmajer et al., 1995) and in developing mouse organs (Ekblom et al., 1994) fibroblasts are the only source of nidogen at the epithelial-mesenchymal interface. An interesting, additional regulatory step of the supramolecular assembly of DEJZ is provided by post-translational proteolytic processing of some components. The trimming is thought to contribute to correct association and folding of the polypeptide subunits, since propeptides can recognize binding sites for accurate alignment of the subunits or prevent premature aggregation of the molecules. Two important components of the anchoring complex are subject to such processing, laminin 5 and collagen VII. The 200 kD α3 chain of laminin 5 is processed to a 145 kD mature chain by cleavage in the C-terminal G-domain, and the 155 kD γ2 chain is cleaved within the N-terminal short arm to yield a 105 kD polypeptide (Marinkovich et al., 1992). The carboxyterminal NC-2 domain of procollagen VII is removed prior to stabilization of the anchoring fibrils (BrucknerTuderman et al., 1995). BMP-1 (bone morphogenetic protein 1), also known as procollagen C-proteinase (Kessler et al., 1996), processes the γ2 chain of laminin 5 (Amano et al., 1997), and possibly procollagen VII, in the extracellular space. Interestingly, BMP-1 is synthesized by fibroblasts, whereas both laminin 5 and collagen VII are epithelial products suggesting that epithelial-mesenchymal interactions play an important regulatory role at this step. The significance of the proteolytic processing for assembly of the DEJZ was demonstrated in BMP-1 knock-out mice which showed a


disorganized basement membrane structure (Suzuki et al., 1996). This is further corroborated by the finding that a deletion of the BMP-1 consensus sequence in human procollagen VII was associated with dystrophic epidermolysis bullosa (Winberg et al., 1997). Yet another form of regulation may be proteolytic cleavage of the extracellular domains of integral type 1 or type 2 transmembrane proteins. The ectodomains of collagen XVII and syndecans are shed from the keratinocyte surface by selective posttranslational proteolysis (Subramanian et al., 1997, Hirako et al., 1998, Schäcke et al., 1998). The process is catalyzed by a group of enzymes collectively referred to as secretases or sheddases which have been only partially characterized, but many can be grouped as metallo- and/or serine proteinases (Hooper et al., 1997). At least in some instances, e.g. by processing of the collagen XVII ectodomain, proprotein convertases of the furin/ PACE-family are involved. Furin mediated proteolysis is also required to yield a heavy and a light chain of the α6 integrin polypeptide, a necessary step for activation of the integrin (Delwel et al., 1997). The proteolytic cleavage of the ectodomains of keratinocyte surface proteins may provide means to regulate cell adhesion, differentiation and migration at the DEJZ, however, the details of these events remain unclear. The biology of the DEJZ is evidently also controlled by growth factors and cytokines. Apart from proteoglycans in the dermis, perlecan and syndecans can trap various factors, act as a reservoir for regulatory signals and release them during physiological and pathological processes, such as development, repair or invasion. Factors relevant for modulation of synthesis and maintenance of the DEJZ include TGF-β, CTGF, KGF, TNFα and interferon-γ (Eckes et al., 1997). Most potent stimulators of laminin 5, collagen VII, collagen XVII, and perlecan expression are TGF-β1 and -β2 isoforms, both of which bind to microfibrils and other extracellular matrix structures in the skin (Raghunath et al., 1998). DISORDERS OF DERMAL-EPIDERMAL ADHESION Many DEJZ macromolecules are targeted in both hereditary and acquired blistering skin diseases. In acquired autoimmune blistering disorders, tissue bound autoantibodies are believed to perturb ligand interactions of the target proteins thereby contributing to blister formation (for review, see Yancey, 1995, Pleyer et al., 1996). In hereditary epidermolysis bullosa, mutated DEJZ components form instable anchoring complexes resulting in detachment of the epidermis from the dermis after minor friction or trauma (Gedde-Dahl and Anton-Lamprecht, 1996, Bruckner-Tuderman, 1993, Christiano and Uitto, 1996). Acquired Disorders of Dermal-Epidermal Adhesion: Autoantibodies to DEJZ Molecules Acquired disorders of dermal-epidermal adhesion are usually autoimmune diseases with circulating and tissue-bound autoantibodies targeting DEJZ molecules. Molecular cloning, protein isolation and generation of recombinant protein fragments have facilitated


Table 7.2 Autoimmunity to DEJZ components

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


or inflammatory cells in degradation of DEJZ components in autoimmune blistering skin diseases (Liu et al., 1998). Somewhat puzzling is the role of autoantibodies to BP230 and plectin (Stanley et al., 1988, Fujiwara et al., 1996), both intracellular proteins, in the pathogenesis of skin blistering. Possibly, the antibody formation is secondary to blistering and exposure of the intracellular epitopes and represents an epiphenomenon. Heritable Disorders of Dermal-Epidermal Adhesion: Epidermolysis Bullosa Mutations in the genes for anchoring complex components impair the dermal-epidermal adhesion and cause increased sensitivity to friction and skin blistering as a consequence of minor trauma. The name epidermolysis bullosa (EB) which refers to the mechanically induced detachment of the epidermis from the dermis, into the blister roof, was coined for these conditions almost 80 years ago (Siemens, 1921). Despite modern knowledge about the genetic and biological heterogeneity of the disease group, the name has stayed. Based on ultrastructural criteria, three main categories of EB have been defined according to the precise level of tissue separation: simplex, junctional and dystrophic subtypes. In EB simplex (EBS) the separation occurs within the basal keratinocytes, as a consequence of cytolysis of the cells; in junctional EB (JEB) the cleavage occurs along the lamina lucida; and in dystrophic EB (DEB), below the basement membrane, within the uppermost dermis (Figure 7.2). Predictably, abnormality of any DEJZ component can lead to impaired interactions, to diminished adhesion of the skin layers and to blistering. The multitude of pathologic alterations is alluded to by the extensive clinical heterogeneity of EB, more than 20 genetic and clinical subtypes are known (Table 7.3; Fine et al., 1991). In Table 7.4, the molecular defects in different EB subtypes are summarized. EBS: a disorder affecting keratin intermediate filaments The first indications for involvement of keratins in the etiopathogenesis of EBS came from ultrastructural observations on clumping of the keratin filament bundles in basal keratinocytes in EBS skin (Fuchs and Yang, 1999). The basal cell keratins 5 and 14, the former a type II and the latter a type I keratin, form as a pair a heterodimer and copolymerize into supramolecular intermediate filaments (Lane, 1993). Consistently, transgenic mice with truncated keratin 14 exhibited a phenotype with tonofilament clumping, basal cell cytolysis and skin blistering (Vassar et al., 1991), and subsequently a large number of mutations in the genes for keratin 5 and 14 have been identified in EBS families. Different mutations in the two genes underlie a variety of clinical EBS phenotypes, i.e. the subtypes Köbner (Bonifas et al., 1991)) Dowling-Meara (Coulomb et al., 1991, Lane et al., 1992), Weber-Cockayne (Chan et al., 1993) and EBS with mottled pigmentation (Uttam et al., 1996). Filament assembly assays with mutated keratins from the patient cells pointed to a tendency of mutations in the highly con server regions of the protein to cause severe disruption of the filament network and more severe phenotypes. In contrast, mutations in the less conserved domains had minor effects on the filament network and were associated with a milder clinical presentation (Uttam et al., 1996).


Figure 7.2 Immunofluorescence staining of DEJZ components in spontaneously separated EB skin. A: Laminin 5 on the floor of a junctional blister. B: Collagen XVII in the roof of a junctional blister. C: Collagen VII on the floor of a junctional blister. D: Lack of collagen XVII in GABEB skin. E: Collagen VII in the roof of a dystrophic blister. F: Intraepidermal accumulation of collagen VII in TBDN.

A ligand of the intermediate filaments, the cytoskeleton-membrane linker protein plectin is involved in the rare autosomal recessive EBS with muscular dystrophy (EBS-MD) (McLean et al., 1996); nonsense mutations in the PLEC1 gene have been disclosed in about a dozen EBS-MD families (Pulkkinen and Uitto, 1999). These lead to lack of plectin in the hemidesmosomal plaque, to defective cytoskeleton anchorage and to cell fragility. Clinically, the disease manifests with trauma-induced non-scarring skin blistering in the neonatal period and late onset muscular dystrophy at the age of 2–30 years. The expression of plectin isoforms in many tissues including epithelia and muscle explains the combined EBS-MD phenotype (Wiche et al., 1983). Mutations of the other intermediate filament connector, BP230, have not been found yet. However, targeted elimination of the gene for BP230 in mice predictably produced a cutaneous phenotype. Beside abnormal hemidesmosome structure, lack of anchorage of


Table 7.3 Subtypes of epidermolysis Bullosa

*AD=autosomal dominant; AR=autosomal recessive; X-=X-chromosomal; EBS-MD=EBS with muscular dystrophy; GABEB=generalised atrophie benign EB; JEB-PA=JEB with pyloric atresia; TBDN=transient bullous dermolysis of the newborn. For more details, and for a revised classification the reader is referred to Fine et al., 2000 and Gedde-Dahl and Anton-Lamprecht, 1996

the cytoskeleton and keratinocyte fragility, the structure, lack of anchorage of the cytoskeleton and keratinocyte fragilityr, the mice unexpectedly developed sensory nerve degeneration (Guo et al., 1995) and muscular dystrophy. This unusual phenotype led to the discovery of a neural isoform of BP230 which also had been inactivated by the targeting vector (Yang et al., 1996). JEB: abnormalities of the anchoring filaments Detachment of the basal keratinocytes from the lamina densa, rudimentary hemidesmosomes and anchoring filaments are ultrastructural hallmarks in JEB (McGrath and Eady, 1997, McMillan et al., 1998). Therefore, molecular components of the anchoring filaments, laminin 5, α6β4 integrin and collagen XVII, were obvious candidates


Table 7.4 Human disease genes in heritable blistering disorders of the skin

Figure 7.3 A similar phenotype with skin blistering and parietal alopecia in two JEB patients with mutations in different genes. On the left, an 18 year-old proband with the homozygous nonsense mutation Q1016X in COL17A1 gene (Schumann et al., 1997). On the right, a 43 year-old patient with the heterozygous LAMBS mutation R635X; the other mutation of this patient remains elusive.

for the different JEB subtypes. Indeed, mutations in the genes encoding these molecules have been shown to cause clinically diverse JEB subtypes, ranging from the lethal Herlitz JEB to mild localized blistering (Gedde-Dahl & Gedde-Dahl and Anton-Lamprecht, 1996, Bruckner-Tuderman, 2000, Pulkkinen and Uitto, 1999). Lack of laminin 5 results in extreme cutaneous and mucosal fragility in JEB Herlitz, the most severe, often postnatally lethal JEB subtype. Homozygous or compound


heterozygous mutations in the LAMAS, LAMBS and LAMC2 genes encoding the α3, β3 and the γ2 chains of laminin 5, respectively, are associated with JEB Herlitz (see Christiano and Uitto, 1996). Mutations causing premature termination codons often lead to nonsense-mediated mRNA decay and to lack of synthesis of the corresponding polypeptide chain, and absence of any one of the three subunit polypeptides prevents assembly and secretion of the entire heterotrimeric laminin 5 molecule. Use of chain specific laminin antibodies is therefore of limited value for determination of the defective gene (McMillan et al., 1997). However, mutation screening is facilitated by the fact that LAMBS mutations account for 80 % of all gene defects in JEB (Christiano and Uitto, 1996). In 50 % of the cases, one of the two common mutations R42X or R635X is seen (Kivirikko et al., 1996). R635X and some other nonsense mutations in combination with missense mutations has been found associated with milder, non-lethal JEB subtypes ( Table 7.4, Figure 7.3). COL17A1 gene mutations underlie most cases of generalized atrophie benign EB, GABEB (McGrath et al., 1995, 1996 a/b, Darling et al., 1997, 1998 a/b, Gatalica et al., 1997, Schumann et al., 1997, Chavanas et al., 1997, Jonkman et al., 1997, Floeth et al., 1998, 1999). This represents a JEB subtype with generalized blistering, skin atrophy, extensive alopecia, scarce eyelashes, eyebrows and body hair, as well as nail dystrophy and dental anomalies (Figure 7.3). Morphological hallmarks are rudimentary hemidesmosomes and anchoring filaments (McGrath and Eady, 1997). Most of the mutations known as far lead to a premature termination codon and absence of collagen XVII in the skin. Missense mutations or missense/nonsense mutation combinations which allowed synthesis of structurally abnormal collagen XVII caused milder JEB subtypes, including the JEB localisata subtype with mostly acral blistering, but not with alopecia (McGrath et al., 1996, Schumann et al., 1997, Floeth et al., 1998, Tasanen et al., 2000). This raises a very intriguing biological quesr tion about the relation of collagen XVII abnormalities with hair growth and loss. Deficiency of α6β4 integrin is associated with abnormal hemidesmosomes, blistering of the skin and pyloric atresia. This JEB subtype often has lethal outcome due to extensive skin fragility (Vidai et al., 1995, Pulkkinen et al., 1997, Ruzzi et al., 1997). Consistently, α6 and β4 integrin knock-out mice exhibited severely attenuated epithelial adhesion in the skin and other organs, and early demise (Van der Neut et al., 1996, Georges-Labouesse et al., 1996). Recently, missense mutations in the gene for integrin β4, ITGB4, were found to underlie pyloric atresia with extremely mild, late onset skin blistering (Pulkkinen et al., 1998). The prognosis in these cases is very good after the pyloric atresia is surgically corrected. DEB: abnormalities of anchoring fibrils In DEB, the cleavage plane is below lamina densa, at the level of the anchoring fibrils. In contrast to normal skin with well-defined slender and centrosymmetrically cross-banded anchoring fibrils (Keene et al., 1997), DEB skin presents with broad, blunt whispy fibrils without cross-banding, or no fibrils at all (McGrath and Eady, 1994, Gedde-Dahl and Anton-Lamprecht, 1996). Complete lack of anchoring fibrils and collagen VII are


characteristic signs of the most severe subtype, DEB mutilans (Bruckner-Tuderman et al., 1989). In milder DEB forms mutated collagen VII polypeptides are synthesized, but the morphology of the anchoring fibrils is abnormal. Over 100 mutations in COL7A1, the gene for collagen VII, have been identified in different DEB subtypes (Christiano et al., 1993, Bruckner-Tuderman et al., 1995, Gardella et al., 1996, Christiano and Uitto, 1996, Dunnill et al., 1996, Hovnanian et al., 1997, Mellerio et al., 1997), but the complexity of the mutation constellations and their biological consequences are only beginning to emerge. Clinically, DEB presents with blistering of the skin followed by scarring. The cleavage below the basement membrane explains the scar formation, since any wound reaching into the dermis heals with a scar. DEB contains two dominantly and four recessively inherited subtypes (Table 7.3). Apart from the inheritance pattern, the subtypes differ in the distribution of the lesions. The spectrum varies from extensive blistering and mutilating scarring to a mild acral affection, or in extreme cases, to mere nail dystrophy. Despite this wide spectrum of phenotypes, all DEB forms are believed to be allelic. COL7A1 mutations have been shown to underlie both recessive and dominant DEB subtypes, and in some families combinations of recessive and dominant mutations lead to unusual and variable phenotypes (Christiano et al., 1996, Winberg et al., 1997, Hammami-Hauasli et al., 1998a). Similarly to the deficiencies of other anchoring complex molecules, collagen VII nullizygotes present with the most severe DEB phenotypes (see Christiano and Uitto, 1996). Milder phenotypes are due to missense mutations or nonsense/ missense mutation combinations. Molecular heterogeneity of DEB Investigation of homozygous and heterozygous COL7A1 mutations and their biological consequences have revealed extensive molecular heterogeneity of collagen VII defects. From a protein chemical point of view, many pathogenetic mechanisms would seem predictable. Dominant negative interference of amino acid substitution or deletion mutations for formation of anchoring fibrils has been documented (Christiano et al., 1996, Sakuntabhai et al., 1998). However, it has come as a surprise that the trimeric triplehelical collagen VII molecule seems to tolerate structural aberrations without obvious functional defects. This is best illustrated by the example of unaffected parents of patients with recessive DEB. These individuals carry heterozygous missense mutations, express the mutated polypeptides and have abnormal collagen VII molecules in the skin, however, without obvious functional deficits. In this context, glycine substitutions are particularly interesting. Usually a substitution of a small glycine residue within the triple-helix of a collagen leads to dominant negative effects (Prockop and Kivirikko, 1995, BrucknerTuderman and Bruckner, 1998, Bruckner-Tuderman et al., 1999). The mutated polypeptides fold together with normal polypeptides into collagen triple-helices, and the structurally aberrant trimeric molecules are incorporated into fibrils. The effects of the initally rather small structural abnormality are thereby accentuated by the supramolecular assembly, and the resulting collagen fibrils are rendered functionally inadequate. This is not the case with collagen VII, as seen by the unaffected carriers of mutations. Recent studies showed that glycine substitutions in critical positions within collagen VII molecule


interfered with the protein folding and suprastructure in a dominant negative manner (Figure 7.4), whereas other glycine substitutions within the distal ends of the triple-helix exhibited no adverse biological effects (Christiano et al., 1996, Hammami-Hauasli et al., 1998 a,b, Terracina et al., 1998).

Figure 7.4 Immunofluorescence staining with antibodies to collagen VII of keratinocytes from a patient with dominant DEB (A) and control (B). The dominant COL7A1 deletion mutation 6081del28 causes in-frame skipping of exon 73 (Sakuntabhai et al., 1998) and interferes with folding and secretion of procollagen VII in a dominant negative manner. This results in intracellular accumulation, partial degradation and delayed secretion of the protein.

Another example of unusual variable phenotypes is modulation of the phenotype by second mutations. We have characterized two families with an exon skipping mutation that prevented normal processing of procollagen VII to collagen VII, i.e. the carboxyterminal propeptide was not removed. The deletion was combined with a different glycine substitution mutation in each family resulting in distinct clinical phenotypes (Winberg et al., 1996). Similarly, in a TBDN family, combination of dominant and recessive glycine substitution mutations in COL7A1 resulted in modulation of the phenotype. Two point mutations caused amino acid substitutions G1519D and G2251E in the triple helical domain of collagen VII. In the heterozygous state the paternal mutation G1519D was silent, and the maternal mutation G2251E led merely to nail dystrophy, but not skin blistering (Figure 7.5). In the proband, compound heterozygosity for the mutations caused massive, transitory retention of collagen VII in the epidermis, its


reduced deposition at the basement membrane zone and extensive dermo-epidermal separation at birth (Hammami-Hauasli et al., 1998b). Discovery of the extensive variability of COL7A1 mutation constellations has consequences for genetic counseling of affected families. Detection of one or two mutations in the index patient does not always allow unambiguous determination of the inheritance pattern or prognostic predictions. Therefore, the counceling should be very cautious. Allelic diseases: heterogeneity of EB phenotypes Molecular analysis of skin diseases has provided unambiguous evidence for the fact that phenotypically different diseases can be allelic disorders. In EBS, keratin 5 and 14 abnormalities underlie at least four different clinical subtypes. DEB patients who are nullizygotes for COL7A1 alleles suffer from a very severe mutilans variant of dystrophic EB with extensive blistering and scarring, while patients with missense mutations in the same gene exhibit the localisata EB dystrophica variant with milder skin affection. Similarly, null alleles of COL17A1 gene underlie GABEB with loss of hair, but missense mutations in the same gene cause junctional EB localisata with blistering at mechanically exposed sites only. As further examples serve LAMBS or ITGB4 gene mutations with lethal JEB phenotypes as a consequence of homozygous or compound heterozygous nonsense mutations and milder clinical affections by missense mutations. The different phenotypes probably reflect perturbation of particular functions exercised by the protein domain affected by the mutation. Recent investigations have revealed an unusual self-limiting postnatal blistering disease, the transient bullous dermolysis of the newborn (TBDN), as allelic to DEB (Figure 7.5). At birth, TBDN is characterized by extensive subepidermal blisters, reduced or abnormal anchoring fibrils, and massive transitory retention of collagen VII in the epidermis. However, within the first months and years of life, TBDN heals or improves dramatically. In two families, compound heterozygosity for dominant and negative glycine substitution mutations in COL7A1 (Hammami-Hauasli et al., 1998b; Shimizu et al., 1999) was found to cause accumulation of collagen VII in keratinocytes, a situation that normalized within approximately two years. The molecular mechanisms of the distinct accumulation of collagen VII in the epidermis and in particular its disappearance from the epidermis and deposition at the DEJZ after a certain time remain elusive at present. Skin Blistering Phenotypes—Lessons for Normal Biology Molecular studies on EB and the genes and proteins involved have produced a wealth of information on the normal biology of the DEJZ. The function of keratins in maintaining structural integrity of cells was established when keratin mutations were shown to lead to skin blistering (Vassar et al., 1991). Since, several keratins and other epidermal proteins have been implicated in heritable disorders of epidermal differentiation (Roop, 1995). The investigations established plectin and BP230 as versatile cytoskeletal linker proteins in the hemidesmosomes and showed that BP230 and plectin isoforms exist in the skin,


Figure 7.5 Modulation of DEB phenotype by a second mutation. A: A proband with the dominant COL7A1 mutation G2251E has toe nail dystrophy, but no skin blistering. B: Her newborn child who had inherited G2251E and another glycine substitution mutation, G1519D, from the father presented with extensive skin blistering. G1519D was silent in heterozygous state, the father was clinically unaffected.

muscle and neural tissues (see Fuchs and Yang, 1999). Disclosure of mutations in JEB has shown that some proteins of the anchoring complex play a more pivotal role in epithelial cell adhesion and epidermal resistance to friction than others. Lack of laminin 5 seems to produce the most devastating, lethal JEB phenotype, whereas lack of another anchoring filament component, collagen XVII, leads to generalized skin blistering with altogether milder manifestations that are well compatible with life (Burgeson and Christiano, 1997, Pulkkinen and Uitto, 1999). The ligand of laminin 5, integrin a6β4, likewise represents an indispensable component of the anchoring complex, since homozygous null alleles of either gene desmonstrated extensive skin blistering with lethal outcome (GeorgesLabouesse et al., 1996, Van der Neut et al., 1996, Pulkkinen et al., 1997, 1998). The association of integrin 64 deficiency with congenital pyloric atresia (Vidai et al., 1995,


Ruzzi et al., 1997) suggests an important role for a6β4 integrin in the development of the gastrointestinal tract. New information was also obtained about the polymerization of collagen VII into anchoring fibrils. Patients with recessive DEB are compound heterozygous or homozygous for COL7A1 gene mutations, and therefore, unaffected parents are obligate carriers of heterozygous mutations (Christiano et al., 1996, Hammami-Hauasli et al., 1998b). This is a situation not observed with other collagens. Heterozygous mutations, typically glycine substitutions, usually cause dominant negative effects which lead to more or less severe disturbances of molecular assembly or supramolecular aggregation of collagens. Collagen VII therefore must use different mechanisms for stabilisation of suprastructures than other collagens, perhaps binding of other, yet unknown components of the basement membrane zone. The functional significance of the anchoring complex proteins is further under-lined by the fact that a quantitative reduction of a protein to 50% is not sufficient to impair dermalepidermal adhesion. This is illustrated by clinically unaffected heterozygous carriers of nonsense mutations in the genes for plectin, a6β4 integrin, collagen XVII, laminin 5 or collagen VII who only express one allelic product, and therefore theoretically synthesize only one-half of the relevant protein. Indeed, keratinocytes from such individuals have been shown to synthesize reduced amounts of protein in vitro (Hilal et al., 1993). Remarkably, we have recently identified a clinically unaffected proband with a heterozygous nonsense mutation in both LAMBS and COL17A1 genes (Floeth and Bruckner-Tuderman, 1999). Therefore, it seems that a drastic reduction in the quantity of the normal DEJZ molecules is required to cause symptoms and the molecular constituents forming the biological suprastructures appear to be, at least in part, functionally redundant. Novel Candidate Genes and DEJZ Proteins The DEJZ is known to contain a number of additional proteins which are candidates for blistering skin disorders. The presence of these components at the DEJZ has been demonstrated mostly with immunohistological techniques, however, their suprastructures, ligands and roles in dermal-epidermal adhesion are not yet well denned. Some of them may be expressed only under certain conditions, such as during development or wound healing, others may have more permanent functions. Examples of such components are are laminins 2 and 10, dystroglycan, syndecans, uncein, the GDA-J/ F3- antigen, fibulins, collagen XVIII, fibrillins, LTBPs and other components of the microfibrils (for reviews, see Beck and Gruber, 1995, Ekblom and Timpl, 1996, Timpl, 1996, Burgeson and Christiano, 1997, Aumailley and Smyth, 1998, Bruckner-Tuderman and Bruckner, 1998, Pulkkinen and Uitto, 1999). Animal Models for Acquired and Heritable Blistering Skin Diseases Animals have been shown to suffer from autoimmune blistering disorders (Iwasaki et al., 1995) similar to the human counter parts, and animal models have helped define some


aspects of dermal-epidermal adhesion. The role of autoantibodies in the pathogenesis of bullous pemphigoid and cicatricial pemphigoid has been demonstrated in a mouse model, using passive transfer of antibodies generated against collagen XVII (Liu et al., 1993) or laminin 5 (Lazarova, 1995). Remarkably, neonatal mice injected with the antibodies developed a blistering disorder that faithfully reproduced the key immunopathological features of bullous pemphigoid or cicatricial pemphigoid, respectively, including circulating autoantibodies, deposition of IgG and complement at the DEJZ, inflammatory infiltration of the upper dermis, and subepidermal blistering in skin and/or mucous membranes. These data suggest that the autoimmune response against the human collagen XVII or laminin 5 is relevant in the pathogenesis of blister formation in patients. Heritable mechanobullous disorders in animals are rare (see Bruckner-Tuderman et al., 1991). Autosomal dominant EBS was reported in bulls, a severe JEB in a toy poodle, and DEB in calves and dogs, but these forms have not been characterized in molecular terms. Severe recessive DEB in inbred sheep was shown to result from lack of collagen VII and anchoring fibrils in the skin, a phenotype similar to the mutilating DEB in humans (Bruckner-Tuderman et al., 1991). More recently, gene ablation in mice has delivered information of the functions of some novel DEJZ proteins. Studies on skin development in integrin α3β1-deficient mice revealed regions of disorganized basement membrane in the skin (DiPersio et al., 1997). In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. The findings support a novel role for α3β1 in establishment and/or maintenance of basement membrane integrity. Ablation of the gene for laminin α5 chain lead to abnormalities in many organs during embryogenesis and early lethality. Among others, the basement membrane of skin and placenta was abnormal, indicating the importance of this laminin chain for the development of the subepithelial basement membranes (Miner et al., 1998). Targeted in activation of the collagen VII gene lead to severe skin blistering and early demise of the animals (Heinonen et al., 1999). Future Perspectives Clarification of molecular pathomechanisms of blistering skin disorders not only extends our knowledge on normal biology of the DEJZ but also forms a basis for novel therapeutic approaches, such as somatic gene therapy. Optimal diseases to be treated with such approaches are blistering skin disorders caused by null alleles, e.g. lack of collagen VII or collagen XVII. The skin is an easily accessible organ for morphologic and biochemical investigations, and keratinocyte culture and transplantation techniques have been well developed for treatment of burns. Gene transfer into human keratinocytes in vitro has been successfully performed in many laboratories, however stable transfection and expression of correctly folded proteins still are problematic (see Khavari, 1998). Future development of diverse successful therapies will also depend on the progress in our understanding of suprastructure formation by structural DEJZ macromolecules. This


not only concerns the mechanisms of aggregate formation but also the structural characteristics unique for each molecule. Further, functional redundancies of molecular components within supramolecular aggregates will have to be defined in greater detail than presently available. This endeavour will not only be assisted by the analysis of aggregate formation by purified matrix macromolecules or their mixtures in vitro, but also by the elucidation of further genetic defects and their consequences in animal or human diseases as well as the generation of transgenic animals as models for human diseases. This combined information will not only help to understand and treat heritable skin diseases but also many common disorders currently considered as acquired. ACKNOWLEDGMENTS The author’s work was supported by grants SFB 492/A3 and SFB 293/B3 from the Deutsche Forschungsgemeinschaft (DFG) and by the University of Münster IZKF 2/D5 from the Ministry for Education and Research. The expert help of Nadja HammamiHauasli and Hauke Schumann with the illustrations is grate-fully acknowledged. REFERENCES Aho, S. and Uitto, J. (1998) Direct interaction between the intracellular domains of bullous pemphigoid antigen 2 (BP180) and beta 4 integrin, hemidesmosomal components of basal keratinocytes. Biochem. Biophys.Res. Commun. 243:694–699 Amano, S., Takahara, K., Gerecke, D.R., Nishiyama, T., Lee, S., Greenspan, D.S., Hogan, B., Birk, D.E., and Burgeson, R.E. (1997) The gamma 2 chain of laminin 5 is processed BMP-1 and processing is essential to basement membrane assembly in vivo. J. Invest. Dermatol 108: 542 (abstract) Andra, K., Lassmann,H., Bittner, R., Shorny, S., Fassler, R., Propst, F., and Wiche, G. (1997) Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture. Genes Dev. 11:3143–3156 Antignac, C. (1995) Molecular genetics of basement membranes: the paradigm of Alport syndrome . Kidney Int. 49:29–33 Aumailley, M. and Smyth, N. (1998) The role of laminins in basement membrane func-tionJ. Anat., 193:1–21 Balding, S.D., Prost, C., Diaz, L.A., Bernard, P., Bédane, C., Aberdam, D., and Giudice, G.J. (1996) Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain. J Invest Dermatol 106:141–146 Beck, K. and Gruber, T. (1995) Structure and assembly of basement membrane and related extracellular matrix proteins. In. P.D.Richardson and M. Steiner (eds.), Principles of cell adhesion. CRC Press, Boca Raton, pp. 219–252 Bonifas, J.M., Rothman, A.L., and Epstein, E.H. Jr. (1991) Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities. Science 254:1202–1205 Borradori, L. and Sonnenberg, A. (1996) Hemidesmosomes: roles in adhesion, signaling and human diseases. Curr. Op. CellBiol 8:647–656. Borradori, L., Koch, P.J., Niessen, C.M., Erkeland, S.van Leusden, M.R., and Sonnenberg, A. (1997) The localization of bullous pemphigoid antigen 180 (BP180) in hemidesmosomes is


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Schaapveld, R.Q.J., Borradori, L., Geerts, D., van Leusden, M.R., Kuikman, L,Nievers, M.G., Niessen, C.M., Steenbergen, R.D.M., Snijders, P.J.F., and Sonnenberg, A. (1998) Hemidesmosome formation is initiated by the 4 integrin subunit, requires complex formation of 4 and HD1/plectin and involves a direct interaction between 4 and bullous pemphigoid antigen 180. J. Cell Biol. 142:271–284 Schäcke, H., Schumann, H., Hammami-Hauasli, N., Raghunath, M., and Bruckner-Tuderman, L. (1998) Two forms of collagen XVII in keratinocytes: a full-length transmembrane protein and a soluble ecto-domain. J. Biol. Chem., 273:25937–25943 Schumann, H., Hammami-Hauasli, N., Pulkkinen, L., Mauviel, A., Küster, W., Lüthi, U., Owaribe, K., Uitto. J., and Bruckner-Tuderman, L. (1997) Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relations to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa. Am J Hum Genet 60:1344–1353 Schumann, H., Baetge, J., Tasanen, K., Wojnarowska, F., Schäcke, H., Zillikens, D., and Bruckner-Tudermann, L. (2000) The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases. Am. J. Pathol. 156:685–695 Shimizv, H., Hammomi-Hauasli, N., Hatta, N., Nishikawa, T., and Bruckner-Tuderman, L. (1999) Compound heterozygosity for silent and dominant glycine substitution mutations in COL7A1 leads to a marked transient intracytoplasmic retention of procollagen VII and a moderately severe dystrophic epidermolysis bullosa phenotype. J. Invest. Dermatol 113:419–421 Siemens, H.W. (1921) Zur Klinik, Histologie und Aetiologie der sogenannten Epidermolysis bullosa traumatica (Bullosis mechanica), mit klinisch-experimentellen Studien ueber die Erzeugung von Reibungsblasen. Arch. Derm. Syph. 134:454–477 Stanley, J.R., Tanaka, T., Muller, S., Klaus-Kovtun, V. and Roop, D. (1988) Isolation of cDNAs for bullous pemphigoid antigen by use of patient autoantibodies. J. Clin. Invest. 82: 1864–1870 Subramanian, S.V., Fitzgerald, M.L., and Bernfield, M. (1997) Regulated shedding of syndecan-1 and -4 ectodomains by thrombin and growth factor receptor activation. J. Biol. Chem 272: 14713–14720 Suzuki, N., Labosky, P.A., Furuta, Y., Hargett, L., Dunn, R., Fogo, A.B., Takahara, K., peters, D.M., Greenspan, D.S., and Hogan-BL (1996) Failure of ventral body wall closure in mouse embryos lacking a procollagen C-proteinase encoded by Bmpl, a mammalian gene related to Drosophila tolloid. Development. 122:3587–3595 Talts, J., Andac, Z., Gohring, W., Brancaccio, A., and Timpl, R. (1999) Binding of the G-domains of laminin alpha 1 and alpha 2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins. EMBOJ. 18:863–870 Tanaka, H., Ishida-Yamamoto, A., Hashimoto, T., Hiramoto, K., Harada, T., Kawachi, Y, Shimizu, H., Tanaka, T., Kishiyama, K., Höpfher, B., lizuka, H., and Bruckner-Tuderman, L. (1997) A novel variant of acquired epidermolysis bullosa with autoantibodies against the central triplehelical domain of type VII collagen. Lab Invest 77:623–632 Tasanen, K., Eble, J.A., Aumailley, M., Schumann, H., Baetge, J., Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) Collagen XVII is destabilized by a glycine substitution mutation in the cell adhesion domain COL15. J. Biol. Chem. 275:3093–3099 Terracina, M., Posteraro, P., Schubert, M., Sonego, G., Atzori, F., Zambruno, G., BrucknerTuderman, L., and Castiglia, D. Compound heterozygosity for a recessive glycine substitution and a splice site mutation in the COL7A1 gene causes an unusually mild form of localized recessive dystrophic epidermolysis bullosa. Submitted for publication. Timpl, R. (1996) Macromolecular organisation of basement membranes. Curr. Op. Cell Biol. 8: 618–624


Uttam, J.Hutton, E., Coulombe, P., Anton-Lamprecht, I., Yu, Q.C., Gedde-Dahl, T.Jr., Fine, J.D., and Fuchs, E. (1996) The genetic basis of epidermolysis bullosa simplex with mottled pigmentation. Proc. NatlAcad. Sci. 93:9079–9084 Van der Neut, R., Krimpenfort, P., Calafat, J., Niessen, C.M., and Sonnenberg, A. (1996) Epithelial detachment due to absence of hemidesmosomes in integrin 4 null mice. Nature Genet13:366–369 Vassar, R., Coulombe, P.A., Degenstein, L., Albers, K., and Fuchs, E. (1991) Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease. Cell 64:365–380 Vidai, F., Aberdam, D., Miquel, C., Christiano, A.M., Pulkkinen, L., Uitto, J., Ortonne, J.-P., and G. Meneguzzi (1995) Integrin 4 mutations associated with junctional epidermolysis bullosa with pyloric atresia. Nature Genet. 10:229–234 Wiche, G., Becker, B., Luber, K., Weitzer, G., Castanon, M.J., Hjauptmann, R., Stratowa, C. and Stewart, M. (1991) Cloning and sequence of rat plectin indicates a 466 kD polypeptide chain with a three domain structure based on central alpha-helical coiled-coil. J. Cell Biol. 114: 83–99 Wiche, G., Krepler, R., Artlieb, U., Pytela R., and Denk, H. (1983) Occurrence and immunolocalization of plectin in tissues. J. Cell Biol. 97:887–901 Williamson, R.A., Henry, M.D., Daniels, K.J., Hrstka, R.F., Lee, J.C., Sunada, Y, Ibraghimov, A., Beskrovnaya, O., and Campbell, K.P. (1997) Dystroglycan is essential for early embryonic development: disruption of Reichert’s membrane in Dagl-null mice. Hum. Mol. Genet. 6:831–841 Winberg, J.-O., Hammami-Hauasli, N., Nilssen, O., Anton-Lamprecht, I., Naylor, S., Kerbacher, K., Zimmermann, M., Krajci, P., Gedde-Dahl, T. Jr., and Bruckner-Tuderman, L. (1997) Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with different missense mutations in the COL7A1 gene. Hum Mol Genet 6: 1125–1135 Woodley, D.T., Burgeson, R.E., Lunstrum, G., Bruckner-Tuderman, L., Reese, M.J., and Briggaman, R.A. (1988) Epidermolysis bullosa acquisita antigen is the globular carboxylterminus of type VII procollagen. J. Clin. Invest. 81:683–687 Yancey, K. (1995) Adhesion molecules: interactions of keratinocytes with epidermal basement membrane. Arch. Dermatol. 104:1008–1014 Yang, Y., Bowling, J., Yu, Q.C., Kouklis, P., Clevelard, D.W. and Fuchs, E. (1996) An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell 86: 655–665 Zillikens, D., Pose, P.A., Balding, S.D., Liu, Z., Olague-Marchan, M., Diaz, L.A., and Giudice, G.J. (1997) Tight Clustering of Extracellular BP180 Epitopes Recognized by Bullous Pemphigoid Autoantibodies. J Invest Dermatol 109:573–579 Zone, J.J., Taylor, T.B., Kadunce, D.P., and Meyer, L.J. (1990) Identification of the cutaneous basement membrane antigen in linear IgA bullous dermatosis. J. Clin. Invest. 85:812–820. Zone, J.J., Taylor, T.B., Meyer, L.J., and Petersen, M.J. (1998) The 97 kDa linear IgA bullous disease antigen is identical to a portion of the extracellular domain of the 180 kDa bullous pemphigoid antigen, BPAg2. J. Invest. Dermatol. 110:207–210.

LEUKOCYTE TRAFFICKING IN SKIN DISEASES

8. INTRODUCTION JONATHAN N.W.N.BARKER

The skin, because of its privileged site, is continuously exposed to a number of potentially injurious stimulae, not encountered in other organs. In much the same way that mucosal structures, such as the gastro-intestinal tract, have developed specialised immunological responses directed to its special needs, for example the production of secretory IgA and mucosal homing T lymphocytes, so the skin has its own specialised structures. Critical amongst these are epidermal antigen presenting Langerhans cells, which form a network throughout the epidermis, and skin homing lymphocytes. Keratinocytes also critically influence skin immune responses and should therefore be considered as specialised immunologically active cells. To reflect the growing evidence for an active immunological role for the skin, Bos coined the term Skin Immune System (SIS), building on skin associated lymphoid tissue (SALT) defined by Streilein (1), to define the multiple cell types and the complexity of immune responses within the skin (2). Circulating blood leucocytes constantly scrutinize potential sites of pathogen entry, rapidly moving from blood into tissue to mediate effective host defenses. The neutrophil influx that characterises the immediate response is superseded by antigen-specific mononuclear cell infiltration. In chronic inflammatory skin diseases such as psoriasis, atopic dermatitis, lichen planus and lupus erythematosus and other T cell mediated conditions including allergic contact dermatitis, this highly tuned surveillance system appears awry, with inflammatory cells infiltrating skin in response to an unidentified “pathogen” or “antigen”. Early vascular dilatation and perivascular accumulation of lymphocytes, monocytes, and macrophages are followed by accumulation of T lymphocytes within epidermis and at the tips of dermal papillae. Thus, mechanisms responsible for leucocyte infiltration into skin are of great relevance to the pathogenesis of the conditions mentioned above and may point to important targets for future treatment strategies. Cohnheim devised an elegant in vivo experimental system using intravital microscopy to visualise the inflammatory response in rabbit ears following topical croton oil application (3). He was the first to observe that passage of leucocytes from blood into tissue occurred in sequential steps: initial vascular dilatation, followed by leucocytes rolling on endothelia, stopping, and finally transendothelial migration towards the inflammatory focus. Over the last 10 years, the molecular basis for this sequence of events has been subject to intense research. Contrary to previous dogma, where postcapillary venules

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were thought to be passive bystanders, these vessels undergo profound morphological and functional changes, actively seeking and filtering out leukocytes of appropriate phenotype in a situation analogous to the role of high endothelial venules in peripheral lymph nodes. Once through the vessel wall, further complex interactions between leucocytes and tissue extracellular matrix determine the speed and direction of leucocyte movement and their subsequent retention in skin or return to the circulation. These events are mediated by a group of receptors/ ligands expressed on endothelial cells, leucocytes, and tissue matrices, known collectively as adhesion molecules. Through multiple, sequential steps these molecules organise and direct cell trafficking in skin in close collaboration with cytokines, particularly chemoattractant chemokines. Three main groups of adhesion molecules are recognised, based on structural and functional characteristics: selectins, integrins and members of the immunoglobulin supergene family (Figure 8.1). SELECTINS Selectins are a group of glycoproteins, characterised by an N-terminal lectin-like domain, homologous to Ca2+ -dependent lectins and a variable number of repeated complement regulatory protein-like residues. To date, three selectins have been identified: E-selectin (endothelial leukocyte adhesion molecule-1), Pselectin (granule membrane protein Mr 140 kDa(GMP-140)), and L-selectin (LEC-CAM-1). P-selectin and E-selectin act as ligands for neutrophils, eosinophils, monocytes, and memory T lymphocytes. Both molecules require endothelial cell activation for expression. P-selectin is present within resting vascular endothelial cells (together with von Willebrand factor), in the form of granules known as Weibel-Palade bodies. Following stimulation by acute inflammatory mediators such as histamine, thrombin or components of complement (C5b-9) these cytoplasmic granules rapidly fuse with the cell surface membrane to reach peak expression at 10 minutes. In contrast, E-selectin expression on human umbilical vein endothelial cells, and in human skin in vivo, reaches peak expression 4 hours after stimulation by interleukin (IL) -1, tumour necrosis factor-α or bacterial lipopolysaccharide(LPS) and requires de novo mRNA and protein synthesis. Selectins bind with low affinity to sialylated carbohydrate moities which are closely related to sialyl lewis x (SLX) and its isomer sialyl lewis a (SLA). These carbohydrate ligands are arranged in clusters, O-linked to much larger, serine-and threonine- rich molecules, which have been likened to mucins due to their similarly extended, Oglycosylated, rod-like, protein structure. P- and E-selectins mediate initial, potentially reversible, leucocyte tethering to, and rolling along, the endothelial cell surface. INTEGRINS Integrins consist of non-covalently linked α and β subunits, with an extracellular ligand binding site and an intracellular portion linked to the cell cytoskeleton. They are subdivided into various families according to the associated β subunit.

Figure 8.1. Structure of selectins, immunoglobulins and integrins.

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Figure 8.2. Molecular basis for leukocyte migration through vascular endothelium.

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

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transcription and protein synthesis. In vitro, mRNA is detectable at 2 hours following cytokine stimulation, and persists for at least 72 hours. ICAM-2 is also strongly constitutively expressed on isolated vascular endothelium, but does not appear to be regulated by cytokines and may therefore have a role in leucocyte trafficking in uninflamed tissue. ICAM-3 acts primarily as a T-cell accessory molecule. VCAM-1 is absent from unstimulated vascular endothelium, but in common with ICAM-1 is upregulated by a number of cytokines including IL-1 and TNF-α. Notably, IL-4 selectively upregulates endothelial VCAM-1 expression, whilst having no demonstrable effect on that of ICAM-1. Further details of the structure and function of the selectins, integrins and immunoglobulins together with detatils of the molecular basis of skin homing are presented in Chapter 9. Dynamic in vitro and in vivo models examining leucocyte adhesion to endothelia under conditions of flow indicate that these different groups of adhesion molecules operate in a sequential, interdependent manner (Figure 8.2). At sites of inflammation, local dilatation of post capillary venules results in margination of leucocytes to the peripheral, slow flowing, blood stream. Vascular P- and E-selectins capture these marginating, nonactivated leucocytes causing them to roll along the luminal surface of the vascular endothelium. Provided the cell is activated, this weak, selectin-mediated adhesion, is superceded by high avidity integrin/immunoglobulin binding at which point the leucocyte becomes stationary (arrest). Diapedesis then occurs through the vessel wall followed by directed migration to the inflammatory focus. Chemokines, released by cells at the inflammatory focus and endothelial cells and bound by extracellular matrix to the luminal surface of the vasculature, play a pivotal role in facilitating the switch between weak, selectin-mediated adhesion, to firm, integrin-mediated adhesion. There is clear evidence that these events are important in skin inflammation and cutaneous chronic inflammatory diseases. E-selectin and ICAM-1 are upregulated on vascular endothelium after epicutaneous application of antigen (4), intradermal injection of tuberculin or exposure to UVB irradiation (5) in a time dependent fashion, parallelling the influx of inflammatory cells. In chronic inflammatory conditions such as atopic dermatitis and psoriasis there is strong and persistent upregulation of ICAM-1 and Eselectin on dermal blood vessels which are themselves surrounded by skin homing lymphocytes (see chapter 9). Furthermore frozen section adhesion studies demonstrate that these vessels can support adhesion of activated T lymphocytes (6). Chemokines, including IL-8 and MCP-1, which are also thought to play a key role in leukocyte trafficking are also upregulated in these same cutaneous conditions (7). In the ensuing chapters, the role of adhesion molecules and cytokines, including chemokines, in the control of leukocyte activity, such as trafficking and activation, within the skin are discussed in detail. The significance of the events detailed are highlighted by the use of animal models to reveal their function in vivo and their importance to human skin disease by the use of leukocyte adhesion and accessory molecules as important immunotherapeutic targets for skin diseases.


REFERENCES: 1. 2. 3. 4.

5.

6. 7.

Streilein, J.W. Lymphocyte traffic, T-cell malignancies and the skin. Journal of Investigative Dermatology 71:167–171, 1978. Bos, J.D., Das, P.K. and Kapsenberg, M.L. The skin immune system. In: Skin Immune System (SIS), edited by Bos, J.D.Boca Raton: CRC, 1997, p. 9–16. Cohnheim, J. The pathology of the circulation. New Sydenham Society 242–382, 1889. Griffiths, C.E., Barker, J.N., Kunkel, S. and Nickoloff, B.J. Modulation of leucocyte adhesion molecules, a T-cell chemotaxin (IL-8) and a regulatory cytokine (TNF-alpha) in allergic contact dermatitis (rhus dermatitis). Brit JDermatol 124:519–526, 1991. Norris, P., Poston, R.N., Thomas, D.S., Thornhill, M., Hawk, J. and Haskard, D.O. The expression of endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in experimental cutaneous inflammation: a comparison of ultraviolet B erythema and delayed hypersensitivity. Journal of Investigative Dermatology 96:763–770, 1991. Barker JNWN, Groves RW, Allen MH and DM MacDonald DM. Adherence of T lymphocytes and neutrophils to psoriatic epidermis. Brit.J. Dermatol.1992, 127:205–211. Schroder, J.M. and Christophers, E. Identification of C5ades arg and an anionic neutrophilactivating peptide (ANAP) in psoriatic scales. Journal of Investigative Dermatology 87:53–58, 1986.

9. SKIN HOMING LYMPHOCYTES CONRAD HAUSER AND RENÉ MOSER

INTRODUCTION The migration of lymphocytes to nonlymphatic tissue is an essential step in the homeostatic adaptation to injury and is involved in many reactive and neoplastic disorders. Besides granulocytes and monocyte/macrophages, which are leukocytes of the innate (non-adaptive) immune system, lymphocytes are cellular elements of the adaptive immune system and play a very important role in defense and disease. The skin is one of the principal organs that delimits the body from the outside world. As many injuries which originate from outside can hit the skin, lymphocyte migration to this organ has to be particularly well assured. Although B and natural killer lymphocytes can infiltrate the skin in various pathologic conditions, little is known about the mechanisms that direct them to the skin. The vast majority of lymphocytes which infiltrate the skin are T cells. Therefore, this chapter deals with the migration of T cells to the skin. LYMPHOCYTES IN NORMAL SKIN Very few or no lymphocytes can be observed in histological sections of normal skin. It has been estimated, however, that the entire skin can contain a large number of lymphocytes —mainly T cells. It is currently unknown whether they migrate there because of environmental stimulation (but without clinical signs) or whether some lymphocytes have an intrinsic capacity to migrate to normal skin. Lymphocytes with intrinsic capacity to migrate to skin are a subset of T cells in the mouse that express the γδ type T cell receptor. This subset of T γδ cells can form a network of dendritically shaped cells, similar to epidermal Langerhans cells. In humans, however, γδ T cells are rarely encountered in normal skin. Extensive analysis of T cells in normal human epidermis has shown that the majority lie within the most basal keratinocyte layer, often in close apposition to Langerhans cells and comprise less than 1 % of all epidermal cells. Among these CD3+ intraepidermal T cells, the majority express the αβ type T cell receptor (Foster et al., 1990). Most of the CD3+ intraepidermal cells bear either CD4 or CD8 and a minority are negative for these 2 markers. About 90% of intraepidermal T cells are CD45RO+RA−, about 75% are CLA− (see below) and about half express the integrin aEb7 (Spetz et al., 1996). Although T cells located in normal dermis have been less

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characterized, they have been estimated to account for >95% of all lymphocytes in normal skin. Most dermal T cells can be observed around postcapillary venules of the papillary vascular plexus (Bos et al., 1987). The majority express the CD45RO+R− phenotype and the CD4/CD8 ratio has been reported to be approximately 1. LYMPHOCYTES IN INFLAMED SKIN Molecules Involved in Lymphocyte Migration During their ontogeny, and in order to exert effector function, lymphocytes migrate between lymphoid organs and nonlymphoid tissues. Precursors from the bone marrow migrate to the thymus and differentiate into mature T cells by negative and positive selection. From there, they arrive via the blood and the high endothelial venules in lymph nodes or spleen. Once activated by the appropriate antigen, they move into the bloodstream and can enter into non-lymphoid tissue. Some of the lymphocytes in peripheral nonlymphoid tissue may regain peripheral lymphoid tissue via the afferent lymphatics. It is possible that an activated or memory cell can circulate between peripheral lymphoid and nonlymphoid tissue more than one round. Postcapillary venules are the principal blood vessel structures that control tissue entry of lymphocytes. Thus, much effort has been invested to study the molecules and biology of lymphocyte— endothelium interaction. Before focusing specifically on T cell migration to the skin, a brief overview on the molecules involved in lymphocyte—endothelium adhesion and transmigration is presented. Selectins and their ligands The selectin family of adhesion molecules is defined by their common protein structure with an extracellular N-terminal C-type lee tin (sugar binding) domain, a single epidermal growth factor -like domain, short consensus repeats, a transmembrane domain, and a short C-terminal cytoplasmic domain. The known members are E-selectin (ELAM-1, CD62E), L-selectin (LECAM-1, LAM-1, CD62L), and P-selectin (PADGEM, GMP-140, CD62P). They bind with their lee tin domain to anionic oligosaccharides related to sialylated Lewis x (sLex, CD15s) (Tedder et al., 1995). The binding is charactericed by fast on- and fast offrates, considerable binding forces when exposed to fluid shear (socalled tensile strength), and Ca++ dependence (for review see Bevilacqua and Nelson, 1993, Kansas, 1996). (i) L-selectin and its ligands L-selectin (CD62L, LAM-1) is constitutively expressed at high levels by most leukocytes except by the majority of activa ted/memory lymphocytes, and is intriguing in mediating tethering and rolling adhesion of the different types of leukocytes to the activated

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endothelium (Knol et al., 1994, Smith et al., 1991, Spertini et al., 1991, Spertini et al., 1992, Tedder et al., 1995). The molecular cloning of L-selectin revealed a sequence encoding a 37 kD core protein with eight possible sites for N-linked glycosylation and contains a highly conserved lectin domain at the NH2-terminus, where the initial nine amino acids are critical for ligand binding. The fact that L-selectin is rapidly shed from the cell surface after leukocyte activation limits the capacity to provide tethering, rolling (Kishimoto et al., 1989), and also arrested adhesion (Moser et al., 1993). The shedding of L-selectin occurs by endoproteolytic cleavage in the membrane-proximal region of the extracellular domain (Chen et al., 1995, Migaki et al., 1995). Bonds between L-selectin and its ligand(s), once initiated, have considerable mechanical strength, allowing initial tethering to the endothelium under significant fluid shear. In addition, fast on and off rates seem to determine the rolling velocity in response to hydrodynamic drag (finger et al., 1996). L-selectin binds in a Ca++ dependent manner to sialylated derivatives of the Lewisx oligosaccharide on leukocytes (Tedder et al., 1995). Sialylation, sulfation, and fucosylation of the oligosaccharides are necessary to bind to Lselectin (Hemmerich et al., 1994, Hemmerich et al., 1994, Hemmerich et al., 1995, Hemmerich and Rosen, 1994). There are different sialylated glycoproteins (SGP) which fulfill these criteria. The most import SGP are the mucin-like SGP 50, GlyCAM-1 that is restricted to high endothelial venules (Lasky et al., 1992), and the more broadly distributed SGP 90, now identified as CD34 (Baumhueter et al., 1994, Baumhueter et al., 1993). To date, the protein core ligand for L-selectin, which is induced after activation of the endothelial cells by IL-1 or TNF (Spertini et al., 1991), and thought to be crucial in recruiting leukocytes at the endothelial lining, is still unknown. One reason for the difficulty in defining such membrane proteins is the relative promiscuity of carbohydrate structures concerning their protein back bones. Recent research into the understanding of the complex protein-oligosaccharide interactions indicates that different mucins, when presented in unique spacing and/or clustered combinations, probably dictated by the polypeptide backbone, may generate functional L-selectin ligands. (ii) E-selectin and its ligands E-selectin (ELAM-1, CD62E) also provides rolling adhesion under conditions of physiologic flow (Spertini et al., 1991) and is expressed on endothelial cells at sites of inflammation, including inflamed skin. It is not expressed in normal tissue but induced by proinflammatory cytokines such as interleukin (IL)-l, tumor necrosis factor-a (TNF-a), lipopolysaccharide (LPS), IL-10 and lymphotoxin (Bevilacqua et al., 1987, Pober et al., 1987, Vora et al., 1996). Recently, E-selectin induction has also been reported by CD40 ligand (Hollenbaugh et al., 1995, Karmann et al., 1995). The expression of E-selectin is downregulated by transforming growth factor-β (TGF-β) (Gamble et al., 1993), IL-4 and IL-13 (Etter et al., 1998). The expression of E-selectin on cultured human umbilical vein endothelial cells (HUVEC) is known to be transient culminating at 4–6 hours, then rapidly declining and disappearing after 24 hours. In vivo, E-selectin is continuously expressed in different inflammatory skin diseases (Groves et al., 1991, Norris et al., 1991,


Groves et al., 1991, Norton et al., 1991, Brasch and Sterry, 1992, Rohde et al., 1992, Norton et al., 1993, Norris et al., 1992, Bradley et al., 1994, Barlow et al., 1994, Wakita and Takigawa, 1994, Menange et al., 1996, Jones et al., 1996, Sais et al., 1997) and correlates with mixed leukocyte infiltrates (Barker et al., 1992). Endothelial E-selectin is induced in experimental delayed hypersensitivity reaction and in late phase skin reactions in atopies (Waldorf et al., 1991, Silber et al., 1994, Leung et al., 1991). Cutaneous graftversus-host disease is associated with expression of E-selectin (Norton et al., 1991, Norton et al., 1993). It can also be induced by injection of TNF- or IL-1 into skin (Groves et al, 1992, Groves et al., 1995). Prolonged expression of E-selectin was found when human umbilical vein endothelial cells were cultured in human serum (Etter et al., 1998). The detection of a factor in human serum providing sustained E-selectin expression supports this observation (Sepp et al., 1994). In addition, monocytes may also provoke late E-selectin expression by a yet unknown mechanism (Rainger et al., 1996). Typically, E-selectin requires fucose for biologically relevant recognition. The efficient binding to the E-selectin N-terminal lectin domain depends on fucosylated tetrasaccharide sialyl LewisX (SLex; NeuAc, α2,3Galβ1,4(Fuc α1,3)GlcNAc; CD15s) or closely related structures (Goelz et al., 1990, Phillips et al., 1990, Tiemeyer et al., 1991, Walz et al., 1990). High levels of SLex and fucosylated lactosamins are constitutively expressed on different leukocytes, including natural killer cells (Munro et al., 1992, Walz et al., 1990), whereas peripheral T and B lymphocyte do not express SLex unless activated ex vivo (Ohmori et al., 1993). CLA, an antibody defined carbohydrate related to sLex and bound to PSGL-1 (CD162), is the major ligand for E-selectin in T cells (see also below). Due to its eminent importance in certain inflammatory skin disorders, CLA is discussed seperately (see below). (iii) P-selectin and its ligand P-selectin (CD62P, PADGEM) GMP-140 (granule membrane protein-140) is also expressed in endothelial cells and provides leukocyte rolling. It is constitutively expressed in these cells and stored in Weibel-Palade bodies from where it can be rapidely translocated to the cell membrane (Bonfanti et al., 1989, McEver et al., 1989). Thrombin and histamine induce such fusions of granule- and cell membranes within seconds to minutes, causing rapid redistribution of P-selectin to the cell surface (Hattori et al., 1989, McEver et al., 1989). In addition, endothelial cells express C5a receptors, which induce Pselectin expression when occupied (Foreman et al., 1994). Similar P-selectin expression is induced by the C5b-9 membrane attack complex (Hattori et al., 1989), oxidized lowdensity lipoprotein (Gebuhrer et al., 1995), and oxygen radicals (Patel et al., 1991). In cultured endothelium, P-selectin induction is transient and downregulated by endocytosis to basal levels within 1 hour (Hattori et al., 1989), whereas IL-4 or oncostatin M induce a prolonged expression (Yao et al., 1996). Transcriptional upregulation of P-selectin by proinflammatory cytokines and lipopolysaccharides points to an additional level of P-selectin regulation in chronic inflammation.


The ligands of P-selectin are the Lewisx (Lex) trisaccharide (CD15) (Larsen et al., 1990), Galβ1,4(Fuc α1,3)GlcNAc and with higher affinity the sialylated tetrasaccharide SLex (Foxall et al., 1992, Handa et al., 1991, Policy et al., 1991, Zhou et al., 1991). Like Eselectin, P-selectin binds also to SLea (Handa et al., 1991). In addition, sulfated glycolipids and certain sulfated polysaccharides, like heparin, bind to the P-selectin lectin domain (Aruffo et al., 1991, Handa et al., 1991, Needham and Schnaar, 1993, Skinner et al., 1989). A major protein carrier of these ligands on T cells and granulocytes is also PSGL-1, which requires O-linked sLex-like structures and sulfated N-terminal tyrosines for optimal binding (Moore et al., 1995). Significant binding to P-selectin requires coexpression of a fucosyl transferase (Sako et al., 1993). The binding of PSGL-1 to Pselectin is calcium dependent and requires presentation of sialyl-Lewisx -type structures on the Olinked glycans of PSGL-1 (Sako et al., 1993). PSGL-1 is widely distributed on different leukocytes and is present on lymphocytes in a nonfunctional form. PSGL-1 can acquire binding activity after cellular stimulation by post-translational modifications (Vachino et al., 1995) such as glycosylation and sulfation (Li et al., 1996). Integrins and their ligands Integrins are adhesion molecules with diverse functions in processes such as embryogenesis, maintenance of tissue integrity and leukocyte migration. They consist of a large family of non-covalently associated α and β subunits generally of 150 and 100 kDa, respectively (Hynes, 1992, Smyth et al., 1993). The β1, β2 and β7 subfamilies play a role in lymphocyte migration. Whereas the role of the β1 and β2 integrins is well established in migration of lymphocytes to skin, the β7 integrins are involved in lymphocyte migration to gut-associated lymphoid tissue. The α4β7 integrin directs homing of lymphocytes to Peyers patches. The αEβ7 intergin (αIELβ7, αHMLβ7)was first described to be involved in interaction with intestinal epithelial cells. In the skin, this integrin is expressed on intraepithelial lymphocytes in normal and inflamed skin as well as by intraepidermal lymphocytes in cutaneous T cell lymphoma with epidermotropic T cell infiltration (Spetz et al., 1996, Dietz et al., 1996). These cells may undergo adhesive interaction with keratinocytes which express E-cadherin, a ligand for αEβ7 intergin (Cepek et al., 1994, Simonitsch et al., 1994). Also dermal lymohocytes have been reported to express this integrin to a significant percentage. An example with dermal αEβ7 integrin positive T cells is atopic dermatitis (de Vries et al., 1997). Only 1–2% of peripheral blood lymphocytes express this integrin which can be upregulated by mitogens or induced by TGF-β1. Expression of integrins at the cell surface can be regulated either by biosynthesis of these molecules or by their transport to the cell surface. In addition, once on the surface, the affinity of integrins for their ligands can be regulated. Various signals can induce a transient increase of affinity and thus permit firmer adhesion. Such signals include activation by the T cell receptor, interaction with CD31, hepatocyte growth factor (HGF) and most importantly chemokines which act via their numerous serpin (7 transmembrane domain) receptors. Many of the previously known chemokines such as interleukin-8 (IL-8, NAP-1), γ-interferon-induced peptide (IP-10), macrophage inflammatory protein-1α and-β (MIP-1α, MIP-1β), RANTES (regulated on activation, normal T-cell


expressed and secreted), and macrophage chemotactic protein-1, -2, -3 (MCP-1, -2, -3) had activity on integrins expressed on T cells. Only very recently, several newly cloned chemokines were shown to arrest T cells much faster (within minutes) on immobilized ICAM-1 than the previously identified members of the chemokine family (Campbell et al., 1998). These include stromal cell-derived factor-1 α (SDF-1α), 6-C-kine and macrophage inflammatory protein-3β (MIP-3β). The latter, in contrast to the former two, was selective for CD4+ activated/memory T cells. (i) b2 integrins and their ligands The expression of β2-integrins is restricted to leukocytes. They consist of three heterodimeric membrane glycoproteins, leukocyte function associated molecule-1 (LFA-1, CD11a/CD18, αLβ2 integrin), Mac-1 (CD11b/CD18, αMβ2 integrin) and p150, 95 (CD11c/CD18, αXβ2 integrin). The CD11 molecules and the common GDIS are the products of different genes, exhibiting distinct though overlapping patterns of tissue- and developmental stage-specific expression. The expression of CD11b and CD11c is almost exclusively restricted to cells of the myeloid lineage, whereas that of CD11a and GD18 is panleukocytic. The α subunits CD11a, CD11b and CD11c have different molecular masses of 180 Kd, 155 Kd, and 150 Kd, respectively. The formation of α/β heterodimers is stabilized by Ca2+ and Mg2+ (for review see Arnaout, 1990). Peripheral blood lymphocytes express LFA-1, whereas neutrophils, monocytes and NK cells all express Mac-1 (Arnaout, 1990). Dendritic cells of the myeloid lineage express CD11c. The density of LFA-1 is increased on activated/memory T cells than on naïve T cells. LFA-1 binds to the intercellular adhesion molecules-1, -2 and -3 (ICAM-1, -2, -3, CD54, CD102, CD50). ICAM-1 belongs to the immunoglobulin (Ig) gene superfamily and plays apivotal role in leukocyte adhesion. Endothelial cells constitutively express ICAM-1 and continuously upregulate it within 24 hour in response to stimulation with pro-inflammatory cytokines and lipopolysaccharides (Thornhill and Haskard, 1990). ICAM-1 is a single copy gene located on chromosome 19, consisting of five Ig-like domains (Katz et al., 1985, Simmons et al., 1988, Staunton et al., 1988). The first domain binds to the A-domain of CD 11a (Staunton et al., 1990), whereas the third domain confers binding to the A-domain of CD11b (Diamond et al., 1991). Only the third domain of ICAM-1 requires N-linked glycosylation to confer binding (Diamond et al., 1991). The cytoplasmic domain of ICAM-1, is connected to the cytoskeleton via α-actinin and thus represents a fixed ligand on the endothelial surface (Simmons et al., 1988, Staunton et al., 1988), whereas a glycophosphatidylinositol-linked variant can diffuse laterally within the plasma membrane (Carpen et al., 1992). As a ligand of LFA-1 and Mac-1, the transmembrane form of ICAM-1 is thought to mediate arrested adhesion and cell-attached migration during the transendothelial migration of leukocytes. ICAM-1 is expressed on both hemapoietic and non-hemapoietic cells, including T cells and endothelial cells. Inflamed endothelium in skin expresses high levels of ICAM-1 and is important for leukocyte recruitment to skin (Griffiths and Nickoloff, 1989, Griffiths et al., 1989, Lisby et al., 1989, Lewis et al., 1989, Majewski et al., 1991, Norris et al., 1991, Norris et al,


1992, Brasch and Sterry, 1992, Das et al., 1994, Jung et al., 1996, Teina et al., 1996, Jones et al., 1996, Dressler et al., 1997, Menage et al., 1996, Bennion et al., 1995, Baranda et al., 1997, Gruschwitz and Vieth, 1997). ICAM-1 is upregulated in dermal delayed hypersensitivity reactions, in human allergic contact dermatitis sites and in the Arthus reaction (Silber et al., 1994, Brasch and Sterry, 1992, Norman et al., 1994). It is also upregulated by injection of TNF-a into human skin (Groves et al., 1995). Its expression can be further induced in cultured endothelial cells by pro-inflammatory cytokines such as IL-1 and TNF-a. ICAM-1 is also expressed in keratinocytes in many inflammatory skin disorders, but its role in skin inflammation has remained elusive (Williams and Kupper, 1994). Resting skin endothelium expresses lower but detectable levels of this adhesion molecules. ICAM-2 (CD102) is also involved in leukocyte adhesion. It is constitutively expressed on endothelial cells at high levels and not upregulated by TNF and IL-1. Human ICAM-2 is a single copy gene localized on chromosome 17 and is composed of only two extracellular Ig-like domains (Hogg et al., 1991, Staunton et al., 1989). ICAM-2 binds to LFA-1 (Staunton et al., 1989) and a peptide derived from ICAM-2 can bind to purified CD11a/CD18 (Li et al., 1993). ICAM-2 also binds to LFA-1 via its A domain (Xie et al., 1995). Like ICAM-1, the cytoplasmic domain of ICAM-2 is linked to a-actinin of the cytoskeleton, suggesting anchored expression at the surface of endothelial cells (Heiska et al., 1996). ICAM-3 (CD50) is constitutively expressed on leukocytes, including resident epidermal Langerhans cells, but not on endothelial cells in normal skin. ICAM-3 was found on skin endothelial cells by immunohistology in only 5% of skin biopsies from various inflammatory and neoplastic skin diseases (Montazeri et al., 1995). (ii) The β1 integrins and their ligands The β1 integrins comprise different receptors for extracellular matrix proteins, including fibronectin, collagen, laminin and vitronectin. CD29 is the common β subunit shared among the different heterodimers. Only one member of the β1 integrin family, the α4β1 heterodimer also called very late antigen-4 (VLA-4, CD49d/CD29), has been shown to confer binding of lymphocytes (Elices et al., 1990, Schwartz et al., 1990, van Kooyk et al., 1993, Vennegoor, et al., 1992, Vonderheide and Springer, 1992), eosinophils (Bochner, et al., 1991, Dobrina et al., 1991, Moser et al., 1992, Moser et al., 1992, Schleimer et al., 1992) monocytes (Carlos et al., 1991, Jonjic et al., 1992), basophils (Bochner et al., 1991, Schleimer et al., 1992) and NK cells (Allavena et al., 1991) to cytokine-activated endothelial cells. Of importance, neutrophils do not express VLA-4 and therefore cannot utilize vascular cell adhesion molecule-1 (VCAM-1), which is the most important ligand of VLA-4 (Elices et al., 1990). Aside from providing firm adhesion to endothelium, α4 (CD49d) integrins, forming α4β1 and α4β7 heterodimers, have recently been shown to initiate lymphocyte tethering under shear and in the absence of a selectin contribution (Alon et al., 1995, Berlin et al., 1995). VLA-4 thus plays a crucial role in primary and secondary adhesion of T cells to inflamed endothelium as well as in transmigration across endothelium (Santamaria Babi et al., 1995a).


VCAM-1 (CD106, INCAM-110) belongs also to the Ig gene superfamily and primarily mediates firm adhesion. In addition, VCAM-1 provides tethering and rolling of VLA-4expressing leukocytes under conditions of physiologic shear forces. It is important to mention that the rolling adhesion on VCAM-1 does not require VLA-4 activation or the presence of an α4 cytoplasmic domain (Alon et al., 1995). For peripheral T lymphocytes, of which the majority lack both Lselectin and the ligand for E-selectin but express VLA-4, rolling on VCAM-1 is thought to be important. In addition, VLA-4 has been shown to participate in lymphocyte tethering to and rolling on VCAM-1 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the absence of L-selectin (Berlin et al., 1995). VCAM-1 can be found on inflamed cutaneous endothelium, but is not detectable in resting skin (Das et al, 1994, Jung et al., 1996, Bradley et al., 1994, Barlow et al., 1994, Jones et al., 1996, Petzelbauer et al., 1996, Wakita et al., 1994, Menage et al., 1996, Sais et al., 1997, Groves et al., 1993, Norris et al., 1991, Norris et al., 1992, Brasch and Sterry, 1992). VCAM-1 is upregulated in vitro by proinflammatory cytokines such as IL-1 and TNF. IL-4, a lymphokine with many anti-inflammatory actions, is also capable of inducing VCAM-1 on endothelial cells (Moser et al., 1992, Schnyder et al., 1996). In contrast to human umbilical vein endothelial cells, dermal microvascular endothelial cells respond only to TNF-α but not IL-1 in induction of VCAM-1, showing that there is heterogeneity among endothelial cells from different sources with regard to cytokine induction of this adhesion molecule (Swerlick et al., 1992). This adhesion molecule can be induced in vivo in human skin by IL-1 and TNF-α (Groves et al., 1992, Groves et al., 1995). In baboons, the injection of TNF-α leads to sustained expression of VCAM-1 on endothelial cells of the skin and correlated anatomically well with accumulation of T cells (Briscoe et al., 1992). LPS injection was not capable of recruiting T cells to skin and this correlated with the failure to induce VCAM-1 expression. It was also shown in this in vivo model that ineffective doses of TNF-α together with IL-4 also induced VCAM-1 on endothelial cells and T cell accumulation in skin (Briscoe et al., 1992). Similar observations were made in pig skin where injection of TNF-α induced VCAM-1 better than IL-1. Interestingly, injection of purified protein derivative (PPD) induced VCAM-1 only in sensitized pigs but not naïve pigs whereas E-selectin was induced in both sensitized and naïve pigs (Harrison, et al., 1997). Similarly, cutaneous delayed hypersensitivity reaction to tuberculin in sensitized rhesus monkey was associated with VCAM-1 induction on endothelial cells (Silber et al., 1994). Induction of VCAM-1 in skin endothelial cells was often associated with induction of this molecule on other perivascular cells such as dendritic cells. MAdCAM-1, a further ligand of VLA-4, is an additional member of the Ig gene superfamily. It was molecularly cloned in 1993 by screening a cDNA library derived from a TNF-activated murine endothelioma cell line (Briskin et al., 1993), and in 1996 the human counterpart was cloned based on sequence homology (Shyjan et al., 1996). MAdCAM-1 recognizes the VLA-4 and supports Lselectin-dependent rolling (Berg et al., 1993, Berlin et al., 1993). MAdCAM-1 is known to be constitutively expressed but seems to depend on TNF and lymphotoxin- (Eugster et al., 1996, Neumann et al., 1996). It participates in migration of lymphocytes to the mucosa-associated lymphoid tissue of the gut (mesenteric lymph nodes, Peyers patches, and intestinal lamina propria).


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


The Dynamic Model of Lymphocyte Endothelium Interaction In principle, leukocyte extravasation, consists of a sequential order of molecular interactions involving different classes of endothelial and leukocyte adhesion molecules (Butcher, 1991). A model of multistep binding of leukocytes to endothelium was postulated by Butcher. It was mostly elaborated with neutrophils but is also generally valid for lymphocytes. Before focusing specifically on T cell migration to the skin, a brief introduction on the different steps guiding the circulating leukocytes to penetrate the endothelium at sites of inflammatory or immune reactions is presented. As for neutrophils, the first adhesive interaction of lymphocytes with endothelium (primary adhesion) is located at the level of postcapillary venules. It is transient, reversible and of low affinity/avidity. Morphologically, it may impress as continuous tethering and rolling of the lymphocytes along the endothelium. Blood vessel dilatation which occurs in many inflammatory conditions may favor this process by lowering the blood flow rate. This first step is mediated by lectin— carbohydrate interactions involving principally the selectin family of adhesion molecules (for review see Tedder et al., 1995). Later, the α4β1 and the a4β7 integrins and CD44 were shown to be involved in rolling adhesion (Berlin et al., 1995). Indeed, lymphocytes roll on E- and P-selectins in vitro under conditions of capillary blood flow (Diacovo et al., 1996, Luscinskas et al., 1995). In this context it appears logical that L-selectin, the a4 integrins and probably PSGL-1 are concentrated on the tips of lymphocyte microvilli (Berlin et al., 1995, Erlandsen et al., 1993, Moore et al., 1995). Reversibility is the major characteristic of rolling unless accompanied by a second event, due to the continuous activation of the leukocyte by focal adhesive contacts to the activated endothelium, local stimuli like chemokines, and platelet-activating factor (Kuijpers et al., 1991, Kuijpers et al., 1990, Lo et al., 1991, Zimmerman et al., 1996). This second event is also referred to as triggering which leads to activation of integrins (see also above). Chemokines may participate in this step not only by being released in soluble form but also by immobilization on carbohydrates such as heparan sulfate (Tanaka et al., 1993). Induction of avidity changes in LFA-1 have been reported by ligation of CD2, CD3, CD43, and CD44 (Shimizu et al., 1992). In addition, binding of T cells to endothelial CD31 (PECAM-1) increased the function of β1 integrins (Tanaka et al., 1992). Further co-stimulatory signals derive from binding to ICAM-1, ICAM-2 and VCAM-1 generating signals for T-cell proliferation and IL-2 secretion (Burkly et al., 1991, Damle and Aruffo, 1991, Damle et al., 1992, Damle et al., 1992, van Seven ter et al., 1991). Neutrophils, as a best characterized example, continuously shed L-selectin, permitting the reversion of primary adhesion, and upregulate Mac-1 during rolling. Rolling-dependent activation triggers a conformational change in the integrin heterodimers which increases the avidity of binding to the endothelial ligands (Ginsberg et al., 1992, Hogg et al., 1993, Hynes, 1992, Sastry and Horwitz, 1993, Schwartz, 1992, Simon et al., 1995, Smyth et al., 1993). Such “inside-out” signalling allows quick transformation of the integrins from low-avidity to a high-avidity state. The increasing formation of integrin-dependent ligations strengthens the binding forces, slowing and, eventually, terminating rolling adhesion. As a result, leukocytes firmly adhere to the


activated endothelium. This third step of firm irreversible adhesion (secondary adhesion) is resistant to shear forces of the bloodflow due to higher affinity/avidity interactions. Integrins, including the α4β1 integrin, the α4β7 integrin, LFA-1 and MAC-1, have been implicated in this step (Arnaout, 1990, Elices et al., 1990, Berlin et al., 1993). As mentioned before, in their activated state, they have higher affinity/avidity for their ligands. The final step, diapedesis or transmigrations across the endothelial lining, begins immediately after firm adhesion. This process, in itself a multistep sequence of events, involves integrins, CD44 and CD31. Induction of a 72 kDa gelatinase via ligation of the α4β1 integrin may help in digesting basement membranes underlying endothelial cells and may facilitate migration into the surrounding extravascular tissue (Romanic and Madti, 1994). It is thought that the combined and possible, sequential action of adhesion molecules on lymphocytes and their corresponding receptors on endothelial cells can confer tissuespecific homing of lymphocytes. Some adhesion molecules such as LFA-1 may have a role in homing to most tissues and thus have a more general function in migration. Others may serve exclusively for migration to one particular organ system. Therefore, a combination of general and tissue selective adhesion receptors should determine the migratory pattern of lymphocytes, especially T cells. Springer has made an analogy of tissue-specific homing of lymphocytes based on a combination of adhesion molecules this with telephone numbers which consist of general codes such as country and area code and specific codes such as the local number. The best characterized homing pathways of lymphocytes include migration of naïve T cells to peripheral lymph nodes. It is directed by L-selectin but LFA-1 and G protein-linked receptors are also involved. The leading molecule for migration to the gut-associated lymphoid tissue is the α4β7 integrin. Migration of T cells to the skin appears to be principally directed by CLA but adhesion molecules with a more general function may also be needed for migration to the skin. Molecules Required for T Lymphocyte Migration to the Skin Before mentioning the adhesion molecules required in vivo for lymphocyte migration to skin it should be mentioned that the vast majority of skin infiltrating T cells in disease and immunopathology are activated/memory cells with an CD45RA−CD45RO+ surface phenotype (Bos et al., 1989, Markey et al., 1990, Sterry and Hauschild, 1991, Frew and Kay, 1991). Numerous studies have directly addressed the question of which adhesion molecules are required for T cell migration to the skin. Most of them investigated the role of adhesion molecules in skin inflammation induced by T cell-dependent skin reactions such as contact hypersensitivity and dermal delayed hypersensitivity reactions. There is good evidence that E- and P-selectin, a4ß1 (integrin/VCAM-1 and LFA-1/ICAM-1 as well as CD44 play an important role in the generation of cutaneous lymphocyte infiltration induced by antigen or hapten challenge in sensitized animals such as mice, pigs and nonhuman primates (Ferguson et al., 1991, Ferguson and Kupper, 1993, 1994, Scheynius et al., 1993, Chisholm et al., 1993, Elices et al., 1993, Issekutz, 1993, Camp et al., 1993, Labwohl et al., 1994, Silber et al., 1994, Kondo et al., 1994, Tipping et al., 1996, Staite et


al., 1996, Binns et al., 1996a, 1996b). In mice, both P- and E-selectin play a role for migration of T cells to skin in delayed hypersensitivity reactions but it is not clear whether the contribution of P-selectin stems from endothelial cells or platelets (Austrup et al., 1997, Borges et al., 1997). In any case, rolling of leukocytes in non-inflamed skin depends in vivo on P-selectin (Nolte et al., 1994). As cutaneous delayed type hypersensitivity reactions can be mediated by CD4+ T cells with a type 1 lymphokine pattern (IL-2+IFN-β +IL- 4-IL-5-, Th1 cells) the migration of Th1 cells was confirmed to depend on both Pand E-selectin. In contrast, CD4+T cells with type 2 lymphokine pattern (IL- 2-IFN-γ-IL-4 +IL-5+, Th2 cells) were unable to migrate to skin and to induce delayed hypersensitivity reaction. The absence of E-selectin and P-selectin ligands on Th2 cells, in contrast to Thl cells which expressed ligands for both selectins, may explain their deficiency to migrate to skin in this murine model (Austrup et al., 1997, Borges et al., 1997). Among the chemokines that may participate in the triggering of integrins, IL-8 and MCP-1 have been shown in vivo to contribute to the migration of T cells to skin (Larsen et al., 1995, Rand et al., 1996). The Cutaneous Lymphocyte-Associated Antigen (CLA) Because CLA is selectively associated with skin-infiltrating T cells in humans and as it represents a ligand for E-selectin, this determinant has stimulated intense research. The results are summarized in a separate paragraph. The mouse homologue of CLA has not yet been identified, although E-selectin ligands on a subpopulation of T cells that migrate to skin and mediate delayed hypersensitivity responses have been demonstrated (Austrup et al., 1997, Borges et al., 1997). Cellular distribution of CLA The term Cutaneous Lymphocyte-associated Antigen (CLA) was coined when Picker et al. found that the monoclonal rat IgM antibody HECA-452 stained the majority of skininfiltrating lymphocytes in a variety of reactive inflammatory skin disorders but only in a subpopulation of circulating blood T cells and in a minority of lymphocytes infiltrating other organs than skin (Picker et al., 1990a). Studies from other labs confirmed the selective association of CLA+ T cells and skin. For example, only GvH of the skin (but not of gut) is associated with infiltration of CLA+ T cells (Davis and Smoller, 1992). Skin of psoriatic patients contained significantly more CLA+ T cells than synovium from patients with psoriatic arthritis or rheumatoid arthritis, and skin DTH reactions from patients with rheumatoid arthritis accumulated more CLA+ T cells than their synovial membranes (Pitzalis et al. 1996, Jones et al., 1997). Positive staining with HECA-452 is also found on circulating monocytes and polymorphonuclear leukocytes. In contrast to these cells, CLA expression in T cells was not uniform. CLA was expressed in about 16% of T cells from the blood, in about 10% of tonsil T cells but was virtually absent in thymocytes. CD45RO- T cells did not express CLA and only a subpopulation of CD45RO+ activated/ memory T cells expressed the determinant, suggesting CLA induction as a consequence of T cell activation (Picker et al.,


1990a). CLA did not appear to be a classical activation marker because activation of peripheral blood mononuclear cells in vitro with phytohemagglutinin and concanavalin-A downregulated CLA expression (Picker et al., 1993b). Further study of CLA expression revealed that CLA+ T cells and those labeling with a mAb to the mucosal homing receptor integrin a4b7 were mutually exclusive in the blood but that the majority of CLA+ cells expressed L-selectin, a homing receptor for lymph nodes (Picker et al., 1990b). Both CD4+ and CD8+ T cells as well as T cells with αβ and γδ T cell receptors could be identified in CLA+ circulating T cells. To obtain more information from T cells infiltrating the skin, suction blister were raised, overlying delayed type hypersensitivity reactions in the skin. The blisters were tapped at different time points and the cellular content analyzed. With this technique, it was confirmed that the majority of skin infiltrating T cells expressed high levels of CLA and L-selectin. In addition, HLA-DR, an activation marker of T cells was mainly expressed on CLAhigh expressing cells, suggesting that activation of T cells in the skin leads to further upregulation of CLA (Picker et al., 1994). In contrast to CLA, the integrins aeb7 and a4b7 in skin blister T cells had a distribution that resembles T cells in the blood. Comparison with lung lavage T cells from normal and diseased respiratory systems confirmed the selective association of CLA with skin (Picker et al., 1994). CLA expression was also compared in T cell clones derived from the skin and the blood. Clones from the skin had a much higher density of CLA and adhered better to E-selectin than those from the blood. In contrast, binding to P-selectin was equal among skin and blood derived clones (Rossiter et al., 1994). Molecular aspects of CLA Initial immunochemical analysis of CLA revealed molecular masses of 200 and 125 Kd. Periodate treatment abrogated immunoreactivity, suggesting that CLA was a carbohydrate-dependent epitope (Picker et al., 1990a). The hypothesis of expression of organ selective adhesion molecules in T cells gained support when Picker showed that CLA+ T cells selectively adhered to E-selectin transfected COS cells (Picker et al., 1991). E-selectin is expressed on endothelial cells in inflamed skin. It was postulated that circulating CLA+ T cells are directed by CLA to migrate and accumulate in the skin. However, many authors could not confirm that endothelial cells of the skin preferentially expressed E-selectin when compared to endothelial cells in other inflamed organs. Thus, the differential expression of CLA alone but not its receptor on endothelial cells could not fully explain the apparent selective migration of CLA+ T cells to the skin. In subsequent studies it was confirmed that the HECA-452 reactive material extracted and affinity purified from tonsils was a ligand for E-selection. It was further shown that the binding of E-selection to HECA-452 antigen was neuraminidasc and Ca- sensitive (Berg et al., 1991). Biochemical and antibody inhibition studies subsequently established the close relation between CLA and sialyl Lewis x (sLex, CD15s), the carbohydrate blood group antigen which is the major E-selectin ligand on neutrophils. E-, P- and L-selectin ligands are complex carbohydrates, glycans which are under the control of an ordered series of glycosylation reactions whereby the terminal steps are controlled by specific fucosyltransferases. The


leukocytes of mice who are deficient in fucosylransferase VII have no ligands for E- and Pselectin, demonstrating that this enzyme was necessary for the build up of ligands for both E-and P-selectin (Mali et al., 1996). On the other hand, the transfection of fucosyltransferase IV and VII into E-selectin negative hemopoiectic cell lines renders them reactive with E-selectin. But only the transfection of fucosyltransferase VII reconstituted the staining with HECA-452, indicating that fucosyltransferase VII was specifically involved in the synthesis of the CLA epitope. Further, fucosyltransferase VII generated Eselectin ligands with higher affinity than fucosyltransferase IV (Wagers et al., 1997). Finally, the protein carrying the CLA carbohydrate epitope was identified as PSGL-1, previously known to bear determinants responsible for binding to P-selectin (Fulbrigge et al., 1997). It thus appears that PSGL-1 can be glycosylated to bear ligands for either Pselectin or both P- and E-selectin. Differential glycosylation of PSGL-1 may thus regulate lymphocyte migration to skin. CLA+ T cells in the circulation mirror their counterparts in the skin The association of CLA and skin was tested in blood T cells. If circulating CLA+ T cells are predestined to migrate to the skin then they should also contain cells that respond to cutaneous antigens. This was tested with nickel, an exclusive skin antigen (hapten) and tetanus toxoid which is not expected to exclusively induce T cells that migrate to skin. CLA+ and CLA− CD45RA− T cells from the blood were incubated with these antigens. The proliferative response to nickel, but not tetanus toxoid, was confined to the CLA+ subset (Santamaria Babi et al., 1995b). On the other hand, the response to the same antigen in diseases with different organ localization was investigated. Asthma and atopic dermatitis are both atopyassociated diseases and affected individuals are often sensitized to house dust mite. The CLA+ blood T cells of patients with atopic dermatitis proliferated more vigorously than CLA- cells in contrast to asthmatics in which CLA- cells responded better than CLA− cells. In addition, expression of HLA-DR, an activation marker in human T cells, and release of IL-4, a Th2 marker lymphokine associated with atopy, were selectively increased in CLA+ cells from patients with atopic dermatitis. Together, these results supported the contention that CLA+ T cells migrate to the skin. Further support was provided when it was shown that the increased HLA-DR expression in circulating CLA+ T cells from patients with atopic dermatitis was reduced after combined UVA and B treatment, as was the extent and severity of this condition (Piletta et al., 1996). Analoguous findings were made for IL-2 receptor and CD30 expression in CLA1 blood T cells with this treatment. When the CLA+ circulating T cells of these patients were further analyzed, they were shown to proliferate spontaneously, to release IL-13 spontaneously, and to stimulate IgE synthesis in autologous B cells (Akdis et al., 1997). Because the cytokine release occured early in culture and was not inhibited by cycloheximide it can be assumed that they have been turned on in vivo. A selective increase of HLA-DR in CLA+ circulating T cells was also found in delayed cutaneous drug reactions (Gonzalez et al., 1997).


The regulation of CLA expression in T cells As previous observations suggested that CLA may be induced in response to activation, the regulation of CLA expression was further studies in vitro. With peripheral blood mononuclear cells. Naïve T cells, which express CD45RA but no CD45RO, go from CD45RA+RO− to a CD45RA+RO+ intermediate to become finally CD45RA-RO+ when activcated (Picker et al., 1993b). Some T cells in the double positive intermediate fraction were CLA+, suggesting that CLA is induced in the conversion from the naïve to activated state (Picker et al., 1993a). This CLA+ intermediate was also identified in lymph node cells. The percentage of CLA+ cells in this fraction was correlated with their anatomic origin: lymph nodes from sites close to skin contained a higher percentage of CLA+ intermediates than lymph nodes from sites remote from the skin (e.g. lymph nodes from the appendix), further supporting the association of CLA and the skin (Picker et al., 1993a). In addition, these data suggested that the conditions for CLA induction were more favorable in skin draining lymph nodes than those draining the gut. Most likely, the microenvironment in the lymph node regulates the induction of CLA. For this reason, the response of naïve T cells to activation using a panel of cytokines was investigated in vitro. TGF-β, and to a lesser extent IL-6, were capable of upregulating CLA on T cells. This was the case for naïve T cells from adults and neonatal chord blood T cells but CLA could also be further upregulated on activated T cells when restimulated and exposed to these cytokines (Picker et al., 1993a). Later it was shown that IL-12, a cytokine that plays an important role in the initiation of immune T cell responses, also upregulated the density of CLA. Inducers of CLA via IL-12 include SEE, TSST-1 and streptococcal pyrogenic exotoxins A and C (Leung et al., 1995) . Recently, it was shown that T cells activated and cultured in fetal calf serum-free media and IL-2 express high levels of CLA on virtually all T cells (Fuhlbrigge et al., 1997). Fetal calf serum therefore appears to have a suppressive effect on CLA expression on T cells. But this suppression seems to be overcome to some extent by IL-6, TGF-β and IL-12. Furthermore, activation of T cells via CD2 induces strong CLA expression in T cells, even in the presence of fetal calf serum. IL-4 inhibited CLA induction under these conditions. In contrast, activation by either CD3 or CD3 plus CD28 or mitogens induces CLA weakly and only transiently. Antigens could also induce CLA in culture (Liu et al., 1996) . When peripheral blood mononuclear cells were cultured with casein or Candida, CLA (but not L-selectin) increased in the T cells exposed to casein. This phenomenon was selective for patients with milk-induced atopic dermatitis but not milk-induced enterocolitis, allergic eosinophilic gastroenteritis or normal controls (Abernathy-carver et al., 1995). CLA in the interaction of T cells with endothelium In vitro studies attempted to clarify the role of CLA in the interaction with endothelial cells. In a static migration assay across endothelial cells, it was demonstrated that CLA+ T cells migrated in greater numbers across IL-1β and TNF-α activated (but not nonactivated) endothelial cell layers than the corresponding CLA− T cells. CLA appeared to be directly involved in enhanced transendothelial migration because HECA-452 blocked

Circulating memory T lymphocytes undergo adhesive interaction with activated endothelium on the level of postcapillary skin venules. The initial adhesive interaction is reversible and impresses as rolling. P- and E-selectin appear primarily to be involved in this step. Subsequent firm adhesive interaction terminates rolling. CLA, an E-selectin ligand, directs T lymphocytes selectively to skin. Activated integrins (LFA-1 and VLA-4) and their respective receptors (ICAM-1 and VCAM-1) appear primarily to be implicated in this step. firm adhesive interaction is triggered by chemokines and other factors via activation of integrins. Once firm adhesion is initiated migration across the inflamed endothelium starts. After completion of this step, T lymphocytes are located in interstitial skin tissue where they can interact with antigen presenting cells such as Langerhans cells. This interaction leads to T cell activation, secretion of lymphokines, and induction of cytotoxicity.

Figure 9.1 A combination of adhesion molecules and their receptors direct T lymphocytes to skin.



to levels of CLA− CD45RA– T cells (Santamaria Babi et al., 1995a). As integrins are involved in adhesion to endothelial cells, the influence of these molecules on endothelial transmigration of CLA+ and CLA− T cells was investigated. Blocking of the VLA-4 and its endothelial receptor VCAM-1 selectively blocked enhanced transmigration of CLA+ T cells across activated endothelial cells. Because CLA+ and CLA− T cells expressed the same density of VLA-4 integrin these results indicated that this integrin was selectively involved in transmigration of CLA+ (but not CLA− T cells) . In contrast, blocking of LFA-1 and its receptor ICAM-1 on endothelial cells inhibited transmigration, regardless of whether CLA + or CLA− T cells and activated or nonactivated endothelial cells were used, demonstrating that these adhesion molecules are not selective for the transmigration of CLA+ T cells (Santamaria Babi, et al., 1995a). As chemokines and their receptors are involved in lymphocyte homing and integrin activation their effect on transmigration of CLA+ and CLA+ T cells and CLA+ and CLA- subclones of the cutaneous T cell lymphoma cell line HUT-78 was studied in this system. Pertussis toxin and antibodies to IL-8 and antibodies to its receptor B (CXCR-1) blocked the enhanced transmigration of CLA+ (but not CLA−) T cells across activated umbilical and skin microvascular endothelial cells, again showing the selectivity of this chemokine and the CXCR1 for CLA+ T cells (Santamaria Babi et al., 1996). Collectively, these transmigration studies under static conditions clearly demonstrate that adhesion molecules and chemokine receptors have a distinctly different role in CLA+ than in CLA− activated/memory T cells. The interaction of CLA expressing T cells and adhesion molecules was also investigated under conditions of flow. CLA+ T cells adhered much better to E-selectin transfectants than CLA− memory T cells, supporting a role for CLA in rolling, the first adhesive step in the interaction with endothelium (Jones et al., 1994, Ronen et al., 1994). To have an in vivo demonstration for the role of CLA in the migration of T cells to the skin, attempts were made with SCID mice transplanted with human skin and the CLA+ and CLA− subclones of the mentioned T cell line. When the grafts were stimulated with TNF-a, the frequency of positive signals for the T cell receptor of the HUT-78 cell line was somewhat higher after intraperitoneal injection of the CLA+ subclone than the CLA− subclone. But the overall positive signal frequency was low in the skin and therefore the difference not statistically significant (Rosenblatt-Velin et al., 1997). Another approach with SCID mice transplanted with human skin and i.p. injection of lymphocytes was more successful. Transplants of skin containing superficial, but not deep, vascular plexus accumulated CLA+ T cells (Kunstfeld et al., 1997). SUMMARY AND CONCLUSION The skin accumulates activa ted/memory effector T lymphocytes in a variety of inflammatory skin diseases. In the last few years, some insight into the mechanisms of T cell migration to skin has been gained. General and tissue-selective adhesion molecules in T lymphocytes and their counter-receptors expressed on inflamed microvascular endothelium in skin determine the migratory capacity of T lymphocytes. These adhesion molecules, together with soluble factors such as chemokines, participate in a complex


process of interaction between T lymphocytes and endothelium. The interaction includes primary adhesion (rolling) of T lymphocytes along endothelium followed by firm adhesion (secondary adhesion), arrest and diapedesis across the endothelial barrier. There is good evidence that on the inflamed endothelium, the classic adhesion principles mediated by Eand P-selectin (primary adhesion), ICAM-1, and VCAM-1 (secondary adhesion) with their respective leukocyte integrins (LFA-1 and VLA-4) drive T cell migration to the skin. On the other hand, circulating T cells appear to be selectively directed to skin by the Eselectin ligand CLA, which is a carbohydrate determinant on PSGL-1 and dependent on fucosyltransferase VII (Figure 9.1). It is possible that the understanding of CLA expression or interference with its binding may provide new strategies for the treatment of T lymphocyte-dependent inflammatory skin diseases. REFERENCES Abernathy-Carver, K.J., Sampson, H.A., Picker, L.J., and Leung, D.Y. (1195) Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen. J Clin Invest, 95:913–918. Akdis, M, Akdis, C.A., Weigl, L., Disch, R, and Blaser, K. (1997) Skin-homing, CLA+ memory T cells are activated in atopic dermatitis and regulate IgE by an IL-13-dominated cytokine pattern: IgG4 counter-regulation by CLA- memory T cells. J Immunol, 159:4611–4619. Albelda, S.M., Muller, W.A., Buck, CA, and Newman, P.J. (1991) Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule. J Cell Biol, 114:1059–1068. Allavena, P., Paganin, C., Martin-Padura, I., Peri, G., Gaboli, M., Dejana, E., Marchisio, P.C., and Mantovani, A. (1991) Molecules and structures involved in the adhesion of natural killer cells to vascular endothelium. J Exp Med, 173:439. Alon, R., Kassner, P.D., Carr, M.W., finger, E.B., Hemler, M.E., and Springer, T.A. (1995) The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J Cell Biol, 128: 1243–1253. Arnaout, M.A. (1990) Structure and function of the leukocyte integrins (CD18/CD11). Blood, 75: 1037–1050. Aruffo, A., Kolanus, W., Walz, G., Fredman, P., and Seed, B. (1991) CD62/P-selection recognition of myeloid and tumor cell sulfatides. Cell, 67:35–44. Arvilommi, A.M., Salmi, M., Kalimo, K., and Jalkanen, S.(1996a) Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular adhesion protein-1 (VAP-1). Eur J Immunol, 26:825–833. Arvilommi, A.M., Salmi, M., Kalimo, K., and Jalkanen S.(1996b) Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular cell adhesion molecule-1 (VAP-1). Eur J Immunol, 26:825–833. Austrup, F., Vestweber, D., Borges, E., Löhning, M., Bräuer, R., Herz, U., Renz, H., Radbruch, A., and Hamann, R. (1997) P-and E-selectin mediate recruitment of T-helper-1 but not Thelper-2 cells into inflamed skin. Nature, 385:81–83. Baranda, L., Torres-Alvarez, B., Cortes-Franco, R., Moncada, B., Portales-Perez, D.P., and Gonzalez-Amaro, R. (1997) Involvement of cell adhesion and activation molecules in the pathogenesis of erythema chronicum perstans (ashy dermatitis). The effect of clofazimine therapy . Arch Dermatol, 133:325–329.


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10. T-CELL ACCESSORY MOLECULES RALF W.DENFELD AND JAN C.SIMON

INTRODUCTION In many inflammatory skin diseases, T cells accumulate around the dermal perivascular plexus and some subsequently migrate into the epidermis. These T cells are thought to make a major contribution to inflammation and tissue damage in these skin diseases. On the other hand, T cells residing in skin are supposed to play an important role in protecting the epidermis from potentially dangerous immune responses. In mice, a unique T-cell subset, namely dendritic epidermal γδ-TCR positive T cells (DETC), has been characterized as being responsible for the maintenance of immunologie homeostasis within skin, however, a human epidermotrophic T-cell analogue has not been identified as of yet (Shiohara and Moriya, 1997). Specialized antigen-presenting cells (APC) resident in skin, namely Langerhans cells, are considered to play a central role in the induction of T cellmediated cutaneous immune responses. Upon antigen-capture, i.e. by epicutaneously applied haptens or invading pathogens, these sentinel cutaneous APC migrate via dermal lymphatics into the T-cell areas of skin-draining lymphoid tissues where T cells are activated. Subsequently, these antigen-specific T cells travel via the blood stream to the original site of antigen-deposition. Also, cutaneous APC, i.e. Langerhans cells and dermal dendritic cells, are well equipped to stimulate resident T cells intracutaneously (Banchereau and Steinman, 1998). However, it must be noted that the integrated immune system within skin is not only comprised of a distinctive population of resident and recirculating T cells and cutaneous APC. Additionally, neighboring cells like keratinocytes, fibroblasts, and mast cells regulate T-cell responses via cytokine-mediated intercellular communication and cell-cell interactions (Takashima and Bergstresser, 1996). Thus, mechanisms of intracutaneous as well as extracutaneous T-cell activation are of central importance for the pathophysiology of inflammatory and some malignant skin diseases. Today it is generally accepted that activation of T cells requires two signals from APC (Bretscher, 1992). The first signal provides specificity via binding of the T-cell receptor (TCR)-CD3 complex to its antigen-MHC ligand. The second signal is mediated by additional costimulatory signals delivered to the T cell via a number of accessory molecules which serve as receptors for specific antigen-independent ligands expressed on APC. Over the years, CD28 and its homologue cytotoxic T lymphocyte antigen-4

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(CTLA-4, CD 152) have emerged as key regulators of T-cell responses. CD28 is the primary T-cell costimulatory receptor, and upon interaction with its ligands, B7–1 (CD80) and B7–2 (CD86), it enhances T-cell proliferation and cytokine synthesis, induces T-cell differentiation and the expression of anti-apoptotic proteins (Lenschow et al., 1996a). By contrast, CTLA-4 functions to inhibit T-cell responses, which is not simply due to competition with CD28 for costimulation (Thompson and Allison, 1997). Furthermore, another key player in T-cell activation is the ligand for CD40 (CD40L, CD154). CD40–CD40L interactions play a pivotal role in the development of T celldependent immune responses by upregulating APC costimulatory molecule expression and sustaining T-cell clonal expansion (Grewal and Flavell, 1998). How the T cell integrates signals through the TCR-CD3 complex, CD28, CTLA-4 and CD40L to initiate, maintain and/or terminate antigen-specific immune responses is currently a central issue in understanding T-cell activation. Furthermore, the knowledge of the mechanisms involved in T-cell activation in inflammatory and malignant skin diseases will have implications for future dermatologic therapy. For example, allergic and autoimmune skin diseases are characterized by imbalanced T-cell differentiation (Th1 vs. Th2) as a result of differential costimulation, offering a potential target for interfering with these pathways. Also, introduction of costimulatory molecules into melanoma cells augmented anti-tumor immunity in mice in vivo. CD28, A POSITIVE REGULATOR OF T-CELL ACTIVATION The CD28 receptor is a member of the immunoglobulin supergene family and exists as a disulfide-linked homodimeric glycoprotein in situ. CD28 is constitutively expressed at the cell surface on the majority of both resting and activated human T cells. The CD28 ligands, B7–1 (CD80) and B7–2 (CD86), are constitutively expressed on resting APC at low levels. Both B7 molecules are upregulated upon activation but with different kinetics. Following APC activation first B7–2 and then B7–1 expression is increased (Lenschow et al., 1996a). T-cell responses require a nonantigen-driven costimulatory signal provided by CD28 (and possibly other accessory molecules), in addition to ligation of the TCR-CD3 complex with CD4 or CD8. Indeed, in vitro interruption of the B7-CD28 pathway combined with TCR occupancy leads to failure of proliferation in human T cells upon restimulation with normal APC plus antigen, a state termed anergy. However, the induction of anergy can be reversed by the addition of exogenous IL-2 or, more importantly, by ligation of CD28 during the initial activation event (Lenschow et al., 1996a). Several other costimulatory molecules have been found to regulate T-cell activation, i.e. integrins (Dubey et al., 1995), CD43 (Sperling et al., 1995), CD44 (Guo et al., 1996), CD47 (Waclavicek et al., 1997), heat-stable antigen (CD24, Wu et al., 1998) and 4–1BB (CDw 137, Kim et al., 1998), but none of these molecules can block the induction of T-cell anergy or regulate T-cell activation as profoundly as CD28. In most T cells, CD28 lowers the threshold needed for activation and increases response longevity, effects linked to enhanced stability and transcription of lymphokine mRNA, in particular those encoding IL-2 and IL-4 (Lenschow et al., 1996a). Analysis of CD28-deficient mice indicated many T cell responses to be impaired, i.e. T-cell responses to superantigens

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(Saha et al., 1996), however, some responses proceed normally (Lenschow et al., 1996a). In addition, it has been shown that CD28-deficient T cells can initiate, but, more importantly, cannot sustain, proliferative responses (Kündig et al., 1996). Furthermore, CD28 costimulation may amplify an immune response by recruitment of T cells to inflammatory sites via the production of chemokines (Herold et al., 1997a). Together, these results suggest that CD28 is critically important for initiating and sustaining T-cell responses. The B7-CD28 pathway also amplifies cytolytic responses in tumor models. The first reports to document the importance of CD28 costimulation in tumor rejection in vivo demonstrated that the murine melanoma K1735 transfected with B7–1 was rejected by syngeneic hosts. This resulted in immunity to rechallenge with the parental, B7–1 negative, tumor cells. Subsequent work demonstrated B7–1 transfected tumor cells to induce regression of established mouse melanoma requiring both CD4+ and CD8+ T cells (Allison et al., 1995), and the effect of B7–2 transfected tumor cells in elicitating antitumor immunity (Allison et al., 1995, Martin-Foncheta et al., 1996). It is well documented that multiple antigenic peptides or tumor antigens presented by the MHC on tumor cells are recognized by T cells. Nevertheless, tumor-infiltrating T cells against most of these epitopes of autologous cancer cells are usually not active in vivo, a phenomenon called immunological ignorance of silent tumor antigens, possibly resulting in tumor escape (Chen, 1998). Recently, it has been shown that, as a result of B7-CD28 costimulation, T-cell responses could be spread to normally silent tumor antigens (Johnston et al., 1996). Despite these successes, early reports showed clearly that the expression of a CD28 ligand on poorly immunogenic tumor cells is not sufficient to induce regression in all tumor models (Allison et al., 1995), suggesting that increased B7CD28 costimulation may not be suitable for conversion of all silent tumor antigens. Instead, tumor immunogenicity, that is threshold of TCR molecules to be engaged with the peptide-MHC-complex, determines the effect of CD28 costimulation via B7 on tumor immunity (Chen, 1998). However, in some models the coexpression of B7 plus other costimulators, i.e. CD48 (Li et al., 1996) and 4–1BB (Melero et al., 1998), induced a vigorous T-cell response to silent epitopes. Taken together, B7-CD28 costimulation provides an important mechanism to enhance immune responses against (silent) tumor antigens for immunotherapy of cancer. Moreover, the relevance of B7-CD28 interactions in vivo in transplantation models and autoimmune diseases is well documented. The first observation was made in mice transplanted with xenogeneic human pancreatic islet cells. Inhibition of B7-CD28 interactions by treatment with CTLA-4 fusion protein (CTLA-4-Ig) resulted in prevention of pancreatic islet rejection and, additionally, led to long-term donor-specific tolerance, which was later confirmed using antibodies (mAb) to B7–1 and B7–2 (Lenschow et al., 1996a). Other investigators have demonstrated that CTLA-4-Ig treatment in cardiac and renal allogeneic transplant models can result in indefinitive graft survival (Sayegh et al., 1995, Sayegh et al., 1997). Similar findings were made in murine graft-versus-host disease (Via et al., 1996, Blazar et al., 1996). Most recently, Olthoff et al. (1998) elegantly demonstrated that following gene transfer of sequences encoding CTLA-4-Ig allogeneic liver transplants produced the recombinant protein shortly after revascularization,


resulting in intact liver function, indefinite allograft survival, and the development of donor-specific unresponsiveness. Furthermore, at least three rodent autoimmune models corroborate the importance of B7-CD28 costimulation in vivo. First, in the spontaneous non-obese diabetic mouse model, blocking the B7-CD28 pathway by CTLA-4-Ig or antiB7–2 mAb greatly reduced the incidence of diabetes when administered before or even after the onset of insulitis (Lenschow et al., 1995), which seems to be IL-4 dependent (Arreaza et al., 1997). Also in streptozotocin-induced diabetes CD28 expression is necessary since CD28-deficient animals and wildtype animals treated with anti-B7– 2 mAb develop neither hyperglycemia nor insulitis (Herold et al., 1997b). Second, in experimental allergic encephalomyelitis (EAE), a rodent model of multiple sclerosis, CTLA-4-Ig protected against EAE induced by either active immunization or adoptive transfer of activated peptide-specific T cells (Cross et al, 1995, Racke et al., 1995, Gallon et al., 1997). The profound inhibition of the clinical and histological manifestations of EAE continued after cessation of CTLA-4-Ig treatment (Cross et al., 1995). Treatment with anti-B7-l mAb ameliorated EAE and resulted in the predominant generation of Th2 clones whose transfer both prevented induction of EAE and abrogated established disease (Kuchroo et al., 1995). Even epitope spreading and clinical relapses in EAE were prevented following treatment with non-cross-linking Fab fragments of anti-B7–1 mAb (Miller et al., 1995). Third, in lupus-prone (NZB/ W Fl) mice, a murine model resembling human systemic lupus erythematosus, CTLA-4-Ig treatment suppressed the lupus-like illness and prolonged life even when treatment was administered late in disease (Finck et al., 1994). However, both anti-B7–1 and anti-B7–2 mAb were needed to prevent the development and progression of lupus, with B7–2 playing a more critical role and contributing to Th2-mediated cytokine production (Nakajima et al., 1995). In summary, the manipulation of the B7-CD28 pathway in vivo can prevent transplant rejection, the initiation of autoimmune responses, as well as suppress ongoing autoimmune processes. Furthermore, costimulatory signals delivered through the CD28 molecule have been shown to have profound effects on the differentiation of Th1 (i.e. IL-2, IFNγ) and Th2 (i.e. IL-4, IL-5) subsets (Mosmann and Sad, 1996). Kucheroo and collegues first suggested that interactions of CD28 with B7–2 but not B7–1 preferentially induce a Th2 response (Kuchroo et al., 1995). Additional in vitro (Freeman et al., 1995) and in vivo (Subramanian et al., 1997, Keane-Myers et al., 1998) studies supported a selective role of B7–2 in Th2 development, indicating B7–1 and B7–2 to deliver qualitatively distinct signals via CD28. However, other groups have not observed significant differences in the induction of Th1/Th2 cytokines when using B7–1 and B7–2 transfectants (Lanier et al., 1995) or APC lacking B7–1 and B7–2 (Schweitzer et al., 1997). In vivo, in the non-obese diabetic mouse strain, early disruption of the B7-CD28 pathway using CD28-deficient mice promoted the development and progression of spontaneous autoimmune diabetes correlating with an enhanced Th1 response (Lenschow et al., 1996b), indicating that the early differentiation of naive diabetogenic T cells into the Th2 subset is dependent upon CD28 signaling. Subsequently, using antigen-specific TCR-transgenic T cells, Rulifson et al. (1997) demonstrated that Th2 but not Th1 cytokine production was highly dependent on CD28 ligation, suggesting that CD28-mediated costimulation can drive the


differentiation of T cells towards a Th2 phenotype. Further evidence for the necessity of CD28 costimulation for Th2 immune responses was obtained in vivo in a Th2-mediated murine model of allergic airway hyperresponsiveness. Blocking the B7-CD28 pathway ablated allergen-induced airway dysfunction concomitant with a significant reduction in the Th2 response (Keane-Myers et al., 1998). However, in other in vivo Th2-mediated immune responses to a nematode parasite no CD28 dependency on T-cell differentiation was found (Cause et al., 1997), implying that the specific kind of immune response determines whether Th2 differentiation is CD28 dependent. Also, the point in time during an ongoing immune response at which CD28 signaling is provided or blocked (Arreaza et al., 1997) and the strength of the TCR signal (Tao et al., 1997) seem to be crucial for the requirement of CD28 costimulation in Th1/Th2 differentiation. Finally, recent reports have suggested a role for CD28 costimulation in regulating Tcell survival (Boise et al., 1995). It has been shown that following a TCR signal, CD28 costimulation dramatically upregulates expression of Bcl-xL on T cells. Indeed, following the initiation of T-cell activation, the amount of cl-xL protein is associated with resistance to Fas- and TNF receptor 2-mediated apoptosis (Boise et al., 1995, Radvanyi et al., 1996, Noel et al., 1996, Lin et al., 1997). Since both, the Fas/Fas ligand system and TNF/TNF receptor system, are the major pathways mediating activation induced T-cell death in the periphery, CD28 costimulation can maintain adequate numbers of functional T cells to ensure the successful outcome of a productive immune response (Boise et al., 1995, Lenardo, 1997). Although CD28 costimulation does not have a significant effect on Bcl-2 expression, it results in significant IL-2 accumulation, which can subsequently upregulate Bcl-2 expression providing a possible additional mechanism for clonal expansion (Mueller et al., 1996). In conclusion, CD28 is a positive regulator of T-cell activation. CTLA-4, A NEGATIVE REGULATOR OF T-CELL ACTIVATION CD28 and CTLA-4 are related glycoproteins of the immunoglobulin supergene family, with both molecules existing as disulfide-linked homodimers. The variable domains of CD28 and CTLA-4 contain conserved sequences necessary for binding their ligands B7–1 and B7–2. However, CTLA-4 has a much higher affinity and avidity for both B7 molecules compared to CD28. Although CTLA-4 and CD28 share a number of common structural properties and have the same ligands, their patterns of expression are quite distinct. Conversly to CD28, CTLA-4 protein levels are low or undetectable on resting T cells, but following activation CTLA-4 expression is maximal at 48 hours at the peak of the T-cell response (Thompson and Allison, 1997). CTLA-4 protein is primarily localized and stored intracellularly in the perinuclear Golgi vesicles, and upon activation of resting T cells, CTLA-4 transits from the internal stores to the cell surface and becomes directed towards the site of TCR engagement (Linsley et al., 1996). These differences in CD28 and CTLA-4 expression patterns have important functional implications. As mentioned earlier, many T-cell responses are impaired in CD28-deficient mice. Similarly, initial experiments demonstrating antibodies to CTLA-4 to enhance T-cell proliferation were interpreted to mean that CTLA-4 synergized with CD28 in enhancing T-cell costimulation (Lenschow et al., 1996a, Thompson and Allison, 1997). This result,


however, may be a consequence of the removal of an inhibitory signal by interrupting B7CTLA-4 interactions. In support of this idea, it was demonstrated that the inhibition of the B7-CTLA-4 pathway with anti-CTLA-4 mAb augmented alloreactive and peptidespecific T-cell responses in vitro (Wahmas et al., 1996). Furthermore, T-cell proliferation induced by CD3 and CD28 cross-linking was inhibited by simultaneous CTLA-4 crosslinking (Krummel and Allison, 1996). These effects were due to the inhibition of cell cycle progression (Krummel and Allison, 1996, Walunas et al., 1996). This was supported by the finding that CTLA-4 ligation diminished IL-2 production and permitted induction and expression of the anti-apoptotic gene Bcl-xL (Blair et al., 1998). Very recently, Walunas and Bluestone (1998) reported that CTLA-4 blockade inhibits tolerance induction, possibly by skewing T-cell differentitation towards a Th2 response. These results indicate that CTLA-4 transduces an inhibitory signal modulating the outcome of CD3 and/or CD28 signalling. The first demonstration that CTLA-4 blockade can augment antigen-specific T-cell responses in vivo was reported by Kearney et al. (1995). Here, adoptively transferred TCR-transgenic T cells expanded in response to immunization with OVA-peptide, a response which was markedly augmented by treatment with antiCTLA-4 mAb. Similarly, CTLA-4 blockade increased T-cell responses to superantigen in vivo (Krummel et al., 1996). Using CTLA-4-deficient mice the importance of CTLA-4 in regulating T-cell activation was shown dramatically. The absence of the CTLA-4 molecule led to a massive T-cell lymphoproliferative disease, i.e. lymphadenopathy and splenomegaly, with early lethality at 3–4 weeks of age (Thompson and Allison, 1997, Chambers et al., 1997). These results demonstrated the essential functional role of CTLA-4 in vivo for maintaining peripheral T-cell homeostasis. Furthermore, the phenotype of CTLA-4-deficient mice is remarkably different from that of CD28-deficient mice (Lenschow et al., 1996a), again indicating that CTLA-4 and CD28 have distinct functions. In addition, the role of the B7-CTLA-4 pathway in autoimmune disease and tumor models has recently been addressed. In EAE, treatment with antibodies to CTLA-4 resulted in exacerbation and, if treatment was initiated during disease remission, relapses of disease, which was associated with enhanced production of the encephalitogenic cytokines TNF-α, IFN-γ, and IL-2 (Thompson and Allison, 1997). In an elegant study, Leach et al. (1996) examined an alternative strategy to augment anti-tumor T-cell responses in vivo by blocking B7-CTLA-4 interactions. Treatment with anti-CTLA-4 mAb enhanced the rejection of B7-transfected tumor cells in vivo. Most strikingly, animals completly rejected the B7 negative parental tumor when treated with anti-CTLA-4 mAb, even when treatment was delayed until a palpable tumor was established (Leach et al., 1996, Yang et al., 1997). Although the exact mechanism for this rejection is not yet known, these results suggest that CTLA-4 blockade can augment T-cell responses to tumor antigens presented by host APC in vivo. In summary, the most compelling interpretation of all this data is that CTLA-4 is a negative regulator of T-cell activation.


THE ROLE OF CD40L IN COSTIMULATION AND T-CELL ACTIVATION CD40 ligand (CD40L, CD154) is a member of the tumor necrosis factor (TNF) cytokine superfamily, including TNF, Lymphotoxin α/β, Fas ligand, as well as the ligands for CD27, CD30, 4–1BB, and OX40. Like other members of this superfamily, CD40L is expressed as a homotrimeric complex of type II integral membrane glycoproteins. In humans, naturally occuring soluble forms of CD40L have also been described. Although the distribution of CD40L is not yet completly denned, it is preferentially expressed on activated CD4+ T cells, but also on mast cells, eosinophils, B cells and dendritic cells. For example, activated CD4+ T cells rapidly express CD40L in response to TCR ligation. The counter-receptor for CD40L is CD40, a member of the TNF receptor superfamily. The type I integral membrane glycoprotein CD40 is found on APC, i.e. B cells, dendritic cells and macrophages, some T cells, but also on non-hematopoetic cells, including endothelial cells, fibroblasts, and epithelial cells, i.e. keratinocytes (Grewal and Flavell, 1998). The importance of the CD40L in humans was revealed in patients suffering from Hyper-IgM syndrome, an immunodeficiency characterized by mutations in the CD40L gene locus, leading to high levels of IgM but severly reduced levels of circulating IgG and IgA, and the absence of germinal centers. Although patients show abnormal antibody responses, i.e. their B cells are unable to switch to produce other Ig-classes and to establish B-cell memory, their B cells are capable of producing normal antibodies in vitro when co-cultured with normal T-helper cells, indicating the deficient ability of T cells to activate B cells. Furthermore, the lack of functional expression of CD40L on activated T cells in Hyper-IgM syndrome patients makes them susceptible to recurrent upperrespiratory-tract and opportunistic infections, i.e. with Pneumocystis carinii, Cryptosporidium, and cytomegalovirus, pathogens usually cleared by T cell-dependent mechanisms. Similarly, CD40L-deficient mice are severely impaired in primary T-cell responses to protein antigens and clonal expansion of CD4+ T cells. Moreover, it is well documented that CD40 signals are required for germinal center formation and antibody isotype class switching (Grewal and Flavell, 1998). Furthermore, CD40– CD40L interactions affect APC function via upregulation of costimulatory and/or adhesion molecule expression, i.e. B7–1, B7–2, ICAM-1, LFA-3, CD44 (Caux et al,, 1994, Kiener et al., 1995, Guo et al., 1996), but also cytokine secretion, i.e. TNF-a, IL-12 (Kiener et al., 1995, Kato et al., 1997). Another important effect of CD40L signaling is its ability to enhance APC longevity by inhibiting either spontaneous or activation-induced cell death. It was shown that CD40 ligation via CD40L promoted sustained viability of dendritic cells and monocytes in culture (Peguet-Navarro et al., 1995, Kiener et al., 1995) and allowed naive and memory B cells as well as dendritic cells to resist T cell-induced Fas-mediated apoptosis (Lagresle et al., 1996, Bjorck et al., 1997), thereby possibly sustaining and amplifying APC-driven T-cell responses. The CD40–CD40L pathway is also critical in T cell-dependent macrophage activation by stimulating the release of proinflammatory cytokines, nitric oxide, and metalloproteinases (Stout and Suttles, 1996), the latter of which are thought to facilitate the penetration of cells into inflammatory sites. There is also evidence for direct effects of the CD40– CD40L pathway on T-cell function. For


example, Blotta et al (1996) showed that crosslinking CD40L on T cells can enhance Tcell proliferation and cytokine production. CD40L-deficient mice have been tested for their ability to resist Leishmania infection, which is believed to be contained by T cell-activated macrophages. Soong et al (1996) found CD40L-deficient mice to have an enhanced susceptibility to Leishmaniasis with higher parasite load associated with low levels of proinflammatory cytokine and nitric oxide production compared to wild type animals. Similar results were obtained by other investigators (Campbell et al., 1996), additionally demonstrating a failure to mount a Th1 type immune response. The impairment of the Th1 response was dependent on the inability of macrophages to produce IL-12, and, thus, could be overcome by administration of exogenous IL-12 to infected mice. Also in hapten-induced colitis, an experimental model for Thl-mediated disease, the early blockade of CD40–CD40L interactions by antibody to CD40L prevented the detrimental Thl response, while IL-12 injections caused an exacerbation of disease (Stüber et al., 1996). The impact of a lack of CD40–CD40L interactions in vivo has also been studied in models of T cell-mediated autoimmune diseases. In type II collagen-induced arthritis the in vivo administration of antibody to CD40L prevented autoimmune disease (Durie et al., 1993). In EAE, a T-cell priming deficiency became apparent, when the CD40L mutation was bred onto encephalitogenic peptide-specific TCR transgenic mice (Grewal et al., 1996): EAE could not be provoked in these mice by immunization with the specific peptide, whereas the same peptide induced EAE in CD40L+ TCR transgenic mice. Also, lupus-prone mice did not develop renal disease when treated with anti-CD40L early in life (Early et al., 1996) or, when treated at the time of established nephritis, showed a decrease in severitiy of renal disease (Railed et al., 1998). Finally, the importance of the CD40–CD40L pathway was demonstrated in several transplantation models. Inhibition of this pathway with antibodies to CD40L dramatically prolonged murine islet and cardiac allograft survival (Parker et al., 1995, Larsen et al., 1996). These results were consistent with findings of other investigators in graft-versushost disease models in which disease was inhibited by antiCD40L mAb or by CD40L gene disruption (Durie et al., 1994, Blazar et al., 1997). More recently, Rirk and collegues (1997) have demonstrated the remarkable potency of blocking CD40–CD40L interactions in a Rhesus renal allograft model. Overall, the data suggest that CD40L is a master regulator of the immune system, with strong influence on T-cell activation, as well as on macrophage, dendritic cell, B-cell and T-cell effector functions. THE COMPLEXITIES OF T-CELL ACTIVATION AND COSTIMULATION It is now more than 25 years since Bretscher and Cohn first proposed that the generation of an antigen-specific T-cell response requires at least two distinct signals from the APC (Bretscher, 1992). We now know that the first signal occurs upon engagement of the antigen-specific TCR-CD3 complex with antigenic peptide bound to major histocompatibility complex (MHC) antigens, while another set of non-cognate cell-cell interactions provides the second, so called costimulatory signal to the T cells via the B7-


CD28 pathway (Lenschow et al., 1996a). Over the years, numerous studies have supported the two-signal hypothesis. Interactions between T cells and APC must be carefully regulated such that activation of T cells is sufficient to generate a necessary immune response, but also that activation of quiescent self-reactive or bystander T cells is avoided. As described earlier in this review, the presence of MHC and CD40 molecules on resting APC, in conjunction with the TCR-CD3 complex and CD28 on resting T cells, is sufficient to trigger an immune response, while the lack of CD40 and B7 costimulation leads to T-cell anergy or tolerance (Lenschow et al., 1996a, Grewal and Flavell, 1998). Cognate interaction followed by CD40–CD40L interaction is necessary for the activation of costimulatory activity on APC, which has been shown for several APC, including B cells, macrophages, and dendritic cells. However, it is the dendritic cell which is implicated in the initiation of an immune response (Banchereau and Steinmann, 1998). And, indeed, DC, generated from human cord blood CD34+ progenitor cells, upregulate B7–1 and B7–2 dramatically, upon activation by CD40L (Caux et al., 1994). But the question remains whether a T cell can activate its APC before the T cell itself is activated. Addressing this issue, it was shown that the upregulation of CD40L on resting T cells requires stimulation via the TCR (signal 1) whereas costimulation (signal 2) is not required (Jaiswal and Croft, 1997). Therefore, a two-step model for the activation of T cells in the initiation of an immune response can be proposed (Grewal and Flavell, 1998). First, dendritic cells take up antigen at the site of injury or infection and migrate to the lymph node, where the antigen is presented to naive T cells. The T cell receives the antigenic signal 1, causing the rapid upregulation of CD40L (step 1). This in turn activates the expression of costimulatory molecules/activity on the APC. In the second phase, the costimulatory signal 2 is received by the T cell via CD28 which drives the T cell into cell cycle and full activation (step 2). These activated T cells can now enter into secondary cognate, antigen-specific CD40–CD40L-dependent and/or B7-CD28-dependent T-cell helper or T-cell effector functions in the effector phase of the immune response. Hence, a reciprocal dialogue between APC and T cell can only follow if either APC or T cell is activated. This two-step model of T-cell activation was confirmed in vivo in the already mentioned EAE model using CD40L-deficient, peptide-specific TCR transgenic mice (Grewal et al., 1996). Here, T cells could be primed to produce IFN-γ and mediate EAE only when B7– 1+ APC were provided by adoptive transfer. These findings suggest that CD40L is required for the induction of in vivo costimulatory activity on APC, which in turn is required for activation and priming of T cells evoking EAE. However, in a transplant model the simultaneous blockade of both the CD40–CD40L and the B7-CD28 pathways was required to permit engraftment of highly immunogenic allografts (Larsen et al., 1996, Konieczny et al., 1998), indicating that B7–1/2 induction and T-cell activation can occur in the absence of CD40–CD40L interactions. Thus, although they are interrelated, both pathways are independent regulators of T-cell-mediated immune responses. Similar observations were made in autoimmune/transplant models where the synergistic effects of a brief simultaneous blockade of CD40–CD40L and B7-CD28 interactions led to longterm inhibition of murine lupus (Daikh et al., 1997), and renal allograft rejection (Kirk et al., 1997), whereas blocking either the CD40– CD40L or B7-CD28 pathway alone was


not sufficient to inhibit disease progression. Taken together, these data suggest that there are both CD40-CD40L-dependent and -independent mechanisms for B7–1 and B7–2 induction in vivo. As described earlier, full T-cell activation occurs in the presence of MHC-peptide ligand complexes and TCR occupancy sufficient for adequate TCR-CD3 complex signalling (signal 1) and increased expression of B7–1 and B7–2, initially on dendritic cells and subsequently on activated B cells, resulting in CD28 costimulation (signal 2). This response is characterized by cell cycle progression, induction of anti-apoptotic proteins, Tcell clonal expansion, T-cell differentiation, and the generation of effector functions, such as cytolytic responses or directed T-cell help by polarized cytokine production. In this setting, CD28-mediated positive costimulation is not only important for initiating but also for sustaining T-cell responses (Kündig et al., 1996). However, longevity and strength of a T-cell response need to be controlled. Here, CTLA-4, the negative regulator of T-cell activation, comes to play its role. Optimal T-cell activation induces the synthesis and peak cell surface expression of CTLA-4. The increased localized density of B7–1, B7–2 and CTLA-4 molecules (Linsley et al., 1996) would facilitate high avidity B7-CTLA-4 interactions. The resultant inhibitory signals could limit T-cell clonal expansion, decrease the duration of the response, and/or influence the effector function, all of which would ultimately lead to downregulation of the ongoing T-cell response necessary for maintaining peripheral T-cell homeostasis. Indeed, in a very recent study by Saito and coworkers (1998) acute graft-versus-host disease (GVHD) was induced by allogeneic CD28-deficient T cells, which was inhibited by treatment with antibody to CD40L, but was exacerbated by treatment with antibodies to B7–1 and B7–2, thereby interrupting B7CTLA-4 interactions. These findings indicate the B7-CTLA-4 pathway to act protectively in the development of GVHD by downregulating the allogeneic T-cell response. Overall, the complex array of cognate and non-cognate interactions, i.e. between the CD40–CD40L, B7-CD28 and B7-CTLA-4 pathways, and the reciprocal dialogue between APC and T cells allow for a well balanced T cell-mediated immune response. Nevertheless, how the T cell integrates these signals to initiate, maintain and/or terminate antigen-specific immune responses still remains one of the central issues in the understanding of T-cell activation. T-CELL ACCESSORY MOLECULE EXPRESSION IN INFLAMMATORY AND MALIGNANT SKIN DISEASES The in situ expression of the receptor/ligand pairs B7-CD28 and CD40-CD40L has been examined in different T-cell-mediated inflammatory skin diseases. In allergic contact dermatitis and lichen planus, we found CD28 to be expressed on the majority of dermal and epidermal T cells. B7–1/2 and CD40 expression was clearly detectable on Langerhans cells, dermal dendritic cells (DC)/ macrophages, and occasionally on dermal T cells. Moreover, strong CD40 staining was observed on endothelial cells (EC) in diseased skin. In normal skin, however, CD28 was observed only occasionally on perivascular T cells, whereas B7–1/2 could not be detected in situ.


Only a faint CD40 staining was found on cutaneous DC, EC and keratinocytes (KG) of the basal layer (Simon et al., 1994, Hollenbaugh et al., 1995 and unpublished results). By contrast, no significant expression of CTLA-4 and CD40L could be detected, most likely due to their limited and transient expression patterns. In experimental murine contact hypersensitivity (CHS), a T-cell-mediated response to epicutaneous hapten sensitization and challange, the in vivo relevance of both pathways has recently been highlighted. Disruption of B7-CD28 interactions in vivo by CTLA-4-Ig or mAb to B7–1 and B7–2 during hapten sensitization inhibits CHS with distinct roles for B7–1 and B7–2 during the priming of CD8+ and CD4+ T cells (Tang et al., 1996, Xu et al., 1997). Furthermore, the additional inhibition of the CD40-CD40L pathway induced tolerance in CHS, but had no effect on the elicitation phase of the CHS response (Tang et al., 1996, Tang et al., 1997), indicating both pathways to be essential for complete sensitization to haptens. In psoriasis, CD28 and B7–1 were found to be expressed by virtually all T cells in the epidermis and dermis of psoriatic lesions (Nickoloff et al., 1994). This expression pattern may permit self-costimulation and thereby contribute to the ongoing T-cell proliferation. Additionally, dermal DC, being more prevalent in psoriatic plaques than in normal skin, have been shown to express high levels of B7–2 and intermediate levels of B7–1 (Mitra et al., 1995). Moreover, in psoriatic skin we have demonstrated a markedly enhanced expression of CD40 on KC in the suprabasal layers of the epidermis as well as on the dermal mononuclear infiltrate and on EC. The CD40 expression co-localized with the enhanced expression of the adhesion molecule ICAM-1 and the anti-apoptotic protein Bclx on KC and an influx of juxtaposed T cells (Denfeld et al., 1996). These findings suggest that direct interactions between infiltrating T cells and KC via the CD40–CD40L pathway might contribute to the induction of CD40, ICAM-1 and Bcl-x on KC and, therefore, could have implications for the pathogenesis of psoriasis. In diseased skin of patients with systemic, subacute cutaneous and chronic discoid lupus erythematosus (SLE, SCLE, CDLE), we detected CD28 expression on most T cells infiltrating the dermis and epidermis. B7–1 and B7–2 were expressed on dermal and epidermal DC, particularly when in apposition to CD28+ T cells, but not on KC. By contrast, in uninvolved skin of SLE, SCLE, and CDLE patients, CD28, B7–1, and B7–2 expression was negligible. Treatment of SLE patients with systemic corticosteroids revealed a correlation between clinical improvement, the resolution of the infiltrate and the reduced expression of B7–1, B7–2, and CD28 in formerly lesional skin. These findings suggest that in LE, B7–1+ and B7–2+ DC could contribute to the intracutaneous activation of CD28+ T cells, thus making an inhibition of B7-CD28 interactions worth considering in the therapy of LE (Denfeld et al., 1997). Indeed, in a murine model resembling SLE, disruption of the B7-CD28 pathway suppressed the lupus-like illness and prolonged life (Finck et al., 1994, Nakajima et al., 1995). Furthermore, it has been shown that T cells of patients suffering of SLE have an increased and prolonged expression of CD40L, documenting an impaired regulation of CD40L expression on T cells in SLE, which might result in abnormal costimulation for autoantibody production (Datta and Kalled, 1997). Specific immunotherapy that blocks CD40-CD40L interactions may thus be of value for treatment of this (and other) autoimmune disease, which has already been


demonstrated in lupus-prone mice (Early et al., 1996, Daikh et al., 1997, Kalled et al., 1998). In addition, EC play an important role in the pathogenesis of T-cell-mediated skin diseases. Although T cells reside within skin under physiological conditions, at sites of cutaneous inflammation effector T cells need to be recruited and activated. Evidence is emerging that CD40 may play an important role in the regulation of leukocyte recruitment into peripheral tissues like the skin. The low level of CD40 expression by EC in situ is enhanced by proinflammatory cytokines, i.e. TNF-α, IL-1β, IFN-γ. Ligation of CD40 on isolated EC induces the expression of the adhesion molecules ICAM-1, VCAM-1 and E-selectin suggesting that signalling through CD40 during T cell-EC interactions may be an important step in leukocyte recruitment (Hollenbaugh et al., 1995, Dechanet et al., 1997). In vivo, we have found a marked upregulation of CD40 expression on EC in T-cell-mediated inflammatory skin disease, such as psoriasis and allergic contact dermatitis, compared to normal skin (Hollenbaugh et al., 1995). Nevertheless, it is not known whether CD40 is actually required for T-cell-dependent EC activation in vivo. For example, although inhibition of CD40–CD40L interactions with antibody against CD40L prolonged allograft survival in several transplantation models including murine skin allografts (Durie et al., 1994, Parker et al., 1995, Larsen et al., 1996, Blazar et al., 1997, Kirk et al., 1997), it is unknown whether such a blockage impedes T-cell recruitment into the graft. In fact, Larsen and coworkers (1996) have demonstrated the expression of B7–1, B7–2 and T-cell cytokine transcripts in anti-CD40L mAb treated allografts suggesting that T cells may still enter the graft in the first days after transplantation. Hence, CD40 expression on EC may not be pivotal for early T-cell recruitment, but the CD40L on T cells may become an important factor in sustaining adhesion molecule expression and leukocyte recruitment during the effector phase of T-cell-mediated inflammatory skin disease. Finally, the expression of costimulatory molecules, especially regarding both, the B7CD28 and the CD40-CD40L pathway, has been studied in skin tumors. In human malignant melanoma, we and others failed to detect B7–1 and B7–2 surface expression on melanoma cells of primary and metastatic malignant melanoma in situ. However, B7–1 and B7–2 expression was found on tumorinfiltrating APC in apposition to CD28+ T cells. Additionally, B7–1 and B7–2 protein expression was at low or undetectable levels in the majority of cell lines derived from cultured primary and metastatic melanomas, while B7–2 RNA was detectable in some cell lines. The important exceptions were primary melanomas with partial spontaneous regression, in which a focal expression of B7–1 and B7–2 was detectable on melanoma cells in situ, suggesting that the absence of B7–1 and B7–2 favors the escape of malignant melanoma from immunosurveillance (Denfeld et al., 1995). In contrast to molecules of the B7 family, CD40 is expressed on melanomas in situ and in vitro following tissue culture (Thomas et al., 1996, van den Oord et al., 1996). Recently, we found that CD40 ligation on human melanoma cells in vitro led to secretion of proinflammatory cytokines, an increased expression of adhesion and MHC molecules, and augmented tumorspecific CTL-mediated lysis and apoptosis of melanoma cells (von Leoprechting et al. submitted). In vivo, CD40-CD40L interactions also contribute to the development of protective immunity against melanoma, since anti-


CD40L mAb treatment inhibited the generation of anti-tumor immune responses in a murine melanoma model (Mackey et al., 1997). Furthermore, skin cancers of KC origin, namely basal cell carcinoma and squamous cell carcinoma, do not express B7 in situ (Simon et al., 1994, Nestle et al., 1997) and exhibit a down-regulation of CD40 compared to the proliferative basal layers of normal skin or benign viral induced skin lesions (Viac et al., 1997). Moreover, only a minority of tumor-associated APC express functional B7–1 and B7–2, whereas CD40 expression was unaffected compared to inflammatory skin disease (Nestle et al., 1997, Viac et al., 1997). Hence, both, the paucity of B7-molecule expression and function of tumor-infiltrating APC as well as the lack of B7 and CD40 expression on epidermal tumor cells, might account for the failure of tumor-infiltrating T cells to become fully activated, thus allowing tumor escape from immunosurveillance. In conclusion, over the years significant evidence has accumulated that both, B7-CD28 and CD40-CD40L interactions play a central role in neoplastic and inflammatory skin diseases. This may bear direct clinical implications since the manipulation of one or both pathways may offer novel and powerful immunotherapeutic strategies by disrupting costimulation in inflammatory skin diseases and enhancing costimulation in malignant skin diseases. ACNOWLEDGEMENTS This work was supported by grants from the Deutsche Forschungsgemeinschaft (Si 397/7– 1, 8–1) REFERENCES Allison, J.P., Hurwitz, AA., and Leach, D.R. (1995) Manipulation of costimulatory signals to enhance antitumor T-cell responses. Curr Opin Immunol 7:682–686. Arreaza, G.A., Cameron, M.J., Jaramillo, A., Gill, B.M., Hardy, D., Laupland, K.B., Rapoport, M.J., Zucker, P., Chakrabarti, S., Chensue, S.W., Qin, H.Y., Singh, B., and Delovitch, T.L. (1997) Neonatal activation of CD28 signaling overcomes T cell anergy and prevents autoimmune diabetes by an IL-4-dependent mechanism. J Clin Invest 100: 2243–2253. Banchereau, J. and Steinman, R.M. (1998) Dendritic cells and the control of immunity. Nature 392: 245–252. Bjorck, P., Banchereau, J., and Flores-Romo, L. (1997) CD40 ligation counteracts Fas-induced apoptosis of human dendritic cells. Int Immunol 9:365–372. Blair, P.J., Riley, J.L., Levine, B.L., Lee, K.P., Craighead, N., Francomano, T., Perfetto, S.J., Gray, G.S., Carreno, B.M., and June, C.H. (1998) CTLA-4 ligation delivers a unique signal to resting human CD4 T cells that inhibits interleukin-2 secretion but allows Bcl-X(L) induction. J Immunol 160:12–15. Blazar, B.R., Sharpe, A.H., Taylor, P.A., Panoskaltsis-Mortari, A., Gray, G.S., Korngold, R, and Vallera, D.A. (1996) Infusion of anti-B7.1 (CD80) and anti-B7.2 (CD86) monoclonal antibodies inhibits murine graft-versus-host disease lethality in part via direct effects on CD4+ and CD8+ T cells. J Immunol 157:3250–3259. Blazar, B.R., Taylor, P.A., Panoskaltsis-Mortari, A., Buhlman, J., Xu, J., Flavell, R.A., Korngold, R., Noelle, R., and Vallera, D.A. (1997) Blockade of CD40 ligand-CD40 interaction impairs


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Lenschow, D.J., Walunas, T.L., and Bluestone, J.A. (1996a) CD28/B7 system of T cell costimulation. Annu Rev Immunol 14, 233–258. Lenschow, D.J., Herold, K.C., Rhee, L., Patel, B., Koons, A., Qin, H.Y., Fuchs, E., Singh, B., Thompson, C.B., and Bluestone, J.A. (1996b) CD28/B7 regulation of Th1 and Th2 subsets in the development of autoimmune diabetes. Immunity 5:285–293. Li, Y., Hellstrom, K.E., Newby, S.A., and Chen, L. (1996) Costimulation by CD48 and B7–1 induces immunity against poorly immunogenic tumors. J Exp Med 183:639–644. Lin, R.H., Hwang, Y.W., Yang, B.C., and Lin, C.S. (1997) TNF receptor-2-triggered apoptosis is associated with the down-regulation of Bcl-xL on activated T cells and can be prevented by CD28 costimulation. J Immunol 158:598–603. Linsley, P.S., Bradshaw, J., Greene, J., Peach, R., Bennett, K.L., and Mittler, R.S. (1996) Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement. Immunity 4:535–543. Mackey, M.F., Gunn, J.R., Ting, P.P., Kikutani, H., Dranoff, G., Noelle, R.J., and Barth, R.J. Jr. (1997) Protective immunity induced by tumor vaccines requires interaction between CD40 and its ligand, CD154. Cancer Res 57:2569–2574. Martin-Fontecha, A., Cavallo, F., Bellone, M., Heltai, S., lezzi, G., Tornaghi, P., Nabavi, N., Forni, G., Dellabona, P., and Casorati, G. (1996) Heterogeneous effects of B7–1 and B7– 2 in the induction of both protective and therapeutic anti-tumor immunity against different mouse tumors. Eur J Immunol 26:1851–1859. Melero, L, Bach, N., Hellstrom, K.E., Aruffo, A., Mittler, R.S., and Chen, L. (1998) Amplification of tumor immunity by gene transfer of the co-stimulatory 4–1BB ligand: synergy with the CD28 co-stimulatory pathway. Eur J Immunol 28:1116–1121. Miller, S.D., Vanderlugt, C.L., Lenschow, D.J., Pope, J.G., Karandikar, N.J., Dal Canto, M.C., and Bluestone, J.A. (1995) Blockade of CD28/B7–1 interaction prevents epitope spreading and clinical relapses of murine EAE. Immunity 3:739–745. Mitra, R.S., Judge, T.A., Nestle, F.O., Turka, L.A., and Nickoloff, B.J. (1995) Psoriatic skinderived dendritic cell function is inhibited by exogenous IL-10. Differential modulation of B7– 1 (CD80) andB7–2 (CD86) expression. J Immunol 154:2668–2677. Mosmann, T.R. and Sad, S.(1996) The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 17:138–146. Mueller, D.L., Seiffert, S., Fang, W., and Behrens, T.W. (1996) Differential regulation of bcl-2 and bcl-x by CD3, CD28, and the IL-2 receptor in cloned CD4+ helper T cells. A model for the long-term survival of memory cells. J Immunol 156:1764–1771. Nakajima, A., Azuma, M., Kodera, S., Nuriya, S., Terashi, A., Abe, M., Hirose, S., Shirai, T., Yagita, H., and Okumura, K (1995) Preferential dependence of autoant body production in murine lupus on CD86 costimulatory molecule. Eur J Immunol 25:3060–3069. Nestle, F.O., Burg, G., Fah, J., Wrone-Smith, T., and Nickoloff, B.J. (1997) Human sunlightinduced basal-cell-carcinoma-associated dendritic cells are deficient in T cell co-stimulatory molecules and are impaired as antigen-presenting cells. Am J Pathol 150: 641–651. Nickoloff, B.J., Nestle, F.O., Zheng, X.G., and Turka, L.A. (1994) T lymphocytes in skin lesions of psoriasis and mycosis fungoides express B7–1: a ligand for CD28. Blood 83: 2580–2586. Noel, P.J., Boise, L.H., Green, J.M., and Thompson, C.B. (1996) CD28 costimulation prevents cell death during primary T cell activation. J Immunol 157:636–642. Olthoff, K.M., Judge, T.A., Gelman, A.E., da Shen, X., Hancock, W.W., Turka, L.A., and Shaked, A. (1998) Adenovirus-mediated gene transfer into cold-preserved liver allografts: survival pattern and unresponsiveness following transduction with CTLA4Ig. Nat Med 4: 194–200.


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11. ANIMAL MODELS OF SKIN INFLAMMATION BENJAMIN E.RICH AND THOMAS S.KUPPER

INTRODUCTION As the major interface with the environment, the skin encounters a steady array of injuries and incursions of foreign materials and pathogens. In response to these continuous challenges, an effective system of cutaneous immunity has evolved to defend the skin against pathogens, to expel foreign materials and to facilitate the repair process. The detection of foreign antigen within tissue initiates a series of reactions that result in a concerted set of physiological changes. Inflammation is the most outwardly evident of these changes. In the normal course of events inflammation serves to protect the organism and initiate the repair of damaged tissue; however, in numerous different circumstances inappropriate inflammation can itself be pathological. The imperative to understand and develop means to control pathological inflammation of skin has led to the study of animals with cutaneous inflammatory disorders. Furthermore, the development of genetic engineering methods has led to the creation of a number of strains of mice in which improper expression of certain genes causes inflammatory disorders. In this chapter we will review the use of such animals to study the process of inflammation in vivo. NORMAL CUTANEOUS INFLAMMATION The normal cutaneous immune response involves at least two qualitatively distinct components. While these components have distinct mechanisms with different triggering events and ultimate outcomes, they each contribute to the successful defense of the cutaneous barrier. One component, called immediate hypersensitivity, can be a nearly instantaneously response to the presence of antigens within the tissue. Immediate hypersensitivity ensues when these antigens are engaged by antigen-specific IgE bound to mast cells. This triggers those mast cells to activate and release factors including vasoactive amines which permeablize cutaneous vessels thereby allowing blood cells to enter the tissue. This initial inflammation subsides within an hour or so, however several hours later a second cellular infiltrate arises at the site of antigen exposure. In this secondary inflammation effector cells, including basophils, neutrophils and eosinophils, seek out and destroy pathogens such as parasites. The immediate hypersensitive response is wholly dependent upon pre-existing antigen-specific IgE molecules. The rapid kinetics

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of the reaction results from the direct activation of mast cells via the IgE molecules. Therefore previous exposure to the antigen is a prerequisite for the response. Indeed, adoptive transfer of antigen-specific IgE molecules from an immunized animal to a naive recipient conveys the ability to mount an immediate hypersensitive response. Unlike the immediate hypersensitive response, the delayed hypersensitive response has no immediate inflammatory phase. Rather, a cellular infiltration and inflammation in response to the detection of foreign antigens within the tissue occurs only after several hours. While this reaction is also dependent upon previous immunization with the antigen, it is unlike the immediate hypersensitive response in that adoptive transfer of cells from an immunized animal can confer reactivity to a naive recipient but serum (cell free immunoglobulins) cannot. The delay in the response is due to the time required for antigen presenting cells (APCs) to take up antigenic molecules, process them internally and display peptides on their major histocompatability complex (MHC) molecules for presentation to T cells. At the same time the APCs migrate to lymphoid tissue where they encounter and activate T cells which facilitate the inflammatory response. SECONDARY EFFECTS OF INFLAMMATION: PERTURBATION OF SKIN ARCHITECTURE In addition to the infiltration of effector cells, the normal process of inflammation also causes secondary effects on the structure of the skin. One of the most prominent effects of inflammation on the structure of the skin involves alterations of the growth and differentiation behavior of epidermal keratinocytes. Epidermal keratinocytes ordinarily divide in the basal layer and undergo a process of differentiation while they move towards the surface as a result of proliferation below and ablation above. Therefore the structure of the epidermis is determined by the balance among the rates at which the keratinocytes divide, differentiate to cornified squamous cells, migrate to the surface and slough off. This epidermal homeostasis is altered when the rate of cell division of basal keratinocytes increases as a result of signals associated with the inflammation, such as cytokines released by infiltrating cells. This phenomenon seems to make sense in the context of a wound or pathogenic intrusion because more keratinocytes may be needed to repair the lesion. However in prolonged pathological inflammations, such as psoriasis, the hyperproliferation of keratinocytes can significantly alter the architecture and functional properties of the skin and become a significant aspect of the pathology. The most prominent perturbation of the epidermis seen in pathological inflammations is an increase in the number of keratinocytes and the thickness of the epidermal layer of living cells. This phenomenon, termed acanthosis, is often accompanied by elongation of the ridges at the dermal/epidermal junction increasing the convolutions and hence the area of the dermal/epidermal interface. This can in turn, provide a greater surface area for the interactions between the dermis and the epidermis. A second alteration of the epidermal structure that is often associated with acanthosis is hyperkeratosis, a thickening of the layer of cornified cells called the stratum corneum. Hyperkeratosis generally appears to be due to an increased rate of production of cornified keratinocytes (corneocytes) however it may also be due to changes in the integrity of the

ANIMAL MODELS OF SKIN INFLAMMATION 223

stratum corneum and a reduced rate of sloughing of the outer cells. This feature is generally the most evident at the macroscopic level. A third morphological feature that is associated with disturbances in epidermal homeostasis is parakeratosis or the presence of imperfections and gaps between cells in the stratum corneum. These perturbations are generally associated with the appearance of incompletely differentiated cells with nuclei in the stratum corneum. This can arise as a result of the program of differentiation of keratinocytes being delayed relative to the proliferation of the basal layer. Parakeratosis may also caused by certain types of trauma which cause cellular injury such as “tape stripping”. Regardless of the etiology, parakeratosis can be associated with degradation of the barrier properties of the skin. Cellular infiltrates are often seen in the dermis and occasionally in the epidermis of inflamed skin. These may consist of myeloid and/or lymphoid cells and are often seen clustered around dermal vessels. Dermal vasculature may be dilated or otherwise compromised. Another mechanism by which inflammation can disrupt the skin architecture is hydrostatic pressure. Perhaps the most clear example of this is in the autoimmune disease bullous pemphigoid in which autoantibodies reactive with the basal layer provoke extensive release of fluid from vessels near the junction between the epidermis and the dermis. This fluid accumulates and forces the epidermis away from the dermis forming blisters. Another example of the effects of hydrostatic pressure is spongiosis where fluid is forced between keratinocytes leaving only desmosomal contacts between the cells. This leads to a characteristic spongy appearance of the tissue in which keratinocytes assume a star-shaped morphology as a result of the tension on the desmosomes. This is distinct from acantholysis in which cell to cell contacts are lost and keratinocytes become round. This can be caused by extreme fluid infiltration, in which case it is called secondary acantholysis. In contrast, primary acantholysis is caused by impairment of the function of cell to cell adhesion molecules by autoantibodies or genetic defects. CONTACT HYPERSENSITIVITY: A MODEL OF SKIN INFLAMMATION Contact hypersensitivity (CHS) is an immunological reaction in which epicutaneous administration of various compounds elicits a T cell mediated inflammatory response in the skin. A standardized experimental method for the generation and measurement of CHS has been developed and utilized extensively. Experimental CHS has proven to be a very useful model for the study of delayed-type hypersensitivity as well as cutaneous immune responses. Detailed methodology of CHS reactions are described elsewhere (1). While some materials that provoke contact hypersensitivity are inherently antigenic, many are chemically reactive compounds, called haptens, that modify skin cell proteins which then become antigenic. Compounds that have been used to provoke CHS include picryl chloride (2,4,6-trinitrochlorobenzene), fluorescein isothiocyanate, oxazalone and dinitrofluorobenzene. In addition to antigenicity, the general irritant properties of the haptenizing compound are important since it has been shown that the simultaneous


application of an unrelated irritant compound with the antigen greatly enhances the inflammatory response to low levels of the antigen (2). Experimental CHS is a cutaneous form of a delayed-type hypersensitive response in that the eliciting exposure to the sensitizing agent does not provoke an immediate response. It requires at least two exposures to the sensitizing agent to cause the reaction and these exposures must be separated by several days to a week. Furthermore, the second, eliciting exposure can be at a site distal to the site of initial sensitization. Therefore it relies upon both afferent and efferent arms of the immune system and is a sensitive measure of their combined action. The inflammatory lesions that arise in response to the second exposure to the sensitizing agent are characterized by edema and perivascular cellular infiltrates. If the second application of the sensitizing agent is applied to an ear, the resulting edema and thickening of the ear provides a convenient index of the extent of the inflammation. The quantitative measurement of the CHS reaction in mouse ears was first described in 1968 (3). Since then it has been exploited extensively as a simple and reliable measure of immunological function. Much of our current understanding of cellular immune responses is derived from experiments using this model. IMMEDIATE HYPERSENSITIVITY: A MODEL OF ATOPIC DERMATITIS Recently there has been increased interest in immediate hypersensitivity provoked by model antigens such as chicken ovalbumin (OVA). This research has focused on airway and cutaneous inflammatory responses after sensitization by direct application of the antigen to the skin. In this method, a concentrated aqueous solution of OVA (1 mg/ml) is absorbed in cotton gauze and held against the skin by an adhesive patch dressing for one week. This sort of topical immunization appears to favor Th2 type responses and generates the requisite antigen specific IgE titers for a constrictive airway response upon subsequent challenge in the lungs. In addition to airway hypersensitivity, Balb/c mice repeatedly challenged by epicutaneous application of OVA develop lesions exhibiting acanthosis and dermal infiltrates of eosinophils, neutrophils and lymphocytes that are very similar to human atopic dermatitis (4). REGULATORY CIRCUITS IN INFLAMMATION In order to maintain healthy skin, cutaneous inflammation must be rapidly inducible to respond effectively to environmental and infectious challenges, but the effects of inflammation can be deleterious, therefore the inflammation must also be controllable. While the large number of cell types and gene products involved in inflammation clearly make the control of inflammation an enormously complex phenomenon, some simple regulatory mechanisms in the skin have been identified. One of these is the autocrine and paracrine induction of proinflammatory genes by interleukin 1α (IL-1α), IL-1β and tumor necrosis factor (TNFα) (5). IL-1α and TNFα stimulate activation of the transcription factor NF-κB in target cells. NF-κB acts directly


to promote transcription of the IL-1α , IL-1β and TNFα genes, causing more release of these factors. Thus a small induction of either IL-1 or TNFα can be amplified and perpetuated within one cell and this induction can be communicated to other adjacent cells by diffusion of the cytokines. This inherently unstable control circuit is modulated by the action of I-κB, an efficient intracellular inhibitor of NF-κB. As discussed below, in the description of motheaten mice, the absence or impaired function of I-κB leads to runaway signaling and profound pathological inflammatory processes. A second regulatory circuit in cutaneous inflammation involves a reciprocal paracrine dialog between keratinocytes and T cells (6, 7). Activated T cells can release interferon γ (IFN-γ) among other products. Keratinocytes exposed to IFN-γ increase transcription of the IL-7 gene and secrete more of this factor. IL-7 is absorbed into extracellular matrix and thus does not diffuse rapidly, though it remains fully active. As a potent mitogen for activated T cells, IL-7 augments their IL-2-mediated autocrine growth stimulation, upregulates expression of IL-2 receptor (IL-2R) and acts to block activation induced programmed cell death. Therefore keratinocytes in the vicinity of activated T cells are stimulated to enhance the activity of those T cells by release of IL-7. Conversely, the expanded population of T cells will release more IFN-γ, perpetuating the cycle. Since this control circuit involves two cell types it is likely to be slower and perhaps less unstable than the IL-1/TNF-α circuit. Nonetheless, this reciprocal positive regulatory loop is likely to have a role in normal inflammation. As described below, deregulated expression of either IL-7 or IFN-γ provokes chronic inflammatory disorders. It remains to be determined to what extent this sort of runaway reciprocal intercellular signaling may contribute to human disease. SPONTANEOUS GENETIC MODELS A number of mutant strains of mice with aberrant cutaneous inflammation have been identified (8). Some of these are similar to human diseases while others are distinct. We present here a brief discussion of a few of the more prominent of these. The Flaky Skin Mouse The flaky skin mouse, which arose spontaneously at The Jackson Laboratory in Bar Harbor, Maine, USA, carries an autosomal recessive mutation, fsn, that causes a cutaneous disorder that resembles human psoriasis. The fsn gene has been mapped to a locus at 56 centimorgans of mouse chromosome 17 but has not been molecularly characterized, flaky skin mice have sparse fur, and a scaly and thickened epidermis. A number of ultrastructural features of the disorder are also found in psoriasis vulgaris (9). The flaky skin phenotype is transferred to SCID mice by transplant of fsn/fsn bone marrow indicating that the defect is primarily in bone marrow derived cells. Like human psoriasis, the lesions of the flaky skin mouse are significantly diminished by administration of cyclosporine. While a direct effect of cyclosporine on keratinocytes has not been ruled out, it appears likely that the interference with the activation of T cells and their production of inflammatory cytokines is the basis of this phenomenon. Therefore, in


addition to the morphological resemblance of the disorder of the flaky skin mouse to human psoriasis, it is likely that both diseases involve some sort of reactive T cells. However, recent reports of extensive systemic autoimmunity in the flaky skin mouse may point to an etiology that is distinct from that of most human psoriasis (10, 11). The Scurfy Mouse A recessive mutation in the murine X-linked scurfy gene, sfy, causes an immunodeficiency with extensive leukocyte infiltration of cutaneous tissues. The scurfy disorder appears to involve improper maturation of T cells since the disease is suppressed in nude mice, can be transmitted to immunodeficient mice by T cell transplants and fails to develop in wildtype mice that have been engrafted with scurfy bone marrow (12). The disorder has been shown to be caused by a class of CD4+ T cells and is associated with increased expression of certain cytokines including IL-4, IL-6, IL-7 and TNF-α (13). The scurfy disease has similarities with the X-linked recessive human immunodeficiency disease Wiskott-Aldrich Syndrome (WAS) and interestingly the scurfy mutation maps very close to the murine homolog of the WAS gene. This has lead to the suggestion that the two disorders may be mechanistically related (14). While this has not been absolutely ruled out, the recent observation that mice with an engineered null mutation of the WAS gene have T cell defects but do not have the characteristic extreme cutaneous inflammation of the scurfy disorder (15) makes it seem likely that these two genes are distinct. The NC/Nga Mouse The inbred NC/Nga strain of mice exhibits spontaneous skin lesions that appear similar to human atopic dermatitis (16). Lesions fail to arise in NC/Nga mice housed under pathogen-free conditions implying that development of these lesions is dependent upon exposure to certain pathogens. The disorder seems mechanistically related to atopic dermatitis in that the levels of circulating IgE in the NC/Nga mice are elevated and individuals with higher IgE levels tend to have more severe dermatitis. This syndrome has been shown to be a multigenic trait in that the dermatitis segregates from the elevated IgE when the NC/Nga mice are crossed with Balb/c mice. The dermatitis appears to be caused by a single recessive gene while the elevated IgE is caused by cooperation of two recessive genes (17). Thus it remains somewhat unclear to what extent the elevated level of IgE in the NC/Nga mice contributes to their disorder. Nonetheless, the recent demonstration that topical application of the immunosuppressive agent FK506 can mitigate the disease of NC/Nga mice lends further support to the role of IL-4 production by Th2 cells in atopy and may lead to the use of this compound to treat human atopic dermatitis (18). The Chronic Proliferative Dermatitis Mouse Chronic proliferative dermatitis (cpdm) is an autosomal recessive mutation that arose spontaneously in C57BL/Ka mice (19). The phenotype associated with this gene is first


evident as inflammatory skin lesions that appear spontaneously on the back and belly at 5 to 6 weeks of age. While the lesions spread progressively over the trunk, the ears, tail and footpads are spared. The epidermis within the lesion is hyperproliferative and parakeratosis and hyperkeratosis are evident. The dermal vasculature in the lesions is enlarged and cellular infiltrates are found in the dermis and epidermis that consist primarily of myeloid cells such as granulocytes, eosinophils and macrophages. Mast cells are found to accumulate in the dermis of lesional skin. Increased numbers of IgE+ mast cells are found in tissues of cpdm/ cpdm mice prior to the development of gross symptoms while the mast cells in wild type mice are IgE− (20). Treatment of the mice with cyclosporin had little effect on the cutaneous symptoms, however corticosteroid treatment was effective in reducing the lesions (21). Transplantation of spleen or bone marrow to syngeneic mice failed to confer the disease on the recipients. Skin grafted from cpdm/ cpdm mice onto unaffected syngeneic or nude mice retained the inflammatory phenotype but did not confer the disorder to host tissue. Conversely, skin from wild type mice grafted onto cpdm/ cpdm mice remained healthy (22). It is clear that the genotype of resident skin cells is critical for the disease to develop and the genotype of the hematopoietic cells is not. The binding of neutrophils to frozen sections of affected skin is elevated while lymphocyte binding to the sections is not (23). This reflects increased levels of expression of ICAM-1 and L-selectin and low expression of E-selectin in the skin. Thus it appears that the defect caused by the cpdm mutation is in resident cells of the skin. This alteration seems to provoke inflammatory behavior in myeloid cells which may, in turn, elicit further abnormalities in the resident skin cells. The Motheaten Mouse Motheaten mice have an autosomal recessive disorder in which cutaneous inflammation is one aspect of a complex immunodeficiency syndrome (24, 25). Their skin abnormality is evident soon after birth as inflammatory cutaneous infiltrates involving granulocytes. These inflammations lead to alopecia and give rise to the characteristic “motheaten” appearance of the mice. Adoptive transfer of splenocytes conveys the disease to syngeneic recipients indicating that the defect is in hematopoietic cells (26). There are two variants of motheaten mice that differ by the severity of their diseases. The original motheaten mice live an average of 22 days while “viable motheaten” mice live an average of 61 days. Both of these were discovered to be caused by mutations in the protein-tyrosine phosphatase gene Hcph (27). The product of this gene appears to act in removing phosphate groups from molecules involved in signal transduction from cytokine receptors, thereby limiting the duration and extent of the signals conveyed. In its absence these signals persist, leading to pathological responses. This is demonstrated by the response of motheaten macrophages to GM-CSF which has been shown to be significantly exaggerated (28). Perhaps the most significant substrate of the phosphatase is IκB, the inhibitor of the transcription factor NF-κB. When IκB is phosphorylated it becomes targeted for proteolytic destruction by ubiquitination. In the absence of IκB, NF-κB is able to translocate to the nucleus and initiate transcription of a number of genes involved


in immune responses, such as cytokines, chemokines, adhesion molecules and enzymes involved in effector functions. Among these are the genes encoding IL-1β and TNF-α. As mentioned above, these two genes have been implicated in the perpetuation of inflammatory reactions since their products activate the NF-κB pathway, creating a positive regulatory loop (5). The activity of this loop is enhanced by the rapid destruction of IκB leading to activation of NF-κB. This model is further supported by the observation of an even more severe, neonatal-lethal inflammatory disorder that arises in gene-targeted mice lacking the IκBα gene (29). Furthermore, transgenic mice expressing TNF-α under the control of the K14 promoter have cutaneous lesions as well as systemic cachexia (30). Recently motheaten mice were used to test the ability of soluble TNFR-1 to block the action of TNF-α in vivo (31). Motheaten mice treated with this compound had twice the lifespan and significant reductions in arthritis, pneumonitis and skin lesions as compared to motheaten mice receiving control treatment. The success of this study confirms the critical role of TNF-α in this inflammatory disorder and illustrates the value of motheaten mice in evaluating candidate therapeutics. ENGINEERED GENETIC MODELS: CYTOKINES EXPRESSED IN SKIN In efforts to elucidate the roles of various biologically active molecules, a number of investigators have created transgenic mice that express these molecules inappropriately. Among the different phenotypes exhibited by these transgenic mice, several of them develop inflammatory disorders of the skin. Foremost among these are transgenic mice expressing cytokines. One of the earliest examples of cutaneous inflammation caused by deregulated cytokine expression is a strain of transgenic mice in which IL-2 was expressed under the control of the MHC class I promoter (H2Kd) in order to test the possibility that broad expression of IL-2 might support indiscriminate maturation of T cells and lead to autoimmunity (32). While no specific autoimmunity was detected in these mice, they were found to have spontaneous alopecia that develops at about 8 weeks of age as well as pneumonia and splenic hypercellularity. The affected skin exhibited thickened epidermis, absent or disfigured hair follicles and sporadic lymphocytic infiltrates. H2Kd-IL2 mice examined at 5 weeks, before the alopecia developed, had increased numbers of Thy-1+ dendritic epidermal T cells (DETC) but very little signs of cutaneous inflammation or any proliferative disorder. The mechanism behind the profound cutaneous inflammation in older H2Kd-IL2 mice is not entirely clear. Since IL-2 acts principally on lymphocytes and is not known to exert any direct proliferative effects on keratinocytes it is likely that the effect on epidermis is indirect and mediated by lymphocytes. When skin grafts were performed between wild type and transgenic mice, only the trans-genic skin became affected; in each configuration the wild-type skin was spared. This demonstrated that the skin disorder is caused by expression of the transgene in resident skin cells and that the effects of the IL-2 expression on circulating cells do not persist when those cells migrate to non-transgenic tissue. The


proinflammatory behavior of the circulating cells must be sustained by localized exposure to IL-2. The specific lymphocytes involved in the infiltrates are also unclear. The authors postulated involvement of Thy-1+ DETC because they are ubiquitous in the epidermis and their numbers are expanded prior to the development of alopecia. On the other hand, the subcutaneous lymphocytic infiltrates were seen only in association with the alopecia and acanthosis. While IL-2 is not directly chemotactic for T cells, it can have an indirect effect in that it blocks their chemotactic responses to certain chemokines (33). Thus T cells passing through tissue in response to relatively long range chemokine gradients may stop when they encounter high levels of IL-2. This might cause T cells to linger and accumulate in the skin of these mice. MHC class I genes are active in a broad spectrum of cells including keratinocytes, and their expression is enhanced by exposure to cytokines such as interferons (IFN), TNF-α and lymphotoxin (LT) that can be produced by activated T cells (34, 35). The IL-2 released by keratinocytes will enhance the proliferation and viability of activated T cells. Those activated T cells can, in turn, release factors that are mitogenic for keratinocytes and also induce higher expression of endogenous MHC genes and the transgenic IL-2. Therefore these reciprocal agonistic signals between lymphocytes and the transgenic keratinocytes may constitute a dialog involving mutual activation of lymphocytes and keratinocytes that leads to inflammation. While exogenous IL-2 will clearly enhance the response of activated T cells, their activation will necessarily depend upon T cell receptor (TCR) mediated signals. No autoreactive antibodies were detected in these mice and adoptive transfer of splenocytes failed to convey the disease to recipients. Thus the role of any autoantigens in this disease is uncertain. IL-7 AND SKIN The IL-7 signaling pathway overlaps the IL-2 signaling pathway in that they both utilize the common gamma chain (γc) which transmits signals via the JAK-3 kinase (36). However, unlike IL-2 which is principally expressed by activated lymphocytes, IL-7 is normally a product of epithelial cells. IL-7 was first identified as a growth factor for immature B lymphocytes that is produced by bone marrow stromal cells (37). Subsequently it was found to stimulate proliferation of thymocytes (38–42) and to be expressed at high levels in thymic epithelial cells (37, 43). Mature B cells generally lose their responsiveness to IL-7 however most T cells remain sensitive to IL-7 (44). The most clearly understood role of IL-7 is in the development of lymphocytes and the maintenance of lymphocyte homeostasis. IL-7 plays a central role in the expansion of early lymphocyte populations and the rearrangement of antigen receptor genes that is essential for generation of their diversity. This is demonstrated by the severely reduced numbers of T and B lymphocytes found in IL-7 deficient mice (45, 46). The IL-7 mediated signaling that controls lymphoid development and homeostasis appears to originate from the thymus and bone marrow. However, in addition to bone marrow and thymic stromal cells, IL-7 has also been shown to be expressed by keratinocytes (47) as well as certain epithelial


cells of the intestine (48). The expression of IL-7 in other epithelial tissues may be more important for regulating the behavior of intraepithelial lymphocytes. γδ T cells, including DETC, are particularly responsive and dependent upon IL-7 (6, 49–51). Much less is known about IL-7 mediated signaling in the immune response. A number of effects of IL-7 signaling on T cell responses have been detected in vitro and in vivo, however the role of IL-7 in reactive immunity has not been well denned. IL-7 stimulates proliferation and enhances the viability of many mature T cells, particularly after TCR stimulation (52). The expression of the IL-2R in T cells is induced by exposure to IL-7 thereby increasing the sensitivity of these cells to IL-2 (53). The responsiveness of T cells to IL-7 is enhanced by their activation which has been shown to bring the IL-7 receptor into association with the γc chain (54). IL-7 also specifically promotes the development and function of lymphokine activated killer (LAK) cells (55–57) and cytotoxic T lymphocytes (CTL) (57, 58). Several observations point to a significant role for IL-7 in skin biology. As discussed above, IL-7 and IFN-γ expression constitute a regulatory circuit involving T cells and keratinocytes. While epidermal keratinocytes appear to express IL-7 constitutively, they are induced to produce higher levels by exposure to IFN-γ (6, 47). The secreted IL-7 supports the viability and prevents apoptosis of DETC and other T cells in the skin. These T cells may indeed be the source of the IFN- γ. Therefore, intraepidermal T cells that become activated and release IFN-γ are likely to encounter reciprocally expressed IL-7 that will in turn enhance their viability. This reciprocal dialog between cutaneous T cells and keratinocytes may be a mechanism that maintains homeostasis of the cutaneous immune system. It may also be a means by which the active participation of keratinocytes enhances the response of intraepithelial T cells (6, 7). Human cutaneous T cell lymphomas (CTCL) are distinguished by their propensity to migrate into the dermis and epidermis and cause pathological inflammatory lesions. CTCL cells have been found to proliferate in response to IL-7 (59–61) and many have detectable autocrine expression of IL-7 (59). However, the expression of the IL-7R by individual tumors does not appear to correlate with their epidermotropism (62). IL-7 is unusual among cytokines in that it can be absorbed into extracellular matrix (ECM) (63–65). Moreover, T cells that encounter IL-7 (either free or matrix-bound) are induced to bind to ECM components such as fibronectin as well as to the adhesion molecule VCAM-1, that is expressed by stimulated endothelial cells (66). This mechanistic link between the IL-7 signaling pathway and T cell extravasation may provide insight into the apparently unique role of IL-7 in the skin. Three different strains of transgenic mice have been generated in which deregulated expression of IL-7 leads to cutaneous infiltrates of lymphocytes and profound cutaneous inflammatory disorders. One of these expresses IL-7 in lymphocytes that are responsive to the IL-7 in an autocrine fashion. A second strain expresses IL-7 under the control of viral sequences that appear to be active in skin. The transgene in third strain directs expression of IL-7 to keratinocytes and therefore delivers IL-7 to responsive cells in a paracrine fashion. Transgenic mice carrying an IL-7 cDNA under the control of immunoglobulin heavy chain promoter and enhancer sequences (EµPµ-IL7) develop a progressive cutaneous


Figure 11.1 Progression of inflammatory disorder in EµPµ-IL7 transgenic mice. Three representative EµPµ-IL7 transgenic mice illustrate the progressive nature of the spontaneous lesions. A 57 day old mouse exhibits minimal skin disorders. Prominent lesions are seen on a 115 day old mouse and a 235 day old mouse is nearly fully denuded with extensive alopecia, inflammation and hyperkeratinization.

disorder involving inflammatory infiltrates leading to alopecia (67). In addition to the cutaneous disorder these mice have expanded populations of lymphocytes and develop sporadic lymphomas that both express and respond to IL-7. The Igµ promoter is active in both T and B cells and therefore both populations are affected. In contrast to the stochastic kinetics of the lymphomas, the cutaneous lesions develop in adult mice in a very uniform fashion. They are first evident at about 12 weeks of age as a thinning of fur on the belly and then progress until the trunk is wholly denuded (see Figure 11.1). Histological examination of the lesions reveals lymphoid infiltrates in the dermis and a hyperproliferative epidermis. Occasional foci of lymphocytes in the epidermis are seen. Acanthosis, hyperkeratosis and parakeratosis are evident and hair follicles appear to be lost in the hyperproliferation (see Figure 11.2). The infiltrating cells are predominately T cells that lack expression of the co-receptors CD4 or CD8. Both αβ and γδ T cells are present in the infiltrates, however αβ cells appear to predominate. When affected skin or dissociated lymphoid tissues were transplanted to syngeneic mice the characteristic skin lesions arose in the recipients. While no clonal TCR gene rearrangements were detected in the affected skin of transgenic mice, TCR-β gene rearrangements were found in the affected skin of the graft recipients. Therefore it appears that the skin infiltrating lymphocytes of the µEµP-IL7 transgenic mice are initially polyclonal, however clonal variants arise and are enriched by selection in transplant experiments. It is clear that the autocrine expression of IL-7 in these mice leads to the development of a class of T lymphocytes that are predisposed to migrate into and accumulate in the dermis. These


Figure 11.2 Histological micrographs of skin from wild type and µEµP -IL7 transgenic mice. Standard paraffin preparations of wild type (left) and µEµP-IL7 transgenic (right) skin were stained with hematoxylin and eosin. The epidermis of wild type skin is only a few cells thick and sections of two hair follicles are present in the field. The epidermis of the µEµP-IL7 transgenic skin has markedly thickened epidermis (acanthosis), parakeratosis and hyperkeratosis. Lymphocytic infiltrates are seen in the dermis and hair follicles are absent.

cells then convey proliferative signals to epidermal cells which respond and thereby disrupt the normal architecture of the skin. Viral transcriptional control elements were used to direct the expression of IL7 to lymphocytes in transgenic mice that develop a similar disorder involving cutaneous lymphoid infiltrates (51). The cellular infiltrates found in the skin lesions of these mice are predominately composed of γδ T cells, however they are polyclonal and are not predominated by the Vγ5 TCR that is found on all DETC. Recently these cells have been shown to express IL-4 (68). Consistent with this is the finding that the serum levels of IgE are elevated in these mice, possibly indicating a Th2 phenotype for the expanded population of lymphocytes in the skin. The effects of deregulated paracrine expression of IL-7 on lymphocytes in the skin was investigated by creating a strain of transgenic mice in which an IL-7 cDNA was placed under the control of the human keratin 14 promoter (K14-IL7) leading to strong expression in basal keratinocytes (69). Much like the autocrine IL-7 transgenic mice, these mice develop inflammatory infiltrates of T lymphocytes that lead to alopecia. However, unlike the autocrine IL-7 transgenic mice, the cutaneous alterations are evident in young animals and appear most severe at about 3 weeks of age. Thereafter they become less intense although a characteristic inflammation of the eyelids remains prominent


throughout life. Many of the lymphocytes that accumulate in the dermis of each of these IL-7 transgenic mice are T cells that express intermediate levels of the TCR complex in addition to the signaling natural killer (NK) cell receptor NK1.1. Thus they appear related to the T cells of similar phenotype termed intermediate T cells that mature without passing through the thymus (70). Indeed, the characteristic skin lesions arise in nude mice bearing the µEµP-IL7 transgene demonstrating that the pathogenic cells are not dependent upon maturation in the thymus (67). However, the autocrine expression of IL-7 in T cells appears to relieve them of the need to mature in the thymus (71). The fact that these cells have the NK1.1 receptor raises the possibility that they may be able to perpetuate inflammatory reactions in the absence of foreign antigens since the NK1.1 receptor is able to convey activation signals when it engages cognate receptor (72). IFN-γ AND SKIN IFN-γ is a key component to the model of keratinocyte—T cell dialog discussed above. To evaluate the effects of constitutive signaling by IFN-γ, transgenic mice were generated in which IFN-γ is expressed under the control of the involucrin promoter which is active in suprabasal keratinocytes (73). These mice have variable skin abnormalities ranging from simple hypopigmentation to gross scaling, flaking and hair loss. The more severely affected skin is characterized by cellular infiltrates in the dermis, dilated dermal capillaries, acanthosis, parakeratosis and hyperkeratosis. The keratinocytes are hyperproliferative and express a proliferation-associated antigen Ki-67 as well as ICAM-1 and MHC class II molecules. The response of these mice to CHS was exaggerated and prolonged. Since IL-7 expression of keratinocytes is stimulated by IFN-γ, it is likely that a significant component of the inflammatory disorder seen in these mice is related to the release of IL-7 by keratinocytes and thus similar to the K14-IL7 transgenic mice discussed above. The phenotypes of the two strains are largely similar, however, hypopigmentation was not seen in the few pigmented K14-IL7 mice that were examined. IL-1 AND SKIN As discussed briefly above, IL-1 is a central signaling molecule of the immune system in that it induces several types of cells to synthesize and release other cytokines and chemokines that recruit effector cells and coordinate inflammation. The high levels of expression of IL-1 and the IL-1R by keratinocytes in inflamed skin led to the speculation that autocrine stimulation of keratinocytes helps to enhance and perpetuate inflammatory reactions in the skin. Strains of transgenic mice expressing IL-1α or the IL-1R in the skin were generated to test the hypothesis that IL-1 signaling can initiate and maintain inflammation. Transgenic mice expressing IL-1α in basal keratinocytes under the control of the human K14 promoter develop a spontaneous skin disorder that is marked by hair loss and hyperkeratosis (74). Foci of inflammatory lesions arise in these mice that have cellular infiltrates and hyperproliferative epidermis leading to acanthosis and parakeratosis. Transgenic mice constitutively expressing the type-1 IL-1R (K14-IL1R1) on their keratinocytes have exaggerated responses to IL-1. When the K14-IL1R1 and K14-IL1α


transgenic lines were crossed together, the resulting mice developed spontaneous inflammation that was more severe than either parental strain and appeared similar to the response to PMA, a potent inducer of protein kinases that stimulates expression of IL-1 in keratinocytes (75). The type 2 IL-1R (IL-1R2) binds IL-1 with high affinity but does not appear to transduce any signals. Moreover, the extracellular domain of the molecule can be shed by cells in a soluble form. Therefore it has been considered an inhibitor of the action of IL-1. The IL-1R2 gene appears to be regulated in parallel with the IL-1 gene in keratinocytes and has also been found to be overexpressed in psoriasis. To test the hypothesis that IL-1R2 modulates the activity if IL-1 in vivo, IL-1R2 was expressed under the control of the K-14 promoter in transgenic mice. Basal keratinocytes of these mice express IL-1R2 constitutively and consequently have a blunted responds to induction of IL-1 by PMA while contact hypersensitivity is not affected (76). CHEMOKINE EXPRESSION IN SKIN Chemokines are a large family of structurally related factors that are principally involved in chemotactic migration of leukocytes (77). The ability of the expression of the chemokine JE in epidermal cells to recruit neutrophils was demonstrated by the characterization of transgenic mice expressing this molecule in basal keratinocytes (78). These K14-JE mice have dramatic increases of neutrophils in their dermis and epidermis but do not exhibit any abnormal inflammation. The chemokines MCP-1 and IP-10 are induced in skin during contact hypersensitive reactions (79) and treatment of mice with anti-MCP-1 antibodies inhibits cutaneous DTH reactions by blocking the recruitment of T cells (80). Tissue specific transgenic expression of MCP-1 recruits macrophages and monocytes to those tissues (81, 82) but these cells cause little inflammation in the absence of antigenic challenge. However, when mice expressing MCP-1 in their basal keratinocytes were challenged by CHS their responses were greatly enhanced (81). Thus the expression of MCP-1 appears to induce leukocytes to travel towards the site of expression without promoting effector function by those cells. However, their presence in greater numbers within the skin provides for a greater inflammatory response. By contrast, expression of IP-10 in keratinocytes of transgenic mice does not affect inflammation spontaneously but it does inhibit wound healing and interfere with repair of vasculature (83). KERATINOCYTE EXPRESSION OF INTEGRINS Integrin molecules expressed on the surface of keratinocytes and other cells interact with extracellular matrix. The β1 integrin dimerizes with the α2 integrin to bind collagen, with the α3 integrin to bind laminin and with the α5 integrin to bind fibronectin. The α2β1 and α3βl integrins are present on normal epidermal keratinocytes while the α5βl complex is expressed at higher levels during wound healing in normal skin and constitutively on keratinocytes in psoriatic skin. To investigate the effects of deregulated expression of these integrin molecules, lines of transgenic mice were created that express α2, α5 or β1 integrins under the control of the involucrin promoter (84). Constitutive expression of


the βl integrin in combination with α2 or α5 or by itself, causes a disorder of the epidermis that involves hyperproliferation, altered keratinocyte differentiation and inflammatory lymphocytic infiltrates. Like the flaky skin mouse, the involucrin- βl transgenic mice have a syndrome that shares features of human psoriasis. CONCLUSIONS As the most studied mammalian model of human biology the mouse is close to an ideal system in which to study cutaneous inflammation. While there are significant differences between human and murine skin, the cellular and molecular principles governing the behavior of the skin and its interactions with the immune system are largely conserved. The development of techniques for genetic manipulation of mice has led to the creation of a large number of different mice in which to study various aspects of cutaneous inflammation. While a number of strains exhibit features of human diseases, few have absolutely identical pathology. Ultimately this may reflect a limitation in the mouse as a model because of inherent differences between the species, however it is clear that the underlying similarities make murine models ideal for investigating cutaneous biology and testing candidate therapeutic regimens. REFERENCES 1. 2.

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12. LANGERHANS CELL MIGRATION GEORG STINGL AND DIETER MAURER

DEVELOPMENT OF LANGERHANS CELLS FROM HEMOPOIETIC PRECURSORS (HPC) (FIGURE) Both in humans and the mouse, Langerhans cells (LC) can already be identified in the fetus. Unlike the fetal murine epidermis, which contains phenotypically immature Ia− LC until the end of gestation (1), HLA-DR+/ATPase+ dendritic cells (DC) can be identified in the human epidermis by 6 to 7 weeks of estimated gestational age (2). These cells must originate from HPC in the yolk sac or fetal liver, the primary sites of hemopoiesis during the embryonic period. Until the twelfth week of pregnancy, these cells are CD1a− and lack Birbeck granules (BG). Thereafter, there occurs a dramatic increase in LC CD1a expression, an event which coincides with the initiation of bone marrow function (2). A major breakthrough in the understanding of LC development came from the observation that the exposure of CD34+ HPC to granulocyte/macrophage colonystimulating factor (GM-CSF) and tumor necrosis factor α (TNF-α) gives rise to a progeny of CD1a+, E-cadherin+, BG-containing cells with immunostimulatory properties strikingly resembling those of LC isolated from human skin (3,4). Subsequent studies have tried to delineate the phenotype of LC progenitors at their various states of maturation/ differentiation. It is now quite clear that, already at the CD34+ HPC stage, cells exist which are committed to the LC lineage. An apparently useful marker to identify these cells is the cutaneous lymphocyte-associated antigen (CLA) which is detectable not only on skin-homing T cells but also on LC in situ. CLA is abundantly expressed by LC precursors rather than by cells giving rise to non-LC DC (5). It has yet to be determined whether this molecule, similar to its function on skin-seeking T cells, helps to direct LC/ LC progenitors to the skin. Around day 4–6 of in vitro culture in GM-CSF- and TNF-α supplemented medium, LC precursors become CD1a+ and, upon prolongation of the culture until day 12–14, preferably on fibronectin-coated tissue culture plates (6), develop into typical DC displaying all the features found in and on epidermal LC. Besides the CD1a+ LC precursor, CD14+ CD1a− cells emerge early during the culture. The lineage commitment of these cells is apparently less restricted as they can give rise to a monocyte/macrophage phenotype when exposed to M-CSF while differentiating into nonLC DC in the presence of GM-CSF and TNF-α(7). Phenotypically, these non-LC DC are characterized by the abundant expression of factor XIIIa, CD1a, CD68, CD11b, CD36

242 GEORG STINGL AND DIETER MAURER

and the virtual absence of E-cadherin and BG and, thus, resemble dermal dendritic cells (DDC). Additional factors governing the development of LC/DC from CD34+ progenitors are stem cell factor (SCF) and flt3-ligand. These cytokines amplify the DC differentiation pathways initiated by GM-CSF and TNF-α without any apparent selectivity for LC or nonLC DC development (8,9). In contrast, TGF-β1 seems to be of unique importance in LC ontogeny. This is evidenced by the lack of LC in TGF-β1−/− mice (10), i.e., mice whose TGF-β1-encoding genes have been deleted by homologous recombination, and by the preferential development of CD1a+, BG+ cells in GM-CSF- and TNF-α-containing, TGFβ1-supplemented serum-free stem cell cultures (11). It is not yet entirely clear which cell types serve as the biologically relevant source of TGF-β1 in LC differentiation. Cell transfer studies in mice suggest that radiation-resistant host cells other than KG are important in this regard (12). Recent studies show that TGF-β1 may have a LCpromoting effect at the CD14+ DC precursor stage (12) and, perhaps even, at the level of peripheral blood monocytes (13). While these cells transform into non-LC DC under the influence of GM-CSF and IL-4 (7,14,15) they upregulate E-cadherin as well as CD la and display BG-like structures when additionally stimulated by TGF-β1 (13). LANGERHANS CELL HOMING TO THE SKIN (FIG.) The exact maturational stage at which LC precursors enter the skin/epidermis is still unknown as are the mechanisms operative in this process. Some evidence exists that various members of the chemokine (CK) system are of importance in this regard. CK constitute a multipartite superfamily of chemoattractant cytokines which, upon binding to G protein-coupled receptor proteins, induce the directional as well as the non-directed migration of leukocytes and other cells (16–18). Currently, chemokines are subdivided into 4 groups according to the position of the first cysteine pair (CXC, CC), the lack of two of the four cysteines (C), or the presence of three spacing amino acids in the first cysteine tandem (CX3C). Up to now, several receptors for CXC (CXCR1 to 5) and CC chemokines (CCR1 to 9) have been identified (18). While experimental data on the chemokine responsiveness of skin-derived LC are still sparse, a considerable amount of information exists concerning the chemokine responses of other DC, e.g., of those generated from monocytes by GM-CSF plus IL-4 (14, 15). These monocyte-derived (md)DC display migratory responsiveness to a broad repertoire of inflammatory CC chemokines, including macrophage chemotactic protein (MCP)-1, MCP-2, MCP-4, regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-5/HCC2, the CXC chemokines IL-8 and stromal cell-derived factor (SDF)-1 (19–23), and macrophagederived chemokine (MDC) (24). In correlation to this response profile, mdDC express receptors for inflammatory chemokines such as CCR1, CCR2, CCR3, CCR5, CXCR1 and constitutively expressed chemokines (i.e., CXCR4 (19, 21, 23, 25)). Interestingly, LC/DC generated from CD34+ stem cells but not mdDC express CCR6 and respond to MIP-3/liver and activation regulated chemokine (LARC) (26,27). In a recent study, we obtained convincing evidence that MIP-3 is indeed a prime candidate involved in the

LANGERHANS CELL MIGRATION 243

recruitment of LC/LC precursors to the skin epidermis (27a). This argument is supported by (i) the sensitivity of CD1a+ LC precursors for MIP-3α, (ii) the expression of CCR6, the specific receptor for MIP-3α, both by LC in vivo and LC generated in vitro, (iii) the lack of CCR6 expression by non-LC DC which are not present in nonperturbed epidermis, (iv) the loss of CCR6 expression by cytokine-matured epidermal LC, thought to be correlates of lymph node-bound LC, and, importantly, by (v) the constitutive expression of MIP-3α in keratinocytes and skin endothelial cells. One should not forget that MIP-3α, although absent from spleen and bone marrow, is also expressed in fetal liver and lung, appendix, thymus, and tonsils (28–31). However, in tonsils (27), the skin, and, perhaps, in the other organs containing epithelial cells (e.g., liver, lung, gut, thymus), MIP-3α is constitutively expressed by ectodermal rather than mesenchymal/ bone marrow-derived cells. It also appears that mechanisms other than MIP-3α: expression determine whether LC precursors appear and populate a given organ. These may include the organ-specific molecular composition of the vascular barrier and the tissuespecific cytokine milieu. LANGERHANS CELL EMIGRATION AND HOMING TO SECONDARY LYMPHOID ORGANS (FIG.) Finally, there remains the question about the ultimate fate of the epidermal LC population. Despite the shedding of ATPase+ dendritic cells into the para-keratotic horny layer after epidermal injury (32), it is unclear whether elimination through the stratum corneum occurs under physiologic conditions. Instead, evidence exists that perturbation of the cutaneous microenvironment leads to major phenotypic changes in the LC population allowing them to leave their cutaneous residence and to travel to the regional lymphoid organs. The phenotypic and functional metamorphosis of LC has first been observed in single epidermal cell suspension cultures. Over the course of only few days, molecules/ structures linked to or responsible for antigen uptake and processing (e.g., FcεRI, FcγRII, Birbeck granules) as well as for the attachment to their symbionts (e.g., E-cadherin) progressively decrease and often disappear. In contrast, their dendricity as well as their expression of surface moieties needed for T cell priming (e.g., MHC class I and class II; costimulatory molecules CD40, CD54, CD58, CD80, CD86) increase sharply (33,34). Thus, LC recovered from epidermal cell cultures are essentially indistinguishable from MHC class II-bearing DC of lymphoid organs known to be potent stimulators of primary and secondary T cell responses (35,36). It is clear that the behavior of LC in epidermal cell cultures is mediated by cytokines provided by non-LC. Whereas LC highly enriched or even purified from epidermal cell suspensions enjoy only a short survival in normal culture medium, the addition of the cytokines TNF-α and GM-CSF prolongs their viable state. In contrast, the induction of phenotypic changes is mediated mainly by GM-CSF and IL-1 but not by TNF-α (37,38). From a (patho)physiologic view-point, it is important to note that these cytokines can be produced by KG either constitutively (IL-1α) or upon stimulation (GM-CSF, TNF-α). In and ex vivo studies in both the murine and human system have convincingly corroborated the biological relevance of the observations made in single epidermal cell


cultures. In skin transplants (39) as well as in skin expiant cultures (39,40), LC begin to enlarge, exhibit increased amounts of surface-bound MHC class II molecules, spontaneously emigrate from the epidermis and, together with DDC, assemble in dermal afferent lymphatics to begin their journey out of the skin. In similar fashion, application of contact sensitizers to the skin results in the consecutive appearance of strongly anti-MHC class II-reactive cells in the dermis (41), of veiled cells in the lymphatics (42) and, finally, of antigen-bearing LC/DC in the draining nodes (43,44). In the past few years, we have learned a great deal concerning the factors governing LC migration from the skin into draining lymph nodes. Antibodies to TNF-α and IL-1β prevent the early migration of LC from the epidermis, the accumulation of DC in lymph nodes, and the development of optimal contact sensitization (45–47). In keeping with these observations is the finding that mice deficient in IL-1β manifest impaired contact hypersensitivity to haptens (48). Conversely, the intradermal injection of TNF-α or IL-1β stimulates the migration of LC out of the epidermis and the accumulation of DC in draining lymph nodes (49). The effect of TNF-α is apparently mediated by the 75 kD TNF-RII as LC migration in TNF-RI gene-targeted knock out mice is unchanged but still sensitive to TNF-α neutralization (50). The further sequence of events occurring in LC emigration include the loosening of the E-cadherin-dependent attachment of LC to neighboring KC which can be, at least partly, explained by a LC maturation-related downregulation of this molecule (51). Recently, two additional LC surface receptors have been implicated as being essential for LC emigration to occur. One is CD44, a hyaluronic acid receptor putatively involved in the tissue homing of leukocytes and certain cancer cells. Antibody blocking studies suggest that an N-terminal epitope of CD44 is involved in LC emigration while the differentiation-related expression of the CD44 splice variant v6 allows for LC binding to T cell rather than to B cell areas of lymph nodes (52). The other LC-bound receptor structure involved in tissue emigration is a heterodimer built up by the integrin chains α6/β1 or α6/β4 (53). Importantly, the prototype cell expressing the latter moiety is the KC. These cells express this receptor in the hemidesmosome where it mediates KC attachment to laminin, a major constituent of the basement membrane. It is conceivable that in vivo-stimulated LC loosen their KC-binding sites, and use 6-containing integrin receptors to specifically recognize basement membrane components. The antigenic stimulus itself, stimulation-induced KC products, or the receptor-mediated interaction with extracellular matrix proteins may then induce LC to secrete proteolytic activity, e.g., type IV collagenase (MMP-9) (54), allowing them to penetrate the basement membrane and to pave their route through the dense dermal network into the lymphatic system. At least upon in vitro maturation, CD34+ HPC-derived LC/DC acquire responsiveness to MIP-3β/Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC) due to the de novo expression of CCR7, a specific receptor for the factors MIP-3β and secondary lymphoid tissue chemokine (SLC; refs. 27, 55, 56). In keeping with an important role of CCR7 signaling for the proper function of mature DC in vivo is the observation that pit mice, which lack expression of SLC, have reduced numbers of mature DC in T cell areas of lymph node but, importantly, display an epidermal LC population that is normal in terms of cell numbers and distribution (57).

E, epidermis; B, basement membrance; HPC, hemopoietic percursor cell; CLA, cutaneous leukocyte antigen; DC, dendritic cell; LC Langerhans cell; DDC, dermal dendritic cell; BM, bone marrow; PB, peripheral blood; LN, lymph node; IL-1β, interleukin-1β; IL-4, interleukin-4; SCF, stem cell factor; GM-CSF, granulocyte/macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; TNF-α, tumor necrosis factor-α; flt-3-L, flt-3 ligand; TGF-β, transforming growth factor-β; CKs, cemokines; MIP-3α, macrophage inflammatory protein 3α; MIP-3β, macrophage inflammatory protein 3β; SLC, secondary lymphoid tissue chemokine; α6/β1, 4, α6/β1 and α6/β4 intergrins

Figure 12.1Ontogenetic, migration and maturation pathways of Langerhans cells and dermal dendritic cells.



Where and how does the life cycle of a LC end? This question has not yet been completely resolved but some evidence exists that DC upon cognate interaction with T cells physically disappear from the lymph nodes (58). It remains to be seen whether this event is due to their emigration from the nodes or, alternatively, due to their elimination by cytolysis or apoptosis induced by responder T cells. ACKNOWLEDGEMENT This work was supported, in part, by grants from Novartis Pharma, Basel, Switzerland, and the Austrian Science Foundation, Vienna, Austria. REFERENCES 1.

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13. LEUKOCYTE ADHESION AND ACCESSORY MOLECULES AS THERAPEUTIC TARGETS FOR INFLAMMATORY SKIN DISEASES KIMBERLY E.FOREMAN AND BRIAN J.NICKOLOFF

INTRODUCTION Given the visibility and accessibility of human skin to investigators interested in inflammatory and immunologically-mediated disorders, it should not be too surprising that dermatological disease-related studies are continually being reported in the worldwide literature. As such, it is difficult to keep pace with developments regarding the pathophysiology and treatment of various skin disorders. Even though many advances have been made regarding; identification of a new virus that may cause skin lesions (i.e. HHV-8) (Chang et al., 1994), characterization of proteins that inhibit bacterial invasion of the skin (Frohm et al., 1997), and gene mutations responsible for causing cutaneous neoplasms such as basal cell carcinoma (Xie et al., 1998; Dahmane et al., 1997; Oro et al., 1997; Fan et al, 1997), this chapter will be devoted exclusively to the molecular and cellular basis for leukocyte (i.e. neutrophil) and immunocyte (i.e. T-cell) adhesion in the skin. Indeed, the past decade has been characterized by rapid progress in elucidating key adhesion molecules, cytokines, and chemotactic polypeptides which govern the nonrandom trafficking of circulating cells into the dermis and epidermis.

Figure 9.1 Optimal stimulation of T-cells by antigen presenting cells requires two signals.

252 KIMBERLY E.FOREMAN AND BRIAN J.NICKOLOFF

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

LEUKOCYTE ADHESION AND ACCESSORY MOLECULES 253

accessory signaling function, as well as certain cytokines—although this latter point is somewhat controversial. Nonetheless, with respect to dermatology, the importance of focusing on signal #2 is very relevant because in many (if not most) inflammatory/ immunological skin diseases, the nature of the inciting agent/antigen is unknown. From a therapeutic perspective, it is thus possible to envision treating these disorders by targeting the second signal aspect of the T-cell activation pathway. Another caveat for this approach is that a drug that can block signal #2 effectively, may be efficacious in a wide variety of skin diseases. In this chapter, adhesion molecules that govern cell: cell interaction and movement of cells into/out of the skin will be examined for their role in pathophysiology and as molecular targets for therapeutic intervention. In addition, the role of accessory molecules that regulate the activation state of the immunocytes once they have entered the skin will be covered in a secondary but complementary fashion. Since adhesion molecules may also mediate cell activation, it is probably that certain drugs may have a dual role of not only blocking adhesion, but also to interrupt the signal #2 component for T-cell activation. It is hoped that after reading this chapter a clear understanding of the importance of adhesion molecules in various skin diseases will be achieved, as well as being integrated with the concept that accessory signaling molecules are excellent targets for therapeutic intervention in dermatology. BASIC REVIEW OF THE ADHESION CASCADE IN THE SKIN Endothelial Cells Trafficking of leukocytes into an area of inflammation is a multi-step sequence of cellular and molecular events that are necessary for an effective inflammatory and/or immunological response to occur. At least four distinct steps have been defined for endothelial cell:leukocyte interactions including (1) attachment and rolling, (2) adhesion triggering, (3) firm adhesion, and (4) transendothelial migration. At sites of cutaneous inflammation, the release of pro-inflammatory mediators, including cytokines, induce changes in the adhesive properties of endothelial cells lining the surrounding vasculature. In the first step of the adhesion cascade, leukocytes attach and begin to roll along the surface of the endothelial cells. This process is mediated by the selectin family of adhesion molecules (see Table 13.1) consisting of L-selectin (constitutively expressed on leukocytes), P-selectin and E-selectin (expressed on activated endothelial cells). Table 13.1


The selectins interact with the carbohydrate ligands CLA (cutaneous lymphoid antigen) and sialyated Lewis X (sLeX) expressed on leukocytes and activated endothelial cells. (Fuhlbrigge et al., 1997) If the stimulus persists, the rolling leukocytes become firmly bound to the activated endothelial cell surface through interactions occurring between members of the immunoglobulin gene superfamily and integrin adhesion molecules (Table 13.1) expressed on leukocytes. Interestingly, there is direct evidence that integrins such as LFA-1 (CD11a/ GDIS) exist in two different affinity states. (Lollo et al., 1993; Carlos and Harlan, 1990). An increase in cell surface expression of these molecules is neither necessary nor sufficient for increased adhesiveness; instead, they must undergo a conformational change to become fully activated. Such activation has been referred to as “inside-out” signaling. Data indicate that integrins also act as classic receptors, and engagement of these molecules results in intracellular signaling or “outside-in signaling”. (Petty and Todd, 1996; Resales et al., 1995). Finally, the leukocytes migrate into the epidermal compartment by moving between the endothelial cells towards the area of inflammation where they can interact with keratinocytes. Keratinocytes In addition to mediating the binding of leukocytes to endothelial cells, adhesive interactions play an important role in the interaction of keratinocytes with T-cells. Previously, epidermal keratinocytes were thought of as merely passive targets in immunologie reactions; however, it has since become clear that keratinocytes can be stimulated by a variety of mediators resulting in upregulation of adhesion molecules, such as ICAM-1, and binding of T-cells. Such interactions facilitate functional participation and an active role for keratinocytes in inflammation, and are important to understand how keratinocytes play a key role in the pathogenesis of many cutaneous diseases. Both in vitro and in vivo studies have indicated that a variety of substances can induce keratinocytes to express ICAM-1 including cytokines (such as TNF-a and IFN-γ), phorbol esters, poison ivy antigen (urushiol), nickel ion, ultraviolet light, irritants, and bacterial derived superantigens. Not only can stimuli induce keratinocyte ICAM-1 expression, but certain stimuli can also suppress keratinocyte ICAM-1 expression in vitro. (Norris et al., 1990) ROLE OF ADHESION MOLECULES IN PSORIASIS Psoriasis is a common, chronic inflammatory skin disease which is characterized by epidermal hyperproliferation, parakeratosis, and the presence of an inflammatory infiltrate consisting of neutrophils and activated T-cells in the dermis. While the precise mechanisms by which these cells are recruited selectively into the dermis is unknown; studies have demonstrated altered expression of adhesion molecules on keratinocytes and endothelial cells in this disease. ICAM-1 is constitutively expressed on endothelial cells in normal human skin and uninvolved skin in psoriatic patients; however, both keratinocytes and endothelial cells upregulate ICAM-1 expression in involved psoriatic lesions. (Barker et al., 1992; Veale et al., 1995) In contrast, E-selectin expression on endothelial cells is minimal in normal skin, and is widely expressed in psoriatic lesions. Similarly, VCAM-1 is


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


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


endothelial cells. A dose-dependent reduction of VCAM-1 expression on human endothelial cells in vitro following Cetrizine treatment has also been reported. (Jean-Louis et al., 1998) Anti-LFA-1 Antibodies Given the importance of adhesive interactions between endothelial cells, keratinocytes, and T-cells that involve LFA-1/ICAM-1, it should not be surprising that anti-LFA-1 monoclonal antibody has been given to psoriasis patients. Previous investigators have observed that the use of monoclonal antibodies against the T-cell surface antigen CD3 or CD4 were associated with improvement of psoriasis skin lesions. (Morel et al., 1992) With the increasing advances made in the production of fully or partially humanized antibodies, more recent clinical trials have replaced mouse monoclonal antibodies directed against various human cell surface adhesion molecules with humanized versions of such blocking antibodies. (Neuberger and Bruggemann, 1997) Preliminary results in 31 patients with moderate to severe psoriasis who received a single intravenous dose of humanized monoclonal antibody (hu 1124) against LFA-1 (CD11a) produced a mean decrease in PASI scores of 33%. (Gottlieb et al., 1998) Biopsies of treated patients revealed decreased numbers of LFA-1 positive T-cells in the epidermis with diminished keratinocytes and endothelial cell ICAM-1 expression. Vitamin D The most active metabolite of vitamin D used to treat psoriasis patients is 1,25dihydroxyvitamin D3. This form of vitamin D is very effective in the treatment of psoriasis, although its precise mechanism of action has yet to be fully characterized. (Smith et al., 1988; Kragballe et al., 1988) Using an in vitro tissue culture system, 1,25dihydroxyvitamin D3 was capable of decreasing IFN-γ induced HLA-DR, but ICAM-1 expression was not significantly reduced on keratinocytes. (Tamaki et al., 1990; Gerritsen et al., 1993) It is possible that this vitamin D derivative improves psoriasis by direct interaction on keratinocytes and T-cells that does not involve modulation of adhesion molecules. Nonsteroidal Anti-inflammatory Drugs (NSAIDs) NSAIDs have been used successfully for years in the treatment of many acute and chronic inflammatory diseases. While it is known that these compounds block cyclooxygenase and inhibit prostaglandin synthesis, this may not be the sole mechanism of action of NSAIDs. (Vane, 1971) Recent studies have indicated that several NSAIDs including indomethacin, diclofenac, aspirin, aceclofenac, mefenamic acid and flufenamic acid strongly inhibit neutrophil adhesion to endothelial cells in vitro. (Diaz-Gonzalez and Sanchez-Madrid, 1998; DiazGonzalez et al., 1995; Gonzalez-Alvaro et al., 1996) This effect appears to result from a decrease in the expression of neutrophil L-selectin, an adhesion molecule involved in the initial (rolling) interaction between these two cell types. (DiazGonzalez


and Sanchez-Madrid, 1998) Studies indicate that the mechanism of action of these NSAIDs is through metalloproteinases, which cleave L-selectin resulting in shedding of this molecule from the neutrophil surface. However, not all NSAIDs function the same way. Piroxicam and meloxicam do not reduce L-selectin expression and do not inhibit initial adhesion of neutrophils to endothelial cells. However, these NSAIDs are able to prevent the activation of integrins and thereby decrease cell adhesiveness. (Garcia-Vicuna et al., 1997; Abramson et al., 1994) CO-STIMULATORY MOLECULES As mentioned earlier, besides an antigen-specific signal (i.e. signal #1), the full activation of T-cells requires a second non-antigen specific signal generally mediated by costimulatory molecules. Three different co-stimulatory molecules will be mentioned which include (1) CD28/B7; (2) LFA-2/LFA-3; and (3) CD44 variant V7. For skin diseases such as psoriasis, the nature of the antigen responsible for inducing T-cell activation/ proliferation is unknown. However, it has been established that the disease is mediated by T-cells since either treatment with a highly specific reagent for activated T-cells (DAB369IL-2 fusion protein that attach to IL-2 receptor bearing T-cells) improved psoriasis, or introduction of pathogenic T-cells into symptomless skin engrafted onto SCID mice produced psoriasis. (Gottlieb et al., 1995; Wrone-Smith and Nickoloff, 1996) The role for co-stimulatory molecules in psoriasis was initially supported by the ability of a fusion protein (i.e. CTLA4Ig) designed to interrupt CD28-B7 interactions to block the ability of psoriatic plaque derived dendritic cells to activate autologous resting T-cell proliferation and cytokine release. (Nestle et al., 1994b; Nestle et al., 1994a) Clinical trials using CTLA4Ig in psoriasis have yielded promising early results. The T-cell surface protein LFA-2 (CD2) and its ligand LFA-3 (CD58) are also involved in co-stimulation of T-cells resulting in activation. (Selvaraj et al., 1987) Recent clinical studies by Magilavy et al. (1998) used a human fusion protein designed to bind to LFA-2 and thereby inhibit T-cell activation via the costimulatory pathway mediated by CD2/ LFA-3 interactions. The results demonstrated a decrease in peripheral lymphocytes in normal volunteers as well as patients with chronic psoriasis with few side effects indicating potential as a treatment for psoriasis. The CD44 gene codes for a family of alternatively spliced, multifunctional adhesion molecules that participate in lymphocyte activation and cell migration. Increased expression of CD44 standard has been shown on the inflammatory and endothelial cells in psoriasis lesions making this molecule a potential target for therapy. (Reichrath et al., 1997) Similarly, recent studies using a murine model for inflammatory bowel disease (remember some patients with psoriasis also suffer from Crohn’s Disease) found that antibody against the CD44 variant V7 was highly efficient at preventing or reversing chronic colitis. (Wittig et al., 1998)


SUMMARY Adhesion molecules provide attractive targets for immimo-interaction strategies for several skin diseases including psoriasis. Indeed, many investigators and pharmaceutical companies interested in adhesion molecules are beginning to use inflammatory skin disease for their phase I and phase II clinical trials given the accessibility of skin and early promising results with several anti-adhesion molecule therapies. Thus, even though skin diseases may not be their ultimate disease target, a demonstration of efficacy in a disorder such as psoriasis can provide compelling proof of concept evidence for novel treatments. REFERENCES Abramson, S.B., Leszczynska-Piziak, J., Clancy, R.M., Philips, M., and Weissmann, G. (1994). Inhibition of neutrophil function by aspirin-like drugs (NSAIDS): requirement for assembly of heterotrimeric G proteins in bilayer phospholipid. Biochem Pharmacol 47:563–572. Baker, B.S., Powles, A.V., Savage, C.R., McFadden, J.P., Valdimarsson, H., and Fry, L. (1989). Intralesional cyclosporin in psoriasis: effects on T lymphocytes and dendritic cell subpopulations. Br J Dermatol 120:207–213. Barker, J.N.W.N., Groves, R.W., Allen, M.H., and MacDonald, D.M. (1992). Preferential adherence of T lymphocytes and neutrophils to psoriatic epidermis. BrJ Dermatol 127: 205–211. Bonifati, C., Trento, E., Carducci, M., Sacerdoti, G., Mussi, A., Fazio, M., and Ameglio, F. (1995). Soluble E-selectin and soluble tumour necrosis factor receptor (60 kD) serum levels in patients with psoriasis. Dermatology, 0:128–131. Butcher, E.G. (1991). Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67:1033–1036. Buurman, W.A., Ruers, T.J., Daemen, I.A., van der Linden, C.J., and Groenewegen, G. (1986). Cyclosporin A inhibits IL 2-driven proliferation of human alloactivated T cells. J Immunol 136: 4035–4039. Gai, J.P., Harris, K., Falanga, V., Taylor, J.R., and Chin, Y.H. (1996). UVB therapy decreases the adhesive interaction between peripheral blood mononuclear cells and dermal microvascular endothelium, and regulates the differential expression of CD54, VCAM-1, and E-selectin in psoriatic plaques. BrJ Dermatol 134:7–16. Caproni, M., Palleschi, G.M., Falcos, D., Papi, C., and Lotti, T. (1995). Pharmacologie modulation by Cetirizine of some adhesion molecules expression in psoriatic skin lesions. Int J Dermatol 34:510–513. Carlos, T.M. and Harlan, J.M. (1990). Membrane proteins involved in phagocyte adherence to endothelium. Immunol Rev 114:5–28. Chang, Y., Cesarman, E., Pessin, M.S., Lee, F., Culpepper, J., Knowles, D.M., and Moore, P.S. (1994). Identification of herpesvirus-like DNA sequences in AIDS- associated Kaposi’s sarcoma. Science 266:1865–1869. Dahmane, N., Lee,J., Robins, P., Heller, P., and Ruiz(1997). Activation of the transcription factor Glil and the Sonic hedgehog signaling pathway in skin tumours. Nature 389: 876–881. Diaz-Gonzalez, F., Gonzalez-Alvaro, L, Campanero, M.R., Mollinedo, F., del, P., MA, Munoz, C., Pivel,J.P., and Sanchez-Madrid, F. (1995). Prevention of in vitro neutrophil-endothelial


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INDEX

acanthosis 58, 222, 232 accessory plaque protein 74–76 adherens junction 1 adhesion cascade in the skin 253–254 adhesion molecule 168 allelic diseases 150–152 allergic contact dermatitis 167, 178, 212 alopecia 231 alpha3beta1 integrin 116, 135, 137 alpha4 integrin 179 alpha4betal integrin 179 alpha6 integrin 108, 110, 146, 146 alpha6beta4 integrin 10, 93, 94–95, 101, 102, 109, 110, 114, 134, 135, 137, 245 mutations 124–125 alphaEbeta7 integrin 177 anchoring complex 133 BMZ associations 99–100 anchoring fibril 107, 108 proteins 140–141 anchoring filament 107, 108 and lamina lucida proteins 138–139 anchoring plaques 90 antigen presenting cell (APC) 203, 222, 251, 252 anti-LFA-1 antibodies 257 Arthus reaction 178 atopic dermatitis 167, 177, 224 autoimmune bullous disease 89 bacterial lipopolysaccharide 168 basal cell carcinoma 251 basement membrane 89–106, 107, 245 ultrastructure 89–90 basophils 221

betal integrin 177, 179–180 beta2 integrin 177, 178–179 beta4 integrin 108, 110, 115, 146 beta4 knock out mice 116 beta7 integrin 177, 251 Birbeck granules 241 blistering skin diseases, animal models 153–154 BP230 135, 143, 146 BPAG1 10, 91, 101, 102, 108, 110, 113, 115, 136 BPAG1 knockout mice 115 BPAG2 10, 108, 110, 113–114, 115 bullous congenital ichthyosiform erythroderma (BCIE) 2, 36, 37, 39–40, 47 bullous pemphigoid 5, 113, 142, 143, 223 bullous SLE 143 cadherin 57 CD28 203, 204–207, 251 CD28-B7 pathway 205, 258 CD29 179 CD31 177 CD34 253 CD40 204 CD40L 204, 208–210 CD43 204 CD44 181, 204, 258 CD47 204 CD50 178 CD54 178 CD80 203, 204 CD86 203, 204 CD102 178 cell adhesion recognition (CAR) 61–62 cell-cell attachment 7–85

INDEX 265

cell-matrix attachment 87–163 cetirizine 256–257 chemoattractant cytokine 168 chemokine 177, 188, 242 expression in skin 234 chronic discoid lupus erythematosus (CDLE) 213 chronic proliferative dermatitis mouse 227 cicatricial pemphigoid 96, 142, 143 CLA 176, 188 collagen 179 fibril 108 collagen IV 99, 100–101, 102, 103, 134, 135, 139 collagen VII 134, 135, 140, 143, 144, 146 collagen XIII 137 collagen XVII 93, 95–97, 101, 102, 103, 134, 135, 136, 143, 144, 146 collagen XVII ectodomain 139 contact hypersensitivity (CHS) 212, 223–224 corneocytes 9, 222 corneodesmosin (CDE) 11, 16–17 cornified cell envelope (CE) 5, 9–25 co-stimulatory molecules 258 CR3 253 CTLA-4 207–208 fusion protein 205 cutaneous lymphocyte-associated antigen (CLA) 183–189, 241, 254 cellular distribution 184–185 in the circulation 185–186 molecular aspects 185 regulation 186–187 and T cell-endothelium interaction 187– 189 cutaneous T cell lymphoma (CTCL) 230 cyclosporin 256 cystatin A (CYA) 11, 114 cytokeratin expression pattern 33 cytotoxic T lymphocyte antigen-4 (CTLA-4) 203, 204 Darier’s disease 10 dendritic cell 241, 245 dendritic epidermal gammadelta-TCR positive T cell (DETC) 203 dermal adhesion 5

dermal connective tissue 1 dermal dendritic cell (DDC) 212, 241, 245 dermal elastin/collagen 102 dermal T cell 212 dermal-epidermal adhesion 133–163 disorders 142–154 aquired 142–143 dermal-epidermal basement membrane zone (BMZ) 89–106 laminins 97–99 ultrastructure 89–90 dermal-epidermal junction (DEJ) 133–142 anchoring fibril 140–141 anchoring filament 138–139 biosynthesis, processing and regulation 141– 142 intracellular proteins 136 lamina densa protein 139–140 molecular components 135–141 morphology and suprastructure 133–135 transmembrane proteins 136–138 dermis 107 desmin 27 desmocalmin 76 desmocollin (Dscs) 4, 10, 57, 58 desmocollin 1 59 desmocollin 2 59 desmocollin 3 10, 59 desmoglea 57 desmoglein (Dsgs) 4, 10, 57, 58 desmoglein 1 59 desmoglein 2 10, 59 desmoglein 3 10, 59 desmoplakin (Dp) 4, 31, 57, 58, 72–73, 91 desmoplakin I 59 desmoplakin II 59 desmosomal glycoprotein 58–67 and cell adhesion 58–63 and cytoplasmic interactions 63–64 in epidermis 64–67 desmosomal plaque 67–76 desmosome 1, 4, 5, 29, 31, 57–85 dystroglycan 138 EHK 10 elafin (ELA) 10, 13–14 endothelial cell (EC) 212, 253–254

266 INDEX

envoplakin (EPL) 11, 15–16, 57, 73–74 eosinophils 221 epidermal differentiation complex (EDC) 10 epidermal keratinocyte 9, 222, 252 epidermis 1, 9, 107, 245 epidermolysis bullosa (EB) 35, 89, 109, 134, 143–152 acquisita 143 dystrophic (DEB) 10, 35, 101, 144, 145, 148–149, 148–150 generalisata 145 inversa 145 localisata 145, 150 mutilans 145, 148, 150 junctional 10, 35, 121, 144, 145, 147–148, 147–148 cicatricans 145 generalisata mitis 145 Herlitz 145, 147 inversa 145 localisata 145, 147, 148 progressiva 145 pyloric atresia 145, 148 recessive forms 38–39 epidermolysis bullosa simplex (EBS) 2, 10, 35– 38, 45, 116, 144, 145–146 Dowling-Meara (EBS-DM) 2, 36, 47, 145, 146 Kallin 145 Kobner (EBS-K) 36, 39, 145, 146 Mendes da Costa 145 mottled pigmentation (EBS-MP) 38, 45, 146 muscular dystrophy (EBS-MD) 116–121, 145, 146 Ogna 145 Weber-Cockayne (EBS-WC) 36, 38, 145, 146 epidermolysis bullosa-pyloric atresia (EB-PA) 118, 124–125 epidermolytic hyperkeratosis (EH) see bullous congenital ichthyosiform erythroderma epidermolytic palmoplantar keratoderma (EPPK) 2, 41 Epstein-Barr virus induced molecule 1 ligand chemokine (ELC) 244 erythrokeratoderma (EK) 2, 10, 18–19

E-selectin (ELAM-1) 168, 169, 171, 174, 175– 176, 188, 253 extracellular matrix 230 fibronectin 170, 179 fibrosarcoma 60 filaggrin 14–15 flaky skin mouse 225–226 focal contact junction 1 focal non-epidermolytic palmoplantar keratoderma (FNEPPK) 42 gamma delta T cell 173 gamma-interferon-induced peptide (IP-10) 177 gap junction I GDA-J/F3 antigen 135, 140 gene therapy 19–21, 45–47 generalized atrophie benign epidermolysis bullosa (GABEB) 95, 117, 121–124, 144 glial fibrillary acidic protein (GFAP) 27 gly-CAM-1 253 GM-CSF 245 graft-versus-host disease 212 granulocyte/mactophage colony stimulating factor (GM-CSF) 241 Hailey-Hailey 10 haptens 223 heat-stable antigen 204 hemidesmosome 1, 5, 90, 101 biology and pathology 107–131 genetic diseases 116–125 inner plaque 108 outer plaque 108 protein-protein interaction 115–116 ultrastructural and molecular features 109– 111 hemopoietic precursor cell 245 hepatocyte growth factor (HGF) 177 heritable skin diseases, clinical implications 125– 126 Herlitz junctional epidermolysis bullosa 5 herpes gestationis 113 HHV-8 251 histamine 176 human umbilical vein endothelial cell (HUVEC) 170, 175, 180

INDEX 267

hyperkeratosis 222, 232 ICAM-1 169, 170, 177, 178, 188, 251, 253 ICAM-2 169, 170, 171, 178, 179, 253 ICAM-3 170, 171, 178, 179, 253 ichthyosis bullosa of Siemens (IBS) 2, 36, 41 IFAP300 108, 109 IL-1 178 IL-10 175 IL-13 175 IL-lbeta 245 IL-2 receptor (IL-2R) 225 IL-4 171, 175, 180, 245 IL-7 and skin 229–233 IL-8 171 immunoglobulin 170–171 immunoglobulin supergene family 168 inflammatory and malignant skin disease 212– 215 inflammatory bowel disease 143, 181 integrin 168–170, 177–180 interferon gamma (IFN-gamma) 171, 225 and skin 233 interleukin 1 alpha (IL-1 alpha) 224 interleukin 1beta (IL-lbeta) 224 interleukin-1 (IL-1) 168, 171, 175 and skin 233–234 interleukin-8 (IL-8) 177 intermediate filament 27, 89, attachment 74–76 hemidesmosome interaction 90–91 molecular structure 27–34 intracellular protein 136 involucrin 10, 12–13 keratin 1 10 keratin 10 10 keratin 14 2, 110, 90, 102, 134, 135, 146 keratin 2e 10 keratin 5 2, 10, 90–91, 102, 134, 135, 146 keratin 9 10 keratin acidic 27 basic 27 disorders 27–55 dominant-negative mutation clusters 46 filament 64

bundles 29 gene mutation 34 genetics research 45–47 intermediate filament 5 medical aspects 34–45 molecular structure 27–34 protein domain structure 30 trichocyte 27 type I intermediate filament 31–34 type II intermediate filament 31–34 keratinocyte 1, 89, 254 differentiation 9 expression of integrins 235 LAD-1 96 lamellar ichthyosis 10, 17–18 lamina densa 90, 107, 108, 134 proteins 139–140 lamina lucida 90, 107, 108, 134 laminin 179 laminin 1 99 laminin 2 135, 139 laminin 5 93, 97–99, 102, 103, 108, 109, 116, 10, 135, 138, 143, 144, 146 laminin 6 93, 97–99, 102, 103, 135, 143 laminin 10 99, 102, 103, 135, 138 Langerhans cell (LC) 167, 173, 188, 203, 212, 245 development from hemopoietic precursors (HPC) 241–242 emigration to secondary lymphoid organs 243–246 homing to the skin 242–243 migration 241–249 leukocyte adhesion and accessory molecules 251–262 leukocyte function associated molecule-1 (LFA-1) 169, 170, 178, 188, 251, 253 leukocyte function associated molecule-2 (LFA-2) 258 leukocyte function associated molecule-3 (LFA-3) 258 leukocyte trafficking in skin diseases 165–262 lichen planus 167, 212 lipopolysaccharide (LPS) 175 liver and activation regulated chemokine (LARC) 242

268 INDEX

liver defects 44 loricrin 2, 10, 12 mutation 18 L-selectin (LAM-1) 168, 169, 174–175, 253 lupus erythematosus 167 lymphocyte endothelium interaction 181–183 in inflamed skin 174–189 migration 174–181 in normal skin 173–174 lymphotoxin 175

NC/Nga mouse 226 nestin 27 neurofilament 27 neutrophils 221 nidogen 99, 102, 103, 135, 140 non-epidermolytic palmoplantar keratoderma (NEPPK) 2, 40–41, 45 non-Herlitz junctional epidermolysis bullosa 5 nonsteroidal anti-inflammatory drugs (NSAIDs) 257–258 novel candidate genes 153

MAC-1 170, 178 macrophage 212 macrophage chemotactic protein-1 (MCP-1) 177, 242 macrophage chemotactic protein-2 (MCP-2) 177, 242 macrophage chemotactic protein-3 (MCP-3) 177 macrophage chemotactic protein-4 (MCP-4) 177, 242 macrophage inflammatory protein-1alpha (MIP-1 alpha) 177, 242 macrophage inflammatory protein-1beta (MIPlbeta) 177, 242 macrophage inflammatory protein-3alpha (MIP-3alpha) 242–243 macrophage inflammatory protein-3beta (MIP-3beta) 177 macrophage inflammatory protein-5 (MIP-5) 242 macrophage-derived chemokine (MDC) 242 MAdCAM-1 169, 180, 253 major histocompatibility complex (MHC) 210, 222, 251, 252 malignant melanoma 214 M-CSF 245 Meesmann’s corneal dystrophy (MCD) 43, 44– 45, 47 MIP-3alpha 245 MIP-3beta 245 monilethrix 2 , 44 motheaten mouse 227–228 MPC-1 171 muscular dystrophy 116

p200, 108, 109 pachyonychia congenita (PC) 2, 41–42 Jackson-Lawler (PC-2) 42 Jadassohn-Lewandowsky (PC-1) 42, 43 papillary dermis 90, 108 parakeratosis 223, 232 PECAM-1 180–181, 253 pemphigoid gestationis 142, 143 pemphigus 4, 58, 66 foliaceous 4, 67 vulgaris 4, 67 antigen 10, 60 periphenin 27 periplakin (PPL) 11, 16, 57, 73–74 perlecan 99, 102, 103, 135, 140 photochemotherapy (PUVA therapy) 256 plakoglobin (Pg) 4, 57, 58, 59, 68–72, 91 plakophilin (Pp) 4, 47, 57, 58, 68–72 plakophilin 1 59, 69, 91 plakophilin 2 59, 69 plasma membrane 108 plectin 10, 47, 73–74, 91–94, 101, 108, 109, 110, 111–113, 115, 134, 135, 136, 143, 146 PPK 10 pre-implantation genetic diagnosis (PGD) 125 prenatal testing 47, 125 profilaggrin (PFN) 10, 11, 14–15 progressive symmetric erythrokeratoderma (PSEK) 18–19 protein-protein interaction 5 P-selectin (PADGEM) 168, 169, 171, 174, 176– 177, 253 PSGL-1 176–177 psoralen 256 psoriasis 167, 213, 254–255

INDEX 269

and anti-LFA-1 antibodies 257 and cetirizine 256–257 and cyclosporin 256 and nonsteroidal anti-inflammatory drugs (NSAIDs) 257–258 and ultraviolet light 255–256 and vitamin D purified protein derivative (PPD) 180

stromal cell-derived factor-1 alpha (SDF-1alpha) 177 subacute cutaneous lupus erythematosus (SCLE) 213 sub-basal dense plate 107 subcutaneous fat 1 syndecans 1 and 4 135, 138 systemic lupus erythematosus (SLE) 213

RANTES 177, 242

T cell 251 accessory molecule 203–220 expression 212–215 activation and costimulation 210–212 mediated skin disease 213 receptor (TCR) 251 CD3 complex 203 T lymphocyte migration to the skin 183 TGF-beta 245 TGF-betal 177 thrombin 176 tight junction 1 TNF-alpha 245 transcription factor NF-kappaB, 225 transforming growth factor-beta (TGF-beta) 175 transforming growth factor-betal (TGF-betal) 175, 242 transglutaminase (TG) 10, 11–12 mutation 17–18 transient bullous dermolysis of the newborn (TBDN) 151 transmembrane proteins of basal keratinocytes 136–138 trichohyalin (THH) 10, 11, 15 tumor necrosis factor alpha (TNF-alpha) 168, 171, 175, 178, 180, 224, 241 type VII collagen 10, 108 tyrosine activation motif (TAM) 94

S100 protein family 11 14 S100A 10 scurfy mouse 226 secondary lymphoid tissue chemokine 245 selectin 168, 174 sialylated Lewis X (sLeX) 168, 174, 254 skin architecture perturbation 222–223 associated lymphoid tissue (SALT) 167 blistering phenotypes 152–153 disease, inflammatory and malignant 212–215 homing lymphocyte 167, 173–201 immune system (SIS) 167 animal models 221–240 contact hypersensitivity 223–224 delayed hypersensitive response 222 engineered genetic models 228–229 immediate hypersensitivity 221, 224 normal cutaneous inflammation 221–222 regulatory circuits 224–225 secondary effects 222–223 spontaneous genetic models 225–228 tumour 214 small proline-rich protein (SPRP) 10, 13 spongiosis 223 SPRR 10 stem cell factor (SCF) 242, 245 stratum basalis 9 stratum corneum 1, 9, 222–223 stratum granulosum 9 stratum lucidum 9 stratum spinosum 9 striate palmoplantar keratoderma 4 stromal cell-derived factor-1 (SDF-1) 242

ultraviolet light 255–256 vascular adhesion molecule-1 (VAP-1) 181 vascular cell adhesion molecule-1 (VCAM-1) 169, 170, 171, 179, 188, 253 vimentin 27, 31, 32 vitamin D 257 vitronectin 179

270 INDEX

VLA-4 (alpha4betal) 169, 170, 179, 188 Vohwinkel’s syndrome 2, 10, 17, 18 Weibel-Palade bodies 168, 176 white sponge nevus of Cannon (WSN) 42–44, 47 x-linked ichthyosis (XLI) 20–21

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