Animal Models of Human Inflammatory Skin Diseases

Animal Models of Human Inflammatory Skin Diseases 1391_C00.fm Page 2 Wednesday, November 19, 2003 10:32 AM Animal M...

Author: Lawrence S. Chan

59 downloads 1321 Views 13MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

Animal Models of Human Inflammatory Skin Diseases

1391_C00.fm Page 2 Wednesday, November 19, 2003 10:32 AM

Animal Models of Human Inflammatory Skin Diseases Edited by

Lawrence S. Chan

CRC PR E S S Boca Raton London New York Washington, D.C.


Library of Congress Cataloging-in-Publication Data Animal models of human inflammatory skin diseases / edited by Lawrence S. Chan. p. cm. Includes bibliographical references and index. ISBN 0-8493-1391-0 (alk. paper) 1. Skin—Inflammation—Animal models. 2. Alopecia areata—Animal models. I. Title. RL231.C48 2003 616.5—dc22 2003060292 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. All rights reserved. Authorization to photocopy items for internal or personal use, or the personal or internal use of specific clients, may be granted by CRC Press LLC, provided that $1.50 per page photocopied is paid directly to Copyright clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA. The fee code for users of the Transactional Reporting Service is ISBN 0-8493-1391-0/04/$0.00+$1.50. The fee is subject to change without notice. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe.

Visit the CRC Press Web site at www.crcpress.com © 2004 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-1391-0 Library of Congress Card Number 2003060292 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper


Dedication This book is dedicated to my former mentors who have participated in building the foundation of my academic career: John H. Rockey, who kindly introduced me to the exciting world of laboratory research; John J. Voorhees, who inspired me with his unwavering dedication to cutaneous biology; Kevin D. Cooper, who gently guided me through the paths of learning through investigation and scientific writing; and David T. Woodley, who enlightened me with his unceasing curiosity in clinical and experimental dermatology. This book is also dedicated to my parents who have instilled in me the characteristics of humility and perseverance; to my wife, who has unselfishly supported my academic career, each step of the way; and to James Bostwick, who has always encouraged me throughout the years.



Preface What is an animal model of human inflammatory skin disease? According to the American Heritage Dictionary, a model is a small object, usually built to scale, that represents some existing object. Likewise, an animal model of inflammatory skin disease is an animal representation of a human inflammatory skin disease. An animal model of human inflammatory disease does not need to be completely identical to that of its human counterpart, nor can it always be. The degree of dissimilarity between the human immune system and that of an animal or between the human skin structure and that of an animal, however small, makes it almost impossible for perfect identity. Nevertheless, sufficient similarity allows investigators who study the animal model to gain insight into the general pathological pathways of a disease process that likely occurs in human patients. In this book, both spontaneously arising and experimentally induced animal models are presented. Why should we study an animal model of human inflammatory skin disease? Indeed there are many outstanding in vitro model systems from which we can gain much insight into the molecular mechanisms of skin inflammation. However, only an animal model can bring our understanding of the disease mechanism to a real-life level. An animal model makes it possible for investigators to study how the immune system relates to the actual tissues of the biggest organ of the body, the skin. Moreover, the actual pathological process of an inflammatory skin disease can only be accurately observed and analyzed in a living animal model, and not in any in vitro model system, however brilliant it may be. By using an animal model, investigators can learn about the step-bystep immunological sequence of events for inflammation induction and progression and the factors contributing to these events. The complexity of the disease process in a living organism simply cannot be adequately understood by any in vitro model system, however sophisticated it may be. Furthermore, the in vivo studies conducted in animal models certainly cannot be carried out in human patients, for practical reasons and for obvious ethical concerns. Finally, an animal model provides an excellent avenue for pharmaceutical companies to test the effectiveness and safety of new anti-inflammatory medications before testing these medications in human patients. How then shall we study the animal model of human inflammatory skin disease? This is indeed the major focus of this book. This book provides both the principles and practices of how we go about studying inflammatory skin diseases using living animal models. On the theoretical side, this book establishes foundations by providing the comparative structure and function of the skin and the comparative immunology system in animal species commonly used as models. This comparative information is provided to help readers analyze the relevance of findings obtained in an animal model with that obtained in human patients. Additionally, the unique immune privileges occurring in the eye and hair follicles are discussed with respect to their possible breakdown that leads to inflammation at these “privilege sites.” On the practical side, this book provides general discussion on methods of experimental animal modeling, as well as specific expert experience from investigators who themselves have successfully generated and studied these models. I have attempted to include the most useful models in this book and have requested that contributors follow a standard format for the sake of uniformity and benefit to readers. In a fast-moving field of biomedical research, I am sure that new information regarding these animal models will surface soon after this book is published. These future new discoveries, along with suggestions from readers, will be used to develop better subsequent editions based on the foundation provided in this first edition.



Editor Lawrence S. Chan, M.D., was born in Hong Kong, the then-British colony. He immigrated to the United States in 1975. After graduating from the Massachusetts Institute of Technology with double bachelor degrees in chemical engineering and life sciences in 1981, he entered medical school at the University of Pennsylvania, where he obtained his M.D. degree in 1985. Dr. Chan then performed his internship at the Cooper Hospital/University Medical Center. Both his dermatology residency and immunodermatology fellowship took place at the University of Michigan Medical Center. After a brief stint on the Wayne State University School of Medicine faculty, he served as assistant professor of dermatology and director of immunodermatology at Northwestern University Medical School from 1993 to 2002. Currently, he is associate professor of dermatology and microbiology/immunology and the director of skin immunology research at the University of Illinois at Chicago, and is supported by three research grants from the National Institutes of Health. Dr. Chan has authored and co-authored 75 peer-reviewed biomedical journal articles and 25 book chapters.



Contributors Grant J. Anhalt, M.D. Professor of Dermatology Johns Hopkins University School of Medicine Baltimore, Maryland

Kenneth B. Gordon, M.D. Associate Professor of Dermatology Loyola University Maywood, Illinois

Joseph M. Carroll, Ph.D. Senior Scientist Millennium Pharmaceuticals Cambridge, Massachusetts

Bruce Hammerberg, Ph.D. Professor of Immunoparasitology North Carolina State University College of Veterinary Medicine Raleigh, North Carolina

Lawrence S. Chan, M.D. Associate Professor of Dermatology and Microbiology/Immunology University of Illinois at Chicago College of Medicine Chicago, Illinois Paul J. Christner, Ph.D. Associate Professor of Medicine Division of Rheumatology Thomas Jefferson University Philadelphia, Pennsylvania Jonathan L. Curry, M.D. Postdoctoral Fellow in Pathology Loyola University School of Medicine Maywood, Illinois Luis A. Diaz, M.D. Professor of Dermatology University of North Carolina Chapel Hill, North Carolina Amos Gilhar, M.D. Associate Professor of Dermatology Laboratory for Skin Research The Bruce Rappaport Faculty of Medicine Technion-Israel Institute of Technology Haifa, Israel Anita C. Gilliam, M.D., Ph.D. Associate Professor of Dermatology, Pathology, and Hematology-Oncology Case Western Reserve University Cleveland, Ohio

Andrew Hillier, MVSc, MACVSc Associate Professor of Dermatology College of Veterinary Medicine The Ohio State University Columbus, Ohio Natsuho Ito, M.D. Research Fellow in Dermatology Department of Dermatology Hautklink Universitaet Hamburg Universitaets-Krankenhaus Eppendorf Hamburg, Germany Taisuke Ito, M.D. Research Fellow in Dermatology Department of Dermatology Hautklink Universitaet Hamburg Universitaets-Krankenhaus Eppendorf Hamburg, Germany Toshiroh Iwasaki, D.V.M., Ph.D. Professor of Veterinary Internal Medicine Department of Veterinary Internal Medicine Tokyo University of Agriculture and Technology Fuchu, Tokyo, Japan Sergio A. Jimenez, M.D. Professor of Medicine and Biochemistry/Molecular Biology Division of Rheumatology Thomas Jefferson University Philadelphia, Pennsylvania


Richard S. Kalish, M.D., Ph.D. Associate Professor of Dermatology Health Science Center State University of New York at Stony Brook Stony Brook, New York

Kevin J. McElwee, Ph.D. Senior Scientist Department of Dermatology Philipp University at Marburg Marburg, Germany

Keiko Kawamoto, D.V.M., Ph.D. Associate Professor Research Center for Animal Hygiene & Food Safety Obihiro University of Agriculture and Veterinary Medicine Obihiro, Japan

Vu Thuong Nguyen, M.D. Assistant Professor of Dermatology University of California at Davis Davis, California

Lloyd E. King, Jr., M.D., Ph.D. Professor of Dermatology Vanderbilt University Nashville, Tennessee Zelmira Lazarova, M.D. Assistant Professor of Dermatology Medical College of Wisconsin Milwaukee, Wisconsin Ning Li, Ph.D. Associate Professor of Dermatology Department of Dermatology University of North Carolina Chapel Hill, North Carolina Xiu-Min Li, M.D. Assistant Professor of Pediatrics Division of Pediatric Allergy and Immunology Mount Sinai School of Medicine New York, New York Zhi Liu, Ph.D. Associate Professor of Dermatology and Microbiology/Immunology University of North Carolina Chapel Hill, North Carolina Hiroshi Matsuda, D.V.M., Ph.D. Professor Laboratory of Clinical Immunology Faculty of Agriculture Tokyo University of Agriculture and Technology Fuchu, Tokyo, Japan

Brian J. Nickoloff, M.D., Ph.D. Professor of Pathology and Microbiology/Immunology Loyola University School of Medicine Maywood, Illinois Jerry Y. Niederkorn, Ph.D. Professor of Ophthalmology University of Texas Southwestern Dallas, Texas Thierry Olivry, Dr. Vet., Ph.D. Associate Professor of Dermatology North Carolina State University College of Veterinary Medicine Raleigh, North Carolina Ralf Paus, M.D. Professor of Dermatology Department of Dermatology Hautklink Universitaet Hamburg Universitaets-Krankenhaus Eppendorf Hamburg, Germany Kalyanasundaram Ramaswamy, Ph.D. Associate Professor of Microbiology/Immunology College of Medicine University of Illinois at Rockford Biomedical Science Rockford, Illinois David S. Rubenstein, M.D., Ph.D. Assistant Professor of Dermatology University of North Carolina Chapel Hill, North Carolina


Hugh A. Sampson, M.D. Professor of Pediatrics Division of Pediatric Allergy and Immunology Mount Sinai School of Medicine New York, New York Jonathan M. Spergel, M.D., Ph.D. Assistant Professor of Pediatrics The Children’s Hospital of Philadelphia University of Pennsylvania School of Medicine Philadelphia, Pennsylvania Birte Steiniger, Ph.D. Professor of Anatomy and Immunobiology Institute of Anatomy and Cell Biology Marburg, Germany John P. Sundberg, D.V.M., Ph.D., Dipl. A.C.V.P. Senior Staff Scientist The Jackson Laboratory Bar Harbor, Maine

Desmond J. Tobin, Ph.D. Associate Professor of Cell Biology University of Bradford West Workshire, England Simon J. Warren, M.D. Assistant Professor of Dermatology and Pathology University of North Carolina Chapel Hill, North Carolina Toshiyuki Yamamoto, M.D. Professor of Dermatology Tokyo Medical and Dental University School of Medicine Bunkyo-ku, Tokyo, Japan Minglang Zhao, M.D. Assistant Professor of Dermatology University of North Carolina Chapel Hill, North Carolina



Table of Contents Part I Comparative Structure and Function of the Skin.........................................................................1 Chapter 1 Comparative Structure and Function of the Skin: Overview of Structures and Components .........3 Lawrence S. Chan Chapter 2 Comparative Structure and Function of the Skin: Epithelial Basement Membrane Zone.............19 Lawrence S. Chan

Part II Comparative Immunology.............................................................................................................31 Chapter 3 Human Immune System...................................................................................................................33 Kalyanasundaram Ramaswamy Chapter 4 Canine Immune System ...................................................................................................................79 Bruce Hammerberg Chapter 5 Rat Immune System.........................................................................................................................91 Kevin J. McElwee and Birte Steiniger Chapter 6 Mouse Immune System .................................................................................................................119 Lawrence S. Chan and Kenneth B. Gordon

Part III Immune Privilege and Skin Inflammation.................................................................................141 Chapter 7 Immune Privilege of the Eye.........................................................................................................143 Jerry Y. Niederkorn Chapter 8 The Theory of Immune Privilege of the Hair Follicle..................................................................155 Ralf Paus, Natsuho Ito and Taisuke Ito


Part IV Methods of Experimental Animal Modeling .............................................................................167 Chapter 9 Passive Transfer and Active Induction of Autoimmune Diseases ................................................169 Zhi Liu, Minglang Zhao and Luis A. Diaz Chapter 10 Adoptive Transfer of Cellular Immunity.......................................................................................179 Lawrence S. Chan Chapter 11 Molecular Biological Manipulation of the Immune System by Transgenic Techniques .............187 Lawrence S. Chan

Part V Inflammatory Skin Disease Models ............................................................................................197

Section A Bullous Pemphigoid......................................................................................................................199 Chapter 12 Natural Bullous Pemphigoid in Companion Animals...................................................................201 Thierry Olivry Chapter 13 Experimental Mouse Model of Bullous Pemphigoid: Passive Transfer of Anti-BP180 Type XVII Collagen, Antibodies ...................................................................................................213 Zhi Liu and Luis A. Diaz

Section B Epidermolysis Bullosa Acquisita.................................................................................................225 Chapter 14 Spontaneous Canine Model of Epidermolysis Bullosa Acquisita ................................................227 Thierry Olivry

Section C Mucous Membrane Pemphigoid .................................................................................................239 Chapter 15 Spontaneous Canine Model of Mucous Membrane Pemphigoid .................................................241 Thierry Olivry


Chapter 16 Experimental Mouse Model of Mucous Membrane Pemphigoid: Passive Transfer of Anti-Laminin 5 Antibodies ............................................................................................................251 Zelmira Lazarova

Section D Pemphigus Vulgaris .....................................................................................................................261 Chapter 17 Spontaneous Canine Model of Pemphigus Vulgaris .....................................................................263 Thierry Olivry Chapter 18 Experimental Mouse Model of Pemphigus Vulgaris: Passive Transfer of Desmoglein-Targeting Antibodies..................................................................................................275 Zelmira Lazarova and Grant J. Anhalt Chapter 19 Experimental Mouse Model of Pemphigus Vulgaris: Passive Transfer of Nondesmoglein 1 and 3 Antibodies ............................................................................................................................285 Vu Thuong Nguyen

Section E Pemphigus Foliaceus ....................................................................................................................307 Chapter 20 Spontaneous Canine Model of Pemphigus Foliaceus ...................................................................309 Toshiroh Iwasaki and Thierry Olivry Chapter 21 Experimental Mouse Model of Pemphigus Foliaceus: Passive Transfer of Desmoglein-Targeting Antibodies..................................................................................................321 David S. Rubenstein, Simon J. Warren, Ning Li, Zhi Liu and Luis A. Diaz

Section F Psoriasis.........................................................................................................................................329 Chapter 22 Experimental Chimeric SCID Mouse/Human Skin Model of Psoriasis: Induction by Transfer of Cellular Immunity..................................................................................................331 Jonathan L. Curry and Brian J. Nickoloff Chapter 23 Experimental Mouse Model of Psoriasis by Transgenic Expression of Integrin .........................341 Joseph M. Carroll


Section G Atopic Dermatitis .........................................................................................................................351 Chapter 24 Spontaneous Canine Model of Atopic Dermatitis.........................................................................353 Andrew Hillier and Thierry Olivry Chapter 25 Spontaneous Mouse Model of Atopic Dermatitis in NC/Nga Mice.............................................371 Keiko Kawamoto and Hiroshi Matsuda Chapter 26 Experimental Mouse Model of Atopic Dermatitis by Transgenic Induction ...............................387 Lawrence S. Chan Chapter 27 Experimental Mouse Model of Atopic Dermatitis: Induction by Oral Allergen..........................399 Xiu-Min Li and Hugh A. Sampson Chapter 28 Experimental Mouse Model of Atopic Dermatitis: Induction by Epicutaneous Application of Allergen......................................................................................................................................417 Jonathan M. Spergel

Section H Alopecia Areata ............................................................................................................................427 Chapter 29 Spontaneous and Experimental Skin-Graft-Transfer Mouse Models of Alopecia Areata ...........429 John P. Sundberg, Kevin J. McElwee and Lloyd E. King, Jr. Chapter 30 Spontaneous Rat Model of Alopecia Areata in the Dundee Experimental Bald Rat (DEBR)..........451 Kevin J. McElwee Chapter 31 Spontaneous Canine Model of Alopecia Areata ...........................................................................469 Desmond J. Tobin and Thierry Olivry Chapter 32 Experimental Chimeric SCID Mouse/Human Skin Model of Alopecia Areata: Induction by Transfer of Cellular Immunity..................................................................................................483 Richard S. Kalish and Amos Gilhar


Section I Scleroderma ..................................................................................................................................493 Chapter 33 Spontaneous Mouse Models of Systemic Scleroderma ................................................................495 Paul J. Christner and Sergio A. Jimenez Chapter 34 Experimental Mouse Model of Scleroderma/Graft versus Host Disease: Induction by Transfer of Cellular Immmunity....................................................................................................517 Anita C. Gilliam Chapter 35 Experimental Mouse Model of Scleroderma: Induction by Bleomycin.......................................535 Toshiyuki Yamamoto Index ..............................................................................................................................................549


1391_C01.fm Page 1 Tuesday, November 18, 2003 4:50 PM

PART I Comparative Structure and Function of the Skin



CHAPTER

1

Comparative Structure and Function of the Skin: Overview of Structures and Components Lawrence S. Chan

CONTENTS I. Introduction ............................................................................................................................3 II. The Epidermis ........................................................................................................................4 A. Langerhans Cells ...........................................................................................................5 B. Melanocytes ...................................................................................................................5 C. Keratinocytes .................................................................................................................7 III. The Dermal–Epidermal Junction (Skin Basement Membrane Zone)...................................9 IV. The Dermis.............................................................................................................................9 V. The Hypodermis...................................................................................................................10 VI. Summary ..............................................................................................................................10 Acknowledgment..............................................................................................................................11 References ........................................................................................................................................11

I. INTRODUCTION The skin is the largest organ of the human body and accounts for about 15% of total body weight [1]. Skin is well known for its functional role as a protective physical barrier: water and electrolytes are kept inside of the body and toxins and pathogens are kept out [1–7]. It is now clear that skin is far more than just a protective physical barrier, but rather a dynamic organ that has three other recognized functions: endogenous homeostasis (e.g., body temperature and fluid regulation); metabolism (e.g., Vitamin D synthesis); and sensory input [8]. More recently, skin has been recognized as actively participating in various immunological regulation processes and responses [8–12]. However, not all immunological reactions occurring in the skin are beneficial to the host. Some harmful reactions result from overreacting to trivial “invaders” such as allergens, and lead to intolerable skin inflammation (contact dermatitis) that requires medication [13–16]. Other reactions probably result from exposing autoreactive lymphocytes to immunologically hidden skin antigens, such as epidermal cell surface component desmoglein 1 (Dsg1) or epithelial basement membrane component type VII collagen, through an “epitope spreading” mechanism or other

0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

3


4

ANIMAL MODELS OF HUMAN INFLAMMATORY SKIN DISEASES

autoimmune mechanisms, which in turn lead to autoimmunity and inflammatory damage to skin structure and functions [17–25]. In this book, the principles and practices of animal models of human inflammatory skin diseases are discussed. Before confident interpretation of clinical and experimental data obtained from animal models is possible, comparative analyses of the structure and function and the immune systems of skin are in order. Demonstrating structural and functional similarities between the human skin and that of mammals used in animal modeling goes a long way in providing a sound scientific basis to interpret these data. Thus, in this chapter, we provide an overview of skin structure and components. In Chapter 2, the detailed structures and functions of the skin basement membrane zone are discussed. In these two chapters, we discuss the comparative structure and function between human skin and that of other mammals, particularly small mammals commonly used in biomedical research laboratories. Part I is not meant to be a comprehensive review of all skin structures and functions; rather, its purpose is to highlight salient features essential for understanding inflammatory skin diseases. Part II is devoted to comparison of the immune systems between humans and small mammals commonly used in animal modeling. These comparative studies help in determining the scientific relevance of findings obtained from animal models in relationship to human inflammatory skin diseases.

II. THE EPIDERMIS There are two types of epithelia in a mammal’s body: the epithelium of the skin (epidermis) provides an external covering for the whole body, and the epithelium of the mucous membrane provides a lubricating lining of an internal surface or an organ, such as oral cavity, conjunctiva, and intestine. The epidermis of the skin differs slightly from the epithelium of the mucous membrane in that stratum cornea (outer keratinized layer) is present only in the former. Besides this difference, it is now clear that the composition of certain epidermal components is also varied. For example, one of the intercellular adhesion molecules that bind epithelial cells together, termed desmoglein 3 (Dsg3), is present primarily in the suprabasal layers of the skin epidermis, but it is present throughout all layers of the mucous membrane epithelium [26]. Furthermore, it has also been reported that the degree of expression of Dsg1, an intercellular adhesion molecule located in the upper epidermis, is lower in the mucous membrane epithelium than that of skin [27]. These distinct tissue distribution patterns of Dsg between skin and mucous membrane are identified in both human and mouse epithelia [26,27]. Correspondingly, these distinct distributions of Dsg seem to explain the clinical phenomenon that patients with the autoimmune skin blistering disease, pemphigus foliaceus, with autoantibodies targeting Dsg1, manifesting with intraepidermal blister primarily on the skin, and not on mucous membranes, is due to the combined effect of compensatory abilities of Dsg3 to maintain upper epidermal adherence in mucous membrane and the low level of Dsg1 expression in mucous membrane, despite Dsg1 being targeted by the autoantibodies. Similarly, this distinct tissue distribution of Dsg3 also seems to explain the clinical observation that patients with another autoimmune skin blistering disease, pemphigus vulgaris, with autoantibodies targeting Dsg3, manifests as intraepidermal blister initially on mucous membranes followed by blister on skin at a later stage [28], as this process is likely due to greater amounts of target antigen (Dsg3) of the autoantibodies present in mucous membranes. In addition, the types of keratins present in the skin epidermis vary somewhat from those in the mucous membrane epithelium [29]. These small variations notwithstanding, the epithelia have similar structures and functions throughout the body. The epidermal cells are primarily composed of keratinocytes, Langerhans cells, and melanocytes, with more than 80% of epidermal cells being keratinocytes [29]. As a continually renewing squamous epithelium, the epidermis is organized into four layers named after their position or structural properties: stratum germinativum (basal layer); stratum spinosum (squamous cell layer); stratum granulosum (granular


COMPARATIVE STRUCTURE AND FUNCTION OF THE SKIN

5

layer); and stratum corneum (horny layer). The thickness of epidermis of a given species varies according to body part. In addition, the thickness of epidermis among different species of the same body part varies. In Figure 1.1, skin and mucous membrane samples obtained from human, porcine, canine, rat, and mouse are illustrated for direct comparison. Due to the small numbers of epidermal layers in rat and mouse, the four organized layers are sometimes not easily discerned. Whereas the human epidermis usually contains seven more layers of epithelial cells, the porcine epidermis contains about five layers (Figure 1.1A and B). The epidermis in canine, rat, and mouse skin is relatively thin, containing only two to three layers (Figure 1.1C, D, and E). The epithelia of the mucous membranes, on the contrary, are substantially thicker (Figure 1.1F, G, and H). Among the porcine, canine, rat, and mouse skin, porcine skin is the closest match to that of humans (Figure 1.1). A. Langerhans Cells Langerhans cells are not original epidermal cells, but are bone marrow–derived cells that migrate into the epidermis during embryonic development [30–32]. Localized in suprabasal layers of the epithelia of skin and mucous membrane, Langerhans cells are dendritic cells with important immune functions. Accounting for about 5% of epidermal cells, they are professional antigen-presenting cells that come into contact with foreign antigens passing through the epidermis [30–32]. Upon internalizing the antigen, the Langerhans cells migrate out from the skin to regional lymph nodes with the help of integrin molecules, where they present the processed antigen to T cells by way of their surface MHC class II molecule and co-stimulatory molecules [30–32]. A unique Langerhanscell cytoplasmic structure known as the Birbeck granule (evocative of a tennis racket), is observable under transmission electron microscope (Figure 1.2) [32]. A Langerhans cell–specific protein termed Langerin/CD207, a type II transmembrane protein essential for Birbeck granule formation, has recently been isolated in human [33,34]. Subsequently, the equivalent of Langerin was identified in mouse [35–37], suggesting a similar functional role between the human Langerhans cells and that of small mammals. The human Langerin shares 66% of overall amino-acid sequence identity with its mouse counterpart, with a 75% amino acid homology at the important carbohydrate recognition domain of Langerin [34,35]. Furthermore, using this specific antibody, the Langerhans cells in both human and mouse have been shown to migrate to draining lymph nodes upon inflammatory stimulation, confirming a similar function between human and mouse Langerhans cells [37,38]. Other support for a similar function between human Langerhans cells and that of mouse is in findings that many important functional cell surface markers are present in both species: MHC class II molecules, and co-stimulatory molecules including B7-1 (CD80), B7-2 (CD86), and other cell surface markers such as CD54 (ICAM-1) and E-cadherin [39–47]. It has recently been demonstrated that human epidermal Langerhans cells are differentiated from dermal resident CD14+ cells [43]. B. Melanocytes Melanocytes are not original epidermal cells, but migrate into the epidermis during embryonic development. Originating in the neural crest, melanocytes are important pigment-producing cells in the skin [48]. Primarily located in the basal layer, melanocytes synthesize pigments in the form of melanosomes, which are then transferred by way of their dendrites to the neighboring suprabasal epidermal layer-located keratinocytes. Melanocytes are present in the epithelia of skin and mucous membrane, as well as in the hair follicle bulb [48]. Similarities of structure and function between human melanocytes and that of mouse are supported by a similar resident location of melanocyte in the epidermis for both species, the presence of essential melanin-synthesis enzymes such as tyrosinase in both species, and the occurrence of genetic pigmentary diseases in both species via the same defect [49–58]. For example, a recessive mutation at the pale ear (ep) locus, a homologue of human HPS gene, on mouse chromosome 19, exhibits abnormalities in melanosmes and platelet-


6

Figure 1.1


Comparative histology of the skin. Normal skin and mucous membrane samples obtained from human newborn foreskin (A), pig chest (B), dog chest (C), rat chest (D), mouse chest (E), pig lip (F), dog lip (G), and rat lip (H) were stained with hematoxylin and eosin, and were photographed. These samples illustrate the relative thickness of epithelium and relative concentration of hair follicles. Bar = 130 mm (A to E), 260 mm (F to H).



Figure 1.2

7

Ultrastructure of Langerhans cell. Transmission electron micrograph of cytoplasm in a human epidermal Langerhans cell, demonstrating the unique cytoplasmic structure termed “Birbeck granules” (white arrows). Bar = 0.4 mm.

dense granules similar to that occurring in human patients with Hermansky-Pudlak syndrome (HPS), an autosomal recessive disease characterized by albinism, bleeding, and lysosomal storage that results from defects of diverse cytoplasmic organelles [49–51]. Similarly, an ocular albinism type 1 (Oa1) gene-deficient mouse line generated by gene targeting has resulted in ocular fundus hypopigmentation, reduction in the size of uncrossed pathway, misrouting of optic fibers at the chiasm, and presence of giant melanosomes in retinal pigment epithelium, findings observed in human patients with ocular albinism type 1 (OA1), an X-linked disorder characterized by severe impairment of visual acuity, retinal hypopigmentation, and macromelanosomes in retinal pigment epithelium [52,53]. Another example is the oculocutaneous albinism type 2 (OCA 2), an autosomal recessive disorder characterized by hypopigmentation in skin, hair, and iris in human patients as a result of mutation in the p gene. Mutations of the homologous p-locus (pink-eyed dilution gene) in mice, has also resulted in a homologous disorder manifested with hypopigmented eye, fur, and skin [54,55]. Another example of structural and functional similarities between human and mouse melanocytes is a newly recognized form of albinism termed Griscelli syndrome, a rare autosomal recessive disease characterized by partial albinism, along with immunological and/or neurological impairments. Mutations in a peripheral melanosome distribution regulatory gene RAB27A and the mouse homologue (ash, Rab27a) have resulted in Criscelli syndrome in human patients and lightened coat color and defects of pigment granule transport in ashen mutant mice [56–58]. C. Keratinocytes Being the predominant cell type in the epidermis, keratinocytes are intuitively considered to be the most important cell type for maintaining epidermal structure integrity. The keratinocyte stem cells are known to locate at the basal layer of the epidermis, from which they continuously divide in order to provide a sufficient number of cells for the purpose of differentiating into the upper layers of suprabasal keratinocytes, granular keratinocytes, and then simply keratins (at the stratum corneum) [59]. As the most important structural cell of the epidermis, keratinocytes form a coherent structural frame by way of desmosome, an intercellular adhesion network [60]. Currently, well-characterized transmembrane desmosomal components include two members of the cadherin


8


supergene family, each having four isoforms: Dsg1, 2, 3, and 4, and desmocollin (Dsc) 1, 2, 3, and 4 [60–69]. On the molecular structure level, human Dsg1 shares 76% identity of amino acid sequence with mouse Dsg1 [61,62], human Dsg2 shares 75% identity of amino acid sequence with mouse Dsg2 [61,63], whereas human Dsg3 shares 85.6% homology of amino acid sequence with its mouse counterpart [64,65]. The newly isolated human Dsg4 shares 79% amino acid identity with that of murine [66]. The genes encoding human Dsc1, mouse Dsc2, and human Dsc3 have also been isolated [67–69]. At the functional level, anti-Dsg1 and anti-Dsg3 autoantibodies that cause acantholysis and intraepidermal blisters in human patients can induce the same pathological effects in newborn mice [70,71]. Furthermore, genetic mutations in Dsg4 have been identified in human patients with inherited hypotrichosis, manifested with sparse scalp hair and eyebrows and bumpy scalp skin, and in the lanceolate hair mouse, manifested with baldness and thickened and fold skin [66]. In addition, targeted disruption of mouse Dsc1 has resulted in acantholysis and separation of epidermis below stratum granulosum, similar to the pathology occurred in the superficial form IgA pemphigus, where IgA autoantibodies targeted the human Dsc1 [72,73]. However, it should also be pointed out that there are dissimilarities in the skin components between different species. For example, there is only one form of Dsg1 in human, whereas there are three isoforms of Dsg1 (a, b, g) in mouse [66]. Being the major structural component of the epidermis, keratinocytes also synthesize keratins, intermediate 10-nm filaments that form network and provide mechanical strength, cellular structure, and assistance in adhesion molecule attachment [74]. In the basal epidermal level, keratinocytes of the keratinized stratified squamous epithelia synthesize a pair of keratins, namely keratins 5 and 14. However, when they differentiate to the suprabasal layers, keratinocytes no longer synthesize keratins 5 and 14, but rather they primarily synthesize another pair of keratins (1 and 10) [75]. The resemblance between keratins and other intermediate filaments in humans and mouse can be stated based on their functional similarities. Functionally, mutant expression of the keratin 14 gene in transgenic mice has resulted in a clinical phenotype of blisters similar to that of human heritable noninflammatory superficial blistering disease, epidermolysis bullosa simplex, due to genetic mutation of keratin 14 [76–80]. Similarly, targeted disruption of the keratin 5 gene in mice has resulted in a neonatal lethality and severe skin fragility resembling human epidermolysis bullosa simplex due to genetic mutation of keratin 5 [81–84]. Furthermore, mutant expression of the keratin 10 gene in transgenic mice has resulted in a clinical phenotype blisters like that of human heritable noninflammatory superficial blistering disease, epidermolytic hyperkeratosis, due to genetic mutation of keratins 10 [85,86]. Another keratinocyte-originated structurally important protein is plectin, which functions to linked components within the desmosomes and hemidesmosomes of skin and muscle [87]. At the molecular structure level, human plectin shares 93% amino-acid sequence identity with that of murine (rat) [88,89]. Functionally, targeted inactivation of the plectin gene in murine (mice) has resulted in a clinical phenotype of blisters similar to the human heritable noninflammatory superficial blistering disease known as epidermolysis bullosa simplex–muscular dystrophy due to genetic mutation of plectin [90–93]. As mentioned above, the skin is now recognized as an important immune organ [8–12]. Being the major cell type in the epidermis, keratinocytes share a substantial part in these immune functions. Keratinocytes are now known to express many different kinds of immune function-related molecules, such as cytokines, chemokines, co-stimulatory molecules, and MHC class II molecules [94–148]. As of this writing, expressions of many of these immune function molecules have been identified, either constitutively or upon activation, in the following human keratinocytes: complement component 3 [94], factor B [94], interleukin (IL)-1a/IL-1b [97,98], IL-3 [101], IL-6 [103], IL-7 [105], IL-8 [107,108], IL-10 [110], IL-12 [111], IL-15 [112], IL-18 [114], IL-20 [115], IL-1 receptor antagonist (IL-1ra) [99], tumor necrosis factor-alpha (TNF-a) [132–134], interferon-alpha (IFN-a) [135], IFN-b [135], CC chemokine receptor 3 (CCR3) [142,143], RANTES [128], eotaxin [144], monocyte chemoattractant protein-1 (MCP-1) [145], macrophage inflammatory protein-3 (MIP-3) [141], macrophage colony stimulating factor (M-CSF) [136], granulocyte macrophage



9

colony stimulating factor (GM-CSF) [139], intercellular adhesion molecule-1 (ICAM-1) [132,146,147], transforming growth factor-alpha (TGF-a) [130], TGF-b [130], vascular endothelia growth factor (VEGF) [116–119], platelet derived growth factor (PDGF) [123], CD14 [127], CD40 [125], toll-like receptor 4 [127], interferon inducible protein 10 (IP-10) [121,122], and MHC class II molecules [137]. Among these immune function molecules identified in human keratinocytes, mouse keratinocytes are confirmed thus far to share expressions of the following molecules with the human counterpart: IL-1a/IL-1b [95,96], IL-3 [102], IL-6 [104], IL-7 [105,106], IL-10 [109], IL-18 [113], IL-1ra [100], TNF-a [133], RANTES [129], M-CSF [136], GM-CSF [140], ICAM-1 [148], TGF-b [131], VEGF [120], PDGF [124], CD40 [126], and MHC class II molecules [138]. Significant similarities have been shown between human and mouse keratinocytes in structure and function. These similarities in intercellular adhesions, structural integrity, and immune function–related molecules strongly indicate that this major cell type of the epidermis in human and small mammals share very similar, if not identical, structural and functional properties.

III. THE DERMAL–EPIDERMAL JUNCTION (SKIN BASEMENT MEMBRANE ZONE) The skin basement membrane zone situated at the dermal–epidermal junction is an interface between the upper skin layer of the epidermis and the lower skin layer of the dermis [29]. The current understanding of the skin basement membrane zone is that it primarily functions to attach epidermis and dermis to one another, support the growth and organization of epidermis, and serve as a semipermeable barrier [29]. Due to the structural and functional complexity of this skin basement membrane zone, Chapter 2 is devoted to the discussion of details.

IV. THE DERMIS The dermis comprises the bulk of the skin and provides its elasticity, tensile strength, and pliability [29]. The major components of the dermis are collagen and elastic connective tissues, with collagen accounting for about 75% of the skin’s dry weight [29]. In fetal human skin, the predominantly expressed collagen is type III collagen [149]. In adult human skin, however, the predominant collagen in the dermis is type I collagen [29]. In adult human skin, the three interstitial collagens — type I collagen (80% to 90%), type III collagen (8 to 12%), and type V collagen (about 5%) — assemble into interwoven fibrous networks and provide strength and elasticity for the skin [29]. The dermal collagens are synthesized by fibroblasts, mesenchymally derived cells and the predominant cell type in the dermis [29]. The similarities between human and mouse dermal fibroblasts are illustrated by the facts that both human and mouse fibroblasts synthesize the major constituents of dermis, type I and type III collagens [150–152]. In addition, human dermal fibroblasts are capable of synthesis and deposit the two essential skin basement membrane-specific collagens, type IV and type VII collagens [153–156]. A mouse fibroblast-derived tumor cell line (EHS fibrosarcoma) is also known to synthesize the basement membrane type IV collagen [157]. Besides the major cell type of fibroblasts, the dermis also houses other cells, such as mast cells, macrophages, and T cells, with the greatest density around papillary dermis and vasculature [29]. The dermis also contains many important structures for the functions of the skin. Among them is the all-important blood vessel, which provides essential nutrients to the skin, temperature and blood pressure regulation, and wound repair, as well as immunological defense for the body. The microvasculatures of the skin include arterioles/terminal arterioles, precapillary sphincters, arterial and venous capillaries, postcapillary venules, and collecting venules [29]. All cutaneous microvasculatures are covered by a special type of cell called veil cells, which define a domain for the dermal microvasculature although they are not part of vessel wall [29]. Microscopic examination of dermal blood vessels of both human and mouse skin, as identified by immunolabeling of factor


10


B

A

Figure 1.3

Microvascular networks in the skin dermis. Immunofluorescence microscopy of normal human newborn foreskin (A) and mouse ear skin (B) using anti-factor VIII–related antigen (A) and antiCD31 (B) illustrates the presence of microvascular networks in the dermis. Bar = 72 mm (A), 50 mm (B).

VIII-related antigen and CD31 (a blood vessel endothelial antigen), respectively, shows similarities of microvasculature in numbers and shape (Figure 1.3). Endothelial cells lining the lumen of dermal vasculature are well known to participate in cutaneous inflammation by their expressions of adhesion molecules that facilitate the migration of inflammatory leukocytes from peripheral blood to the skin sites. It is now clear that both human and mouse dermal vascular endothelial cells are capable of expressing these inflammation-related adhesion molecules: ICAM-1, VCAM-1, E-selectin, and P-selectin [158–161]. In addition, the dermis also houses nerves and receptors of the skin and the epidermis-derived appendages, including hair follicles, sebaceous glands, eccrine (sweat) glands, and apocrine glands, and lymphatic vessels [29].

V. THE HYPODERMIS The tissue below the dermis is called hypodermis, which is abruptly distinct from the upper layer dermis histologically [29]. The hypodermis is an adipose tissue-predominant region, but is functionally integrated with the upper layer dermis through nerve, microvascular, and lymphatic networks and the continuity of hair follicles into the hypodermis [29]. Mesenchymally derived, the adipocyte is the predominant cell type in the hypodermis. Adipocytes are organized into lobular structures outlined by fibrous connective tissues [29]. The tissues in the hypodermis serve as an energy supplier, insulator, cushion, and protector of the skin, and also provide mobility over the underlying structures [29]. Scleroderma in human patients as well as a spontaneous mouse model of scleroderma (tight-skin mouse) are characterized by the presence of autoantibodies to extracellular matrix, major microfibrillar-component protein fibrillin 1, increased biosynthesis of type I collagen, and clinical improvement by intravenous immunoglobulin treatment. In both cases, hypodermal adipose tissues are infiltrated by mononuclear cells in early lesions, and are replaced by dense fibrous connective tissue in late lesions, resulting in taut skin [162–168].

VI. SUMMARY Comparative analyses of skin structure and function allow us to determine that there are substantial similarities in both structural and functional properties of human skin and that of small



11

mammals. These similarities are particularly well characterized for human skin and mouse skin, in part due to the greater availability of research reagents for studies of mouse skin components. Thus, these similarities allow readers to interpret the observations in animal models of inflammatory skin diseases with greater confidence, knowing that the extrapolations have a strong scientific basis.

ACKNOWLEDGMENT This work is supported by NIH grants R01 AR47667, R03 AR47634, and R21 AR48438 (Lawrence S. Chan).

REFERENCES 1. Kanitakis, J., Anatomy, histology, and immunohistochemistry of normal human skin, Eur. J. Dermatol., 12, 390, 2002. 2. Nemes, Z. and Steinert, P.M., Bricks and mortar of the epidermal barrier, Exp. Mol. Med., 31, 5, 1999. 3. Wertz, P.M., Lipids and barrier function of the skin, Acta Derm. Venereol., 208, 7, 2000. 4. Forslind, B., The skin barrier: analysis of physiologically important elements and trace elements, Acta Derm. Venereol., 208, 46, 2000. 5. Bouwstra, J. et al. New aspects of the skin barrier organization, Skin Pharmacol. Appl. Skin Physiol., 14 (Suppl. 1), 52, 2001. 6. Bouwstra, J. et al., Structure of skin barrier and its modulation by vesicular formulations, Prog. Lipid Res., 42, 1, 2003. 7. Tsuruta, D. et al., The barrier function of skin: how to keep a tight lid on water loss, Trends Cell Biol., 12, 355, 2002. 8. Spellberg, B., The cutaneous citadel. A holistic view of skin and immunity, Life Sci., 67, 477, 2000. 9. Bos, J.D., The skin as an organ of immunity, Clin. Exp. Immunol., 107 (Suppl. 1), 3, 1997. 10. Robert, C. and Kupper, T.S., Inflammatory skin diseases, T cells, and immune surveillance, N. Engl. J. Med., 341, 1817, 1999. 11. Chuong, C.M. et al., What is the “true” function of skin? Exp. Dermatol., 11, 159, 2002. 12. Hayday, A. and Viney, J.L., The ins and outs of body surface immunology, Science, 290, 97, 2000. 13. Beltrani, V.S. and Beltrani, V.P., Contact dermatitis, Ann. Allergy Asthma Immunol., 78, 160, 1997. 14. Bernstein, D.I., Allergic reactions to workplace allergens, JAMA, 278, 1907, 1997. 15. Sussman, G.L., Beezhold, D.H., and Kurup, V.P., Allergens and natural rubber proteins, J. Allergy Clin. Immunol., 110 (Suppl. 2), 33, 2002. 16. Kimbe, I. et al., Allergic contact dermatitis, Int. Immunopharmacol., 2, 201, 2002. 17. Chan, L.S. et al., Eptiope spreading: lessons from autoimmune skin diseases, J. Invest. Dermatol., 110, 103, 1998. 18. Koulu, L. et al., Human autoantibodies against a desmosomal core protein in pemphigus foliaceus, J. Exp. Med., 160, 1509, 1984. 19. Eyre, R.W. and Stanley, J.R., Identification of pemphigus vulgaris antigen extracted from normal human epidermis and comparison with pemphigus foliaceus antigen, J. Clin. Invest., 81, 807, 1988. 20. Anhalt, G.J. et al., Paraneoplastic pemphigus: an autoimmune mucocutaneous disease associated with neoplasia, N. Engl. J. Med., 323, 1729, 1990. 21. Woodley, D.T. et al., Identification of the skin basement membrane autoantigen in epidermolysis bullosa acquisita, N. Engl. J. Med., 310, 107, 1984. 22. Labib, R.S. et al., Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting, J. Immunol., 136, 1231, 1986. 23. Zone, J.J. et al., Identification of the cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis, J. Clin. Invest., 85, 812, 1990. 24. Domloge-Hultsch, N. et al., Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease, J. Clin. Invest., 90, 1628, 1992.


12


25. Chan, L.S. et al., Identification and partial characterization of a novel 105-kDalton lower lamina lucida autoantigen associated with a novel immune-mediated subepidermal blistering disease, J. Invest. Dermatol., 101, 262, 1993. 26. Mahoney, M.G. et al., Explanations for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris, J. Clin. Invest., 103, 461, 1999. 27. Shirakata, Y. et al., Lack of mucosal involvement in pemphigus foliaceus may be due to low expression of desmoglein 1, J. Invest. Dermatol., 110, 76, 1998. 28. Sirois, D., Leigh, J.E., and Sollecito, T.P., Oral pemphigus vulgaris preceding cutaneous lesions: recognition and diagnosis, J. Am. Dent. Assoc., 131, 1156, 2000. 29. Haake, A.R. and Holbrook, K., The structure and development of skin, in Fitzpatrick’s Dermatology in General Medicine, Freedberg, I.M. et al., Eds., McGraw-Hill, New York, 1999, chap. 7. 30. Katz, S.I. et al., Epidermal Langerhans cells are derived from cells originating in the bone marrow, Nature, 282, 324, 1979. 31. Castellino, F. et al., Antigen presentation by MHC class II molecules: invariant chain function, protein trafficking, and the molecular basis of diverse determinant capture, Hum. Immunol., 54, 159, 1997. 32. Stingl, G. et al., The epidermis: an immunologic microenvironment, in Fitzpatrick’s Dermatology in General Medicine, Freedberg, I.M., et al., Eds., McGraw-Hill, New York, 1999, chap. 28. 33. Valladeau J. et al., The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface, Eur. J. Immunol., 29, 2695, 1999. 34. Valladeau, J. et al., Langerin, a novel C-type lectin specific to Langerhans cells, is an enodocytic receptor that induces the formation of Birbeck granules, Immunity, 12, 71, 2000. 35. Valladeau, J. et al., Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues, J. Immunol., 168, 782, 2002. 36. Takahara, K. et al., Identification and expression of mouse langerin (CD207) in dendritic cells, Int. J. Immunol., 14, 433, 2002. 37. Stoitzner, P. et al., Visualization and characterization of migratory Langerhans cells in murine skin and lymph nodes by antibodies against langerin/CD207, J. Invest. Dermatol., 120, 266, 2003. 38. Geissmann, F. et al., Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin, J. Exp. Med., 196, 417, 2002. 39. Borkowski, T.A. et al., Expression of E-cadherin by murine dendritic cells: E-cadherin as a dendritic cell differentiation antigen characteristic of epidermal Langerhans cells and related cells, Eur. J. Immunol., 24, 2767, 1994. 40. Blauvelt, A., Katz, S.I., and Udey, M.C., Human Langerhans cells express E-cadherin, J. Invest. Dermatol., 104, 293, 1995. 41. Salgado, C.G. et al., Functional CD40 ligand is expressed on epidermal Langerhans cells, J. Leukoc. Biol., 66, 281, 1999. 42. Salgado, C.G. et al., Differential effects of cytokines and immunosuppressive drugs on CD40, B7-1, and B7-2 expression on purified epidermal Langerhans cells1, J. Invest. Dermatol., 113, 1021, 1999. 43. Adriana, T. et al., Dermal-resident CD14+ cells differentiate into Langerhans cells, Nat. Immunol., 2, 1151, 2001. 44. Teunissen, M.B. et al., Human epidermal Langerhans cells undergo profound morphologic and phenotypical changes during in vitro culture, J. Invest. Dermatol., 94, 166, 1990. 45. Tang, A. and Udey, M.C., Inhibition of epidermal Langerhans cell function by low dose ultraviolet B radiation. Ultraviolet B radiation selectively modulates ICAM-1 (CD54) expression by murine Langerhans cells, J. Immunol., 146, 3347, 1991. 46. Epstein, S.P., Baer, R.L., and Belsito, D.V., Effect of triggering epidermal Fc gamma receptors on the interleukin-2- and interleukin-6-induced upregulation of Ia antigen expression by murine epidermal Langerhans cells: the role of prostaglandins and cAMP, J. Invest. Dermatol., 97, 461, 1991. 47. Chang, C.H., Furue, M., and Tamaki, K., B7-1 expression of Langerhans cells is up-regulated by proinflammatory cytokines, and is down-regulated by interferon-gamma or by interleukin-10, Eur. J. Immunol., 25, 394, 1995. 48. Jimbow, K. et al., Biology of melanocytes, in Fitzpatrick’s Dermatology in General Medicine, Freedberg, I.M., et al., Eds., McGraw-Hill, New York, 1999, chap. 15. 49. Gahl, W.A. et al., Genetic defects and clinical characteristics of patients with a form of oculocutaneous albinism (Hermansky-Pudlak syndrome), N. Engl. J. Med., 338, 1258, 1998.



13

50. Gardner, J.M. et al., The mouse pale ear (ep) mutation is the homologue of human Hermansky-Pudlak syndrome, Proc. Natl. Acad. Sci. U. S. A., 94, 9238, 1997. 51. Oh, J. et al., Positional cloning of a gene for Hermansky-Pudlak syndrome, a disorder of cytoplasmic organelles, Nat. Genet., 14, 242, 1996. 52. Bassi, M.T. et al., Cloning of the gene for ocular albinism type 1 from the distal short arm of the x chromosome, Nat. Genet., 10, 13, 1995. 53. Incerti, B. et al., Oa1 knock-out: new insights on the pathogenesis of ocular albinism type 1, Hum. Mol. Genet., 9, 2781, 2000. 54. Rosemblat, S. et al., Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene, Proc. Natl. Acad. Sci. U. S. A., 91, 12071, 1994. 55. Spritz, R.A. et al., Frequent intragenic deletion of the P gene in Tanzanian patients with type II oculocutaneous albinism (OCA2), Am. J. Hum. Genet., 56, 1320, 1995. 56. Wilson, S.M. et al., A mutation in Rab27a causes the vesicle transport defects observed in ashen mice, Proc. Natl. Acad. Sci. U. S. A., 97, 7933, 2000. 57. Hume, A.N. et al., Rab27a regulates the peripheral distribution of melanosomes in melanocytes, J. Cell Biol., 152, F21, 2001. 58. Anikster, Y. et al., Evidence that Criscelli syndrome with neurological involvement is caused by mutations in RAB27A, not MYO5A, Am. J. Hum. Genet., 71, 407, 2002. 59. Lavker, R.M. and Sun, T.T., Epidermal stem cells, J. Invest. Dermatol., 81, 121s, 1983. 60. Green, K.J. and Jones, J.C., Desmosomes and hemidesmosomes: structure and function of molecular components, F.A.S.E.B. J., 10, 871, 1996. 61. Mahoney, M.G. et al., Interspecies conservation and differential expression of mouse desmoglein gene family, Exp. Dermatol., 11, 115, 2002. 62. Liu, Z. et al., cDNA cloning of a novel human ubiquitin carrier protein. An antigenic domain specifically recognized by endemic pemphigus foliaceus autoantibodies is encoded in a secondary reading frame of this human epidermal transcript, J. Biol. Chem., 267, 15829, 1992. 63. Schafter, S., Koch, P.J., and Franke, W.W., Identification of the ubiquitous human desmoglein, Dsg2, and the expression catalogue of the desmoglein subfamily of desmosomal cadherins, Exp. Cell Res., 211, 391, 1994. 64. Ishikawa, H. et al., Cloning of the mouse desmoglein 3 gene (Dsg3): interspecies conservation within the cadherin superfamily, Exp. Dermatol., 9, 229, 2000. 65. Amagai, M., Klaus-Kovtun, V., and Stanley, J.R., Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion, Cell, 67, 869, 1991. 66. Kijuic, A. et al., Desmoglein 4 in hair follicle differentiation and epidermal adhesion: Evidence from inherited hypotrichosis and acquired pemphigus vulgaris, Cell, 113, 246, 2003. 67. King, I.A. et al., Cloning of the cDNA (DSC1) coding for human type 1 desmocollin and its assignment to chromosome 18, Genomics, 18, 185, 1993. 68. Lorimer, J.E. et al., Cloning, sequence analysis and expression pattern of mouse desmocollin 2 (DSC2), a cadherin-like adhesion molecule, Mol. Membr. Biol., 11, 229, 1994. 69. King, I.A. et al. The desmocollins of human foreskin epidermis: identification and chromosomal assignment of a third gene and expression patterns of the three isoforms, J. Invest. Dermatol., 105, 314, 1995. 70. Anhalt, G.J. et al., Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease, N. Engl. J. Med., 306, 1189, 1982. 71. Roscoe, J.T. et al., Brazilian pemphigus foliaceus autoantibodies are pathogenic to BALB/c mice by passive transfer, J. Invest. Dermatol., 85, 538, 1985. 72. Chidgey, M. et al., Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation, J. Cell Biol., 155, 821, 2001. 73. Hashimoto, T. et al., Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis type of IgA pemphigus, J. Invest. Dermatol., 109, 127, 1997. 74. Goldman, R.D. et al., The function of intermediate filaments in cell shape and cytoskeletal integrity, J. Cell Biol., 134, 971, 1996. 75. Latkowski, J-A.M. and Freedberg, I.M. Epidermal cell kinetics, epidermal differentiation, and keratinization, in Fitzpatrick’s Dermatology in General Medicine, Freedberg, I.M. et al., Eds., McGrawHill, New York, 1999, chap. 9.


14


76. Vassar, R. et al., Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease, Cell, 64, 365, 1991. 77. Coulombe, P.A. et al., Point mutation in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses, Cell, 66, 1301, 1991. 78. Bonifas, J.M., Rothman, A.L., and Epstein, E.H., Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities, Science, 254, 1202, 1991. 79. Hovanian, A. et al., A missense mutation in the rod domain of keratin 14 associated with recessive epidermolysis bullosa simplex, Nat. Genet., 3, 327, 1993. 80. Rugg, E.L. et al., A functional “knockout” of human keratin 14, Genes Dev., 8, 2563, 1994. 81. Peters, B. et al., Complete cytolysis and neonatal lethality in keratin 5 knockout mice reveal its fundamental role in skin integrity and in epidermolysis bullosa simplex, Mol. Biol. Cell, 12, 1775, 2001. 82. Lane, E.B. et al., A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering, Nature, 356, 244, 1992. 83. Dong, W., Ryynanen, M., and Uitto, J., Identification of a leucine-to-proline mutation in the keratin 5 gene in a family with the generalized Kobner type of epidermolysis bullosa simplex, Hum. Mutation, 2, 94, 1993. 84. Chan, Y.M. et al., Mutations in the non-helical linker segment L1-2 of keratin 5 in patients with Weber-Cockayne epidermolysis bullosa simplex, J. Cell Sci., 107, 765, 1994. 85. Fuchs, E., Esteves, R.A., and Coulombe, P.A., Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for epidermolytic hyperkeratosis, Proc. Natl. Acad. Sci. U. S. A., 89, 6906, 1992. 86. Chen, J. et al., The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes, Cell, 70, 811, 1992. 87. Wiche, G., Role of plectin in cytoskeleton organization and dynamics, J. Cell Sci., 111, 2477, 1998. 88. Liu, C.G. et al. Human plectin: organization of the gene, sequence analysis, and chromosome localization (8q24), Proc. Natl. Acad. Sci. U. S. A., 93, 42278, 1996. 89. Wiche, G. et al. Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil, J. Cell Biol., 114, 83, 1991. 90. Andra, K. et al., Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle and heart cytoarchitecture, Genes Dev., 11, 3143, 1997. 91. Smith, F.J.D. et al., Plectin deficiency results in muscular dystrophy with epidermolysis bullosa, Nat. Genet., 13, 450, 1996. 92. McLean, W.H.I. et al., Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization, Genes Dev., 10, 1724, 1996. 93. Gache, Y. et al., Defective expression of plectin in epidermolysis bullosa simplex with muscular dystrophy, J. Clin. Invest., 87, 2289, 1996. 94. Pasch, M.C. et al., Synthesis of complement component C3 and factor B in human keratinocytes is differentially regulated by cytokines, J. Invest. Dermatol., 114, 78, 2000. 95. Luger, T.A. et al., Murine epidermal cell-derived thymocyte-activating factor resembles murine interleukin-1, J. Immunol., 128, 2147, 1982. 96. Ansel, J.C. et al. The expression and modulation of IL-1 alpha in murine keratinocytes, J. Immunol., 140, 2274, 1988. 97. Kupper, T.S. et al., Human keratinocytes contain mRNA indistinguishable from monocyte interleukin1 alpha and beta mRNA. Keratinocyte epidermal cell-derived thymocyte-activating factor is identical to interleukin-1, J. Exp. Med., 164, 2095, 1986. 98. Bell, T.V. et al., Expression of mRNA homologous to interleukin 1 in human epidermal cells, J. Invest. Dermatol., 88, 375, 1987. 99. Hammerberg, C. et al., Interleukin-1 receptor antagonist in normal and psoriatic epidermis, J. Clin. Invest., 90, 571, 1992. 100. La, E. and Fischer, S.M., Transcriptional regulation of intracellular IL-1 receptor antagonist gene by IL-1 alpha in primary mouse keratinocytes, J. Immunol., 166, 6149, 2001. 101. Danner, M. and Luger, T.A., Human keratinocytes and epidermoid carcinoma cell lines produce a cytokine with interleukin 3-like activity, J. Invest. Dermatol., 88, 353, 1987. 102. Luger, T.A. et al., Keratinocyte-derived interleukin 3, Ann. N. Y. Acad. Sci., 548, 253, 1988.



15

103. Yoshizaki, K. et al., Interleukin 6 and expression of its receptor on epidermal keratinocytes, Cytokine, 2, 381, 1990. 104. Sprecher, E. and Becker, Y., Detection of IL-1 beta, TNF-alpha, and IL-6 gene transcription by the polymerase chain reaction in keratinocytes, Langerhans cells and peritoneal exudate cells during infection with herpes simplex virus-1, Arch. Virol., 126, 253, 1992. 105. Heufler, C. et al., Interleukin 7 is produced by murine and hman keratinocytes, J. Exp. Med., 178, 1109, 1993. 106. Ariizumi, K. et al., IFN-gamma-dependent IL-7 gene regulation in keratinocytes, J. Immunol., 154, 6031, 1995. 107. Barker, J.N. et al., Modulation of keratinocyte-derived interleukin-8 which is chemotactic for neutrophils and T lymphocytes, Am. J. Pathol., 139, 869, 1991. 108. Kristensen, M.S. et al., Quantitative determination of IL-1 alpha-induced IL-8 mRNA levels in cultured human keratinocytes, dermal fibroblasts, endothelial cells, and monocytes, J. Invest. Dermatol., 97, 506, 1991. 109. Enk, A.H. and Katz, S.I. Identification and induction of keratinocyte-derived IL-10, J. Immunol., 149, 92, 1992. 110. Enk, A.H. et al., Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro, J. Immunol., 154, 4851, 1995. 111. Muller, G. et al., Identification and induction of human keratinocyte-derived IL-12, J. Clin. Invest., 94, 1799, 1994. 112. Blauvelt, A. et al., Interleukin-15 mRNA is expressed by human keratinocytes, Langerhans cells, and blood-derived dendritic cells and is downregulated by ultraviolet B radiation, J. Invest. Dermatol., 106, 1047, 1996. 113. Stoll, S. et al., Production of IL-18 (IFN-gamma-inducing factor) messenger RNA and functional protein by murine keratinocytes, J. Immunol., 159, 298, 1997. 114. Koizumi, H.,et al., Distribution of IL-18 and IL-18 receptor in human skin: various forms of IL-18 are produced in keratinocytes, Arch. Dermatol. Res., 293, 325, 2001. 115. Grone A., Keratinocytes and cytokines, Vet. Immunol. Immunopathol., 88, 1, 2002. 116. Ballaum, C. et al., Human keratinocyte express the three major splice forms of vascular endothelial growth factor, J. Invest. Dermatol., 104, 7, 1995. 117. Frank, S. et al., Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair, F.A.S.E.B. J., 13, 2002, 1999. 118. Diaz, B.V. et al., Regulation of vascular endothelial growth factor expression in human keratinocytes by retinoids, J. Biol. Chem., 275, 642, 2000. 119. Sen, C.K. et al., Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing, J. Biol. Chem., 277, 33284, 2002. 120. Larcher, F. et al., Up-regulation of vascular endothelial growth factor/vascular permeability factor in mouse skin carcinogenesis correlates with malignant progression state and activated H-ras expression levels, Cancer Res., 56, 5391, 1996. 121. Sarris, A.H. et al., Human interferon-inducible protein 10: expression and purification of recombinant protein demonstrate inhibition of early human hematopoietic progenitors, J. Exp. Med., 178, 1127, 1993. 122. Boorsma DM et al., Chemokine IP-10 expression in cultured human keratinocytes, Arch. Dermatol. Res., 290, 335, 1998. 123. Ansel, J.C. et al., Human keratinocytes are a major source of cutaneous platelet-derived growth factor, J. Clin. Invest., 92, 671, 1993. 124. Beer, H.D., Longaker, M.T., and Werner, S., Reduced expression of PDGF and PDGF receptors during impaired wound healing, J. Invest. Dermatol., 109, 132, 1997. 125. Denfeld, R.W. et al., CD40 is functionally expressed on human keratinocytes, Eur. J. Immunol., 26, 2329, 1996. 126. Coutant, K.D. et al., Early changes in murine epidermal cell phenotype by contact sensitizers, Toxicol. Sci., 48, 74, 1999. 127. Song, P.I. et al., Human keratinocytes express functional CD14 and toll-like receptor 4, J. Invest. Dermatol., 119, 424, 2002.


16


128. Li, J. et al., Epidermal and oral keratinocytes are induced to produce RANTES and IL-8 by cytokine stimulation, J. Invest. Dermatol., 106, 661, 1996. 129. Frank, S. et al., Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression, Biochem. J., 347, 265, 2000. 130. Gaido, K.W. et al., 2,3,4,8-Tetrachlorodibenzo-p-dioxin-dependent regulation of transforming growth factors-alpha and -beta2 expression in a human keratinocyte cell line involves both transcriptional and post-transcriptional control, J. Biol. Chem., 267, 24591, 1992. 131. Bascom, C.C. et al., Complex regulation of transforming growth factor beta 1, beta 2, and beta 3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factor beta 1 and beta 2, Mol. Cell Biol., 9, 5508, 1989. 132. Pastore, S. et al., Interferon-gamma promotes exaggerated cytokine production in keratinocytes cultured from patients with atopic dermatitis, J. Allergy Clin. Immunol., 101, 538, 1998. 133. Kolde, G. et al., Immunohistological and immunoelectron microscopic identification of TNF alpha in normal human and murine epidermis, Arch. Dermatol. Res., 284, 154, 1992. 134. Matsuura, K., Otsuka, F., and Fujisawa, H., Effects of interferons on tumor necrosis factor alpha production from human keratinocytes, Cytokine, 10, 500, 1998. 135. Fujisawa, H. et al., The expression and modulation of IFN-alpha and IFN-beta in human keratinocytes, J. Interferon Cytokine Res., 17, 721, 1997. 136. Chodakewitz, J.A. et al., Macrophage colony-stimulating factor production by murine and human keratinocytes. Enhancement by bacterial lipopolysaccharide, J. Immunol., 144, 2190, 1990. 137. Basham, T.Y. et al., Recombinant gamma interferon differentially regulates class II antigen expression and biosynthesis on cultured normal human keratinocytes, J. Interferon Res., 5, 23, 1985. 138. Jun, B.D., Krueger, G.G., and Roberts, L.K., Differential expression of Ia by murine keratinocytes and gut epithelium in response to recombinant gamma-interferon, J. Invest. Dermatol., 93, 33, 1989. 139. Kupper, T.S. et al., Interleukin 1 binds to specific receptors on human keratinocytes and induces granulocyte macrophage colony-stimulating factor mRNA and protein. A potential autocrine role for interleukin 1 in epidermis, J. Clin. Invest., 82, 1787, 1988. 140. Chodakewitz, J.A., Kupper, T.S., and Coleman, D.L., Keratinocyte-derived granulocte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages, J. Immunol., 140, 832, 1988. 141. Tohyama, M. et al., Differentiated keratinocytes are responsible for TNF-alpha regulated production of macrophage inflammatory protein 3alpha/CCL20, a potent chemokine for Langerhans cells, J. Dermatol. Sci., 27, 130, 2001. 142. Wakugawa, M. et al., Expression of CC chemokine receptor 3 on human keratinocytes in vivo and in vitro-upregulation by RANTES, J. Dermatol. Sci., 25, 229, 2001. 143. Petering, H. et al., Characterization of the CC chemokine receptor 3 on human keratinocytes, J. Invest. Dermatol., 116, 549, 2001. 144. Jean-Baptiste, S. et al., Expression of eotaxin, an eosinophil-selective chemokine, paralleles eosinophil accumulation in the vesiculobullous stage of incontinentia pigmenti, Clin. Exp. Immunol., 127, 470, 2002. 145. Li, J., Farthing, P.M., and Thornhill, M.H., Oral and skin keratinocytes are stimulated to secrete monocyte chemoattractant protein-1 by tumor necrosis factor-alpha and interferon gamma, J. Oral Pathol. Med., 29, 438, 2000. 146. Griffiths, C.E., Voorhees, J.J., and Nickoloff, B.J., Gamma interferon induces different keratinocyte cellular patterns of expression of HLA-DR and DQ and intercellular adhesion molecule-1 (ICAM-1) antigens, Br. J. Dermatol., 120, 1, 1989. 147. Albanesi, C., Cavani, A., and Girolomoni, G., IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha, J. Immunol., 162, 494, 1999. 148. Carroll JM et al., Transgenic mice expressing IFN-gamma in the epidermis have eczema, hair hypopigmentation, and hair loss, J. Invest. Dermatol., 108, 412, 1997. 149. Sandberg, M. et al., Construction of a human pro alpha 1(III) collagen cDNA clone and localization of type III collagen expression in human fetal tissues, Matrix, 9, 82, 1989.



17

150. Liau, G., Yamada, Y., and de Crombrugghe, B., Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts, J. Biol. Chem., 260, 531, 1985. 151. Lambert, C.A. et al., Coordinated regulation of procollagens I and III and their post-translational enzymes by dissipation of mechanical tension in human dermal fibroblasts, Eur. J. Cell Biol., 80, 479, 2001. 152. Dumas, M. et al., In vitro biosynthesis of type I and type III collagens by human dermal fibroblasts from donors of increasing age, Mech. Ageing Dev., 73, 179, 1994. 153. Olsen, D.R., Chu, M.L., and Uitto, J., Expression of basement membrane zone gene coding for type IV procollagen and laminin by human skin fibroblasts in vitro: elevated alpha 1 (IV) collagen mRNA levels in lipoid proteinosis, J. Invest. Dermatol., 90, 734, 1988. 154. Markinkovich, M.P. et al., Cellular origin of the dermal-epidermal basement membrane, Dev. Dyn., 197, 255, 1993. 155. Woodley, D.T. et al., Epidermolysis bullosa acquisita antigen, a major cutaneous basement membrane component, is synthesized by human dermal fibroblasts and other cutaneous tissues, J. Invest. Dermatol., 87, 227, 1986. 156. Chen, M. et al., Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa, Nat. Genet., 32, 670, 2002. 157. Schwarz, U. et al., Structure of mouse type IV collagen. Amino-acid sequence of the C-terminal 511residue-long triple-helical segment of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain, Eur. J. Biochem., 157, 49, 1986. 158. Sigurdsson, V. et al., Expression of VCAM-1, ICAM-1, E-selectin, and P-selectin on endothelium in situ in patients with erythroderma, mycosis fungoides and atopic dermatitis, J. Cutan. Pathol., 27, 436, 2000. 159. Harari, O.A. et al., Endothelial cell E- and P-selectin up-regulation in murine contact sensitivity is prolonged by distinct mechanisms occurring in sequence, J. Immunol., 163, 6860, 1999. 160. Saloga, J. et al., Cutaneous exposure to the superantigen staphylococcal enterotoxin B elicits a T-celldependent inflammatory response, J. Invest. Dermatol., 106, 982, 1996. 161. Zhou, L. et al., Tepoxalin blocks neutrophil migration into cutaneous inflammatory sites by inhibiting Mac-1 and E-selectin expression, Eur. J. Immunol., 26, 120, 1996. 162. Tu, J.H. and Eisen, A.Z., Scleroderma, in Fitzpatrick’s Dermatology in General Medicine, Freedberg, I.M. et al., Eds., McGraw-Hill, New York, 1999, chap. 174. 163. Christner, P.J. et al., The tight skin 2 mouse: an animal model of scleroderma displaying cutaneous fibrosis and mononuclear cell infiltration, Arthritis Rheum., 38, 1791, 1995. 164. Arnett, F.C. et al., Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin 1, in patients with localized scleroderma, Arthritis Rheum., 42, 2656, 1999. 165. Tan, F.K. et al., Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin 1, in patients with scleroderma and other connective tissue diseases, J. Immunol., 163, 1066, 1999. 166. Murai, C. et al., Spontaneous occurrence of anti-fibrillin-1 autoantibodies in tight-skin mice, Autoimmunity, 28, 151, 1998. 167. Blank, M. et al., The role of intravenous immunoglobulin therapy in mediating skin fibrosis in tight skin mice, Arthritis Rheum., 46, 1689, 2002. 168. Levy, Y. et al., Skin score decrease in systemic sclerosis patients treated with intravenous immunoglobulin: a preliminary report, Clin. Rheumatol., 18, 207, 2000.



CHAPTER

2

Comparative Structure and Function of the Skin: Epithelial Basement Membrane Zone Lawrence S. Chan

CONTENTS I. Overview of Skin Basement Membrane Zone ....................................................................19 II. Structures of Skin Basement Membrane Zone ...................................................................20 III. Skin Basement Membrane Components .............................................................................20 A. Type VII Collagen .......................................................................................................20 B. Laminins ......................................................................................................................24 C. Integrin Subunits..........................................................................................................24 D. Type XVII Collagen (BP180) .....................................................................................25 E. Other Skin BMZ Components ....................................................................................25 IV. Summary ..............................................................................................................................26 Acknowledgment..............................................................................................................................26 References ........................................................................................................................................26 I. OVERVIEW OF SKIN BASEMENT MEMBRANE ZONE Situated between the epidermis (the upper layer of the skin) and the dermis (the middle layer of the skin), the specific location of the skin basement membrane zone (BMZ) conveys an intuitive view of its functional role as a connecting network that anchors the epidermis to the dermis. This view has been supported by cumulative scientific evidence, both direct and indirect. For example, when one or more of the components of the skin BMZ is defective due to genetic mutation, skin fragility and associated blisters were observed [1–3]. Conversely, restoration of expression of these defected BMZ components by molecular biology techniques results in restoration of skin BMZ structure and function [4–6]. Similarly, when autoantibodies target one or more of the components of the skin BMZ, inflammation and the accompanied blistering diseases are detected [7–12]. In addition, in vitro studies demonstrate that individual components of the skin BMZ have the capacity to form strong binding to one another or to other structures, thus providing evidence to support their roles in forming a connecting network for the purpose of anchoring the epidermis to the dermis [13–19]. In the following sections, the detailed skin BMZ ultrastructures and the individual skin BMZ components that are relevant to the inflammatory skin diseases described in this book will be discussed. 0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

19


20

Figure 2.1


Transmission electron microscopy of a normal human skin basement membrane zone. The epidermis is located above the basement membrane zone, while the dermis is located below it. Short white arrows point to lamina densa, long white arrows to hemidesmosomes, and black arrowheads to anchoring fibrils. Asterisks indicate the areas of lamina lucida. Bar = 0.1 mm.

II. STRUCTURES OF SKIN BASEMENT MEMBRANE ZONE The structure of skin BMZ is best visualized under transmission electron microscopy (TEM). An ultra-thin section of the skin viewed under TEM delineates a linear electron translucent zone, which has been termed lamina lucida (LL) (asterisks, Figures 2.1 and 2.2). Below the LL on the dermis side lies a gray electron-dense (nontranslucent) linear zone, which has been termed lamina densa (LD) (short white arrows, Figures 2.1 and 2.2). Connecting beneath the LD, strings of gray structures known as anchoring fibrils are observed (black arrowheads, Figures 2.1 and 2.2). On the upper LL, dark gray electron-dense bodies that lie parallel to the skin BMZ are named hemidesmosomes, onto which the tonofilaments of the basal keratinocytes are attached (long white arrows, Figures 2.1 and 2.2). Careful examination within the LL space reveals fine string-like gray structures termed anchoring filaments that span the entire width of LL (Figures 2.1 and 2.2). It is now recognized that type VII collagen is the major component of anchoring fibril [8,19–21], which is also linked to LD. type IV collagen appears to be the major component of LD [22]. The compositions of anchoring filaments within the LL appear to include type XVII collagen and laminin-5 [23–25]. On the ultrastructural level, human skin BMZ (Figure 2.1) seems to be identical to canine (Figure 2.2). A schematic representation of skin BMZ is illustrated in Figure 2.3.

III. SKIN BASEMENT MEMBRANE COMPONENTS A. Type VII Collagen Located in the lamina densa and sub–lamina densa areas of the skin BMZ, type VII collagen is a homotrimer of alpha-1 (COL7A1) chains [20]. Each of the alpha-1 chain monomers is composed of a 145-kDa centrally located collagenous triple-helical domain flanked by a 145-kDa N-terminallocated noncollagenous domain 1 (NC1) and a 34-kDa C-terminal-located noncollagenous domain


EPITHELIAL BASEMENT MEMBRANE ZONE

Figure 2.2

21

Transmission electron microscopy of a normal canine skin basement membrane zone. The epidermis is located above the basement membrane zone, while the dermis is located below it. Short white arrows point to lamina densa, long white arrows to hemidesmosomes, and black arrowheads to anchoring fibrils. Asterisks indicate the areas of lamina lucida. Bar = 0.12 mm.

Epidermis α6β4 integrin Collagen 17 Hemidesmosome Anchoring Filaments

LL

LD (Collagen 4)

Dermis

Figure 2.3

Laminin-1 Laminin-5 Laminin-6

Anchoring Fibrils (Collagen 7)

Schematic representation of skin BMZ. LL, lamina lucida; LD, lamina densa.

2 (NC2) [19,20]. The gene encoding human type VII collagen was the first to be delineated, followed by the genes encoding mouse homologue and the partial gene encoding canine homologue [26–29]. It is now known that the NC1 domain is the major target region of the autoantibodies in human patients with an inflammatory blistering skin disease termed epidermolysis bullosa acquisita [30]. Homology analyses of the NC1 domain revealed 87% and 83% amino acid identity between humans and canine and between humans and murine, respectively [29]. In certain well-characterized regions of antigenic epitope within the NC1 domain, as defined by autoantibodies of human patients with epidermolysis bullosa acquisita, the amino acid identities between humans and those of canine and murine are greater than 90% [29]. This high degree of amino acid identity in these antigenic epitope regions is correlated with the positive labeling to the skin BMZ of human, pig, dog, rat, and mouse by an antibody raised against a full-length recombinant human type VII collagen NC1 domain (Figure 2.4). Indirect immunofluorescence microscopy illustrating the binding to the skin basement membranes from different species by the same human autoantibodies against type VII collagen

Indirect immunofluorescence microscopy illustrates the binding to the skin basement membranes of various species by an antibody raised against the human type VII collagen NC1 domain. This antihuman type VII collagen antibody positively labels the skin BMZ of human (A), pig (B), dog (C), rat (D), and mouse (E), as well as the BMZ of mucous membrane (lip) in pig (F), dog (G), and rat (H). Bar = 80 mm (A through H).

H

G

F

E

Figure 2.4

D

C

B

22

A



Figure 2.5

A C

D

Indirect immunofluorescence microscopy illustrating the binding to the skin basement membranes from different species by the same autoantibodies (A and B) and the binding to the same skin basement membrane by autoantibodies from patients of different species (C and D). The antitype VII collagen IgG autoantibodies from a human patient affected with epidermolysis bullosa acquisita positively label the floor of salt-split skin BMZ of human (A) and dog specimens (B). Similarly, the antitype XVII collagen (BPAG2) IgG autoantibodies from a pig patient (C) and a human patient (D) suffering from bullous pemphigoid label the roof of salt-split human skin samples. Stars indicate the lamina lucida space split by 1.0-M NaCl salt solution. The arrows point to the binding sites of IgG autoantibodies. Bar = 100 mm (A through D).

B


EPITHELIAL BASEMENT MEMBRANE ZONE 23


24


NC1 domain further supports the amino acid homology between different species (Figure 2.5, A and B). Functionally, scientific data seems to support a similar and essential role of type VII collagen in connecting the epidermis from the underlying dermis in both humans and small animals [31–36]. In human patients suffering from a noninflammatory heritable blistering skin disease termed dystrophic epidermolysis bullosa, mutations at various areas of type VII collagen gene have been identified [31–33]. Similarly, targeted disruption of type VII collagen gene in mice has resulted in an identical skin blistering phenotype [34]. In addition, a sheep patient suffered from a similar blistering disease also exhibited type VII collagen defect [35]. Further support for this functional similarity between human type VII collagen and that of mammals is the report of a canine equivalent of human epidermolysis bullosa acquisita characterized by autoantibodies targeting the NC1 domain of type VII collagen [36]. B. Laminins Located in the lamina lucida region, well-characterized laminin isoforms that are present in the skin BMZ include laminin-1, laminin-5, and laminin-6 [37–40]. Laminin molecules are heterotrimers each is composed of an alpha, a beta, and a gamma chain, linked together by disulfide bonds [37–40]. Different compositions of monomers constitute different isoforms of laminin. Whereas laminin-1 is composed of alpha-1, beta-1, and gamma-1 chains, laminin-5, also known as nicein/kalinin/epiligrin, is composed of alpha-3, beta-3, and gamma-2 chains. Similar to laminin1 and laminin-5, laminin-6 (originally termed k-laminin), is composed of alpha-3, beta-1, and gamma-1 chains [37–40]. The genes encoding laminin-5 chains have been isolated for human and mouse [41–43]. Homology analyses of the alpha-3 chain revealed 77% amino acid homology between human and mouse [42]. Laminin-5 and laminin-6 have been identified as the target antigens of the autoantibodies from a subgroup of human patients suffered from an inflammatory blistering skin disease named mucous membrane pemphigoid, whereas laminin-1 has not been implicated as target antigen in any human skin disease [10,44–48]. Specifically, the antigenic epitopes of these patients’ autoantibodies recognize alpha-3, beta-3, and gamma-2 chains of laminin-5 and laminin6 [10,44–48]. The functional similarity between the human laminin-5 and that of the mammals lies in the findings that genetic mutation of laminin-5 could result in a spontaneously arising noninflammatory blistering skin disease called “junctional epidermolysis bullosa” in both human patients [49–52] and in large and small mammals [53,54]. Moreover, targeted disruption of the laminin 5 gene in mice has resulted in a similar clinical phenotype as in human patients [55]. Additional support for this functional similarity between human laminin-5 and that of mammals comes from findings that the presence of autoantibodies to laminin-5 could lead to similar inflammatory blistering skin disease in both human patients [10,44–48] and small mammal [56]. Furthermore, passive transfer of antibodies to laminin-5 could induce inflammatory blistering skin disease similar to human mucous membrane pemphigoid in newborn mice [57]. C. Integrin Subunits Integrin molecules, the principal receptors for extracellular matrix, are heterodimers consisting of noncovalently linked one alpha and one beta subunits [13]. Located in the upper lamina lucida and basal epithelial cell area, major integrin subunits alpha-6 and beta-4 are essential elements for the assembly of hemidesmosome of the skin BMZ [13]. They are functionally associated with the laminin molecules and with type XVII collagen [16,17,58]. The genes encoding these skin BMZlocated integrin subunits for human, mouse, and rat have been isolated [59–64]. Sequence analyses of beta-4 subunit determined 95.1% and 87.5% identity in amino acids between mouse and rat, and between human and rat, respectively [64]. Similarly, the identity of alpha-6 subunit amino acid sequence between human and mouse was determined to be 93% [63]. In addition to the structural



25

similarity between the human alpha-6 beta-4 integrin subunits and that of small mammals [59–64], there is evidence indicating their functional similarity [65–70]. In human patients suffering from a severe heritable noninflammatory blistering skin disease known as junctional epidermolysis bullosa with pyloric atresia, beta-4 subunit gene mutation has been identified [65–67]. Likewise, the absence of alpha-6 beta-4 subunits in knockout mice results in the phenotype of a severe epidermis detachment from the underlying BMZ, which closely resembles that observed in human patients [68–70]. Furthermore, mucous membrane pemphigoid, a human inflammatory blistering skin disease, has been observed as a result of autoantibodies targeting these BMZ-located integrin subunits, although animal models of this inflammatory blistering skin disease due to anti-integrin autoantibodies have not yet been reported [71–74]. D. Type XVII Collagen (BP180) Located in the upper lamina lucida and basal epidermal cell areas, type XVII collagen is a hemidesmosomal component of the skin BMZ [75–77]. As a transmembranous protein, type XVII collagen is composed of a C-terminal–located intracellular domain, a transmembrane domain, and an N-terminal–located domain composed of many collagenous and 16 noncollagenous domains [75–77]. NC16A, the largest of all the noncollagenous domains, located just outside the basal epidermal cell membrane, is now known to be the major target antigenic site of the autoantibodies from patients with an inflammatory blistering skin disease named bullous pemphigoid [78]. The genes encoding type XVII collagen have been identified in human, mouse, and partially in canine [75–77,79]. Homology analyses of the NC16A domain revealed 58% and 57% amino acid identity between human and canine, and between human and mouse, respectively [79]. Interestingly, the amino acid identity of the transmembrane domain reaches 100% between human and canine and between human and mouse [79]. These high homology data correlate positively with binding to the same skin basement membrane by anti-BP180 IgG autoantibodies from patients of different species (Figure 2.5C and D). Functionally, scientific data seem to support a similar and essential role of type XVII collagen in connecting the epidermis from the underlying dermis in both humans and other mammals [80–88]. In human patients suffering from generalized atrophic benign epidermolysis bullosa, a noninflammatory junctional form of heritable blistering skin disease, mutations at various areas of type XVII collagen gene have been identified [80–82]. The cellular defects of this mutation are reported to be restorable by in vitro gene delivery [4]. In addition, spontaneous inflammatory blistering skin disease like that of human bullous pemphigoid has been observed in horses, pigs, dogs, and cats, showing similar clinical phenotype, histopathological findings, IgG autoantibody binding to the skin BMZ, and IgG autoantibodies targeting type XVII collagen [83–86]. Furthermore, passive transfer experiments using rabbit antibodies against mouse recombinant type XVII collagen reproduce a similar skin blistering disease phenotype as observed in humans [87,88]. E. Other Skin BMZ Components Besides type VII collagen, laminin-5, laminin-6, alpha-6 and beta-4 integrin subunits, and type XVII collagen, there are other well-characterized skin BMZ components, such as type IV collagen, laminin-1, nidogen, and heparan sulfate proteoglycan [1]. Although these components are essential elements for the skin BMZ, they have not been targeted in any inflammatory skin diseases, with the noticeable exception of two patients suffering from inflammatory skin blistering disease, renal insufficiency, and autoantibodies targeting the alpha-5 chain of type IV collagen (COL4A5) in skin BMZ and renal glomerular BMZ [89]. These BMZ components are beyond the scope of this book, and are not discussed here.


26


IV. SUMMARY Cumulative scientific data from comparative studies on the structure and function of skin BMZ supports a close resemblance between human skin and that of other mammals, particularly the small mammals commonly used in the laboratories of biomedical researchers. This close resemblance of skin BMZ structures and functions thus provides investigators who study the animal models of inflammatory skin diseases with a useful tool in interpreting research results concerning the relevance to human diseases.

ACKNOWLEDGMENT This work is supported by NIH grants R01 AR47667, R03 AR47634, and R21 AR48438 (Lawrence S. Chan).

REFERENCES 1. Marinkovich, M.P., The molecular genetics of basement membrane diseases, Arch. Dermatol., 129, 1557, 1993. 2. Christiano, A.M. and Uitto, J., Molecular complexity of the cutaneous basement membrane zone. Revelation from the paradigms of epidermolysis bullosa, Exp. Dermatol., 5, 1, 1996. 3. Korge, B.P. and Krieg, T., The molecular basis for inherited bullous diseases, J. Mol. Med., 74, 59, 1996. 4. Seitz, C.S. et al., BP180 gene delivery in junctional epidermolysis bullosa, Gene. Ther., 6, 42, 1999. 5. Dellambra, E. et al., Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly, J. Biol. Chem., 276, 41336, 2001. 6. Chen, M. et al., Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa, Nat. Genet., 32, 670, 2002. 7. Labib, R.S. et al., Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting, J. Immunol., 136, 1231, 1986. 8. Woodley, D.T. et al., Identification of the skin basement membrane autoantigen in epidermolysis bullosa acquisita, N. Engl. J. Med., 310, 107, 1984. 9. Zone, J.J. et al., Identification of the cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis, J. Clin. Invest., 85, 812, 1990. 10. Domloge-Hultsch, N. et al., Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease, J. Clin. Invest., 90, 1628, 1992. 11. Chan, L.S. et al., Identification and partial characterization of a novel 105-kDalton lower lamina lucida autoantigen associated with a novel immune-mediated subepidermal blistering disease, J. Invest. Dermatol., 101, 262, 1993. 12. Chan, L.S. et al., The first international consensus on mucous membrane pemphigoid: definition, diagnostic criteria, pathogenic factors, medical treatment, and prognostic indicators, Arch. Dermatol., 138, 370, 2002. 13. Jones, J.C., Hopkinson, S.B., and Goldfinger, L.E., Structure and assembly of hemidesmosomes, Bioessays, 20, 488, 1998. 14. Maldonado, B.A. and Furcht, L.T., Involvement of integrins with adhesion-promoting, heparin-binding peptides of type IV collagen in culture human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci., 36, 364, 1995. 15. Thie, M. et al., Cell adhesion to the apical pole of epithelium: a function of cell polarity, Eur. J. Cell Biol., 66, 180, 1995. 16. Tozeren, A. et al., Integrin alpha 6 beta 4 mediates dynamic interactions with laminin, J. Cell Sci., 107, 3153, 1994.



27

17. Niessen, C.M. et al., The alpha 6 beta 4 integrin is a receptor for both laminin and kalinin, Exp. Cell Res., 211, 360, 1994. 18. Chen, M. et al., a2b1 integrin mediates dermal fibroblast attachment to type VII collagen via a 158amino-acid segment of the NC1 domain, Exp. Cell Res., 249, 231, 1999. 19. Chen, M. et al., The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa, J. Biol. Chem., 277, 2118, 2002. 20. Burgeson, R.E. et al., The structure of type VII collagen, Ann. N. Y. Acad. Sci., 460, 47, 1985. 21. Chen, M. et al., The carboxyl terminus of type VII collagen mediates antiparallel dimmer formation and constitutes a new antigenic epitope for epidermolysis bullosa acquisita autoantibodies, J. Biol. Chem., 276, 21649, 2001. 22. Kühn, K., Basement membrane (Type IV) collage, Matrix Biol., 14, 439, 1994. 23. Marinkovich, M.P. et al., LAD-1, the linear IgA bullous dermatosis autoantigen, is a novel 120-kDa anchoring filament protein synthesized by epidermal cells, J. Invest. Dermatol., 106, 734, 1996. 24. Schäcke, H. et al., Two forms of collagen XVII in keratinocytes: a full-length transmembrane protein and a soluble ectodomain, J. Biol. Chem., 273, 25937, 1998. 25. Rousselle, P. et al., Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments, J. Cell Biol., 114, 567, 1991. 26. Parente, M.G. et al., Human type VII collagen: cDNA cloning and chromosomal mapping of the gene, Proc. Natl. Acad. Sci. U.S.A., 88, 6931, 1991. 27. Christiano, A.M. et al., Cloning of human type VII collagen: complete primary sequence of the alpha 1 (VII) chain and identification of the intragenic polymorphisms. J. Biol. Chem., 269, 20256, 1994. 28. Kivirikko, S. et al., Cloning of mouse type VII collagen reveals evolutionary conservation of functional protein domains and genomic organization, J. Invest. Dermatol., 106, 1300, 1996. 29. Xu, L. et al., Molecular cloning and characterization of a cDNA encoding canine type VII collagen non-collagenous (NC1) domain, the target antigen of autoimmune disease epidermolysis bullosa acquisita (EBA), Biochim. Biophys. Acta, 1408, 25, 1998. 30. Lapiere, J-C. et al., Epitope mapping of type VII collagen. Identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa, J. Clin. Invest., 92, 1831, 1993. 31. Christiano, A.M. et al., A missense mutation in type VII collagen in two affected siblings with recessive dystrophic epidermolysis bullosa, Nat. Genet., 4, 62, 1993. 32. Hovnanian, A. et al., Characterization of 18 new mutations in COL7A1 in recessive dystrophic epidermolysis bullosa provides evidence for distinct molecular mechanisms underlying defective anchoring fibril formation, Am. J. Hum. Genet., 61, 599, 1997. 33. Jarvikallio, A., Pulkkinen, L., and Uitto, J., Molecular basis of dystrophic epidermolysis bullosa: mutations in the type VII collagen gene (COL7A1), Hum. Mutation, 10, 338, 1997. 34. Heinonen, S. et al., Targeted inactivation of the type VII collagen gene (Col7a1) in mice results in severe blistering phenotype: a model for recessive dystrophic epidermolysis bullosa, J. Cell Sci., 112, 3641, 1999. 35. Bruckner-Tuderman, L., Guscetti, F., and Ehrensperger, F., Animal model for dermolytic mechanobullous disease: sheep with recessive dystrophic epidermolysis bullosa lack collagen VII, J. Invest. Dermatol., 96, 452, 1991. 36. Olivry, T. et al., Canine epidermolysis bullosa acquisita: circulating autoantibodies target the aminoterminal non-collagenous (NC1) domain of collagen VII in anchoring fibrils, Vet. Dermatol., 9, 19, 1998. 37. Burgeson, R.E. et al., A new nomenclature for the laminins, Matrix Biol., 14, 209, 1994. 38. Timpl, R. and Brown, J.C., The laminins. Matrix Biol., 14, 275, 1994. 39. Aumailley, M. and Krieg, T., Laminins: a family of diverse multifunctional molecules of basement membranes, J. Invest. Dermatol., 106, 209, 1996. 40. Marinkovich, M.P. et al., The dermal-epidermal junction of human skin contains a novel laminin variant, J. Cell Biol., 119, 695, 1992. 41. Ryan, M.C. et al., Cloning of the LamA3 gene encoding the a3 chain of the adhesive ligand epiligrin, J. Biol. Chem., 269, 22779, 1994. 42. Galliano, M.F. et al., Cloning and complete primary structure of the mouse laminin alpha 3 chain. Distinct expression pattern of the laminin alpha 3A and alpha 3B chain isoforms, J. Biol. Chem., 270, 21820, 1995.


28


43. Utani, A. et al., Mouse kalinin B1 (laminin beta 3 chain): cloning and tissue distribution, Lab. Invest., 72, 300, 1995. 44. Chan, L.S. et al., Laminin-6 and laminin-5 are recognized by autoantibodies in a subset of cicatricial pemphigoid, J. Invest. Dermatol., 108, 848, 1997. 45. Kirtschig, G. et al., Anti-basement membrane antibodies in patients with anti-epiligrin cicatricial pemphigoid bind the a subunit of laminin 5, J. Invest. Dermatol., 105, 543, 1995. 46. Lazarova, Z. et al., Anti-epiligrin cicatricial pemphigoid represents an autoimmune response to subunits present in laminin 5 (a3b3g2), Br. J. Dermatol., 139, 791, 1998. 47. Nousari, H.C. et al., Anti-epiligrin cicatricial pemphigoid with antibodies against the g2 subunit of laminin 5, Arch. Dermatol., 135, 173, 1999. 48. Leverkus, M. et al., Anti-epiligrin cicatricial pemphigoid: an underdiagnosed entity within the spectrum of scarring autoimmune subepidermal bullous dermatoses, Arch. Dermatol., 135, 1091, 1999. 49. Baudoin, C. et al., Herlitz junctional epidermolysis bullosa keratinocytes display heterogeneous defects of nicein/kalinin gene expression, J. Clin. Invest., 93, 862, 1994. 50. Pulkkinen, L. et al., Mutations in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa, Nat. Genet., 6, 293, 1994. 51. Takizawa, Y. et al., Compound heterozygosity for a point mutation and a deletion located at splice acceptor sites in the LAMB3 gene leads to generalized atrophic benign epidermolysis bullosa, J. Invest. Dermatol., 115, 312, 2000. 52. Nakano, A. et al., Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs. non-Herlitz phenotypes, Hum. Genet., 110, 41, 2002. 53. Spirito, F. et al., Animal models for skin blistering conditions: absence of laminin 5 causes hereditary junctional mechanobullous disease in the Belgian horse, J. Invest. Dermatol., 119, 684, 2002. 54. Kuster, J.E. et al., IAP insertion in the murine LamB3 gene results in junctional epidermolysis bullosa, Mamm. Genome, 8, 673, 1997. 55. Ryan, M.C. et al., Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells, J. Cell Biol., 145, 1309, 1999. 56. Olivry, T. et al., Laminin-5 is targeted by autoantibodies in feline mucous membrane (cicatricial pemphigoid), Vet. Immunol. Immunopathol., 88, 123, 2002. 57. Lazarova, Z. et al., Passive transfer of anti-laminin 5 antibodies induces subepidermal blisters in neonatal mice, J. Clin. Invest., 98, 1509, 1996. 58. Schaapveld, R.Q. et al., Hemidesmosome formation is initiated by the beta 4 integrin subunit, requires complex formation of beta4 and HD1/plectin, and involves a direct interaction between beta4 and the bullous pemphigoid antigen 180, J. Cell Biol., 142, 271, 1998. 59. Hogervorst, F. et al., Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain, EMBO J., 9, 765, 1990. 60. Suzuki, S. and Naitoh, Y., Amino acid sequence of a novel integrin beta 4 subunit and primary expression of the mRNA in epithelial cells, EMBO J., 9, 757, 1990. 61. Hogervorst, F. et al., Molecular cloning of the human alpha 6 integrin subunit. Alternative splicing of alpha 6 mRNA and chromosomal localization of the alpha 6 and beta 4 genes, Eur. J. Biochem., 199, 425, 1991. 62. Kennel, S.J. et al., Sequence of a cDNA encoding the beta4 subunit of murine integrin, Gene, 130, 209, 1993. 63. Hierck, B.P. et al., Variants of the alpha 6 beta 1 laminin receptor in early murine development: distribution, molecular cloning and chromosomal localization of the mouse integrin alpha 6 subunit, Cell Adhes. Commun., 1, 33, 1993. 64. Feltri, M.L. et al., Cloning and sequence of the cDNA encoding the beta 4 integrin subunit in rat peripheral nerve, Gene, 186, 299, 1997. 65. Vidal, F. et al., Integrin b4 mutations associated with junctional epidermolysis bullosa with pyloric atresia, Nat. Genet., 10, 229, 1995. 66. Pulkkinen, L. et al., Genomic organization of the integrin beta 4 gene (ITGB4): a homozygous splicesite mutation in a patient with junctional epidermolysis bullosa associated with pyloric atresia, Lab. Invest., 76, 823, 1997.



29

67. Takizawa, Y. et al., Novel ITGB4 mutations in a patient with junctional epidermolysis bullosa-pyloric atresia syndrome and altered basement membrane zone immunofluorescence for the alpha6beta4 integrin, J. Invest. Dermatol., 108, 943, 1997. 68. van der Neut, R. et al., Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice, Nat. Genet., 13, 366, 1996. 69. Dowling, J., Yu, Q.C., and Fuchs, E., Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival, J. Cell Biol., 134, 559, 1996. 70. Georges-Labouesse, E. et al., Absence of integrin alpha 6 leads to epidermolysis bullosa and neonatal death in mice, Nat. Genet., 13, 370, 1996. 71. Tyagi, S. et al., Ocular cicatricial pemphigoid antigen: partial sequence and biochemical characterization, Proc. Natl. Acad. Sci. U. S. A., 93, 14714, 1996. 72. Chan, R.Y. et al., The role of antibody to human beta4 integrin in conjunctival basement membrane separation: possible in vitro model for ocular cicatricial pemphigoid, Invest. Ophthalmol. Vis. Sci., 40, 2283, 1999. 73. Bhol, K.C. et al., The autoantibodies to alpha 6 beta 4 integrin of patients affected by ocular cicatricial pemphigoid recognize predominantly epitopes within the large cytoplasmic domain of human beta 4, J. Immunol., 165, 2824, 2000. 74. Bhol, K.C. et al., Autoantibodies to human alpha 6 integrin in patients with oral pemphigoid, J. Dent. Res., 80, 1711, 2001. 75. Hopkinson, S.B. et al., Cytoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: molecular and cell biological characterization, J. Invest. Dermatol., 99, 264, 1992. 76. Giudice, G.J., Emery, D.J., and Diaz, L.A., Cloning and primary structural analysis of the bullous pemphigoid autoantigen, BP-180, J. Invest. Dermatol., 99, 243, 1992. 77. Li, K. et al., Cloning of type XVII collagen. Complementary and genomic DNA sequences of mouse 180-Kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5'-end of the gene and 3'-untranslated region of the mRNA, J. Biol. Chem., 268, 8825, 1993. 78. Giudice, G.J. et al., Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain, J. Immunol., 151, 5742, 1993. 79. Xu, L. et al., Molecular cloning of canine bullous pemphigoid antigen 2 cDNA and immunomapping of NC16A domain by canine bullous pemphigoid autoantibodies, Biochim. Biophys. Acta, 1500, 97, 2000. 80. McGrath, J.A. et al., Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa, Nat. Genet., 11, 83, 1995. 81. Chavanas, S. et al., A homozygous in-frame deletion in the collagenous domain of bullous pemphigoid antigen BP180 (type XVII collagen) causes generalized atrophic benign epidermolysis bullosa, J. Invest. Dermatol., 109, 74, 1997. 82. Pulkkinen, L. et al., Compound heterozygosity for novel splice site mutations in the BPAG2/COL17A1 gene underlies generalized atrophic benign epidermolysis bullosa, J. Invest. Dermatol., 113, 1114, 1999. 83. Iwasaki, T. et al., Canine bullous pemphigoid (BP) — Identification of the 180 kd canine BP antigen by circulating autoantibodies, Vet. Pathol., 32, 387, 1995. 84. Olivry, T. et al., Novel feline autoimmune blistering diseases resembling bullous pemphigoid in humans: IgG autoantibodies target the NC16A ectodomain of type XVII collagen (BP180/BPAG2), Vet. Pathol., 36, 328, 1999. 85. Olivry T. et al., A spontaneously arising porcine model of bullous pemphigoid, Arch. Dermatol. Res., 292, 37, 2000. 86. Olivry, T. et al., Equine bullous pemphigoid IgG autoantibodies target linear epitopes in the NC16A ectodomain of collagen XVII (BP180, BPAG2), Vet. Immunol. Immunopathol., 73, 45, 2000. 87. Liu, Z. et al., A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180, J. Clin. Invest., 92, 2480, 1993.


30


88. Liu, Z. et al., The role of complement in experimental bullous pemphigoid, J. Clin. Invest., 95, 1539, 1995. 89. Ghohestanti, R.F. et al., The alpha 5 chain of type IV collagen is the target of IgG autoantibodies in a novel autoimmune disease with subepidermal blisters and renal insufficiency, J. Biol. Chem., 275, 16002, 2000.


PART II Comparative Immunology



CHAPTER

3

Human Immune System Kalyanasundaram Ramaswamy

CONTENTS I. Overview of Immune System ..............................................................................................34 II. Innate Immunity...................................................................................................................35 A. Phagocytes ...................................................................................................................36 1. Mannose and CR3 Receptors in Phagocytosis .....................................................36 2. Scavenger Receptors and Phagocytosis ................................................................36 3. Toll-Like Receptors in Phagocytosis.....................................................................36 4. CD14 Receptors and Phagocytosis .......................................................................37 5. Fc Receptors and Phagocytosis .............................................................................37 B. Neutrophils...................................................................................................................37 C. Natural Killer Cells .....................................................................................................38 D. Eosinophils...................................................................................................................39 E. Mast Cells and Basophils ............................................................................................40 F. Complement.................................................................................................................41 III. Adaptive Immunity ..............................................................................................................44 A. B Lymphocytes ............................................................................................................44 B. B1 Cells .......................................................................................................................44 C. Immunoglobulins .........................................................................................................45 1. IgM.........................................................................................................................46 2. IgD .........................................................................................................................46 3. IgG .........................................................................................................................46 4. IgA .........................................................................................................................47 5. IgE..........................................................................................................................47 6. Immunoglobulin Gene Regulation and Class Switch ...........................................48 D. Major Histocompatibility Complex.............................................................................48 1. Structure of Class I MHC Molecules ...................................................................49 2. Structure of Class II MHC Molecules ..................................................................49 3. Peptide Binding to MHC Molecules.....................................................................49 E. Minor Histocompatibility (H) Antigens ......................................................................50 F. Antigen Processing and Presentation ..........................................................................50 1. Antigen Processing and Presentation on Class I MHC Molecules ......................50

0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

33


34


2. Antigen Processing and Presentation on Class II MHC Molecules.....................51 3. MHC Tetramers .....................................................................................................51 G. Langerhans Cells and Other Dendritic Cells ..............................................................52 H. T Cells..........................................................................................................................53 I. Cytokines .....................................................................................................................53 1. Interferons ..............................................................................................................54 2. Tumor Necrosis Factor ..........................................................................................54 3. Interleukins ............................................................................................................55 a. IL-1 Family of Cytokines................................................................................55 b. IL-2 Family of Cytokines and Common Cytokine Receptor Gamma Chain ..................................................................................................57 c. IL-3 and IL-5 Family of Cytokines ................................................................59 d. IL-6 and gp130 Family of Cytokines..............................................................59 e. IL-8 ..................................................................................................................61 f. IL-10 Family of Cytokines..............................................................................61 g. IL-12 Family of Cytokines..............................................................................62 h. IL-13 ................................................................................................................63 i. IL-14 ................................................................................................................63 j. IL-16 ................................................................................................................64 k. IL-17 Family of Cytokines..............................................................................64 l. IL-28 and IL-29 Family of Cytokines ............................................................64 4. Growth Factors ......................................................................................................65 a. Transforming Growth Factor...........................................................................65 b. Stem Cell Factor ..............................................................................................65 c. Leukemia Inhibitory Factor.............................................................................65 d. Platelet-Derived Growth Factor.......................................................................65 J. Chemokines..................................................................................................................66 IV. Summary ..............................................................................................................................68 References ........................................................................................................................................68

I. OVERVIEW OF IMMUNE SYSTEM The primary role of the immune system is surveillance and destruction of molecules or substances that are foreign to the body. To accomplish these functions the immune system has developed a sophisticated network of cells and soluble molecules that are distributed strategically throughout the body. These effector mechanisms of the immune system are highly capable of destroying a wide variety of cells including microorganisms such as bacteria, viruses, parasites, and fungi that invade the body. A critical aspect of the effector function of the immune system is to avoid harming self, or the body’s own cells. The immune effector cells achieve this function elegantly by discriminating self from non-self. However, occasional failure to discriminate self can lead to tolerance (of harmful non-self), self-destruction, or autoimmune diseases. Based on the type of effector interaction, the cells and molecules within the immune network can be grouped into two major classes: one responds specifically (adaptive immunity) and another that responds nonspecifically (innate immunity) toward the invading organisms or molecules. Both of these arms of immunity consist of several cells and molecules that often have overlapping functions (Figure 3.1). For purposes of simplicity, each is described separately in this chapter.


HUMAN IMMUNE SYSTEM

35

Immune system

Innate Figure 3.1

Adaptive

Schematic representations of the two major arms of the immune system. Effector cells and molecules that function in a nonspecific fashion are grouped under innate immunity and those functions specifically are grouped as adaptive immunity, with significant functional overlaps between them.

II. INNATE IMMUNITY Innate immunity refers to the first line of natural defense offered by the body. A variety of factors contribute to innate immunity, including anatomical barrier, molecules, and cells. Resistance offered by the anatomical barrier includes the body surfaces at the skin, gastrointestinal tract, urogenital tract, mammary gland, and respiratory tract. The skin is the largest organ of the human body. In addition to providing a mechanical barrier, the skin also houses a variety of cells such as keratinocytes, mast cells, tissue macrophages, sebaceous glandular cells, natural killer (NK) cells, and endothelial cells that actively participate in innate immunity [1]. Some of these cells, such as tissue macrophages, can engulf foreign material and destroy or eliminate it from the body by producing proteolytic enzymes and reactive oxygen radicals [2,3]. However, certain antimicrobial peptides (AMPs) and proteins produced by the cells of the innate immune system in the skin have a major role to play in the initial defense against invading gram-positive or gram-negative bacteria, fungi, and certain viruses [4–6]. The AMPs have a cationic charge and can interact with bacterial membranes through hydrophobic amino acids. Some of the AMPs identified in the skin include cathelicidins, defensins, dermcidin, granulysin, adrenomedullin, cystatin, and secretory leukocyte protease inhibitor [7,8]. The cathelicidins and defensins probably play the major role in host defense [9]. Cathelicidins are proteins approximately 37 amino acids long, and are highly expressed by human keratinocytes under certain inflammatory conditions such as psoriasis. Defensins, on the other hand, are cationic peptides 28 to 44 amino acids long. Defensins contain six to eight cysteine residues that form three characteristic intramolecular disulfide bonds [6,10]. Three forms of defensins — a-defensin, bdefensin and q-defensin — have been described to date and each consists of several molecules. There are at least six different a-defensins. Four of these, designated as human neutrophil peptides (HNP1 through 4), are produced by neutrophils, and the other two a-defensins are highly expressed in the Paneth’s cells of the small intestinal crypts and the epithelial cells of the female urogenital tract. These are called human defensins 5 (HD-5) and HD-6. Similarly, at least four human bdefensins (HBD1 through 4) have been described to date. These AMPs participate in innate immunity by directly killing microorganisms or by indirectly activating cells that are involved in innate immunity or adaptive immunity. For example, HBD2 may participate in cutaneous allergic reactions by inducing the release of histamine and prostaglandin D2 from mast cells. Similarly, HBD can bind to the chemokine receptor CCR6 and attract immature dendritic cells and memory T cells to the site. HNP1-3 can increase the expression of tumor necrosis factor (TNF)-a and interleukin (IL)-1 from human macrophages.


36


A. Phagocytes Phagocytes are specialized professional cells that can engulf large particles including microorganisms. These engulfed particles are then destroyed or degraded by powerful enzymes within the cytoplasm of these cells. This specialized function originally described by Metchnikoff a century ago is referred to as phagocytosis [11]. In humans, several cell types including macrophages, monocytes, neutrophils, eosinophils, basophils, mast cells, platelets, and B lymphocytes possess the abilities for phagocytosis. The process of phagocytosis begins when the foreign particle or certain regions of the particle (called ligands) bind to specific receptors on the surface of phagocytes. Medzhitov and Janeway [12] proposed the term “pattern-recognition receptors” (PRRs) for these molecules on the surface of the phagocytes and “pathogen-associated molecular patterns” (PAMPs) for the ligands on the surface of microbes that bind to these receptors. Binding of PAMP to PRR triggers rearrangement of actin in the cytoskeleton leading to pseudopodial formations that engulf and internalize the particle. The PRRs involved in phagocytosis can be classified into two groups based on whether they induce opsonic phagocytosis (type I phagocytosis) or nonopsonic phagocytosis (type II phagocytosis). Receptors that mediate type I phagocytosis bind to the integral surface components of the particles. Some of the PRRs in this category include macrophage mannose receptor (MR); complement receptor 3 (CR3, Mac-1, or integrin CD11b/CD18); scavenger family of receptors (class A through class F); toll-like receptors (TLR 2 and TLR 4); and the GPIanchored receptor, CD14. Type II phagocytosis is mediated by Fc receptors. 1. Mannose and CR3 Receptors in Phagocytosis The mannose receptor (MR) family of proteins consists of four multifunctional multidomain glycoproteins that are type I transmembrane receptors with an N-terminal cysteine-rich domain. MR is involved in both phagocytosis and endocytosis [13,14]. The CR3 is a versatile multipurpose adhesion and recognition receptor. Common ligands for CR3 consist of complement component C3bi (see below) and several molecules of microbial organisms. In addition, CR3 is an integrin that can mediate migration of phagocytes, activate them through a variety of signaling pathways, and induce cytoskeleton rearrangement. Several studies show that CR3 can mediate both type I and type II phagocytosis [15–18]. 2. Scavenger Receptors and Phagocytosis The scavenger receptors (SR) are multidomain transmembrane glycoproteins that can bind with high affinity to a broad range of ligands including lipopolysaccharide (LPS) of bacteria, phosphatidylserine of apoptotic cells, and the cell surface of certain tumors. Therefore, SR are often described as “molecular flypaper” [19]. One of the unique properties of SR is that they can bind to chemically modified low-density lipoproteins and promote their uptake by phagocytes [11]. The SR family of proteins includes the following receptors: class A (type I and II macrophage scavenger receptors, and MARCO); class B (CD36, scavenger receptor class B1); class C (scavenger receptor class C1); class D (CD68/macrosialin); class E (the endothelial lectin-like oxidized LDL receptor 1/LOX-1); and class F (scavenger receptor from endothelial cells/SREC). 3. Toll-Like Receptors in Phagocytosis The Toll-like receptors (TLR) are key molecules involved in the recognition of pathogens by the innate immune system [3,20,21]. TLR consist of a family of proteins first identified on the basis of sequence similarity with the Drosophila protein Toll. Ten members of the TLR family (TLR1 through TLR10) have been identified in the human. TLR have an extracellular domain with leucine-rich repeats, and an intracytoplasmic region with significant sequence similarity to the IL-1


HUMAN IMMUNE SYSTEM

37

and IL-18 receptors. This suggests that binding of ligand to the TLR triggers a common pathway of intracellular signal-transduction cascade. Some of the major ligands of TLR include LPS, bacterial lipoproteins, peptidoglycan, and bacterial DNA [22]. Engagement of TLR by pathogenassociated ligands results in the activation of the phagocytes leading to phagocytosis, production of reactive oxygen and nitrogen intermediates (ROI and RNI), proinflammatory cytokines, and upregulation of co-stimulatory molecules [21,23]. Thus, TLR signaling represents a key component of the innate immune response to microbial infection [22]. 4. CD14 Receptors and Phagocytosis The CD14 is a multiligand PRR that recognizes apoptotic cells and certain surface components of bacteria such as LPS [24,25]. Although CD14 binds to LPS and apoptotic cells, it cannot initiate a transmembrane activation signal because it is a glycosylphosphatidyl-inositol (GPI)-anchored protein [26]. Therefore, CD14 must interact with other receptor proteins such as TLR2 or TLR4 to mediate cell signaling in the phagocytes [27–29]. 5. Fc Receptors and Phagocytosis Fc receptors (FcRs) mediate type II phagocytosis of IgG-coated particles [27]. FcRs that participate in this type of phagocytosis include FcgR1, FcgRIIA, and FcgRIIIA. FcgRIIA is a singlechain protein with an extracellular Fc-binding domain, a transmembranous domain, and a cytoplasmic tail containing two tyrosine activation motifs. The tyrosine activation motif is important for the phagocytic function. When two FcgRIIA are cross-linked by the IgG-coated particles, phosphorylation of the tyrosine motif occurs resulting in the activation of the phagocyte. A member of the src family of tyrosine kinase, this protein is believed to initiate the phosphorylation followed by Syk kinases that causes transcriptional activation and cytoskeletal rearrangement leading to phagocytosis of the IgG-coated particle. FcgRI and FcgRIIIA have extracellular Fc-binding domains similar to FcgRIIA. However, they lack the tyrosine motifs on their cytoplasmic tail. Therefore, these receptors must form dimers with another receptor that has the tyrosine motif for their phagocytic function. Phagocytes can also internalize IgA-coated particles [30]. B. Neutrophils Neutrophils are one of the major phagocytic cells in peripheral circulation besides monocytes and mononuclear phagocytes that act as frontline defenders against invading bacteria [31]. Neutrophils are generated in the bone marrow and are released into the peripheral circulation from where they migrate to the tissues and mucosal surfaces. Neutrophils comprise approximately 70% of total circulating leukocytes, and about 100 billion neutrophils enter and leave the circulation daily in a normal adult. Neutrophils are the initial cells that arrive at sites of inflammation and thus play a central role in innate immunity [32,33]. The exit of neutrophils from the peripheral circulation into the tissue is facilitated by chemokines and expression of adhesion molecules on the surface of endothelial cells. Neutrophils bind to these molecules via the CD11/CD18 complex expressed on their surface. A defect in the expression of the CD11/CD18 complex leads to a condition called “leukocyte adhesion deficiency” (LAD) type I syndrome, where neutrophils fail to adhere to endothelium and are thus unable to enter the tissue. After adhering to the endothelium, the neutrophils emigrate to the tissue via the intercellular tight junctions of the endothelium. This process, called transendothelial migration, is facilitated by several cytokines and chemokines. Once in the tissue, the neutrophils get activated at the site of inflammation and release toxic oxygen radicals and their azurophilic granular content. The release of toxic oxygen radicals, called respiratory bursts, are characterized by increased production of superoxide (O2-) and hydrogen peroxide. In addition, the granules contain several degradative enzymes (such as acid hydrolases,


38


myeloperoxidases), lactoferrin, and a number of cationic proteins including defensins and other antibacterial agents that destroy the invading pathogen or molecule [34]. Neutrophils thus play an important role in innate immunity. C. Natural Killer Cells NK cells are large lymphocytes containing azurophilic granules, and account for nearly 10 to 20% of peripheral blood lymphocytes. NK cells do not express receptors for antigens but carry typical surface markers CD3-, CD56+, and CD16+ [35]. As the name suggests, NK cells are killer cells and are one of the most important effector cells of innate immunity [36]. The targets of NK cells include a variety of cells that carry intracellular pathogens (infected cells) and tumor cells. The effector function of NK cells is spontaneous and requires only a weak stimulation or activation. However, the pathways leading to NK cell activation are poorly characterized. Nevertheless, recognition of the absence of class I human leukocyte antigens (HLA) on the surface of target cells is a critical event in the activation of NK cells. All human nucleated cells carry class I HLA molecules on cell surfaces. A single cell such as a lymphocyte can express several thousands of these molecules. However, certain viral infections or tumor transformations of cells can cause a decrease in the number of class I HLA expressions on the surface of affected cells. NK cells can discriminate and recognize these cells with no or low levels of class I HLA. Certain molecules expressed on the surface of NK cells such as CD94/NKG2 (natural killer group 2) and the killer immunoglobulin (Ig)-like family of receptors (KIR or CD158) can detect the density of class I HLA molecules on normal cells. Transient engagement of these receptors to the class I HLA molecule delivers inhibitory signals to the NK cells, thereby preventing them from killing normal cells. However, the lack of or insufficient amounts of class I molecules on the surface of potentially dangerous cells, such as tumor cells or cells that are infected with viruses, leads to activation of NK cells, which then produce molecules that kill the tumor cells or infected cells. This target recognition is referred to as the “missing-self” hypothesis. In addition to CD94/NKG2 and KIR, NK cells also carry several target recognition receptors such as NK cell-receptor protein (NKRP)1A, killer cell lectin-like receptor (KLR)F1, leukocyte-associated Ig-like receptors (LAIR), lectin-like transcript (LLT)1, activation-induced C-type lectin (AICL), C-type lectin-like receptor (CLEC), and natural cytotoxicity receptors (NKp46, NKp30, and NKp44) that participate in the effector function [37–39]. NK cells are rapidly recruited from the peripheral blood to the site of tissue injury or infection by chemokines (CXCR1, CX3CR1, fractalkine) [40] and are then activated by cytokines such as IL-12, type 1 interferon (IFN), IL-15 [41], IL-18, and IL-2 [36]. Dendritic cells (DC), which can interact with NK cells, are another cell type drawn to the initial site of injury [39]. This interaction involves certain cytokines and chemokines released by activated NK cells, which in turn promotes maturation of immature DC. Mature DC can then elicit cognate adaptive immune responses. However, when the ratio of DC to NK cells in the microenvironment decreases, NK cells gain the upper hand and lyse the DC, which turns off the immune responses [42]. Thus, NK cells play a major role in the control switch of innate immunity. Chronic NK-cell lymphocytosis (CNKL) is an abnormal chronic proliferation of NK cells in humans characterized by neutropenia, anemia, fever, cutaneous vasculitis, and autoimmune disorders. NK cells are activated when the activation receptors (such as CD16) are cross-linked [43]. Upon activation, there is an increase in Ca2+ flux and inositol phosphate turnover leading to granule exocytosis, cytokine secretion, and transcription of effector proteins. The key molecular players in the effector function of NK cells are the pore-forming protein perforin and a family of granulebound serine proteases called granzymes [44,45]. These granules are preformed and are stored within the cytoplasm of NK cells. Thus, NK cells are armed and can kill target cells within minutes (Figure 3.2). Exocytosis of these granules results in the release of perforin, granzymes, and granulysin into the tight intracellular junction formed between the NK cell and target cell. Phosphocholine present on the target cell membrane acts as a specific calcium-dependent receptor for


HUMAN IMMUNE SYSTEM

39

Granzyme

CI-MPR

Target cell Death

Cleave Procaspase 3

Perforin

Figure 3.2

Activated granzyme

Molecular mechanism of NK cell-mediated killing of target cells. Upon activation, NK cells release the pore-forming protein perforin to bind on target cell surface, lyse the membrane and open the pore, allowing the entry of granzyme. The perforin-activated granzyme then cleaves procaspase 3, leading to target cell death.

perforin. Receptor-bound perforin polymerizes and damages the target cell membrane. Certain tumor cells that lack these receptors resist the perforin-mediated membrane damage and are thus resistant to immune killing by NK cells. Similarly, a defect in the perforin gene in humans is associated with familial hemophagocytic lymphohistiocytosis (FHL), a disease characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines. Through studies using synthetic peptides and recombinant perforins, it has been suggested that the N-terminal region of the perforin molecule is an important domain responsible for the lytic activity. To complete NK-cell–mediated killing, the perforin has to combine with granzymes (A and B). As perforin traverses the cell membrane and enters the cell, granzymes also enter the target cell by endocytosis after binding to its cation-independent mannose-6-phosphate receptor (CI-MPR) on the surface of target cells. Binding of perforin to granzyme B in the cytoplasm activates the granzyme, which then cleave procaspase 3 leading to DNA fragmentation and programmed cell death or apoptosis of the target cell. Cathepsin C (DPPI) is a critical molecule required for the activation of granzyme. A deficiency in DPPI leads to Papillion–Lefevre syndome in humans, also known as keratosis palmoplanterus with periodontopathia, a disease characterized by premature loss of teeth and thickening of the skin [46]. D. Eosinophils Eosinophils are granulocytes that exhibit typical bilobar nucleus and carry electron-dense granules in their cytoplasm [47,48]. These granules contain cationic proteins that easily stain with the acid aniline dye, eosin, and hence the name. Eosinophils are generated in the bone marrow from CD34+ hematopoietic stem cells and mature eosinophils leave the bone marrow and enter peripheral circulation. In healthy individuals, only a few eosinophils (less than 4% of total leukocytes) are present in circulation. However, in helminth infection, allergy, and asthma very high numbers of eosinophils (up to 40% of total leukocytes) appear in peripheral circulation and tissues [49]. These allergic conditions and parasitic infections are associated with high levels of the cytokine IL-5 that promotes differentiation, proliferation, and release of eosinophils from the bone marrow. Circulating eosinophils are then recruited to the site of allergy. The mechanism of eosinophil trafficking into the tissue from peripheral circulation is well characterized [47]. The adhesion molecule very late antigen (VLA)-4 expressed on the surface of eosinophils interacts first with


40


certain receptors (called selectins) expressed on the surface of endothelial cells causing rolling motion of the eosinophils. This rolling is arrested by a firm adhesion of VLA-4 to the vascular cell-adhesion molecule (VCAM)-1 on the surface of endothelial cells. Subsequently, a concentration gradient formed by specific CC chemokines such as eotaxins (CCL11, CCL24, and CCL26), and mediators such as leukotriene B4 (LTB4) and platelet-activating factor (PAF) at the site of allergy promotes focused chemotaxis of eosinophils through the blood vessel wall into the tissue. Several cytokines such as IL-4, IL-13, and TNF-a play a central role in this eosinophil chemotaxis by up-regulating VCAM-1 on endothelial cells. Eosinophils migrated into the tissue can then be primed or activated to induce its effector function. Mediators such as IL-3, IL-5, GM-CSF, CC chemokines, and PAF prime eosinophils by promoting their survival in the tissue. Primed or activated eosinophils exhibit lower density than resting eosinophils and are thus called hypodense eosinophils. Cross-linking of receptors (for IgG or IgA) on the surface of the hypodense eosinophils causes the release of eosinophil granular contents, including an array of proinflammatory cytokines into the microenvironment. One of the major components of the eosinophil granules is the major basic protein (MBP) that has a potent toxic function against several helminth parasites and respiratory epithelial cells [50]. A significant increase in the levels of MBP occurs during asthma, which is responsible for the pathological changes during bronchial hyperresponsiveness and bronchoconstriction. Another important constituent of the granular content of eosinophils is the eosinophil-derived neurotoxin (EDT) that is highly toxic to myelinated neurons and has RNAse activity. Eosinophil granules also contain eosinophil cationic proteins (ECPs) that are toxic to bacteria, helminths, and epithelial cells of the body. ECPs also possess RNAse activity such as EDT. These molecules can thus kill singlestranded RNA-pneumoviruses, such as respiratory syncytial viruses. Another constituent of eosinophil granules is eosinophil peroxidase, which is capable of generating hypohalous (such as hypochlorous and hypobromous) acids from hydrogen peroxide and halides. Eosinophil peroxide is toxic to helminths, protozoan parasites, bacteria, tumor cells, and several other cells of the body. Since eosinophil granules also contain histaminase that can degrade histamine, it was proposed that eosinophils are recruited into the tissue to mop up the histamine released by mast cells and basophils. Eosinophils can also produce and release a variety of mediators such as LTC4, IL-1, transforming growth factor (TGF)-b, IL-3, IL-4, IL-5, IL-8, and TNF that can participate in allergic inflammatory reactions [51]. Human eosinophils also contain a 17-kDa hydrophobic protein that forms Charcot–Leydon crystals often found in the sputum, feces, and tissue of patients with allergic asthma and other eosinophil-related diseases. E. Mast Cells and Basophils Mast cells and basophils play an important role in innate and adaptive immune responses by virtue of their ability to secrete a plethora of mediators, including histamine, that are stored in their cytoplasmic granules [52–54]. An important effector role for these two cell types in allergic reactions and parasitic infections is well established; however, their contribution to innate immunity is becoming more apparent as their biology and functions are better understood [55–57]. Both mast cells and basophils originate from CD34+ hematopoietic stem cells in the bone marrow and express the high-affinity receptor for IgE (FceRI) on their surface. Cross-linking of the FceRIs by antigenbound IgEs activates these cells to release their granules, which contain preformed mediators such as histamine, proteoglycans, and neutral proteases. Human mast cells contain approximately 2 to 5 pg of histamine per cell. In addition, activated mast cells and basophils also synthesize de novo certain mediators, such as LT C4, PAF, IL-4, and IL-13. Activated mast cells, but not basophils, produce heparin, prostaglandin D2, serotonin, and a variety of cytokines such as IL-1, IL-3, IL-5, IL-6, IL-8, IL-10, IL-16, RANTES, IFN-g, TGF-b, TNF, and granulocyte-macrophage-colonystimulating factor (GM-CSF), as well as chemokines such as C-C chemokines, macrophage inflammatory protein (MIP)-1a, and monocyte chemoattractant protein (MCP)-1. Mast cell–derived


HUMAN IMMUNE SYSTEM

41

serotonin is associated with contact dermatitis. Similarly, TNF and LT produced by mast cells play a major role in the development of septic peritonitis and graft-versus-host disease (GVHD) reaction [52,58–60]. Mast cells can be triggered to release TNF through a variety of receptors expressed on their surface [61]. The released TNF then activates endothelial cells resulting in neutrophil and macrophage recruitment into the microenvironment. Vasoactive amines released from these activated mast cells cause vasodilatation, increased mucus secretion, and massive flux of chloride secretion resulting in serum exudation and edema. Thus, mast cells may play an essential role in the initiation of an immune response. To perform this unique function, mast cells are placed strategically throughout the mucous membrane and different organs that are highly vascularized. There are two different phenotypes of human mast cells described based on the type of neutral protease they carry and their location. Mast cells found in the mucosa of intestinal tissue and lungs (MCT) carry mostly tryptase, whereas mast cells found in the skin and submucosa of the small intestine (MCTC) carry both tryptase and chymase as well as carboxypeptidase A and cathepsin G. Mast cell precursors generated in the bone marrow are recruited to various tissues where they mature into MCT or MCTC types. On the other hand, basophils mature in the bone marrow and are released into the circulation from where they are recruited into the tissue. Although activation of mast cells and basophils via the IgE-mediated pathway is well characterized, both mast cells and basophils can be activated through an IgE-independent pathway. This activation may occur through a variety of receptors on the surface of these cells including CD48; CD88 (complement component C3a and C5a receptors); TrkA (nerve growth factor receptor); FcgRI (IgG receptor); cytokine receptors (IL-4R, IL-5R, IL-9R, IL-10R, GM-CSFR, IFN-gR); chemokine receptors (CCR3, CCR5, CXCR2, CXC4); and CD8-like molecules on mast cells. Mast cells carry a receptor (Kit) for stem cell factor (SCF). In the presence of SCF mediator, the release from mast cells is enhanced several fold [62]. Receptors on basophils include cytokine receptors (IL-3R, IL-5R, GM-CSFR); chemokines receptors (CCR2, CCR3); complement receptors (CD11b, CD11c, CD35, CD88); prostaglandin receptors (CRTH2); and FcgR1I. Certain molecules secreted by bacteria, viruses, and parasites can activate mast cells and basophils directly through these non-FceRI pathways that lead to release of mediators including histamine and TNF [63–65]. Mast cell activation and release of mediators together initiates a cascade of events resulting in an immediate hypersensitivity reaction as well as a late-phase reaction [66,67]. The immediate hypersensitivity reaction in the skin appears as erythema, edema, and itch; in the upper airways as sneezing, rhinorrhea, and mucus secretion; in the gastrointestinal tract as nausea, vomiting, diarrhea, and cramping. These symptoms coincide with release of histamine, PGD2, and LTC4. This immediate reaction is then followed 6 to 24 hours later by persistent edema and leukocytic infiltration. Additional mediators released by mast cells and inflammatory mediators released by accumulating leukocytes initiate the late-phase reaction, which is believed to be a major contributor of persistent skin inflammation and asthma. Mast cells can also down-regulate an inflammatory reaction in the skin and lungs by releasing IL-1 receptor antagonist, IL-10, and heparin. In addition, both mast cells and basophils can phagocytose bacteria and destroy them. Subjects with a mutation in the Kit gene have increased numbers of mast cells in their body, a pathological condition called mastocytosis. Patients with mastocytosis exhibit a typical skin lesion called “urticaria pigmentosa,” along with elevated serum levels of tryptase, episodes of unexplained flushing, and anaphylaxis [68]. F.

Complement

The term “complement” was coined by Paul Ehrlich in 1899 to describe a group of factors in the serum that can lyse bacteria. After more than a decade of research we now know that the complement system consists of over 30 different proteins, some present as soluble proteins in the plasma and the others as bound in the cell membrane [6,69]. The soluble components in plasma consist of complement proteins numbered from C1 through C9. C3 is present in the highest concentration in plasma. The membrane-bound complement proteins are named based on their


42


Alternate Pathway

C5 C3a

C3

C3b

C3b,Bb (C3 convertase)

Classic Pathway By way of activating C3 to become C5 Convertase

C1s C1r

C4 C4a Fc

C4b Immunoglobulin

C4b,C2a (C3 Convertase)

Antigen

C2

C2b C5b

Mannose-binding pathway

MBL (Mannose-binding Lectin)

C6, C7, C8, C9

MAC (Membrane attack complex)

Figure 3.3

Schematic representations of the three major pathways of complement activation: classic, alternate and mannose-binding.

function (e.g., decay accelerating factor), or biochemical structure, such as those belonging to the cluster of differentiation (CD) system or as complement receptor proteins (CR1, CR2, CR3, and CR4). Approximately 90% of the plasma-complement proteins are synthesized in the liver. Other sources of complement include monocytes, macrophages, endothelial cells, lymphocytes, glial cells, astrocytes, renal epithelium, and reproductive organs. Acute-phase mediators (such as IL-1, IL-6, TNF, IL-11) and IFN-g stimulate the synthesis of complement following tissue injury. The released complement proteins are then activated through a systematic cascade of sequential proteolytic cleavage of components (the sequence being C1-C4-C2-C3-C5-C6-C7-C8-C9) [70]. The proteolytic cleavage leaves a smaller and a bigger fragment designated as “a” for smaller and “b” for bigger fragment, respectively, except for C2a, which is a larger fragment. This proteolytic cleavage and subsequent activation sequence are collectively called the “complement activation pathway.” There are three different pathways of complement activation: alternative, mannose-binding lectin, and classical (Figure 3.3). Activation of C3 by cleavage to C3b is an important and common reaction in all three activation cascades. The enzyme that catalyzes C3 fragmentation to C3b is the C3 convertase (also called C3b,Bb). C3b contains an intramolecular thioester bond that is activated upon cleavage and removal of the C3a fragment. The reactive thioester acts as an acceptor site that can form covalent bonds with several targets, including OH, NH2, and H2 molecules. The alternative pathway of complement activation is phylogenetically the oldest C3-activating pathway, and is one of the first lines of defense against invading microorganisms. This pathway is activated instantaneously when the acceptor site of activated C3b fragment comes into contact with a foreign molecule that is not self (activator surface). Factors B, D, and P present in the plasma then associate with the C3b and convert it into C3b,Bb (C3 convertase), which in turn activates fresh C3 resulting in the amplification of complement activation. However, binding of the C3


HUMAN IMMUNE SYSTEM

43

acceptor site to a host cell membrane (self) triggers the factors H and I that disassociate from the C3b,Bb, thus down-regulating the alternative pathway of complement activation. The classical pathway of complement activation consists of C1, C4, C2, and C3 proteins. C1 is a complex 750-kDa protein consisting of two C1r and two C1s molecules noncovalently associated with a C1q molecule [71]. The C1q molecule consists of six identical subunits, each with three homologous chains that form a globular domain at the C-terminal that can bind to the Fc portion of Ig [72]. The C1r and C1s molecules are placed inside the cone shaped region of the C1q molecule [73]. Both C1r and C1s contain a serine protease domain and a contact domain. Antigenbound IgG and IgM are the primary triggers for C1 complex activation, although bacterial LPS, acute-phase mediators, myelin, polyanionic compounds, and some viruses can activate C1 complex. C1q binds to Fc portions of IgG (IgG3> IgG1> IgG2>IgG4, affinity) and IgM that has undergone conformational changes as a result of antigen binding. At least two of the six globular domains have to be occupied by the Ig to trigger the activation of C1. Engagement of C1q to Fc portion of Ig activates C1r, which in turn activates C1s. The activated C1s will then cleave the C4 complement protein into C4a and C4b fragments. The C4b fragment then refolds to expose the thiol ester region, which can form covalent ester and amide bonds with several proteins, carbohydrates, and water. C1 activation to C4b binding occurs in microseconds. Complement protein C2 then form complexes with C4b, allowing cleavage of C2 into C2a and C2b by C1s. The C2b is then released with a kinin-like activity. The C2a bound to C4b, results in an enzymatically active fragment, C4b,C2a, which acts as the C3 convertase for the classical pathway [74,75]. The mannose-binding lectin (MBL) pathway consists of a C-type lectin (MBL) that can bind to mannose and a variety of carbohydrate molecules in a Ca+-dependent fashion [76]. The structure of MBL is very similar to C1q molecule with six globular domains that bind to carbohydrates. Two MBL-associated serine proteases, MASP-1 and MASP-2, are noncovalently associated with MBL in analogy with the C1r and C1s proteins. Active MASP-2 can cleave C4 similar to C1s, leading to the formation of the C3 convertase C4b,C2a as described above. Thus, there are two C3 convertases (C3b,Bb and C4b,C2a) formed as a result of the three different pathways. The C3 convertases then initiate cleavage of the C5 component of the complement resulting in the formation of a small C5a peptide, a powerful chemoattractant for inflammatory cells and a bigger fragment C5b, which is important in the formation of the membrane attack complex (MAC). C5b in turn activates C6, C7, C8, and C9 nonenzymatically. C9 molecules are cylinders that get inserted into the membrane of the target cell. Depending on the availability of C9 molecules in the microenvironment, several C9 can bind to the MAC, thus increasing the strength of the deathblow. Several cells in the body, including erythrocytes, lymphocytes, macrophages, monocytes, dendritic cells, eosinophils, and neutrophils, can express receptors for complement and their active fragments. Binding of the fragments to these receptors can also activate the complement cascade. The major receptors of complement are CR1, CR2, CR3, and CR4. The CR1 serves as a receptor for C3b-bound toxin–antitoxin or antigen–antibody immune complexes. Cells expressing the CR1bound complexes are then phagocytosed by macrophages or if the complex is bound to the CR1 of a phagocyte, it acts as a phagocytic stimulus. Human CR2 can bind fragments of C3, envelop protein (gp350/220) of Epstein–Barr virus, and the low-affinity receptor for IgE (CD23) on B cells. CR3 (also called Mac-1, CD11b/CD18) is expressed on dendritic cells, phagocytes, neutrophils, NK cells, and mast cells. The most important function of CR3 is phagocytosis. Some bacteria and yeast can bind directly to the lectin domain of the receptor without the need for complement. Triggering of CR3 via its lectin domain can cause oxidative burst in neutrophils and phagocytes. Other ligands for CR3 include intercellular adhesion molecule (ICAM)-1, fibrinogen, and clotting factor X. CR4 (also known as p150/95 and CD11c/CD18) has similar function as CR3 on phagocytes. In addition, several cells express receptors for C5a, C3a, C1q, and factor H. Among these, the receptor for C5a (C5aR) is probably the most important because binding of C5a to C5aR triggers severe inflammatory reaction including smooth muscle contraction and vascular permeability. C5a


44


can also trigger mast cell degranulation and cause chemotaxis of neutrophils. Thus, complement plays a central role in linking innate immunity with adaptive immunity [77].

III. ADAPTIVE IMMUNITY Adaptive immunity refers to the specific immune response to a microbe, an antigen or foreign particle through an acquired pathway. The adaptive immune response is expressed by two different mechanisms; one involves the B cells and immunoglobulin, known as humoral immunity, and the other involves T cells and various mediators produced by the T cells, which primarily functions via a cell-mediated mechanism. An important feature of the adaptive immune response is memory. This allows the body to respond to a given antigen in a specific way at a later time (also known as recall responses) when the body re-encounters the antigen. Adaptive immunity is the underlying principle for vaccine development [78]. A. B Lymphocytes B lymphocytes are cells derived from the bone marrow; hence the name. The progenitors of B cells in the bone marrow have a typical set of surface markers of CD19+, CD10+, and CD34+. These cells develop into precursor cells or pre-B cells with a phenotype of CD19+, CD10+, and CD34-. The number of these progenitor and precursor cells decline with age. Nevertheless, B cells are generated throughout the life cycle, mainly from bone marrow [79,80]. Pre-B cells can be found in fetal liver by 8 weeks of gestation. This suggests that the B-cell compartment is formed well before birth. B cells produce immunoglobulins that participate in the humoral arm of adaptive immunity. After generation in bone marrow, B cells exit into peripheral circulation as immature B cells. These immature B cells are highly susceptible to inactivation upon antigen contact [81]. This is probably one of the mechanisms by which immune tolerance to an antigen is developed. About 1 week after entering the periphery, B cells mature and become competent cells that can make antibodies when they encounter antigens. Binding of T-cell–independent antigens triggers signaling through B-cell receptors (BCRs), resulting in B-cell activation that is characterized by an upregulation of class II MHC molecule expression and cell proliferation. BCRs consist of a complex formed by membrane immunoglobulin (mIg) that is associated with at least two transmembranous polypeptide chains called Ig-a and Ig-b [82]. Binding of antigen to the BRC and subsequent crosslinking trigger a cascade of intracellular events, leading to internalization of antigens. The internalized antigens are then degraded into small peptides inside the cytoplasm and are expressed in the class II MHC groove for presenting the antigen to helper T cells in lymphoid organs. Interacting helper T cells express CD40 ligand (CD40L) molecules and secrete cytokines that in turn activate the B cells to proliferate and terminally differentiate into antibody-secreting plasma cells. These plasma cells then migrate from the T-cell areas to follicular areas and initiate formation of germinal centers. Immature B cells that are not recruited into the long-lived follicular region will die within a week. Inside these germinal centers, the B cells activate the somatic hypermutation of their immunoglobulin genes (see below) to create an antibody with high affinity to the antigen. The germinal center is thus the major site where plasma cells that produce high-affinity antibodies are generated. These cells subsequently develop into memory B cells. B. B1 Cells B1 cells are a separate lineage of B cells that are present predominantly in the peritoneal and pleural cavities. B1 cells express high levels of IgM, and low levels of IgD and Mac1, but do not express CD23. A subset of B1 cells expresses low levels of CD5 [83]. Thus, based on the expression of CD5, B1 cells are classified into B1a (that express a low level of CD5) and B1b (that do not


HUMAN IMMUNE SYSTEM

45

express CD5). B1 cells are generated during fetal life and are characterized by restricted IgH gene rearrangements that primarily use only the VH repertoire. Thus, they have limited ability to rearrange their antibody. A unique property of B1 cells is that they have the potential to self-replicate indefinitely. Although the functions of B1 cells are not fully understood, these cells can remarkably produce IgM antibodies against common bacterial proteins within 48 hours of exposure. C. Immunoglobulins Humoral immune responses in humans are essentially mediated by immunoglobulins (Igs), which are specialized globular glycoproteins produced by B cells, more specifically plasma cells. There are five major classes or isotypes of Igs: IgM, IgD, IgG, IgA, and IgE. An Ig generated against a specific antigen is called an antibody. Classic studies by Edelman and Porter in the 1950s and subsequent crystal structure analysis of Igs show that the different classes of Igs share a basic structure. Thus, an Ig molecule consists of two heavy chains and two light chains that are held together by interchain disulfide bonds and hydrophobic interactions (Figure 3.4). The heavy chain of Ig has a molecular mass of approximately 55 kDa, and the light chain has a molecular mass of 25 kDa. One light chain pairs with one heavy chain. Disulfide interaction between the two heavy chains results in the formation of a heterodimeric structure that contains the two light chains and two heavy chains. Studies by Edelman and Porter in the 1950s using IgG molecule showed that papain digestion yields two monovalent antigen-binding fragments called Fab and an easily crystalizable fragment called Fc. Digestion of IgG molecule with the enzyme pepsin yields a divalent antigen-binding fragment F(ab)'2. Subsequent treatment of the F(ab)'2 with mercaptoethenol yielded the monmeric form. These findings were subsequently confirmed by the crystal structure analysis of IgG, suggesting the significance of the interchain disulfide bonds in holding the heterodimer together. The amino terminal of light and heavy chains express two to five unique domains called Ig domains consisting of approximately 110 amino acids. These Ig domains are the antigen-binding sites of an Ig, and are thus the fundamental units of an antibody. The Ig domains of all Ig classes of proteins exhibit a common structural motif called the Ig fold, which is formed by two b-pleated sheets oriented in a sandwich-like structure enclosing a hydrophobic core. The amino acid sequences within each of these Ig domains show considerable variation between diverse Ig molecules and between light and heavy chains. This region is therefore collectively called as the variable (V) region of the Ig, and is responsible for antigenic specificity and for diversity of the antibodies. For a given Ig, more than 100 genes code the V regions, thus amplifying the permutation combination

VH VL C1 Hinge region

C2 C3 Figure 3.4

Structure of a typical immunoglobulin (Ig) molecule consists of two heavy chains (H) and two light chains (L) held together by interchain disulfide bonds and hydrophobic interactions. The variable regions of heavy chain (VH) and light chain (VL) are responsible for antigen specificity and antibody diversity; whereas the constant regions (C) show little sequence variation. Hinge region is a flexible part of Ig.


46


of the antigen-binding sites. Variable regions of light chain are designated as VL and the variable regions of heavy chain are designated as VH. The carboxyl termini of light and heavy chains show very little sequence variation within a given class of Ig. Therefore, this region is referred to as the constant (C) region. The light chain has one constant region (CL), whereas the heavy chain has more than one constant region designated as CH1, CH2, CH3, and so on, with CH1 being close to the V region. The C region of an Ig is responsible for binding of the Ig to its receptor (Fc), serves as a binding site (fixation) for complement, confers ability to form Ig multimers, and allows certain classes of Igs to be secreted into the mucosal site. Between the CH1 and CH2 regions is a hinge region that permits considerable flexibility to the V regions. The CH3 domain of the heavy chain shows significant variation that gives the unique characteristics to each class of Ig. Thus, there are five different CH3 domains: mu (m), delta (d), gamma (g), alpha (a), and epsilon (e), which are associated with the IgM, IgD, IgG, IgA, or IgE isotype, respectively. There are two classes of light chain constant regions, Ck and Cl, whose functions are poorly understood at the present time. 1. IgM Developmentally, the heavy chain of IgM (m chain) is the first Ig isotype to be synthesized by B cells, and IgM antibodies are the first to be produced during a primary immune response [84]. Approximately 10% of circulating Igs are of IgM isotype, which have a short half-life of 5 days. IgM exists in two forms: membrane bound monomeric and secreted pentameric. The membrane bound form of IgM is expressed on the surface of immature B cells and serves as an important molecule for B-cell activation upon antigen binding [85]. IgM has four constant regions in its heavy chains, and binding to its Fc receptor occurs through the CH4 (Cm4) region. The secreted pentameric form of IgM consists of five monomeric IgM molecules held together by a J chain. The J chain can interact with a molecule called secretory component (SC), which is expressed on the basal surface of epithelial cells [86]. Pentameric IgM bound to the SC are then transported across the epithelial cells into the mucosal surface, including breast milk. Because of multiple antigen-binding sites, the pentameric IgM has higher avidity for antigens than the monomeric form. Similarly, the pentameric secretory IgM can efficiently bind complement fragments of the classical pathway. Thus, pentameric IgM are the first line of defense against invading organisms at the mucosal surfaces. Memory B cells are poor sources of secretory IgM; therefore, an elevation in the levels of IgM is an indication of recent antigen activation. 2. IgD Less than 0.5% of IgD is present in the circulation in any given time. In part, this may be because of its short half-life and susceptibility to proteolytic degradation. Interestingly, the C region genes of both m and d are transcribed together in B cells. Thus, the mature B cells that migrate out of bone marrow are IgD+/IgM+ and constitute nearly 90% of circulating B cells [87]. Although a specific immunological function is not ascribed to IgD, activation of the cells through the membrane bound IgD can up-regulate certain molecules such as B7-1 and B7-2 on the surface of B cells and thus promote antigen presentation. Activation through IgD can also increase the secretion of IgE from B cells. Another important function of IgD is that it can fix complement, and thus participate in innate immunity [88]. 3. IgG Nearly 75% of serum Igs are of the IgG isotype, and are distributed equally between intravascular and extravascular serum pools [89]. IgG is also present in the lymph, peritoneal, and cerebrospinal fluid. IgG has a molecular mass of approximately 150 kDa and exists in four different


HUMAN IMMUNE SYSTEM

47

forms or subclasses: IgG1, IgG2, IgG3, and IgG4. An important functional characteristic of IgG (especially IgG1, IgG3, and IgG4) is its ability to cross the placenta with the ability of conferring protection to the fetus and newborn. The half-life of circulating IgG is about 7 to 24 days depending on the subclass of IgG. IgG3 has an extended hinge region that makes it more amenable to proteolytic degradation resulting in a shorter half-life (7 days) compared to the other subclasses. Increases in the levels of high-affinity antigen-specific IgG occur during a secondary immune response [90]. These antigen-coupled IgGs can then bind to FcgR expressed on a variety of cells such as B cells, macrophages, granulocytes, and cytotoxic lymphocytes, resulting in cell activation, cytokine production, expression of receptors, phagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC). Among the various subclasses, IgG1 has the highest efficiency in inducing ADCC than the other Igs (IgG3, IgG2, or IgG4) [91]. This difference is reflected in their differential binding to FcgR, which is attributable to a sequence difference in their Cg2 region [92]. Another important function of IgG is its ability to activate complement via the classical pathway. Again, the ability of each subclass to activate complement varies, with IgG3 having the highest capacity, followed by IgG1 and IgG2. These differences appear to be due to a structural variation at the hinge and Cg2 regions. IgG4 has a compact structure and appears to activate complement via the alternate pathway better than the classical pathway. Another interesting fact is that because of its compact nature, IgG4 antibodies are functionally monovalent and are believed to possess the ability of scavenging allergenic antigens. Binding of these IgG4 antibodies to mast cells and basophil surfaces are believed to down-regulate IgE-mediated hypersensitivity reactions. Thus, IgG4 antibodies can serve as blocking antibodies [93–95]. 4. IgA IgA is the major immunoglobulin present in mucosal surfaces, and is especially abundant in the saliva, tears, intestinal mucus secretion, bronchial secretion, nasal mucosa, colostrum, prostatic fluid, and vaginal secretions [96]. In the circulatory system, IgA constitutes approximately 15% of total serum immunoglobulins. IgA exists as a monomeric or polymeric form. Over 90% of IgAs present in circulation are in monomeric form, whereas IgAs present in the mucous surface are primarily polymeric [97,98]. These polymeric secretory IgAs are formed from two monomeric IgA molecules linked together by a J chain that is attached to an SC. Polymorphic IgA is secreted into the mucosal surface, similar to IgM. The molecular mass of a monomeric IgA is 160 kDa, whereas the molecular mass of a polymeric IgA is 400 kDa. The two subclasses of IgAs present in humans are IgA1 and IgA2. The two subclasses differ in their amino acid sequences in the hinge and Ca regions. IgA1 is susceptible to proteolytic degradation, whereas IgA2 lacks the 13 amino acid proteolytic-sensitive domains in its hinge region, and is resistant to this degradation. This unique feature makes IgA2 more resistant to enzymatic cleavage by a variety of bacteria (Clostridium sp. Haemophysalis influenzae, Neisseria gonorrhoeae, Streptococcus pneumoniae, Streptococcus sanguis, Neisseria meningitides, and so on). IgA2 is present in two allotypic forms: IgA2m(1) and IgA2m(2). One of the major functions of IgA appears to block uptake of bacterial and viral antigens by inflammatory cells, thereby limiting the inflammatory responses [99–101]. However, by binding to certain bacteria and parasites, IgA can trigger the ADCC mechanism via FcaR in some effector lymphocytes. In general, IgA is a poor activator of complement via the classical pathway. However, IgA can activate complement via the alternate pathway [102,103]. 5. IgE IgE is present in very small amounts in the circulation of healthy individuals and comprises less than 0.004% of total serum immunoglobulins. IgE has a major function in allergic reactions and parasitic infections [104–106]. Helminth infections can trigger production of large quantities of polyclonal IgE antibodies. Cross-linking of two IgE molecules bound to its high-affinity receptor


48


(FceRI) on mast cells or basophils can trigger the release of a variety of vasoactive mediators by these cells leading to allergy and hypersensitivity reaction. However, binding of monomeric IgE to FceRI on mast cells can render these cells resistant to apoptosis [107]. IgE lacks the J segment, yet in some parasitic infections IgE is present as a secretory antibody in the bronchial and intestinal mucous secretions [108,109]. In addition to mast cells and basophils, the high-affinity receptor for IgE (FceRI) is also present on skin Langerhans cells (LCs). A low-affinity receptor for IgE (CD23, or FceRII) is present on a variety of cells including B cells, monocytes, and macrophages. CD23 has been shown to be involved in the regulation of IgE synthesis in B cells [110]. 6. Immunoglobulin Gene Regulation and Class Switch The regulation and rearrangement of Ig gene are highly complex events. In immature B cells, the various regions of the Ig genes are not assembled. As the B cells mature in the follicle, a unique somatic hypermutation event occurs within the B cells that can rearrange various units of the Ig genes to produce an antibody with high specificity to the antigen [111]. Such a rearrangement can create antibodies with more than 108 different types of specificities. As mentioned above there are two families of Ig light chains (k and l) and one family of heavy chain (H), each consisting of its own set of V genes and C genes. Each family resides in different chromosomes. The process of assembling various chains within the activated B cells is called the recombination event leading to rearrangement of various chains by allelic exclusion [112]. First, recombination occurs on the V(D)J segment of the heavy chain, which then pairs with the VL CL units of k or l light chain to generate antibody diversity. Finally, the type of CH gene recombination will define the isotype of antibody. A single B cell can produce only one isotype of antibody at any given time. However, depending on the environmental stimuli and differentiation, B cells can switch from one isotype to another antibody isotype. This process is called class switching or isotype switching [113–116]. Class switching occurs only in the CH gene. Thus, during class switching a single VH gene may combine with more than one CH gene, which allows generation of antibodies with different isotype but same specificity. Initially, B cells generate Cm. During class switching, the Cm region is deleted allowing combination with other CH genes such as Cg,Ce, and Ca. D. Major Histocompatibility Complex Major histocompatibility complex (MHC) is a collection of closely linked genes located in the human chromosome 6. MHC proteins encoded by these genes are vital for the induction and regulation of specific immune responses. On chromosome 6, these MHC genes are organized into three major regions that are broadly designated as class I, class II, and class III (Figure 3.5). The class III region is located between class I and class II regions, and contains genes that code for some of the complement proteins, soluble serum proteins, and TNF [117]. Within the class I and class II MHC regions are the loci for the human leukocyte antigens (HLA). Class I region encodes

Genes

Class II

Locus

DP DQ DR

Gene products Figure 3.5

αβ

αβ

αβ

Class III

Class I

C4 C2 BF

B C

A

B

A

Complement

TNF

Organization of human MHC genes on chromosome 6.

C


HUMAN IMMUNE SYSTEM

49

for the a or heavy chain of three major subclasses of HLA (HLA-A, HLA-B, and HLA-C). In addition, the class I region also contains several nonclassical MHC class 1b genes such as HLAG and MHC class I chain–related (MIC) genes that are believed to be important in immune evasion [118,119]. Class I region also contains several pseudogenes that are essentially nonfunctional. On the other hand, class II genes code for both a and b chains of three HLA subclasses (HLA-DP, HLA-DQ, and HLA-DR). The class II genes are further subdivided into A or B depending on whether they code for a or b chains. Thus, there are four DP genes (A1, B1, A2, B2), five DQ genes (A1, B1, A2, B2, B3), and several DR genes (A, B1 to B9), depending on the haplotype. 1. Structure of Class I MHC Molecules Class I MHC molecule is expressed on all human nucleated cells although their number may vary from cell to cell. Lymphocytes express the highest number (> 5 ¥ 105 molecules per cell) of class I MHC molecules, whereas liver cells and neuronal cells express the least number. A typical structure of a human class I MHC molecule consists of a polymorphic transmembranous glycoprotein heavy chain (~44 kDa) bound noncovalently to a nonpolymorphic 12-kDa light-chain protein called b2 microglobulin or b2m. The a chain of the class I molecule has three external domains (a1, a2, and a3), each with approximately 90 amino acids, a transmembranous domain that is 40 amino acids long, and a cytoplasmic tail of 30 amino acids. There is significant sequence homology among the a3 domain, b2m, and the C region domain of Ig. Therefore, the class I molecule belongs to the Ig superfamily of proteins. The a1 and a2 domains of class I MHC interact with each other to form a cleft or groove that holds a short peptide of eight to ten residues. These peptides are usually derived from endogenous proteins, infectious agents, or tumors. The sides of the groove are formed by two a-helical regions, and the base of the groove where peptide attaches is formed by a b sheet with eight antiparallel b strands. The a3 domain is highly conserved among all three classes of MHC class I molecules and contains the binding site for the CD8 membrane receptor on T cells. 2. Structure of Class II MHC Molecules Class II MHC molecules are typically expressed by antigen-presenting cells, including dendritic cells, macrophages, thymic epithelial cells, and mature B cells. The human class II MHC molecule is a heterodimeric glycoprotein comprised of a a chain (33 kDa) and a b chain (28 kDa) that are noncovalently associated with each other [120]. Both a and b chains have an external domain, a transmembranous domain, and a cytoplasmic tail similar to the class I molecule. The external domain of the a and b chain has significant sequence homology to the Ig-fold domain. Thus, the class II MHC molecule also belongs to the Ig superfamily of proteins. The external domain of each chain is comprised of two distinct regions: a1 and a2 domains and b1 and b2 domains. The a1 and b1 domains interact with each other to form a groove that is similar to the class I groove but is much shallower with one end relatively open. This allows binding of slightly bigger (13 to 25 residues) peptides to the class II MHC molecule. The external domain of the class II MHC molecule contains the binding site for the CD4 membrane receptor on T cells. 3. Peptide Binding to MHC Molecules Both class I and class II MHC molecules are promiscuous as far as peptide binding is concerned. A single MHC molecule can bind several different types of peptides; similarly, a single peptide can bind to several different MHC molecules. This unique function is due to significant structural similarity between the class I and class II peptide–binding grooves. Nearly all the peptides that bind to the class I MHC molecule share similar amino acid residues (also called anchor residues) at their amino and carboxyl termini with which they bind to the class I MHC molecule. Since the


50


peptide-binding sites on the class I MHC molecule clusters to the two ends of the groove, the peptide that binds to the class I molecule arches away from the groove exposing the middle region of the peptide for T-cell presentation. On the other hand, the class II MHC groove has several peptide-binding sites and uses hydrophobic interaction to bind peptides. Because of the diversity in the MHC molecule, a normal individual can express up to six different class I molecules and 12 different class II molecules. Peptides binding to the grooves of these few numbers of class I or class II molecules are responsible for almost all T-cell activation in the body and subsequent initiation of cell-mediated and humoral immune responses against a variety of antigens. E. Minor Histocompatibility (H) Antigens Minor histocompatibility antigens or minor H antigens play a major role in graft rejection [121,122]. Minor H antigens are short MHC-bound peptides derived from an allelic variation (usually a single amino acid change) of endogenous self-peptides. Genes of minor H antigen have been traced to the mitochondrial genome, the Y chromosome, and the autosome [123]. The first minor H peptide to be sequenced was HA-2, which was found to be associated with HLA-A2.1 and graft-versus-host disease (GVHD) [124]. HA-2 sequence was found to be homologous to myosin heavy-chain proteins. The existence of minor H antigens was first described in inbred mice by Snell [125], who demonstrated the ability of inbred mice to reject skin grafts and tumor cells from H2 (MHC) matched donors. Subsequently, the presence of minor H antigens in humans was established when skin grafts or stem cells from HLA-identical siblings was rejected [126]. It is now well established that mismatched H antigens can cause GVHD, graft-versus-leukemia (GVL) effect and host-versusgraft (HVG) reaction in humans [127,128]. An increased frequency of skin allograft rejection may be due to a higher level of H antigens being presented to T cells by LCs in the skin. Both CD4 and CD8 T cells can respond to peptides presented by H antigens. In some cases, recognition of minor H peptides by CD8+ T cells can lead to tolerance. An important feature of T-cell responses to H antigen is the requirement of prior exposure of T cells to the antigenic peptide, unlike those that naïve T cells can respond to when presented by MHC. Thus, prior exposure to minor H antigen creates a memory pool of T cells that are involved in subsequent graft rejection or tolerance. Substantial clinical data are accumulating on the role of minor H antigen on the rejection of various allografts including cornea grafts [122]. This exciting field is fast evolving, especially with respect to transplantation immunology. F.

Antigen Processing and Presentation

T-cell activation of antigen requires that small peptides derived from the antigen be displayed within the groove of a MHC molecule expressed on the surface of antigen-presenting cells (APCs). The sequence of events involved in the formation of the peptide–MHC complex is called antigen processing [129]. The antigen processing occurs within the cytoplasm of APCs via a cytosolic pathway for class I molecules and via an endocytic pathway for class II molecules. The processed peptide–MHC complex is then transported to the exterior and presented on the surface of the APC. This process is designated as antigen presentation [130]. 1. Antigen Processing and Presentation on Class I MHC Molecules Cytosolic pathway of antigen presentation on class I MHC molecule occurs when intracellular soluble proteins or endogenous antigens are degraded, and the peptides of such degraded proteins are expressed on the class I MHC molecule [131–133]. Examples of endogenous antigens include proteins released by viruses, bacteria, or intracellular protozoans invading an infected cell. In some cases, the endogenous antigens can be normal cellular proteins. The mechanism of antigen


HUMAN IMMUNE SYSTEM

51

processing begins when a small group of proteins within the cytoplasm called ubiquitin binds to the intracellular antigen. Several ubiquitin molecules can bind to a single protein. This process is called ubiquitylation or multiubiquitin tagging of a protein. When an antigen or protein is ubiquitinated, it becomes a target for degradation by a specialized ATP-dependent, multisubunit complex of proteases called the proteasome. Proteasomes are a large group of proteolytic enzymes enclosed inside a cylindrical enzymatic chamber with a central channel of 10 to 20 Å. Proteasomes are found in several compartments of a cell and are responsible for degrading proteins that are no longer necessary for the cells, including damaged proteins or proteins that are folded incorrectly. The multiubiquitin tagged proteins to be degraded are selectively guided into the enzymatic chamber by a 19S ATPases-containing subunit of proteasome, and the proteins are degraded inside the chamber. Thus, other proteins within the cell are prevented from proteolysis. Peptides generated by a typical proteasomes are not always antigenic. However, the cytokine interferon-g induces transcription of specialized subunits of proteasome, which creates an immunoproteasome that can now generate antigenic peptides. These antigenic peptides are then transported into the rough endoplasmic reticulum (ER) by a transporter protein called TAP (transporters associated with antigen processing) that has a high affinity for peptides with 8 to 13 amino acid residues. TAP is a membrane-spanning heterodimer consisting of proteins TAP1 and TAP2. The life of a class I MHC molecule begins with the co-translational translocation of a and b2-microglobulin across the ER. Several chaperone proteins such as calnexin, calreticulin, tapasin, and the ERp57 are involved in this assembly initially. Tapasin binds the class I molecule to the ER and mediates physical association of TAP to the class I molecule. The TAP then loads the peptide onto the class I MHC groove. Tapasin releases the peptide-loaded class I molecule into the ER lumen, initiating the transportation process [134]. Several viral proteins are known to block the release of class I MHC molecule from ER by modulating the tapasin functions. The mature peptide containing class I MHC molecule is then transported to the surface of the cell where it presents the antigenic peptide to CD8+ T cells. 2. Antigen Processing and Presentation on Class II MHC Molecules Endocytic pathway or exogenous antigen presentation occurs when a foreign antigen is internalized by APCs through endocytosis (receptor mediated or pinocytosis) or phagocytosis. The internalized antigen is then degraded into short peptides (of 13 to 25 residues) within the endosomes or phagocytic vesicle by several hydrolytic enzymes functioning at three different pH ranges (6.0 to 6.5, 5.0 to 6.0, and 4.5 to 5.0). These antigenic peptides are now ready for loading on to new class II MHC molecules. Similar to the class I molecule, the assembly of class II molecules also occurs in the ER. During the assembly of class II molecules, they associate with another protein called invariant chain (Ii), which stabilizes the class II molecule and prevents the binding of any peptide to the class II groove. In addition, the Ii chain also directs the movement of class II molecules from ER to golgi apparatus and from there to the endosomes, where the antigenic peptide fragments are made. Once inside the endosome, the hydrolytic enzymes will cleave the Ii chain, leaving a small region of the invariant chain called CLIP (class II–associated invariant chain peptide) bound to the class II groove. CLIP remains associated to the class II molecule until it is actively removed by HLA-DM, a class II MHC-like molecule present only within the endosomal vesicles. Once the CLIP is removed, peptide can bind to the class II groove, and the class II MHC molecule is then transported to the surface where antigen is presented to CD4+ T cells. 3. MHC Tetramers The development of class I and class II tetramers has provided invaluable insight into the identification and characterization of MHC-restricted CD8 and CD4 cells in the immune responses against complex antigens [135]. Class I tetramers are generated by expressing the respective a and


52


b2-microglobulin (for class I) and a and b chains (for class II) in Escherichia coli. The recombinant proteins are then refolded in the presence of an antigenic peptide of interest [136]. The MHC–peptide complex is then biotinylated (a process that adds biotin molecule to the protein using the enzyme Bir A) and aggregated into tetramers by the addition of phycoerythrin-labeled streptavidin that binds to the biotin molecule. Tetramers generated in this fashion are stable for at least 1 year. These tetramers can be then used to estimate the precursor frequency of antigen-specific T cells in the peripheral blood, to map the T-cell epitopes of complex antigens, and study T-cell response against a wide variety of antigens including infectious agents, tumors, or autoimmune diseases [137,138]. Application of this MHC tetramer technology to vaccine development will bring this powerful tool to the forefront of human immunology in the next decade. G. Langerhans Cells and Other Dendritic Cells Langerhans cells (LCs) are bone marrow–derived dendritic cells that reside in the skin epidermal layer and play a central role in both innate and adaptive immunity [139,140]. LC was originally discovered by Paul Langerhans; hence the name. LCs are potent APCs that express high levels of class II MHC molecules and CD1a molecules; thus, LCs play an important role in the skin immune system [141–143]. Activated LCs can express other surface markers, such as CD4, CD40, CD14, CD15, CD23, CD33, FcgR, complement receptors, ICAM-1, B7-1, B7-2, E-cadherin, E-selectin, and very late antigen (VLA) molecules. LCs also express the high-affinity FceRI, and may thus participate in IgE-mediated allergic diseases. LCs carry a unique organelle in their cytoplasm called Birbeck granules that can be identified under the electron microscope. The function of these granules is not fully understood. LCs have the ability to process antigens in the periphery and transport it to the draining lymph nodes where they are able to cluster with and activate antigen specific naïve T cells. Thus, the ability of LCs to migrate from the epidermis to regional lymph nodes is of pivotal importance to the induction of primary immune responses [144–146]. In the skin, LCs are normally found in an inactive state. However, upon interaction with a foreign antigen or stimuli, they get activated and differentiate to express various molecules on their cell surface typical of an APC [147]. These activated cells then disassociate themselves from the epidermis and migrate to the regional lymph nodes to initiate the immune response [148]. During migration, LCs undergo further phenotypic and functional changes, which enable them to perform their immune function [149,150]. The journey that the LC has to make from the skin has a number of requirements. Several cytokines, chemokines, and their interaction with appropriate receptors orchestrate this mobilization. Initially, it is necessary that the LCs disassociate from surrounding keratinocytes. This requires downregulation of E-cadherins. This response is mediated by TNF binding to TNF-R2 on the surface of LCs. There is also a second requirement involving IL-1b produced by LC binding to IL-1R1 in an autocrine fashion. For this reason, both TNF and IL-1b are essential for the migratory function of LCs and they in turn up-regulate other molecules such as ICAM-1 and CD44 (including exonsplice isoforms of CD44) on the surface of LCs. Absence of any one of these molecules may interfere with LC migration. Once dislodged from keratinocytes, migrating LCs must successfully traverse the basement membrane of the dermal–epidermal junction. This is facilitated by expression of very late antigen 6 (VLA-6, a6 integrin) that confers laminin-binding activity. Passage across the basement membrane will also require proteolysis. TNF induces matrix metalloprotienase-9 (MMP-9) and MMP-3 expression on the surface of LC that help in this function. CD40–CD40 ligand interaction is also critical for the migration of antigen-bearing LCs from the skin to the draining lymph nodes. Once in the dermis, the LCs make their way via afferent lymphatics into the draining lymph nodes. This migration is facilitated by expression of CCR7 chemokine receptors. Chemokines such as MIP-3a, MCP-1, MRP-1, and secondary lymphoid organ chemokine (SLC) can enhance migration of LCs. The only known cytokine that can block LC migration from the skin is IL-10. There are other less well-characterized dendritic cells including dendritic epidermal


HUMAN IMMUNE SYSTEM

53

T cells, dermal dendrocytes, and dermal Langerhans-like cells in the skin [151,152]. Although there is no evidence that dendritic epidermal T cells can present antigen or migrate to lymph nodes, they do influence the intensity of cutaneous immune responses to chemical haptens. Thus, LCs are probably the most important immune cells in the skin that can activate naïve T cells and initiate a primary immune response. Because of this unique property, LCs are a major target for vaccine and immune modulation [153–156]. H. T Cells T lymphocytes undergo development, differentiation, and maturation in the thymus; hence the name. Similar to B cells, T cells are also generated in the bone marrow from where they migrate to the thymus for phenotypic development and maturation [157]. Like B cells, T cells also possess a membrane-bound antigen receptor, which is structurally different from Ig, but share some common features with the Ig molecule. Called T-cell receptor (TCR) [158], the receptor can only recognize antigens presented by membrane-bound MHC displayed by several APCs. The APCs phagocytose antigens, degrade them in their cytoplasmic compartment, and express fragments of these antigens on the MHC groove. When naïve T cells recognize the presence of these antigenic peptides within the MHC molecules, the T cells get activated, which results in proliferation and differentiation of T cells leading to the formation of memory cells for that particular antigen. These T cells can then differentiate into various effector cells. Thus, the fundamental difference between humoral and cellmediated response is that the B cells can bind to soluble antigens using the Ig on their surface, whereas T cells require that the antigen be processed and presented to them by the APCs [159]. Based on the expression of the TCR molecules, there are at least two well-defined groups of T cells: ab T cells (these include CD4+ and CD8+ cells) and gd T cells. The ab T cells are responsible for most of the cell-mediated immune responses. Based on the cytokine they produce, the ab T cells can be further divided into T helper cells Th1 and Th2. Th1 cells secrete proinflammatory cytokines such as interferon-g and TNF, whereas Th2 cells secrete cytokines such as IL-4, IL-5, IL-6, IL-9, and IL-10 that provide help for the synthesis of antibodies [160,161]. During an immune response, cytokines generated by these two subsets of Th cells orchestrate the outcome of the immune reaction. I.

Cytokines

Cytokines are short-acting, low-molecular-weight protein messengers that orchestrate an immune response, and are analogous to hormones in the endocrine system. Cytokines are produced and released by a variety of cells that participate in immune defense. For its function, a cytokine has to bind to a specific receptor on the surface of target cells. In this way, cytokines can have an autocrine (on the same cell that produced it) or paracrine action (on other cells in the microenvironment), thus regulating the immune and inflammatory responses associated with innate and adaptive immunity. Pleiotropism, redundancy, and synergy are typical characteristics of cytokines. A single cell can produce several different types of cytokines depending on the stimuli, and can express receptors for more than one cytokine simultaneously, often with opposing actions. Thus, a single cell can be regulated differently by different cytokines. Similarly, some cytokines have functions on various cells bringing about diverse biological effects. In the skin, keratinocytes are a major source of cytokines, although other cells such as LCs, lymphocytes, fibroblasts, and endothelial cells can also release a wide array of cytokines and chemokines that participate in the cutaneous inflammatory and immune responses [162]. A typical inflammatory/immune response is characterized by participation of several cytokines with redundant and synergistic functions. The balance between the functions of these various cytokines largely dictates the outcome of an inflammatory/immune response. Broadly, the cytokines that participate in the inflammatory/immune reactions can be grouped into four major groups: interferons, tumor necrosis factors, interleukins, and growth factors.


54


1. Interferons Interferons (IFNs) are glycoproteins with potent antitumor and antiviral properties, and are produced by a variety of cells. Type I IFNs include IFN-a, IFN-b, IFN-W, IFN-k, and IFN-tau. Type II IFNs consists of IFN-g only [163]. Among these, IFN-a, IFN-b, and IFN-g play a major role in host defense and immune homeostasis. In general, IFNs are immunoregulators that can alter cell growth, differentiation, gene transcription, and translation. Binding IFNs to their receptors triggers transmembrane signaling and transcription of certain proteins, collectively known as IFNinduced proteins (IPs). More than 25 IPs have been identified to date. IPs mediate most of IFN functions [164,165]. Generally, IFNs are growth inhibitors for a variety of cells including tumor cells. IFN-a is produced by B cells, T cells, macrophages, NK cells, and large granular lymphocytes in response to viruses such as Newcastle disease virus and Sendai virus, and bacteriophages, bacterial products, polynucleotides, tumor cells, and allogeneic cells [166–168]. Glucocorticoid hormones and viral RNA can up-regulate synthesis of IFN-a. The binding of IFN-a to its receptor on fibroblasts, tumor cells, B cells, monocytes, or hematopoietic progenitor cells causes selective growth inhibition and stimulation of class I MHC molecules. In response to IFN-a, tumor cells express increased amounts of tumor antigen on the surface for targeted cytotoxicity, B cells stop antibody production, and the bone resorption function of osteoclasts is inhibited. Because of its antitumor effect, IFN-a has been approved for therapy against hairy cell leukemia and Kaposi’s sarcoma. IFN-a is also effective against Mycobacterium leprae and Plasmodium falciparum. Interestingly, levels of IFN-a are increased in certain autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, and acquired immune deficiency syndrome (AIDS). IFN-b is secreted by fibroblasts and some epithelial cells. Infections with viruses or doublestranded RNA up-regulate synthesis of IFN-b. The IFN-b binds to the same receptor as IFN-a and has essentially the same function as IFN-a. IFN-b has potent antiviral activity against both RNA and DNA viruses and against the protozoan parasite Toxoplasma gondii. IFN-b also inhibits growth of tumor cells, up-regulates class I MHC molecules and increases NK cell and cytotoxic cell activity similar to IFN-a. IFN-b therapy is indicated in a variety of tumor conditions (bladder carcinoma, bronchiogenic carcinoma, renal carcinoma, malignant glioma, hairy cell leukemia, T-cell leukemia, lymphomas); for viral infections (hepatitis B and C, herpes zoster, herpes simplex, cytomegalovirus, HIV); and multiple sclerosis. IFN-g is mainly produced by Th1-type lymphocytes and NK cells, although several other cells can also secrete IFN-g. The secretion of IFN-g is induced by antigens, endotoxin, and other cytokines such as IL-2. IFN-g receptors are present in almost all nucleated cells. Binding of IFN-g to its receptor induces inhibition of cell growth in a variety of cells including tumor cells. IFN-g upregulates class I and class II MHC molecules, and enhances NK and cytotoxic cell activity. In addition, IFN-g also has potent antiviral function similar to other IFNs. Clinical application of IFN-g includes chronic granulomatous diseases, as an antiviral agent, against tumors and in autoimmune diseases. 2. Tumor Necrosis Factor Tumor necrosis factor (TNF) is a 26-kDa transmembrane protein belonging to a large TNF ligand family of proteins that are characterized by a unique b structure formed by two b-pleated sheets and two anti-parallel b strands [169–171]. The human TNF gene is located within the class III MHC region on chromosome 6. The TNF ligand family of cytokines has potent proinflammatory and antitumor activity. They exert their biological effects by binding to a family of receptors, collectively called the TNF-R [172]. Two such receptors — TNF-R1 and TNF-R2 — can bind TNF that is either membrane-bound or free in a soluble form [173]. TNF-R1 is constitutively expressed in nearly all tissues, whereas TNF-R2 is highly regulated and found only on cells of the immune


HUMAN IMMUNE SYSTEM

55

system [174,175]. Cross-linking and activation of TNF-R occur when three molecules of TNF bind to the receptors to form a homotrimer. TNF is mainly produced by macrophages, although a variety of cells such as neutrophils, mast cells, fibroblasts, NK cells, astrocytes, endothelial cells, and smooth muscle cells can also produce TNF. Although TNF has potent antitumor properties, it is best known for its ability to induce a variety of inflammatory cytokines and acute phase proteins, resulting in fever, release of prostaglandin E2, synthesis of collagen, resorption of bone and cartilages, inhibition of lipoprotein lipases, production of complement fragments, tissue necrosis, neurodegeneration, cachexia, and septic shock. Another important function of TNF is its ability to induce programmed cell death (apoptosis) through its TNF-R1. The molecular mechanism of TNF-induced apoptosis is now well characterized. Binding of TNF to TNF-R1 recruits the adaptormolecule TNF receptor-associated death domain (TRADD), which in turn recruits another adaptormolecule Fas-associated death domain (FADD). These receptor-bound death domains interact with the proteases caspase-8 and caspase-10, initiating the cascade of caspase activation leading to DNA fragmentation and cell death. There is also a TNF-related apoptosis-inducing ligand (TRAIL) that can bind to receptors DR4 and DR5 and induce apoptosis [176]. Binding of TRAIL to two TNF family decoy receptors DcR1 and DcR2 prevents apoptosis, as these receptors lack the death domains. A third decoy TNF receptor, DcR3, which binds to Fas ligand (FasL), is constitutively expressed at high levels in keratinocytes. Interestingly, soluble forms of DcR3 can profoundly modulate dendritic cell maturation and differentiation in the skin. The homotrimeric cytokines, lymphotoxin (LT)-a and LT-b, are structurally and functionally homologous to TNF. LT-a and LT-b are produced by activated T cells, B cells, and NK cells. LT-a homotrimer can bind to TNF-R1 and TNF-R2 and transduce signals similar to TNF. LT-b has a different receptor, LT-bR, and hence has a different biological function. 3. Interleukins Interleukins (ILs) are cytokines with a plethora of functions in the immune system, and are the major mediators of cell-mediated immunity. To date there are 29 different interleukins described in the literature. They are designated as IL-1 through IL-29. Several of these interleukins have overlapping functions, mainly because they use common receptor complexes to transduce intracellular signals in the effector cells. Based on receptor use, the interleukins can be grouped into the IL-1 family of cytokines, IL-2 family of cytokines that use receptors containing a g chain (gc), IL-3 and IL-5 family, IL-6 and gp130 family, IL-8, IL-10 family, IL-12 family, IL-13, IL-14, IL-16, IL-17 family, and IL-28 and IL-29 family of cytokines. a. IL-1 Family of Cytokines The IL-1 family of cytokines consists of four distinct but structurally related molecules: IL-1a, IL-1b, IL-1 receptor antagonist (IL-1ra), and IL-18 [177]. Since IL-1ra can bind to IL-1 receptors without activating them, IL-1ra is a natural blocking agent of IL-1–induced function on target cells [178]. Activated mononuclear phagocytes are the major cellular source of IL-1, although several activated cells other than the mononuclear phagocytes can also secrete IL-1 in response to infection, products of infectious agents (bacterial LPS, superantigens, viruses, antigens of parasites), tissue injury, inflammatory process, or specific endogenous mediators (LTs, complement fragment C5a, TNF, GM-CSF). The nature of the stimulus will determine whether IL-1 accumulates intracellularly or is secreted. Despite the fact that IL-1 is secreted, it lacks the typical signal sequence required for secretion, suggesting that IL-1 utilizes a pathway different from the classical secretory pathway used by other secreted proteins. Both IL-1a and IL-1b are synthesized as a 31-kDa–precursor protein (pro–IL-1a and pro–IL-1b), which is then proteolytically cleaved to generate the 17-kDa mature form. Most of IL-1a is stored in the cytoplasm as pro–IL-1a. Calpain and other extracellular proteolytic enzymes cleave the pro–IL-1a to the mature form. The pro–IL-1b on the other hand is


56


cleaved by an enzyme called IL-1b converting enzyme (ICE), which is not effective for converting pro–IL-1a [179]. LPS and IFN-g up-regulate the expression of ICE. Both pro–IL-1a and membranebound mature IL-1a are biologically active, whereas pro–IL-1b has no biological function. Therefore, pro–IL-1b has to be cleaved by ICE to be functional. The third member of the IL-1 family, IL-1ra, is produced mainly by tissue macrophages and keratinocytes. Two forms of IL-1ra are synthesized by these cells: an intracellular form that lacks the signal sequence (icIL-1ra) and a secreted form that shows varying levels of glycosylation and possesses a signal sequence. Keratinocytes express mainly IL-1a and icIL-1ra. IL-1 induces its effect by binding to its receptor (IL-1R), which is expressed on a number of cell types [180]. There are two distinct types of IL-1R (IL-1RI and IL-1RII) and they belong to the Ig superfamily [181]. IL-1RI is an 80-kDa transmembrane protein with a long cytoplasmic tail that participates in signal transduction. The extracellular IL-1 binding site consists of three Ig-like domains. The IL-1RII is a 60-kDa transmembrane protein that possesses similar extracellular domains but lacks the long cytoplasmic tail and is incapable of signal transduction. Therefore, IL1RII acts as a decoy receptor for IL-1 and thus down-regulates IL-1–induced functions. There is also a transmembrane IL-1 receptor accessory protein (IL-1R-AcP) that can complex with IL-1RI and can bind IL-1 and IL-1ra. Since fewer IL-1RI receptors are expressed by cells, a majority of the IL-1–induced effects are mediated through the IL-1RI/IL-1R-AcP complex. IL-1RI is expressed on T cells, endothelial cells, fibroblasts, keratinocytes, and hepatocytes, whereas IL-1RII is expressed on neutrophils, B cells, and monocytes. IL-1a binds to IL-1RI with high affinity but has low affinity to IL-1RII. IL-1b binds to IL-1RII with high affinity. IL-1ra binds to both receptors with 10- to 50-fold higher affinity than IL-1 and does not transduce any signals. Thus, IL-1ra is a potent inhibitor of IL-1 function. However, under physiological conditions, an excess of 10- to 500-fold of IL-1ra is required in the microenvironment to completely block the IL-1–mediated responses. Activated neutrophils and monocytes can shed the extracellular domain of IL-1RI and IL-1RII. The soluble IL-1R can then bind to IL-1 and block their function. Certain viruses such as vaccinia and cowpox can secrete proteins with structures similar to soluble IL-1R, and are thus capable of modulating the IL-1 function. Similarly, certain viruses can inhibit the function of ICE, thereby affecting the cleaving of pro–IL-1b. Signals transduced through IL-1R activate the nuclear transcription factors NF-kB and AP-1 that translocate to the nucleus and activate several IL-1–inducible cytokine genes for IL-6, IL-8, TNF, granulocyte-colony–stimulating factor (G-CSF), plateletderived growth factor (PDGF), IL-11, IL-2 receptor, IFN-g receptor, IL-3 receptor, ICAM-1, and other adhesion molecules on endothelial cells. IL-1 also acts as an autocrine or paracrine costimulant of early inflammatory immune responses. When released in large quantities, IL-1 can activate the fever center in the brain, induce production of leptins, stimulate the hypothalamuspituitary-adrenal axis to release several hormones, and induce production of acute-phase proteins. IL-1 acts a comitogen by up-regulating IL-2 production and IL-2 receptor expression on T cells. Increased IL-1 secretion in the thymus helps maturation and differentiation of CD4+CD8+ cells. Similarly, IL-1 can also induce maturation of B cells, resulting in augmented B-cell proliferation, surface IgM expression, and antibody production [182]. IL-1 on antigen-presenting cells is also a co-stimulator for Th2-type cells that are being activated through the T-cell receptor. IL-1 stimulates fibroblast proliferation and secretion of collagenases. In addition, IL-1 also induces cycloxygenase synthesis and prostaglandin release from fibroblasts. Intracellular IL-1b can inhibit Fas-mediated apoptosis. Thus, IL-1 plays a central role in regulating immune responses, essentially by inducing release of other mediators and cytokines. IL-18 was originally identified as an IFN-g–inducing factor [183,184]. IL-18 is expressed in a wide range of cells including Kupffer cells, macrophages, T cells, B cells, osteoblasts, keratinocytes, dendritic cells, astrocytes, and microglia [185]. IL-18 shares biological property with IL-12, including stimulation of IFN-g production, NK cell activation, and stimulation of Th1 cell differentiation. Despite their functional similarity, IL-18 is not related to IL-12. In terms of structure, IL-18 and


HUMAN IMMUNE SYSTEM

57

IL-1b share primary amino acid sequences of the so-called “signature sequence” motif and are similarly folded as all-beta–pleated sheet molecules. Also similar to IL-1b, IL-18 is synthesized as a pro–IL-18 in the cells and is then converted to mature IL-18 by the intracellular cysteine proteinase ICE [181]. The activity of mature IL-18 is closely related to that of IL-1. Several cells including macrophages produce pro–IL-18. LPS activates ICE; therefore, LPS activation of these cells may contribute to IL-18 secretion. IL-18 induces gene expression and synthesis of TNF, IL-1, FasL on NK cells, and several chemokines. IL-18 is also involved in endotoxin-induced liver injury and inhibits osteoclast formation. IL-18 induces these biological effects by binding to its receptor, IL18R complex. This IL-18R complex is made up of a binding chain termed IL-18Ralpha, a member of the IL-1 receptor family previously identified as the IL-1 receptor–related protein (IL-1Rrp), and a signaling chain, also a member of the IL-1R family. The IL-18R complex recruits the IL1R–activating kinase (IRAK) and TNFR-associated factor-6 (TRAF-6), which phosphorylates NFkB-inducing kinase (NIK) with subsequent activation of NFkB. Although IL-18 is a potent inducer of the Th1 cytokine IFN-g, recent studies show that IL-18 can also induce IL-13 from NK and T cells in the absence of IFN-g. Thus, IL-18 can promote both Th1 and Th2 cytokines depending on the microenvironment milieu. b.

IL-2 Family of Cytokines and Common Cytokine Receptor Gamma Chain

The IL-2 family of cytokines plays a major role in immune system development and modulation of lymphocyte activities during immune responses [186,187]. The IL-2 family of cytokines includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, which share the common cytokine receptor gamma chain (gc) [188–190]. The gene for gc is localized to human chromosome 13 in the region previously determined to be a locus for X-linked severe combined immunodeficiency (XSCID). Subsequently, a mutation in the gc gene was determined in human XSCID [191]. XSCID is a disease characterized by significant decrease in the numbers of T cells and NK cells, but normal numbers of nonfunctional B cells. In addition to gc, the IL-2 family of cytokines also requires a specific receptor (such as IL-2Rb, IL-4a, IL-7a, and IL-9a) for their intracellular signal transduction [192]. IL-15 shares IL-2Rb and hence both IL-2 and IL-15 have overlapping biological functions [193]. In addition, both IL-2 and IL-15 require a a chain (IL-2Ra and IL-15Ra) for higher binding affinity to respective receptors, but these a chains do not participate in signal transduction [194]. A mutation in the IL-2Ra chain can lead to increased susceptibility to bacterial, fungal, and viral infections in humans. Signals that are dependent on gc have been shown to be important for the development of the T-cell compartment, playing an important role not only in inducing expansion and preventing apoptosis of peripheral T cells, but also in supporting various stages of thymocyte development. The IL-2 family of cytokines can also act on other lineage of cells in addition to the cells of T-cell lineages [195]. IL-2 induces proliferation of T cells and augmentation of NK cell activity [196,197]. IL-2 also plays an important role in eliminating autoreactive T cells from circulation and can promote Ig synthesis by B cells. Interaction of IL-2 with its receptor complex (IL-2a/IL-2Rb/gc) signals activation of the receptor-associated tyrosine kinases, Jak1 and Jak3. These kinases in turn activate other signaling molecules such as Shc, signal transducer and activators of transcription (STAT)5a and STAT5b, which then dock to the cytoplasmic tail of IL-2Rb, triggering the Ras-Raf-Map kinasesignaling pathway [186]. Ubiquitin/proteasome-mediated degradation of STAT proteins attenuates IL-2–induced responses. IL-4 is a critical mediator that promotes differentiation of T-cell precursor cells into the Th2 type phenotype. The IL-4 gene is located on chromosome 5 (q23-31). Like IL-2, IL-4 is produced by activated CD4+cells and uses the common gc chain in its receptor complex [198]. IL-4 can also be secreted by mast cells, basophils, and keratinocytes [199,200]. IL-4 is the major B-cell growth factor and a vital cytokine for Ig class switching leading to IgE production [94]. In addition, IL4 can up-regulate class II MHC and CD23 expressions on B cells. IL-4 also has effects on


58


macrophages, fibroblasts, stromal cells, and hematopoietic precursor cells. Furthermore, IL-4 can potentiate T-cell apoptosis via IL-2–dependent mechanisms [201]. A novel cytokine-like molecule designated FISP (IL-4–induced secreted protein) is secreted by Th2 cells and requires signaling through both TCR and STAT6-dependent IL-4R [202]. IL-7 is a tissue-derived cytokine produced primarily by MHC class II+ cortical epithelial cells in the thymus and bone marrow [203]. The bone marrow–derived dendritic cells are a minor source of IL-7. Additional sources of IL-7 include keratinocytes, intestinal epithelial cells, fetal liver, dendritic cells, and follicular dendritic cells [204]. The gene for IL-7 is located on chromosome 8 (q12-13). IL-7 can bind extensively to extracellular matrix–associated glycosaminoglycan (GAG), heparin sulfate, and fibronectin. This binding allows an increase of IL-7 concentration in the microenvironment. IL-7 and transforming growth factor (TGF)-b maintain a reciprocal relationship, wherein one down-regulates the expression of the other. Like other members of the family, IL-7 transduces its intracellular signal through a receptor complex formed by its alpha chain (IL-7Ra) and the gc. IL-7 can signal through a number of nonreceptor tyrosine kinase pathways that bind to the cytoplasmic tail of IL-7Ra. These pathways include the Jak/STAT pathway, phosphatidylinositol 3-kinase pathway, and Src family tyrosine kinases. IL-7 was originally isolated as a growth factor for B-cell precursors [205]. Subsequently, IL-7 was shown to be an important cytokine for T-cell development in the thymus, for development and maturation of dendritic cells in various tissues, for modulating T-cell function and homeostasis, and as an antitumor agent by virtue of its ability to down-regulate TGF-b [206]. IL-9 is a multifunctional cytokine produced by activated memory (CD45 RO+) Th2 type T cells, eosinophils, and mast cells [207]. IL-1, IL-4, and IL-10 augment IL-9 production. The IL-9 gene is located on chromosome 5 (q31-35). IL-9 inhibits cytokine production by IFN-g–producing CD4+ cells and promotes proliferation of CD8+ T cells. IL-9 is also an eosinophil and mast cell growth factor that can promote IgE production by B cells and induce chemokine and mucus secretion by bronchial epithelial cells [208]. An important role for IL-9 in the pathology of asthma has been established due to its action on mast cells, eosinophils, neutrophils, and airway epithelium. The existence of an IL-9-mediated autocrine loop has been suggested for some malignancies such as Hodgkin’s disease. The IL-9 receptor is a member of the hematopoietin receptor superfamily and consists of a ligand-specific a subunit (IL-9Ra) and the gc [195]. Signal transduction through this receptor is dependent on Jak/STAT-1, STAT-3, and STAT-5 pathways. IL-15 is a 15-kDa cytokine expressed in a broad range of tissues and cells such as activated monocytes, dendritic cells, osteoblasts, and fibroblasts [41]. The receptor for IL-15 consists of a trimeric IL-15 receptor (IL-15R) formed by IL-2Rb, gc, and the IL-15Ra chain, which is alternatively spliced to generate three active forms that can bind IL-15 with high-affinity transducing signals through Jak1/3 and STAT3/5 [194,195,209]. Since IL-15 uses IL-2Rb, some of the functions of IL-15 overlap with that of IL-2. IL-15Ra is expressed in various tissues and cells including activated T cells [210]. Some functional activities of IL-15 overlap with those of IL-2. IL-15 is a potent initiator of the innate immune system by activating NK cells and CD8 cells [41]. In addition, IL-15 also plays a major role in the development and survival of NK cells, development of TCRgd intestinal intraepithelial lymphocytes, and functional maturation of macrophages and dendritic cells [211]. IL-15 is also important for the maintenance and proliferation of the memory phenotype of CD8+ T cells that express IL-2Rb, CD44, and Ly6C. Thus, IL-15Ra signaling is an important event in contributing to CD8+ T-cell memory responses and proliferation of NK cells [212]. IL-21 is a recently discovered cytokine that uses the common gc in the formation of its active heterodimeric receptor complex [213]. Like other cytokines in this family, IL-21 influences the proliferation of T cells and B cells, augments the cytolytic activity of NK cells, and up-regulates genes associated with innate immunity [214,215]. IL-21 may also play a role in regulating B-cell homeostasis and Ig production [216,217].


HUMAN IMMUNE SYSTEM

c.

59

IL-3 and IL-5 Family of Cytokines

IL-3 and IL-5 belong to a family of hemopoietic cytokines that includes the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) and are involved in the differentiation and activation of cells in the myeloid compartment [218]. All three cytokines are comprised of four a helices packed as a bundle, which is dimerized in the case of IL-5. These cytokines utilize a receptor complex consisting of respective a subunits and a b common chain (designated bc). The a chains of these cytokines have a unique N-terminal Ig-like domain that is not seen in other types of cytokine receptors. The a subunit binds to its cognate ligand with low affinity and is insufficient for full receptor activation by itself. The bc subunit is a member of the type 1 cytokine receptor superfamily that contains conserved extracellular domains called cytokine receptor modules (CRMs). Each CRM consists of two repeats of a fibronectin type III–like domain. Each domain is made up of approximately 100 amino acids and 7 b strands with intervening loops arranged into a b barrel structure. These repeats carry two sets of conserved motifs typical of this family of receptors. The first repeat contains four cysteines with conserved spacing, while the second repeat contains a WSXWS motif. Binding of IL-3, IL-5, and GM-CSF to their respective receptors results in the activation of Jak2 and phosphorylation of tyrosine and serine residues of the bc cytoplasmic tail [219]. Disulfide-mediated dimerization of the signaling units is unique to this family of cytokines. Eosinophils express receptors for all three cytokines, whereas monocytes express only receptors for GM-CSF and IL-3. Thus, all three cytokines can activate eosinophils, but IL-5 cannot activate monocytes. These cytokines thus contribute to inflammatory diseases such as atopic dermatitis, allergic rhinitis, and asthma. Subjects with low levels of bc expression on peripheral blood cells exhibit a condition called “pulmonary alveolar proteinosis” characterized by reduced numbers of eosinophils in the lungs. IL-3, IL-5, and GM-CSF are produced by activated T cells. IL-3 is a multilineage hemopoietic regulator that promotes the survival, proliferation, and development of a wide variety of myeloid progenitors such as hematopoietic stem cells and committed progenitor cells of the granulocyte-macrophage, erythrocyte, eosinophil, basophil, megakaryocyte, mast, and lymphocyte lineage cells [220]. Because of these functions, IL-3 is therapeutically useful in primary marrow disorders, including myelodysplastic syndromes and aplastic anemia [221]. IL-3 also enhances phagocytosis and cytotoxic function of myeloid cells and promotes activation of basophils and eosinophils. In addition, IL-3 potentiates IL-2–dependent growth of normal T cells and IL-2–dependent secretion of IgG by activated B cells [222]. IL-5 is the predominant cytokine that acts on eosinophils [223]. The pro–eosinophilic effects of IL-5 include enhanced replication and differentiation of eosinophilic myelocytes, enhanced degranulation of eosinophils, prolonged survival time of eosinophils, and enhanced adhesion of eosinophils [224]. Thus, blocking of IL-5 can have significant advantages therapeutically to block atopic conditions mediated by eosinophils [225]. GM-CSF is an 85-kDa glycoprotein that interacts with stem cells to differentiate and promote clonal development of granulocyte and dendritic cell progenitors [226]. Activated T cells, B cells, macrophages, mast cells, endothelial cells, and fibroblasts produce GM-CSF in response to a variety of stimuli. Receptors of GM-CSF are expressed on the surface of monocytes, neutrophils, eosinophils, granulocyte progenitors, and fibroblasts, and on endothelial cells. GM-CSF thus essentially functions as a survival factor for hematopoietic progenitor cells. Binding of GM-CSF to its receptor on phagocytes activates these cells to enhance their phagocytosis. In addition, GM-CSF mediates ADCC reaction and stimulates secretion of IL-1 and TNF. Clinically, GM-CSF is administered to ameliorate neutropenia following cancer chemotherapy and bone marrow transplantation. d. IL-6 and gp130 Family of Cytokines The IL-6 family of cytokines consists of IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and a novel


60


neurotropin-1/B-cell stimulatory factor 3 (NNT-1/BSF-3). These cytokines are structurally and functionally related and share gp130 as a critical component in their receptor complexes for signal transduction [227,228]. The gp130 molecule has no IL-6 binding capability by itself but plays an important role in the formation of high-affinity IL-6 binding sites by associating with the IL-6/IL-6R complex in the transduction of IL-6 signals [229]. The gp130 also forms a similar receptor complex with the other IL-6 family of cytokines [230]. Cytoplasmic signaling through gp130 is mediated by tyrosine phosphorylation of STAT by Jak kinases [231,232]. IL-6 was originally identified as a factor that stimulates and differentiates B cells to produce Ig. Now we know that IL-6 is a pleiotropic cytokine with a wide range of biological functions. These include inducing proliferation of megakaryocyte progenitors, differentiation of macrophages and cytotoxic cells, stimulation of hepatocytes, induction of expression of various acute-phase proteins, differentiation of neuronal cells, and stimulation of secretion of anterior pituitary hormones [231]. Because of its effect on acute-phase proteins, IL-6 also increases body temperature. Other cytokines in the IL-6 family have biological functions that overlap those of IL-6. This functional redundancy is due to gp130, which is an essential component of the receptor complex of all members of the IL-6 family of cytokines [233]. A secretory form of IL-6 receptor (sIL-6R) is present in healthy human sera at a concentration of approximately 80 ng/ml. Since sIL-6R can promote formation of osteoclast-like cells, an increase in circulating sIL-6R is associated with increased bone absorption as seen in osteolytic diseases such as multiple myeloma and juvenile rheumatoid arthritis [234,235]. Similarly, clinical studies have shown that the circulating levels of IL-6 and other cytokines in the IL-6 family are increased in patients with congestive heart failure [236]. IL-11 is a multifunctional cytokine that uses gp130 and has activities on a broad range of hematopoietic cells including primitive stem cells and mature progenitor cells [237,238]. IL-11 supports the growth of colony-forming units in megakaryocytes. Administration of IL-11 increases two- to three-fold circulating platelets, stimulates bone marrow and spleen megakaryocyte progenitor numbers, and enhances megakaryocyte maturation [239]. Thus, IL-11 is therapeutically highly effective for myelosuppression and thrombocytopenia associated with cancer chemotherapy and bone marrow transplantation. IL-11 has potent anti-inflammatory activity and can suppress TNF and IL-12 expression in activated macrophages [238]. IL-11 down-regulates glucocorticoid receptors in a dose-dependent fashion, whereas IL-6 up-regulates the expression of glucocoticoid receptors. Similarly, IL-1b up-regulates IL-11 secretion. LIF and OSM are closely related cytokines in both function and structure [240,241]. LIF was first identified as a factor that inhibits the growth of leukemia cell lines, and OSM was identified as a factor that inhibits growth of human melanoma cells [242]. Several cells, including lymphocytes, monocytes, and bone marrow stromal and osteoblastic cells can produce LIF in addition to certain malignant cells. Osteoblasts appear to be the major target of LIF and OSM. Both LIF and OSM induce an anti-apoptotic effect on these cells and promote proliferation and differentiation [243]. Their effects on osteoclasts are not fully understood but both can activate osteoclasts and cause hypercalcemia. LIF is important for embryo implantation [244]. Lack of the LIF gene can lead to female infertility [245]. LIF and OSM induce their effect by binding to gp130-associated receptors [246]. In addition, LIF and OSM also bind to a low-affinity receptor, LIFR, whose structure is closely related to that of gp130. LIFR then becomes heterodimerized with gp130 to form the high-affinity and signaling-competent complex. OSM can also bind to OSM-specific receptor component (OSMR) to form a heterodimer complex with gp130 [247]. The LIFR/gp130 complex is also utilized by CNTF, CT-1, and NNT-1/BSF-3 to transduce their signals. However, these cytokines have to bind to the a chain of their respective receptors such as CNFR and CT-1R. Although gp130 and LIFR are expressed in nearly all organs, expression of the a chain of IL-6R, IL-11R, CNTFR, and CT-1R is limited, suggesting that the cellular responsiveness to these cytokines is largely determined by the regulated expression of their specific receptor chains. Inflammatory responses can up-regulate the expression of the respective receptor a chains.


HUMAN IMMUNE SYSTEM

61

CNTF enhances the survival of neuronal cells and has been investigated as a therapeutic agent for motor neuronal diseases [248]. CNTF is also shown to have a promising role in the treatment of obesity and diabetes, and is recognized as a major protective factor in demyelinating central nervous system diseases [249]. To exert its biological effect, CNTF has to bind to its nonsignaling specific receptor, CNTFR, which then associates with the gp130/LIFR complex to induce the Jak/STAT signaling pathway [250]. CT-1 is a cytokine that induces cardiac myocyte hypertrophy [251]. Serum levels of CT-1 are increased in patients with chronic heart failure. Cardiac myocytes and fibroblasts are a major source of CT-1. The CT-1 can also induce angiotensinogen expression in cardiac myocytes [252]. These functions of CT-1 are regulated by its binding to the gp130 receptor complex [253]. Thus, gp130 appears to play an important role in cardiomyocyte regulation, including survival and proliferation during development and induction of hypertrophy after birth [254]. NNT-1/BSF-3 is a new addition to the IL-6 family of proteins that uses the LIFR/gp130 complex to transduce its signal [244]. NNT-1/BSF-3 was identified first in the activated human Jurkat T-cell lymphoma cells. Similar to other IL-6 members, the NNT-1/BSF-3 also has a neurotropic effect in addition to being a potent B-cell stimulator to produce IgG and IgM. e. IL-8 IL-8 was originally discovered as a neutrophil chemotactic factor in the supernatants of activated human monocytes [255]. IL-8 is the founding member of the CXC chemokine superfamily [256]. One of the major biological functions of IL-8 is that it is an activator and chemoattractant for neutrophils. Expression of IL-8 considerably varies. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by a factor of 10 to 100 in response to cellular stress, proinflammatory cytokines such as TNF or IL-1, and microbial agents such as bacteria and viruses [257,258]. IL-8 is also frequently expressed by tumor cells. In fact, IL-8 expression was first demonstrated at high levels in human melanomas [259]. Subsequently, increased IL-8 expression has been found in several tumors such as acute myelogenous leukemia, B-cell chronic lymphocytic leukemia, brain tumors, breast cancer, colon cancer, cervical cancer, gastric cancer, Hodgkin’s disease, lung cancer, mesothelioma, ovarian cancer, pituitary adenomas, prostrate cancer, renal cell carcinoma, pancreatic tumors, and thyroid tumors. IL-8 has been shown to be motogenic (chemotactic effect), mitogenic, and angiogenic, and thus plays an important role in human tumor progression by an autocrine loop. IL-8 expression can be induced by numerous stress factors present in the tumor environment, such as hypoxia, acidosis, hyperglycemia, hyperosmotic pressure, high cell density, hyperthermia, radiation, and chemotherapeutic agents. Because of its critical role in tumor metastasis, IL-8 is one of the major targets for cancer treatment [260]. IL-8 also plays an important role in the pathophysiology of pulmonary diseases [261,262]. The receptor for IL-8 is CXCR2, which is expressed on various cells including macrophages [258]. f.

IL-10 Family of Cytokines

The IL-10 family of cytokines consists of IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26 [263]. The genes for IL-19, IL-20, and IL-24 are clustered together with IL-10 on chromosome 1, whereas the genes for IL-22 and IL-26 are located close to the IFN-g genes on chromosome 12. Originally discovered as an inhibitory factor for the production of Th1 type cytokines, IL-10 is a pleiotropic inhibitor of several cell types. Subsequent studies showed that IL-10 can also act as a survival and differentiation factor for B cells. Thus, IL-10 can function both as an immunosuppressive and immunostimulatory cytokine. IL-10 is produced by a variety of cells including activated monocytes, T cells, and keratinocytes. The existence of a family of IL-10 cytokines came from studies analyzing the genome of several viruses such as human g1-herpesvirus, Epstein–Barr virus (EBV), monkey yatapoxvirus, and human and simian cytomegaloviruses. The genome of these viruses contains


62


proteins that show functional sequence homology to human IL-10. Furthermore, these viral IL-10 homologues were shown to bind to IL-10 receptors and mediate IL-10 functions. Using subtractive cDNA hybridization with these sequences, five human paralogues of IL-10 have been discovered that show 20% to 83% sequence homology to human IL-10. The IL-10 family of cytokine consists of the melanoma differentiation-associated antigen (designated as IL-24); IL-10 related T-cell–derived inducible factor (IL-22); AK155 (IL-26); IL-19; and IL-20. IL-19 is expressed by activated monocytes [264]. Although the cellular source of IL-20 is not established yet, IL-20 appears to have a significant role in the development of psoriatic lesions. IL-22 is produced by CD4+ cells, and this cytokine mediates acute-phase response signals in hepatocytes [264]. IL-24 is expressed by differentiated melanoma cells and Th2 type lymphocytes [265]. IL-24 has antitumor activity. IL-26 is highly expressed by T cells transformed with herpes virus. IL-10 and IL-24 have significant therapeutic potential as immunosuppressive agents for organ transplantation and tumor therapy [264]. The three-dimensional structure of IL-10 shows that the molecule forms V-shaped dimers and each arm of the dimer consists of six a helices, four originating from one subunit and two from the other subunit [263]. Four of the a helices form a bundle similar to other helical cytokines such as IL-4 and GM-CSF. The overall topology of IL-10 closely resembles that of IFN-g, suggesting a close relationship between these two cytokines. The IL-10 family of cytokines exerts effects by forming a complex with the IL-10 receptors (IL-10R). The IL-10R consists of a longer chain, IL-10R1, which is the major signaling component of the receptor, and a shorter membrane-spanning receptor chain, IL-10R2 (also called cytokine receptor family 2, CRF2) with a short intracellular segment. Dimeric IL-10 molecules first bind to the IL-10R1 with high affinity and subsequently, IL-10R2 binds to the IL-10/IL-10R1 complex with low affinity to form the IL-10/IL-10R1/IL10R2 complex. Other members of the family also form similar complexes by binding to CRF2. Thus, IL-20 binds to IL-20R1 and IL-20R2 on the surface of keratinocytes, IL-22 binds to the long chain of IL-10R (IL-10R1) and IL-22 binding protein (also called IL-22RA2), and IL-19 and IL-24 use IL-20R1/IL-20R2 heterodimers. Similarly, IL-20 and IL-24 can bind to the IL-10R1/IL22RA2 and IL-10R1/IL-20R2 complexes. Thus, CRF2 receptors show certain degree of promiscuity for binding of the IL-10 family of cytokines. Signaling through this receptor complex activates Jak kinases and STAT-dependent transcription of genes that encode suppression of cytokine signaling. g. IL-12 Family of Cytokines The IL-12 family of cytokines consists of the two subunits of IL-12 (p35 and p40), IL-23 and IL-27 [266]. IL-12 is a heterodimeric cytokine composed of two subunits, p35 and p40. P35 is constitutively expressed by a number of cells. The p40 chain exists as a soluble monomer, a homodimer (with another p40), or as a heterodimer (with p35 or other proteins). For expression of IL-12 (IL-12p70 or IL-12p75), both p35 and p40 subunits have to express in the same cell. Activated macrophages and dendritic cells secrete significant amounts of monomeric and homodimeric p40. The heterodimer formed by p40 and a p35-related protein p19 is designated as IL-23. The heterodimer formed by a p40-related protein plus a p28 protein is designated as IL-27. All these proteins critically influence the induction and maintenance Th1 type responses. IL-12 is an immunoregulatory cytokine that promotes cell-mediated immunity, specifically by promoting secretion of IFN-g from NK cells [267]. The p35 subunit of IL-12 has significant homology to the IL-6 family of cytokines, and the p40 subunit has homology to the extracellular domain of IL-6R1a and CNTFR. The biological function of IL-12 is mediated by the high-affinity receptor IL-12R, which is formed of two subunits b1 and b2. The IL-12R is a member of the class I cytokine receptor family and is closely related to the glycoproteins gp130 and LIFR. NK cells and T cells can express both the subunits of IL-12R and are required for IL-12 bioactivity. The homodimeric p40 subunit acts as an antagonist for IL-12 bioactivity by binding to the b1 subunit


HUMAN IMMUNE SYSTEM

63

of the IL-12R. Homodimeric p40 is also an efficient chemoattractant for macrophages and can promote inducible nitric oxide synthetase (iNOS) expression in these cells. The proinflammatory function of IL-12 as well as its ability to stimulate the Th1 type response play an essential role in the defense against many intracellular protozoa, and fungal and bacterial pathogens. Antigens from microbial agents induce primarily IL-12p40. Subsequent cross-linking of CD40 on antigen-activated dendritic cells by CD40L-expressing T cells amplifies IL-12 production by increasing the synthesis of IL-12p35. As mentioned above, IL-23 consists of the p40 subunit and a p19 protein [268]. The p19 protein has considerable sequence homology to p35 and is functionally inactive by itself. IL-23 binds to IL-12Rb1 and a b2-like receptor subunit designated as IL-23R for its biologic function. The p19 component is produced in large amounts by activated macrophages, dendritic cells, T cells, and endothelial cells. Th1 cells express larger amounts of p19 than Th2 cells. However, only activated macrophages and dendritic cells concomitantly express IL-12 p40, the other molecule required for IL-23 signal transduction. The p19 expression is up-regulated by bacterial products that signal through toll-like receptors 2 and 4. IL-23 induces proliferation of memory T cells, and production of IFN-g from memory and naïve T cells. IL-23 may play a significant role in eczematous skin diseases. IL-27 is a heterodimeric protein that consists of EBV-induced gene 3 (EBI3), a p40-related protein, and p28, a polypeptide related to IL-12p35 [269]. Monocytes, macrophages, fetal cells, and placental trophoblasts express EBI3. IL-27 appears to be produced early by APCs. IL-27 can thus induce clonal proliferation of naïve but not memory CD4+ T cells, and can augment IL-12-induced IFN-g production by naïve CD4+ T cells. The receptor for IL-27 appears to be a recently identified orphan receptor TCCR that is shown to play an important role in the early initiation of Th1 type responses, but is not required for the maintenance of Th1 responses. Therefore, it is possible that IL-27 and IL-12 act sequentially in generating and maintaining the Th1 responses. h. IL-13 IL-13 is a key mediator in the pathogenesis of allergic inflammation, and is produced by activated Th2 cells [270]. The IL-13 gene is located on chromosome 5 (q31). Subsequently, IL-13 was shown to up-regulate MHC class II expression on B cells and monocytes, promote IgE class switching in B cells, and inhibit inflammatory cytokine production. Thus, IL-13 was originally thought to be functionally redundant with IL-4. However, several recent studies suggest that IL-13 is a pleotropic cytokine with several unique biological functions. IL-13 plays a significant role in resistance against bacteria, intracellular protozoa, and helminth parasites. In addition, IL-13 is an anti-apoptotic cytokine that promotes tumor growth. IL-13 has a central role in the pathogenesis of asthma by virtue of its ability to activate eosinophils, induce mucus secretion, and cause airway hyperreactivity. IL-13 can up-regulate extracellular matrix, thereby promoting tissue fibrosis. Thus, IL-13 is a potent inducer of tissue fibrosis in schistosomiasis and asthma. To transduce signals, IL-13 first binds to its high-affinity receptor formed by IL-4Ra and IL13Ra1 [271]. This heterodimer then binds to the decoy receptor IL-13Ra2 to form the receptor–ligand complex. IL-13Ra1 is expressed on a wide variety of cells such as B cells, basophils, eosinophils, mast cells, epithelial cells, endothelial cells, fibroblasts, monocytes, smooth muscle cells, and macrophages. However, functional IL-13R has not been demonstrated on T cells. Thus, unlike IL-4, IL-13 does not activate CD4+ T cells. IL-13Ra1 and a2 expression are up-regulated by IL-3, IL-4, and IL-10, whereas IFN-g down-regulates the expression of IL-13R. i.

IL-14

IL-14 is a high-molecular-weight B-cell growth factor produced by malignant B cells and T cells [272]. IL-14 can induce proliferation of activated B cells, stimulate resting B cells, and induce


64


synthesis or inhibition of antibodies by B cells [273]. IL-14 shares various functional activities with IL-4 such as long-term growth of B cells and increasing cAMP in B cells. IL-14 induces this effect by binding to its receptor IL-14R. Peripheral blood B cells and T cells from patients with systemic lupus erythematosus (SLE) and B-cell type non-Hodgkin’s lymphoma spontaneously produce significant amounts of IL-14 that may contribute to disease pathology. j.

IL-16

IL-16 is a 14-kDa cytokine initially described as a T-cell chemoattractant [274,275]. Subsequent studies show that IL-16 is a chemoattractant for a variety of CD4+ immune cells [276]. IL-16 acts as an immunoregulator for selective trafficking of CD4+ cells into the inflammatory immune site. IL-16 expression is up-regulated during asthma and autoimmune diseases [277,278]. k.

IL-17 Family of Cytokines

IL-17 is a 17-kDa proinflammatory cytokine produced and released by CD45RO+ memory CD4+ T cells and, under certain conditions, from CD8+ T cells [279,280]. In the lungs, eosinophils are the major source of IL-17 during asthma. IL-17 expression is elevated in rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, and transplantation rejection [281,282]. IL-17 is characterized by the presence of four cysteines in its protein structure. Cytokines with similar structures are grouped under the IL-17 family of cytokines [283]. There are four such cytokines in the IL-17 family of cytokines: IL-17B, IL-17C, IL-17F, and IL-25. IL-17 induces its biological functions by binding to IL-17R, which is expressed in nearly all cells. IL-17 shares transcriptional pathways similar to IL-1 and TNF. P38 and NF-kB are the key transcriptional factors for IL-17 function and require toll-like receptor 4 signaling. IL-17 can promote the production of TNF, IL-1b, IL-6, IL-8, and G-CSF. IL-17B and C, however, do not bind to IL-17R, and only promote TNF and IL-1b production. IL-17F is produced by memory T cells in patients with asthma and contributes to the pathology of asthma. IL-17F can also activate bronchial epithelial cells to release the neutrophil chemoattractant IL-8 and neutrophil-activating factor IL-6. In addition, IL-17F can induce IFN-g in the lung. Local production of IL-17 is a critical event in the host defense against gram-negative bacteria. IL-25 is a cytokine with significant sequence homology to IL-17 [284]. However, in the lungs, IL-25 promotes production of Th2 cytokines, IL-4, IL-15, IL-13, and eotaxins. The source of these IL-25–induced cytokines in the lungs appears to be mast cells [285]. By promoting Th2 cytokine secretion, IL-25 is contributing to the pathology of asthma by inducing eosinophil infiltration, mucus production, and airway hyperreactivity reactions [286]. IL-25 expression is also significantly increased in the gut and lungs during gastrointestinal parasitic infections. l.

IL-28 and IL-29 Family of Cytokines

This family consists of three cytokines: IL-28A, IL-28B, and IL-29 [287]. These cytokines represent an evolutionary link between IL-10 and the type 1 IFN family of cytokines. At the amino acid level, IL-28 and IL-29 are related to type I IFN, whereas at the genomic structure level, IL-28 and IL-29 are more similar to members of the IL-10 gene family. Like type 1 IFN, IL-28 and IL29 are induced by viral infection and show antiviral activity. However, for signal transduction, both IL-28 and IL-29 use the heterodimeric class II cytokine receptor that consists of IL-10Rb and an orphan class II receptor chain designated IL-28Ra. Therefore, IL-28 and IL-29 serve as an alternative to type 1 IFNs in providing immunity to viral infections.


HUMAN IMMUNE SYSTEM

65

4. Growth Factors a. Transforming Growth Factor Transforming growth factor (TGF)-b is a 25-kDa multifunctional cytokine with proinflammatory and immunosuppressive activities [288]. TGF-b is a potent stimulator of cell growth and is produced by a variety of cells including tumor cells. Many cells, including platelets, macrophages, B cells, T cells, fibroblasts, epithelial cells, endothelial cells, astrocytes, microglial cells, osteoblasts, and osteoclasts express TGF-b constitutively. The three major isomeric forms of TGF-b in humans — TGF-b1, b2 and b3 — are induced by a variety of agents, including steroids, oncogenes, epidermal growth factor, nerve growth factor, and IL-1. TGF-b2 and b3 are involved in embryogenesis, whereas TGF-b1 has several immunological functions. The secreted form of TGF-b is biologically inactive. Enzymes such as plasmin, cathepsin D, thrombospondin, or heat activation and low pH can activate TGF-b. Activated TGF-b then binds to specific receptors expressed universally on the surface of cells except on some neoplastic cells. Three classes of TGF-b receptors have been identified and they are designated as type I, type II, and type III TGF-bR. Circulating TGF-b has a plasma half-life of 2 to 3 seconds. Despite the short half-life, the effects of TGF-b on various tissues are remarkable, the most important of which is its effect on extracellular matrix. TGF-b increases synthesis and secretion of matrix proteins that are important in cell migration, wound healing on soft and hard tissues, embryogenesis, carcinogenesis, and fibrotic diseases in various organs. Despite its potent stimulatory activity, TGF-b suppresses the growth of all lymphocyte lineage cells and thus mediates immunosuppression. b.

Stem Cell Factor

Stem cell factor is a growth factor that promotes growth and differentiation of hematopoietic cells, melanocytes, and mast cells. Stem cell factor is produced by cells in the bone marrow, liver, lung, kidney, brain, placenta, testis, and fibroblasts. The receptor for stem cell factor is a c-kit protooncogene that has tyrosine kinase activity. IL-3, GM-CSF, and erythropoietin modulate expression of c-kit. c.

Leukemia Inhibitory Factor

Leukemia inhibitory factor (LIF) is a cytokine that promotes differentiation of embryonic stem cells, hepatocytes, adipocytes, neurons, and some hematopoietic cells [246]. Alloreactive T cells, and certain tumor cells can produce LIF. Clinically, LIF is useful in differentiation of cells, stimulation of platelet formation, and enhanced local bone healing. d. Platelet-Derived Growth Factor Platelet-derived growth factor (PDGF) is a cytokine released from platelets that stimulates proliferation and viability of a variety of cells. PDGF is synthesized as a heterodimeric protein in megakaryocytes that is stored in platelet a granules and released from platelets at the site of blood vessel injury [289]. Serum levels of PDGF range from 15 to 60 ng/ml in normal individuals. Gastric epithelial cells can secrete PDGF-like peptides that promote stromal cell growth through paracrine mechanisms [290]. PDGF receptors are expressed on fibroblasts, and binding of PDGF to its receptor induces tyrosine kinase activity leading to several PDGF-inducible genes such as JE, KC.


66


J. Chemokines In order to participate in an acute or chronic inflammatory immune process, a great majority of the cells in the immune system must emigrate out of the circulation. Initial events that promote these processes include various integrins and their ligands that cause tethering (rolling), which leads to firm adhesion of the immune cells to the endothelial cells in areas where inflammatory immune responses are occurring in tissue. The immune cells are then drawn to the site of action from the endothelial surface via several factors, of which the chemokines are an important component. Thus, chemokines are chemical agents that attract cells (or chemoattractants) to a specific site. Unlike other chemoattractants that have a broad spectrum of activity, the chemotactic activity of chemokines is cell type specific. In addition to targeted trafficking, chemokines also promote angiogenesis, lymphopoiesis, and hematopoiesis. Chemokines are produced by a wide variety of cells including leukocytes in response to irritants, mitogens, endogenous cytokines, and antigens. Chemokines belong to a large supergene family of low-molecular-weight (8 to 10 kDa), heparinbinding proteins [162,291]. More than 50 chemokines have been reported to date and the list is growing (Table 3.1). Based on unique sequence homology and position of cysteine residues, chemokines have been divided into four subfamilies CXC (a), CC (b), C (g), and CX3C. The CXC subfamily comprises of nearly 15 chemokines designated CXCL1 through 15. Except for CXCL12, all CXC genes are located on chromosome 4 (q12-21). CXC chemokines have four conserved cysteine residues in their sequences; of these the first two cysteine residues are separated by a single amino acid (X); hence the name. The CC chemokine subfamily consists of a large number of chemokines; 11 are located on chromosome 7 (q11-21), and others are located on chromosomes 2, 9, and 16. The CC chemokines have their first two cysteine residues close together without any amino acid residues between them. Disufide bonds formed between cysteine residue 1 and 3 and between 2 and 4 provide a stable tertiary structure to the chemokine molecules. The C chemokine subfamily consists of a 16-kDa protein XCL1 whose gene is located on chromosome 11. XCL1 has only two cysteine residues (1 and 3) located near the amino terminus. The CX3C chemokine subfamily has a single 38-kDa protein, CX3CL1, whose gene is located on chromosome 16. CX3CL1 has three amino acids between the first two cysteine residues. CC and CXC chemokines are produced by a wide array of cell types. The CC chemokines attract monocytes, dendritic cells, eosinophils, lymphocytes, and NK cells, whereas CXC chemo ines preferentially attract neutrophils besides lymphocytes and NK cells. XCL1 chemokine is produced by NK cells and is chemoattractive for NK cells and T cells. The CXCL1 has a hydrophobic membrane-anchoring motif at its carboxyl terminal, with which the molecule attaches to endothelial cells. Enzymatic cleaving of the amino termini produces a soluble form of CXCL1 that is 80 amino acids long. The soluble form has potent chemotactic activity for T cells, monocytes, and activated NK cells. The membrane bound form helps in the adhesion of these leukocytes to the endothelium. The carboxyl terminus of chemokines possesses a low-affinity heparin-binding property that allows the chemokines to bind to GAG and other negatively charged sugar molecules on the surface of cells and to tissue matrix glycoproteins. This property allows the chemokines to be adsorbed to endothelial surface, connective tissue, and extracellular matrices, where it acts as a focus for attracting cells that roll along the GAG-coated surfaces. Chemokines bind with high affinity to a subfamily of homologous seven-transmembrane G-protein–coupled receptors that constitute the chemokine receptors. As the list of chemokines is expanding, newer receptors are being identified. However, a great majority of chemokines share receptors. Thus, there are six receptors (designated CXCR1 through 6) identified for the CXC subfamily of chemokines, 11 receptors (designated CCR1 through 11) for CC subfamily of receptors, and one receptor each for C chemokines (designated as XCR1) and for CX3C chemokines (designated as CX3CR1). Chemokine receptors are expressed on the surface of leukocytes and are regulated by exogenous and endogenous stimuli. Generally, activated cells express greater number of these receptors than resting cells. Similarly, one cell can express more than one type of receptor, thus


HUMAN IMMUNE SYSTEM

67

Table 3.1 Chemokines and Their Receptors Chemokines

Receptors C — Chemokines

XCL1 (Lymphotactin a) XCL2 (Lymphotactin b)

XCR1 XCR1

CC — Chemokines CCL1 (TCA-3) CCL2 (MCP-1) CCL3 (M1P-1a) CCL4 (MIP-1b) CCL5 (RANTES) CCL6 (C10) CCL7 (MCP-3) CCL8 (MCP-2) CCL9/10 (MIP-1g) CCL11 (Eotaxin) CCL12 (MCP-5) CCL13 (MCP-4) CCL14 (HCC-1) CCL15 (HCC-2) CCL16 (HCC-4) CCL17 (TARC) CCL18 (MIP-4) CCL19 (MIP-3b) CCL20 (MIP-3a) CCL21 (ECkine) CCL22 (MDC) CCL23 (MPIF-1) CCL24 (Eotaxin-2) CCL25 (TECK) CCL26 (Eotaxin-3) CCL27 (CTACK)

CCR8 CCR2, 10, 11 CCR1, 5 CCR5, 8 CCR1, 3, 5, 11 Not identified CCR1, 2, 3, 10, 11 CCR2, 3, 11 CCR1 CCR3 CCR2 CCR2, 3, 11 CCR1 CCR1 Not identified CCR4, 8 Not identified CCR7 CCR6 CCR7 CCR4 CCR1 CCR3 CCR9 CCR3 CCR10 CXC — Chemokines

CXCL1 (GROa) CXCL2 (GROb) CXCL3 (GROg) CXCL4 (PF-4) CXCL5 (ENA-78) CXCL6 (GCP-2) CXCL7 (NAP-2) CXCL8 (IL-8/NAP-1) CXCL9 (MIG) CXCL10 (IP-10) CXCL11 (H174/IP-9) CXCL12 (SDF-1/PBSF) CXCL13 (BLC/BCA-1) CXCL14 (BRAK) CXCL15 (Lungkine)

CXCR1, 2 CXCR2 CXCR2 Not known CXCR2 CXCR1, 2 CXCR2 CXCR1, 2 CXCR3 CXCR3 CXCR3 CXCR4 CXCR4, 5 Not known Not known

CX3C — Chemokine CX3CL1 (Fractalkaine)

CX3CR1

Note: Old names for respective chemokines are in parentheses.


68


having the ability to respond to different chemokines. Signal transduction through chemokine receptors requires an initial coupling with G proteins, a large gene family coding for at least 16 a subunits, four b subunits and multiple g subunits. Uncoupling of G protein inhibits chemokine receptor-mediated responses. Binding of a chemokine to its G-protein coupled receptor initiates the cascade of cell signaling, resulting in an increase in intracellular calcium, actin polymerization, reconfiguration of adhesion proteins, and other cellular responses contributing to cell migration. Many pathogens use the chemokine receptors to their advantage, such as the malaria parasite (Plasmodium vivax), and certain viruses (HIV-1, CMV) use chemokine receptors for entering the cell. Similarly, the human herpes virus up-regulates CXCR2 and binding of CXC to this receptor, which promotes angiogenesis. CXC chemokines can be divided into two groups based on the presence or absence of the amino acid residues Glu-Leu-Arg (ELR) at positions 4, 5, and 6 near the amino termini. This ELR sequence binds to CXCR2 receptors expressed on neutrophils, thus preferentially attracting them to the inflammatory loci. At higher concentrations, these ELR+ chemokines can activate the neutrophils to release oxygen radicals and their granular contents, thus amplifying the inflammatory responses. ELR- chemokines are not chemotactic for neutrophils, but are chemotactic for monocytes and T lymphocytes. Thus, chemokines and their receptors play an important role in both innate and adaptive immune responses.

IV. SUMMARY The immune system is the sentinel that constantly maintains vigilance over the various portals of the body and destroys microbes and foreign molecules or substances that gain access. To perform these functions, the immune system has developed a sophisticated network of cells and complex molecules that interact with the foreign substance or with each other to elicit their functions. There are two principal pathways of defense orchestrated by the immune system. The defense mechanism that functions nonspecifically toward the foreign substance is called the innate immunity, and the defense mechanism reacting very specifically toward the foreign substance is called the adaptive immunity. Participants of innate immunity include phagocytes, neutrophils, NK cells, eosinophils, mast cells, basophils, and complement. These participants are called for action early in the process of an inflammatory/immune response and are thus vital for maintaining health. Molecules and cells that participate in the adaptive immunity are highly adapted for variability. This adaptive property allows them to modify their artillery to suit the need. Participants in the adaptive immune system include B lymphocytes and their specific immunoglobulins and antibodies, T lymphocytes (especially CD4 and CD8 cells), and an extensive network of mediators that includes the cytokines, chemokines, and growth factors. These cells and mediators of adaptive immunity destroy and eliminate foreign molecules and substances. A remarkable feature of adaptive immunity is memory. Both innate and adaptive immunity rely on their ability to recognize and differentiate self from non-self. Dysfunction in this recognition can lead to tolerance, self-destruction, or autoimmunity.

REFERENCES 1. Bos, J.D., The skin as an organ of immunity, Clin. Exp. Immunol., 107, 3, 1997. 2. Bogdan, C. et al. The role of nitric oxide in innate immunity, Immunol. Rev., 173, 17, 2000. 3. Curry, J.L. et al., Innate immune-related receptors in normal and psoriatic skin, Arch. Pathol. Lab. Med., 127, 178, 2003. 4. Gallo, R.L. et al., Biology and clinical relevance of naturally occurring antimicrobial peptides, J. Allergy Clin. Immunol., 110, 823, 2002. 5. Sallenave, J.M., Antimicrobial activity of antiproteinases, Biochem. Soc. Trans., 30, 111, 2002.


HUMAN IMMUNE SYSTEM

69

6. Bos, J.D. et al., Defensins and complement systems from the perspective of skin immunity and autoimmunity, Clin. Dermatol., 19, 563, 2001. 7. Schroder, J.M. and Harder, J., Human beta-defensin-2, Int. J. Biochem. Cell Biol., 31, 645, 1999. 8. Dorschner, R.A. et al., Neonatal skin in mice and humans expresses increased levels of antimicrobial peptides: innate immunity during development of the adaptive response, Pediatr. Res., 53, 566, 2003. 9. Di Nardo, A. et al., Cutting edge: mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide, J. Immunol., 170, 2274, 2003. 10. Fellermann, K. et al., Antimicrobial peptides in the skin, N. Engl. J. Med., 348, 361, 2003. 11. Underhill, D.M. and Ozinsky, A., Phagocytosis of microbes: complexity in action, Annu. Rev. Immunol., 20, 825, 2002. 12. Medzhitov, R. and Janeway, C., Jr., Innate immune recognition: mechanisms and pathways, Immunol. Rev., 173, 89, 2000. 13. East, L. and Isacke, C.M., The mannose receptor family, Biochim. Biophys. Acta., 1572, 364, 2002. 14. Apostolopoulos, V. and McKenzie, I.F., Role of the mannose receptor in the immune response, Curr. Mol. Med., 1, 469, 2001. 15. Ehlers, M.R., CR3: a general purpose adhesion-recognition receptor essential for innate immunity, Microb. Infect., 2, 289, 2000. 16. Reichert, F. and Rotshenker, S., Complement-receptor-3 and scavenger-receptor-AI/II mediated myelin phagocytosis in microglia and macrophages, Neurobiol. Dis., 12, 65, 2003. 17. Taborda, C.P. and Casadevall, A., CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are involved in complement-independent antibody-mediated phagocytosis of Cryptococcus neoformans, Immunity, 16, 791, 2002. 18. Le Cabec, V. et al., Complement receptor 3 (CD11b/CD18) mediates type I and type II phagocytosis during nonopsonic and opsonic phagocytosis, respectively, J. Immunol., 169, 2003, 2002. 19. Gough, P.J. and Gordon, S., The role of scavenger receptors in the innate immune system, Microbes Infect., 2, 305, 2000. 20. Heine, H. and Lien, E., Toll-like receptors and their function in innate and adaptive immunity, Int. Arch. Allergy Immunol., 130, 180, 2003. 21. Werling, D. and Jungi, T.W., TOLL-like receptors linking innate and adaptive immune response, Vet. Immunol. Immunopathol., 91, 1, 2003. 22. Barton, G.M. and Medzhitov, R., Toll-like receptors and their ligands, Curr. Top. Microbiol. Immunol., 270, 81, 2002. 23. Takeda, K. and Akira, S., Toll receptors and pathogen resistance, Cell Microbiol., 5, 143, 2003. 24. Dobrovolskaia, M.A. and Vogel, S.N., Toll receptors, CD14, and macrophage activation and deactivation by LPS, Microbes Infect., 4, 903, 2002. 25. Gregory, C.D., CD14-dependent clearance of apoptotic cells: relevance to the immune system, Curr. Opin. Immunol., 12, 27, 2000. 26. Triantafilou, M. and Triantafilou, K., Lipopolysaccharide recognition: CD14, TLRs and the LPSactivation cluster, Trends Immunol., 23, 301, 2002. 27. Aderem, A. and Underhill, D.M., Mechanisms of phagocytosis in macrophages, Annu. Rev. Immunol., 17, 593, 1999. 28. Baldini, M. et al., CD14: an example of gene by environment interaction in allergic disease, Allergy, 57, 188, 2002. 29. Schmitz, G. and Orso, E., CD14 signalling in lipid rafts: new ligands and co-receptors, Curr. Opin. Lipidol., 13, 513, 2002. 30. Geissmann, F. et al., A subset of human dendritic cells expresses IgA Fc receptor (CD89), which mediates internalization and activation upon cross-linking by IgA complexes, J. Immunol., 166, 346, 2001. 31. Allen, L.A., The role of the neutrophil and phagocytosis in infection caused by Helicobacter pylori, Curr. Opin. Infect. Dis., 14, 273, 2001. 32. Burg, N.D. and Pillinger, M.H., The neutrophil: function and regulation in innate and humoral immunity, Clin. Immunol., 99, 7, 2001. 33. Fierer, J., Polymorphonuclear leukocytes and innate immunity to Salmonella infections in mice, Microb. Infect., 3, 1233, 2001.


70


34. Levy, O., A neutrophil-derived anti-infective molecule: bactericidal/permeability-increasing protein, Antimicrob. Agents Chemother., 44, 2925, 2000. 35. Moretta, L. et al., Natural killer cells: a mystery no more, Scand. J. Immunol., 55, 229, 2002. 36. Lieberman, N. and Mandelboim, O., The role of NK cells in innate immunity, Adv. Exp. Med. Biol., 479, 137, 2000. 37. Yokoyama, W.M. and Plougastel, B.F., Immune functions encoded by the natural killer gene complex, Nat. Rev. Immunol., 3, 304, 2003. 38. Boyington, J.C. and Sun, P.D., A structural perspective on MHC class I recognition by killer cell immunoglobulin-like receptors, Mol. Immunol., 38, 1007, 2002. 39. Moretta, A., Natural killer cells and dendritic cells: rendezvous in abused tissues, Nat. Rev. Immunol., 2, 957, 2002. 40. Robertson, M.J., Role of chemokines in the biology of natural killer cells, J. Leukoc. Biol., 71, 173, 2002. 41. Ohteki, T., Critical role for IL-15 in innate immunity, Curr. Mol. Med., 2, 371, 2002. 42. Zitvogel, L., Dendritic and natural killer cells cooperate in the control/switch of innate immunity, J. Exp. Med., 195, F9, 2002. 43. Yokoyama, W.M. and Scalzo, A.A., Natural killer cell activation receptors in innate immunity to infection, Microb. Infect., 4, 1513, 2002. 44. Griffiths, G.M., Endocytosing the death sentence, J. Cell Biol., 160, 155, 2003. 45. Trapani, J.A., Granzymes: a family of lymphocyte granule serine proteases, Genome Biol., 2, 3014, 2001. 46. Van Steensel, M.A. et al., New syndrome of hypotrichosis, striate palmoplantar keratoderma, acroosteolysis and periodontitis not due to mutations in cathepsin C, Br. J. Dermatol., 147, 575, 2002. 47. Robinson, D.S. et al., Eosinophils, Clin. Allergy Immunol., 16, 43, 2002. 48. Gleich, G.J. et al., The biology of the eosinophilic leukocyte, Annu. Rev. Med., 44, 85, 1993. 49. Capron, M. and Goldman, M., The eosinophil, a cell with multiple facets, Therapie, 56, 371, 2001. 50. Plager, D.A. et al., Human eosinophil granule major basic protein and its novel homolog, Allergy, 53, 33, 1998. 51. Wardlaw, A.J., Eosinophils in the 1990s: new perspectives on their role in health and disease, Postgrad. Med. J., 70, 536, 1994. 52. Prussin, C. and Metcalfe, D.D., IgE, mast cells, basophils, and eosinophils, J. Allergy Clin. Immunol., 111, S486, 2003. 53. Abraham, S.N. and Malaviya, R., Mast cells in infection and immunity, Infect. Immun., 65, 3501, 1997. 54. Gurish, M.F. and Boyce, J.A., Mast cell growth, differentiation, and death, Clin. Rev. Allergy Immunol., 22, 107, 2002. 55. Taylor, M.L. and Metcalfe, D.D., Mast cells in allergy and host defense, Allergy Asthma Proc., 22, 115, 2001. 56. Abraham, S.N. and Arock, M., Mast cells and basophils in innate immunity, Semin. Immunol., 10, 373, 1998. 57. Henz, B.M. et al., Mast cells as initiators of immunity and host defense, Exp. Dermatol., 10, 1, 2001. 58. Malaviya, R. and Abraham, S.N., Mast cell modulation of immune responses to bacteria, Immunol. Rev., 179, 16, 2001. 59. Hirji, N.S. et al., Novel CD8 molecule on macrophages and mast cells: expression, function and signaling, Int. Arch. Allergy Immunol., 118, 180, 1999. 60. Arock, M. et al., Phagocytic and tumor necrosis factor alpha response of human mast cells following exposure to gram-negative and gram-positive bacteria, Infect. Immun., 66, 6030, 1998. 61. Varadaradjalou, S. et al., Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells, Eur. J. Immunol., 33, 899, 2003. 62. Kassel, O. et al., The stem cell factor, its properties and potential role in the airways, Pulm. Pharmacol. Ther., 14, 277, 2001. 63. Gnanasekar, M. et al., Molecular characterization of a calcium binding translationally controlled tumor protein homologue from the filarial parasites Brugia malayi and Wuchereria bancrofti, Mol. Biochem. Parasitol., 121, 107, 2002. 64. Rao, K.V. et al., Cloning and characterization of a calcium-binding, histamine-releasing protein from Schistosoma mansoni, J. Biol. Chem., 277, 31207, 2002.


HUMAN IMMUNE SYSTEM

71

65. MacDonald, S.M. et al., Immune mimicry in malaria: Plasmodium falciparum secretes a functional histamine-releasing factor homolog in vitro and in vivo, Proc. Natl. Acad. Sci. U. S. A., 98, 10829, 2001. 66. Charlesworth, E.N., The role of basophils and mast cells in acute and late reactions in the skin, Allergy, 52, 31, 1997. 67. Krishnaswamy, G. et al., The human mast cell: functions in physiology and disease, Front. Biosci., 6, D1109, 2001. 68. Escribano, L. et al., Mastocytosis: current concepts in diagnosis and treatment, Ann. Hematol., 81, 677, 2002. 69. Mastellos, D. and Lambris, J.D., Complement: more than a “guard” against invading pathogens? Trends Immunol., 23, 485, 2002. 70. Gal, P. and Ambrus, G., Structure and function of complement activating enzyme complexes: C1 and MBL-MASPs, Curr. Protein Peptide Sci., 2, 43, 2001. 71. Kishore, U. and Reid, K.B., C1q: structure, function, and receptors, Immunopharmacology, 49, 159, 2000. 72. Kishore, U. et al., Recent progress in the understanding of the structure-function relationships of the globular head regions of C1q, Immunobiology, 205, 355, 2002. 73. Crawford, K. and Alper, C.A., Genetics of the complement system, Rev. Immunogenet., 2, 323, 2000. 74. Dodds, A.W., Which came first, the lectin/classical pathway or the alternative pathway of complement? Immunobiology, 205, 340, 2002. 75. Arlaud, G.J. et al., Structural biology of the C1 complex of complement unveils the mechanisms of its activation and proteolytic activity, Mol. Immunol., 39, 383, 2002. 76. Hajela, K. et al., The biological functions of MBL-associated serine proteases (MASPs), Immunobiology, 205, 467, 2002. 77. Carroll, M.C. and Prodeus, A.P., Linkages of innate and adaptive immunity, Curr. Opin. Immunol., 10, 36, 1998. 78. Esser, M.T. et al., Memory T cells and vaccines, Vaccine, 21, 419, 2003. 79. Galloway, T.S. et al., Regulation of B lymphocytes in health and disease. Meeting review, Mol. Immunol., 39, 649, 2003. 80. Defrance, T. et al., The life and death of a B cell, Adv. Cancer Res., 86, 195, 2002. 81. Cyster, J.G. et al., Traffic patterns of B cells and plasma cells, Adv. Exp. Med. Biol., 512, 35, 2002. 82. Turner, M., B-cell development and antigen receptor signaling, Biochem. Soc. Trans., 30, 812, 2002. 83. Raman, C., CD5, an important regulator of lymphocyte selection and immune tolerance, Immunol. Res., 26, 255, 2002. 84. Boes, M., Role of natural and immune IgM antibodies in immune responses, Mol. Immunol., 37, 1141, 2000. 85. Pike, K.A. and Ratcliffe, M.J., Cell surface immunoglobulin receptors in B cell development, Semin. Immunol., 14, 351, 2002. 86. Brandtzaeg, P., The secretory immunoglobulin system: regulation and biological significance. Focusing on human mammary glands, Adv. Exp. Med. Biol., 503, 1, 2002. 87. Preud'homme, J.L. et al., Structural and functional properties of membrane and secreted IgD, Mol. Immunol., 37, 871, 2000. 88. Vladutiu, A.O., Immunoglobulin D: properties, measurement, and clinical relevance, Clin. Diagn. Lab. Immunol., 7, 131, 2000. 89. Papadea, C. and Check, I.J., Human immunoglobulin G and immunoglobulin G subclasses: biochemical, genetic, and clinical aspects, Crit. Rev. Clin. Lab. Sci., 27, 27, 1989. 90. Ferrante, A. et al., IgG subclass distribution of antibodies to bacterial and viral antigens, Pediatr. Infect. Dis. J., 9, S16, 1990. 91. Clark, M.R., IgG effector mechanisms, Chem. Immunol., 65, 88, 1997. 92. Jefferis, R. and Lund, J., Interaction sites on human IgG-Fc for FcgammaR: current models, Immunol. Lett., 82, 57, 2002. 93. Leynadier, F. et al., Blocking antibodies to inhalant allergens and asthma, Allergy Immunol. (Paris)., 23, 341, 1991. 94. Vercelli, D. et al., To E or not to E? Can an IL-4-induced B cell choose between IgE and IgG4? Int. Arch. Allergy Immunol., 116, 1, 1998.


72


95. Scott, J.R., A review of in-vitro assays for IgG and IgG4 antibodies: concept and potential applications, N. Engl. Reg. Allergy Proc., 8, 385, 1987. 96. van Egmond, M. et al., IgA and the IgA Fc receptor, Trends Immunol., 22, 205, 2001. 97. Rojas, R. and Apodaca, G., Immunoglobulin transport across polarized epithelial cells, Nat. Rev. Mol. Cell Biol., 3, 944, 2002. 98. Phalipon, A. and Corthesy, B., Novel functions of the polymeric Ig receptor: well beyond transport of immunoglobulins, Trends Immunol., 24, 55, 2003. 99. Noguera-Obenza, M. and Cleary, T.G., The role of human milk secretory IgA in protecting infants from bacterial enteritis, Adv. Nutr. Res., 10, 213, 2001. 100. Fagarasan, S. and Honjo, T., Intestinal IgA synthesis: regulation of front-line body defences, Nat. Rev. Immunol., 3, 63, 2003. 101. Pilette, C. et al., Lung mucosal immunity: immunoglobulin-A revisited, Eur. Respir. J., 18, 571, 2001. 102. Zhang, W. et al., Complement in IgA immune-complex-induced neutrophil activation, Biochem. Soc. Trans., 25, 462, 1997. 103. Russell, M.W. et al., IgA antibody as a non-inflammatory regulator of immunity, Biochem. Soc. Trans., 25, 466, 1997. 104. Vercelli, D., Immunoglobulin E and its regulators, Curr. Opin. Allergy Clin. Immunol., 1, 61, 2001. 105. Ebo, D.G. and Stevens, W.J., IgE-mediated food allergy — extensive review of the literature, Acta Clin. Belg., 56, 234, 2001. 106. Heyman, B., IgE-mediated enhancement of antibody responses: the beneficial function of IgE? Allergy, 57, 577, 2002. 107. Novak, N. et al., IgE receptors, Curr. Opin. Immunol., 13, 721, 2001. 108. Ramaswamy, K. et al., Evidence for an interleukin 4-inducible immunoglobulin E uptake and transport mechanism in the intestine, J. Exp. Med., 180, 1793, 1994. 109. Ramaswamy, K. and Befus, D., IgE antibody responses in bronchoalveolar spaces of rats infected with Nippostrongylus brasiliensis, Exp. Parasitol., 76, 23, 1993. 110. Vercelli, D., The regulation of IgE synthesis, Clin. Allergy Immunol., 16, 179, 2002. 111. Papavasiliou, F.N. and Schatz, D.G., Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity, Cell, 109, S35, 2002. 112. Kinoshita, K. and Honjo, T., Linking class-switch recombination with somatic hypermutation, Nat. Rev. Mol. Cell. Biol., 2, 493, 2001. 113. Stavnezer, J., Immunoglobulin class switching, Curr. Opin. Immunol., 8, 199, 1996. 114. Manis, J.P. et al., Mechanism and control of class-switch recombination, Trends Immunol., 23, 31, 2002. 115. Vercelli, D., Novel insights into class switch recombination, Curr. Opin. Allergy Clin. Immunol., 2, 147, 2002. 116. Zhang, K., Immunoglobulin class switch recombination machinery: progress and challenges, Clin. Immunol., 95, 1, 2000. 117. Kumánovics, A. et al., Genomic organization of the mammalian MHC, Annu. Rev. Immunol., 21, 629, 2003. 118. Seliger, B. et al., HLA-G and MIC expression in tumors and their role in anti-tumor immunity, Trends Immunol., 24, 82, 2003. 119. Rouas-Freiss, N. et al., HLA-G in transplantation: a relevant molecule for inhibition of graft rejection? Am. J. Transplant., 3, 11, 2003. 120. Mach, B. et al., Regulation of MHC class II genes: lessons from a disease, Annu. Rev. Immunol., 14, 301, 1996. 121. Simpson, E. and Roopenian, D., Minor histocompatibility antigens, Curr. Opin. Immunol., 9, 655, 1997. 122. Streilein, J.W. et al., The role of minor histocompatibility alloantigens in penetrating keratoplasty, Dev. Ophthalmol., 36, 74, 2003. 123. Roopenian, D. et al., The immunogenomics of minor histocompatibility antigens, Immunol. Rev., 190, 86, 2002. 124. Falkenburg, J.H. et al., Minor histocompatibility antigens as targets of graft-versus-leukemia reactions, Curr. Opin. Hematol., 9, 497, 2002. 125. Snell, G.D., Recent advances in histocompatibility immunogenetics, Adv. Genet., 20, 291, 1979.


HUMAN IMMUNE SYSTEM

73

126. Ceppellini, R. et al., Experimental allotransplantation in man. I. The role of the HL-A system in different genetic combinations, Transplant. Proc., 1, 385, 1969. 127. Otten, H.G. et al., Prevention of acute graft-versus-host-disease by selective depletion of T cells reactive with minor histocompatibility antigens on epithelial cells, Leuk. Lymphoma, 40, 385, 2001. 128. Simpson, E. et al., Minor H antigens: genes and peptides, Transpl. Immunol., 10, 115, 2002. 129. McDevitt, H.O., Discovering the role of the major histocompatibility complex in the immune response, Annu. Rev. Immunol., 18, 1, 2000. 130. Watts, C., Capture and processing of exogenous antigens for presentation on MHC molecules, Annu. Rev. Immunol., 15, 821, 1997. 131. Rock, K.L. and Goldberg, A.L., Degradation of cell proteins and the generation of MHC class Ipresented peptides, Annu. Rev. Immunol., 17, 739, 1999. 132. Solheim, J.C., Class I MHC molecules: assembly and antigen presentation, Immunol. Rev., 172, 11, 1999. 133. Engelhard, V.H. et al., Insights into antigen processing gained by direct analysis of the naturally processed class I MHC associated peptide repertoire, Mol. Immunol., 39, 127, 2002. 134. Gruhler, A. and Fruh, K., Control of MHC class I traffic from the endoplasmic reticulum by cellular chaperones and viral anti-chaperones, Traffic, 1, 306, 2000. 135. Nepom, G.T., MHC multimers: expanding the clinical toolkit, Clin. Immunol., 106, 1, 2003. 136. Sun, M.Y. and Bowness, P., MHC class I multimers, Arthritis Res., 3, 265, 2001. 137. Kwok, W.W. et al., Use of class II tetramers for identification of CD4+ T cells, J. Immunol. Methods, 268, 71, 2002. 138. Buckner, J.H. et al., Defining antigen-specific responses with human MHC class II tetramers, J. Allergy Clin. Immunol., 110, 199, 2002. 139. Kanitakis, J., Anatomy, histology and immunohistochemistry of normal human skin, Eur. J. Dermatol., 12, 390, 2002. 140. Katz, S.I., The role of Langerhans’ cells in immunity, Arch. Dermatol., 116, 1361, 1980. 141. Carr, M.M. et al., MHC class II antigen expression in normal human epidermis, Immunology, 59, 223, 1986. 142. Cruz, P.D., Jr. and Bergstresser, P.R., Antigen processing and presentation by epidermal Langerhans cells. Induction of immunity or unresponsiveness, Dermatol. Clin., 8, 633, 1990. 143. Gielen, V. et al., HLA class I antigen (heavy and light chain) expression by Langerhans cells and keratinocytes of the normal human epidermis: ultrastructural quantitation using immunogold labelling procedure, Arch. Dermatol. Res., 280, 131, 1988. 144. Cumberbatch, M. et al., Langerhans cell migration, Clin. Exp. Dermatol., 25, 413, 2000. 145. Sallusto, F., Origin and migratory properties of dendritic cells in the skin, Curr. Opin. Allergy Clin. Immunol., 1, 441, 2001. 146. Romani, N. et al., Migration of dendritic cells into lymphatics: the Langerhans cell example: routes, regulation, and relevance, Int. Rev. Cytol., 207, 237, 2001. 147. Pieri, L. et al., Langerhans cells differentiation: a three-act play, Ital. J. Anat. Embryol., 106, 47, 2001. 148. Udey, M.C., Cadherins and Langerhans cell immunobiology, Clin. Exp. Immunol., 107, 6, 1997. 149. Lanzavecchia, A. and Sallusto, F., Regulation of T cell immunity by dendritic cells, Cell, 106, 263, 2001. 150. Lanzavecchia, A., Dendritic cell maturation and generation of immune responses, Haematologica, 84, 23, 1999. 151. Bergstresser, P.R. et al., Dendritic epidermal T cells: lessons from mice for humans, J. Invest. Dermatol., 100, 80S, 1993. 152. Boismenu, R. et al., Molecular and cellular biology of dendritic epidermal T cells, Semin. Immunol., 8, 323, 1996. 153. Kumamoto, T. et al., Recent advances in dendritic cell vaccines for cancer treatment, J. Dermatol., 28, 658, 2001. 154. Chen, D. and Payne, L.G., Targeting epidermal Langerhans cells by epidermal powder immunization, Cell Res., 12, 97, 2002. 155. Hammond, S.A. et al., Transcutaneous immunization: an emerging route of immunization and potent immunostimulation strategy, Crit. Rev. Ther. Drug Carrier Syst., 18, 503, 2001.


74


156. Luger, T.A. et al., Cutaneous immunomodulation and coordination of skin stress responses by alphamelanocyte-stimulating hormone, Ann. N. Y. Acad. Sci., 840, 381, 1998. 157. Starr, T.K. et al., Positive and negative selection of T cells, Annu. Rev. Immunol., 21, 139, 2003. 158. Anton van der Merwe, P. and Davis, S.J., Molecular interaction mediating T cell antigen recognition, Annu. Rev. Immunol., 21, 659, 2003. 159. Guermonprez, P. et al., Antigen presentation and T cell stimulation by dendritic cells, Annu. Rev. Immunol., 20, 621, 2002. 160. Mosmann, T.R. and Coffman, R.L., TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties, Annu. Rev. Immunol., 7, 145, 1989. 161. Romagnani, S., Biology of human TH1 and TH2 cells, J. Clin. Immunol., 15, 121, 1995. 162. Uchi, H. et al., Cytokines and chemokines in the epidermis, J. Dermatol. Sci., 24, S29, 2000. 163. Brierley, M.M. and Fish, E.N., Review: IFN-alpha/beta receptor interactions to biologic outcomes: understanding the circuitry, J. Interferon Cytokine Res., 22, 835, 2002. 164. Deonarain, R. et al., Interferon-alpha/beta-receptor interactions: a complex story unfolding, Curr. Pharm. Design, 8, 2131, 2002. 165. Grossberg, S.E. et al., Interferon receptors and their role in interferon action, Experientia, 45, 508, 1989. 166. Brassard, D.L. et al., Interferon-alpha as an immunotherapeutic protein, J. Leukoc. Biol., 71, 565, 2002. 167. Fitzgerald-Bocarsly, P., Natural interferon-alpha producing cells: the plasmacytoid dendritic cells, Biotechniques, 22 (Suppl. 16–22), 24, 2002. 168. Pestka, S., The human interferon alpha species and receptors, Biopolymers, 55, 254, 2000. 169. Locksley, R.M. et al., The TNF and TNF receptor superfamilies: integrating mammalian biology, Cell, 104, 487, 2001. 170. Zhou, T. et al., Immunobiology of tumor necrosis factor receptor superfamily, Immunol. Res., 26, 323, 2002. 171. Tracey, K.J. and Cerami, A., Tumor necrosis factor: an updated review of its biology, Crit. Care Med., 21, S415, 1993. 172. Wajant, H. et al., Tumor necrosis factor signaling, Cell. Death Differ., 10, 45, 2003. 173. Olsson, I. et al., Tumour necrosis factor (TNF) binding proteins (soluble TNF receptor forms) with possible roles in inflammation and malignancy, Eur. Cytokine Network, 4, 169, 1993. 174. Ryffel, B. and Mihatsch, M.J., TNF receptor distribution in human tissues, Int. Rev. Exp. Pathol., 34, 149, 1993. 175. Cosman, D., A family of ligands for the TNF receptor superfamily, Stem Cells, 12, 440, 1994. 176. Baetu, T.M. and Hiscott, J., On the TRAIL to apoptosis, Cytokine Growth Factor Rev., 13, 199, 2002. 177. Roux-Lombard, P., The interleukin-1 family, Eur. Cytokine Network, 9, 565, 1998. 178. Arend, W.P., The balance between IL-1 and IL-1Ra in disease, Cytokine Growth Factor Rev., 13, 323, 2002. 179. Dinarello, C.A., Interleukin-1 beta, interleukin-18, and the interleukin-1 beta converting enzyme, Ann. N. Y. Acad. Sci., 856, 1, 1998. 180. Daun, J.M. and Fenton, M.J., Interleukin-1/Toll receptor family members: receptor structure and signal transduction pathways, J. Interferon Cytokine Res., 20, 843, 2000. 181. Sims, J.E., IL-1 and IL-18 receptors, and their extended family, Curr. Opin. Immunol., 14, 117, 2002. 182. Fitzgerald, K.A. and O’Neill, L.A., The role of the interleukin-1/toll-like receptor superfamily in inflammation and host defense, Microb. Infect., 2, 933, 2000. 183. Akira, S., The role of IL-18 in innate immunity, Curr. Opin. Immunol., 12, 59, 2000. 184. Dinarello, C.A., Interleukin-18, Methods, 19, 121, 1999. 185. Lebel-Binay, S. et al., Interleukin-18: biological properties and clinical implications, Eur. Cytokine Network, 11, 15, 2000. 186. Lin, J.X. and Leonard, W.J., The role of Stat5a and Stat5b in signaling by IL-2 family cytokines, Oncogene, 19, 2566, 2000. 187. Kuziel, W.A. and Greene, W.C., Interleukin-2 and the IL-2 receptor: new insights into structure and function, J. Invest. Dermatol., 94, 27S, 1990. 188. Nelson, B.H. and Willerford, D.M., Biology of the interleukin-2 receptor, Adv. Immunol., 70, 1, 1998. 189. Semenzato, G. et al., The interleukin-2/interleukin-2 receptor system: structural, immunological, and clinical features, Int. J. Clin. Lab. Res., 22, 133, 1992.


HUMAN IMMUNE SYSTEM

75

190. Sugamura, K. et al., The interleukin-2 receptor gamma chain: its role in the multiple cytokine receptor complex and T cell development in XSCID, Annu. Rev. Immunol., 14, 179, 1996. 191. Schmalstieg, F.C. and Goldman, A.S., Immune consequences of mutations in the human common gamma-chain gene, Mol. Genet. Metab., 76, 163, 2002. 192. Kono, T. et al., The interleukin-2 receptor complex and signal transduction: role of the beta-chain, Semin. Immunol., 5, 299, 1993. 193. Minami, Y. et al., The IL-2 receptor complex: its structure, function, and target genes, Annu. Rev. Immunol., 11, 245, 1993. 194. Di Santo, J.P. et al., Common cytokine receptor gamma chain (gamma c)-dependent cytokines: understanding in vivo functions by gene targeting, Immunol. Rev., 148, 19, 1995. 195. Demoulin, J.B. and Renauld, J.C., Signalling by cytokines interacting with the interleukin-2 receptor gamma chain, Cytokines Cell Mol. Ther., 4, 243, 1998. 196. Gillis, S., Interleukin 2: biology and biochemistry, J. Clin. Immunol., 3, 1, 1983. 197. Smith, K.A., Interleukin-2: Inception, impact, and implications, Science, 240, 1169, 1988. 198. Nelms, K. et al., The IL-4 receptor: Signaling mechanisms and biologic functions, Annu. Rev. Immunol., 17, 701, 1999. 199. Ackermann, L. et al., Staphylococcal enterotoxin B inhibits the production of interleukin-4 in a human mast-cell line HMC-1, Immunology, 94, 247, 1998. 200. Viac, J. et al., Interleukin-4 and interferon-gamma interactions in the induction of intercellular adhesion molecule-1 and major histocompatibility complex class II antigen expression of normal human keratinocytes, Exp. Dermatol., 3, 72, 1994. 201. Zhang, J. et al., IL-4 potentiates activated T Cell apoptosis via an IL-2-dependent mechanism, J. Immunol., 170, 3495, 2003. 202. Schaefer, G. et al., FISP (IL-4-Induced Secreted Protein), a novel cytokine-like molecule secreted by Th2 cells, J. Immunol., 166, 5859, 2001. 203. Appasamy, P.M., Biological and clinical implications of interleukin-7 and lymphopoiesis, Cytokines Cell Mol. Ther., 5, 25, 1999. 204. Moller, P. et al., Interleukin-7. Biology and implications for dermatology, Exp. Dermatol., 5, 129, 1996. 205. Mertsching, E. et al., Interleukin-7, a non-redundant potent cytokine whose over-expression massively perturbs B-lymphopoiesis, Int. Rev. Immunol., 16, 285, 1998. 206. Appasamy, P.M., Interleukin-7: biology and potential clinical applications, Cancer Invest., 11, 487, 1993. 207. Demoulin, J.B. and Renauld, J.C., Interleukin 9 and its receptor: an overview of structure and function, Int. Rev. Immunol., 16, 345, 1998. 208. Soussi-Gounni, A. et al., Role of IL-9 in the pathophysiology of allergic diseases, J. Allergy Clin. Immunol., 107, 575, 2001. 209. Waldmann, T.A., The IL-2/IL-15 receptor systems: targets for immunotherapy, J. Clin. Immunol., 22, 51, 2002. 210. Lodolce, J. et al., Interleukin-15 and the regulation of lymphoid homeostasis, Mol. Immunol., 39, 537, 2002. 211. Waldmann, T.A. and Tagaya, Y., The multifaceted regulation of interleukin-15 expression and the role of the cytokine in NK cell differentiation and host response to intracellular pathogens, Annu. Rev. Immunol., 17, 19, 1999. 212. Weng, N.P. et al., IL-15 is a growth factor and an activator of CD8 memory T cells, Ann. N. Y. Acad. Sci., 975, 46, 2002. 213. Habib, T. et al., The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3, Biochemistry, 41, 8725, 2002. 214. Parrish-Novak, J. et al., Interleukin-21 and the IL-21 receptor: novel effectors of NK and T cell responses, J. Leukoc. Biol., 72, 856, 2002. 215. Strengell, M. et al., IL-21 up-regulates the expression of genes associated with innate immunity and Th1 response, J. Immunol., 169, 3600, 2002. 216. Mehta, D.S. et al., IL-21 Induces the apoptosis of resting and activated primary B cells, J. Immunol., 170, 4111, 2003. 217. Ozaki, K. et al., A critical role for IL-21 in regulating immunoglobulin production, Science, 298, 1630, 2002.


76


218. Guthridge, M.A. et al., Mechanism of activation of the GM-CSF, IL-3, and IL-5 family of receptors, Stem Cells, 16, 301, 1998. 219. Woodcock, J.M. et al., The functional basis of granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 receptor activation, basic and clinical implications, Int. J. Biochem. Cell Biol., 31, 1017, 1999. 220. Morris, C.F. et al., Molecular and cellular biology of interleukin-3, Immunol. Ser., 49, 177, 1990. 221. Sunderland, M.C. and Roodman, G.D., Interleukin-3. Its biology and potential uses in pediatric hematology/oncology, Am. J. Pediatr. Hematol. Oncol., 13, 414, 1991. 222. Yang, Y.C. and Clark, S.C., Interleukin-3: molecular biology and biologic activities, Hematol. Oncol. Clin. North Am., 3, 441, 1989. 223. Weltman, J.K. and Karim, A.S., IL-5: biology and potential therapeutic applications, Expert Opin. Invest. Drugs, 9, 491, 2000. 224. Lalani, T. et al., Biology of IL-5 in health and disease, Ann. Allergy Asthma Immunol., 82, 317, 1999. 225. Mordvinov, V.A. and Sanderson, C.J., Regulation of IL-5 expression, Arch. Immunol. Ther. Exp (Warsz), 49, 345, 2001. 226. Herbst, B. et al., GM-CSF promotes differentiation of a precursor cell of monocytes and Langerhanstype dendritic cells from CD34+ haemopoietic progenitor cells, Br. J. Haematol., 101, 231, 1998. 227. Nakashima, K. and Taga, T., gp130 and the IL-6 family of cytokines: signaling mechanisms and thrombopoietic activities, Semin. Hematol., 35, 210, 1998. 228. Hirano, T., Interleukin 6 and its receptor: ten years later, Int. Rev. Immunol., 16, 249, 1998. 229. Bravo, J. and Heath, J.K., Receptor recognition by gp130 cytokines, E.M.B.O. J., 19, 2399, 2000. 230. Grotzinger, J. et al., IL-6 type cytokine receptor complexes: hexamer, tetramer or both? Biol. Chem., 380, 803, 1999. 231. Chow, D.C. et al., A structural template for gp130-cytokine signaling assemblies, Biochim. Biophys. Acta., 1592, 225, 2002. 232. Heinrich, P.C. et al., Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway, Biochem. J., 334, 297, 1998. 233. Taga, T. and Kishimoto, T., Gp130 and the interleukin-6 family of cytokines, Annu. Rev. Immunol., 15, 797, 1997. 234. Montero-Julian, F.A., The soluble IL-6 receptors: serum levels and biological function, Cell Mol. Biol., 47, 583, 2001. 235. Heymann, D. and Rousselle, A.V., gp130 Cytokine family and bone cells, Cytokine, 12, 1455, 2000. 236. Wollert, K.C. and Drexler, H., The role of interleukin-6 in the failing heart, Heart Fail. Rev., 6, 95, 2001. 237. Leng, S.X. and Elias, J.A., Interleukin-11, Int. J. Biochem. Cell Biol., 29, 1059, 1997. 238. Trepicchio, W.L. and Dorner, A.J., Interleukin-11. A gp130 cytokine, Ann. N. Y. Acad. Sci., 856, 12, 1998. 239. Turner, K.J. et al., The role of recombinant interleukin 11 in megakaryocytopoiesis, Stem Cells, 14, 53, 1996. 240. Knight, D., Leukaemia inhibitory factor (LIF): a cytokine of emerging importance in chronic airway inflammation, Pulm. Pharmacol. Ther., 14, 169, 2001. 241. Bruce, A.G. et al., Oncostatin M, Prog. Growth Factor Res., 4, 157, 1992. 242. Grant, S.L. and Begley, C.G., The oncostatin M signalling pathway: reversing the neoplastic phenotype? Mol. Med. Today, 5, 406, 1999. 243. Miyajima, A. et al., Role of Oncostatin M in hematopoiesis and liver development, Cytokine Growth Factor Rev., 11, 177, 2000. 244. Robb, L. et al., Leukemia inhibitory factor and interleukin-11: cytokines with key roles in implantation, J. Reprod. Immunol., 57, 129, 2002. 245. Lass, A. et al., Leukemia inhibitory factor in human reproduction, Fertil. Steril., 76, 1091, 2001. 246. Gadient, R.A. and Patterson, P.H., Leukemia inhibitory factor, Interleukin 6, and other cytokines using the GP130 transducing receptor: roles in inflammation and injury, Stem Cells, 17, 127, 1999. 247. Gomez-Lechon, M.J., Oncostatin M: signal transduction and biological activity, Life Sci., 65, 2019, 1999. 248. Ip, N.Y., The neurotrophins and neuropoietic cytokines: two families of growth factors acting on neural and hematopoietic cells, Ann. N. Y. Acad. Sci., 840, 97, 1998.


HUMAN IMMUNE SYSTEM

77

249. Lesser, S.S. and Lo, D.C., CNTF II, I presume? Nat. Neurosci., 3, 851, 2000. 250. Inoue, M. et al., Activating mechanism of CNTF and related cytokines, Mol. Neurobiol., 12, 195, 1996. 251. Latchman, D.S., Cardiotrophin-1: a novel cytokine and its effects in the heart and other tissues, Pharmacol. Ther., 85, 29, 2000. 252. Latchman, D.S., Cardiotrophin-1 (CT-1): a novel hypertrophic and cardioprotective agent, Int. J. Exp. Pathol., 80, 189, 1999. 253. Wollert, K.C. and Chien, K.R., Cardiotrophin-1 and the role of gp130-dependent signaling pathways in cardiac growth and development, J. Mol. Med., 75, 492, 1997. 254. Pennica, D. et al., Cardiotrophin-1: a multifunctional cytokine that signals via LIF receptor-gp 130 dependent pathways, Cytokine Growth Factor Rev., 7, 81, 1996. 255. Baggiolini, M. et al., Neutrophil activation and the effects of interleukin-8/neutrophil-activating peptide 1 (IL-8/NAP-1), Cytokines, 4, 1, 1992. 256. Baggiolini, M. and Clark-Lewis, I., Interleukin-8, a chemotactic and inflammatory cytokine, F.E.B.S. Lett., 307, 97, 1992. 257. Hoffmann, E. et al., Multiple control of interleukin-8 gene expression, J. Leukoc. Biol., 72, 847, 2002. 258. Mukaida, N. and Matsushima, K., Regulation of IL-8 production and the characteristics of the receptors for IL-8, Cytokines, 4, 41, 1992. 259. Shi, Q. et al., Regulation of interleukin-8 expression by tumor-associated stress factors, J. Interferon Cytokine Res., 21, 553, 2001. 260. Xie, K., Interleukin-8 and human cancer biology, Cytokine Growth Factor Rev., 12, 375, 2001. 261. Hay, D.W. and Sarau, H.M., Interleukin-8 receptor antagonists in pulmonary diseases, Curr. Opin. Pharmacol., 1, 242, 2001. 262. Strieter, R.M., Interleukin-8: a very important chemokine of the human airway epithelium, Am. J. Physiol. Lung Cell Mol. Physiol., 283, L688, 2002. 263. Fickenscher, H. et al., The interleukin-10 family of cytokines, Trends Immunol., 23, 89, 2002. 264. Kotenko, S.V., The family of IL-10-related cytokines and their receptors: related, but to what extent? Cytokine Growth Factor Rev., 13, 223, 2002. 265. Sarkar, D. et al., mda-7 (IL-24): signaling and functional roles, Biotechniques, 30 (Suppl. 1), 30, 2002. 266. Brombacher, F. et al., Novel IL-12 family members shed light on the orchestration of Th1 responses, Trends Immunol., 24, 207, 2003. 267. Trinchieri, G., Interleukin-12 and the regulation of innate resistance and adaptive immunity, Nat. Rev. Immunol., 3, 133, 2003. 268. Frucht, D.M., IL-23: a cytokine that acts on memory T cells, Sci. S.T.K.E., 2002, 114, 2002. 269. Pflanz, S. et al., IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells, Immunity, 16, 779, 2002. 270. Hershey, G.K., IL-13 receptors and signaling pathways: an evolving web, J. Allergy Clin. Immunol., 111, 677, 2003. 271. Zheng, T. et al., Cytokine regulation of IL-13Ralpha2 and IL-13Ralpha1 in vivo and in vitro, J. Allergy Clin. Immunol., 111, 720, 2003. 272. Ford, R. et al., Identification of B-cell growth factors (interleukin-14; high molecular weight-B-cell growth factors) in effusion fluids from patients with aggressive B-cell lymphomas, Blood, 86, 283, 1995. 273. Ambrus, J.L., Jr. et al., A Potential Role for PGE and IL-14 (HMW-BCGF) in B-Cell Hyperactivity of Patients with Systemic Lupus Erythematosus, Am. J. Ther., 2, 933, 1995. 274. Center, D.M. et al., Interleukin-16, Int. J. Biochem. Cell Biol., 29, 1231, 1997. 275. Cruikshank, W.W. et al., Interleukin-16, J. Leukoc. Biol., 67, 757, 2000. 276. Center, D.M. et al., Interleukin 16: implications for CD4 functions and HIV-1 progression, Immunol. Today, 21, 273, 2000. 277. Kornfeld, H. and Cruikshank, W.W., Prospects for IL-16 in the treatment of AIDS, Expert Opin. Biol. Ther., 1, 425, 2001. 278. Little, F.F. et al., Immunomodulatory effect of interleukin-16 on allergic airway inflammation, Chest, 123, 431S, 2003. 279. Albanesi, C. et al., IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha, J. Immunol., 162, 494, 1999.


78


280. Fossiez, F. et al., Interleukin-17, Int. Rev. Immunol., 16, 541, 1998. 281. Miossec, P., Interleukin-17 in rheumatoid arthritis: if T cells were to contribute to inflammation and destruction through synergy, Arthritis Rheum., 48, 594, 2003. 282. Laan, M. and Linden. A., IL-17 as a potential target for modulating airway neutrophilia, Curr. Pharmacol. Design, 8, 1855, 2002. 283. Aggarwal, S. and Gurney, A.L., IL-17: prototype member of an emerging cytokine family, J. Leukoc. Biol., 71, 1, 2002. 284. Hurst, S.D. et al., New IL-17 family members promote Th1 or Th2 responses in the lung: in vivo function of the novel cytokine IL-25, J. Immunol., 169, 443, 2002. 285. Ikeda, K. et al., Mast cells produce interleukin-25 upon Fcepsilon RI-mediated activation, Blood, 101, 3594, 2003. 286. Fort, M.M. et al., IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo, Immunity, 15, 985, 2001. 287. Sheppard, P. et al., IL-28, IL-29 and their class II cytokine receptor IL-28R, Nat. Immunol., 4, 63, 2003. 288. Dhainaut, J.F. et al., Transforming growth factor-beta: A mediator of cell regulation in acute respiratory distress syndrome, Crit. Care Med., 31, S258, 2003. 289. Heldin, C.H. and Westermark, B., Mechanism of action and in vivo role of platelet-derived growth factor, Physiol. Rev., 79, 1283, 1999. 290. Wada, K. et al., Gastric epithelial cells secrete a PDGF-like peptide, a potent mitogen for human gastric fibroblasts, Proc. Soc. Exp. Biol. Med., 217, 109, 1998. 291. Matsukawa, A. et al., Chemokines and innate immunity, Rev. Immunogenet., 2, 339, 2000.


CHAPTER

4

Canine Immune System Bruce Hammerberg

CONTENTS I. II. III. IV. V.

Introduction ..........................................................................................................................79 Major Histocompatibility Complex (MHC) ........................................................................80 Complement System ............................................................................................................80 Antibodies ............................................................................................................................81 Immune Cells .......................................................................................................................82 A. Mast Cells ....................................................................................................................82 B. Neutrophils...................................................................................................................82 C. Lymphocytes ................................................................................................................83 D. Dendritic Cells .............................................................................................................84 VI. Cytokines and Chemokines .................................................................................................84 References ........................................................................................................................................85

I. INTRODUCTION Distinct from conventional laboratory animal models, the characterization of the canine immune system has been driven in large part by a need to understand the pathogenesis and develop effective therapies for naturally occurring diseases in the dog, in addition to the need to find research models for the human immune system. This is true for antibody production, the complement system, immune cells, cytokines and chemokines. Consequently, where functionality of the canine and human immune systems is coincidental, this has been discovered because of identical or very similar pathogenic mechanisms in naturally occurring diseases. The power of this type of discovery is in its ability to allow manipulation of a complex natural process to instruct rather than depending upon the preconceptions of how the researcher may think a pathogenic mechanism may be functioning to create a complex disease model. This is particularly important for the complex disease processes considered in this review.

0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

79


80


II. MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) Characterization of the dog MHC, or dog leukocyte antigen (DLA) as it is officially referred to by the International Society for Animal Genetics (ISAG) [1], was begun more than 25 years ago [2] and has been vigorously pursued primarily by researchers using the dog as a model for transplantation studies. In the last 15 years much progress has been made and earlier serological study results have been recently correlated, where possible, with sequence data [1]. Four complete canine class II genes have been characterized: DLA-DRA1 [3], DLA-DQA1 [4], DLA-DRB1 [5], and DLA-DQB1 [6]. More recently, four complete class I genes have been sequenced and characterized with regard to polymorphism: DLA-12, DLA-79, DLA-88, and DLA-64 [7,8]. Even though the DLA has been characterized primarily for transplantation research, just as in humans and mice the functions of MHC class I and II are associated with the immune system, including the recognition of self- and non-self antigens. This function makes MHC important to autoimmune and infectious diseases. The organization of the canine major histocompatibility complex and a comparison to human MHC has been reviewed by Wagner et al. [9]. As with human class I molecules, HLA-A, HLA-B, and HLA-C, the dog DLA-A molecules are typical of class I in their noncovalent association with b2 microglobulin. Although the tissue distribution and function of DLA class I genes are not yet known, it can be inferred from other species that DLA class I surface proteins could be the targets of cytotoxic T lymphocytes [9]. In contrast to class I genes in families that have independently come about in various mammalian orders, the class II genes are highly conserved and analogies exist between human and canine [9]. Thus, names are shared between canine and human analogous genes of class II. Even though class II molecules are presented on antigen-presenting cells as part of their well known function in antigen processing, one very interesting difference between human and canine class II gene expression is that almost all canine T lymphocytes in peripheral circulation express class II antigens, whereas human T lymphocytes display this molecule only after activation [10]. Similarly, many other mammals, such as the horse [11], pig [12], and dolphin [13] also express class II molecules on nonactivated lymphocytes. The role of MHC genes in human familial systemic lupus erythematosus (SLE) has yet to be defined because of suspected multiple gene contribution and environmental stimuli [14]. Naturally occurring canine SLE has an association with DLA-A7 [15], and dogs share many of the same habitat stimuli as their human owners. Manipulation of these stimuli in dogs with high risk of SLE may reveal important clues to identifying environmental stimuli in susceptible populations. Naturally occurring immunopathological diseases in dogs are potentially important models for the identification of MHC ancestral haplotypes (AHs) recently suggested to be associated with these diseases in humans. Ancestral haplotypes are defined by their high degree of conservation and combinations of alleles at multiple loci that are strongly associated with immunopathological diseases [16]. One of these haplotypes, designated as 8.1, includes the genes HLA-A1, CW7, B8, BfS, C4AQ0, C4B1, DR3, and DQ2. AH 8.1 is associated with several immunopathological diseases including SLE [17] and dermatitis herpetiformis [18]. Do AHs exist in dogs that are associated with immunopathological diseases as in humans?

III. COMPLEMENT SYSTEM Early-stage second and fourth components of the complement cascade, C2 and C4, have loci within the MHC and are associated with human familial autoimmune diseases [19] and may be similarly associated in canine familial autoimmunity [15,20], including SLE. No genetic deficiency of C2 or C4 has been described in dogs to date; however, there is one report of statistically lower levels of C2 in collie breed dogs compared to noncollie breeds [21]. Canine C4 is polymorphic with eight electrophoretically detectable variants and an additional three allotypes due in part to


CANINE IMMUNE SYSTEM

81

neutral sugar variations in the a chain and microheterogeneity in the g chain [22]. Although no disease manifestation has been associated with C2 variation in dogs, there is a reported association of C4 phenotype 4 with idiopathic cardiomyopathy where 30% of the affected dogs had antimitochondrial antibody and 47% had serum antinuclear antibody [23]. The roles of C2 and C4 deficiencies or abnormal function in human autoimmune disease pathogenesis are not known, but it is suggested that C4 binding to immune complexes inhibits formation of insoluble forms of these complexes [24]. Complement protein C3 has been described in all vertebrate animals and as early in the phylogenetic tree as deuterostomes such as sea urchins [25]. Its function is innate resistance and greatly predates the appearance of adaptive immunity in evolutionary development. C3 is converted to opsonic and chemotactic forms by serine protease activity, which can be generated by the classical pathway cascade through C1, C2, and C4, by the alternative pathway using the C3 convertase activity of factor B or by the mannose-binding lectin (MBL) pathway–associated proteases [26]. C3-activated forms can be detected where immune complexes, certain foreign elements, or MBL binding are present in tissues, and this is used as an indicator of pathological changes. Although C3 localization in tissues is widely used as an indicator of pathological change, the role of C3 in pathogenesis is the result of a balance between its role in clearance and processing of immune complexes and its inflammatory activities [27]. The only naturally occurring nonhuman, complete C3 deficiency reported to date is in a colony of Brittany spaniel dogs [28]. This deficiency is the consequence of a deletion of a cytosine at position 2136 resulting in a stop codon 11 amino acids downstream from codon 712 [29]. These dogs demonstrated increased frequency of bacterial infections [30] and type I membranoproliferative glomerulonephritis [31]. The complexity of C3 involvement in pathogenesis is illustrated by the observation that the renal disease associated with C3 deficiency was made worse when C3deficient dogs were given normal plasma to replace C3 [31]. Canine C3-cDNA sequence alignment with human sequences demonstrated that the regions most critical for C3 function and post-translational changes are highly conserved between dog and human [29]. Deficiencies and polymorphisms of individual complement proteins in the dog are well characterized and provide avenues to discovering insights about the complex balance between complement roles of homeostasis and inflammatory response.

IV. ANTIBODIES Characterization of canine immunoglobulins was initiated by Patterson et al. [32–37] in the early 1960s partly because of interest in the dog as a model for allergic disease due to its naturally occurring pollen sensitivity [33,34,37]. A systematic approach to characterize dog immunoglobulins with rabbit antisera and physicochemical separation by molecular weight and charge and electrophoretic mobility demonstrated three major and one minor gamma-migrating subclasses of IgG, as well as subclasses that were analogous to human IgM and IgA [38,39]. Initial evidence for canine reagenic antibody, obtained from clinically seasonal allergic dogs, being analogous to human IgE was presented by Patterson et al. [36] and this subclass was further characterized as analogous to human IgE by Halliwell et al. [40,41]. Serum, colostral, and fecal levels of canine IgGa and IgGb, which could be recognized as immunologically distinct proteins but not separable, IgGc, IgGd (IgG1g), IgA, and IgM were done by Reynolds and Johnson [42] using radial immunodiffusion. Subsequent publications used corresponding designations as follows: IgGa = IgG2a, IgGb = IgG2b, IgGc = IgG2c, IgGd (7Sg1) = IgG1. More recently, monoclonal antibodies have been developed against four subclasses of canine IgG separated on the basis of binding to protein A and protein G [43,44]. These reagents were used to rename the IgG subclasses and relate them to human IgG subclasses based on serum levels and electrophoretic migration. Thus, as with humans, canine IgG2 and IgG4 migrate anodally, canine


82


IgG1 and IgG3 migrate cathodally; and, canine IgG1 and IgG2 occur at higher levels in normal dog serum than IgG3 and IgG4 [44,45]. Further characterization of these canine IgG subclasses has not been reported with regard to complement fixing and opsonic activity, nor has there been any association of these subclasses with polar T-helper cytokine profiles that currently link production of human IgG1 and IgG3 with IL-10 and IgG4 with IL-4 and IL-13 [46]. Major progress toward affirming the presence of four different canine immunoglobulin g chains was made by sequence analysis of cDNA from spleen cells and lymphoma cells that demonstrated much similarity in CH1, CH2 and CH3 domains but great distinction in hinge region sequences [47]. Three of four primers specific to canine IgG hinge region sequences demonstrated detection of IgG g chain mRNA expression by peripheral blood mononuclear cells. Because there was a higher degree of sequence homology within species than between species comparing dog with human and mouse the designation of IgG A–D was used instead of IgG 1–4. The relationship of the hinge region differences to monoclonal antibody–defined subclasses may be possible if those monoclonal antibodies made against Fab fragments happen to be specific for hinge region epitopes. Derived amino-acid sequence analysis of canine IgG subclasses and studies of canine myeloma IgG proteins should shed light on the functional requirements for opsonic, complement fixing and FcgRII-binding activities across species and identify tertiary structures essential for these functions. One recent application of knowing the derived amino-acid sequence of canine IgE and having canine IgE available from a heterohybridoma was the development of a peptide vaccine that induced nonanaphylactoic, autoantibodies against IgE that greatly reduced serum IgE levels in immunized dogs [48]. Heterohybridomas resulting from the fusion of mouse myeloma cells with canine B cells specific for filarial nematode antigens have been produced that yield monoclonal canine IgG from stable cell lines [49]. These monoclonal canine IgGs have yet to be characterized regarding function, nor has the IgG cDNA from the cell lines been sequenced.

V. IMMUNE CELLS A. Mast Cells The field of immune cell function in pathogenesis has benefited much from studies using canine in vitro and in vivo approaches. The most valuable and often pathbreaking work has involved canine mast cells and neutrophils. Some of the earliest mast cell lines of mammals to be studied were developed from canine mastocytomas [50–52]. These cell lines are valuable for having fully functional high-affinity receptors for IgE [51], 2 decades of work have characterized a vast array of canine mast cell line cell-derived inflammatory mediators [53–56]. The availability of canine mast cells lines and a heteromyeloma-derived canine IgE of known allergen specificity [49] greatly facilitate the study of spontaneous allergic diseases in dogs that closely parallel like diseases in humans [57–59]. In addition, the complexity of the mast cell heterogeneity and its relationship to tissue location is well documented for the dog by the comprehensive histochemical study of Kube et al. [60]. B. Neutrophils Similarly, although not to the same degree, the function of canine neutrophils have been studied for their role in acute inflammatory disease, and most importantly in the canine cardiac reperfusion injury model [61]. From this work, much is known about canine neutrophil responses to chemokine factors and integrins [62–69], and the canine model has demonstrated the role that neutrophilderived inflammatory mediators play in cardiac transplantation medicine. Transfer of what has been



83

learned about canine neutrophil activity in heart, lung, and liver pathogenesis to dermal pathogenesis may suggest new roles for neutrophils in the pathogenesis of dermatitis. Many studies have focused on the role of the surface complement CR3 receptor CD11b/CD18 (Mac-1) in activation and adhesion of canine neutrophils, but most recently the respiratory burst response of neutrophils interacting with the intercellular adhesion molecule-1 (ICAM-1 or CD54) was found to involve both CD11b/CD18 and the lymphocyte function–associated antigen-1 (LFA-1 or CD11a/CD18); the former requires chemotactic stimulation and the latter not [70]. Canine neutrophil priming and function has been demonstrated to be enhanced by pituitary growth hormone, as in humans, and this connection between immune and neuroendocrine systems [71] should not be overlooked in experimental design. Finally, canine leukocyte adhesion deficiency (CLAD) has been described in Irish setters and shown to be the result of a single missense mutation of a crucial cysteine residue in the beta-2 integrin gene encoding the CD18 subunit [72,73]. C. Lymphocytes Regarding lymphocytes, the classic subsets of B cells, CD3+ T helper cells (distinguished by CD4 and CD8, and cytokine production profiles) and NK cells are well characterized in the dog. Several studies have been conducted to find cross-reactive antihuman leukocyte monoclonal antibodies that react with canine cells [74–76]. Brodersen et al. [77] surveyed 213 monoclonal antibodies and reviewed previously published findings on cross-reactive antibodies. Important to the function of T cells in responding to antigen-specific stimulatory signals from antigen-presenting cells are the co-stimulatory molecules CD28 and CTL-4. The balance of signals from CD28 and CTL-4 following binding with B7 (CD80) and B7-2 (CD86) determine whether the T-cell response is anergic (absence of CD28 signal), or potentiates autoimmunity (absence of CTL-4) or an appropriate protective immune response (early CD28 signaling followed by CTL-4 signals) [78]. The cDNA that encodes the full length of the canine CD28 molecule has been cloned and sequenced [79]. Both nucleic acid and derived amino acid sequences show about 80% homology with human CD28, and the hexapeptide motif, MYPPPY, inside the “V”-like domain is identical with human CD28 and a similar site in the published sequence of canine CTL-4 (GenBank accession number AF143204). Although canine CD80 and CD86 have been sequenced, and m-RNA expression for membrane-bound and secreted forms have been demonstrated to occur in peripheral blood mononuclear cells (PBMC) [80], the corresponding proteins have yet to be identified on cells or in serum. In a survey of commercially available anti-human leukocyte monoclonal antibodies by Lilliehook et al. [81], it was found that surface glycoproteins of canine granulocytes, including neutrophils and eosinophils, are recognized by monoclonal antibodies specific for human CD11b, CD18, and CD49d. Neutrophils, but not eosinophils, are recognized by antibodies specific for CD16 and CD32. Anti-CD9 demonstrated no binding to neutrophils but bound to a subset of eosinophils from four of seven dogs. These cross-reactive monoclonal antibodies identify cell adhesion and Fcg-receptors as follows: CD11b and CD18 are adhesion molecules, CD49d and CD29 make up the very late activation antigen 4 (VLA-4), and CD16 and CD32 are Fcg-receptors. Thus, it appears that surface molecules delineating granulocytes are more highly conserved than those of lymphocytes among mammals [81]. Early life age-related changes in canine PBMC neutrophil:lymphocyte ratios and in subsets of lymphocytes identified by CD3, CD4, CD8, CD21, and gd-TCR were recently reported [82,83]. Total lymphocyte counts were markedly higher in dogs under 2 months of age compared to adults and this was reflected in neutrophil/lymphocyte ratios of 1:1.5 compared to 2.4 for adults. The percentage of CD8 positive cells increased from very low levels at birth of about 3% to 10% to 14% at 1 to 3 months of age before obtaining near 20% as adults in beagles. In this same group of dogs, B cells identified by CD21 decreased from levels between 30% and 40% at 2 months. In breeds comparing beagles, dachshunds, German shepherds, and dalmatians; for example, dalmatians


84


showed the lowest percentage of CD3 positive cells with the lowest CD4/CD8 ratio and the highest percentage of CD21 positive cells, and these values were significantly different from beagles. D. Dendritic Cells Dendritic cells (DCs), as described in humans and mice, exist as several distinguishable types based on phenotype, function, and location in tissues [84]. This heterogeneity is thought to be due to both lineage differences and maturational stages, such that lymphoid [85] and myeloid lineages [86,87] have been described that produce cells found to be phenotypically different based on tissue location [88]. These differences are also reflected in cell function where the CD11c- lymphoid lineage, or plasmocytoid DCs, produce alpha interferon (IFN-a) and limited amounts of IL-12, in contrast to myeloid lineage cells that can be subdivided into dermal or interstitial DCs and Langerhans cell (LC)–derived DCs and that do produce IL-12 but no IFN-a [88]. Within the human myeloid lineage, dermal DCs produce IL-10 whereas LC-derived DCs do not [89]. Canine dendritic cells from peripheral blood were distinguished as a subset of immune stimulatory cells distinct from monocytes as early as 1985 [90,91]. Cells of similar morphology, called veiled cells, were found in lymph drainage from skin [92] and these cells were further characterized as being antigen-presenting cells [93]. As with human DCs, and unlike rodent DCs, canine cells can express the high-affinity receptor for IgE and the group I CD1 surface glycoproteins, CD1a and CD1c, as shown in the dermis and epidermis [94] and in peripheral blood [95]. The lack of group I CD1 in mice makes extrapolations of DC function from mice to human difficult given the demonstrated importance of this group of surface molecules in immune responses [96]. Thus, the dog would appear not to suffer from this potential defect as a model for human immune-mediated diseases. In addition, the importance of the canine model for studies of DC function has been enhanced by the demonstration that DC populations can be expanded ex vivo from bone marrow cells by treatments with Fms-like tyrosine-kinase receptor 3 ligand (Flt3L), granulocyte/macrophage colonystimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-a) [97]. More recently, DCs have been generated from canine PBMC by culture with Flt-3L, GM-CSF, and interleukin-4 (IL-4) [98].

VI. CYTOKINES AND CHEMOKINES As noted above in the review of canine neutrophils, much of the data regarding canine chemokines derive from studies of cardiac reprefusion done in dogs. Thus, the m-RNA expression of the CC chemokine CCL2 (MCP)-1 [99], and more recently CXCL 10 or IP-10 [100], have been measured in endothelial cells of various tissues where infiltrates of mononuclear leukocytes have been observed in response to various inflammatory stimuli. CCL2 expression in reperfused canine heart-venule endothelium has been found to be closely associated with concentrations of reactive oxygen intermediates and inhibitable with antioxidants [101]. An indication of the shifting application of canine chemokine reagents from modeling of transplantation and cardiac research to that of spontaneous allergic disease research in the dog is the recent report of thymus and activationregulated, chemokine (TARC) m-RNA expression in lesional skin during atopic dermatitis manifestation [102]. The lack of reagents for detecting and measuring canine cytokines has been a major barrier to the fulfillment of the potential for learning from naturally occurring canine analogues to human diseases. This barrier is being removed rapidly by the application of molecular biology to the development of canine-specific reagents, and the systematic discovery of many human-specific reagents that cross-react with canine cytokines having a high degree of homology with human counterparts.



85

Thus, a recent survey of commercially available monoclonal antibodies (mAb) made against a variety of human and animal cytokines reported cross-reactivity for canine IL-8, IL-4, and IFN-g determined by flow cytometric staining of canine PBMC intracellular cytokines [103]. Even where cross-reactive mAb reagents may be lacking, it is likely that bioassay systems will be functional in cross-reactive assays, as indicated from a survey of 47 cytokines known to cross-react with cells from another species. In this survey, species biological cross-reactivity occurred when cytokine amino-acid sequence identity between two species was above 60% [104]. Because of the often highly conserved nature of many cytokines between dog and human, it has been possible to use consensus sequences, sometimes degenerate, in polymerase chain reaction (PCR) detection of mRNA expression. Thus, commercially available services for the detection of canine IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, GM-CSF, TNF-a, TNF-b, IFNg and IFN-a can be found at the University of California, Davis TaqMan‚ Service (www.vetmed.ucdavis.edu/vme/taqmanservice). Additionally, the measurement of expression of canine IL-5 mRNA has been reported in comparison of AD lesional and nonlesional skin from dogs [105] and recently canine IL-5 has been expressed as a biologically active recombinant protein [106]. Other canine cytokines that have been expressed as biologically active recombinant proteins include IL13 [107], and IL-4, IL-10, and IFN-g (available from R&D Systems, Inc. at www.RnDSystems.com). As the sequences of canine cytokines become available there are likely to be important observations to be made in how amino acid sequence relates to function in the parallel pathogenesis of disease between dog and human. One example of this may be in the sequence polymorphism of IL-13 in humans where it has been reported that a substitution of the amide amino acid, glutamine, for the base amino acid, arginine, at position 130 is associated with elevated serum total IgE and allergen-specific IgE [108]. An analysis of the amino acid sequence of canine IL-13, having 61.8% identity with human IL-13, that was derived from a cDNA library of canine PBMC [109] showed that at position 130 this canine IL-13 contained the amide amino acid, asparagine. It is not known to what degree there is polymorphism in canine IL-13 or at what frequency asparagine is present at this position. However, it would be interesting to know if the relatively high level of serum IgE often reported for dogs [110,111] could be associated with an amino acid polymorphism at IL-13 position 130 similar to that in humans.

REFERENCES 1. Kennedy, L.J. et al., Nomenclature for factors of the dog major histocompatibility system (DLA), 1998: First report of the ISAG DLA Nomenclature Committee, Anim. Genet., 31, 52, 2000. 2. Vriesendorp, H.M. et al., Joint report of 1st International Workshop on Canine Immunogenetics, Tissue Antigens, 3, 145, 1973. 3. Wagner, J.L. et al., Nucleotide sequence and polymorphism analysis of canine DRA cDNA clones, Tissue Antigens, 45, 284, 1995. 4. Wagner, J.L. et al., Molecular analysis and polymorphism of the DLA-DQA gene, Tissue Antigens, 48, 199, 1996. 5. Wagner, J.L., Burnett, R.C., and Storb, R., Molecular analysis of the DLA DR region, Tissue Antigens, 48, 549, 1996. 6. Wagner, J.L. et al., Molecular analysis and polymorphism of the DLA-DQB genes, Tissue Antigens, 52, 242, 1998. 7. Burnett, R.C. et al., Molecular analysis of six dog leukocyte antigen class I sequences including three complete genes, two truncated genes and one full-length processed gene, Tissue Antigens, 49, 484, 1997. 8. Graumann, M.B. et al., Polymorphism analysis of four canine MHC class I genes, Tissue Antigens, 51 (4 Pt 1), 374, 1998.


86


9. Wagner, J.L., Burnett, R.C., and Storb, R., Organization of the canine major histocompatibility complex: current perspectives, J. Hered., 90, 35, 1999. 10. Doxiadis, I. et al., Biochemical evidence that the DLA-B locus codes for a class II determinant expressed on all canine peripheral blood lymphocytes, Exp. Clin. Immunogenet., 6, 219, 1989. 11. Crepaldi, T. et al., Equine T lymphocytes express MHC class II antigens, J. Immunogenet., 13, 349, 1986. 12. Saalmuller, A., Weiland, F., and Reddehase, M.J., Resting porcine T lymphocytes expressing class II major histocompatibility antigen, Immunobiology, 183, 102, 1991. 13. Romano, T.A., Ridgway, S.H., and Quaranta, V., MHC class II molecules and immunoglobulins on peripheral blood lymphocytes of the bottlenosed dolphin, Tursiops truncatus, J. Exp. Zool., 263, 96, 1992. 14. Eroglu, G.E. and Kohler, P.F., Familial systemic lupus erythematosus: the role of genetic and environmental factors, Ann. Rheum. Dis., 61, 29, 2002. 15. Teichner, M. et al., Systemic lupus erythematosus in dogs: association to the major histocompatibility complex class I antigen DLA-A7, Clin. Immunol. Immunopathol., 55, 255, 1990. 16. Price, P. et al., The genetic basis for the association of the 8.1 ancestral haplotype (A1, B8, DR3) with multiple immunopathological diseases, Immunol. Rev., 167, 257, 1999. 17. Christiansen, F.T. et al., Major histocompatibility complex (MHC) complement deficiency, ancestral haplotypes and systemic lupus erythematosus (SLE): C4 deficiency explains some but not all of the influence of the MHC, J. Rheumatol., 18, 1350, 1991. 18. Wilson, A.G. et al., Comparative genetic association of human leukocyte antigen class II and tumor necrosis factor-alpha with dermatitis herpetiformis, J. Invest. Dermatol., 104, 856, 1995. 19. Dawkins, R.L. et al., Disease associations with complotypes, supratypes and haplotypes, Immunol. Rev., 70, 1, 1983. 20. Day, M.J. and Penhale, W.J., A review of major histocompatibility complex-disease associations in man and dog, Vet. Res. Commun., 11, 119, 1987. 21. Hargis, A.M. et al., Complement levels in dogs with familial canine dermatomyositis, Vet. Immunol. Immunopathol., 20, 95, 1988. 22. Doxiadis, G. et al., Statement on the nomenclature of dog C4 allotypes, Immunogenetics, 25, 167, 1987. 23. Day, M.J., Inheritance of serum autoantibody, reduced serum IgA and autoimmune disease in a canine breeding colony, Vet. Immunol. Immunopathol., 53, 207, 1996. 24. Schifferli, J.A., Ng, Y.C., and Peters, D.K., The role of complement and its receptor in the elimination of immune complexes, N. Engl. J. Med., 315, 488, 1986. 25. Nonaka, M., Evolution of the complement system, Curr. Opin. Immunol., 13, 69, 2001. 26. Sato, T. et al., Molecular characterization of a novel serine protease involved in activation of the complement system by mannose-binding protein, Int. Immunol., 6, 665, 1994. 27. Winkelstein, J.A., Sullivan, K.E., and Colten, H.R., Genetically determined disorders of the complement system., in The Metabolic and Molecular Basis of Inherited Disease, Scriver, C.R. et al., Eds., McGraw-Hill, New York, 1995, p. 3911. 28. Winkelstein, J.A. et al., Genetically determined deficiency of the third component of complement in the dog, Science, 212, 1169, 1981. 29. Ameratunga, R. et al., Molecular analysis of the third component of canine complement (C3) and identification of the mutation responsible for hereditary canine C3 deficiency, J. Immunol., 160, 2824, 1998. 30. Blum, J.R. et al., The clinical manifestations of a genetically determined deficiency of the third component of complement in the dog, Clin. Immunol. Immunopathol., 34, 304, 1985. 31. Cork, L.L.C. et al., Membranoproliferative glomerulonephritis in dogs with a genetically determined deficiency of the third component of complement, Clin. Immunol. Immunopathol., 60, 455, 1991. 32. Patterson, R., Chang, W.W.Y., and Pruzansky, J.J., Some characteristics of canine precipitating and non-precipitating antibody, Immunology, 7, 150, 1964. 33. Patterson, R. et al., The immunologic resonse of dogs to soluble protein antigens., J. Immunol., 91, 129, 1963. 34. Patterson, R., Pruzansky, J.J., and Chang, W.W.Y., Spontaneous canine hypersensitivity to ragweed. Characterization of the serum factor transferring skin, bronchial and anaphylactic sensitivity., J. Immunol., 90, 35, 1963.



87

35. Patterson, R., Pruzansky, J.J., and Janis, B., Biologic activity of canine nonprecipitating and precipitating antibody, J. Immunol., 93, 51, 1964. 36. Patterson, R., Roberts, M., and Pruzansky, J.J., Types of canine serum immunoglobulins, J. Immunol., 101, 684, 1968. 37. Patterson, R. and Sparks, D.B., The passive transfer to normal dogs of skin reactivity, asthma and anaphylaxis from a dog with spontaneous ragweed pollen hypersensitivity., J. Immunol., 88, 262, 1962. 38. Johnson, J.S. and Vaughan, J.H., Canine immunoglobulins. I. Evidence for six immunoglobulin classes, J. Immunol., 98, 923, 1967. 39. Johnson, J.S., Vaughan, J.H., and Swisher, S.N., Canine immunoglobulins. II. Antibody activities in six immunoglobulin classes, J. Immunol., 98, 935, 1967. 40. Halliwell, R.E. et al., Physicochemical properties of canine IgE, Transplant. Proc., 7, 537, 1975. 41. Halliwell, R.E., Schwartzman, R.M., and Rockey, J.H., Antigenic relationship between human IgE and canine IgE, Clin. Exp. Immunol., 10, 399, 1972. 42. Reynolds, H.Y. and Johnson, J.S., Quantitation of canine immunoglobulins, J. Immunol., 105, 698, 1970. 43. Mazza, G. et al., The separation and identification by monoclonal antibodies of dog IgG fractions, J. Immunol. Methods, 161, 193, 1993. 44. Mazza, G. et al., Preparation of monoclonal antibodies specific for the subclasses of canine IgG, Res. Vet. Sci., 57, 140, 1994. 45. Mazza, G. et al., Development of an enzyme-linked immunosorbent assay for the detection of IgG subclasses in the serum of normal and diseased dogs, Res. Vet. Sci., 57, 133, 1994. 46. Briere, F. et al., Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1 and IgG3, J. Exp. Med., 179, 757, 1994. 47. Tang, L. et al., Cloning and characterization of cDNAs encoding four different canine immunoglobulin gamma chains, Vet. Immunol. Immunopathol., 80, 259, 2001. 48. Wang, C.Y. et al., Synthetic IgE peptide vaccine for immunotherapy of allergy, Vaccine, 21, 1580, 2003. 49. Gebhard, D. et al., Canine IgE monoclonal antibody specific for a filarial antigen: production by a canine x murine heterohybridoma using B cells from a clinically affected lymph node, Immunology, 85, 429, 1995. 50. Lazarus, S.C., Mast cell-derived mediators and their role in cell-to-cell interactions in the lung, Respiration, 50 (Suppl. 2), 17, 1986. 51. DeVinney, R. and Gold, W.M., Establishment of two dog mastocytoma cell lines in continuous culture, Am. J. Respir. Cell Mol. Biol., 3, 413, 1990. 52. Lazarus, S.C. et al., Isolated canine mastocytoma cells: propagation and characterization of two cell lines, Am. J. Physiol., 251 (6 Pt 1), C935, 1986. 53. Caughey, G.H., Raymond, W.W., and Wolters, P.J., Angiotensin II generation by mast cell alpha- and beta-chymases, Biochim. Biophys. Acta., 1480, 245, 2000. 54. Vanderslice, P. et al., Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation, Biochemistry, 28, 4148, 1989. 55. Wolters, P.J. et al., Dipeptidyl peptidase I cleaves matrix-associated proteins and is expressed mainly by mast cells in normal dog airways, Am. J. Respir. Cell Mol. Biol., 22, 183, 2000. 56. Fang, K.C. et al., Mast cell expression of gelatinases A and B is regulated by kit ligand and TGFbeta, J. Immunol., 162, 5528, 1999. 57. Hammerberg, B., Olivry, T., and Orton, S.M., Skin mast cell histamine release following stem cell factor and high-affinity immunoglobulin E receptor cross-linking in dogs with atopic dermatitis, Vet. Dermatol., 12, 339, 2001. 58. Jackson, H.A. and Hammerberg, B., Evaluation of a spontaneous canine model of immunoglobulin E-mediated food hypersensitivity: dynamic changes in serum and fecal allergen-specific immunoglobulin E values relative to dietary change, Comp. Med., 52, 316, 2002. 59. Brazis, P. et al., IgE enhances Fc epsilon RI expression and IgE-dependent TNF-alpha release from canine skin mast cells, Vet. Immunol. Immunopathol., 85, 205, 2002. 60. Kube, P. et al., Distribution, density and heterogeneity of canine mast cells and influence of fixation techniques, Histochem. Cell Biol., 110, 129, 1998. 61. Dreyer, W.J. et al., Neutrophil activation and adhesion molecule expression in a canine model of open heart surgery with cardiopulmonary bypass, Cardiovasc. Res., 29, 775, 1995.


88


62. Burns, A.R. et al., Chemotactic factors stimulate CD18-dependent canine neutrophil adherence and motility on lung fibroblasts, J. Immunol., 156, 3389, 1996. 63. Manning, A.M. et al., Cloning and comparative sequence analysis of the gene encoding canine intercellular adhesion molecule-1 (ICAM-1), Gene, 156, 291, 1995. 64. Dore, M. et al., P-selectin mediates spontaneous leukocyte rolling in vivo, Blood, 82, 1308, 1993. 65. Entman, M.L. et al., Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18dependent mechanism, J. Clin. Invest., 85, 1497, 1990. 66. Hawkins, H.K. et al., Acute inflammatory reaction after myocardial ischemic injury and reperfusion. Development and use of a neutrophil-specific antibody, Am. J. Pathol., 148, 1957, 1996. 67. Shappell, S.B. et al., Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen peroxide production by human and canine neutrophils, J. Immunol., 144, 2702, 1990. 68. Abbassi, O. et al., Canine neutrophil margination mediated by lectin adhesion molecule-1 in vitro, J. Immunol., 147, 2107, 1991. 69. Kukielka, G.L. et al., Role of early reperfusion in the induction of adhesion molecules and cytokines in previously ischemic myocardium, Mol. Cell. Biochem., 147, 5, 1995. 70. Lu, H., Ballantyne, C., and Smith, C.W., LFA-1 (CD11a/CD18) triggers hydrogen peroxide production by canine neutrophils, J. Leukoc. Biol., 68, 73, 2000. 71. Petersen, T.K., Smith, C.W., and Jensen, A.L., Characterization of the priming effect by pituitary canine growth hormone on canine polymorphonuclear neutrophil granulocyte function, Clin. Diagn. Lab. Immunol., 7, 226, 2000. 72. Kijas, J.M. et al., A missense mutation in the beta-2 integrin gene (ITGB2) causes canine leukocyte adhesion deficiency, Genomics, 61, 101, 1999. 73. Giger, U. et al., Deficiency of leukocyte surface glycoproteins Mo1, LFA-1, and Leu M5 in a dog with recurrent bacterial infections: an animal model, Blood, 69, 1622, 1987. 74. Chabanne, L. et al., Screening of 78 monoclonal antibodies directed against human leukocyte antigens for cross-reactivity with surface markers on canine lymphocytes, Tissue Antigens, 43, 202, 1994. 75. Cobbold, S. and Metcalfe, S., Monoclonal antibodies that define canine homologues of human CD antigens: summary of the First International Canine Leukocyte Antigen Workshop (CLAW), Tissue Antigens, 43, 137, 1994. 76. Williams, D.L., Studies of canine leukocyte antigens: a significant advance in canine immunology, Vet. J., 153, 31, 1997. 77. Brodersen, R. et al., Analysis of the immunological cross reactivities of 213 well characterized monoclonal antibodies with specificities against various leucocyte surface antigens of human and 11 animal species, Vet. Immunol. Immunopathol., 64, 1, 1998. 78. Chambers, C.A. and Allison, J.P., Costimulatory regulation of T cell function, Curr. Opin. Cell. Biol., 11, 203, 1999. 79. Khatlani, T.S. et al., Molecular cloning and sequencing of canine T-cell costimulatory molecule (CD28), Vet. Immunol. Immunopathol., 78, 341, 2001. 80. Yang, S. and Sim, G.K., New forms of dog CD80 and CD86 transcripts that encode secreted B7 molecules, Immunogenetics, 50, 349, 1999. 81. Lilliehook, I., Johannisson, A., and Hakansson, L., Expression of adhesion and Fcgamma-receptors on canine blood eosinophils and neutrophils studied by anti-human monoclonal antibodies, Vet. Immunol. Immunopathol., 61, 181, 1998. 82. Faldyna, M. et al., Lymphocyte subsets in peripheral blood of dogs — a flow cytometric study, Vet. Immunol. Immunopathol., 82, 23, 2001. 83. Toman, M. et al., Postnatal development of leukocyte subset composition and activity in dogs, Vet. Immunol. Immunopathol., 87, 321, 2002. 84. Banchereau, J. and Steinman, R.M., Dendritic cells and the control of immunity, Nature, 392, 245, 1998. 85. Banchereau, J. et al., Immunobiology of dendritic cells, Annu. Rev. Immunol., 18, 767, 2000. 86. Caux, C. et al., GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells, Nature, 360, 258, 1992. 87. Romani, N. et al., Proliferating dendritic cell progenitors in human blood, J. Exp. Med., 180, 83, 1994. 88. Pulendran, B. et al., Modulating the immune response with dendritic cells and their growth factors, Trends Immunol., 22, 41, 2001.



89

89. de Saint-Vis, B. et al., The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation, J. Immunol., 160, 1666, 1998. 90. Goodell, E.M., Blumenstock, D.A., and Bowers, W.E., Canine dendritic cells from peripheral blood and lymph nodes, Vet. Immunol. Immunopathol., 8, 301, 1985. 91. Aprile, J. and Deeg, H.J., Ultraviolet irradiation of canine dendritic cells prevents mitogen-induced cluster formation and lymphocyte proliferation, Transplantation, 42, 653, 1986. 92. Galkowska, H. and Olszewski, W.L., Cellular composition of lymph in experimental lymphedema, Lymphology, 19, 139, 1986. 93. Dabrowski, M.I., Galkowska, H., and Olszewski, W.L., Functional characteristics of veiled cells from canine prenodal lymph, Immunobiology, 178, 316, 1989. 94. Olivry, T. et al., Langerhans cell hyperplasia and IgE expression in canine atopic dermatitis, Arch. Dermatol. Res., 288, 579, 1996. 95. Jackson, H.A., Orton, S.M., and Hammerberg, B., IgE is present on peripheral blood monocytes and B cells in normal dogs and dogs with atopic dermatitis but there is no correlation with serum IgE concentrations, Vet. Immunol. Immunopathol., 85, 225, 2002. 96. Porcelli, S.A. and Modlin, R.L., The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids, Annu. Rev. Immunol., 17, 297, 1999. 97. Hagglund, H.G. et al., Ex vivo expansion of canine dendritic cells from CD34+ bone marrow progenitor cells, Transplantation, 70, 1437, 2000. 98. Catchpole, B., Stell, A.J., and Dobson, J.M., Generation of blood-derived dendritic cells in dogs with oral malignant melanoma, J. Comp. Pathol., 126, 238, 2002. 99. Birdsall, H.H. et al., Complement C5a, TGF-beta 1, and MCP-1, in sequence, induce migration of monocytes into ischemic canine myocardium within the first one to five hours after reperfusion, Circulation, 95, 684, 1997. 100. Frangogiannis, N.G. et al., Induction of the synthesis of the C-X-C chemokine interferon-gammainducible protein-10 in experimental canine endotoxemia, Cell Tissue Res., 302, 365, 2000. 101. Lakshminarayanan, V. et al., Reactive oxygen intermediates induce monocyte chemotactic protein-1 in vascular endothelium after brief ischemia, Am. J. Pathol., 159, 1301, 2001. 102. Maeda, S. et al., Lesional expression of thymus and activation-regulated chemokine in canine atopic dermatitis, Vet. Immunol. Immunopathol., 88, 79, 2002. 103. Pedersen, L.L.G. et al., Identification of monoclonal antibodies that cross-react with cytokines from different animal species, Vet. Immunol. Immunopathol., 88, 111, 2002. 104. Scheerlinck, J.P., Functional and structural comparison of cytokines in different species, Vet. Immunol. Immunopathol., 72, 39, 1999. 105. Olivry, T. et al., Toward a canine model of atopic dermatitis: amplification of cytokine-gene transcripts in the skin of atopic dogs, Exp. Dermatol., 8, 204, 1999. 106. Yang, S. et al., Canine interleukin-5: molecular characterization of the gene and expression of biologically active recombinant protein, J. Interferon Cytokine Res., 21, 361, 2001. 107. Tang, L. et al., Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergenspecific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs, Vet. Immunol. Immunopathol., 83, 115, 2001. 108. Leung, T.F. et al., A polymorphism in the coding region of interleukin-13 gene is associated with atopy but not asthma in Chinese children, Clin. Exp. Allergy, 31, 1515, 2001. 109. Yang, S., Boroughs, K.L., and McDermott, M.J., Canine interleukin-13: molecular cloning of fulllength cDNA and expression of biologically active recombinant protein, J. Interferon Cytokine Res., 20, 779, 2000. 110. Hammerberg, B. et al., Auto IgG anti-IgE and IgG x IgE immune complex presence and effects on ELISA-based quantitation of IgE in canine atopic dermatitis, demodectic acariasis and helminthiasis, Vet. Immunol. Immunopathol., 60, 33, 1997. 111. DeBoer, D.J., Ewing, K.M., and Schultz, K.T., Production and characterization of mouse monoclonal antibodies directed against canine IgE and IgG, Vet. Immunol. Immunopathol., 37, 183, 1993.



CHAPTER

5

Rat Immune System Kevin J. McElwee and Birte Steiniger

CONTENTS I. II. III. IV.

Introduction ..........................................................................................................................91 The Laboratory Rat..............................................................................................................92 Major Histocompatibility Complex .....................................................................................94 Immune Organs....................................................................................................................95 A. Thymus ........................................................................................................................95 B. Spleen...........................................................................................................................96 V. Antibodies ............................................................................................................................98 VI. Complement System ............................................................................................................99 VII. Immune Cells .......................................................................................................................99 A. Mast Cells ..................................................................................................................100 B. Natural Killer Cells (NK Cells) ................................................................................100 C. Lymphocytes ..............................................................................................................101 D. Monocytes/Macrophages ...........................................................................................101 E. Dendritic Cells ...........................................................................................................102 VIII. Cytokines and Chemokines ...............................................................................................103 IX. Conclusions ........................................................................................................................105 References ......................................................................................................................................105

I. INTRODUCTION Rats belong to the family Muridae with the brown, or Norway, rat classified as Rattus norvegicus and the black, or house, rat as Rattus rattus. The Norway rat is thought to have originated in temperate Asia and is noted for being extremely aggressive. The two species cannot interbreed, and ultimately the Norway rat replaced the smaller and less aggressive black rat in Europe. Norway rats made their way to the United States by ship around 1775, again replacing the already established black rat. Soon after the introduction of the Norway rat into Western Europe, albino mutants made their appearance in wild populations. This spontaneous coat color mutation is a fairly common occurrence in wild mammals and particularly in rats. Rats initially entered captivity in the early 1800s as large numbers were collected for the blood sport of rat baiting. The hobby of rat breeding developed later with albino and mutant coat color 0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

91


92


rats likely derived from these wild mutants collected for baiting. Rats were the first mammalian species domesticated for use in scientific research [1]. The studies of adrenalectomized rats by Philipeaux [2] in France in 1856 and the neuroanatomical studies by Hatai at the University of Chicago in the early 1890s represent the first known biomedical experiments using rats. The albino rats used in these early studies probably originated from European stocks at the University of Geneva brought to America by the neuropathologist Adolf Meyer [3]. It is speculated that these albino rats of European origin eventually comprised the foundation colony for the Wistar Institute in Philadelphia. However, it has also been indicated that the origins of the Wistar Institute rat colony may have been mutants selected from wild rats captured in the United States [4]. In 1906, the Wistar Institute initiated the standardization of albino and coat color variants that produced the first inbred laboratory rat strains. The first inbred rat strain is generally regarded as the “King albino,” now called the PA strain, produced at the Wistar Institute. Although rat research was initiated in Europe some time before the development of the Wistar Institute rat colonies, the Wistar bloodline is the basis of most laboratory rat strains used today (Table 5.1). II. THE LABORATORY RAT Until relatively recently, rats were the primary laboratory animals employed in experimental research and there is an extensive repertoire of rat reagents available for studies [5], but with the advent of molecular genetics, mice have recently become the species of choice for laboratory research. After the mouse, the laboratory rat is still the most extensively used experimental animal, particularly in the research fields of pharmacology, physiology, neuroscience, aging, transplantation, and immunology. There are many outbred and inbred strains of laboratory rats with over 200 listed [6–8] and likely many more that are currently unlisted. The most frequently used inbred strains include the Fisher 344 (F344), Brown Norway (BN), Lewis (LEW), and Wistar-Furth (WF). A few of the more common outbred strains include the Sprague–Dawley (SD), Wistar (WI) and Long–Evans (LE); the latter is often called the hooded (or piebald) rat. However, most strains are comprised of small, isolated colonies used only by a small number of investigators. In contrast to mouse strains, rat strain distribution is geographically limited with just a handful of strains distributed globally, primarily by commercial entities, and used regularly by investigators. This presents the problem that many strains with unique characteristics of importance to particular fields of research are not readily available to scientists. To remedy this situation, the National Institutes of Health in collaboration with academic and commercial interests, launched the Rat Resource and Research Center (RRRC). While only established in 2001 and still in an embryonic phase, the RRRC should eventually serve as a centralized repository for the distribution of characterized inbred, hybrid, and mutant rats to investigators (www.radil.missouri.edu/rrrc/). The RRRC and other online initiatives for developing and distributing laboratory rat information, such as the rat genome sequencing project (www.hgsc.bcm.tmc.edu/projects/rat/), rat genome database (rgd.mcw.edu/), “RatMap” (ratmap.gen.gu.se/), and others [9,10] may lead to an expansion in rat strains and more rat-focused research in the future. The rat’s larger body size enables microsurgical manipulation that is difficult or impossible in mice. For this reason, rat models dominated in organ transplantation and rejection studies until quite recently [11]. Mouse models generally dominate in fields of tumor research, as most rat strains are relatively less susceptible to spontaneous tumorigenesis and have a longer latent period for tumor induction, particularly skin cancers, compared to rabbits and mice [12]. There are, however, a few notable exceptions. F344 rats are relatively susceptible to spontaneous leukemia, pituitary, and testicular tumors [13,14]. The BDII rat strain is susceptible to the development of endometrial carcinomas [15]. BN rats have a high bladder tumor incidence along with a variety of other tumors [16]. The Tsc2 knockout (Eker) rat is an important renal cell carcinoma model [17]. In addition,


RAT IMMUNE SYSTEM

93

Table 5.1 Rat Strain Derivation ABH AO AS B BB BRUFO BuF GH GK JC K KYN LEW AGUS APR BDE BD I – BD X BDE BS A28807 A7322 A990 A35322 ACH BN ALB CAS CAR DA FH NEDH OM PVG TO

Rat Strains Directly Descended from Wistar Institute Stocks LOU SR MNE W MNR WA MR WAB MHS WAG MNS WBN MW WE ODU WF OKA WKA PA WKY RA WM SHR WN Rat Strains Descended from Crosses between Wistar and Other Stocks DEBR MAXX GHA OLTEF IS SS LE McCollum (outbred) LGE Sprague-Dawley (outbred) LN Rat Strains Descended from Columbia University Stocks ACI COP ACP F344 AUG M520 Avon 34986 Z61 Other Non–Wistar Derived Stocks Descended from Philadelphia (?), USA Descended from Albany Medical College, USA Descended from Michigan State University, USA Descended from Michigan State University, USA Descended from Oak Ridge National Laboratory, USA Descended from University of Michigan, USA Descended from University of Chicago, USA Descended from Connecticut Agricultural Experiment Station, USA Descended from King’s College of Household Science, UK Descended from Hokkaido University, Japan

Sources: From Lindsey, J.R., in The Laboratory Rat, Vol. 1, Biology and Diseases, Academic Press, New York, 1979 [3], and Greenhouse D.D. et al., in Genetic Monitoring of Inbred Strains of Rats, Gustav Fischer, Stuttgart, 1990 [8], with permission.

WF rats and outbred SD rats have a relatively high incidence of mammary and pituitary tumors [18–23]. The rat is widely used to study mechanisms involved in human disease pathogenesis. While laboratory rats have disadvantages as compared to mice, particularly limited transgenic rat development and availability, there are a significant number of rat models for inflammatory and autoimmune diseases for which there is no direct mouse equivalent. Lewis rats (LEW) are susceptible to several autoimmune conditions including experimental autoimmune encephalomyelitis (EAE) [24], autoimmune complex nephritis (AIC), experimental allergic neuritis (EAN) [25,26], and experimental autoimmune uveitis (EAU) [27]. Rat models dominate over mouse models in chronic relapsing EAE (CREAE) [28] and myasthenia gravis (EAMG) [29]. Several rat models for induced arthritis are available [30,31], and the BioBreeding (BB) rat is a popular model for spontaneous type 1 diabetes [32].


94


III. MAJOR HISTOCOMPATIBILITY COMPLEX The locus of the alloantigenic system in the rat is designated by RT followed by a number. The numbers are generally assigned in the order of the loci discovery. The rat major histocompatibility complex (MHC) is officially designated RT1. In early literature, RT1 was also designated as Ag-B or H-1. Haplotypes of inbred strains of rats are designated by superscripted lower-case letters from a to u omitting r (e.g., RT1a). In early literature the same haplotype designations followed H-1 (e.g., H-1a), but with Ag-B numbers were given such that the new designation of RT1a equates to the original designation of Ag-B4 (RT to Ag-B equivalents: 1=1, a=4, b=6, c=5, d=9, f=10, g=7, u=2). Used alone, m indicates the haplotype of the MNR strain (RT1m). When used with another haplotype symbol, m indicates a mutant form of that haplotype (e.g., RT1lm1). A recombinant is designated by the superscript haplotype symbol r followed by a series number (e.g., RT1r1) and a variant haplotype is designated by adding the letter v, and a series number to the haplotype (e.g., F344 = RT1lv1). A haplotype of a wild rat is designated by a superscript w, followed by a series number (e.g., RT1w1). Individual loci are designated by a capital letter and the allele is designated by a superscript denoting the haplotype from which the locus originated. The order in which the letters are written indicates the sequence of loci on the chromosome, as determined by mapping studies (e.g., RT1.AaBaDaEaCa). While RT1 refers to the MHC, RT2 and RT3 are expressed on erythrocytes and are primarily used for monitoring inbred strain purity. RT8 is also an erythrocyteexpressed antigen used to differentiate SHR substrains. RT6 and RT7 are lymphocyte-expressed antigens. These and other details on nomenclature conventions in the laboratory rat can be found in several publications [33,34]. The MHC loci designation for some of the most popular rat strains are provided in Table 5.2. The MHC plays a central role in the regulation of immune activity [35–37]. As with the MHC of mice and humans, rat MHC genes function by presenting antigenic peptides to the immune system by MHC class I or MHC class II cell-surface molecules [38]. In rats, MHC class I and II expressions become evident in the 2nd to 3rd month postpartum [39]. The MHC region is located on rat chromosome 20 on the telomeric part of the short arm and is 3.7 Mb in size [40–42]. Although the overall genetic diversity between inbred rat strains is higher than that of mouse strains [43], the polymorphism for the MHC across rat strains is lower than that observed in mice and humans [44,45]. Analysis of the rat MHC was based on inbred strains, RT1 congenic strains, and recombinants in the same way as for the mouse MHC (H-2) complex. As with other species, the rat MHC contains equivalent class I, II, and III regions and shows a large degree of genomic conservation with both mice and humans [38]. Similar to mice, but in contrast to humans, the rat MHC contains two class I regions. The rat’s telomeric class I region is equivalent to the human class I HLA while the second Table 5.2 Rat MHC Specificities Strain Designation A28807 ALB BDIX1 BN BUF DA F344

RT1

Strain Designation

RT1c RT1b RT1d RT1n RT1b RT1av1 RT1lv1

LE LEW LOU PVG SHR WF WKY2

RT1 RT1uv2 RT1l RT1u RT1c RT1k RT1u RT1k or RT1l

Notes: BDIX does not express RT1.B [289]. Some substrains of WKY are RT1k while others are RT1l, suggesting that contamination of some inbred substrains has occurred in their history. Sources: Kren, V., Transplantation, 17, 148, 1974 [290]; Kunz, H.W., I.L.A.R. News, 33, 1, 1991 [291]; and Arenas, O. et al., J. Immunogenet., 8, 307, 1981 [292], with permission.


RAT IMMUNE SYSTEM

Figure 5.1

95

Schematic structure of the MHC in rats compared to humans.

class I region is located centromerically from the classical class II region [46] (Figure 5.1). The detailed structure of the rat MHC and comparison to mice and humans is comprehensively described elsewhere [38,42,46–51].

IV. IMMUNE ORGANS In terms of cell volume, rat lymph nodes contain on average 1.8 ¥ 109 cells per organ, the spleen 1.0 ¥ 109, Peyer’s patches 0.3 ¥ 09 per organ, and the thymus 1.1 ¥ 109 [52–57]. By weight, 3% of the adult rat is bone marrow [58]. B and T lymphocytes proliferate locally, both in central lymphoid organs such as the thymus and the bone marrow, and in peripheral lymphoid organs such as the spleen, lymph nodes, and Peyer’s patches. Studies on immune cell migration are predominantly derived from rat studies with the first understanding of lymphocyte recirculation published in 1959 [59]. The time required for most lymphocytes to cross lymph nodes from blood to lymph ranges from 4 to 18 hours. On average, lymphocytes take 5 to 10 minutes to cross high endothelial venules when entering lymph nodes from the blood [60–62]. T lymphocytes quickly take up residence in the paracortex, while migrating B cells may shuttle between the paracortex and cortex [63,64]. Migration from these organs to the periphery usually takes less than 24 hours [65]. A. Thymus Since the first suggestion that the thymus could be involved in the maturation of immune cells, this organ has been identified as a key organ in immune system function. The thymus shapes the nature of the immune system through T-cell production, “education,” and programmed cell death [66–68]. The immature stages of T-cell differentiation occur in the thymic cortex while final maturation into T-cell receptors (TCR) expressing CD4+ or CD8+ cells occurs in the medulla [69]. Recent studies have explored the changes that occur within the rat thymus with age [70,71]. The changes involve a complex remodeling over time that activates a series of interactions between various cell populations that ultimately results in progressive thymic involution. The expression pattern of cell surface receptors can be used to define the development of T cells in the thymus. In all mammals, the development of thymocytes follows the same general course employing similar cell surface receptors and differentiation programs as demonstrated when rat T cells successfully complete full differentiation in the thymus of SCID mice [72]. T cells in their most immature form are characteristically TCR-/CD4-/CD8- triple negative. These cells


96


become positive for a single marker, either CD4-/CD8+ or CD4+/CD8-, and then progress to doublepositive CD4+/CD8+ cells. Mature CD4+/CD8- or CD4-/CD8+ cells develop from these doublepositive cells and are released to the periphery [73,74]. In rats, CD4-/CD8+ cells can differentiate into CD4+/CD8+ cells in vitro in the absence of stimulation, and CD4+/CD8+ cells can be induced to develop into mature CD4-/CD8+ cells through IL-2 [75]. This is distinct from mouse thymocyte development, where IL-2 is suggested to play no significant role in cell maturation [76]. Ligation of TCR can induce mouse CD4+/CD8+ cells to develop into mature CD4+/CD8- lymphocytes [77]. These and other studies are suggestive that while mouse CD4+/CD8+ thymocytes are committed to becoming CD4+ cells, rat CD4+/CD8+ thymocytes are committed to becoming mature CD8+ cells in the absence of external education [78]. It is likely that rat and mouse thymocytes interpret the same external stimuli in different ways. Stimulation of mouse or rat thymocytes with PMA and ionomycin to bypass TCR signaling results in downregulation of CD8 marker in mice, but CD4 in rats. B. Spleen The spleen of rats and other mammals is a secondary lymphatic organ that harbors more or less motile leukocytes and erythrocytes in a reticular connective tissue stroma composed of fibroblasts and reticular fibers. The spleen consists of two large microanatomic compartments, the white pulp and red pulp. The white pulp is formed by dense accumulations of different lymphocyte populations, while the red pulp is composed of connective tissue cords and sinuses. The rat spleen has a unique circulatory system that differs from all other organs. There are two parallel pathways of blood flow: closed and open circulation. In the closed system, the finer branches of the arterial vessels are supposed to supply the splenic sinuses, a specialized form of capillaries without continuous basement membrane and with slits between the endothelial cells [79]. These sinuses then continue into the venous part of the splenic vasculature. The open circulation on the other hand terminates in arterioles that pour blood into the open spaces of the splenic red pulp cords. In this compartment, the blood percolates through the labyrinth of the reticular connective tissue without an endothelial barrier and thus comes into direct contact with a large population of macrophages inhabiting the cords. The splenic cords either drain through the slits into the splenic sinuses or their blood may even pass directly into the open beginnings of red pulp venules and veins [80]. In rats, the white pulp consists of three different lymphocyte compartments arranged around the finer branches of the splenic artery, the so-called central arterioles [80]. These vessels are covered by a concentric sheath composed primarily of T lymphocytes, called the periarteriolar lymphatic sheath (PALS). At regular intervals, hemispherical accumulations of migratory B lymphocytes are attached to this T-cell sheath, called the follicles. Finally, both the periarteriolar lymphatic sheath and the follicles are delimited from the red pulp by a broad sheath of specialized B lymphocytes, the marginal zone. Thus, the white pulp consists of a central T-cell region and two more peripherally situated B-cell regions of a different shape. In comparison to mice and humans, the marginal zone is especially prominent in rats [81]. In rats and mice, but not in humans, this compartment is clearly delimited from the T-cell region and the follicles by a tortuous, capillarylike blood vessel, the marginal sinus. The outer border of the marginal zone to the open spaces of the red pulp is, however, indistinct. Thus, the rat marginal zone always contains a number of evenly distributed erythrocytes and platelets. Some authors tend to regard it as a separate compartment somehow related to the open splenic circulation. There are three essential cell types that can be distinguished within the PALS: T lymphocytes, sessile stromal cells or fibroblasts (formerly designated as fibroblastic reticulum cells), and interdigitating dendritic cells (IDCs) derived from the bone marrow. In addition, the outer PALS is also a migration compartment for B lymphocytes in rats [82–84] and it may contain substantial numbers of B cells among the always predominating T cells. The evenly distributed branched IDCs are the


RAT IMMUNE SYSTEM

97

main cell population positive for MHC class II antigens in the inner PALS. They are regarded as the antigen-presenting cells of the T-cell area, although their function may also comprise the downmodulation of T-cell functions [85]. The fibroblasts of the PALS are poorly defined. In mice and humans, they have been shown to express certain adhesion molecules involved in lymphocyte homing and to secrete a number of chemokines attracting T cells and dendritic cells from the blood [86,87]. The follicles are occupied by migratory B cells. It is believed that the decisive stromal cells of this compartment are the follicular dendritic cells (FDCs), which secrete chemokines attracting recirculating B cells and certain T cells [88]. However, the derivation of the FDCs is not entirely clear. FDCs express complement and Fc receptors and are able to retain immune complexes on their surfaces for astonishingly prolonged periods [89]. Follicles may either appear as primary follicles without any special internal structure or as secondary follicles with pale-staining germinal centers. In secondary follicles the germinal center displaces the remainder of the primary follicle (the small recirculating B lymphocytes) to the follicular periphery. This shell of small B cells is then called the mantle zone or corona. Germinal centers arise when B lymphocytes have met their cognate antigen somewhere in the body. Fragments of this antigen may also have been presented to antigen-specific T cells in the PALS by MHC class II–positive IDCs. This leads to T-cell activation and movement of T cells to the outer PALS. Antigenprimed B cells expressing MHC class II molecules may then also enter the outer PALS from the marginal zone, where they arrive from the blood (see below). The antigen-specific interaction of activated T cells and antigen-presenting B cells in the outer PALS provokes B-cell differentiation. These B cells then develop into low affinity plasma cells at the outer border of the PALS and secrete imunoglobulin for a short period [90,91]. The immunoglobulin forms antigen-antibody complexes that are deposited on the surface of FDCs. If primed recirculating B cells enter follicles containing their cognate antigen in immune complexes, they are arrested in the follicle and start cell division, giving rise to a germinal center. Full-blown germinal centers consist of a dark zone with centroblasts and a light zone containing centrocytes. During division, centroblasts hypermutate the variable antigen-binding regions of their immunoglobulin genes [92]. They may then develop into centrocytes if — by chance — the hypermutation process has led to the expression of surface immunoglobulin with an increased affinity for antigen. Cells with reduced or unchanged affinity of immunoglobulin undergo apoptosis and are phagocytosed by the so-called tingible body macrophages of the germinal center. With the help of germinal center T cells and FDCs, centrocytes further develop into B-memory cells or into plasma cell precursors and then leave the germinal center to distribute in the body. B-memory cells may colonize the splenic marginal zone and several other locations [91], while the majority of plasma cells reside in the bone marrow. Thus, the germinal centers are compartments for immunoglobulin affinity maturation in the spleen and in other secondary lymphatic organs after initial low-affinity immunoglobulin has been produced and distributed systemically [90,93,94]. In rats and mice the marginal zone is a dual-purpose compartment. First, the marginal zone serves as an entry site for antigens and for migratory T and B lymphocytes arriving from the blood stream via the marginal sinus. Second, it harbors a specialized type of more sessile B cells, the marginal zone B lymphocytes, which are recruited from recirculating precursors [91,95–99]. Recirculating B and T lymphocytes are assumed to cross the leaky outer wall of the marginal sinus to first enter the marginal zone. From there T cells move into the PALS to rest for a shorter or longer period, while B cells migrate along the outer PALS to enter the primary follicles or the mantle zone of secondary follicles [91]. The typical marginal zone B cells have a phenotype different from that of small recirculating B cells [91,98]. In rats, marginal zone B cells may function as Bmemory cells for T-cell–dependent antibody reactions, but they are also decisive as precursors of plasma cells for immediate antibody formation against so-called T-independent type 2 antigens [96,100]. These antigens are distinguished by repetitive polysaccharide epitopes such as those present in the capsules of certain gram-negative bacteria that may cause a fulminant septic syndrome


98


in splenectomized humans [101,102]. In addition, the marginal zone may also contain polyreactive, autoreactive and xenoreactive B cells recognizing sugar and lipid determinants [99,103–105]. On confrontation with specific antigen or after polyclonal activation, marginal zone B cells quickly leave their compartment, enter the outer PALS, and differentiate into plasmablasts [106–108]. From the outer PALS the plasmablasts enter the splenic red pulp or other locations and give rise to IgMproducing plasma cells. Thus, marginal zone B cells play a decisive role in the immediate production of large amounts of IgM antibodies to particulate antigens or certain bacteria and viruses present in the blood. The marginal zone consists of ill-defined stromal cells, various slightly activated types of B cells such as polysaccharide-specific, polyreactive, and memory-B cells, recirculating naïve T and B cells, and immature dendritic cells arriving from the blood. In addition, there are two phenotypically defined populations of macrophages, the marginal metallophilic macrophages and the marginal zone macrophages. The marginal metallophilic macrophages line the marginal sinus, while the marginal zone macrophages are evenly scattered throughout the marginal zone. In mice, both populations can be differentially stained with monoclonal antibodies, while in rats [109,110] they both react with antibodies directed against sialoadhesin (CD169). These macrophages phagocytose fluorescent polysaccharides injected intravenously in rats, but whether and how they interact with the special marginal zone B cells remains unknown [111,112]. A minimum of four or five monoclonal antibodies permits visualization of the compartments mentioned above and are recommended for a first overview of rat spleens. Monoclonal antibody R73, which is directed against a constant epitope on the T-cell antigen-receptor beta chain [113] may be used for demonstration of the PALS. Follicles and marginal zone react with antibody Ox33 specific for the B-cell restricted form of the leukocyte common antigen CD45R [114]. Finally, red pulp macrophages and an ill-defined macrophage population at the outer border of the PALS are revealed by antibody ED2 detecting a member of the scavenger receptor family CD163 [109,115,116]. Marginal metallophilic macrophages and marginal zone macrophages stain with antibody ED3 against sialoadhesin [109,110] or, alternatively, with antibody KiM9R of unknown specificity [117]. In rats, mice, and humans, the spleen is the decisive organ for immunologic control of the blood. The marginal zone, which is most easily studied in rats because of its prominence in tissue sections, is regarded as a spleen-specific compartment involved in this function. Marginal zone B lymphocytes are capable of almost immediate plasma cell differentiation and antibody secretion against certain T-cell independent antigens and thus enable the spleen to start a unique emergency reaction before the more slowly progressing reactions against T-cell–dependent protein antigens take over. In addition, the red pulp of the spleen provides a large phagocytic compartment directly in contact with blood flowing slowly in the open circulation. This compartment may contribute decisively to remove opsonized bacteria and other pathogens as well as blood cells with altered surface composition.

V. ANTIBODIES Rat immunoglobulins (Igs) are similar to those identified in other mammals. Rats produce IgA, IgM, IgE, IgD, and IgG, which can be separated into four subcategories: IgG1 and IgG2a are similar to mouse IgG1, and rat IgG2b and IgG2c are the equivalents of mouse IgG2a/IgG2b and IgG3, respectively [118,119]. The concentrations of Ig types in normal rat serum are in the range of 0.15 to 0.9 mg/ml for IgM, 0.13 to 0.18 mg/ml for IgA, 0.0005 to 0.02 mg/ml for IgE, 0.5 to 7.0 mg/ml for IgG1, and 7.0 to 8.0 mg/ml for IgG2a [118,120–123]. In comparison, normal human serum may contain 0.5 to 3.3 mg/ml IgM, 0.6 to 3.1 mg/ml IgA, 24 to 430 mg/ml IgE, and 6.1 to 13.0 mg/ml of total IgG or around 9 mg/ml of IgG1, 3 mg/ml of IgG2, 1 mg/ml of IgG3, and 0.5 mg/ml of IgG4 [124,125].


RAT IMMUNE SYSTEM

99

The repertoire of the Ig light chain variable region in rats is quite extensive. Although rats express Igs at levels no higher than mice, rat variable light chain loci are considerably more complex than those of laboratory mice. Their diversity reflects the products of gene duplications that predate the time of primate/rodent divergence [126]. Significant variations in allotypes can be observed within and between different strains of rat [127–132]. In terms of eliciting phagocytic action by macrophages, rat IgM and IgG1 are efficient, while IgG2a is less so [133–135]. Human antibody isotypes IgG1, IgG3, and to a lesser extent, IgG2 and IgA, elicit phagocytic action [125]. Experimentally, rat IgG2b has a particularly high affinity for human Fc g receptors [136], including receptors on NK cells [137,138]. IgG1 type rat antibody complexes display a low complement-activating capacity compared with IgM, IgG2b, IgG2c, or IgG2a, of which IgG2a is most efficient in complement activation [139,140]. IgA and IgE are much less capable of activating complement [141,142]. Similarly, human IgG1, IgG3, and IgM are most efficient at complement activation while IgG2 and IgG4 have much less effect, and IgA, IgD, and IgE have no significant complement-binding activation ability [125].

VI. COMPLEMENT SYSTEM The complement system is highly conserved across mammalian species, and simpler complement systems may also be found in many invertebrates [143]. The mammalian complement system has three pathways; classical, alternative and lectin, which together jointly function to amplify an initiating signal through feedback systems of serine protease activities. The immediate outcomes of these pathways are similar with each generating a proteolytic enzyme complex, called C3 convertase, which cleaves the C3 protein to C3a and C3b. One of the central functions of complement activation is to “tag” foreign particles for destruction through augmented targeting and phagocytosis of the foreign particle. While C3b tags the target membrane, C3a acts as a potent proinflammatory mediator. In addition, the C3-based tags activate the terminal or lytic pathway that results in the formation of the membrane attack complex [144,145]. Only subtle differences in rat versus human complement systems are apparent and the primary focus is on system regulatory proteins and their distribution [146]. In humans, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) are the most important proteins for controlling complement activation and amplification of the cascade through blockade of C3 cleavage and deposition. Another inhibitor of complement activation termed Crry has been identified on rat, but not human, cell membranes [147]. Crry is broadly distributed and is a functional and structural analogue of human DAF and MCP. Rodent cells also express DAF and MCP homologues, although the tissue distribution of MCP is more restricted [148].

VII. IMMUNE CELLS Fixed values for parameters that can be universally applied to all rodent strains under all environmental conditions are rare, and immune system parameters are no exception. However, some basic data provide a general framework. Rat blood volume is on average 6.4 ml per 100 g of body weight [149,150] with a pH of 7.4 [151]. Erythrocyte counts for adult rats are typically 6 to 10 ¥ 106/ml [152] and change in size dependant on age [153] while platelets are 4 to 10 ¥ 105/ml in frequency [152]. Rat blood total leukocyte counts have a wide range of 3 to 17 ¥ 103/ml, of which neutrophils constitute 14 to 27%, lymphocytes 65 to 83%, monocytes 0 to 4.0%, eosinophils 0 to 4.0%, and basophils 0 to 1.0% [152,154–158]. In comparison, human blood samples have a typical erythrocyte count range of 4.2 to 5.4 ¥ 106/ml, platelet count of 1.4 to 4.4 ¥ 105/ml and a leukocyte count range of 4.8 to 10.8 ¥ 103/ml. Neutrophils constitute 40 to 74%, lymphocytes 19 to 48%, monocytes 3.4 to 9.0%, eosinophils 0 to 7.0%, and basophils 0 to 1.5% [124,158].


100


A. Mast Cells Mast cells, basophils, and eosinophils are all bone marrow–derived cells that contribute allergic responses and other immune mechanisms [159–161]. An initial allergic reaction involves the interaction of allergen with specific IgE antibodies attached to high-affinity receptors (FceRI) on tissue mast cells [162]. This typically takes place on mucosal surfaces and in connective tissue. The subsequent recruitment of other cells, such as eosinophils and basophils, to the affected tissue sites expands the spectrum of triggering cells beyond mast cells in the mediation of chronic inflammatory processes [163]. Rat eosinophils have collagenase activity and are associated with areas of morphologically altered collagen fibers in incisional skin wounds suggesting that rat eosinophils may contribute to the remodeling of connective tissue [164,165]. Basophils are derived from different progenitor cells than mast cells and for the most part it is agreed that basophils more closely resemble eosinophils than mast cells, but the two cell types share several properties such as the expression of FceRI and histamine-containing granules [166]. From studies with human cells, it is now apparent that mast cells, basophils, and eosinophils share several recruitment pathways and inflammatory responses with one another, but each cell type possesses unique adhesion and migration responses that can contribute to their preferential accumulation in an immune response [163]. Rat mast cells can be divided into two basic types based on their histochemical and biochemical characteristics and are called connective tissue type mast cells (atypical) or mucosal mast cells (typical). The two mast cell types were initially defined by their toluidine or alcain blue dye–binding properties. Rat mast cells have two types of proteases designated as rat mast cell protease I and II, the differential expression of which also defines connective tissue derived or mucosal derived mast cells, respectively [167]. Rat mast cells are relatively abundant, and their activation and release of mediators such as prostaglandin D2 [168], leukotrienes [169], platelet-activating factor [170], histamine [171,172], heparin [173], and chondroitin sulfate [174] are considered central to the pathophysiology of allergic diseases in rodents. However, there are several differences between human and rodent mast cells that may make information derived from rodent studies difficult to extrapolate to human disease [175]. For example, rodent mast cell subsets store multiple chymase isoforms and rarely contain tryptase, while in contrast, only one chymase isoform has been identified in a subset of human mast cells and tryptase may comprise up to 25% of a human mast cells’ protein content [176]. Thus, while the fundamental functions of rat mast cells are similar to that of humans, subtle differences may complicate the comparison of rat research to humans. B. Natural Killer Cells (NK Cells) NK cells are bone marrow–derived lymphocytes that share a common progenitor with T cells [177]. As a component of the innate immune response, the primary function of NK cells is to monitor cells for the presence of crucial self markers [178]. In the absence of these markers, and in the absence of inhibitory factors, NK cells target the offending cell. As such, the most important role of NK cells in host defence may be in anti-viral immunity as well as bacterial and parasitic infections [179]. The potency of the NK cell response towards cells with an altered target phenotype means their involvement in autoimmune disease is also likely [180]. Certain T-cell responses in rats can be inhibited by NK cells suggesting an immunoregulatory role [181,182], while in disease models NK cells can be highly reactive to the target cell type. For example, as with human diabetes, BB rats have a significant deficiency in NK cells that may lead to reduced immune system regulation [183], but other studies demonstrate NK cells in BB rats are highly responsive to islet cells [184]. Rat NK cells are morphologically defined as large granular lymphocytes (LGL). These cells express asialo-GM1, laminin, and CD8 but do not express CD5, OX19, CD4, or surface Igs [185–187]. From the LGL population, lymphokine activated killer cells can be produced whereas T-cell populations devoid of LGL activity cannot produce lymphokine-activated killer cells


RAT IMMUNE SYSTEM

101

[187–189]. These properties of rat NK cells are in line with observations for mouse and human NK cells [190–192]. While NK cells are stimulated by cytokines such as IL-15, IL-12, IL-10, IL2 and IFN [193], NK cells themselves produce cytokines such as IL-12 and IFN-a capable of promoting Th1 type lymphocytes. Recognition of, and response to, allogeneic and xenogeneic cells by NK cells is mediated by the opposing effects of various activation and inhibitory receptors that are less well defined in the rat than in humans and mice [194,195]. Rat NK cells recognize MHC class I molecules encoded by both the classical (RT1-A) and non-classical (RT1-C/E/M) MHC class I (MHC-I) regions [196,197]. Responsiveness is accentuated by the presence of complement. C. Lymphocytes Mature rat lymphocytes range in size from 6 to 15 mm much as is observed in humans. However, rat lymphocyte size is biased more towards the lower end of the scale while in humans larger lymphocytes are more common. The majority of peripheral T cells are of two main phenotypes; CD4+ and CD8+ cells. CD4+ and CD8+ T cells identified in rats carry out essentially the same functions as equivalent cells in mice and humans [198,199]. The ratio of CD4 to CD8 cells varies considerably between rat strains [200]. The peripheral CD4/CD8 ratio variability is genetically determined much as is observed in humans [201]. In contrast to mice, the MHC haplotype of rats plays a dominant role in determining the peripheral CD4/CD8 ratio probably through positive and negative selection [200,202]. In rats and humans the CD4 antigen is expressed on both T helper cells and macrophages. In contrast, CD4 is apparently absent from mouse macrophages [203–205]. CD4+ T cells are classified into two subpopulations depending on the expression of CD45R. CD45RC- cells (CD45RC in rats equates to CD45RB in mice) are viewed as memory, or regulatory, T cells with a capacity to produce IL-4 [206]. CD45RC+ cells are naïve T cells with a propensity for IL-2 and IFNg production. Both memory and naïve rat CD4 cells migrate into lymph nodes via high endothelial venules but their rate of passage through the lymph node differs, with memory cells migrating faster than naïve cells [207]. With the discovery of CD25 as a potential marker for regulatory cells in mice [208], rat CD4 cells have also been examined for expression of CD25 and a regulatory phenotype. Rat CD4 cells expressing CD25 with a regulatory phenotype were also identified as low expressors of CD45RC. However, other cell subpopulations negative for CD25 expression have also been identified in rats with a regulatory cell phenotype [209]. Nevertheless, overall there is compelling evidence that essentially the same regulatory cells exist in rats, mice and humans. In rats, humans, and mice T cells can be divided into two main populations based on their TCR properties, specifically a/b TCR and g/d TCR. Most peripheral rat T cells express a/b TCR, but g/d TCR expressing cells are present at low levels with widespread distribution, including the skin [210,211]. Analysis of TCR transcripts demonstrates a high degree of similarity in both number and sequence between rat and mouse TCR genes [212,213]. However, in contrast to mouse and human a/b TCR cells that are typically CD4+, rat g/d TCR cells are CD8+ [214]. D. Monocytes/Macrophages Classically the definition of monocytes and macrophages has been defined by their relative physical location with monocytes restricted to the blood and macrophages resident in peripheral tissues. However, this distinction is not clear cut as, in contrast to humans, mature intravascular macrophages can be identified in some species [215] and in response to liver damage, intravascular macrophages may develop in some rat models [216]. A strict functional segregation of monocytes from macrophages is also difficult as any in vivo stimulation will affect both monocytes and macrophages, while in vitro monocytes quickly differentiate into macrophages upon adherence to the culture vessel. In non antigen challenged rats, monocytes represent 0–4% of white blood cells in the blood while in humans and mice the level can be somewhat higher at 5–10%. Monocytes,


102


derived from hematopoietic bone marrow stem cells, are continuously released into the blood where they reside for 24 hours before migrating, differentiating, and taking up residence in peripheral rat organs [217,218]. The most widely used reagents for visualizing tissue macrophages in rats are monoclonal antibodies ED1, ED2 and ED3 [109]. The target structure of ED1 has not been entirely characterized up to now. It is a primarily intracellular lysosomal antigen which may be similar to CD68 [219]. ED2 obviously binds to a member of the family of scavenger receptor molecules, potentially to rat CD163 [116]. ED3 is supposed to detect sialoadhesin (CD169) in rats [110,220]. While the ED1 target is present in the majority of tissue macrophages and monocytes [116], ED2 and ED3 antibodies only react with certain macrophage populations, but not with normal monocytes. The molecule identified by ED2 is present in interstitial macrophages in many, but not all, organs such as Kupffer cells, splenic red pulp macrophages, interstitial macrophages in the heart and perivascular macrophages in the brain. Strong reactivity for ED3 is found in marginal metallophilic macrophages and marginal zone macrophages of the spleen and in subsinusoidal and medullary macrophages of the lymph nodes. In addition, reactivity with ED2 and ED3 may be induced in macrophages under conditions of inflammation in vivo and experimentally in vitro [116]. Interestingly, none of the three antibodies label normal microglia cells in the brain. Up to now the antigens detected by antibodies ED1, ED2 and ED3 have not been detected outside the monocyte/macrophage and dendritic cell system to any larger extent. There is, however, a substantial number of further antibodies reacting with monocytes/macrophages and additional cells in rats [221]. The vast majority of blood monocytes can be detected by antibodies ED9 or Ox41, which are directed against different epitopes on a molecule of the signal-regulatory protein (SIRP) family [222]. If these reagents are used in flow cytometry, granulocytes have to be removed before due to potential crossreactivity. Two other antigens, the ED1 target structure and CD11b, detected by mAb Ox42 [223], are also present in all normal blood monocytes. The latter two reagents are, however, not well-suited to detect monocytes by flow cytometry, because staining for ED1 needs permeabilization of cell membranes and CD11b is also present in certain lymphocytes. Several other antigens of immunologic interest are expressed by subpopulations of normal ED9-positive rat monocytes including CD4, CD8, CD43, CD62L and CD161 [224]. These antigens may be either upregulated (CD161, CD62L, and CD8) or down-modulated (CD43 and CD4) by monocytes during immune reactions in vivo [224]. Interestingly, in LEW rats kept under specified pathogen-free conditions, MHC class II antigens are only present in a very low number of blood monocytes. This number does, however, increase during immune reactions. E. Dendritic Cells Since their first identification [225], dendritic cells have been characterized as key players in the adaptive immune system. Their ability to sample antigens [226] and present them to activate naïve lymphocytes, both CD4 and CD8, [227,228] dictates the nature of the immune response to an antigenic challenge [229]. In humans and mice the dendritic cell populations have a heterogeneous presentation of cell surface markers and rat dendritic cells exhibit similar heterogeneity. No one marker identifies all rat dendritic cells and it is likely that expression of dendritic cells changes depending on the degree of maturity, activation, and the surrounding environment of the cell [230,231]. A variety of antigens are used to identify rat dendritic cells, often employing several simultaneously, such as CD11c, MHC class II, ae-integrin and signal inhibitory regulatory proteins [232–236]. Rat dendritic cells can also variably express other antigens that are used to identify their source and lymphocyte stimulatory capacity including; CD4, Thy-1, CD2, CD25, MHC class I, and CD11b [236–238]. Of particular note, while CD4 is expressed on dendritic cell subpopulations from intestine and respiratory tract epithelia [239], CD4 is virtually absent from rat skin Langerhans cells [233].


RAT IMMUNE SYSTEM

103

Studies on dendritic cells are most advanced in humans and mice [240,241] while rat dendritic cell research is far less developed. However in one particular research area, namely the understanding of dendritic cell migration in vivo, rat models excel. Much detailed research with mice and humans involves ex vivo and in vitro models. The larger size of rats enables the development of in vivo models using, for example, cannulation of lymphatic vessels [242], a procedure for which mice are too small. Thus, rat research provides one of the primary sources for understanding in vivo cell migration dynamics. While some suggest dendritic cells only migrate from the periphery after acquiring foreign antigen and receiving appropriate stimuli [243], research from rat models indicate continuous migration of dendritic cells from the periphery regardless of stimuli, although stimuli enhance migration [236,238,244,245]. Most other cells migrating through peripheral tissues in adult animals either have an activated or memory phenotype [246]. Once the lymph node is reached, dendritic cells enter the paracortex and reside as interdigitating cells [247]. This continuous dendritic cell migration from the periphery to lymph nodes, and residency in the paracortex in close association with T lymphocytes [248], is suggested as one mechanism for the maintenance of self tolerance, and the immunostimulatory role of dendritic cells is only initiated in direct response to antigenic challenge. Rat dendritic cells are capable of inducing Th1 or Th2 type responses after antigenic challenge depending on the cytokine environment [249]. For a continuous migration of dendritic cells from the periphery to lymph nodes to be maintained, the departing cells must be continuously replaced. Dendritic cells are ultimately replaced with cells derived from bone marrow hematopoietic stem cells [250,251], but the nature of the transformation from stem cells to differentiated, mature peripheral tissue dendritic cell is poorly understood in all species. The immediate precursor cells of tissue dendritic cells are likely derived from the spleen and lymph nodes [252]. The rate of dendritic cell turnover in the periphery varies with tissue type. Rat dendritic cells reside for an average of 3 days in the intestines and respiratory tract before migrating to lymph nodes [244,253] while dendritic cells in the skin, kidneys, and heart remain resident for between 2 and 4 weeks [232]. These differences suggest that the rate of dendritic cell turnover may reflect the degree of antigenic load to which the cells are exposed in different tissues.

VIII. CYTOKINES AND CHEMOKINES Interleukin-1 (IL-1) is produced during infection, injury, or immunological challenge. IL-1 promotes a variety of systemic effects such as fever, sleep, ACTH release, and increased sodium excretion. IL-1 activates both T and B lymphocytes and induces production of various lymphokines, interferons, and other cytokines, particularly tumor necrosis factor [254]. IL-1 is a general name for at least two distinct proteins, IL-1a and IL-1b, considered to be the first of a small (but growing) family of regulatory and inflammatory cytokines [255]. There is a 78% amino acid identity between mouse and human mature IL-1b [256]. Full length IL-1a shows 54% amino acid identity and mature IL-1a shows 58% amino acid identity between mouse and human. Both human and rat IL-1 are active on mouse cells [257,258]. See Table 5.3 for human/rat cytokine homology. Interleukin-2 (IL-2) is a potent immunoregulatory cytokine that plays a central role in a number of T-cell functions. Although IL-2 has traditionally been grouped with the Th1 cytokines, it is now thought that IL-2 is not a true Th1 cytokine since naïve CD4+ T cells are known to produce it [259]. Mouse IL-2 is approximately 63% identical to human IL-2, but contains a unique stretch of repeated glutamine residues [260]. Rat IL-2 is similar to mouse and human IL-2 in its biochemical properties [261–264]. There at least 2 forms of rat IL-2, while at least 5 forms have been identified in different mouse strains [265,266]. Interleukin-4 (IL-4) was originally described as a growth factor for B cells stimulated with antiIgM antibodies. Subsequent investigation has revealed an abundance of other functions including


104


Table 5.3 Human/Rat Cytokine Homology Cytokine or Receptor

Human Chromosome Location

IL-1a IL-1b IL-1r IL-2 Il2ra Il2rb IL-4 IL-4r IL-6 Il6r Il10 Il10ra IL10rb IL12a IL12b IFNg IFNgr TNFa

2q14 2q14 2q12 4q26-q27 10p15-p14 10p15-p14 5q31.1 16p11.2-12.1 7p21 1q21 1q31-q32 11q23 21q22.11 3p12-q13.2 5q31.1-q33.1 12q14 6q23-q24 6p21.3

Rat Chromosome Location

Human/Rat Protein Homology

3q36 3q36 2q24 17q12.3 7 10 1q33 4 2q34 13q11 8q22 11q11-q22 2q31 10q21 7q21 1q11 20p12

64% 66% 67% 64% 60% 60% 43% 49% 40% 54% 73% 61% 58% 66% 39% 48% 76%

Cross-Species Reactivity Yes Yes — Yes — — No — Partial — Partial — — No No No — Partial

References 293

261,294,295 296 296–298 267,299 269,270,300–302 302–305 274,275 279 280 306,307 284,308,309

Note: Italics indicate predicted position not yet confirmed. General Sources: Makalowski and Boguski, Proc. Natl. Acad. Sci. U.S.A., 95, 9407, 1998 [310], and Locuslink, available at www.ncbi.nlm.nih.gov/LocusLink/, accessed April 2003.

the ability to induce or enhance the expression of MHC class II molecules and CD23 on B cells, its own receptor on lymphocytes and VCAM-1 on endothelial cells. In addition, IL-4 can induce the secretion of IgG1and IgE while restricting the secretion of other Ig isotypes as well as acting as a growth factor for T cells and mast cells. The predicted amino acid sequence of the rat IL 4 gene shows low homology (57%) with the mouse homologue [267]. IL4 receptor (IL4r) comprises an overall identity of 52% and 78% to its human and mouse homologues, respectively [268]. Mouse and rat IL-6 also have been cloned and are approximately 40% identical to human IL6 at the amino acid level [269,270]. Unlike human IL-6, mouse and rat IL-6 lack potential N-linked glycosylation sites, but may be O-glycosylated [269]. The presence or absence of glycosylation, however, has no effect on bioactivity. Human IL-6 is active on both mouse and rat cells [270,271] while the effects of rat IL-6 on human cells has apparently not been examined, mouse IL-6 has no activity on human cells [270,272]. Interleukin-10 (IL-10) is primarily an anti-inflammatory, immunoregulatory cytokine [273]. The active form of IL-10 is a noncovalent homodimer, exhibiting species-specificity both with respect to structure and biological activity. Human and rat IL-10 exhibit 81% sequence identity at the amino acid level, and share 73% identity at the cDNA level [274]. Most notably, in addition to the two disulphide bonds also present in human IL-10, rat IL-10 has a fifth unpaired cysteine that may increase its biological activity [275]. Rat IL-10 may play a role in resistance to autoimmune diseases in rats [276], may be used to aid allograft survival [277], and reduces rat NK cell sensitivity to tumor cells [278]. The nucleic acid coding sequence for rat IL-10r exhibits 88% and 68% homology with the mouse and human IL-10 receptor sequences respectively, and the translated protein exhibited 83% and 61% homology with the mouse and human IL-10 receptor proteins respectively [279]. Interleukin 12 (IL-12) is involved in the stimulation and maintenance of Th1 cellular immune responses, including the normal host defence against various intracellular pathogens. IL-12 also has an important role in Th1 disease pathogenesis, such as in inflammatory bowel disease and multiple sclerosis. Bioactive IL-12 was identified as a disulfide-linked heterodimer, composed of


RAT IMMUNE SYSTEM

105

a heavy chain of 40 kDa (p40) and a light chain of 35 kDa (p35) encoded by separate genes. Human and rat IL-12 share 65% and 58% amino acid sequence homology in their p40 [280] and p35 subunits, respectively. Human IL-12 shows minimal activity in rats. Tumor necrosis factor (TNF) is a pleiotropic mediator contributing to cellular signaling pathways and cell growth, immune responses, angiogenesis, as well as modulating viral replication, growth of bacteria and other parasites [281]. TNFb and TNFa share 30% amino acid homology and have similar biological activities [282]. Unlike human TNFa, mouse TNFa is glycosylated [283]. The propeptide of rat TNFa as well as the biologically active TNFa possess a homology of 92% and 76% to mouse and human TNFa, respectively [284].

IX. CONCLUSIONS The evolutionary divergence between rats and mice is proposed to be about 20 to 40 million years [285–287], while the evolutionary distance from rats to humans may be around 100 million years [288]. Despite this long time period for evolutionary divergence, the genetic difference between rodents and humans is limited. Rats, mice and other rodents exhibit the same basic immunologic system as humans. Minor modifications are apparent, but fundamentally the innate and adaptive arms of the rat immune system operate much the same way as is observed in humans. The advantages of rodent research, their small size, their rapid breeding speed, the availability of genetically identical inbred stains, the abundance of immune reagents, and the absence of the ethical limitations as in human experimentation, naturally favor the development that much of our understating of immune system function in health and dysfunction in immunologic diseases is derived from rodent research. Mice now far exceed rats in their frequency of use in research and correspondingly much more is now known about mouse immunology. However, rats still retain a significant role and with the further development of production and distribution facilities, rat strains may regain their previous popularity as a prominent laboratory research model.

REFERENCES 1. Richter, C.P., The effects of domestication and selection on the behavior of the Norway rat. J. Natl. Cancer Inst., 15, 727, 1954. 2. Philipeaux, J.M., Note sur l’extirpation des capsules survenales chez les rats albinos (Mus rattus), C. R. Habd. Séances Acad. Sci., 43, 904, 1856. 3. Lindsey, J.R., Historical foundations, in The Laboratory Rat, Vol. 1, Biology and Diseases, Baker, H.J., Lindsey, J.R., and Weisbroth S.H., Eds., Academic Press, New York, 1979, p. 1. 4. Donaldson, H.H., The history and zoological position of the albino rat, J. Acad. Natl. Sci., 15, 365, 1912. 5. van den Berg, T.K. et al., Monoclonal antibodies against rat leukocyte surface antigens, Immunol. Rev., 184, 109, 2001. 6. Festing, M.F.W., Inbred strains, in The Laboratory Rat, Vol. 1, Biology and Diseases, Baker, H.J., Lindsey, J.R., and Weisbroth S.H., Eds., Academic Press, New York, 1979, p. 55. 7. Festing, M.F.W., Inbred Strains in Biomedical Research, Macmillan, London, 1979. 8. Greenhouse, D.D. et al., Catalogue of inbred strains of rats, in Genetic Monitoring of Inbred Strains of Rats, Hedrich, H.J., Ed., Gustav Fischer, Stuttgart, 1990, p. 410. 9. Jacob, H.J. and Kwitek, A.E., Rat genetics: attaching physiology and pharmacology to the genome, Nat. Rev. Genet., 3, 33, 2002. 10. Twigger, S. et al., Rat Genome Database (RGD): mapping disease onto the genome, Nucleic Acids Res., 30, 125, 2002. 11. Steiniger, B. et al., Monocytes in the rat: phenotype and function during acute allograft rejection, Immunol. Rev., 184, 38, 2001.


106


12. Bickerton, R.K., Spontaneous tumors and related pathologic changes, in Carcinogenesis Testing of Chemicals, Goldberg, L., Ed., CRC Press, Cleveland, 1974, p. 55. 13. Sher, S.P., Jensen, R.D., and Bokelman, D.L., Spontaneous tumors in control F344 and charles riverCD rats and Charles River CD-1 and B6C3HF1 mice, Toxicol. Lett., 11, 103, 1982. 14. Maekawa, A. et al., Spontaneous tumors in F-344/DuCrj rats, Gann, 74, 365, 1983. 15. Deerberg, F. and Kaspareit, J., Endometrial carcinoma in BD II/Han rats: model of a spontaneous hormone-dependent tumor, J. Natl. Cancer Inst., 78, 1245, 1987. 16. Burek, J.D. and Hollander, C.F., Incidence patterns of spontaneous tumors in BN/Bi rats, J. Natl. Cancer Inst., 58, 99, 1977. 17. McDorman, K.S. and Wolf, D.C., Use of the spontaneous Tsc2 knockout (Eker) rat model of hereditary renal cell carcinoma for the study of renal carcinogens, Toxicol. Pathol., 30, 675, 2002. 18. Kim, U.K, Clifton H., and Furth J., A highly inbred strain of Wistar rats yielding spontaneous mammosomatropic pituitary and other tumors, J. Natl. Cancer Inst., 24, 1031, 1960. 19. Schardein, J.L., Fitzgerald, J.E., and Kaump, D.H., Spontaneous tumors in Holtzman-source rats of various ages, Pathol. Vet., 5, 238, 1968. 20. Prejean, J.D. et al., Spontaneous tumors in Sprague-Dawley rats and Swiss mice, Cancer Res., 33, 2768, 1973. 21. Trouillas, J. et al. Spontaneous pituitary tumors in the Wistar/Furth/Ico rat strain. An animal model of human prolactin adenoma, Am. J. Pathol., 109, 57, 1982. 22. Trouillas, J. and Girod, C., Animal models of human pituitary tumors, Pathol. Res. Pract., 183, 638, 1988. 23. Nakazawa, M. et al., Spontaneous neoplastic lesions in aged Sprague-Dawley rats. Exp. Anim., 50, 99, 2001. 24. Swanborg, R.H., Experimental autoimmune encephalomyelitis in the rat: lessons in T-cell immunology and autoreactivity, Immunol. Rev., 184, 129, 2001. 25. Brosnan. J.V. et al., Disease patterns in experimental allergic neuritis (EAN) in the Lewis rat. Is EAN a good model for the Guillain-Barre syndrome? J. Neurol. Sci., 88, 261, 1988. 26. Maurer, M. and Gold, R., Animal models of immune-mediated neuropathies, Curr. Opin. Neurol., 15, 617, 2002. 27. Sasamoto, Y. et al., Interphotoreceptor retinoid-binding protein derived peptide can induce experimental autoimmune uveoretinitis in various rat strains, Curr. Eye Res., 13, 845, 1994. 28. Lorentzen, J.C. et al., Protracted, relapsing and demyelinating experimental autoimmune encephalomyelitis in DA rats immunized with syngeneic spinal cord and incomplete Freund’s adjuvant, J. Neuroimmunol., 63, 193, 1995. 29. Link, H. and Xiao, B.-G., Rat models as tool to develop new immunotherapies, Immunol. Rev., 184, 117, 2001. 30. Holmdahl, R. et al., Arthritis induced in rats with nonimmunogenic adjuvants as models for rheumatoid arthritis, Immunol. Rev., 184, 184, 2001. 31. Waksman, B.H., Immune regulation in adjuvant disease and other arthritis models: relevance to pathogenesis of chronic arthritis, Scand. J. Immunol., 56, 12, 2002. 32. Ramanathan, S. and Poussier, P., BB rat lyp mutation and type 1 diabetes, Immunol. Rev., 184, 161, 2001. 33. Festing M.F.W. and Staats J., Standardized nomenclature for inbred strains of rats: Fourth listing, Transplantation, 16, 221, 1973. 34. Committee on Rat Nomenclature, Definition, nomenclature, and conservation of rat strains, I.L.A.R. News, 34, s1, 1992. 35. Bogden, A.E. and Aptekman, P.M., The “R-1” factor is a histocompatibility antigen in the rat, Cancer Res., 20, 1272, 1960. 36. Würzburg, U., Correlation between the immune response to an enzyme and histocompatibility in rats, Eur. J. Immunol., 1, 496, 1971. 37. Gill, T.J., 3rd et al., The rat as an experimental animal, Science, 245, 269, 1989. 38. Günther, E. and Walter, L., The major histocompatibility complex of the rat (Rattus norvegicus), Immunogenetics, 53, 520, 2001. 39. Metzger, R. et al., Organ-specific maturation of the major histocompatibility antigens in rats, Pediatr. Surg. Int., 18, 640, 2002.


RAT IMMUNE SYSTEM

107

40. Locker, J. et al., The rat MHC and cystathionine beta-synthase gene are syntenic on chromosome 20, Immunogenetics, 31, 271, 1990. 41. Helou, K. et al., Cytogenetic orientation of the rat major histocompatibility complex (MHC) on chromosome 20, Immunogenetics, 47, 166, 1998. 42. Walter, L. et al., Physical mapping of the major histocompatibility complex class II and class III regions of the rat, Immunogenetics, 54, 268, 2002. 43. Canzian, F., Phylogenetics of the laboratory rat Rattus norvegicus, Genome Res., 7, 262, 1997. 44. Hedrich, H.J., Catalogue of mutant genes and polymorphic loci. RT1, in Genetic Monitoring of Inbred Strains of Rats, Hedrich, H.J., Ed., Gustav Fischer, Stuttgart, 1990, p. 381. 45. Vestberg, M. et al., Limited polymorphism in the first domain of the rat MHC class II RT1-D molecule, Immunogenetics, 48, 344, 1998. 46. Walter, L. and Günther, E., Physical mapping and evolution of the centromeric class I gene-containing region of the rat MHC, Immunogenetics, 51, 829, 2000. 47. Gill, T.J., 3rd et al., Current status of the genetic and physical maps of the major histocompatibility complex in the rat, Transpl. Proc., 29, 1657, 1997. 48. Helou, K. et al., Cytogenetic orientation of the rat major histocompatibility complex (MHC) on chromosome 20, Immunogenetics, 47, 166, 1998. 49. Günther, E. and Walter, L., Comparative genomic aspects of rat, mouse and human MHC class I gene regions, Cytogenet. Cell Genet., 91, 107, 2000. 50. Dressel, R., Walter, L., and Gunther, E., Genomic and funtional aspects of the rat MHC, the RT1 complex, Immunol. Rev., 184, 82, 2001. 51. Ioannidu, S. et al., Physical map and expression profile of genes of the telomeric class I gene region of the rat MHC, J. Immunol., 166, 3957, 2001. 52. Monden, Y., Quantitative evaluation of total cell number and cellular density of the thymolymphatic organs of young albino rats by means of DNA determinations, Okajimas Folia Anat., 32, 193, 1959. 53. Lundin, P.M. and Angervall, L., Effect of insulin on rat lymphoid tissue, Pathol. Eur. 5, 273, 1970. 54. McLean, J.M., Mosley, J.G., and Gibbs, A.C., Changes in the thymus, spleen and lymph nodes during pregnancy and lactation in the rat, J. Anat., 118, 223, 1974. 55. Trepel. F., Number and distribution of lymphocytes in man. A critical analysis, Klin. Wochenschr., 52, 511, 1974. 56. Forster, P.M., McLean, J.M., and Gibbs, A.C., Lymphoid response to pregnancy and pseudopregnancy in the rat, J. Anat., 128, 837, 1979. 57. Klausen, B. and Hougen, H.P., Quantitative studies of lymphoid organs, blood and lymph in inbred athymic and euthymic LEW rats under germfree and specified-pathogen-free conditions, Lab. Anim., 21, 342, 1987. 58. Schermer, S., The Blood Morphology of Laboratory Animals, 3rd ed., F.A. Davis Co., Philadelphia, 1967. 59. Gowans, J.L., The recirculation of lymphocytes from blood to lymph in the rat, J. Physiol., 146, 54, 1959. 60. Fossum, S., Smith, M.E., and Ford, W.L., The recirculation of T and B lymphocytes in the athymic, nude rat, Scand. J. Immunol., 17, 551, 1983. 61. Fossum, S., Smith, M.E., and Ford, W.L., The migration of lymphocytes across specialized vascular endothelium VII. The migration of T and B lymphocytes from the blood of the athymic, nude rat, Scand. J. Immunol., 17, 539, 1983. 62. Smith, M.E. and Ford, W.L., The recirculating lymphocyte pool of the rat: a systematic description of the migratory behaviour of recirculating lymphocytes, Immunology, 49, 83, 1983. 63. Westermann, J. et al., Random entry of circulating lymphocyte subsets into peripheral lymph nodes and Peyer’s patches: no evidence in vivo of a tissue-specific migration of B and T lymphocytes at the level of high endothelial venules, Eur. J. Immunol., 22, 2219, 1992. 64. Blaschke, V. et al., Lymphocyte traffic through lymph nodes and Peyer’s patches of the rat: B- and T-cell-specific migration patterns within the tissue, and their dependence on splenic tissue, Cell Tissue Res., 282, 377, 1995. 65. Westermann, J. et al., Proliferation of lymphocyte subsets in the adult rat: a comparison of different lymphoid organs, Eur. J. Immunol., 19, 1087, 1989. 66. Quaglino, D. and Ronchetti, I. P., Cell death in the rat thymus: a minireview, Apoptosis, 6, 389, 2001.


108


67. Miller, J.F., The discovery of thymus function and of thymus-derived lymphocytes, Immunol. Rev., 185, 7, 2002. 68. Sprent, J. and Kishimoto, H., The thymus and negative selection, Immunol. Rev., 185, 126, 2002. 69. Hünig, T. et al., A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation. Differential reactivity with subsets of immature and mature T lymphocytes, J. Exp. Med., 169, 73, 1989. 70. Quaglino, D. et al., Age-dependent remodeling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age, Eur. J. Cell Biol., 76, 156, 1998. 71. Capri, M. et al., A cytofluorimetric study of T lymphocyte subsets in rat lymphoid tissues (thymus, lymph nodes) and peripheral blood: a continuous remodelling during the first year of life, Exp. Gerontol., 35, 613, 2000. 72. Surh, C.D. and Sprent, J., Long-term xenogeneic chimeras. Full differentiation of rat T and B cells in SCID mice, J. Immunol., 147, 2148, 1991. 73. Paterson, D.J. and Williams, A.F., An intermediate cell in thymocyte differentiation that expresses CD8 but not CD4 antigen, J. Exp. Med., 166, 1603, 1987. 74. Lawetzky, A., Kubbies, M., and Hünig, T., Rat “first-wave” mature thymocytes: cycling lymphoblasts that are sensitive to activation-induced cell death but rescued by interleukin 2, Eur. J. Immunol., 21, 2599, 1991. 75. Park, J.H. et al., T cell receptor ligation induces interleukin (IL) 2R beta chain expression in rat CD4,8 double positive thymocytes, initiating an IL-2-dependent differentiation pathway of CD8 alpha+/betaT cells, J. Exp. Med., 177, 541, 1993. 76. Schorle, H. et al., Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting, Nature, 352, 621, 1991. 77. Bommhardt, U. et al., Signals through CD8 or CD4 can induce commitment to the CD4 lineage in the thymus, Eur. J. Immunol., 27, 1152, 1997. 78. Mitnacht, R. et al., Opposite CD4/CD8 lineage decisions of CD4+8+ mouse and rat thymocytes to equivalent triggering signals: correlation with thymic expression of a truncated CD8 alpha chain in mice but not rats, J. Immunol., 160, 700, 1998. 79. Drenckhahn, D. and Wagner, J., Stress fibres in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility, J. Cell Biol., 102, 1738, 1986. 80. Steiniger, B. and Barth, P., Microanatomy and function of the spleen, Adv. Anat. Embryol. Cell Biol., 151, 1, 2000. 81. Kumararatne, D.S., Bazin, H., and MacLennan, I.C.M., Marginal zones: the major B cell compartment of rat spleens, Eur. J. Immunol., 11, 858, 1981. 82. Brelinska, R. and Pilgrim, C., Macrophages and interdigitating cells: their relationship to migrating lymphocytes in the white pulp of rat spleen, Cell Tissue Res., 233, 671, 1983. 83. Nieuwenhuis, P. and Ford, W.L., Comparative migration of B- and T-lymphocytes in the rat spleen and lymph nodes, Cell Immunol., 23, 254, 1976. 84. van Ewijk, W. and Nieuwenhuis, P., Compartments, domains and migration pathways of lymphoid cells in the splenic pulp, Experimentia, 4, 199, 1985. 85. Probst, H.C. et al., Inducible transgenic mice reveal resting dendritic cells as potent inducers of CD8+ T cell tolerance, Immunity, 18, 713, 2003. 86. Steiniger, B., Barth, P., and Hellinger, H., The perifollicular and marginal zones of the human splenic white pulp. Do fibroblasts guide lymphocyte immigration? Am. J. Pathol., 159, 501, 2001. 87. Förster, R. et al., CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs, Cell, 99, 23, 1999. 88. Ansel, K.M. et al. A chemokine-driven positive feedback loop organizes lymphoid follicles, Nature, 406, 309, 2000. 89. Tew, J.G. et al., Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells, Immunol. Rev., 156, 39, 1997. 90. Jacob, J. and Kelsoe, G., In situ studies of the primary immune response to (4-hydroxy-3-nitophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers, J. Exp. Med., 176, 679, 1992. 91. MacLennan, I.C.M. et al., The changing preference of T and B cells for partners as T-dependent antibody responses develop, Immunol. Rev., 156, 53, 1997.


RAT IMMUNE SYSTEM

109

92. Küppers, R. et al., Tracing B cell development in human germinal centres by molcular analysis of single cells picked from histological sections, EMBO J., 12, 4955, 1993. 93. Smith, K.G.C. et al., The phenotype and fate of the antibody-forming cells of the splenic foci, Eur. J. Immunol., 26, 444, 1996. 94. Toellner, K-M. et al., Immunoglobulin switch transcript production in vivo related to the site and time of antigen-specific B cell activation, J. Exp. Med., 183, 2303, 1996. 95. Kraal, G., Cells in the marginal zone of the spleen, Int. Rev. Cytol., 132, 31, 1992. 96. Liu, Y-J., Oldfield, S., and MacLennan, I.C.M., Memory B cells in T cell-dependent antibody responses colonize the splenic marginal zones, Eur. J. Immunol., 18, 355, 1988. 97. Liu, Y-J., Sites of B lymphocyte selection, activation, and tolerance in spleen, J. Exp. Med., 186, 625, 1997. 98. Gray, D. et al., Migrant m+d+ and static m+ d- B lymphocyte subsets, Eur. J. Immunol., 12, 564, 1982. 99. Dammers, P.M. et al., The origin of marginal zone B cells in the rat, Eur. J. Immunol., 29, 1522, 1999. 100. Liu, Y-J. et al., Sites of specific B cell activation in primary and secondary responses to T celldependent and T cell-independent antigens, Eur. J. Immunol., 21, 2951, 1991. 101. Hosea, S.W. et al., Impaired immune response of splenectomised patients to polyvalent pneumococcal vaccine, Lancet, 1, 804, 1981. 102. Timens, W., The human spleen and the immune system: not just another lymphoid organ. Res. Immunol., 142, 316, 1991. 103. Dammers, P.M. et al., Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected, J. Immunol., 165, 6156, 2000. 104. Chen, X. et al., Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone, Int. Immunol., 9, 27, 1997. 105. Candinas, D. et al., T cell independence of macrophage and natural killer cell infiltration, cytokine production, and endothelial activation during delayed xenograft rejection, Transplantation, 62, 1920, 1996. 106. Gray, D. et al., Relation of intra-splenic migration of marginal zone B cells to antigen localization on follicular dendritic cells, Immunology, 52, 659, 1984. 107. Pettersen, J.C., Borgen, D.F., and Graupner, K.C., A morphological and histochemical study of the primary and secondary immune responses in the rat spleen, Am. J. Anat., 121, 305, 1967. 108. Steiniger, B. and van der Meide, P.H., High-dose interferon-gamma alters the distribution of B lymphocytes and macrophages in rat spleen and lymph nodes, Immunology, 78, 461, 1993. 109. Dijkstra, C.D. et al., The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3, Immunology, 54, 589, 1985. 110. van den Berg, T.K. et al., Sialoadhesin on macrophages: its identification as a lymphocyte adhesion molecule. J. Exp. Med., 176, 647, 1992. 111. Humphrey, J.H., Splenic macrophages: antigen presenting cells for TI-2 antigens. Immunol. Lett., 11, 149, 1985. 112. Humphrey, J.H. and Grennan, D., Different macrophage populations distinguished by means of fluorescent polysaccharides. Recognition and properties of marginal-zone macrophages, Eur. J. Immunol., 11, 221, 1981. 113. Hünig, T. et al., A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation, J. Exp. Med., 169, 73, 1989. 114. Woollett, G.R. et al., Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes, Eur. J. Immunol., 15, 168, 1985. 115. Matsuno, K., Ezaki, T., and Kotani, M., Splenic outer periarterial lymphoid sheath (PALS): an immunoproliferative microenvironment constituted by antigen-laden marginal metallophils and ED2positive macrophages in the rat, Cell Tissue Res., 257, 459, 1989. 116. van den Berg, T.K., Döpp, E.A., and Dijkstra, C.D., Rat macrophages: membrane glycoproteins in differentiation and function. Immunol. Rev., 184, 45, 2001. 117. Wacker, H.H., Sinuswandzellen. Immunakzessorische Zellen des Lymphknotensinus, Prog. Pathol., 143, 1, 1994. 118. Bazin, H., Beckers, A., and Querinjean, P., Three classes and four (sub)classes of rat immunoglobulins: IgM, IgA, IgE and IgG1, IgG2a, IgG2b, IgG2c, Eur. J. Immunol., 4, 44, 1974.


110


119. Bruggemann, M., Evolution of the rat immunoglobulin gamma heavy-chain gene family, Gene, 74, 473, 1988. 120. McGhee, J.R., Michalek, S.M., and Ghanta, V.K., Rat immunoglobulins in serum and secretions: purification of rat IgM, IgA and IgG and their quantitation in serum, colostrum, milk and saliva, Immunochemistry, 12, 817, 1975. 121. Pauwels, R. et al., The measurement of total serum IgE levels in rats, J. Immunol. Methods, 18, 133, 1977. 122. Rieh, B. and Dietzel-Rohl, L., Immunoglobulin G2a levels in SPF rats. 3. Selection of samples for quality control with special reference to the neonatal group, Z. Versuchstierkd., 24, 318, 1982. 123. Bazin, H. et al., Genetic regulation of immunoglobulin synthesis: selection of two rats' lines with respectively a high or low IgM serum level, J. Immunogenet. 10, 293, 1983. 124. Kratz, A. and Lewandrowski, K.B., Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Normal reference laboratory values, N. Engl. J. Med., 339, 1063, 1998. 125. van Dijk, M.A. and van de Winkel, J.G., Human antibodies as next generation therapeutics, Curr. Opin. Chem. Biol., 5, 368, 2001. 126. Aguilar, B.A. and Gutman, G.A., Transcription and diversity of immunoglobulin lambda chain variable genes in the rat, Immunogenetics, 37, 39, 1992. 127. Bazin, H. et al., Allotypes of rat immunoglobulins. I. An allotype at the alpha-chain locus, J. Immunol., 112, 1035, 1974. 128. Beckers, A., Querinjean, P., and Bazin, H., Allotypes of rat immunoglobulins. II. Distribution of the allotypes of kappa and alpha chain loci in different inbred strains of rats, Immunochemistry, 11, 605, 1974. 129. Rokhlin, O, V. and Nezlin, R.S., RL allotypes of light chains of rat immunoglobulins: ratio in heterozygous rats and distribution among inbred and random-bred rats, Scand. J. Immunol., 3, 209, 1974. 130. Gutman, G.A., Loh, E., and Hood, L., Structure and regulation of immunoglobulins: kappa allotypes in the rat have multiple amino-acid differences in the constant region, Proc. Natl. Acad. Sci., 72, 5046, 1975. 131. Nezlin, R. and Rokhlin, O., Allotypes of light chains of rat immunoglobulins and their application to the study of antibody biosynthesis, Contemp. Top. Mol. Immunol., 5, 161, 1976. 132. Gutman, G.A., Genetic and structural studies on rat kappa chain allotypes, Transpl. Proc., 13, 1483, 1981. 133. Medgyesi, G.A. et al., Fc receptors of rat peritoneal macrophages: immunoglobulin class specificity and sensitivity to drugs affecting the microfilament or microtubule system, Immunology, 40, 317, 1980. 134. Tolnay, M. et al., Interaction between rat peritoneal macrophages and sensitised erythrocytes: dependence on IgG subclass, antibody density and the degree of hapten conjugation, Mol. Immunol., 29, 385, 1992. 135. Miklos, K. et al., Rat IgG subclasses mediating binding and phagocytosis of target cells by homologous macrophages, Mol. Immunol., 30, 1273, 1993. 136. van de Winkel, J.G., and Capel, P.J., General characteristics of human IgG Fc receptors, in Human IgG Fc Receptors (Molecular Biology Intelligence Unit), van de Winkel, J.G. and Capel, P.J., Eds., Chapman & Hall, London, 1996, p. 7. 137. Hale, G., Clark, M., and Waldmann, H., Therapeutic potential of rat monoclonal antibodies: isotype specificity of antibody-dependent cell-mediated cytotoxicity with human lymphocytes, J. Immunol., 34, 3056, 1985. 138. Chassoux, D.M. et al., K-cell-mediated cytotoxicity induced with rat monoclonal antibodies. I. Antibodies of various isotypes differ in their ability to induce cytotoxicity mediated by rat and human effectors, Immunology, 65, 623, 1988. 139. Medgyesi, G.A. et al., Classes and subclasses of rat antibodies: reaction with the antigen and interaction of the complex with the complement system, Immunology, 43, 171, 1981. 140. Farkas, A.I. et al., Immunogenicity of antigen complexed with antibody. I. Role of different isotypes, Immunology, 45, 483, 1982. 141. Rits, M. et al., Complement-mediated solubilization of rat IgA immune precipitates, Mol. Immunol., 24, 1047, 1987.


RAT IMMUNE SYSTEM

111

142. Pluschke, G. et al., Isolation of rat IgM to IgG hybridoma isotype switch variants and analysis of the efficiency of rat Ig in complement activation, Eur. J. Immunol., 19, 131, 1989. 143. Smith, L.C., Azumi, K., and Nonaka, M., Complement systems in invertebrates. The ancient alternative and lectin pathways, Immunopharmacology, 42, 107, 1999. 144. Tomlinson, S., Complement defense mechanisms, Curr. Opin. Immunol., 5, 83, 1993. 145. Volanakis, J.E., The role of complement in innate and adaptive immunity, Curr. Top. Microbiol. Immunol., 266, 41, 2002. 146. Miwa, T., Song, W.C., Membrane complement regulatory proteins: insight from animal studies and relevance to human diseases, Int. Immunopharmacol., 1, 445, 2001. 147. Caragine, T.A. et al., Expression of rat complement control protein Crry on tumor cells inhibits rat natural killer cell-mediated cytotoxicity, Blood, 100, 3304, 2002. 148. Mead, R., Hinchliffe, S.J., and Morgan, B.P., Molecular cloning, expression and characterization of the rat analogue of human membrane cofactor protein (MCP/CD46), Immunology, 98, 137, 1999. 149. Brooks, S., A method for measuring the normal blood volume of the rat using (125-I)labelled albumin, Aust. J. Exp. Biol. Med. Sci., 49, 241, 1971. 150. Yale, C.E. and Torhorst, J.B., Critical bleeding and plasma volumes of the adult germfree rat, Lab. Anim. Sci., 22, 497, 1972. 151. Libermann, I.M. et al., Blood acid-base status in normal albino rats, Lab. Anim. Sci., 23, 862, 1973. 152. Ringler, D.H. and Dabich, L., Hematology and clinical biochemistry, in The Laboratory Rat, Vol. 1, Biology and Diseases, Baker, H.J., Lindsey, J.R., and Weisbroth S.H., Eds., Academic Press, New York, 1979, p. 105. 153. Valet, G. et al., Der nachweis verscheidener erythrozyten populationen bei der ratte, Blut, 24, 42, 1972. 154. Schermer S., The Blood Morphology of Laboratory Animals, 3rd ed., F.A. Davis Co., Philadelphia, 1967. 155. Wolford, S.T. et al., Reference range data base for serum chemistry and hematology values in laboratory animals, J. Toxicol. Environ. Health, 18, 161, 1986. 156. Wolford, S.T. et al., Age-related changes in serum chemistry and hematology values in normal Sprague-Dawley rats, Fundam. Appl. Toxicol., 8, 80, 1987. 157. Matsuzawa, T., Nomura, M., and Unno, T., Clinical pathology reference ranges of laboratory animals. Working Group II, Nonclinical Safety Evaluation Subcommittee of the Japan Pharmaceutical Manufacturers Association, J. Vet. Med. Sci., 55, 351, 1993. 158. Geaghan, S.M., Hematologic values and appearances in the healthy fetus, neonate, and child, Clin. Lab. Med., 19, 1, 1999. 159. Marone, G. et al., Molecular and cellular biology of mast cells and basophils, Int. Arch. Allergy Immunol., 114, 207, 1997. 160. Metcalfe, D.D., Baram, D., and Mekori, Y.A., Mast cells, Physiol. Rev., 77, 1033, 1997. 161. Woolley, D.E., The mast cell in inflammatory arthritis, N. Engl. J. Med., 348, 1709, 2003. 162. Ishizaka, T. et al., A new concept of triggering mechanisms of IgE-mediated histamine release, J. Allergy Clin. Immunol., 61, 320, 1978. 163. Bochner, B.S. and Schleimer, R.P., Mast cells, basophils, and eosinophils: distinct but overlapping pathways for recruitment, Immunol. Rev., 179, 5, 2001. 164. Bassett, E.G. et al., Comparison of collagenase activity in eosinophil and neutrophil fractions from rat peritoneal exudates, Aust. J. Exp. Biol. Med. Sci., 54, 459, 1976. 165. Bassett, E.G., Baker, J.R., and de Souza, P., A light microscopical study of healing incised dermal wounds in rats, with special reference to eosinophil leucocytes and to the collagenous fibres of the periwound areas, Br. J. Exp. Pathol., 58, 581, 1977. 166. Kitamura, Y. et al., Development of mast cells and basophils: processes and regulation mechanisms, Am. J. Med. Sci. 306, 185, 1993. 167. Gibson, S. et al., Phenotypic expression of mast cell granule proteinases. Distribution of mast cell proteinases I and II in the rat digestive system, Immunology, 62, 621, 1987. 168. Urade, Y. et al., Mast cells contain spleen-type prostaglandin D synthetase, J. Biol. Chem., 265, 371, 1990. 169. Heavey, D.J. et al., Generation of leukotriene C4, leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells, J. Immunol., 140, 1953, 1988.


112


170. Hogaboam, C.M. et al., Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds, Br. J. Pharmacol., 105, 87, 1992. 171. Moretti, G. et al., The quantitative behavior of cutaneous histamine and mast cells in the hair cycles of rats, J. Invest. Dermatol., 46, 231, 1966. 172. Fredholm, B. and Hagermark, O., Studies on histamine release from skin and from peritoneal mast cells of the rat induced by heat, Acta Derm. Venereol., 50, 273, 1970. 173. Yurt, R.W., Leid R.W., and Austen K.F., Native heparin from rat peritoneal mast cells, J. Biol. Chem., 252, 518, 1977. 174. Seldin, D.C., Austen, K.F., and Stevens, R.L., Purification and characterization of protease-resistant secretory granule proteoglycans containing chondroitin sulfate di-B and heparin-like glycosaminoglycans from rat basophilic leukemia cells, J. Biol. Chem., 260, 11131, 1985. 175. Kirshenbaum, A., Regulation of mast cell number and function, Hematol. Oncol. Clin. North Am., 14, 497, 2000. 176. Beil, W.J., Schulz, M., and Wefelmeyer, U., Mast cell granule composition and tissue location-a close correlation, Histol. Histopathol., 15, 937, 2000. 177. Spits, H., Lanier, L.L., and Phillips, J.H., Development of human T and natural killer cells, Blood, 85, 2654, 1995. 178. Kärre, K. et al., Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy, Nature, 319, 675, 1986. 179. Biron, C.A., Activation and function of natural killer cell responses during viral infections, Curr. Opin. Immunol., 9, 24, 1997. 180. Baxter, A.G. and Smyth, M.J., The role of NK cells in autoimmune disease, Autoimmunity, 35, 1, 2002. 181. Matsumoto, Y. et al., Role of natural killer cells and TCR gamma delta T cells in acute autoimmune encephalomyelitis, Eur. J. Immunol., 28, 1681, 1998. 182. Smeltz, R.B., Wolf, N.A., and Swanborg, R.H., Inhibition of autoimmune T cell responses in the DA rat by bone marrow-derived NK cells in vitro: implications for autoimmunity, J. Immunol., 163, 1390. 1999. 183. Iwakoshi, N.N. et al., Diabetes prone BB rats are severely deficient in natural killer T cells, Autoimmunity, 31, 1, 1999. 184. Nakamura, N. et al., Intrinsic cytotoxicity of natural killer cells to pancreatic islets in vitro, Diabetes, 39, 836, 1990. 185. Reynolds, C.W. et al., Natural killer activity in the rat. II. Analysis of surface antigens on LGL by flow cytometry, J. Immunol., 127, 2204, 1981. 186. Woda, B.A. et al., Separation and isolation of rat natural killer (NK) cells from T cells with monoclonal antibodies, J. Immunol., 132, 2183, 1984. 187. Vujanovic, N.L. et al., Lymphokine-activated killer cells in rats: analysis of progenitor and effector cell phenotype and relationship to natural killer cells, Cancer Res., 48, 884, 1988. 188. Maghazachi, A.A. et al. Lymphokine-activated killer cells in rats. IV. Developmental relationships among large agranular lymphocytes, large granular lymphocytes, and lymphokine-activated killer cells, J. Immunol., 140, 2846, 1988. 189. Sarneva, M. et al., Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells, Cell. Immunol., 118, 448, 1989. 190. Lanier, L.L. et al., Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex, Fed. Proc., 45, 2823, 1986. 191. Salup, R.R., Mathieson, B.J., and Wiltrout, R.H., Precursor phenotype of lymphokine-activated killer cells in the mouse, J. Immunol., 138, 3635, 1987. 192. Mason, L.H., Mathieson, B.J., and Ortaldo, J.R., Natural killer (NK) cell subsets in the mouse. NK1.1+/LGL-1+ cells restricted to lysing NK targets, whereas NK-1.1+/LGL-1- cells generate lymphokine-activated killer cells, J. Immunol., 145, 751, 1990. 193. Kuribayashi, K. et al., Murine NK cell cultures: effects of interleukin-2 and interferon on cell growth and cytotoxic reactivity, J. Immunol., 126, 2321, 1981. 194. Lanier, L.L., NK cell receptors, Ann. Rev. Immunol., 16, 359, 1998. 195. Lanier, L.L., Activating and inhibitory NK cell receptors, Adv. Exp. Med. Biol., 452, 13, 1998.


RAT IMMUNE SYSTEM

113

196. Rolstad, B. et al., Positive and negative MHC class I recognition by rat NK cells, Immunol. Rev., 155, 91, 1997. 197. Rolstad, B. et al., Rat natural killer cell receptor systems and recognition of MHC class I molecules, Immunol. Rev., 181, 149, 2001. 198. Mason, D.W. et al., Functions of rat T-lymphocyte subsets isolated by means of monoclonal antibodies, Immunol. Rev., 74, 57, 1983. 199. Powrie, F. et al., Lineage relationships and functions of CD4+ T-cell subsets in the rat, Res. Immunol., 142, 54, 1991. 200. Damoiseaux, J.G. et al., A dominant role for the thymus and MHC genes in determining the peripheral CD4/CD8 T cell ratio in the rat, J. Immunol., 163, 2983, 1999. 201. Amadori, A. et al., Genetic control of the CD4/CD8 T-cell ratio in humans, Nat. Med., 1, 1279, 1995. 202. van Meerwijk, J.P. et al., Thymic lineage commitment rather than selection causes genetic variations in size of CD4 and CD8 compartments, J. Immunol., 160, 3649, 1998. 203. Barclay, A.N., The localization of populations of lymphocytes defined by monoclonal antibodies in rat lymphoid tissues, Immunology, 42, 593, 1981. 204. Jefferies, W.A., Green, J.R., and Williams, A.F., Authentic T helper CD4 (W3/25) antigen on rat peritoneal macrophages, J. Exp. Med., 162, 117, 1985. 205. Crocker, P.R. et al., Species heterogeneity in macrophage expression of the CD4 antigen, J. Exp. Med., 166, 613, 1987. 206. Powrie, F. and Mason, D., Subsets of rat CD4+ T cells defined by their differential expression of variants of the CD45 antigen: developmental relationships and in vitro and in vivo functions, Curr. Top. Microbiol. Immunol. 159, 79, 1990. 207. Westermann, J. et al., CD4+ T cells of both the naive and the memory phenotype enter rat lymph nodes and Peyer’s patches via high endothelial venules: within the tissue their migratory behavior differs, Eur. J. Immunol., 27, 3174, 1997. 208. Sakaguchi, S. et al., Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases, J. Immunol., 155, 1151, 1995. 209. Stephens, L.A. and Mason, D., Characterisation of thymus-derived regulatory T cells that protect against organ-specific autoimmune disease, Microbes Infect., 3, 905, 2001. 210. Kuhnlein, P. et al., Identification and characterization of rat gamma/delta T lymphocytes in peripheral lymphoid organs, small intestine, and skin with a monoclonal antibody to a constant determinant of the gamma/delta T cell receptor, J. Immunol., 153, 979, 1994. 211. Kuhnlein, P. et al., Gamma/delta T cells in fetal, neonatal, and adult rat lymphoid organs, Dev. Immunol., 4, 181, 1995. 212. Kuhnlein, P. et al., The canonical T cell receptor of dendritic epidermal gamma delta T cells is highly conserved between rats and mice, Eur. J. Immunol., 26, 3092, 1996. 213. Watson, D., Ando, T., and Knight, J.F., Sequence and diversity of the rat delta T-cell receptor, Immunogenetics, 51, 714, 2000. 214. Lawetzky, A. et al., Identification and characterization of rat T cell subpopulations expressing T cell receptors alpha/beta and gamma/delta, Eur. J. Immunol., 20, 343, 1990. 215. Winkler, G.C., Pulmonary intravascular macrophages in domestic animal species: review of structural and functional properties, Am. J. Anat., 181, 217, 1988. 216. Miot-Noirault, E. et al., Scintigraphic in vivo assessment of the development of pulmonary intravascular macrophages in liver disease: experimental study in rats with biliary cirrhosis, Chest, 120, 941, 2001. 217. van Furth, R. and Cohn, Z.A., The origin and kinetics of mononuclear phagocytes, J. Exp. Med., 128, 415, 1968. 218. Volkman, A., The origin and fate of the monocyte, Ser. Haematol., 3, 62, 1970. 219. Damoiseaux, J.G.M.C. et al., Rat macrophage lysosmal membrane antigen recognized by monoclonal antibody ED1, Immunology, 83, 140, 1994. 220. van den Berg, T.K., Döpp, E.A., and Dijkstra, C.D., Cellular binding mechanism on rat macrophages for sialylated glycoconjugates, inhibited by the monoclonal antibody ED3, J. Leukoc. Biol., 49, 434, 1991.


114


221. van den Berg, T.K. et al., Monoclonal antibodies against rat leukocyte surface antigens, Immunol. Rev., 184, 109, 2001. 222. Adams, S. et al., Signal-regulatory protein is selectively expressed by myeloid and neuronal cells, J. Immunol., 161, 1853, 1998. 223. Robinson, A.P., White, T.M., and Mason, D.W., Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3, Immunology, 57, 239, 1986. 224. Steiniger, B. et al., Monocytes in the rat: phenotype and function during acute allograft rejection, Immunol. Rev., 184, 38, 2001. 225. Steinman, R.M. and Cohn, Z.A., Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution, J. Exp. Med., 137, 1142, 1973. 226. Lanzavecchia, A., Mechanisms of antigen uptake for presentation, Curr. Opin. Immunol., 8, 348, 1996. 227. Inaba, K. et al., Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells, J. Exp. Med., 188, 2163, 1998. 228. Albert, M.L. et al., Immature dendritic cells phagocytose apoptotic cells via alphaVbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes, J. Exp. Med., 188, 1359, 1998. 229. Banchereau, J. and Steinman, R.M., Dendritic cells and the control of immunity, Nature, 392, 245, 1998. 230. MacPherson, G.G. and Pugh, C.W., Heterogeneity amongst lymph-borne “dendritic” cells, Immunobiology, 168, 338, 1984. 231. Nelson, D.J. et al., Development of the airway intraepithelial dendritic cell network in the rat from class II major histocompatibility (Ia)-negative precursors: differential regulation of Ia expression at different levels of the respiratory tract, J. Exp. Med., 179, 203, 1994. 232. Hart, D.N. and Fabre, J.W., Demonstration and characterization of Ia-positive dendritic cells in the interstitial connective tissues of rat heart and other tissues, but not brain, J. Exp. Med., 154, 347, 1981. 233. Robinson, A.P., White, T.M., and Mason, D.W., Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3, Immunology, 57, 239, 1986. 234. Brenan, M. and Puklavec, M., The MRC OX-62 antigen: a useful marker in the purification of rat veiled cells with the biochemical properties of an integrin, J. Exp. Med., 175, 1457, 1992. 235. Brenan, M. and Rees, D.J., Sequence analysis or rat integrin alphaE1 and alphaE2 subunits: tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph, Eur. J. Immunol., 30, 1527, 2000. 236. Huang, F.P. et al., A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes, J Exp Med., 191, 435, 2000. 237. Liu, L.M. and MacPherson, G.G., Rat intestinal dendritic cells: immunostimulatory potency and phenotypic characterization, Immunology, 85, 88, 1995. 238. Liu, L. et al., Dendritic cell heterogeneity in vivo: two functionally different dendritic cell populations in rat intestinal lymph can be distinguished by CD4 expression, J. Immunol., 161, 1146, 1998. 239. Schon-Hegrad, M.A. et al., Studies on the density, distribution, and surface phenotype of intraepithelial class II major histocompatibility complex antigen (Ia)-bearing dendritic cells (DC) in the conducting airways, J. Exp. Med., 173, 1345, 1991. 240. Banchereau, J. et al., Immunobiology of dendritic cells, Ann. Rev. Immunol., 18, 767, 2000. 241. Banchereau, J., The long arm of the immune system, Sci. Am., 287, 52, 2002. 242. Pugh, C.W. and MacPherson, G.G., Non-lymphoid cells from rat intestinal lymph, Adv. Exp. Med. Biol., 149, 781, 1982. 243. Janeway, C.A., Jr., The immune system evolved to discriminate infectious nonself from noninfectious self, Immunol. Today, 13, 11, 1992. 244. Pugh, C.W., MacPherson, G.G., and Steer, H.W., Characterization of nonlymphoid cells derived from rat peripheral lymph, J. Exp. Med., 157, 1758, 1983. 245. MacPherson, G.G. et al., Endotoxin-mediated dendritic cell release from the intestine. Characterization of released dendritic cells and TNF dependence, J. Immunol., 154, 1317, 1995. 246. Mackay, C.R. et al., Lymphocyte subsets show marked differences in their distribution between blood and the afferent and efferent lymph of peripheral lymph nodes, J. Exp. Med., 167, 1755, 1988.


RAT IMMUNE SYSTEM

115

247. Fossum, S., Lymph-borne dendritic leucocytes do not recirculate, but enter the lymph node paracortex to become interdigitating cells, Scand. J. Immunol., 27, 97, 1988. 248. Blaschke, V. et al., Lymphocyte traffic through lymph nodes and Peyer’s patches of the rat: B- and T-cell-specific migration patterns within the tissue, and their dependence on splenic tissue, Cell Tissue Res., 282, 377, 1995. 249. Stumbles, P.A. et al., Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity, J. Exp. Med., 188, 2019, 1998. 250. Klinkert, W.E., LaBadie, J.H., and Bowers, W.E., Accessory and stimulating properties of dendritic cells and macrophages isolated from various rat tissues, J. Exp. Med., 156, 1, 1982. 251. Bowers, W.E. and Berkowitz, M.R., Differentiation of dendritic cells in cultures of rat bone marrow cells, J. Exp. Med., 163, 872, 1986. 252. O’Keeffe, M. et al., Mouse plasmacytoid cells: long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8(+) dendritic cells only after microbial stimulus, J. Exp. Med., 196, 1307, 2002. 253. McWilliam, A.S., Nelson, D.J., and Holt, P.G., The biology of airway dendritic cells, Immunol. Cell Biol., 73, 405, 1995. 254. Dinarello, C.A. and Savage, N., Interleukin-1 and its receptor, Crit. Rev. Immunol., 9, 1, 1989. 255. Bazan, J.F., Timans, J.C., and Kastelein, R.A., A newly defined interleukin-1? Nature, 379, 591, 1996. 256. Gray, P.W. et al., Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1 beta, J. Immunol., 137, 3644, 1986. 257. March, C.J. et al., Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs, Nature, 315, 641, 1985. 258. Bergsteinsdottir, K. et al., Rat Schwann cells produce interleukin-1, J. Neuroimmunol., 34, 15, 1991. 259. O’Garra, A., Cytokines induce the development of functionally heterogeneous T helper cell subsets, Immunity, 8, 275, 1998. 260. Kashima, N. et al., Unique structure of murine interleukin-2 as deduced from cloned cDNAs, Nature, 313, 402, 1985. 261. Gillis, S., Smith, K.A., and Watson, J., Biochemical characterization of lymphocyte regulatory molecules. II. Purification of a class of rat and human lymphokines, J. Immunol., 124, 1954, 1980. 262. Mochizuki, D.Y., Watson, J., and Gillis, S., Biochemical separation of interleukin 2, J. Immunol. Methods, 39, 185, 1980. 263. Di Sabato, G., Purification and initial characterization of rat interleukin 2, Proc. Natl. Acad. Sci. 79, 3020, 1982. 264. McKnight, A.J. and Classon, B.J., Biochemical and immunological properties of rat recombinant interleukin-2 and interleukin-4, Immunology, 75, 286, 1992. 265. Thompson, L.P. and Di Sabato, G., Purification and characterization of two forms of rat interleukin2, Cell Immunol., 114, 12, 1988. 266. Matesanz, F., Alcina, A., and Pellicer, A., Existence of at least five interleukin-2 molecules in different mouse strains, Immunogenetics, 38, 300, 1993. 267. Richter, G., Blankenstein, T., and Diamantstein, T., Evolutionary aspects, structure, and expression of the rat interleukin 4 gene, Cytokine, 2, 221, 1990. 268. Richter, G. et al., The rat interleukin 4 receptor: coevolution of ligand and receptor, Cytokine, 7, 237, 1995. 269. van Snick, J. et al., cDNA cloning of murine interleukin-HP1: homology with human interleukin 6, Eur. J. Immunol., 18, 193, 1988. 270. Chiu, C.P. et al., Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells, Proc. Natl. Acad. Sci., 85, 7099, 1988. 271. Northemann, W. et al., Structure of the rat interleukin 6 gene and its expression in macrophage-derived cells, J. Biol. Chem., 264, 16072, 1989. 272. Coulie, P.G., Stevens, M., and Van Snick, J., High- and low-affinity receptors for murine interleukin 6.Distinct distribution on B and T cells, Eur. J. Immunol., 19, 2107, 1989. 273. Lalani, I., Bhol, K., and Ahmed, A.R., Interleukin-10: biology, role in inflammation and autoimmunity, Ann. Allergy Asthma Immunol., 79, 469, 1997.


116


274. Feng, L. et al., Molecular cloning of rat cytokine synthesis inhibitory factor (IL-10) cDNA and expression in spleen and macrophages, Biochem. Biophys. Res. Commun., 192, 452, 1993. 275. Ball, C. et al., Rat interleukin-10: production and characterisation of biologically active protein in a recombinant bacterial expression system, Eur. Cytokine Netw., 12, 187, 2001. 276. Sun, B. et al., Evaluation of in vivo cytokine expression in EAU-susceptible and resistant rats: a role for IL-10 in resistance? Exp. Eye Res., 70, 493, 2000. 277. David, A. et al., Interleukin-10 produced by recombinant adenovirus prolongs survival of cardiac allografts in rats, Gene Ther., 7, 505, 2000. 278. Tsuruma, T. et al., Interleukin-10 reduces natural killer sensitivity and downregulates MHC class I expression on H-ras-transformed cells, Cell Immunol., 184, 121, 1998. 279. Ward, H. et al., The rat interleukin 10 receptor: cloning and sequencing of cDNA coding for the alphachain protein sequence, and demonstration by western blotting of expression in the rat brain, Cytokine, 15, 237, 2001. 280. Khalife, J. et al., Molecular cloning and sequencing of the rat interleukin-12 p40 gene, Eur. Cytokine Netw., 9, 69, 1998. 281. Aggarwal, B.B. and Natarajan, K., Tumor necrosis factors: developments during the last decade, Eur. Cytokine Netw., 7, 93, 1996. 282. Nedwin, G.E. et al., Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization, Nucleic Acids Res., 13, 6361, 1985. 283. Pennica, D. et al., Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor, Proc. Natl. Acad. Sci. U. S. A., 82, 6060, 1985. 284. Estler, H.C. et al., Rat tumor necrosis factor-alpha. Transcription in rat Kupffer cells and in vitro posttranslational processing based on a PCR-derived cDNA, Biol. Chem. Hoppe Seyler, 373, 271, 1992. 285. Kumar, S. and Hedges, S.B., A molecular timescale for vertebrate evolution, Nature, 392, 917, 1998. 286. Catzeflis, F.M. et al., DNA-DNA hybridization evidence of the rapid rate of muroid rodent DNA evolution, Mol. Biol. Evol., 4, 242, 1987. 287. O’hUigin, C. and Li, W.H., The molecular clock ticks regularly in muroid rodents and hamsters, J. Mol. Evol., 35, 377, 1992. 288. Li, W.H. et al., Molecular phylogeny of Rodentia, Lagomorpha, Primates, Artiodactyla, and Carnivora and molecular clocks, Proc. Natl. Acad. Sci. U. S. A., 87, 6703, 1990. 289. Male, D.K., Pryce, G., and Butcher, G.W., Serological evidence for a defect in RT1.B (I-A) expression by the BDIX rat strain, J. Immunogenet., 14, 301, 1987. 290. Kren, V., The major histocompatibility system (H-1) alleles of some British rat strains, Transplantation, 17, 148, 1974. 291. Kunz, H.W., RT1A (MHC), RT2, and RT3 (blood group) specificities of 44 inbred and congenic rat strains, I.L.A.R. News, 33, 1, 1991. 292. Arenas, O. et al., Comparison of the haplotypes of the major histocompatibility complex in the rat. V. A serological, genetic and numerical study of the RT1.A region antigens, J. Immunogenet., 8, 307, 1981. 293. Kato, H. et al., Molecular cloning of equine interleukin-1 alpha and -beta cDNAs, Vet. Immunol. Immunopathol., 48, 221, 1995. 294. McKnight, A.J., Mason, D.W., and Barclay, A.N., Sequence of rat interleukin 2 and anomalous binding of a mouse interleukin 2 cDNA probe to rat MHC class II-associated invariant chain mRNA, Immunogenetics, 30, 145, 1989. 295. Matesanz, F., Alcina, A., and Pellicer, A., Existence of at least five interleukin-2 molecules in different mouse strains, Immunogenetics, 38, 300, 1993. 296. Page, T.H. and Dallman, M.J., Molecular cloning of cDNAs for the rat interleukin 2 receptor alpha and beta chain genes: differentially regulated gene activity in response to mitogenic stimulation, Eur. J. Immunol., 21, 2133, 1991. 297. Bucan, M. et al., Comparative mapping of 9 human chromosome 22q loci in the laboratory mouse, Hum. Mol. Genet., 2, 1245, 1993. 298. Watanabe, T.K. et al., A radiation hybrid map of the rat genome containing 5,255 markers, Nat. Genet., 22, 27, 1999. 299. McKnight, A.J., Barclay, A.N., and Mason, D.W., Molecular cloning of rat interleukin 4 cDNA and analysis of the cytokine repertoire of subsets of CD4+ T cells, Eur. J. Immunol., 21, 1187, 1991.


RAT IMMUNE SYSTEM

117

300. Sehgal, P.B. et al., Human chromosome 7 carries the beta 2 interferon gene, Proc. Natl. Acad. Sci. U. S. A., 83, 5219, 1986. 301. Levan, G. et al., The gene map of the Norway rat (Rattus norvegicus) and comparative mapping with mouse and man, Genomics, 10, 699, 1991. 302. Szpirer, J. et al., The interleukin-6-dependent DNA-binding protein gene (transcription factor 5: TCF5) maps to human chromosome 20 and rat chromosome 3, the IL6 receptor locus (IL6R) to human chromosome 1 and rat chromosome 2, and the rat IL6 gene to rat chromosome 4, Genomics, 10, 539, 1991. 303. Baumann, M., Baumann, H., and Fey, G.H., Molecular cloning, characterization and functional expression of the rat liver interleukin 6 receptor, J. Biol. Chem., 265, 19853, 1990. 304. Sugita, T. et al., Functional murine interleukin 6 receptor with the intracisternal A particle gene product at its cytoplasmic domain. Its possible role in plasmacytomagenesis, J. Exp. Med., 171, 2001, 1990. 305. Kluck, P.M.C. et al., The human interleukin-6 receptor alpha-chain gene is localized on chromosome 1 band q21, Hum. Genet., 90, 542, 1993. 306. Dijkema, R. et al., Cloning and expression of the chromosomal immune interferon gene of the rat, EMBO J., 4, 761, 1985. 307. Dijkema, R. et al., Cloning, expression, and purification of rat IFN-gamma, Methods Enzymol., 119, 453, 1986. 308. Kirisits, M.J. et al., Mapping of the TNFA locus in the rat, Immunogenetics, 39, 59, 1994. 309. Masuyama, T. et al., An improved genetic linkage map of rat chromosome 20, Comp. Med., 50, 369, 2000. 310. Makalowski, W. and Boguski, M.S., Evolutionary parameters of the transcribed mammalian genome: an analysis of 2,820 orthologous rodent and human sequences, Proc. Natl. Acad. Sci. U. S. A., 95, 9407, 1998.



CHAPTER

6

Mouse Immune System Lawrence S. Chan and Kenneth B. Gordon

CONTENTS I. Introduction ........................................................................................................................120 II. Innate Immune System ......................................................................................................120 A. Membrane Receptors, Transcriptional Factors, and Soluble Factors .......................120 1. Toll-Like Receptors .............................................................................................120 a. TLR4 ..............................................................................................................121 b. TLR2 (TLR1 and TLR6)...............................................................................121 c. TLR5 ..............................................................................................................121 d. TLR9 ..............................................................................................................121 e. TLR3 ..............................................................................................................121 f. TLR7 ..............................................................................................................121 2. NF-kB Family of Transcriptional Factors...........................................................122 a. NF-kB ............................................................................................................122 b. IkB Proteins ...................................................................................................122 c. NF-kB Activation ..........................................................................................122 3. Defensins..............................................................................................................122 4. Cytokines .............................................................................................................123 a. Th1 vs. Th2....................................................................................................123 b. Cytokine Receptor Superfamilies..................................................................123 c. Cytokines in Mouse Immune System ...........................................................124 5. Chemokines..........................................................................................................124 6. Adhesion Molecules ............................................................................................125 7. Complement.........................................................................................................126 B. Immune Cells.............................................................................................................126 1. Natural Killer Cells .............................................................................................127 2. Mast Cells ............................................................................................................127 3. B-1 Cells and Natural Antibodies .......................................................................128 4. Interferon-Producing Cells ..................................................................................128 5. Gamma/Delta T Cells (gd T Cells) .....................................................................128 6. Intraepithelial Lymphocytes ................................................................................129 III. Adaptive Immune System..................................................................................................129 A. T Lymphocytes ..........................................................................................................129 0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

119


120


B. B Lymphocytes (B-2 Cells).......................................................................................129 C. Antigen-Presenting Cells ...........................................................................................130 D. Major Histocompatibility Complex (MHC)..............................................................130 IV. Mice as an Essential Tool for Animal Modeling of Human Immune-Mediated Diseases ..............................................................................................................................130 A. Development and Nomenclature of Inbred Mouse Strains ......................................131 B. The Use of Inbred Mouse Strains .............................................................................131 C. Inbred Mouse Strains and Disease Susceptibility.....................................................132 D. Naturally Occurring Mutations of Mouse Immune System .....................................133 1. Severe Combined Immunodeficiency (SCID) Mice ...........................................133 2. Nude Mice ...........................................................................................................134 E. Experimentally Induced Alteration of Mouse Immune System ...............................134 1. RAG KO Mice.....................................................................................................134 2. Cytokine KO Mice ..............................................................................................134 3. Adhesion Molecule KO Mice .............................................................................134 4. Immune Cell KO Mice........................................................................................135 V. Conclusion..........................................................................................................................135 Acknowledgments ..........................................................................................................................135 References ......................................................................................................................................135

I. INTRODUCTION The mouse immune system is remarkably similar to that of humans, despite the existence of some variations [1–45]. In many ways, the mouse immune system is understood to a greater extent than the human system, due to the ability of researchers to use transgenic and knockout methods in mice to investigate the in vivo functions of immune components, one at a time. The organization and function of the human immune system have been described in substantial detail in Chapter 3. To avoid unnecessary repetition, the goal of this chapter is to highlight the essential facts about the mouse immune system in fulfilling the purpose of a comparative analysis between the mouse and human immune systems, without significant elaboration. However, features of mouse immune system distinct from the human counterpart, will be discussed in greater detail.

II. INNATE IMMUNE SYSTEM The innate (natural) immune system is traditionally considered to be the front line of immune defense that is instantly available without the need of prior encounter, as required in the adaptive (acquired) immune system. This division, however, is somewhat artificial. The current understanding of the innate immune system has led us to conclude that the innate and adaptive immune systems are not mutually exclusive, whether in components or in functions, but rather that they are strongly linked to provide an optimal immune defense for the entire body [34,46,47]. Many of the components described in the innate system actually actively participate in development and regulation of the adaptive system [3,7,8,10,11,13–15,17,26,33–36,38]. A. Membrane Receptors, Transcriptional Factors, and Soluble Factors 1. Toll-Like Receptors Toll-like receptors (TLRs) are considered to the primary sensors of the innate immune system and are important in the host defense against pathogenic microorganisms, as they are capable of


MOUSE IMMUNE SYSTEM

121

recognizing conserved motifs in pathogens known as pathogen-associated molecular patterns (PAMPs) in bacteria, fungi, and viruses [4,6,7]. The first human homologue of the Drosophila protein Toll was identified in 1997 — hence the term, “Toll-like receptor.” Since then, a total of ten mammalian TLRs have been identified, TLR1 through TLR10 [4,7]. As a family of conserved innate immune recognition receptors, TLRs share similarity in a cytoplasmic Toll/IL-1 receptor domain with that of the IL-1 receptor family, and are characterized by a distinct extracellular domain containing leucine-rich repeats [7]. Thus, TLRs and the IL-1 receptor family exert their functions by the same signaling pathways, including MyD88, IL-1 receptor–associated kinase (IRAK), TNF receptor-associated factor 6 (TRAF 6), mitogen-activated protein (MAP) kinases, and nuclear factor kappa B (NF-kB) [7]. a. TLR4 Experimental data from knockout mice have shown that TLR4 is essential for Gram- bacterial, outer-membrane component lipopolysaccharide (LPS) signaling. In addition, interaction of LPS with TLR4 requires another molecule, MD-2, which is unique for TLR4 [7,48,49]. Mouse TLR4 differs from human TLR4 in that the ligands lipid A analog, lipid IVa, and plant-derived reagent taxol act as LPS mimetics in the mouse system, whereas they act either as an antagonist or a nonfunctional entity in the human system [7]. b.

TLR2 (TLR1 and TLR6)

TLR2 recognizes Gram+ bacterial peptidoglycan, bacterial lipoproteins and lipopeptides, Trypanosoma glycophosphatidylinositol anchors, Mycobacterium lipoarabinomannan, Neisseria porins, and yeast cell-wall component zymosan [7]. Analyses of knockout mice confirmed that TLR2 ligand recognition is formed by way of heterodimers between TLR2 and other TLRs [7,50]. c.

TLR5

Expressed on the basolateral, but not apical, surface of intestinal epithelia, TLR5 recognizes a 55-kDa flagellin monomer, a rod-like propelling appendage of Gram- bacteria [7,51]. d. TLR9 The important role of TLR9 lies in its recognition of bacterial DNA, viral DNA, and synthetic oligodeoxynucleotides containing unmethylated CpG (CpG DNA). Due to amino acid sequence differences between the extracellular domains of the human and mouse TLR9, the optimal immunostimulatory CpG DNA motifs are distinct between humans and mice [7,52]. e. TLR3 Data from TLR3 knockout mice showed a reduced response to double-stranded RNA, suggesting the role of TLR3 in recognition of double-stranded RNA, which is a common result of viral replication within infected cells [7,53]. f.

TLR7

Rather than being known for its recognition of pathogen motifs, TLR7 is known for its ability to recognize imidazoquinolines. One of these molecules termed imiquimod, a topical antihuman papilloma virus medication, has the ability to induce Th1-type cytokines IL-12 and IFN-a via the TLR7/MyD88 pathway [7,54].


122


2. NF-kB Family of Transcriptional Factors a. NF-kB When LPS or other pathogen components induce signaling to the nucleus of macrophages of the innate immune system, these signaling pathways are mediated by NF-kB and other stressresponsive transcriptional factors (SRTFs), which in turn lead to expressions of multiple inflammatory genes. The NF-kB is normally present in the cytoplasm during inactivated state and is associated with inhibitors of NF-kB (IkBs). When activated by inducers, IkBs are phosphorylated, ubiquitylated, and degraded by proteasome, allowing the translocation and entrance of NF-kB to the nucleus. The binding of NF-kB to cognate DNA-binding sites in the nucleus allows transcriptional activation of more than 100 genes encoding mediators of inflammatory and immune responses [10,11]. In the mouse immune system, five members of the NF-kB family have been identified, including NF-kB1, NF-kB2, c-REL, RELB, and RELA [11]. All five members have a structurally conserved amino-terminal Rel-homology domain, which contains three subdomains: dimerization, nuclear-localization, and DNA-binding subdomains. In addition, the c-REL, RELB, and RELA proteins also have a carboxy-terminal nonhomologous transactivation domain. RELB protein has an additional leucine-zipper motif [11]. Target disruption of the genes encoding these five members in mouse models has led to understanding of the define roles of these NF-kB proteins in both innate and adaptive immune systems [11]. While mice deficient in RELA resulted in embryonic death, due to liver degeneration, mice lacking one of the other four members are immunodeficient, but without developmental defects. Interestingly, mice deficient in more than one member have more severe clinical phenotypes than those deficient in single protein, indicating the existence of functional redundancy among these five members [11,55,56]. b.

IkB Proteins

Three common forms of IkB proteins are identified, including IkBa, IkBb, and IkBe [11,55]. Results obtained from knockout mice experiments indicate distinct and redundant functions of these proteins [11]. Whereas IkBa is responsible for regulating transient activation of NF-kB, IkBb involves persistent activation of NF-kB [11]. c.

NF-kB Activation

NF-kB is activated by many pathways, including bacterial LPS, TNF/IL-1, and T-cell receptor (TCR) signaling [11]. Regardless which of the pathways is initiated, they all converge through the IkB kinase (IKK) complex, including IKKa, IKKb, and IKKg; the latter is also known as NF-kB essential modulator (NEMO) [11]. Activation of the IKK complex leads to phosphorylation and degradation of IkB proteins, resulting in exportation of NF-kB to the nucleus with the subsequent induction of expressions of multiple inflammatory genes [11]. 3. Defensins Mammalian defensins are small, cationic, cysteine-rich peptides with important antimicrobial functions [13,14]. They are an important member of the innate immunity, but also argument-adaptive immune responses [13,14]. Three major groups of defensins are known: a-defensins, b-defensins, and circular defensins. Existing data indicate a structural diversity of defensins between human and mouse immune systems. While a-defensins are noticeably absent in mouse neutrophils, they are prominently present in human neutrophils, with four of the six a-defensins are primarily expressed in human granulocytes and certain lymphocytes and are also termed human neutrophil peptides [13]. The only known mouse a-defensins are expressed in the intestinal Paneth cells [13].


MOUSE IMMUNE SYSTEM

123

On the other hand, at least five members of b-defensins are known in mouse, with partially overlapping tissue distributions between them [13,57–61]. While several forms of b-defensins are identified in human, circular defensins have only been identified in primates thus far [13]. Being a positively charaged cationic molecule, defensin attaches and attacks microbial negatively charged surface components by way of electrostatic interactions. The attacking sites of defensins include anionic membrane phospholipids such as phosphotidyl glycerol and cardiolipin, lipoteichoic acids, and lipid A moiety of LPS [13]. By interacting with chemokine receptors on dendritic cells and T cells, defensins may also participate in the regulation of host adaptive immunity in protecting against microbial invasion [13,14]. 4. Cytokines Cytokines are defined as soluble proteins or glycoproteins produced by leukocytes and other cells and function as chemical communicators between cells or as self-communicator (autocrine function). Although they carry many different functions, a unifying characteristic of most cytokines is that they regulate host defense against pathogens and inflammatory responses. It is now clear that cytokines are involved in both innate and adaptive immune functions; some cytokines, however, are more restricted to the adaptive immune system. While most cytokines are secreted, some are expressed on cell surface and others are stored in extracellular matrix reservoirs. Whereas some cytokines are constitutively expressed, most cytokines are expressed only under stimulation by infectious microorganisms such as bacteria, fungi, viruses, and parasites, toxin, and tissue damage. Cytokines exert their functions by binding to specific target cell-surface receptors that coupled with intracellular signal transduction and second messenger pathway. Most cytokines exhibit four essential features: (1) pleiotropy, having more than one action; (2) redundancy, having biological effects observed in other cytokine; (3) potency, having functional activity in nanomolar to femtomolar concentration; and (4) teamwork, participating, often synergistically, as a part of cascade of cytokines released in succession, and counter-balanced by antagonist inhibition [15–17]. a. Th1 vs. Th2 One well-characterized cytokine network pattern is the polarization of cytokine production by helper T-cell subset 1 (Th1) and subset 2 (Th2), which was defined in the mouse system by Fitzgerald et al. [16] and Mossman et al. [62]. This clear-cut polarization is not as obvious in the human system. Experimental data indicate that the cytokines in Th1 and Th2 subsets carry distinct immunological roles and are functionally antagonistic to each other [16,62]. While the roles of Th2 cytokines (IL-4, IL-5, IL-6, IL-10, and IL-13) were assigned to predominantly humoral immunity and allergy, the roles of Th1 cytokines (IL-12, INF-g, and TNF-b) were skewed toward primarily cellular immunity, inflammation, and organ-specific autoimmunity [16,62]. Table 6.1 depicts the current understanding on cytokine molecules as well as other characteristics of Th1 and Th2 Cells. b.

Cytokine Receptor Superfamilies

The cloning of cytokine receptors and analysis of their primary structures allow them to be grouped into families based on common homology regions. The major cytokine receptor superfamilies recognized are the hematopoietic receptor (also known as type I cytokine receptor), the interferon receptor (also named type II cytokine receptor), the TNF receptor, the IL-1/Toll-like receptor, the tyrosinase kinase receptor, and the chemokine receptor superfamilies [16]. The signal transductions by the hematopoietic receptor and the interferon receptor superfamilies are through the Janus kinases (Jaks) and signal transducers of activated transcription (STATs) [16,63,64]. The signal transductions by the TNF receptor and the IL-1/Toll-like receptor superfamilies that lead to inflammation take place in part through the NF-kB pathway [16].


124


Table 6.1 Characteristics of Th1 and Th2 Cells Characteristics

Th1 Cells

Signals Transcriptional factors Cell surface molecules

JNK, p38 MAP kinase T-bet, STAT4, NF-kB LAG-3, IL-12R-b2, CCR5, CXCR3

Activators Inhibitors Cytokine produced

IL-12, IL-18, IL-23, CD28 IL-4, IL-10 IL-2, IL-18, IFN-g, TNF-b

Th2 Cells STAT6, GATA-3, c-Maf, JunB, NFATc ICOS, CD30, CD62, CCR3, CCR4, CCR8, CXCR4 IL-4, ICOS IFN-g IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-25

Sources: From Fitzgerald, K.A. et al., The Cytokine Facts Book, Academic Press, San Diego, 2001 [16]; Mosmann, T.R. et al., J. Immunol. 136, 2348, 1986 [62]; Oppmann, B. et al., Immunity, 13, 715, 2000 [70]; Fort, M.M. et al., Immunity, 15, 985, 2001 [74]; Hurst, S.D. et al., J. Immunol., 169, 443, 2002 [75]; Paul, W.E. and Seder, R.A., Cell, 76, 241, 1994 [84]; and Mosmann, T.R. and Coffman, R.L., Annu. Rev. Immunol., 7, 145, 1989 [94], with permission.

c.

Cytokines in Mouse Immune System

To illustrate the similarities in cytokines between human and mouse immune systems, the followings are a list of all known interleukins and some common cytokines and cytokine receptors that are confirmed to be present in both human and mouse systems [16]: Interleukin-1a (IL-1a), IL-b, IL-1 receptor antagonist (IL-1Ra), type I and II IL-1 receptors, IL-2, IL-2 receptor, IL-3, IL3 receptor, IL-4, IL-4 receptor, IL-5, IL-5 receptor, IL-6, IL-6 receptor, IL-7, IL-7 receptor, IL-8 receptor (CXCR2), IL-9, IL-9 receptor, IL-10, IL-10 receptor, IL-11, IL-11 receptor, IL-12 (p35 and p40 chains), IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-16, IL-17, IL-17 receptor, IL-18, IL-18 receptor, angiostatin, epidermal growth factor (EGF), EGF receptor, Fas ligand (FasL), FasL receptor (Fas/Apol, CD95), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), FGF receptors (types 1, 2, 3, and 4), granulocyte colony-stimulating factor (G-CSF), G-CSF receptor, granulocyte/macrophage colony-stimulating factor (GM-CSF), GM-CSF receptor, IFN-a, IFN-a receptor (IFNAR-1), IFN-b, IFN-g, IFN-g receptor, lymphotoxin (LT, TNF-b), macrophage colony-stimulating factor (M-CSF), M-CSF receptor, nerve growth factor (NGF), neurotrophin 3 (NT-3), NT-3 receptor, platelet-derived growth factor (PDGF), PDGF receptor, stem cell factor (SCF), SCF receptor, transforming growth factor b1, 2, 3 (TGFb1, 2, 3), TGFb receptor (type I), tumor necrosis factor a (TNFa), and TNFa receptors (type I and II). Worth noticing are the facts that there is no obvious mouse homologue of human IL-8 and IL-14 [16]. The direct comparison of the physiochemical properties of some major human cytokines with that of mice is depicted in Table 6.2. More recently, several novel cytokines have been identified in both human and mouse, with the respective percentage sequence identities at the amino acid levels: IL-19 (71%) [65]; IL-20 (76%) [66]; IL-21 (79%) [67,68]; IL-22 [69]; IL-23 [70–72]; IL-24 [73]; and IL-25 (80%) [74,75]. 5. Chemokines Chemokines are basically chemotactic cytokines and are members of the chemokine superfamily within the cytokine superfamilies [16,18]. They primarily act on immune cells such as neutrophils, eosinophils, monocytes/macrophages, and lymphocytes, thus exerting an essential role in host defense, both innate and adaptive [18,76]. Their fundamental roles are for the development, homeostasis, and function of the immune system [76]. Chemokines are characterized by their abilities to deliver cell-specific migratory signals. Chemokines are primarily divided into three families, a (CXC), b (CC), and g (C), based on the presence and position of the conserved cysteine amino acid residues [16,18]. A new classification has been proposed recently to include a new family termed CX3C [76]. Generally speaking, a chemokines are chemotactic to granulocytes, including neutrophils, eosinophils, and basophils, whereas b chemokines are chemotactic to mononuclear


MOUSE IMMUNE SYSTEM

125

Table 6.2 Comparative Physiochemical Properties of Human and Mouse Cytokines Cytokine

PI

AAa

Mrb

N-linked G Sitec

DS bondsd

h IL-1a m IL-1a h IL-1b m IL-1b h IL-2 m IL-2 h IL-3 m IL-3 h IL-4 m IL-4 h IL-5 m IL-5 h IL-6 m IL-6 h IL-7 m IL-7 h IL-10 m IL-10 h IL-12 p35 m IL-12 p35 h IL-12 p40 m IL-12 p40 h IL-13 m IL-13 h TNF-a m TNF-a h IFN-g m IFN-g

5 5 7 7 8.2 ? 4-8 4-8 10.5 6.5 7 7.8 6.2 6.5 9 8.7 8 8.1 6.5 8.2 5.4 6 8.69 8.34 5.6 5.6 7-9 5.5-6

159 156 153 159 133 149 133 140 129 120 115 113 183 187 152 129 160 160 196 193 306 313 112 113 157 156 143 133

18 18 17.4 17.4 15.4 17.2 15.1 15.7 15 13.6 13.1 13.1 20.8 21.7 17.4 14.9 18.6 18.8 22.5 21.7 34.7 35.8 12.3 12.4 17.4 17.3 17.1 15.9

2 3 1 2 0 0 2 4 2 3 2 3 2 0 3 2 1 2 3 1 4 5 4 3 0 1 2 2

0 0 0 0 1 1 1 2 3 3 2 2 2 2 3 3 2 2 3 3 5 6 2 ? 1 1 0 0

Note: h, human; m, mouse; PI, isoelectric point. a b c d

Amino acids after signal peptide removal. Predicted molecular ratio. Potential N-linked glycosylation site. Disulfide bonds.

cells, such as lymphocytes and monocytes/macrophages. However, there are some exceptions [18]. To demonstrate the similarities in chemokines between human and mouse immune systems, the followings are a list of chemokines confirmed to be present in both human and mouse systems [18,76]: melanoma growth stimulatory activity (MGSA), gamma interferon–inducible protein-10 (IP-10), macrophage inflammatory protein-2 (MIP-2), stromal cell derived factor-1 (SDF-1), monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, macrophage inflammatory protein-1a (MIP-1a), MIP-1b, MIP-3a (LARC), MIP-3b (EBI-1 ligand chemokine, ELC), RANTES (regulated on activation, normal T cell expressed and secreted), I-309/TCA-3, lymphotactin (Lptn), eotaxin-1, eotaxin-2, eotaxin-3, B-cell–attracting chemokine-1 (BCA-1), 6Ckine, cutaneous T-cell–attracting chemokine (CTACK), granulocyte chemotactic protein-2 (GCP-2), migration inhibition factor (MIF), monokine induced by interferon g (MIG), thymus expressed chemokine (TECK), PF4, BRAK, HCC4/LCC-1, TARC, macrophage-derived chemokine (MDC)/ABCD-1, and the new CX3C chemokine fractalkine/neurotactin. 6. Adhesion Molecules Adhesion molecules are cell-surface components that mediate adhesive functions between cells or between cells and extracellular matrix. Adhesion molecules are very important in the inflammatory


126


process during which immune cells migrate out of the blood vessels and moved toward the inflammatory sites. The majority of adhesion molecules can be categorized into four families based on both the structural and functional similarities of the family members: cadherins, integrins, selectins, and syndecans [24]. An additional family is called the immunoglobulin superfamily. The adhesion molecules of the cadherins families that are components of epidermis and which are discussed in Chapters 1 and 2, will not be further described here. To illustrate the similarities in adhesion molecules between human and mouse immune systems, the following components have been confirmed to be present in both human and mouse systems [24]: E-cadherin, N-cadherin, P-cadherin, R-cadherin, VE-cadherin, K-cadherin, cadherin-8, OB-cadherin, Br-cadherin, H-cadherin, Ksp-cadherin, cadherin-14, LI-cadherin, ALCAM (CD166), CD22, CD31 (PECAM-1), CD33, CD147, CEACAM (CD66), contactin-1, ICAM-1 (CD54), ICAM-2 (CD102), ICAM-3 (CD50), junctional adhesion molecule (JAM), mucosal addressin cell-adhesion molecule 1 (MadCAM-1), neural cell adhesion molecule (NCAM, CD56), sialoadhesin (CD169), VCAM-1 (CD106), integrin aL (CD11a, LFA-1), integrin aM (CD11b), integrin a1 (CD49a), integrin a2 (CD49b), integrin a3 (CD49c), integrin a4 (CD49d), integrin a5 (CD49e), integrin a6 (CD49f), integrin a7, integrin a8, integrin a9, integrin aIIb (CD41), integrin av (CD51), integrin aE (CD103), integrin b1 (CD29), integrin b2 (CD18), integrin b3 (CD61), integrin b4 (CD104), integrin b5, integrin b6, integrin b7, integrin b8, E-selectin, L-selectin, P-selectin, syndecan-1 (CD138), syndecan-2 (HSPG), syndecan-3, syndecan-4, CD6, CD23, CD34, CD36, CD39, CD43, CD44, CD98, E-selectin ligand (ESL-1), and P-selectin ligand (PSGL-1, CD162). 7. Complement The complement system consists of a group of soluble molecules that function to defend against invading pathogens [25]. The complement system can be activated by three pathways: classical, mannose-binding lectin (MBL), and alternative pathways [25]. In the classical antibody-dependent pathway, the C1 complex (C1q, C1r, C1s) initiates the activation by the globular domain of C1q to the Fc region of immunoglobulin (IgG or IgM), followed by activation of C4, C2, and then C3 [25]. In the MBL antibody-independent pathway, the MBL/mannose-associated serine protease 1,2 (MASP-1, 2) initiates the activation by binding to mannose-containing proteins or carbohydrates on the surface of bacteria or viruses, in much the same way as the C1 complex, followed by activation of C4, C2, and then C3 [25]. The alternative antibody-independent pathway activation is based on a continuous low-grade hydrolysis of serum C3, with binding by factor B, and activation of factor D, can form a unstable C3 convertase, cleaving C3 to C3a and a labile C3b*, which degrades rapidly. If one of a variety of pathogen components is present, such as Gram- bacterial LPS, Gram+ bacterial cell wall teichoic acid, or zymosan, C3b* can bind to these components and activate factor B and properdin, leading to stable C3 convertase and C3 activation [25]. After the activation of C3, the common converging point for all three pathways, the activation of the other components — C5, C6, C7, C8, and C9 — follow, resulting in the formation of membrane attack complex (MAC). The immune defense functions of the complement system include opsonization (by C3b and C4b), release of anaphylatoxin (C3a and C5a), and lysis of bacterial membrane (by MAC) [25]. The genes encoding the following components of the complement system have been identified in both human and mouse [25]: C1q, MBL, MASP-1, factor B, factor D, factor H, factor I, properdin, C2, C3, C4, C5, C6, C7, C8, and C9 [25]. B. Immune Cells The immune cells in the mouse innate immune system include neutrophils, eosinophils, monocytes/macrophages, natural killer cells, mast cells, basophils, B-1 cells, interferon-producing cells, gamma/delta T cells, and intraepithelial lymphocytes. Obviously, some of these “innate immune


MOUSE IMMUNE SYSTEM

127

cells” do participate in the adaptive immune functions. The distinct features of mouse immune system of selected cell types are discussed below: 1. Natural Killer Cells Natural killer (NK) cells are large bone marrow–derived granular lymphocytes that are important for innate immune defense against a variety of “unwanted” cells, such as transformed cells, stressed cells, and infected cells, by their abilities to recognize and lyse them [28–30]. NK cells distinct from B and T lymphocytes in that they do not require gene rearrangement to assemble their receptor genes, yet they have definite abilities to discriminate between the “unwanted cells” and normal cells of the body [28–30]. These discriminatory abilities of NK cells are the current subject of intense investigations. The current data suggest that NK cells utilize both inhibitory recognition receptors as well as stimulatory receptors for their discriminatory actions. The inhibitory or negative recognition receptors function to prevent NK cells from lysing self-cells that express normal levels of each self-MHC class Ia molecule. This concept is supported by the fact that most self-cells that express normal levels of class Ia molecules are spared from NK cell killing. However, it is also apparent that some cells express normal levels of class Ia become targets of NK cells while some cells express low levels of class Ia are spared from the NK cell killing. It turns out that NK cells also utilize stimulatory receptor as a positive target-cell recognition mechanism. In the mouse system, three families of inhibitory MHC class I-recognizing receptors were expressed by NK cells: Ly49 (a C-type lectin-like protein), CD94/NKG2A, NKG2B heterodimers, and immunoreceptor tyrosine-based inhibitory motif (ITIM) [29]. In addition, two major groups of stimulatory receptors are recognized in mice: MHC class I recognizing and non-MHC class I recognizing. Several ligands have so far been identified for the mouse stimulatory receptors. H2-D has been identified as a ligand for Ly49D, a MHC class I-recognizing receptor. The retinoic acid early-1 protein (Rae 1) and H60 minor histocompatibility antigen have been identified as ligands for NKG2D, a non-MHC class I-recognizing receptor. Expression of Rae 1 or H60 on target cells has led to induction of strong NK cytotoxic killing activities [29,77,78]. Interestingly, human NK cells have no functional Ly49 receptor, but have an alternative receptor named killer cell immunoglobulin-like receptor (KIR) [29]. In addition to their cytotoxic effector functions, NK cells are capable of producing abundant cytokines such as IFN-g, TNF-a, GM-CSF, MIP-1, and RANTES [29]. 2. Mast Cells Mouse mast cells are bone marrow–derived cells originated from Thy-1lo c-kithi cells, which upon circulating in the blood and lymphatics migrate into tissues, where they mature with distinct morphological and functional features under the influence of local microenvironment [31–33]. Thus, mast cells are a heterogeneous population, exhibiting histochemical heterogeneity based on the cytoplasmic granule protein content [32]. The proliferation and maturation of rodent mast cells are primarily promoted by IL-3 and SCF, whereas IL-3 has a minimal direct effect on human mast cells [32]. Mast cells are well recognized as critical effector cells in the Th2-mediated, IgE-directed immediate hypersensitive responses [32]. Since they are strategically located at the interface between environmental and mucosal surfaces, mast cells are also thought to play roles in “immune surveillance” and effector roles in defense against bacterial and viral infection, based on some recent evidence [33]. For example, TLRs, such as TLR2, TLR4, and TLR6 have been identified in mouse mast cells [33]. Moreover, mast cells are capable of phagocytose and kill bacteria and participate in leukocyte recruitment by increasing vascular endothelial cell expressions of E-selectin, ICAM, and VCAM through the release of various cytokines during degranulation [32]. In addition, mast cells are capable of release TNF-a in response to Gram- bacteria [33]. Both human and rodent mast cells have been reported to express MHC class II molecules, and to process and present immunogenic antigens to CD4+ Th cells [32]. Mast cells are primarily activated by their surface


128


receptor FceRI, a high affinity receptor for IgE. Noticeably, the FceRI system in mouse cells is structurally different from that of humans [79]. In the mouse system, FceRI is primarily expressed in mast cells and basophils with a tetrameric structure consisting of one a chain, one b chain, and two identical g chains [79]. In human system, FceRI is expressed not only in mast cells, basophils, but also epithelial Langerhans cells, eosinophils, certain dendritic cells, and some monocytes [79]. However, the FceRI on human Langerhans cells, dendritic cells, and monocytes carries no b chain [79]. In addition, mouse mast cells are now known to express IgG receptors FcgRII and FcgRIII, and be activated by FcgRIII or by direct contact with microorganisms [32]. 3. B-1 Cells and Natural Antibodies Natural or spontaneous antibodies to toxins, bacteria, and red blood cells are present in sera of normal, nonimmunized humans and mice. Peritoneal CD5+ B1 cells are the major producers of natural IgM antibodies; however, natural antibodies can be synthesized by splenic CD5- B2 cells as well [35,80–82]. The production of natural antibodies is not dependent on internal or external antigenic stimulation, and the majority of natural antibodies are in the IgM class, with some in IgG and IgA classes [35,82]. Natural antibodies act as the first line of defense against infections by the following three major mechanisms. First of all, they can directly neutralize the incoming pathogen. Moreover, natural antibodies can eliminate the pathogens by three possible usage of activating the complement system: (1) complement-mediated lysis of pathogens; (2) T-cell–independent antibody response by targeting the antigen to splenic marginal zone; and (3) T-cell–depedent antibody response by stimulating B cells. Finally, they can also form antibody–antigen complex, thus preventing pathogen spread and enhancing immune responses in lymphoid organs [35]. 4. Interferon-Producing Cells Interferon-producing cells (IPC), found in the human system 2 decades ago, have been identified in the murine system only recently. IPC correspond to the previously named “plasmacytoid cells” identified in human lymph nodes during infections [36]. They are a small population of leukocytes that secrete high levels of type I IFN (IFN-a and IFN-b) in response to viral infection, thus playing a role in innate immunity and in shaping T-cell responses [36,37]. 5. Gamma/Delta T Cells (gd T Cells) These T cells are referred to T cells that express T-cell receptor gamma/delta chains (TCR gd) and participate in both innate and adaptive immune functions [38–41]. These gd T cells, together with B cells and ab T cells, are the three vertebrate cell types that use somatic DNA rearrangement to assemble the genes encoding their cell-surface receptors [83]. The gd T cells utilize variable (V), diversity (D), and joining (J) gene segments conferring on TCR gd in such a way that they are expected to contribute to adaptive immune response like that of B cells and ab T cells, but instead their role remains unclear [83]. TCR d knockout (KO) mice showed some degrees of immunodeficiency to infection by Listeria, vesicular stomatitis virus, and malaria parasites, usually confined to the early points of the infection, suggesting that the gd are designed to be a player in fast-acting innate response [83]. The gd T cells are shown to play an immunoregulatory role, as the gd T-cell–deficient mice have exaggerated and accelerated immunopathology due to an increase of CD4+ ab T cells [83]. The gd T cells are also known for their association with tissues [83], the most obvious example of which is their disproportionate enrichment in the epithelia [43–45,83] and will be described in the section below.


MOUSE IMMUNE SYSTEM

129

6. Intraepithelial Lymphocytes Intraepithelial lymphocytes (IEL) are locally resided, potently cytolytic, and immunoregulatory lymphocytes containing primarily T cells [42]. Unlike normal human skin, in which small numbers of T cells are residing predominantly in the dermis, normal mouse skin harbors an extensive network of IEL of dendritic morphology, known as dendritic epidermal T cells (DETCs) [42,83]. Essentially, all mouse DETCs express TCR gd, express CD8aa (a homodimer) or CD4-/CD8- (double negative), and respond to antigens not restricted by conventional MHC [42]. In all animals examined, DETCs are some of the earliest developed T-cell subsets, equipping them to respond to hostile environmental challenges faced by the newborns. Functionally, DETCs respond to heat-shocked autologous keratinocytes with cytokine secretion and kill the target cells. In addition, DETCs target Rae-1+ (a mouse MICA equivalent) transformed cells during the skin chemical carcinogenesis process. These findings suggest that one of the roles of DETCs may be to eradicate infected or transformed epidermal cells before they disseminate systemically [42]. In fact, DETCs are considered to be “revertants,” meaning immune cells that are functionally reversed from the adaptive to the innate response, using gene rearrangement, a marker of the adaptive response, to generate receptors for conserved autoantigens specifying infection, cell transformation, or other dysfunctions within local tissues [42].

III. ADAPTIVE IMMUNE SYSTEM A. T Lymphocytes Like human T cells, the mouse T cells are bone marrow–derived and thymus-matured mononuclear cells. All mouse T cells are CD3+. The majority of CD3+ T cells have either CD4+ or CD8+ surface markers, which they acquired during their maturation in the thymus. Whereas the mouse CD4+ helper T cells (Th) can be further divided into Th1 or Th2 based on the types of cytokines they secreted, this clear-cut division is not as obvious in the human system. Under the influence of IL-12, uncommitted Th cells (Th0) become committed Th1 cells, capable of secreting IL-2, IL-18, IFN-g, and TNF-b [16,62,84,85]. Similarly, under the influence of IL-4, Th0 cells become committed Th2 cells, with the ability to secret IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, and IL-25 [16,62,74,75,84,85] (Table 6.1). It is now clear that the T-box transcription factor T-bet is central for Th1 cell development [86]. Similarly, Th2 cell development is primarily dependent on a zincfinger transcription factor GATA-3 [87–89]. Although many outstanding studies have shown the in vitro functional roles of human T cells, the in vivo functions of T cells in inducing inflammatory and autoimmune diseases have been demonstrated only in small mammals such as rats and mice, into which the adoptively transferred pathogenic T lymphocytes reproduce the clinical disease phenotype in the immunocompetent recipient mice [90–92]. B. B Lymphocytes (B-2 Cells) Since the term “B-1 cell” is now assigned to a subset of B cells that synthesize and secrete natural antibodies for the innate immune system, the conventional B lymphocyte is now termed “B-2 lymphocytes” or B-2 cells. The mouse B-2 lymphocytes, like those of humans, are derived from the liver during mid- to late fetal life and matured in bone marrow after birth [93]. However, the products of B-2 cells are classified distinctly from those of humans [94]. Unlike the human IgG antibodies, which are subclassed as IgG1, IgG2, IgG3, and IgG4, mouse IgG antibodies are subclassed as IgG1, IgG2a, IgG2b, and IgG3 [94]. For T-cell–dependent antibody production, it is driven by the interaction between the co-stimulatory molecules CD40 ligand (CD40L) on activated T cells and CD40 on B cells, plus the interaction of TCR of activated T cells and MHC-linked


130


antigenic peptide on B cells. Furthermore, the cytokine environment is of particular importance for class switching and has been established within the murine immune system. Mature, IgM-expressing B cells will alter their heavy-chain isotype when restimulated with antigen or a polyclonal activator like LPS. The class of heavy chain that they will switch to is dependent upon the cytokine environment. Cytokines from Th1-helper T cells, primarily IFN-g will induce the class to switch to IgG2a. The presence of IL-4 from Th2-type helper T cells will induce a class switch to IgG1 or IgE [16,62,84,94]. Both of the major phenotypes of T-helper cells can induce antibody responses in mice. However, it is possible to determine the predominant helper T-cell type by a close examination of the isotype of antibody being produced. While this identification has been postulated in humans, it is not as clearly demonstrated as with mice. C. Antigen-Presenting Cells In the skin, the most important antigen presenting cells are the Langerhans cells (LCs), which are present in both human and mouse epidermis [95–99]. Both human and mouse LCs are dendritic in form, and a LC-specific protein termed Langerin/CD207, a type II transmembrane protein essential for Birbeck granule formation, has recently been discovered in humans [95,96]. Subsequently, the equivalent of Langerin was identified in the mouse system [97–99], suggesting a similar functional role between the human LCs and that of mice. The human Langerin shares 66% overall sequence identity at the amino acid level with its mouse counterpart, with a 75% amino acid homology at the important carbohydrate recognition domain of Langerin [96,97]. Using this specific antibody, the LCs in both human and mouse have been shown to migrate to draining lymph nodes upon inflammatory stimulation, confirming a similar function between human and mouse LCs [99,100]. Both human and mouse LCs have the following surface markers: MHC class II molecule, E-cadherin, CD40L co-stimulatory molecules B71, B72, and ICAM-1 [101–109]. In the human system, LCs are recognized to have cell-surface FceRI, the high-affinity receptor for IgE, whereas such receptor has not yet been identified in the mouse system [110,111]. Mouse LCs, however, do possess a low-affinity receptor for IgE (FceRII/CD23) [112]. D. Major Histocompatibility Complex (MHC) The mouse MHC genes, located in chromosome 17, are organized slightly differently from those of humans, which are located in chromosome 6 [113]. Figure 6.1 illustrates the main genetic regions of the mouse MHC in comparison with the human counterpart.

IV. MICE AS AN ESSENTIAL TOOL FOR ANIMAL MODELING OF HUMAN IMMUNE-MEDIATED DISEASES Mice have been used as experimental animal models of human immune diseases for many decades and these models have provided us with significant insight into the function and dysfunction of the human immune system. While the mouse immune systems varies somewhat from that of humans, the mice play important roles in the animal modeling of human disease due to their small size (thus less expensive for keeping), their relatively short gestation period (thus less time required for data collection), and their ability to breed large numbers (thus more suitable for obtaining statistically significant results). However, the greatest advantage of mouse models of human inflammatory diseases, as opposed to humans or other mammals, stems from the development of immunologically identical strains of mice. These syngeneic mouse strains have consistent and reproducible immune responses that can be studied in detail to give us significant information that is applicable to mammalian immunity in general. Thus, it is central to understand the function of the immune


MOUSE IMMUNE SYSTEM

H:

DP

M:

K

131

DQ DR

A

E

C

C

HSP TNF etc

HSP TNF etc

B

C

D

A

L

Class I: Class II: Class III: Figure 6.1

Main genetic regions of human and mouse MHC. H, human; M, mouse; C, complement components; HSP, heat shock protein; TNF, tumor necrosis factor.

system in inbred mouse strains to decipher the importance of murine models of inflammatory disease. A. Development and Nomenclature of Inbred Mouse Strains The concept of mating between litter mates for establishing reproducible immunological identity or nonidentity between different groups of mice was developed in the mid-20th century. It was long known that grafting of skin from one mouse to another would induce rejection of the graft. However, the likelihood of rejection and the rate of rejection was less between littermates. After multiple rounds of inbreeding (about 20), multiple strains of mice were identified that would tolerate skin transplantation between members of the same strain. After some time, it was determined that theses strains were syngeneic, that is, they shared the same alleles in the genes that regulated graft rejection or acceptance. The genes associated with graft rejection were termed the histocompatibility locus and were localized to chromosome 17. Later, another genetic area locus associated with syngeneic immune responses was identified and referred to as immune-response genes. It was determined that immunologically identical, syngeneic strains would have identical alleles in both of these determinant areas. After the major histocompatibility genes in humans were identified, it became clear that the histocompatibility locus in mouse, or H-2, corresponded to the MHC class I determinant in humans, while the mouse immune response gene region, or Ir gene, corresponded to the human MHC class II. The nomenclature for histocompatibility in mice is based on these early experiments. The H-2 gene group in mice has two initially identified alleles, K and D. The specific gene allele is associated with the initial strain in which it was identified and is designated as a small letter. Thus, for the MHC I gene group, a mouse from the b strain for the K and D alleles would be identified as H2Kb and H-2Db, respectively. Similarly, the Ir groups have two specific loci, A and E, identified as I-A and I-E loci. Again, the specific allele that confers acceptance of grafts within the d strain is identified as a small letter superscript as in I-Ad, I-Ed. B. The Use of Inbred Mouse Strains When it was recognized that T-cell recognition and activation with appropriate peptide antigens required the peptides to be bound to these MHC sequences, it became clear that the inbred strains


132


of mice held many potential experimental advantages. In immune responses that are driven by specific antigens, the recognition of specific peptide antigens that may bind to a certain strain’s MHC locus would facilitate experiments to identify pathogenic proteins and peptide antigens as well as learning how to induce tolerance to these peptides. In experimental mouse models of human disease, like experimental autoimmune encephalomyelitis (EAE) or collagen-induced arthritis (CIA), immunization of specific strains of mice with adjuvant can lead to the phenotypic expression of the disease. Through the use of inbred strains of mouse with reproducible antigenic binding to the MHC locus, specific peptides that are the primary, secondary, or even tertiary in nature are recognized. These observations have led to significant insight into the nature of autoimmune disease. For example, the hierarchy of immunogenic peptides has resulted in the concept of “epitope spreading,” where waxing and waning autoimmune disease is related to newer responses to other peptides and proteins [114–116]. Moreover, antigen-specific tolerance to a disease can be conferred by inducing tolerance to a peptide in this hierarchy. In a virus-induced autoimmune disease, Theiler’s murine encephalomyelitis virus infection, the infection-inducing cell damage has been determined as an initiating factor that is responsible for a specific progression of antigenic peptide recognition, leading to an immune-mediated disease process [117–119]. This evidence strongly suggests that the nature of this virus-induced disease is not molecular mimicry between microorganism antigen and self-antigen as had previously been thought. Rather, the induction of an immune response in a genetically susceptible individual is brought on by a viral infection. These significant observations were only possible because of the existence of inbred strains of mouse. The second major advantage of using inbred strains of mice is the ability to transplant specific cells from one animal to another within the same strain. Specifically, it is possible to determine the specific cell type that may be inducing a specific immune-mediated disease through this process of adoptive transfer. For example, in mice with EAE, it is possible to prime an individual mouse with proteolipid protein and remove the draining lymph nodes prior to the onset of clinically apparent disease. Specific CD4+ T cells can then be isolated, restimulated in vitro with syngeneic antigen-presenting cells and the appropriate peptide, and then transferred to a naïve mouse of the same strain. The recipient mouse will, in turn, develop clinical signs of EAE. With this technique made possible by the use of syngeneic mice, it is now feasible to perform numerous experiments to determine pathological cell types, along with potential pathogenic cytokines, and so on. C. Inbred Mouse Strains and Disease Susceptibility While it was predictable that the development of mouse strains with consistent MHC I and II molecules would lead to new discoveries in antigen processing and presentation and information about autoimmunity, other experiments demonstrated that the differences among the strains were beyond the MHC locus. Many of these discoveries have led to important information about immunity and highlight differences among mouse strains that can be used for experimental models. One of the most significant discoveries with inbred strains of mice may be the finding that some specific strains are susceptible to specific infectious organisms while others are not. Almost all inbred strains of mice are resistant to infection with Leishmania major, a protozoum that can also be infectious in humans. The classic response is seen in the C57BL/6 mouse, which has an initial swelling response in the footpad after the injection of the parasite followed by resolution. However, the BALB/c strain seems to lack this resistance and will die within a few weeks after injection of the parasite. The resistance gene or genes for differences among these inbred strains was not determined until it became clear that the CD4+ T cells from resistant mice tended to produce primarily IFN-g upon injection of L. major while the BALB/c mice predominantly produced IL-4. Multiple experiments demonstrated that altering this IFN-g: IL-4 ratio, either by treatment with anti–IFN-g antibodies or with recombinant IL-4 in resistant mice, or by treating with IL-12 in susceptible mice, altered this pattern. Thus, it became clear that the immunological differences among inbred strains


MOUSE IMMUNE SYSTEM

133

Table 6.3 Immune Mutant Mouse Strains in C57BL/6Ja Background (Jackson Laboratory) Strain Name nu

B6.Cg-Foxn1 B6.CB17-Prkdcscid/SzJ B6.129S7-Rag1tm1Mom B6.129S2-Cd4tm1Mak B6.129S2-Cd8atm1Mak B6.129S4-Cd80tm1Shr B6.129S4-Cd86tm1Shr B6.129S6-Rac2tm1Mddw B6.129P2-Il4tm1Cgn B6.129S1-Il12atm1Jm B6.129S4-Icam1tm1Jcgr B6.129S7-Selptm1Bay

Stock No.

Deficiencyb

000819 001913 002096 002663 002665 003611 003609 004197 002253 002692 002867 002289

T cells, partial B cells T and B cells Matured T and B cells Helper T cells CD8+ T cells MLR, partial B cells Partial B cells Neutrophils, mast cells, phagocytosis IL-4, IgG1, IgE IL-12, DTH ICMA-1, neutrophil migration, DTH, MLR P-selectin, leukocyte rolling and migration

Refs. 127, 128 122 124 130 131 132 133 134 135 136 137 138

Note: MLR, mixed lymphocyte response; DTH, delayed-type hypersensitivity. a b

The control strain is C57BL/6J (stock no. 000664). In most cases, the identifiable deficiencies are not 100%.

of mice were more significant than simply the binding of antigen but included patterns of response. These findings were seminal in the development of the Th1/Th2 theory of T-cell responses in mice and were later applicable to human immunology with regard to mycobacterial infections [120,121]. These findings also give investigators important clues on which strains of mice to initially use when trying to develop models of human inflammatory diseases that may have a Th1 or Th2 tendency. D. Naturally Occurring Mutations of Mouse Immune System While there are many naturally occurring mouse strains that may be of importance in the development of new models of human autoimmunity and inflammation, there is one that merits special discussion. The severe combined immunodeficiency mouse can be used as a base to study many human immune processes. Table 6.3 lists some major spontaneously arising immune mutant mice available at Jackson Laboratory in Bar Harbor, ME (www.jax.org), in the C57BL/6J background. 1. Severe Combined Immunodeficiency (SCID) Mice The naturally occurring mouse strain with major implications for animal models of immune disease is the SCID. SCID mice have a genetic defect in a DNA repair gene that is central to recombination for both the T-cell receptor and immunoglobulin genes. This defect does not allow for normal production of these proteins, which causes elimination of cells within the T- and B-cell lineages. Thus, while these mice have intact neutrophils, monocytes, and other cells of the innate immune system, they have no adoptive immune system, such as T and B lymphocytes [122]. There are multiple possible uses of these mice in the study of human immune mediated diseases. The first area of study involves the transfer of human tissue to the SCIDs. As would be expected from animals that lack adaptive immunity, xenografts are not rejected [123]. Thus, it is possible to transfer skin from a susceptible human to the mouse to allow for the induction of inflammatory disease. Likewise, to study immune responses in human cancers, human tumor cells can be transferred to the SCID mice, followed by the transfer of human immunocytes. Manipulations of these models may lead to new insights in both the down-regulation of the immune system to treat autoimmune disease as well as up-regulation of the transferred immunity in the treatment of tumors. The second method of studying immune-mediated diseases in SCID mice is the partial constitution of the immune system with cells from patients suffering from autoimmune diseases. This method has been studied with a number of systems including multiple sclerosis and rheumatoid arthritis.


134


There are, however, a number of difficulties with using SCID mice for the development of models for human immune-mediated diseases. The first is that the SCID defect is in a DNA repair gene. This repair gene has implications for the normal homeostasis of the mouse’s genome. This complicates potential analyses of the meaning of experimental findings. Moreover, there is “leakiness” in the SCID system [122]. In other words, some mice will be able to produce some level of an adaptive immune system over time. These difficulties are overcome by using a strain of mouse that has a genetic knockout at recombination activating gene (RAG) locus [124–126]. It is becoming clear that RAG knockout (RAG NO) mice will likely replace SCID mice in future mouse models of immune disease. 2. Nude Mice Nude mice are T-cell–deficient, with partial defect in B-cell development [127–129]. Jackson Laboratory has several strains of nude mice available for investigators: stock no. 000819 (in C57BL/6J background); stock no. 000711 (in BALB/cByJ background); and stock no. 100402 (in CByB6F1/J background). E. Experimentally Induced Alteration of Mouse Immune System There are now many experimentally induced mouse lines that have been established for the purpose of experimental modeling for human inflammatory diseases. Most of these lines have a single immune component “knocked out,” so that immunological researchers can study the role of these “knocked out” components in certain inflammatory diseases. Table 6.3 lists some major knockout (KO) immune mutant mice available at Jackson Laboratory in the C57BL/6J background. 1. RAG KO Mice The establishment of a functional immune system with diverse antigen receptors such as that in adaptive systems depends on the V(D)J recombination–activating gene products Rag-1 and Rag-2, which compose the key components for the activation of antigen receptor rearrangement [124]. These gene products are essential for the catalysis of the initial stages of V(D)J recombination and functional disruption of them leads to lymphoid arrest before the recombination of the antigen receptor loci takes place [124]. As a result of the absence of antigen receptors, both B- and T-cell differentiation is terminated, leading to the absence of the major conventional populations of matured B and T cells [124–126]. Jackson Laboratory has several strains of RAG-1 Nmice available for investigators: RAG-1-/- (in BALB/cJ background, stock no. 003145); and RAG-1-/- (in C57BL/6J background, stock no. 002096). (See Table 6.3.) 2. Cytokine KO Mice Jackson Laboratory has a collection of many cytokines KO mouse lines developed in certain mouse strains. Important examples include IL-4-/- (in C57BL/6J background, stock no. 002253) and IL-12 a-chain-/- (in C57BL/6J background, stock no. 002692). (See Table 6.3.) 3. Adhesion Molecule KO Mice Jackson Laboratory has a collection of many adhesion molecules KO mouse lines developed in certain mouse strains. A few important examples include: ICAM-1-/- (in C57BL/6J background, stock no. 002867); P-selectin-/- (in C57BL/6J background, stock no. 002289); and ICAM-1/Pselectin-/- (in C57BL/6J background, stock no. 002285). (See Table 6.3.)


MOUSE IMMUNE SYSTEM

135

4. Immune Cell KO Mice Jackson Laboratory has a collection of many immune cell that Kline developed in certain mouse strains. These include neutrophil/mast cell-/- (in C57BL/6 background, stock no. 004197); CD4 T cell-/- (in C57BL/6J background, stock no. 002663); and CD8 T cell-/- (in C57BL/6J background, stock no. 002665). (See Table 6.3.)

V. CONCLUSION The use of mouse models to derive a better understanding of immune mediated diseases requires information on the genetic makeup of inbred strains of mice. Clearly, many models have been developed that take advantage of the unique circumstances of inbred mouse strains. It is important to remember, however, that while there are many stable breeding colonies of mice in the world, the specifics of mouse immunity are not static. Crossbreeding of different strains and manipulation of the immune system through the use of adjuvant has greatly enhanced the development of many models. Thus, the future development of models of inflammatory skin disease does not rest simply on the use of existing inbred strains of mice but in the development of new manipulations to best take advantages of their genetics.

ACKNOWLEDGMENTS This work is in part supported by National Institutes of Health grants (R01 AR47667, R03 AR47634, and R21 AR48438, L.S.C.).

REFERENCES 1. Janeway, C.A. Jr. and Medzhitov, R., Innate immune recognition, Annu. Rev. Immunol., 20, 197, 2000. 2. Aderen, A. and Ulevitch, R.J., Toll-like receptors in the induction of the innate immune response, Nature, 406, 782, 2000. 3. Akira, S., Takeda, K., and Kaisho, T., Toll-like receptors: critical proteins linking innate and acquired immnity, Nat. Immunol., 2, 675, 2001. 4. Means, T.K., Golenbock, D.T., and Fenton, M.J., Structure and function of Toll-like receptor proteins, Life Sci., 68, 241, 2000. 5. O’Neill, L.A., Toll-like receptor signal transduction and the tailoring of innate immunity: a role or Mal? Trends Immunol., 23, 296, 2002. 6. Beutler, B., Toll-like receptor: how they work and what they do, Curr. Opin. Hematol., 9, 2, 2002. 7. Akira, S., Mammalian Toll-like receptors, Curr. Opin. Immunol., 15, 5, 2003. 8. Caamano, J. and Hunter, C.A., NF-kappaB family of transcription factors: central regulators of innate and adaptive immune functions, Clin. Microbiol. Rev., 15, 414, 2002. 9. Karin, M. and Lin, A., NF-kappaB at the crossroads of life and death, Nat. Immunol., 3, 221, 2002. 10. Hawiger, J., Innate immunity and inflammation: a transcriptional paradigm, Immunol. Res., 23, 99, 2001. 11. Li, Q. and Verma, I.M., NF-kappa regulation in the immune system, Nat. Rev. Immunol., 2, 725, 2002. 12. Bell, S. et al., Involvement of NF-kappaB signaling in skin physiology and disease, Cell Signal, 15, 1, 2003. 13. Lehrer, R.I. and Ganz, T., Defensins of vertebrate animals, Curr. Opin. Immunol., 14, 96, 2002. 14. Yang, D. et al., Mammalian defensins in immunity: more than just microbicidal, Trends Immunol., 23, 291, 2002. 15. Trinchieri, G., Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific immunity, Annu. Rev. Immunol., 13, 251, 1995.


136


16. Fitzgerald, K.A. et al., The Cytokine Facts Book, Academic Press, San Diego, 2001. 17. Banyer, J.L. et al., Cytokines in innate and adaptive immunity, Rev. Immunogenet., 2, 359, 2000. 18. Vaddi, K., Keller, M., and Newton, R.C., The Chemokine Facts Book, Academic Press, San Diego, 1997. 19. Rossi, D. and Zlotnik, A., The biology of chemokines and their receptors, Annu. Rev. Immunol., 18, 217, 2000. 20. Power, C.A., Knock out models to dissect chemokine receptor function in vivo, J. Immunol. Methods, 273, 73, 2003. 21. McColl, S.R., Chemokines and dendritic cells: a crucial alliance, Immunol. Cell Biol., 80, 489, 2002. 22. Kruger-Krasagakes, S. et al., Chemokines and mast cells, in Chemokines and Skin, Kownatzki, E. and Norgauer, J., Eds., Birkhauser, Boston, 1998, p. 75. 23. Schroder, J.M., Chemokines and eosinophils, in Chemokines and Skin, Kownatzki, E. and Norgauer, J., Eds., Birkhauser, Boston, 1998, p. 89. 24. Isacke, C.M. and Horton, M.A., The Adhesion Molecule Facts Book, Academic Press, San Diego, 2000. 25. Morley, B.J. and Walport, M.J., The Complement Facts Book, Academic Press, San Diego, 2000. 26. Carroll, M.C., The role of complement in B cell activation and tolerance, Adv. Immunol., 74, 61, 2000. 27. Barclay, A.N. et al., The Leukocyte Antigen Facts Book, Academic Press, San Diego, 1997. 28. Smith, H.R., Idris, A.H., and Yokoyama, W.M., Murine natural killer cell activation receptors, Immunol. Rev., 181, 115, 2001. 29. Diefenbach, A. and Raulet, D.H., Strategies for target cell recognition by natural killer cells, Immunol. Rev., 181, 170, 2001. 30. Middleton, D., Curran, M., and Maxwell, L., Natural killer cells and their receptors, Transpl. Immunol., 10, 147, 2002. 31. Abraham, S.N. and Arock, M., Mast cells and basophils in innate immunity, Semin. Immunol., 10, 373, 1998. 32. Mekori, Y.A. and Metcalfe, D.D., Mast cells in innate immunity, Immunol. Rev., 173, 131, 2000. 33. Marone, G., Galli, S.J., and Kitamura, Y., Probing the roles of mast cells and basophils in natural and acquired immunity, physiology and disease, Trends Immunol., 23, 425, 2002. 34. Carroll, M.C. and Prodeus, A.P., Linkages of innate and adaptive immunity, Curr. Opin. Immunol., 10, 36, 1998. 35. Ochsenbein, A.F. and Zinkernagel, R.M., Natural antibodies and complement link innate and acquired immunity, Immunol. Today, 21, 624, 2000. 36. Colonna, M., Krug, A., and Cella, M., Interferon-producing cells: on the front line in immune responses against pathogens, Curr. Opin. Immunol., 14, 373, 2002. 37. Le Page, C. et al., Interferon activation and innate immunity, Rev. Immunogenet., 2, 374, 2000. 38. Ziegler, H.K., and Skeen, M.J., and Pearce, K.M., Role of alpha/beta T and gamma/delta T cells in innate and acquired immunity, Ann. N. Y. Acad. Sci., 730, 53, 1994. 39. Boismenu, R. and Havran, W.L., An innate view of gamma delta T cells, Curr. Opin. Immunol., 9, 57, 1997. 40. Welsh, R.M. et al., Alpha beta and gamma delta T-cell networks and their roles in natural resistance to viral infections, Immunol. Rev., 159, 79, 1997. 41. Carding, S.R., Role of gamma delta T cells in immunity to infectious diseases and the regulation of hematolymphoid cell development, Immunol. Rev., 17, 13, 1998. 42. Hayday, A. et al., Intraepithelial lymphocytes: exploring the third way in immunology, Nat. Immunol., 2, 997, 2001. 43. Holtmeier, W. et al., The TCRd repertoire in normal human skin is restricted and distinct from the TCRd repertoire in the peripheral blood, J. Invest. Dermatol., 116, 275, 2001. 44. Stingl, G. et al., Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor g chain, Proc. Natl. Acad. Sci. U. S. A., 84, 4586, 1987. 45. Asarnow, D. et al., Limited diversity of gd antigen receptor genes of Thy-1+ dendritic epidermal cells, Cell, 55, 837, 1988. 46. Lo, D. et al., Integrating innate and adaptive immunity in the whole animal, Immunol. Rev., 169, 225, 1999. 47. Jack, R.S., Schütt, C., and Bröker, B.M., The guardians at the gate, Trends Immunol., 23, 423, 2002.


MOUSE IMMUNE SYSTEM

137

48. Poltorak, A. et al., Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLr4 gene, Science, 282, 2085, 1998. 49. Nagai, Y. et al., Essential role of MD-2 in LPS responsiveness and TLR4 distribution, Nat. Immunol., 3, 667, 2002. 50. Alexopoulou, L. et al., Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans in in TLR1- and TLR2-deficient mice, Nat. Med., 8, 878, 2002. 51. Hayashi, F. et al., The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5, Nature, 410, 1099, 2001. 52. Bauer, S. et al., Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition, Proc. Natl. Acad. Sci. U. S. A., 98, 9237, 2001. 53. Alexopoulou, L. et al., Recognition of double-stranded RNA and activation of NF-kB by Toll-like receptor 3, Nature, 413, 732, 2001. 54. Hemmi, H. et al., Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway, Nat. Immunol., 3, 196, 2002. 55. Gerondakis, S. et al., Genetic approaches in mice to understand Rel/NF-kB and IkB function: transgenics and knock outs, Oncogene, 18, 6888, 1999. 56. Ghosh, S., May, M.J., and Kopp, E.B., NF-kB and Rel proteins: evolutionarily conserved mediators of immune responses, Annu. Rev. Immunol., 16, 225, 1998. 57. Morrison, G.M. et al., Mouse b defensin-1 is a functional homolog of human b defensin-1, Mamm. Genome, 9, 453, 1998. 58. Morrison, G.M., Davidson, D.J., and Dorin, J.R., A novel mouse b defensin, Defb2, which is upregulated in the airways by lipopolyasccharide, F. E. B. S. Lett., 442, 112, 1999. 59. Bals, R. et al., Mouse b-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs, Infect. Immun., 67, 3542, 1999. 60. Jia, H.P. et al., A novel murine b-defensin expressed in tongue, esophagus, and trachea, J. Biol. Chem., 275, 33314, 2000. 61. Yamaguchi, Y. et al., A novel mouse b-defensin, mBD-6, predominantly expressed in skeletal muscle, J. Biol. Chem., 276, 31510, 2001. 62. Mosmann,T.R. et al., Two types of murine helper T cell clone. I. Definition according to profiles of lymphocyte activities and secreted proteins, J. Immunol., 136, 2348, 1986. 63. Ihle, J.N., The Janus protein tyrosine kinase family and its role in cytokine signaling, Adv. Immunol., 60, 1, 1995. 64. Leonard, W.J. and O’Shea, J.J., Jaks and STATs: biological implications, Annu. Rev. Immunol., 16, 293, 1998. 65. Liao, Y.C. et al., IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TFN-alpha, J. Immunol., 169, 4288, 2002. 66. Blumberg, H. et al., Interleukin-20: discovery, receptor identification, and role in epidermal function, Cell, 104, 9, 2001. 67. Dumoutier, L. et al., Human interleukin-10-related T cell-derived inducible factor: molecular cloning and functional characterization as an hepatocyte-stimulating factor, Proc. Natl. Acad. Sci. U. S. A., 97, 10144, 2000. 68. Parrish-Novak, J. et al., Interleukin 21 and its receptor are involved in NK cells expansion and regulation of lymphocyte function, Nature, 408, 57, 2000. 69. Dumoutier, L. et al., IL-TIF/IL-22: genomic organization and mapping of the human and mouse genes, Genes Immun., 1, 488, 2000. 70. Oppmann, B. et al., Novel p19 protein engages IL12 p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12, Immunity, 13, 715, 2000. 71. Belladonna, M.L., IL-23 and IL-12 have overlapping, but distinct, effects on murine dendritic cells, J. Immunol., 168, 5448, 2002. 72. Cua, D.J. et al., Interelukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain, Nature, 421, 744, 2003. 73. Kotenko, S.V., The family of IL-10-related cytokines and their receptors: related, but to what extent? Cytokine Growth Factor Rev., 13, 223, 2002. 74. Fort, M.M. et al., IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathogenesis in vivo, Immunity, 15, 985, 2001.


138


75. Hurst, S.D. et al., New IL-17 family members promote Th1 or Th2 responses in the lung: in vivo function of the novel cytokine IL-25, J. Immunol., 169, 443, 2002. 76. Zlotnick, A. and Yoshie, O., Chemokines: a new classification system and their role in immunity, Immunity, 12, 121, 2000. 77. Gerwenka, A. et al., Retinoic acid early inducible gene define a ligand family for the activating NKG2D receptor in mice, Immunity, 12, 721, 2000. 78. Malarkannan, S. et al., The molecular and functional characterization of a dominant minor H antigen, H60, J. Immunol., 161, 3501, 1998. 79. Kinet, J.P., The high-affinity IgE receptor (FceRI): from physiology to pathology, Annu. Rev. Immunol., 17, 931, 1999. 80. Baumgarth, N. et al., B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection, J. Exp. Med., 192, 271, 2000. 81. Herzenberg, L.A. and Kantor, A.B., B-cell lineages exist in the mouse, Immunol. Today, 14, 79, 1993. 82. Avrameas, S., Natural autoantibodies: from “horror autotoxicus” to “gnothi seauton”, Immunol. Today, 12, 154, 1991. 83. Hayday, A. and Tigelaar, R., Immunoregulation in the tissues by gd T cells, Nat. Rev. Immunol., 3, 233, 2003. 84. Paul, W.E. and Seder, R.A., Lymphocyte responses and cytokines, Cell, 76, 241, 1994. 85. Murphy, K.M. and Reiner, S.L., The lineage decisions of helper T cells, Nat. Rev. Immunol., 2, 933, 2002. 86. Szabo, S.J. et al., A novel transcription factor, T-bet, directs TH1-lineage commitment, Cell, 100, 655, 2000. 87. Quyang, W. et al., Stat6-independent GATA-3 autoactivation directs IL-4-independent TH2 development and commitment, Immunity, 12, 27, 2000. 88. Zheng, W. and Flavell, R.A., The transcription factor GATA-3 is necessary and sufficient for TH2 cytokine gene expression in CD4 T cells, Cell, 89, 587, 1997. 89. Lee, G.R., Fields, P.E., and Flavell, R.A., Regulation of IL-4 gene expression by distal regulatory elements and GATA-3 at the chromatin level, Immunity, 14, 447, 2001. 90. Braley-Mullen, H. and Sharp, G.C., Adoptive transfer murine model of granulomatous experimental autoimmune thyroiditis, Int. Rev. Immunol., 19, 535, 2000. 91. Archelos, J.J. et al., Inhibition of experimental autoimmune encephalomyelitis by an antibody to the intercellular adhesion molecule ICAM-1, Ann. Neurol., 34, 145, 1993. 92. Zaccone, P. et al., The involvement of IL-12 in murine experimentally induced autoimmune thyroid disease, Eur. J. Immunol., 29, 1933, 1999. 93. Hardy, R.R. and Hayakawa, K., B-cell development pathways, Annu. Rev. Immunol., 19, 595, 2001. 94. Mosmann, T.R. and Coffman, R.L., TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties, Annu. Rev. Immunol., 7, 145, 1989. 95. Valladeau J. et al., The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface, Eur. J. Immunol., 29, 2695, 1999. 96. Valladeau, J. et al., Langerin, a novel C-type lectin specific to Langerhans cells, is an enodocytic receptor that induces the formation of Birbeck granules, Immunity, 12, 71, 2000. 97. Valladeau, J. et al., Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues, J. Immunol., 168, 782, 2002. 98. Takahara, K. et al., Identification and expression of mouse langerin (CD207) in dendritic cells, Int. J. Immunol., 14, 433, 2002. 99. Stoitzner, P. et al., Visualization and characterization of migratory Langerhans cells in murine skin and lymph nodes by antibodies against langerin/CD207, J. Invest. Dermatol., 120, 266, 2003. 100. Geissmann, F. et al., Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin, J. Exp. Med., 196, 417, 2002. 101. Borkowski, T.A. et al., Expression of E-cadherin by murine dendritic cells: E-cadherin as a dendritic cell differentiation antigen characteristic of epidermal Langerhans cells and related cells, Eur. J. Immunol., 24, 2767, 1994. 102. Blauvelt, A., Katz, S.I., and Udey, M.C., Human Langerhans cells express E-cadherin, J. Invest. Dermatol., 104, 293, 1995.


MOUSE IMMUNE SYSTEM

139

103. Salgado CG et al., Functional CD40 ligand is expressed on epidermal Langerhans cells, J. Leukoc. Biol., 66, 281, 1999. 104. Salgado CG et al., Differential effects of cytokines and immunosuppressive drugs on CD40, B7-1, and B7-2 expression on purified epidermal Langerhans cells1, J. Invest. Dermatol., 113, 1021, 1999. 105. Adriana, T. et al., Dermal-resident CD14+ cells differentiate into Langerhans cells, Nat. Immunol., 2, 1151, 2001. 106. Teunissen, M.B. et al., Human epidermal Langerhans cells undergo profound morphologic and phenotypical changes during in vitro culture, J. Invest. Dermatol., 94, 166, 1990. 107. Tang, A. and Udey, M.C., Inhibition of epidermal Langerhans cell function by low dose ultraviolet B radiation. Ultraviolet B radiation selectively modulates ICAM-1 (CD54) expression by murine Langerhans cells, J. Immunol., 146, 3347, 1991. 108. Epstein, S.P., Baer, R.L., and Belsito, D.V., Effect of triggering epidermal Fc gamma receptors on the interleukin-2- and interleukin-6-induced upregulation of Ia antigen expression by murine epidermal Langerhans cells: the role of prostaglandins and cAMP, J. Invest. Dermatol., 97, 461, 1991. 109. Chang, C.H., Furue, M., and Tamaki, K., B7-1 expression of Langerhans cells is up-regulated by proinflammatory cytokines, and is down-regulated by interferon-gamma or by interleukin-10, Eur. J. Immunol., 25, 394, 1995. 110. Wang, B. et al., Epidermal Langerhans cells from normal human skin bind monomeric IgE via Fc epsilon RI, J. Exp. Med., 175, 1353, 1992. 111. Hayashi, S. et al., Mouse Langerhans cells do not express the high-affinity receptor for IgE, Arch. Dermatol. Res., 291, 241, 1999. 112. Nagaoka, Y. et al., Identification and characterization of the low-affinity receptor for immunoglobulin E (FcepsilonRII/CD23) on murine Langerhans cells, J. Invest. Dermatol., 119, 130, 2002. 113. Roitt, I.M. and Delves, P.J., Roitt’s Essential Immunology, 10th ed., Blackwell Scientific, London, 2001. 114. McRae, B.L. et al., Functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis, J. Exp. Med., 182, 75, 1995. 115. Miller, S.D. et al., Blockade of CD28/B7-1 interaction prevents epitope spreading and clinical relapses of murine EAE, Immunity, 3, 739, 1995. 116. Vanderlugt, C.L. and Miller, S.D., Epitope spreading in immune-mediated diseases: implications for immunotherapy, Nat. Rev. Immunol., 2, 85, 2002. 117. Miller, S.D. et al., Epitope spreading leads to myelin-specific autoimmune responses in SJL mice chronically infected with Theiler’s virus, J. Neurovirol., 3, S62, 1997. 118. Miller, S.D. et al., Persistent infection with Theiler’s virus leads to CNS autoimmunity via epitope spreading, Nat. Med., 3, 1133, 1997. 119. Tompkins, S.M., Fuller, K.G., and Miller, S.D., Theiler’s virus-mediated autoimmunity: local presentation of CNS antigens and epitope spreading, Ann. N. Y. Acad. Sci., 958, 26, 2002. 120. Yamamura, M. et al., Defining protective responses to pathogens: cytokine profiles in leprosy lesions, Science, 254, 277, 1991. 121. Salgame, P. et al., Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones, Science, 254, 279, 1991. 122. Bosma, M.J. and Carroll, A.M., The SCID mouse mutant: definition, characterization, and potential use, Annu. Rev. Immunol., 9, 323, 1991. 123. Mosier, D.E., Immunodeficient mice xenografted with human lymphoid cells: new models for in vivo studies of human immunobiology and infectious disease, J. Clin. Immunol., 10, 185, 1990. 124. Spanopoulou, E., Cellular and molecular analysis of lymphoid development using Rag-deficient mice, Int. Rev. Immunol., 13, 257, 1996. 125. Guidos, C.J. et al., Development of CD4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor beta-chain-independent pathway, J. Exp. Med., 181, 1187, 1995. 126. Falk, I. et al., Immature T cells in peripheral lymphoid organs of recombinase-activating gene-1/-2deficient mice. Thymus dependence and responsiveness to anti-CD3 epsilon antibody, J. Immunol., 156, 1362, 1996. 127. Flanagan, S.P., “Nude,” a new hairless gene with pleiotropic defects in the mouse, Genet. Res., 8, 295, 1966.


140


128. Wortis, H.H., Nehlsen, S., and Owen, J.J., Abnormal development of the thymus in “nude” mice, J. Exp. Med., 134, 681, 1971. 129. Kaushik, A., Kelsoe, G., and Jaton, J.C., The nude mutation results in impaired primary antibody repertoire, Eur. J. Immunol., 24, 631, 1995. 130. Rahemtulla, A. et al., Mice lacking CD4 have normal development and function of CD8+ cells but have markedly decreased helper cell activity, Nature, 353, 180, 1991. 131. Fung-Leung, W.P. et al., CD8 is needed for development of cytotoxic T cells but not helper T cells, Cell, 65, 443, 1991. 132. Freeman, G.T. et al., Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice, Science, 262, 907, 1993. 133. Borriello, F. et al., B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation, Immunity, 6, 303, 1997. 134. Roberts, A.W. et al., Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense, Immunity, 10, 183, 1993. 135. Kuhn, R., Rajewsky, K., and Muller, W., Generation and analysis of interleukin-4 deficient mice, Science, 254, 707, 1991. 136. Mattner, F. et al., Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response, Eur. J. Immunol., 26, 1553, 1996. 137. Sligh, J.E. et al., Inflammatory and immune responses are impaired in mice deficient in intercellular adhesion molecule 1, Proc. Natl., Acad. Sci., U. S. A., 90, 8529, 1993. 138. Bullard, D.C. et al., P-selectin/ICAM-1 double mutant mice: acute migration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli, J. Clin. Invest., 95, 1782, 1995.


PART

III

Immune Privilege and Skin Inflammation



CHAPTER

7

Immune Privilege of the Eye Jerry Y. Niederkorn

CONTENTS I. Background ........................................................................................................................143 II. Anatomical and Structural Factors That Contribute to Ocular Immune Privilege...........144 III. Anti-Inflammatory and Immunosuppressive Factors Within the Intraocular Milieu ........................................................................................................145 A. Soluble Factors ..........................................................................................................145 B. Cell Membrane–Bound Factors.................................................................................145 IV. ACAID: A Dynamic Down Regulation of Systemic Immune Responses to Ocular Antigens .............................................................................................................146 V. Immune-Mediated Ocular Diseases: A Breakdown in Immune Privilege? ......................147 A. Intraocular Immune-Mediated Diseases....................................................................147 B. Immune-Mediated Diseases of the Cornea and Conjunctiva ...................................148 C. Ocular Cicatricial Pemphigoid ..................................................................................149 VI. Immune Privilege of the Hair Follicle...............................................................................149 Acknowledgments ..........................................................................................................................150 References ......................................................................................................................................150

I. BACKGROUND Although the human eye is only a few centimeters in diameter, it is composed of a diverse array of tissues, including some cellular and noncellular elements found nowhere else in the body [1]. There are approximately 1 million ganglion cells in the retina that transmit 500 electrical signals per second, which is roughly equivalent to 1.5 ¥ 109 bits of computer information [1]. The extraordinary complexity of the retina and its neurological communications with the brain are meaningless if the transparency of the cornea and the aqueous humor are compromised by trauma, infection, or inflammation. The eye is an extension of the brain and like other elements of the central nervous system; cells in the retina and the corneal endothelium cannot regenerate. Unlike other organs, the eye cannot tolerate even mild inflammation without jeopardizing its only known function, vision. Thus, tight regulation of ocular inflammation and immune responses in the eye is crucial for maintaining the integrity and function of the visual axis. Ocular immune privilege is believed to be an adaptation for restricting the expression of potentially damaging immune-mediated 0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

143


144


responses that indiscriminately inflict injury to ocular cells that possess limited regenerative properties [2–5]. The phenomenon that we know as ocular immune privilege was recognized over a century ago by van Dooremaal, who demonstrated that tumors transplanted into the anterior chamber of the rabbit eye survived for prolonged periods of time [6]. However, the significance of graft survival in the anterior chamber (AC) was not fully appreciated until the seminal studies of Medawar and colleagues ushered in the age of transplantation immunology [7–9]. Medawar recognized that the prolonged survival of foreign tissue grafts in the AC of the eye was a departure from the expected fate of tissue grafts transplanted to other body sites [9]. Since the eye lacks patent lymphatic drainage, it was widely believed that foreign tumor and tissue grafts placed into the AC were sequestered from the peripheral immune apparatus. The notion that immune privilege in the AC was simply a case of immunological ignorance went unchallenged until the late 1970s when Kaplan and Streilein demonstrated that alloantigenic cells introduced into the AC of the rat were indeed perceived by the peripheral immune apparatus [10,11]. Not only did ocular antigens enter peripheral lymphoid tissues, but they also evoked an antigen-specific down regulation of cell-mediated immunity and a concomitant activation of humoral antibody production. This pattern of immune responses was a radical deviation from the normal responses evoked by antigens introduced by other routes. Subsequent studies in mice using allogeneic tumors and minor histocompatibility antigens demonstrated a similar spectrum of suppressed cell-mediated immunity and a preservation of humoral antibody responses [12,13]. Accordingly, the deviant immune response induced by antigens introduced into the AC was termed “anterior chamber-associated immune deviation” (ACAID) to connote the dynamic nature of this immune phenomenon and its relationship with the AC [14]. Since its initial description, ACAID has been demonstrated by numerous laboratories using a wide range of antigens including viral antigens [15], major and minor histocompatibility antigens [12,16–18], soluble proteinaceous antigens [19–21], haptenated cells [22], and tumor antigens [23]. To date, all of the soluble antigens tested have induced ACAID [4]. Although ACAID plays a central role in the maintenance of ocular immune privilege, other properties of the eye also limit the induction and expression of immune-mediated inflammation. These include: (a) anatomical barriers that limit the entry of inflammatory cells into the eye; (b) the low expression of major histocompatibility complex (MHC) antigens on various ocular tissues; (c) a potpourri of anti-inflammatory and immunosuppressive molecules in the aqueous humor; and (d) cell membrane-bound molecules that inactivate immune effector elements that may enter the eye. Thus, ocular immune privilege is a product of multiple anatomical, physiological, and immunoregulatory processes that conspire to limit the induction and expression of immune-mediate inflammation.

II. ANATOMICAL AND STRUCTURAL FACTORS THAT CONTRIBUTE TO OCULAR IMMUNE PRIVILEGE The simplest, and initially the most appealing, explanation for ocular immune privilege was based on the unique absence of major, patent lymphatics draining the interior of the eye, thus creating an afferent blockade of antigen egression to the immune apparatus. However, subsequent studies in mice demonstrated that antigens deposited into the anterior chamber could be detected in the submandibular lymph node as early as three days after intracameral injection [24]. It has been suggested that the tight junctional complexes in the endothelial cells of iris and retinal blood vessels limits the egression of macromolecules and cells from the blood into the eye. This would seemingly prevent the entry of systemically generated immune effector elements from entering the eye. However, passive transfer of immune serum, lymphocytes, or cytotoxic T-lymphocyte (CTL) clones results in the prompt rejection of some intraocular tumors in mice [25–27]. Thus, immune elements have the capacity to enter the eye and exert their effector functions.


IMMUNE PRIVILEGE OF THE EYE

145

Although MHC class I molecules are expressed on virtually all nucleated cells in the body, their expression is greatly reduced or frankly absent on many cells within the eye [28]. It is noteworthy that the corneal endothelium and the retina are incapable of regeneration and coincidentally express little or no MHC class I antigens. The absence of MHC class I antigens would protect these cells from cytolysis by MHC class I restricted CTL in the event of viral infection or when these tissues are used as allografts. The absence of MHC class II antigens on all three corneal cell layers allows MHC class II-mismatched corneal allografts to escape immune recognition and rejection in 80-90% of the hosts [29,30]. Even more impressive is the absence or dramatic reduction in the expression of both MHC class I and class II antigens on the corneal endothelium [5,31–33].

III. ANTI-INFLAMMATORY AND IMMUNOSUPPRESSIVE FACTORS WITHIN THE INTRAOCULAR MILIEU A. Soluble Factors The aqueous humor (AH) that fills the AC possesses remarkable anti-inflammatory and immunomodulatory properties. AH inhibits lymphoproliferative responses in vitro [34,35] and suppresses the expression of delayed type hypersensitivity (DTH) in vivo [36,37]. At least four different AHborne factors inhibit the expression of DTH within the eye: a) TFG-b, b) a-melanocyte-stimulating hormone (a-MSH), c) vasoactive intestinal peptide (VIP), and d) calcitonin gene-related peptide (CGRP) [38-44]. The AH also contains a mice, J. Invest. Dermatol., 117, 1357, 2001. 70. Colombe, B.W. et al., HLA class II antigen associations help to define two types of alopecia areata, J. Am. Acad. Dermatol., 33, 757, 1995. 71. Van der Steen, P. et al., The genetic risk for alopecia areata in first degree relatives of severely affected patients. An estimate, Acta Derm. Venereol., 72, 373-, 1992. 72. Green, J. and Sinclair, R.D., Genetics of alopecia areata, Australas. J. Dermatol., 41, 213, 2000. 73. Barsky, S. and Gigli, I., Alopecia areata in twins, Arch. Dermatol., 83, 224, 1961. 74. Bonjean, M., Prime, A., and Avon, P., Pelade chez deux jumeaux homozygotes (sic), Lyon Med., 219, 1852, 1968. 75. Cole, G.W. and Herzlinger, D., Alopecia universalis in identical twins, Int. J. Dermatol., 23, 283, 1984. 76. Hendren, S., Identical alopecia areata in identical twins, Arch. Dermatol., 60, 793, 1949. 77. Weidman, A.I., Zion, L.S., and Mamelok, A.E., Alopecia areata occurring simultaneously in identical twins, Arch. Dermatol., 74, 424, 1956. 78. Werth, V.P. et al., Incidence of alopecia areata and lupus erythematosus, Arch. Dermatol., 128, 368, 1992. 79. Insler, M.S. and Helm, C.J., Alopecia areata including the cilia and eyebrows of two sisters, Ann. Ophthalmol., 21, 451, 1989. 80. Hordinsky, M.K. et al., Familial alopecia areata. HLA antigens and autoimmunity formation in an American family, Arch. Dermatol., 120, 464, 1984. 81. Dawn, G. and Kumar, B., Profile of alopecia areata in northern India, Int. J. Dermatol., 35, 22, 1996. 82. Theofilopoulos, A.N., The basis of autoimmunity: Part II. Genetic predisposition, Immunol. Today, 16, 150, 1995. 83. Blum, A. and Miller, H., The major histocompatibility complex and inflammation, South. Med. J., 93, 169, 2000. 84. Campbell, R.D. and Milner, C.M., MHC genes in autoimmunity, Curr. Opin. Immunol., 5, 887, 1993. 85. Corzo, D. et al., Advances in HLA genetics, Exp. Clin. Immunogenet., 12, 156, 1995.


446


86. Tomlinson, I.P. and Bodmer, W.F., The HLA system and the analysis of multifactorial genetic disease, Trends Genet., 11, 493, 1995. 87. Thompson, G., HLA disease associations: models for the study of complex human genetic disorders, Crit. Rev. Clin. Lab. Sci., 32, 183, 1995. 88. Welsh, E.A. et al., Human leukocyte antigen-DQB01 03 alleles are significantly associated with alopecia areata, J. Invest. Dermatol., 103, 758, 1994. 89. Morling, N. et al., DNA polymorphism of HLA class II genes in alopecia areata, Dis. Markers, 9, 35, 1991. 90. Kavak, A. et al., HLA and alopecia areata, Int. J. Dermatol., 39, 589, 2000. 91. Kianto, U. et al., HLA-B12 in alopecia areata, Arch. Dermatol., 113, 1716, 1977. 92. Kuntz, B.M. et al., HLA antigens in alopecia areata, Arch. Dermatol., 113, 1717, 1997. 93. Hacham-Zadeh, S. et al., HLA and alopecia areata in Jerusalem, Tissue Antigens, 18, 71, 1981. 94. Averbakh, E.V. and Pospelo, L.E., HLA antigens in patients with alopecia areata, Vestn. Dermatol. Venereol., 1, 24, 1986. 95. Frentz, G. et al., HLA-DR4 in alopecia areata, J. Am. Acad. Dermatol., 14, 129, 1986. 96. Orecchia, G. et al., Human leukocyte antigen region involvement in the genetic predisposition to alopecia areata, Dermatologica, 175, 10, 1987. 97. Duvic, M. et al., HLA-D locus associations in alopecia areata: DRw52a may confer disease resistance, Arch. Dermatol., 127, 64, 1991. 98. Duvic, M. et al., Analysis of HLA-D locus alleles in alopecia areata patients and families, J. Invest. Dermatol., 104 (Suppl.), 5s, 1995. 99. Colombe, B.W. et al., HLA class II alleles in long-standing alopecia totalis/alopecia universalis and long-standing patchy alopecia areata differentiate these two clinical groups, J. Invest. Dermatol., 104 (Suppl.), 4, 1995. 100. Hang, L. and Nakamura, R.M., Current concepts and advances in clinical laboratory testing for autoimmune diseases, Crit. Rev. Clin. Lab. Sci., 34, 275, 1997. 101. Sundberg, J.P. et al., Major locus on mouse chromosome 17 and minor locus on chromosome 9 are linked with alopecia areata in C3H/HeJ mice, J. Invest. Dermatol., 120, 771, 2003. 102. Gollinck, H. and Orfanos, C.E., Alopecia areata: pathogenesis and clinical picture, in Hair and Hair Diseases, Orfanos, C.E. and Happle, R., Eds., Springer-Verlag, Berlin, 1990, p. 529. 103. Muller, S.A. and Winkelmann, R.K., Alopecia areata. An evaluation of 736 patients, Arch. Dermatol., 88, 290, 1963. 104. Shellow, W.V., Edwards, J.E., and Koo, J.Y., Profile of alopecia areata: a questionaire analysis of patient and family, Int. J. Dermatol., 31, 186, 1992. 105. Puavilai, S., Puavilai, G., and Charuwichitratana, S., Prevalence of thyroid diseases in patients with alopecia areata, Int. J. Dermatol., 33, 632, 1994. 106. Schallreuter, K.U. et al., Vitiligo and other diseases: coexistence or true association? Hamburg study of 321 patients, Dermatology, 188, 269, 1994. 107. Price, V.H. and Khoury, E.L., Alopecia areata, Prog. Dermatol., 25, 1, 1991. 108. Conroy, J.D., Alopecia areata, in Spontaneous Animal Models of Human Disease, Andrews, E.J., Ward, B.C. and Altman, N.H., Eds., Academic Press, New York, 1979, p. 30. 109. Muller, G.H., Kirk, R.W., and Scott, D.W., Small Animal Dermatology, 3rd ed., W.B. Saunders, Philadelphia, 1983. 110. Paradis, M., Fecteau, G., and Scott, D.W., Alopecia areata (pelade) in a cow, Can. Vet. J., 29, 727, 1988. 111. Scott, D.W. and Guard, C.L., Alopecia areata in a cow, Agri-practice, 9, 16, 1998. 112. Sundberg, J.P. et al., Alopecia areata in humans and other mammalian species, J. Invest. Dermatol., 104, 32, 1995. 113. McElwee, K.J. et al., Comparison of alopecia areata in human and nonhuman mammalian species, Pathobiology, 66, 90, 1998. 114. Smyth, J.R. and McNeil, M., Alopecia areata and universalis in the Smyth chicken model for spontaneous autoimmune vitiligo, J. Invest. Dermatol. Symp. Proc., 4, 211, 1999. 115. Michie, H.J. et al., Immunobiological studies on the alopecic (DEBR) rat, Br. J. Dermatol., 123, 557, 1990. 116. Michie, H.J. et al., The DEBR rat: an animal model of human alopecia areata, Br. J. Dermatol., 125, 94, 1991.


SKIN-GRAFT-TRANSFER MOUSE MODELS OF ALOPECIA AREATA

447

117. Oliver, R. et al., The DEBR rat model for alopecia areata, J. Invest. Dermatol., 96, 97, 1991. 118. Yanagisawa, H. et al., Characterization of psoriasiform and alopecic skin lesions in HLA-B27 transgenic rats, Am. J.Pathol., 147, 995, 1995. 119. Bullard, D.C. et al., A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice, Proc. Natl. Acad. Sci. U. S. A., 93, 2116, 1996. 120. Sundberg, J.P., Vallee, C.M., and King, L.E., Alopecia areata in aging C3H/HeJ mice, in Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools, Sundberg, J.P., Ed., CRC Press, Boca Raton, FL, 1994, p. 499. 121. Safavi, K.H. et al., Incidence of alopecia areata in Olmstead County, Minnesota 1975-1989, Mayo Clin. Proc., 70, 628, 1995. 122. Friedman, P.S., Clinical and immunological association of alopecia areata, Semin. Dermatol., 4, 9, 1985. 123. Tobin, D.J., Fenton, D.A., and Kendall, M.D., Ultrastructural observations on the hair bulb melanocytes and melanosomes in acute alopecia areata, J. Invest. Dermatol., 94, 803, 1990. 124. Tobin, D.J., Fenton, D.A., and Kendall, M.D., Cell degeneration in alopecia areata: an ultrastructural study, Am. J. Dermatopathol., 13, 248, 1991. 125. Shapiro, J. et al., Alopecia areata-like hair loss in C3H/HeJ mice and DEBR rats can be reversed using topical diphencyprone, J. Invest. Dermatol. Symp. Proc., 4, 239, 1999. 126. Freyschmidt-Paul, P. et al., Induction of hair regrowth in C3H/HeJ alopecia areata by treatment with the topical immunosuppressant FK506, J. Invest. Dermatol., 114, 871, 2000. 127. Freyschmidt-Paul, P. et al., Current and potential therapeutic agents for the treatment of alopecia areata, Curr. Pharm. Des., 7, 213, 2001. 128. Gardner, S.P. et al., Normalisation of hair follicle morphology in C3H/HeJ alopecia areata mice after treatment with squaric acid dibutylester, Eur. J. Dermatol., 10, 443, 2000. 129. Gardner, S.H., The Role of Autoantibodies to Hair Follicles in Alopecia Areata, Ph.D. thesis, University of Bradford, UK, 2001. 130. Sundberg, J.P. et al., Inflammatory bowel disease involving the cecum in C3H/HeJ mice, Gastroenterology, 107, 1726, 1994. 131. Grp Italian Std Epidemiologici in Derm, Epidemiological evidence of the association between lichen planus and two immune-related diseases: alopecia areata and ulcerative colitis, Arch. Dermatol., 127, 688, 1991. 132. Treem, W.R. et al., Ulcerative colitis and total alopecia in a mother and her son, Gastroenterology, 104, 1187, 1993. 133. Brandwein, S.L. et al., Spontaneously colitic C3H/HeJBir mice demonstrate selective antibody reactivity to antigens of the enteric bacterial flora, J. Immunol., 159, 44, 1997. 134. Gilhar, A. et al., Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice, J. Clin. Invest., 101, 62, 1998. 135. McElwee, K.J. et al., Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts, J. Invest. Dermatol., 111, 797, 1998. 136. Zoller, M. et al., Transient CD44 variant isoform expression and reduction in CD4+/CD25+ regulatory T cells in C3H/HeJ mice with alopecia areata, J. Invest. Dermatol., 118, 983, 2002. 137. McElwee, K.J. et al., Alopecia areata in C3H/HeJ mice involves leucocyte mediated root sheath disruption in advance of overt hair loss, Vet. Pathol., in press. 138. Freyschmidt-Paul, P. et al., Treatment with an anti-CD44v10-specific antibody inhibits the onset of alopecia areata in C3H/HeJ mice, J. Invest. Dermatol., 115, 653, 2000. 139. McElwee, K.J. et al., Resistence to alopecia areata in C3H/HeJ mice is associated with increased expression of regulatory cytokines and a failure to recruit CD4+ and CD8+ cells, J. Invest. Dermatol., 119, 142b, 2002. 140. Grabbe, S. et al., B2 integrins are required for skin homing of primed T cells but not for priming naive T cells, J. Clin. Invest., 109, 183, 2002. 141. Borson, N.D. et al., Temporal sequence of transcription of perforin, fas ligand, and tumor necrosis factor-a genes in rejecting skin allografts, Transplantation, 67, 672, 1999. 142. Dieleman, L.A. et al., Kinetics of cytokine expression during healing of acute colitis in mice, Am. J. Physiol., 271, G130, 1996.


448


143. Hubner, G. et al., Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice, Cytokine, 8, 548, 1996. 144. Parenteau, G.L. et al., Prolongation of skin allografts by recombinant tumor necrosis factor and interleukin-1, Ann. Surg., 221, 572, 1995. 145. Cumberbatch, M. and Kimber, I., Dermal tumour necrosis factor-a induces dendritic cell migration to draining lymph nodes, and possibly provides one stimulus for Langerhans’ cell migration, Immunology, 75, 257, 1992. 146. Cumberbatch, M., Fielding, I., and Kimber, I., Modulation of epidermal Langerhans’ cell frequency by tumour necrosis factor-a, Immunology, 81, 395, 1994. 147. Cotran, R.S. and Pober, J.S., Cytokine-endothelial interactions in inflammatory immunity and vascular injury, J. Am. Soc. Nephrol., 1, 225, 1990. 148. Poper, J.S. and Cotran, R.S., Cytokines and endothelial cell biology, Physiol. Rev., 70, 427, 1990. 149. Kainulainen, V. et al., Suppression of syndecan-1 expression in endothelial cells by tumor necrosis factor-a, J. Biol. Chem., 271, 18759, 1996. 150. Slowik, M.R. et al., Tumor necrosis factor activates human endothelial cells through the p55 tumor necrosis factor but the p75 receptor contributes to activation at low tumor necrosis factor concentration, Am. J. Pathol., 143, 1724, 1993. 151. Galbraith, G.M.P. and Pandey, J.P., Tumor necrosis factor alpha (TNF-alpha) gene polymorphism in alopecia areata, Hum. Genet., 96, 433, 1995. 152. Bugeon, L. and Dallman, M.J., Costimulation of T cells, Am. J. Respir. Crit. Care. Med., 162, S164, 2000. 153. Freyschmidt-Paul, P. et al., Fas-deficient C3H.MRL-Fas mice are not susceptible to alopecia areata after grafting of alopecia areata-affected skin from C3H/HeJ mice, J. Invest. Dermatol., in press. 154. McElwee, K.J. et al., Melanocyte and gonad activity as potential modifying factors in C3H/HeJ mouse alopecia areata, Exp. Dermatol., 10, 420, 2001. 155. Oh, H.S. and Smart, R.C., An estrogen receptor pathway regulates the telogen-anagen hair follicle transition and influences epidermal differentiation, Proc. Natl. Acad. Sci. U. S. A., 93, 12525, 1996. 156. Stenn, K.S., Paus, R., and Filippi, M., Failure of topical estrogen receptor agonists and antagonists to alter murine hair follicle cycling, J. Invest. Dermatol., 110, 95, 1998. 157. Smart, R.C., Oh, H.-S., and Robinette, C.L., Effects of 17-B-Estradiol and ICI 182 780 on hair growth in various strains of mice, J. Invest. Dermatol. Symp. Proc., 4, 285, 1999. 158. Freyschmidt-Paul, P. et al., Successful treatment of alopecia areata-like hair loss with the contact sensitizer squaric acid dibutylester (SADBE) in C3H/HeJ mice, J. Invest. Dermatol., 113, 61, 1999. 159. Tang, L. et al., Topical mechlorethamine autoimmune-arrested follicular activity in mice with an alopecia areata-like disease by targeting infiltrated lymphocytes, J. Invest. Dermatol., 120, 400, 2003. 160. Gibson, D.M., Maclean, S.J., and Cherry, M., Recombination between kappa chain genetic markers and the Lyt-3 locus, Immunogenetics, 18, 111, 1983. 161. Galbraith, G.M.P. and Pandey, J.P., Km1 allotype associated with one subgroup of alopecia areata, Am. J. Hum. Genet., 44, 426, 1989. 162. Galbraith, G.M.P., Thiers, B.H., and Pandey, J.P., Gm allotype associated resistance and susceptibility to alopecia areata, Clin. Exp. Immunol., 56, 149, 1984. 163. Paus, R., Christoph, T., and Muller-Rover, S., Immunology of the hair follicle: a short journey into terra incognita, J. Invest. Dermatol. Symp. Proc., 4, 226, 1999. 164. Ahmad, W. et al., Alopecia universalis associated with a mutation in the human hairless gene, Science, 279, 720, 1998. 165. Sundberg, J.P., Dunstan, R.W., and Compton, J.G., Hairless Mouse, HRS/J hr/hr, Springer-Verlag, Heidelberg, 1989. 166. Sundberg, J.P., The hairless (hr) and rhino (hrrh) mutations, chromosome 14, in Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools, Sundberg, J.P., Ed., CRC Press, Boca Raton, FL, 1994, p. 291. 167. Sundberg, J.P. et al., The “hairless” gene in mouse and man, Arch. Dermatol., 135, 718, 1999. 168. Ahmad, W. et al., Molecular basis for the rhino-8J mouse mutation: a nonsense mutation in the mouse hairless gene, Genomics, 53, 383, 1998.


SKIN-GRAFT-TRANSFER MOUSE MODELS OF ALOPECIA AREATA

449

169. Christiano, A.M. et al., Molecular Pathology of Papular Atrichia: Hairless Gene Mutations in Humans and Rhino Mice, paper presented at Third International Alopecia Areata Workshop, Washington, DC, 1998. 170. Panteleyev, A.A. et al., Molecular basis for the rhino Yurlovo (hrrhY) phenotype: severe skin abnormalities and female reproductive defects associated with an insertion in the hairless gene, Exp. Dermatol., 7, 281, 1998. 171. Ahmad, W.A. et al., Molecular basis of a novel rhino (hrrh-Chr) phenotype: a nonsense mutation in the mouse hairless gene, Exp. Dermatol., 7, 298, 1998. 172. Panteleyev, A.A. et al., Molecular and functional aspects of the hairless (hr) gene in laboratory rodents and humans, Exp. Dermatol., 7, 249, 1998. 173. Ahmad, W., Panteleyev, A.A., and Christiano, A.M., The molecular basis of congenital atrichia in humans and mice: mutations in the hairless gene, J. Invest. Dermatol. Symp. Proc., 4, 240, 1999. 174. McElwee, K.J. et al., Alopecia areata versus juvenile alopecia in C3H/HeJ mice: tools to dissect the role of inflammation in focal alopecia, Exp. Dermatol., 8, 354, 1999. 175. Colombe, B.W., Lou, C.D., and Price, V.H., The genetic basis of alopecia areata: HLA associations with patchy alopecia areata versus alopecia totalis and alopecia universalis, J. Invest. Dermatol. Symp. Proc., 4, 216, 1999. 176. deAndrade, M. et al., Alopecia areata in families: association with the HLA locus, J. Invest. Dermatol. Symp. Proc., 4, 220, 1999. 177. Tarlow, J.K. et al., Severity of alopecia areata is associated with a polymorphism in the interleukin1 receptor antagonist gene, J. Invest. Dermatol., 103, 387, 1994. 178. Galbraith, G.M. et al., Contribution of interleukin 1 beta and KM loci to alopecia areata, Hum. Hered., 49, 85, 1999. 179. Tazi-Ahnini, R. et al., Association analysis of IL1A and IL1B variants in alopecia areata, Heredity, 87, 215, 2001. 180. Cox, A. et al., An analysis of linkage disequilibrium in the interleukin-1 gene cluster, using a novel grouping method for multiallelic markers, Am. J. Hum. Genet., 62, 1180, 1998. 181. Dugoujon, J.M. et al., Gm and Km allotypes in autoimmune diseases, G. Ital. Cardiol., 22, 85, 1992. 182. Dugoujon, J.M. and Cambon-Thomsen, A., Immunoglobulin allotypes (GM and KM) and their interactions with HLA antigens in autoimmune diseases: a review, Autoimmunity, 22, 245, 1995. 183. Tazi-Ahnini, R. et al., Structure and polymorphism of the human gene for the interferon-induced p78 protein (MX1): alopecia areata association with the Down syndrome region, J. Invest. Dermatol., 114, 827, 2000. 184. Gilhar, A. et al., Alopecia areata is a T-lymphocyte mediated autoimmune disease: lesional human T-lymphocytes transfer alopecia areata to human skin grafts on SCID mice, J. Invest. Dermatol. Symp. Proc., 4, 207, 1999.



CHAPTER

30

Spontaneous Rat Model of Alopecia Areata in the Dundee Experimental Bald Rat (DEBR) Kevin J. McElwee

CONTENTS I. II. III. IV. V. VI.

History ................................................................................................................................451 Laboratory Animals: Model Establishment and Development .........................................452 Disease Susceptibility ........................................................................................................453 Course of Disease ..............................................................................................................453 Genetics ..............................................................................................................................455 Assessment of Disease.......................................................................................................456 A. Clinical Manifestation ...............................................................................................456 B. Histopathology Examination .....................................................................................457 C. Immunophenotyping ..................................................................................................458 D. Autoantibodies ...........................................................................................................459 VII. Therapeutic Responses.......................................................................................................461 A. Corticosteroids ...........................................................................................................461 B. Contact-Sensitizing Agents........................................................................................461 C. Cyclosporin A ............................................................................................................462 D. FK506 ........................................................................................................................462 E. In Vivo Lymphocyte Depletion..................................................................................462 VIII. Lessons Learned.................................................................................................................463 IX. Conclusion..........................................................................................................................465 Acknowledgments ..........................................................................................................................465 References ......................................................................................................................................465

I. HISTORY Human alopecia areata (AA) is a nonscarring, inflammatory based hair loss disease that is relatively common in the dermatology clinic, accounting for 2% of new dermatology outpatients in the United Kingdom and United States [1]. The life-time risk for AA development in the general population is 1.7% and at any one time between 0.05% and 0.1% of the population will express the lesion, AA [2]. Although not a life-threatening condition, the increasingly image-oriented society 0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

451


452


in which we live makes hair loss an emotionally disturbing event for many people with AA [3]. In response, national and international support groups have been organized, primarily by AA-affected patients themselves, such as the National Alopecia Areata Foundation (NAAF) in the United States, Alopecia Areata Deutschland (AAD) in Germany, and the Association Alopecia Areata (AAA) in France. These groups are involved in patient support, information distribution and education of the general population, and fund-raising for research. Such groups have facilitated an increase in awareness and understanding of AA, and have catalyzed AA research through the development of research guidelines and organization of research workshops [4–6]. In addition, most animal models for AA thus far utilized in research have been financially supported by the National Alopecia Areata Foundation and largely owe their existence to these support and pressure groups. Circumstantial evidence suggests that AA may have an autoimmune pathogenesis where anagenstage hair follicles become the target for an immune cell attack [7]. The evidence primarily consists of observation of a mononuclear cell infiltrate in and around dystrophic hair follicles [8,9] and the knowledge that some people with AA can regrow hair when undergoing immunosuppressive therapy [10]. More recently, production of autoantibodies specific to hair follicle antigens have been identified with greater frequency in AA-affected individuals [11]. These and other circumstantial evidence are of some assistance in understanding the pathogenesis of AA [12]. However, detailed characterization and functional studies are required to demonstrate the significance of these circumstantial observations and to elucidate disease mechanisms. Primarily due to ethical limitations, functional studies cannot readily be conducted in humans. Consequently, animal models of human AA are required. AA is a relatively rare form of hair loss in nonhuman mammals and few cases have been identified beyond reasonable doubt. Isolated examples of AA-like disease have been found in dogs, cats, horses, and nonhuman primates [13,14]. However, the use of such affected individual animals in AA research is restricted due to their limited numbers, genetic variability, and scattered geographical distribution. More recently, an AA-like condition associated with a vitiligo-like disease has been identified in an inbred chicken strain [15]. However, rodents are the most popular species from which to derive animal models of human diseases due to their small size, the inbred nature of many strains, and extensive availability of rodent reagents for use in scientific research [16]. Inbred rodents living in a controlled environment provide useful models to explore specific aspects of disease and to perform controlled experiments that cannot be conducted with humans. With information derived from animal models, studies on humans may be designed to confirm animal model observations. Monitoring of mice has identified a low frequency of spontaneous AA-like conditions in multiple strains [17], but to be of practical value a relatively high incidence of spontaneous or induced disease expression is required. There are two spontaneous rodent models of spontaneous AA that have been employed in laboratory research that involve a relatively high disease phenotype penetrance: the C3H/HeJ mouse [18] and the Dundee Experimental Bald Rat (DEBR). The DEBR is described here.

II. LABORATORY ANIMALS: MODEL ESTABLISHMENT AND DEVELOPMENT The DEBR is a hooded rat strain that was originally developed in 1984 and maintained at the University of Dundee, Scotland [19,20]. Initially, spontaneous alopecia was identified in an inbred stock of BDIX rats at the Ninewells Hospital and University Medical School in Dundee. The University of Dundee BDIX colony was derived from the Medical Research Council Laboratory of Animal Health (LAH) center at Carshalton, Surry, England. In turn, the LAH Carshalton center colony was derived from a cross between BDI and BDVIII with subsequent selection of brother–sister pairs for agouti coat color and dark, pigmented eyes by Druckrey [21] and arrived at the LAH Carshalton center in or before 1977. The LAH Carshalton center became the Experimental Embryology and Teratology Unit in 1981 and was finally closed in 1988. Consequently, the original BDIX


SPONTANEOUS RAT MODEL OF ALOPECIA AREATA IN THE DUNDEE EXPERIMENTAL BALD RAT

453

colony from which DEBRs are derived no longer exists. The Babraham Institute in Cambridge, England indicated expression of similar hair-loss lesions in an LAH Carshalton-derived BDIX colony in 1994, but this colony also no longer exists. Attempts to locate other BDIX colonies and substrains derived from the LAH Carshalton center have not been successful. Existing strains of BDIX rats are all apparently derived from a single primary source, the BDIX/Han substrain from the Medizinische Hochschule in Hannover, Germany, which obtained the BDIX strain direct from Druckrey in 1979. Currently, no hereditary nonscarring, inflammatory alopecia lesions are known to be reported in the BDIX/Han strain or derivate colonies and substrains. The original BDIX colony maintained at the University of Dundee suffered from exceptionally poor fecundity. To rescue the phenotype for future research use, two BDIX rats were crossed with Wistar rats (Charles River, Kent, England) and the DEBR descendants of this cross were derived from full-sibling matings. Two separate DEBR substrains — one black hooded and one brown hooded — exist. Maintained as distinct colonies, the black and brown hooded substrains exhibit similar gross morphological and histological features.

III. DISEASE SUSCEPTIBILITY The hooded rat phenotype was introduced onto the normal, agouti-haired BDIX rats when the initial Wistar cross and subsequent sib matings introduced the recessive “extension factor” hooded gene (H) on rat Chromosome 14 [22]. The recessive gene introduced a pelage coat of white hair on the flanks and abdomen and pigmented hair on the head/neck with extension along the dorsal midline in DEBR rats (Figure 30.1A). Two separately inbred DEBR substrains exist, one black hooded and the other brown hooded, but both substrains have essentially the same AA phenotype properties. DEBR rats develop a full coat of hair within the first 2 weeks of life and hair growth continues normally. Hair loss follows the normal pattern of hair replacement in rats with the earliest expression observed in the third replacement-coat generation [19]. Spontaneous hair loss in the first or second pelage coat generation has never been observed. The first onset of overt hair loss can occur from age 4 months of in females and from age 6 months in males. The initiation of overt hair loss onset may occur at any time in adult rats, although onset is most commonly observed in females aged 5 to 8 months and in males aged 7 to 10 months. If hair loss has not initiated by 12 months of age, the rats are very unlikely to develop AA. Spontaneous expression of AA is apparent in 42% of individuals by age 18 months in a colony with equal frequencies of each sex. However, females are more likely to be affected than males by a ratio of approximately 4:1. Universal alopecia may be observed in approximately 15% of females affected by AA and 5% of affected males. Spontaneous hair regrowth can be observed in approximately 15% of AA-affected rats, although spontaneous regrowth is rarely observed before age 12 months. Twenty-four percent of DEBR rats have nail deformities with the frequency of expression equally distributed between males and females.

IV. COURSE OF DISEASE Occasionally, AA-affected rats may present with a general diffuse thinning of the pelage coat instead of the more distinctive patchy hair-loss presentation. However, hair loss most typically develops as multiple lesions and progresses with approximate lateral symmetry. Hair loss onset can be associated with, and occasionally preceded by, a slight loss of pigmentation in hair in the hooded region. Hair loss is almost always initiated first on the face around the nose and vibrissae follicles. These alopecic lesions typically spread back over the head around the eyes (Figure 30.1B). The vibrissae will continue to grow hair for some months despite the presence of dystrophic


454

Figure 30.1


A

B

C

D

E

F

Gross presentation of hair loss in DEBR. Normal-haired DEBR (A) typically first lose hair on the face around the myastacial pads and eyes (B) with first onset observed from age 4 months in females. This initial presentation quickly progresses and separate lesions develop on the flanks, (C) typically at age 6 to 10 months in females. Flank hair loss may expand to leave a collar of hair (D) and sometimes a strip of hair along the mid-dorsum. Approximately 20% of AA-affected rats develop a near-universal hair loss at age 12 to 18 months (E). Spontaneous hair regrowth can occur in a subset of aged, AA-affected DEBR (F). Hair regrowth is nonpigmented, and rarely regains color. Any areas of pigmented hair, in this case on the head between the ears (F), is usually hair that survived the initial lesion.

interdigitating pelage follicles. However, eventually the vibrissae too will become dystrophic, visible as kinked, short hair fibers, and eventually production of vibrissa fibers may cease altogether. Hair loss separately occurs on the flanks, most usually as two separate areas with approximate bilateral symmetry that expand and which may coalesce with time (Figure 30.1C and D). The maximum extent of lesion expression can be very heterogeneous, even between littermates, but overall, female rats are more commonly affected than males, as they lose hair more rapidly and to a greater extent. The most common end-stage presentation involves almost complete loss of hair from the head and extensive loss on the flanks, sometimes with lesions separated by a collar of hair growth around the neck and forelimbs (Figure 30.1D), a strip of hair growth along the dorsal midline, and some limited diffuse hair growth on the ventral surface. Some rats may progress further with loss of the hair collar but retain hair along the dorsal midline and diffuse ventral hair growth. Notably, the area of hair most resistant to AA is located between the anus and the base of



A

Figure 30.2

455

B

Nail presentation in DEBR. Onchogryphosis is a common feature of the digits of the hind paws (a) and is less frequently observed on the forepaws of DEBR. The aberrant nail presentation does not correlate with the degree of hair loss. Onchogryphosis is possible in the absence of any overt hair loss. Histology shows aberrant nails to be devoid of apparent inflammation (b). Bar = 2 mm.

the tail in both males and females, and typically this is the last area of hair to be lost in the minority of rats that progress to universal alopecia (Figure 30.1E). During the progressive alopecia development phase, regions of bald skin containing hair follicles that originally produced pigmented hair can usually be identified by a bluish hue, a result of melanin deposition in the dystrophic anagen follicles. In aged rats with long-term, extensive alopecia, melainin incontinence is not apparent in the skin and a distinction between pigmented and nonpigmented regions cannot easily be made by gross observation (compare Figure 30.1D and E). If spontaneous hair regrowth occurs in a region of skin that contained pigmented hair before lesion development, the hair regrowth is predominantly white and rarely regains normal pigmentation (Figure 30.1F). Where present, dystrophic nails typically present as exceptionally long and twisted (onchogryphosis) with minor irregularities on the nail surface (Figure 30.2). Although females show a greater frequency of hair loss, there is no sex bias or alteration of how the nail abnormalities present. In addition, it is not necessary for rats to have extensive hair loss for nail deformities to occur. Some rats with only limited hair loss on the head can have severe onchogryphosis. Unlike human AA, extreme nail involvement resulting in near absence of recognizable, cornified material has not been observed in DEBR. No other lesions have been identified in rats with AA; however, beyond age 2 years, rapid weight loss and pursuant death is common with AA-lesion–affected DEBR as compared to normal-haired DEBR of the same age. Both black- and brown-hooded DEBR substrains demonstrate a high degree of sensitivity to anesthetic such that anesthetic application carries an increased risk of death as compared to PVG/OlaHsd and Wistar rat strains. This can present significant problems in long-term studies involving repeated anesthetic exposure.

V. GENETICS Studying inbred rodent models offers significant advantages when considering genomewide screens to identify AA susceptibility and severity-modifying genes. Their small size, rapid proliferation, controlled cross-breeding, and availability of microsatellite markers, enables a highly accurate genetic comparison of AA-affected and nonaffected rodent offspring. In inbred strains, a disease should be monomorphic between individuals and represent a specific type of AA if the disease is due to a single dominant or recessive gene. However, the unpredictable onset, variability


456


in the phenotype presentation, and variable phenotype expression frequency and distribution in rodent models, indicate that AA is a complex, polygenic trait and likely involves significant gene–environment interaction [23]. Two separately inbred DEBR substrains exist: one black hooded with a BDIX-derived RT1dv1 haplotype [24] and the other brown hooded with a Wistar-derived RT1l haplotype. Both substrains are inbred to generation F40 or more as of August 2002. Notably, the original BDIX strain from which the DEBR substrains have been derived has a defect in the expression of RT1.B, apparently due to absence of the structural or controlling gene, while RT1.D locus class II molecules are more strongly expressed in BDIX than in other strains [25]. Whether this defect persists in the DEBR substrains is not known. Preliminary breeding investigations show the disease to be genetically dominant and polygenic with incomplete penetrance. DEBR of the brown-hooded substrain have been crossbred with PVG/OlaHsd rats yielding 21% F1 offspring with an overt hairloss phenotype and 26% expression in the F2 generation. The frequency of F1 and F2 rats with a subclinical AA state — that is, no hair loss but limited focal hair follicle inflammation — is significantly higher. Phenotype segregation observation suggests that five gene loci are involved with inheritance of the alopecia phenotype. On the assumption that the DEBR alopecic phenotype is a polygenic disease, the phenotype penetrance rate in the primary DEBR strains is exceptionally high. Genomewide analysis of an F2 intercross between DEBR and PVG/OlaHsd is currently underway. Spontaneous rodent models of AA will play an important role in recognizing candidate gene loci. The location of AA susceptibility genes in the rodent genomes should predict the location of homologous genes in the human genome by the use of comparative maps for mice and humans (available at www.jax.org). Various rodent models may contain common key dominant-susceptibility genes but different minor genetic-susceptibility or severity-modifying genes that may be associated with various phenotype characteristics or involve different interactions with environmental triggers. When the locations of these genetic factors are known in humans, rodent genetics will continue to be useful in the long-term goal of defining gene–gene and gene–environment interactions and identifying candidate severity-modifying genes. In the long term, manipulation of AA models to create transgenic and knockout rodents will define the significance of AA-associated genes. VI. ASSESSMENT OF DISEASE A. Clinical Manifestation A hair-pull test at the periphery of lesions may not be positive in the same way as with humans, as the high ratio of telogen- to anagen-stage hair follicles in rodents is the reverse of what is observed in humans without alopecia. However, examination of hair pulled from the periphery of an AA lesion in DEBR can variably reveal typical telogen club hairs, fiber with tapered, proximal ends, and fibers with irregular form (Figure 30.3). Hair fibers from the hooded region may also exhibit disruption of pigment incorporation with irregular pigment deposits along the length of hair fibers. The majority of hair pulled from the periphery of lesions presents as normal. The repetitive grooming habits of rodents likely remove loose and defective hair fibers quickly. Finding exclamation-mark hairs in a clinical examination of human hair loss is taken as a useful, although not exclusive, diagnostic feature of AA [26]. Very occasionally, exclamation-mark hairs can be identified in AA-affected DEBR, but these are not common. All types of hair follicles present in rodents can become affected including pelage (awl, auchene, zigzag, and guard hairs), vibrissae, eyelid cilia, and tail follicles. Observation of the skin surface in AA lesions within the hooded region reveals apparent exudation of keratinized, pigmented material from some hair follicles. Internal organ examination reveals the presence of pigment in enlarged skin-draining lymph nodes. The spleen may also be enlarged as compared to normal-haired, non-AA-affected DEBR.



A

Figure 30.3

457

B

Hair fiber defects associated with AA in DEBR. Hair fibers obtained from the periphery of active AA lesions in DEBR show a range of aberrant presentations under scanning electron microscopy. A constriction in the diameter of a probable guard hair is a candidate focal point for future breakage (A). Other pelage hairs may show additional focal points of dystrophy (B). Bar = 20 mm.

Skin grafts from lesions transferred to prelesional DEBR rats enabled regrowth of normal hair from initially dystrophic hair follicles. A reciprocal study resulted in haired, prelesional skin grafts swiftly developing a dystrophic state when transferred to mature, lesional animals. Lesional skin grafted to nude mice regrew normal-length hair. These simple studies suggest the importance of systemic influence over the localized affected hair follicles, potentially by the immune system, and that AA-like lesions in DEBR rats are a true, nonscarring, reversible form of AA. B. Histopathology Examination Histopathological features of human AA include peri- and intrafollicular lymphocytic infiltrates of anagen-stage hair follicles, while catagen- and telogen-stage hair follicles remain unaffected. In association with this focal inflammation, severe hair follicle dystrophy, including root sheath and hair cortex disruption, is observed and subsequent miniaturization of these follicles may occur (Figure 30.4). Although there is severe disruption of hair follicle organization, total destruction of hair follicles, or scarring, usually does not occur in AA, and hair regrowth is possible either spontaneously or due to a successful treatment. For the histopathology of the DEBR model, the biopsy position and lesion duration are important in defining infiltrate intensity. Biopsies from the center of long-standing lesions show a significantly reduced infiltrate in comparison to biopsies taken from the edge of an actively advancing bald patch, as is similarly observed with human AA. The intensity of inflammation around anagen-stage hair follicles can also be variable from rat to rat. It is important to bear this in mind when utilizing the DEBR model. In comparison to normal hair production in unaffected rats or in rats prior to the onset of AA, hair follicles are dystrophic in lesional DEBR. The degree of dystrophy varies within each rat. Most typically, hair follicles at the edge of an advancing area of alopecia are most severely inflamed and correspondingly have the greatest degree of morphological disruption (Figure 30.4B and E). With increasing age and stabilization of the alopecia, the degree of inflammation around anagenstage hair follicles is significantly reduced while hair follicles may still exhibit extensive dystrophy


458

Figure 30.4


A

B

C

D

E

F

Anagen-stage hair follicle dystrophy and inflammation in DEBR. Prior to hair loss, anagen-stage pelage (A) and guard hair follicles (D) demonstrate normal morphology and no discernable aberrations. However, anagen-stage hair follicles at the periphery of advancing AA lesions may exhibit extensive dystrophy associated with an intense peri- and intra-follicular inflammation (B, E). In long-term lesions, anagen-stage pelage (C) and guard hair follicles (F) are often smaller with hair bulbs located in the upper dermis, and exhibit extensive dystrophy and no significant hair fiber production, but the degree of leukocyte inflammation is typically much reduced. Bar = 25 mm.

(Figure 30.4C and F). As the lesions progress, those normally pigmented hair follicles in the hooded region exhibit the phenomenon of melanin, or pigmentary, incontinence. Pigment normally incorporated into the hair fiber can be observed at the base of hair follicles beside the dermal sheath and occasionally within the dermal papilla of vibrissae, pelage, and pigmented tail follicles. Macrophages containing melanin can be identified in regional skin-draining lymph nodes. Samples taken from AA lesions for transmission electron microscopic examination reveal prominent lymphocyte and macrophage infiltration to peri- and intrafollicular locations (Figure 30.5). C. Immunophenotyping Development of hair loss is correlated with peri- and then intrafollicular mononuclear cell infiltration of anagen-stage vibrissae and pelage follicles. Well before the onset of hair loss, such lymphocytic activity is foreshadowed by the development of an increased perifollicular presence of MHC class II+ cells, macrophages, and particularly CD8+ cells [27,28]. In lesional follicles where there is overt disruption of the hair follicle, the interfollicular infiltrate is primarily composed of CD4+ cells, but CD8+ cells and macrophages constitute the intrafollicular infiltrate [28]. The CD4+ to CD8+ cell ratio of perifollicular cells is around 2:1 and coincides with up-regulation of MHC class II expression in the hair follicle, similar to that observed in humans [27,28]. It is probable that the density of the mononuclear infiltrate and the ratio of CD4+:CD8+ cells varies with time.



A

459

K

B H

L

L L K C

V

K

F

F

L L

Figure 30.5

Peri- and intrafollicular infiltration of anagen-stage hair follicles in AA-affected DEBR. Transmission electron microscopy reveals a leukocyte (L) presence in the periphery in blood vessels (V) between collagen bundles (C) and adjacent to dystrophic hair follicles (H). Some leukocytes penetrate to intrafollicular locations (B), in this example interdigitating between fibroblast-like cells (F) of the dermal sheath and and keratinocytes (K) of the outer root sheath. Bar = 2 mm.

Prior to and during very early onset of hair loss in DEBR rats, the dominant infiltrate cell type is CD8+ [28]. CD4+ cells predominate later in lesion development. The most intense mononuclear infiltrate occurs in rats and mice with active disease progression. The infiltrate is significantly reduced in rats, mice, and humans with long-term stable lesions. The localized hair follicle infiltrate is of greater significance in understanding AA pathogenesis, as AA is a broadly organ-specific disease for the hair follicle and its appendages. One probable explanation for the various ratios of the hair follicle infiltrate is that the majority of infiltrating cells are secondary and have no specificity for any hair follicle epitope, as has been suggested for other autoimmune diseases. Only a minority of the infiltrating cells may actually be specific to hair follicles and promote destruction; the CD4+ and CD8+ cell populations are unlikely to be functionally homogeneous. The majority of the cells may be irrelevant to AA pathogenesis other than indirect effects via cytokine production. These changes in ratios over time may also reflect potential changes in the disease pathogenesis. The initiation mechanism need not be the same as the mechanism involved in progressive expansion of AA, which in turn may be different from immune activity in chronic, stable lesions. D. Autoantibodies Comparison of hair follicle specific–autoantibodies using a refined indirect immunofluorescent technique revealed that individual serum from DEBR rats contain hair follicle-specific IgG autoantibodies [29–31]. While individual sera revealed detailed differences, the target tissues identified were hair cortex and cuticle and the inner root sheath, especially the Henle’s layer. Notably, targeting of the outer root sheath or sebaceous glands was not apparent (Figure 30.6). The morphological targets are the same in different hair follicle types. In published studies, vibrissae are utilized in the assay due to their large size and corresponding ease of observation. However, the same morphological targets are observed in pelage follicles. In DEBR, autoantibodies were found in highest concentration from animals in a progressive, active lesional state, although around 30% of rats aged less than 4 months and in advance of any obvious hair loss also expressed low levels of hair follicle–specific autoantibodies. Some sera also


460


A

M

Figure 30.6

B

C

IRS

M

C

IRS

Hair follicle autoantibodies in DEBR. Blood serum from AA-affected DEBR contains autoantibodies sepecific for hair follicle structures discernable by indirect immunofluoresence. However, the morphological structures targeted by autoantibodies in samples taken from different rats is highly variable. One sera (A) may target the differentiating hair matrix (M) and hair cuticle (C), while serum from a littermate (B) may target the hair matrix (M) and inner root sheath (IRS). Various combinations of targets are apparent in different serum samples with no apparent correlation to the nature of the AA lesions. Bar = 10 mm.

contained autoantibodies specific to skeletal muscle and cell nuclear components. Hair follicle–specific autoantibodies were absent from other rat strains (PVG/OlaHsd, Wistar) that did not exhibit AA. Examination of sera from patients with alopecia areata using the same technique revealed similar patterns of hair follicle structures and cell nuclear-component staining, but in addition the outer root sheath, sebaceous glands and epidermis were also affected [31]. Preliminary Western blot analysis of DEBR sera suggest several targets in the 25- to 65-kDa range with occasional, additional targets in the 110- to 220-kDa range (K.J.M., personal observations, 1995). The production of hair follicle–specific autoantibodes further supports the notion that AA is an autoimmune disease. However, the significance of these autoantibodies has often been brought into question. There are three main possible modes of action for hair follicle–specific autoantibodies. The autoantibodies may be the prime initiators of AA, similar in their pathogenicity to autoantibodies produced in pemphigus vulgaris or myasthenia gravis. Alternatively, they may actually be of advantage to the patient with AA as opsonizers for removal of debris from around dystrophic hair follicles and/or in the bloodstream. Another possibility is that the autoantibodies may arise secondarily to a prior immune, cell-mediated insult, and further promote pathogenic destruction either via C3-complement pathway and/or by opsonization of hair follicle components for future leukocyte-mediated attack. Not all antibodies may be relevant. Different types may have different pathogenic abilities, depending on structure and the specific antigen epitope that the autoantibodies bind. For other autoimmune diseases, over time the range of autoantibody targets can increase due to epitope spreading. As chronic disease progresses and more antigenic debris is produced, a wider range of B-cell clones are promoted to antibody production. Autoantibodies before and during early disease onset will likely be of greater significance in defining disease initiation.



461

VII. THERAPEUTIC RESPONSES Patients with AA are capable of regrowing hair even after many years of hair loss, and spontaneous regrowth can occur in people with AA. However, there is no permanent cure for AA and there is no universally proven therapy for inducing remission. There are a range of therapies for which at least partial success has been claimed. All current modalities used are more effective in those with milder forms of the disease than in people with extensive hair loss. Popular treatments include nonspecific immune inhibitors such as systemic or locally injected corticosteroids, contact sensitizers, such as dinitrochlorobenzene (DNCB); squaric acid dibutyl ester (SADBE); diphencyprone (DPCP); UV light treatment, particularly in conjunction with 8-methoxypsoralen in PUVA therapy; minoxidil; and experimental use of specific immunosuppressants [32]. The DEBR model has also been examined for hair growth in response to various therapeutic modalities [33]. A. Corticosteroids Topical corticosteroids are commonly used for the treatment of limited, focal AA although only limited data provide evidence in support of their use. Only two placebo-controlled studies with small numbers of patients reported treatment response in about 60% of cases, but this response rate has not been confirmed by other studies [34–36]. This may in part be due to insufficient penetration of topically applied drugs to the hair bulb. Controlled studies for the use of intralesional and systemic corticosteroids are also lacking. However, published studies suggest a greater hair growth response rate to intralesional injection [37–39]. AA has been treated with systemic corticosteroids since 1952 [40]. Initially, corticosteroids were applied orally daily or every second day for several months, but this continuous application of corticosteroids is regarded as inappropriate today. The doses that are needed to gain hair regrowth in AA range from 30 to 150 mg daily, thus leading to side effects such as hypertension, diabetes, immunosuppression, osteoporosis, and tendency to thrombosis. More recently, pulsed administration of corticosteroids has been employed to treat severe AA [41–44]. Pulsed application of corticosteroids in single doses, given once monthly until successful hair regrowth is achieved, may reduce the side effects of corticosteroids to an acceptable level. Small-scale, unpublished studies on the DEBR model have shown a hair growth response to systemic corticosteroids at equivalent doses per kilogram of rat weight as used in humans. B. Contact-Sensitizing Agents DNCB, DPCP, also called diphencyprone (DCP), and (SADBE) are topical contact sensitizers. They are primarily used for people with extensive scalp hair loss. The reaction to the chemical(s) can result in good hair regrowth with claims of a 38% success rate for DPCP [45], 63% for DNCB [46], and up to 70% for SADBE [47]. The variability in success rates is due in part to investigators’ different definitions of what constitutes successful regrowth. Given that these treatments are mostly used by people with long-term extensive hair loss, each has a relatively good success rate in growing cosmetically acceptable hair when treatment is given by a dermatologist experienced in its use. How contact-sensitizing agents modulate AA and permit or promote hair growth is not known, but may involve the promotion of regulatory immune cells that supercede the activity of pathogenic cells in the skin. Untreated AA is characterized by an increased expression of the Th1 cytokines IFN-g, IL-2, and IL-1b, whereas after successful treatment with a contact sensitizer, there is increased expression of the Th2 cytokine IL-10 and of TGF-b and TNF-a [48]. These cytokines may be secreted during the down-regulatory phase of the contact dermatitis that occurs as a process of disease self-limitation after the allergen is removed. In unpublished studies, DEBR responded with various degrees of hair growth to the application of DNCB. However, the adverse reaction to


462


treatment in DEBR was severe, involving extensive skin vesicle and pustule formation. As a result, studies of the DEBR using DNCB were abandoned at an early stage. Whether lower dosage or the use of DCP or SADBE would induce hair regrowth with reduced side effects in DEBR is not known. C. Cyclosporin A Cyclosporin A (CyA) is a specific inhibitor of lymphocyte activation, and in principle, it may be useful in treating patients with AA. CyA has been reported to restore hair growth in a subset of patients when administered orally to humans [10,49,51]. However, while systemic immune suppression may release AA-affected hair follicles from their dystrophic state, it can potentially leave the patient partially exposed to infection and side effects, including neurotoxicity and impairment of renal function. Such a side effect profile renders widespread use of oral CyA for AA unacceptable. Attempts to use topical formulations of CyA for AA, and thereby avoid side effects seen with systemic application, have been largely unsuccessful thus far [52–55]. As with humans, CyA in standard formulations must be given systemically to DEBR rats to promote hair growth [56]. At 10 mg/kg, 5 days/week for 7 weeks, CyA in olive oil quickly reversed established AA lesions in DEBR displaying extensive areas of hair loss. New hairs appeared after 10 days and there was simultaneous regrowth of hair over the whole body with restoration of a full pelt by 5 weeks [57,58]. Semiquantitative histological examination of flank skin biopsies revealed early reduction of the cellular infiltrate associated with conversion of dystrophic anagen follicles to normal, hair-producing follicles. Topical application of 0.5% CyA in ethanol has little effect on the hair-loss state. However, recent research suggests that AA-affected DEBR can be successfully treated topically with 0.5% CyA in a unique lipsomal formulation [59]. This suggests that the vehicle used in topical application has a significant impact on the penetration of the drug to the depth of dystrophic hair follicles, and special formulations for CyA and other similar drugs such as FK506 may be required for the successful treatment of human AA. D. FK506 FK506 (tacrolimus) suppresses IL2 production and release in activated T cells by inhibiting calcineurin. Subsequently, activation and proliferation of T cells is inhibited [60]. Because T-cell activation and proliferation as well as an overexpression of HLA are likely significant in the pathogenesis of AA, FK506 is a promising candidate treatment. In the DEBR model for AA, oral application promoted little response [56]. However, topically applied FK506 in both acetone (Figure 30.7) and a propylene glycol vehicle successfully induced hair regrowth [61]. Routine histology (Figure 30.8) and immunohistochemical studies showed reduced peri- and intrafollicular infiltrates of CD4+ and CD8+ T cells in rats treated successfully with FK506 as compared to controlvehicle treated skin. These encouraging results in animal models and recent studies in neurodermitis patients that have demonstrated the safety of treatment with topical FK506 [62–64], suggest that topical application of FK506 could also be a safe and effective treatment of AA in humans. E. In Vivo Lymphocyte Depletion Although CD4+ cell depletion has been seriously investigated as a therapy for multiple sclerosis, systemic lupus erythmatoses and psoriasis in humans with some success, CD4+ cell depletion is unlikely to become a practical therapy for AA in humans, given that CD4+ cell depletion may leave individuals susceptible to infection. However, studies on the activity of CD4+ and CD8+ cells in DEBR lesion perpetuation have included selective in vivo depletion using monoclonal antibody injections. Lesional DEBR rats were given intraperitoneal injections of a CD4+ cell-depleting monoclonal antibody (MAb) cocktail or a CD8 cell-depleting MAb over a 15-day therapy course. Control groups were injected with equivalent volumes of an irrelevant MAb. Removal of CD4+ or



A

Figure 30.7

B

463

C

Topical FK506 treatment of AA in DEBR. In topical treatment studies, 2-cm2 areas of bald flank skin are marked by tattoos, and rats receive the drug to one flank and the control vehicle to the other as an intraindividual control (A). FK506 application in acetone 5 days per week (80 ml of 0.1% FK506 per unit area per day) promotes hair regrowth within the area of drug application, clearly visible by 29 days (B) and progressing to a near-normal pelage density and hair length by day 56 (C).

CD8+ T cells using this approach, confirmed by flow cytometric analysis of blood samples and immunohistology of skin biopsies, enables good hair regrowth in lesional animals [65,66]. The studies suggest that close interaction and stimulus between CD4+ and CD8+ cells is required to perpetuate the AA lesion, possibly with CD8+ cells in their typical cytotoxic role and CD4+ cells in their helper support role. However, the activation events for CD4+ and CD8+ cells in DEBR remain unknown.

VIII. LESSONS LEARNED Human AA is a complex disease in which clinical and pathological presentation varies considerably among individuals. In part, this reflects the mixed genetic background and variable environmental influences on AA-affected patients. Inbred rodents living in a controlled environment provide useful models to explore specific aspects of the disease and to perform experiments that cannot be conducted on humans. Information gained from such models will help define how AA develops and provide clues as to new and improved treatments. Despite being a congenic strain, DEBR rats demonstrate many forms of hair loss which is similar to human lesion progression, including clinical presentation of circumscribed patchy alopecia that may wax, wane, and migrate, and can become more extensive, up to and including universal hair loss. Rat hair loss can occur in any region of skin and affect any type of hair. Hair fibers become dystrophic and exclamation-mark hairs are sometimes present at the periphery of bald patches. Hair follicle dystrophy is associated with a peri- and intra-follicular mononuclear cell infiltrate with CD4+ and CD8+ lymphocytes as the dominant cell types. Only anagen hair follicles are targeted; telogen and catagen hair follicles escape the attention of the inflammatory infiltrate. Hair follicle-specific IgG autoantibodies are produced in association with rat AA and the autoantibodies target the lower hair follicle and bulb, with morphological targets apparently well conserved between species. AA is a nonscarring form of hair loss in all species with the possibility of spontaneous regrowth and a hair growth response to the same immunomodulatory therapies.


464

Figure 30.8


A

B

C

D

Histology after treatment of AA in DEBR with topical FK506. Topical application of FK506 leads to hair growth and a relatively normal pelage (A) and guard hair follicle (C) morphology with few inflammatory cells by day 56. In contrast, flank skin from the same rat receiving the acetone vehicle contains pelage (B) and guard hair follicles (D) with considerable dystrophy and inflammation. Bar = 25 mm.

From our current understanding of rodent models, the development of AA relies upon a general genetic susceptibility. Major rodent AA-susceptibility genes may be supplemented by a variety of minor disease-severity–modifying genes. However, the actual onset of the disease, and its extent, persistence, and resistance to treatment in a given individual rodent may potentially be modulated by epigenetic factors, such as diet, hormones, general viral load, and other environmental influences. The events that lead to human AA development also likely involve both genetic and environmental factors that influence general susceptibility and severity of disease. Both genetic and environmental factors will modulate AA at three levels: overall reactivity of the immune system, specific inciting antigens and how they are presented to the immune system, and the target issue itself [67]. The mechanism of rodent AA seems to be fundamentally, but not exclusively, Th1 cells mediated with CD8+ cells being key instigators of hair follicle disruption. Antigen presentation within the skin itself and within immune system organs likely incite reactive leukocyte clones. The onset of disease is dependent on several factors including the breakdown of immune privilege in hair follicles and perhaps other hair follicle-specific defects, presence of autoreactive lymphocytes, appropriate antigen presentation in association with co-stimulation, and a deficiency of lymphocyte regulation.



465

Without knowing the source of the specific antigen epitopes that trigger AA onset, it is not possible to conclusively define AA as fundamentally autoimmune in nature. However, rodent model research has produced evidence consistent with this hypothesis.

IX. CONCLUSION The AA model in DEBR reveals a phenotype closely resembling human AA, and has shown promise as a model to investigate the pathogenesis of AA. Given the limited understanding of AA initiation and progression and the limited success of current therapeutic modalities, animal models will prove vital to determine pathogenesis and genetic susceptibility to AA. Recent data have strengthened the argument that AA is an immunologically mediated condition. Nonhuman species expressing AA also show promise as models for development and evaluation of new and existing therapies for AA. Ultimately, the models may be used to determine methods of permanent reversal of this presumptive autoimmune disease, which may have implications for research beyond the field of hair biology.

ACKNOWLEDGMENTS I am grateful to R. Oliver, J.P. Sundberg, and R. Hoffmann for their support, and to D. Black, L. Bechtold, S. Kissling, E. Wenzel, and A. Huth for their technical assistance. The work was supported in part by the National Alopecia Areata Foundation. I am a recipient of the Alfred Blaschko Memorial Fellowship, Marburg.

REFERENCES 1. Dawber, D., de Berker, D., and Wojnarowska, F., Disorders of hair: alopecia areata, in Rook/Wilkinson/Ebling Textbook of Dermatology, Champion, R.H. et al., Eds., Blackwell Science, Oxford, 1998, p. 2919. 2. Safavi, K.H. et al., Incidence of alopecia areata in Olmsted County, Minnesota 1975 through 1989, Mayo Clin. Proc., 70, 628, 1995. 3. Thompson, W. and Shapiro, J., Alopecia Areata: Understanding and Coping with Hair Loss, Johns Hopkins University Press, Baltimore, 1996, p. 2. 4. Goldsmith, L.A., Summary of alopecia areata research workshop and future research directions, J. Invest. Dermatol., 96, 98, 1991. 5. McElwee, K.J., Third International Research Workshop on Alopecia Areata, J. Invest. Dermatol., 112, 822, 1999. 6. Olsen, E. et al., Alopecia areata investigational assessment guidelines. National Alopecia Areata Foundation, J. Am. Acad. Dermatol., 40, 242, 1999. 7. McDonagh, A.J. and Messenger, A.G., The pathogenesis of alopecia areata, Dermatol. Clin., 14, 661, 1996. 8. Perret, C., Wiesner-Menzel, L., and Happle, R., Immunohistochemical analysis of T-cell subsets in the peribulbar and intrabulbar infiltrates of alopecia areata, Acta Derm. Venereol., 64, 26, 1984. 9. Ranki, A. et al., Immunohistochemical and electron microscopic characterization of the cellular infiltrate in alopecia (areata, totalis, and universalis), J. Invest. Dermatol., 83, 7, 1984. 10. Gupta, A.K. et al., Oral cyclosporine for the treatment of alopecia areata. A clinical and immunohistological analysis, J. Am. Acad. Dermatol., 22, 242, 1990. 11. Tobin, D.J. et al., Antibodies to hair follicles in alopecia areata, J. Invest. Dermatol., 102, 721, 1994. 12. McElwee, K.J. et al., Alopecia areata: an autoimmune disease? Exp. Dermatol., 8, 371, 1999. 13. Conroy, J.D., Alopecia areata, in Spontaneous Animal Models of Human Disease, Andrew, E.J., Ward, B.C., and Altman, N.H., Eds., Academic Press, New York, 1979, p. 30.


466


14. McElwee, K.J. et al., Comparison of alopecia areata in human and nonhuman mammalian species, Pathobiology, 66, 90, 1998. 15. Smyth, J.R. Jr. and McNeil, M., Alopecia areata and universalis in the Smyth chicken model for spontaneous autoimmune vitiligo, J. Investig. Dermatol. Symp. Proc., 4, 211, 1999. 16. Sundberg, J.P. and King, L.E. Jr., Mouse mutations as animal models and biomedical tools for dermatological research, J. Invest. Dermatol., 106, 368, 1996. 17. McElwee, K.J. et al., Spontaneous alopecia areata-like hair loss in one congenic and seven inbred laboratory mouse strains, J. Investig. Dermatol. Symp. Proc., 4, 202, 1999. 18. Sundberg, J.P., Cordy, W.R., and King, L.E. Jr., Alopecia areata in aging C3H/HeJ mice, J. Invest. Dermatol., 102, 847, 1994. 19. Michie, H.J. et al., The DEBR rat: an animal model of human alopecia areata, Br. J. Dermatol., 125, 94, 1991. 20. Oliver, R.F. et al., The DEBR rat model for alopecia areata, J. Invest. Dermatol., 96, 97, 1991. 21. Druckery, H., Genotypes and phenotypes of ten inbred strains of BD-rats, Arnzneim-Forsch., 21, 1274, 1971. 22. Levan, K.K. et al., Rat gene map, Rat Genome, 2, 30, 1996. 23. McElwee, K.J. et al., Genetic susceptibility and severity of alopecia areata in human and animal models, Eur. J. Dermatol., 11, 11, 2001. 24. Kendall, C. et al., Characterization of strain specific typing antisera for genetic monitoring of inbred strain of rats, Lab. Anim. Sci., 35, 364, 1985. 25. Male, D.K., Pryce G., and Butcher G.W., Serological evidence for a defect in RT1.B (I-A) expression by the BDIX rat strain, J. Immunogenet., 14, 301, 1987. 26. Peerboom-Wynia, J.G.R. et al., Scanning E.M.comparing exclamation mark hairs in alopecia areata with normal hair fibres mechanically broken by traction, Clin. Exp. Dermatol., 14, 47, 1989. 27. Michie, H.J. et al., Immunobiological studies on the alopecic (DEBR) rat, Br. J. Dermatol., 123, 557, 1990. 28. Zhang, J.G. and Oliver, R.F., Immunohistological study of the development of the cellular infiltrate in the pelage follicles of the DEBR model for alopecia areata, Br. J. Dermatol., 130, 405, 1994. 29. McElwee, K.J., Pickett, P., and Oliver, R.F., Hair follicle autoantibodies in DEBR rat sera, J. Invest. Dermatol., 104, 34, 1995. 30. McElwee, K.J., Pickett, P., and Oliver, R.F., Autoantibodies to the hair follicle in sera from the DEBR rat model for Alopecia Areata, Br. J. Dermatol., 134, 55, 1996. 31. McElwee, K.J., Drummond, S., and Oliver, R.F., Hair follicle specific autoantibodies associated with alopecia areata in sera from the DEBR rat model and humans, Excerpta Medica Int. Congress, 1111, 253, 1996. 32. Freyschmidt-Paul, P. et al., Current and potential agents for the treatment of alopecia areata, Curr. Pharmacol. Design, 7, 213, 2001. 33. Sundberg, J.P. et al., Alopecia areata in humans and other mammalian species, J. Invest. Dermatol., 104, 33s, 1995. 34. Pascher, F., Kurtin, S., and Andrade, R., Assay of 0.2% fluocinolone acetonide cream for alopecia areata and totalis, Dermatologica, 141, 193, 1970. 35. Leyden, J.J. and Kligman, A.M., Treatment of alopecia areata with steroid solution, Arch. Derm., 106, 924, 1972. 36. Lehnert, W., Zur Lokalbehandlung schwerer Formen der Alopecia Areata mit Kortikosteroidsalbe, Derm. Mschr., 160, 396, 1974. 37. Kalkoff, K.W. and Macher E., Über das Nachwachsen der Haare bei der Alopecia areata und maligna nach intracutaner Hydrocortisoninjektion, Hautarzt, 9, 441, 1958. 38. Orentreich, N. et al., Local injection of steroids and hair regrowth in alopecias, Arch. Dermatol., 82, 894, 1960. 39. Abell, E. and Munro D.D., Intralesional treatment of alopecia areata with triamcinolone acetonide by jet injector, Br. J. Dermatol., 88, 55, 1973. 40. Dillaha, C.J. and Rothman, S., Therapeutic experiments in alopecia areata with orally administered cortisone, JAMA, 150, 546, 1952. 41. Burton, J.L. and Shuster, S., Large doses of glucocorticoid in the treatment of alopecia areata, Acta Dermatoven., 55, 493, 1975.



467

42. Perriard-Wolfensberger, J. et al., Pulse of methylprednisolone in alopecia areata, Dermatology, 187, 282, 1993. 43. Sharma, V.K., Pulsed administration of corticosteroids in the treatment of alopecia areata, Int. J. Dermatol., 35, 133, 1996. 44. Friedli, A. et al., Pulse methyprednisolone therapy for severe alopecia areata: an open prospective study of 45 patients, J. Am. Acad. Deramtol., 39, 597, 1998. 45. Shapiro, J. et al., Treatment of chronic severe alopecia areata with topical diphenylcyclopropenone and 5% minoxidil: a clinical and immunopathologic evaluation, J. Am. Acad. Dermatol., 29, 729, 1993. 46. Swanson, N.A. et al., Topical treatment of alopecia areata, Arch. Dermatol., 117, 384, 1981. 47. Flowers, F.P. et al., Topical squaric acid dibutylester therapy for alopecia areata, Cutis, 30, 733, 1982. 48. Hoffmann, R. et al., Cytokine mRNA levels in Alopecia areata before and after treatment with the contact allergen diphenylcyclopropenone, J. Invest. Dermatol., 103, 530, 1994. 49. Paquet, P. et al., Oral cyclosporin and alopecia areata, Dermatology, 185, 314, 1992. 50. Ferrando, J. and Grimalt, R., Partial response of severe alopecia areata to cyclosporine A, Dermatology, 199, 67, 1999. 51. Parodi, A. et al., Alopecia universalis and cyclosporin A, Br. J. Dermatol., 135, 657, 1996. 52. Mauduit, G. et al., Treatment of severe alopecia areata with topical applications of cyclosporin A, Ann. Dermatol. Venereol., 114, 507, 1987. 53. Parodi, A. and Rebora, A., Topical cyclosporine in alopecia areata, Arch. Dermatol., 123, 165, 1987. 54. de Prost, Y. et al., Placebo-controlled trial of topical cyclosporin in severe alopecia areata, Lancet, 2, 803, 1986. 55. Gilhar, A., Pillar, T., and Etzioni, A., Topical cyclosporin A in alopecia areata, Acta. Derm. Venereol., 69, 252, 1989. 56. Sainsbury, T.S.L. et al., Differential effects of FK506 and cyclosporine on hair regrowth in the DEBR model of alopecia areata, Transplant. Proc., 23, 3332, 1991. 57. Oliver, R.F. and Lowe, J.G., Oral cyclosporin A restores hair growth in the DEBR rat model for alopecia areata, Clin. Exp. Dermatol., 20, 127, 1995. 58. McElwee, K.J., Lowe, J.G., and Oliver, R.F., Effects of potent immunotherapies, oral cyclosporin A and topical FK506, in the DEBR rat model for alopecia areata, Excerpta Medica Int. Congress, 1111, 259, 1996. 59. Verma, D.D. et al., Treatment of alopecia areata in the DEBR model using Cyclosporin A lipid vesicles, J. Exp. Dermatol., in press. 60. Schreiber, S.L. and Crabtree, G.R., The mechanism of action of cyclosporin A and FK506, Immunol. Today, 13, 136, 1992. 61. McElwee, K.J. et al., Topical FK506: a potent immunotherapy for alopecia areata? Studies using the Dundee experimental bald rat model, Br. J. Dermatol. 137, 491, 1997. 62. Nakagawa, H. et al., Tacrolimus ointment for atopic dermatitis, Lancet, 344, 883, 1994. 63. Ruzicka, T. et al., A short-term trial of tacrolimus ointment for atopic dermatitis. European Tacrolimus Multicenter Atopic Dermatitis Study Group, N. Engl. J. Med., 337, 8161, 1997. 64. Alaiti, S. et al., Tacrolimus (FK506) ointment for atopic dermatitis: a phase I study in adults and children, J. Am. Acad. Dermatol., 38, 69, 1998. 65. McElwee, K.J., Spiers, E.M., and Oliver, R.F., In vivo depletion of CD8+ T cells restores hair growth in the DEBR model of alopecia areata, Br. J. Dermatol., 135, 211, 1996. 66. McElwee, K.J., Spiers, E.M., and Oliver, R.F., Partial restoration of hair growth in the DEBR model for Alopecia areata after in vivo depletion of CD4+ T cells, Br. J. Dermatol., 140, 432, 1999. 67. Marrack, P., Kappler, J., and Kotzin, B.L., Autoimmune disease: why and where it occurs, Nat. Med., 7, 899, 2001.



CHAPTER

31

Spontaneous Canine Model of Alopecia Areata Desmond J. Tobin and Thierry Olivry

CONTENTS I. History ................................................................................................................................469 A. Background ................................................................................................................469 B. Spontaneous Alopecia Areata in Nonhuman Mammals ...........................................470 C. Canine Homologue of Human AA............................................................................470 II. Animals ..............................................................................................................................470 III. Epidemiology .....................................................................................................................471 IV. Course of Disease ..............................................................................................................471 V. Assessment of Disease.......................................................................................................471 A. Clinical Manifestation ...............................................................................................471 B. Histopathological Examination .................................................................................472 C. Immunopathological Data .........................................................................................473 1. Immunophenotyping ............................................................................................473 2. Direct Immunofluorescence Microscopy ............................................................474 3. Indirect Immunofluorescence Microscopy ..........................................................474 4. Immunoblotting....................................................................................................475 5. Selective Immunoprecipitation ............................................................................475 6. Passive Transfer of Canine Anti–HF IgG ...........................................................477 D. Immunogenetics.........................................................................................................478 VI. Therapeutic Responses.......................................................................................................478 VII. Expert Experience ..............................................................................................................478 VIII. Lessons Learned.................................................................................................................478 IX. Conclusion..........................................................................................................................480 References ......................................................................................................................................480

I. HISTORY A. Background Alopecia areata (AA) is a common cause of hair loss afflicting approximately 1.7% of the general human population [1]. At least 0.1% of the human population will express the disorder at 0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

469


470


any one time [2], and it is likely that minor forms of the disorder go unnoticed. It also may not be uncommon among nonhuman mammals (reviewed in McElwee et al. [3]). AA results from selective, largely reversible, damage to the anagen hair follicle (HF) [4–8]. Thus, the manifestation of patchy hair loss is predominantly seen on anagen-bearing skin, such as the scalp in humans. However, other body parts can be affected and the disease can progress to complete loss of all body hair. While not life threatening, the disease is nonetheless serious because it is disfiguring and in humans can cause severe psychological problems and loss of employment [9]. Affected animals may experience a reduction in their monetary value. The etiology of AA remains unclear. Hair loss is associated with an immune-mediated pathology and an autoimmune basis is now strongly suspected (reviewed in McElwee et al. [10]). The recent availability of accepted research guidelines, drawn up by the National Alopecia Areata Foundation (San Rafael, CA [11]), will help avoid addition to the rather confusing AA literature. Much of the earlier data was often based on small numbers of patients, poorly controlled or uncontrolled studies, on differences whose significance were not analyzed statistically, and on results that were often contradictory. However, part of this apparent confusion may be due to the possibility that not all cases of AA represent manifestations of the same disease. Much of the recent progress in AA research is due to the development of two animal models for human AA: the inbred rodent models of the C3H/HeJ mouse [12] and the DEBR rat [13]. Both models have provided very useful data not only on the genetics of AA but also on the associated immune perturbations [14–16] and the assessment of various treatment modalities [17,18]. B. Spontaneous Alopecia Areata in Nonhuman Mammals Only a limited number of case reports and letters on spontaneous AA-like hair loss in dogs appear in the pre-1990s literature. Moreover, it is not always clear if the diagnosis was unequivocally that of AA. AA-like hair loss has been reported in many breeds of dogs including Bernese mountain [19], Dachshund [20–23], Doberman pinscher [19,24], German shepherd dog [24,25], Magyar viszla [24], miniature poodle [21], and in several mixed breeds [24–29]. Recently, we have described a dog with AA-like hair loss in which intrabulbar cytotoxic T lymphocytes infiltrated lower hair follicles, and we demonstrated that this was associated with the alopecia [25]. Moreover, in a recent study of four dogs with progressive, nonscarring, AA-like hair loss we reported that sera of affected dogs contained anti-HF IgG [29]. AA-like disease has been reported in other mammals including cats [21], horses [21,30,31], cows [32,33], and nonhuman primates [21,34]. The poor characterization of many of these cases prevents assessment of their scientific or clinical value. However, animal models, including outbred animals, are likely to be valuable for the dissection of the common pathways underlying the pathogenesis of AA and for the development and evaluation of therapies for AA. C. Canine Homologue of Human AA Our recent immunopathological analyses of dogs with AA-like hair loss [25,29] raised the possibility that AA may not only be more widespread than previously thought, but that a more comprehensive characterization of AA-like hair loss in this species may support the use of canine AA as a homologue for the human condition.

II. ANIMALS As mentioned above, AA-like hair loss has been reported in several canine species including Bernese mountain [19], dachshund [20–23], Doberman pinscher [24–19], German shepherd [24,25], Magyar viszla [24], miniature poodle [21], and several mixed breed dogs [24–29]. In a


SPONTANEOUS CANINE MODEL OF ALOPECIA AREATA

471

recent large-scale study [35], AA-like hair loss in 25 client-owned dogs was systematically assessed using AA-associated clinical, histological, and immunological criteria [11]. Nineteen of the 25 dogs with AA were reported as purebred by their owners and included four German shepherd and crosses, three dachshunds, two beagles, two setters, and two Labrador retriever crosses. The remaining subjects represented crossbred dogs. In this group of dogs with AA, the age of onset of hair loss varied between 1 and 13 years (median, 5 years). Of interest is that dachshunds usually appeared to develop the disease at an earlier age than dogs of other breeds (median, 2 years). The male-tofemale sex ratio was 0.79, and most subjects had been either castrated or spayed. Even though reference populations are not available for comparison, the dachshund breed appears predisposed to develop AA, as this breed seems overrepresented when all cases reported previously are examined altogether.

III. EPIDEMIOLOGY At the time of this writing, all descriptions of canine AA have been of single cases. One case study of 25 patients has been submitted for publication recently [35]. This study regrouped patients from Europe and North America that had been diagnosed with AA between 1994 and 1999. Unfortunately, because of the widespread distribution of the origin of the subjects, an estimation of prevalence and incidence could not be made. It is assumed, however, that spontaneously occurring canine AA is a rare disease.

IV. COURSE OF DISEASE The age of onset of alopecia varies greatly. In one recent study of 25 dogs with AA [35], age of onset ranged from 1 to 11 years (median, 5 years). A minority of dogs had developed AA by age 2 years (young adults). Estimation of odds ratios for disease predisposition has proved difficult, since dogs are often assembled from various North American and European locations and a reference population was not accessible. Where information on disease outcome was available, spontaneous and complete hair regrowth was found to occur in the majority of subjects. Interestingly, regrown hair was commonly white, a feature also seen in humans with AA [36].

V. ASSESSMENT OF DISEASE A. Clinical Manifestation The initial lesion reported by the owners is alopecia. In a minority of cases the growth of white hair, that is, leukotrichia, may precede the first alopecic lesion and supports the growing belief that the hair bulb melanocytes are a target in AA [5,36,37]. Lesions usually develop first on the face (Figure 31.1) and head (muzzle, chin [Figure 31.2], forehead, periocular, ears), in common with the bias toward scalp and beard in human AA. Similarly, in subjects with variable coat color, hair loss usually occurs first in dark brown or black areas, again presumed to correlate with the preferential targeting of pigmented hair and the relative sparing of nonpigmented hair in humans with AA [6,36]. At the time of diagnosis, lesions usually consist of spontaneously arising and well-demarcated alopecic patches. Facial lesions usually exhibit a bilateral symmetry. Hair loss may also be seen on the legs (Figure 31.3) and in some cases hair loss can progress to a more generalized distribution in some patients.


472


Figure 31.1

Sharp-edged alopecic patch with skin hyperpigmentation on the face of a Labrador retriever with AA.

Figure 31.2

Alopecia and hyperpigmentation on the chin (same dog as in Figure 31.1).

B. Histopathological Examination The histological hallmark of human AA is the so-called “swarm of bees” mononuclear cell infiltrate focused at the anagen hair bulb [8] (Figure 31.4a and b). Hematoxylin and eosin-stained sections of canine AA skin biopsy specimens usually reveal an anagen population of approximately 75%, catagen 10%, and a raised telogen count of around 15%. Telogen counts are also high in human AA [38]. Bulbitis is observed in a majority of lesional anagen HF, although, perhaps



Figure 31.3

Patchy hair loss on the dorsal aspect of the forefoot (same dog as in Figure 31.1).

Figure 31.4

Lymphocytic bulbitis and peribulbitis in a dog with AA. Bar = 130 mm (a) and 30 mm (b).

473

surprisingly, not in all HFs. The extent of the folliculotropic infiltration studied is variable and within the lesion around/in various affected HFs. In a recent large-scale study [35], the extent of the mononuclear cell infiltrate has been graded as mild in about 50% of HFs, moderate in 10%, and severe in 3% of follicles. Similarly, peribulbitis was seen around 44% of HFs, and was graded as mild in 33%, moderate in 10%, and severe in 1% of HFs studied. Distorted dystrophic hair shafts were a feature in about a quarter of the affected dogs examined (Figure 31.5). C. Immunopathological Data 1. Immunophenotyping Examination of skin sections immunostained for the pan T-cell marker CD3 reveals a predominantly T-lymphocyte infiltration. T cells are not usually found in/at the HF infundibulum, while the incidence of T lymphocytes increases proximally from the HF isthmus (in about 50% of dogs),


474

Figure 31.5


Lymphocyte infiltration in the bulb is associated with dystrophic shaft formation (arrowhead). Bar = 25 mm.

inferior HF segment (in the vast majority of dogs), to the hair bulb in all dogs. T-lymphocyte infiltration of the perifollicular dermis is also present in all dogs and can extend some distance from the immediate HF area in a few cases. The severity of infiltration is usually mild in the isthmus and inferior segments, but can be mild to severe in hair bulbs and around lower HF structures. To date only limited lymphocyte immunophenotyping has been performed on lesional canine AA skin [25]. In reports containing this information, there appears to be a 1:3 to 1:4 ratio of hair follicle infiltrating CD8+ to CD4+ cells (reviewed in McElwee et al. [3]). However, T cells invading growing hair follicles are more commonly CD8+, while CD4+ cells predominate in the peribulbar T-cell infiltrates and in this way also reflect the human condition. Careful selection of the biopsy site is required and in some cases more than one biopsy is required to fully appreciate the extent of the follicular infiltrate. In a recent study [35] intra- and peri-bulbar lymphocytes expressed the ab T-cell receptor and were either CD8 or CD4. 2. Direct Immunofluorescence Microscopy Deposition of immunoglobulin and complement around HF, especially at the edge of active lesions has been associated with AA [39]. Direct immunofluorescence (DIF) staining for IgG on paraffin-embedded sections of lesional canine AA skin reveals the common ex vivo deposition of IgG in HF structures [35]. Deposits of IgG are usually found on the lower HF glassy membrane and less often within the follicular papilla, medulla, outer root sheath (ORS), or hair matrix. IgG deposits may also be detected around the HF including between collagen fibers and in dermal plasma cells. 3. Indirect Immunofluorescence Microscopy Antibodies to HF-specific antigens are much more common, and present in higher titers, in patients with AA than in control patients, including those with scarring alopecia [40,41]. Moreover, the anti-HF antibodies that have been detected in rats, mice, horses, and dogs (reviewed in McElwee et al. [3]) with AA-like disease appear to target similar HF proteins, and thus suggest a high degree



475

IRS

Epi HF

Hb

Figure 31.6

Indirect immunofluorescence analysis of antibodies to HF in canine AA. Canine IgG antibodies also react specifically with HFs in murine back skin, and may target inner root sheath and medulla in the mid to lower HF. Note lack of reactivity to the upper HF, epidermis (Epi), and dermis. Bar = 50 mm. Hb, hair bulb.

of epitope conservation across species. In a recent study of four dogs with progressive, nonscarring, AA-like hair loss, we reported that sera of affected dogs contained IgG antibodies (some up to titers of 1:2560) that reacted with components of the lower anagen HF (Figure 31.6), especially the inner root sheath (IRS) and medulla [29]. No reactivity was detected in normal healthy dogs or in sera from dogs with inflammatory skin disease due to demodicosis. Moreover, a similar pattern of reactivity was found when canine AA sera were incubated with normal human and murine skin. Furthermore, in a larger study of 14 dogs with AA-like hair loss [35], canine AA sera contained high titer antibodies (>1:500) that reacted to multiple HF structures including IRS, ORS, matrix, and precortex. In addition, the serum from canine AA patients contain antibodies that label the same structure of the HF as that targeted by a monoclonal antibody against trichohyalin (AE15) (Figure 31.7). 4. Immunoblotting Immunoblotting analysis of circulating serum antibodies provides the possibility to gain further information regarding the nature of the target proteins in this disease [14,41,42]. To date, antibody reactivity in whole canine AA sera has been assessed against electroblotted 6-M urea-extractable human HF protein [29]. In a recent study of four dogs with progressive, nonscarring, AA-like hair loss, we reported that sera of affected dogs contained IgG antibodies that reacted intensely with HF proteins of 200 to 220 kDa and also with multiple antigens in the 50 to 60 kDa range (Figure 31.8). Co-migration studies suggested that reactivity to the 200- to 220-kDa doublet reflected in turn reactivity to the IRS and medulla-rich protein trichohyalin [29], while some of the targeted proteins in the 50- to 60-kDa range were likely to represent keratins. We have recently expanded on this study and assessed anti-HF antibody reactivity in a large number of dogs affected with AA-like hair loss [35]. Confirming the previous study, this analysis revealed a strong and heterogeneous pattern of anti-HF reactivity in canine AA serum IgG against multiple antigens between 40 to 60 kDa and to a 200- to 220-kDa doublet. 5. Selective Immunoprecipitation While immunoblotting analysis can provide information on the molecular weight of antibodytargeted HF antigens, this method does not easily reveal antigen identity per se. Therefore, we have


476


IRS IRS

Hb a Figure 31.7

Hb b

Co-localization of AA-target HF antigens in canine AA. The pattern of immunoreactivity with this canine AA serum (a) is very similar to that of a monoclonal antibody AE 15 to trichohyalin (b). Note that both antisera target the inner root sheath. Bar = 150 mm. Hb, hair bulb.

200

50

A Figure 31.8

B

Western immunoblotting analysis of HF antigens defined by IgG antibodies in canine AA sera. (A) 6 M urea–extractable proteins were obtained from normal human scalp anagen HFs and separated by SDS-8% PAGE and immunoblotted with canine AA and (B) control canine sera. Note that AA serum reactivity to HF antigens of 40 to 60 kDa and 200 to 220 kDa. Control sera, including sera from dogs with demodicosis, are minimally reactive/nonreactive to these proteins.



477

200

A Figure 31.9

B

C

Selective immunoprecipitation of trichohyalin protein from 6 M urea–extractable proteins (obtained from normal human scalp anagen HFs) by canine AA sera (B). Positive control (A) was AE15 monoclonal antibody to trichohyalin. Control canine sera failed to immunoprecipitate this HF antigen (C).

exploited the ability of antibodies to immunoprecipitate their specific target antigens using a selective immunoprecipitation assay [29,42]. We have to date performed two studies [29,35], in which we have shown that canine AA serum IgG antibodies can immunoprecipitate trichohyalin protein (200 to 220 kDa) from crude extracts of total HF protein (Figure 31.9). By contrast, normal canine sera failed to immunoprecipitate this antigen. 6. Passive Transfer of Canine Anti–HF IgG Unlike autoantibodies in pemphigus vulgaris and myasthenia gravis, the pathogenic potential of anti-HF autoantibodies in AA remains to be shown. As these may occur prior to the onset of clinical hair loss [14], it is possible that they may not in some cases be formed secondarily to a preexisting immune insult (e.g., cell mediated). We have recently made two attempts to assess the pathogenic potential of anti-HF antibodies in equine and canine sera via their passive transfer into anagen skin of naïve mice [31,35]. In preparation for passive transfer, high-titer anti–HF IgG was purified from the sera of AAaffected dogs. Control IgG was purified normal canine serum. Purified AA IgG reacted with HF, including IRS, ORS, matrix, and HS in murine anagen skin, indicating not only retained reactivity to HFs but also confirming significant cross-species conservation of HF target epitopes. No reactivity to HF was found in control IgG. Moreover, purified AA IgG showed very strong reactivity to a 200- to 220-kDa antigen band, that is, presumptive trichohyalin and weaker reactivity also to multiple antigens between 40 to 60 kDa [35]. Twice weekly intradermal administration of AA and control IgG into anagen III back skin of four C57BL/10scsn mice was performed for 1 month. While both IgG from AA and normal dogs delayed regrowth of hair in and immediately around the site of IgG administration, AA IgG administration resulted in an increased delay of anagen reentry of up to 2 weeks compared with control normal IgG. Limited serum IgG in this study and the resultant low numbers of mice included in the study prevented a more complete assessment of the pathogenic potential of anti-HF IgG in canine AA. However, it should be noted that high-titer anti-HF equine IgG also disrupted hair regrowth (via extension of telogen) when passively transferred to C57BL/6 mice [31].


478


D. Immunogenetics At this time, there is no information on immunogenetic typing of AA-affected canine patients.

VI. THERAPEUTIC RESPONSES As stated above, spontaneous remission occurs in most canine patients with AA. When natural hair regrowth does not occur, treatment with oral glucocorticoids or cyclosporine is initiated. In a recent case series [35], treatment with immunosuppressive doses of prednisone (1 to 2 mg per kg per day) or cyclosporine (5 to 10 mg per kg per day) was reported to lead to remission of clinical signs. In some patients, the treatment could be discontinued after a few months, and lesions did not recur. In rare individuals left untreated, hair loss can remain permanent.

VII. EXPERT EXPERIENCE Spontaneous canine AA is a disease that is seen rarely by veterinarians. The diagnosis of canine AA is not difficult and is based on a combination of clinical signs supported by histopathological examination of lesional and perilesional skin biopsy specimens. Clinical differential diagnoses for canine AA includes pseudopelade, demodicosis, dermatophytosis, staphylococcal folliculitis, and ischemic alopecias such as dermatomyositis and rabies-vaccine reactions. The main histopathological differential diagnosis is canine pseudopelade, but in the latter, lymphocyte epidermotropism targets the mid-HF sections, and there is involution of lower HF segments. Such cases, albeit rare, can be available by contacting veterinary dermatologists worldwide. Access to a veterinary dermatology Internet list (contact T. Olivry) allows easy dissemination of the request and increases the availability of canine AA case material that can be provided to interested researchers.

VIII. LESSONS LEARNED Studies to date support the use of dogs with AA as a potential outbred model for human AA. These studies, using both pure and mixed breeds, have identified strong similarities between canine AA and the disorder in human patients. Important features of canine AA being compatible with human AA include similarities in clinical presentation (including spontaneous remissions), distribution and phenotype of HF-associated mononuclear cell infiltrates, preferential targeting of anagen HFs, presence of anti-HF IgG antibodies that target similar HF components, and HF proteins of similar molecular weight. Taken together, these studies reflect a remarkably strong correspondence between canine AA and human AA on all major themes. Studies to date indicate that canine AA most commonly presents as a patchy alopecia on the scalp and face, as is the case in the human disorder [43]. The appearance of lesions earlier in pigmented areas in some dogs of variable coat color concurs with evidence that human AA may also preferentially target pigmented hair and spare white hair [44], although, like human AA, this is not an absolute feature in canine AA. The finding that spontaneous hair regrowth occurs in the majority of AA-affected dogs is a notable one, as the majority of untreated human AA patients will also regrow their hair within 1 year [45]. Moreover, like the human correlate [46], immunosuppressive therapy is successful in stimulating hair regrowth in dogs, including those not showing spontaneous regrowth. Hair regrowth in human AA is often initially white, confirming the involvement of melanogenic melanocytes



479

located in the anagen hair bulb in this disorder [5,36,37]. Similarly, regrowing white hair occurs in the majority of canine AA cases examined to date, suggesting that target cell(s), and antigens therein are likely to be conserved in nature. It is perhaps noteworthy that a recent study [35] reported that the melanocyte defect may be more severe/long-lasting in canine AA, as regrowing hair may fail to regain pigmentation even after several molts. Canine AA exhibits the histological hallmark of human AA — the so-called “swarm of bees” mononuclear cell infiltrate focused at the anagen hair bulb [8]. Not only are anagen HFs preferentially targeted by the AA disease insult in both human [7] and canine [35] AA, but a nonscarring (peri)bulbitis is a feature in all cases studied to date. Indeed, the T-cell–rich infiltrate in canine AA locates principally in/around the hair bulb and thus correlates even more closely with human AA than does the C3H/HeJ mouse AA model [12]. As is the case in human AA [47], T-cell phenotyping of canine AA reveals that greater numbers of CD8+ T cells invade the HF bulb epithelium while more CD4+ T cells were distributed peribulbarly [25,35]. The role of humoral factors in the pathogenesis of alopecia areata remains controversial. It is in dissecting this aspect of AA that animal models may prove very useful, given the ethical considerations involved in using human subjects for this type of manipulation [3,48] Even if AA IgG antibodies do not prove to be pathogenic, autoantibodies, via the epitopes they target, are likely to tell us a great deal about the HF autoantigen(s) in AA. One of the most unifying features of AA in humans and other mammals is the presence of antibodies directed to HF-specific target proteins detected using direct and indirect immunofluorescence and by immunoblotting [14,26,29,31,40–42,50]. Circulating IgG antibodies in canine AA patients react with multiple components of anagen HFs, again corresponding to the situation in humans [14,31,40]. It is of note that the hair bulb and precortex, the site of significant keratinocyte and melanocyte differentiation, is most commonly targeted in both species. In this way, humoral factors could disturb cell differentiation and so compromise hair formation/cycling. Like all other species examined to date, canine AA sera contain IgG antibodies that preferentially reacted with antigens in the 40- to 60-kDa and 200- to 220-kDa molecular weight range [14,26,29,31,40–42,50]. Keratins are well represented in the former and trichohyalin (the IRS-rich protein) in the latter. Indeed, human AA but not control sera (including from scarring alopecias), was able to selectively immunoprecipitate the 46/47-kDa HF-specific keratin recognized by AE14 [42]. We have previously reported that canine AA sera can immunoprecipitate trichohyalin [29] and this was confirmed in further cases of canine AA [35]. As a member of the intermediate filament-associated protein family, trichohyalin is thought to be necessary for correct alignment of keratin filaments of the IRS and medulla [50]. Thus, it is likely that disruption of IRS differentiation during anagen (AA-targeted phase of the hair cycle) will result in defective hair shaft formation. The precise role of anti-HF antibodies in the pathogenesis remains unknown. Clearly, high-titer antibodies to HFs are not observed in normal individuals despite the release of HF antigens during the normal HF cycle or in even in scarring alopecias with their associated tissue damage [48]. Moreover, an abnormal autoantibody response to HF is present both in affected mice and to a lesser degree in their, as yet, clinically unaffected littermates in the C3H/HeJ mouse model of AA [14]. This suggests that the presence of antibodies to HF appears before the onset of hair loss and so may not be produced as a secondary response to HF damage in AA. Purified IgG from an AAaffected horse has been reported to retard hair regrowth when passively transferred to normal mice [31]. However, an earlier study reported the failure of whole AA serum to inhibit hair growth when passively transferred into human scalp skin grafted onto nude mice [51]. Our recent attempt to passively transfer purified canine AA IgG into naïve mice suggests that AA IgG retards re-entry of telogen HF into anagen for longer than in skin treated with normal canine IgG [35]. However, the AA IgG failed to prevent hair regrowth, and histologically there was no bulbitis. These studies need to be repeated with larger numbers of animals in order to reach statistical significance.


480


IX. CONCLUSION In conclusion, the current literature provides good evidence that dogs with AA-like disease exhibit very similar clinical, histological, and immunopathological features to those seen in AA in humans. Given the outbred nature of canine AA, these findings provide us with a new model system to evaluate the pathology of AA in humans and also to assess treatment modalities.

REFERENCES 1. Safavi, K.H. et al., Incidence of alopecia areata in Olmsted County, Minnesota, 1975–1989, Mayo Clin. Proc., 70, 628, 1995. 2. Safavi, K.H., Prevalence of alopecia areata in the first national health and nutrition examination survey, Arch. Dermatol., 128, 702, 1992. 3. McElwee, K.J. et al., Comparison of alopecia areata in human and non-human mammalian species, Pathobiology, 66, 90, 1998. 4. Tobin, D.J., Morphological analysis of hair follicles in alopecia areata, Microsci. Res. Tech., 38, 443, 1997. 5. Tobin, D.J., Fenton, D.A., and Kendall, M.D., Ultrastructural observations on the hair bulb melanocytes and melanosomes in acute alopecia areata, J. Invest. Dermatol., 94, 803, 1990. 6. Goos, M., Zur histopathologie der alopecia areata, Arch. Derm. Forsch., 240, 160, 1971. 7. Messenger, A.G., Slater, D.N., and Bleehen, S.S., Alopecia areata: alterations in the hair growth cycle and correlation with follicular pathology, Br. J. Dermatol., 114, 337, 1986. 8. Van Scott, E.J., Morphologic changes in pilosebaceous units and anagen hairs in alopecia areata, J. Invest. Dermatol., 31, 35, 1958. 9. Thompson, W. and Shapiro, J., Alopecia areata, in Understanding and Coping with Hair Loss, John Hopkins University Press, Baltimore, 1996, p. 1. 10. McElwee, K.J. et al., Alopecia areata: an autoimmune disease? Exp. Dermatol., 8, 371, 1999. 11. Olsen, E. et al., investigational assessment guidelines. National Alopecia Areata Foundation, J. Am. Acad. Dermatol., 40 (2 Pt 1), 242, 1999. 12. Sundberg, J.P., Cordy, W.R, and King, L.E., Alopecia areata in aging C3H/HeJ mice, J. Invest. Dermatol., 102, 847, 1994. 13. Michie, H. J. et al., The DEBR rat: an animal model of human alopecia areata, Br. J. Dermatol., 125, 94, 1991. 14. Tobin, D.J. et al., Autoantibodies to hair follicles in C3H/HeJ mice with alopecia areata-like hair loss, J. Invest. Dermatol., 109, 329, 1997. 15. Michie, H.J. et al., Immunobiological studies on the alopecic (DEBR) rat, Br. J. Dermatol., 123, 557, 1990. 16. McElwee, K.J. et al., Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts, J. Invest. Dermatol., 111, 797, 1998. 17. Tobin, D.J. et al., Diphencyprone immunotherapy alters anti-hair follicle antibody status in patients with alopecia areata, Eur. J. Dermatol., 12, 327, 2002. 18. Freyschmidt-Paul, P. et al., Treatment of alopecia areata in C3H/HeJ mice with the topical immunosuppressant FK506 (Tacrolimus), Eur. J. Dermatol., 11, 405, 2001. 19. Scarff, D.H. Disorders of pigmentation, in Manual of Small Animal Dermatology, Locke, P.H., Harvey, R.G., and Mason, I.S., Eds., British Small Animal Veterinary Association, Looke Printer Poole, 1993, chap. 3. 20. Conroy, J.D., Alopecia in dogs and cats, J. Am. Anim. Hosp. Assoc., 4, 200, 1968. 21. Conroy, J.D., Alopecia areata, in Spontaneous Animal Model of Human Disease, Andrews, E.J., Ward, B.C., and Altman, N.H., Eds., Academic Press, New York, 1979, p. 30. 22. Muller, G.H., Kirk, R.W., and Scott, D.W., Small Animal Dermatology, W.B. Saunders, Philadelphia, 1983.



481

23. Gross, T.L., Ihrke, P.J., and Walder, E.J., Alopecia areata, in Veterinary Dermatopathology: A Macroscopic and Microscopic Evaluation of Canine and Feline Skin Disease, Gross, T.L., Irhke, P.J., and Walder, E.J., Eds., Mosby Year Book, St. Louis, 1992, p. 291. 24. Tobin, D.J. et al., Hair follicle-specific antibodies in mammalian species with alopecia areata, J. Invest. Dermatol., 108, 654, 1997. 25. Olivry, T. et al., Antifollicular cell-mediated and humoral immunity in canine alopecia areata, Vet. Dermatol., 7, 67, 1996. 26. Guernsey, G.E., Alopecia areata in a dog, Can. Vet. J., 26, 403, 1985 (letter). 27. Yager, J. and Wilcock, B.P., Lymphocytic folliculitis resembling human alopecia areata and universalis, in Color Atlas and Text of Surgical Pathology of the Dog and Cat, Mosby Year Book Europe Limited (Wolfe), St. Louis, 1994, p. 196. 28. Scott, D.W., Miller, W.H., and Griffin, C.E., Small Animal Dermatology, 5th ed., W.B. Saunders, Philadelphia, 1995. 29. Tobin, D.J., Olivry, T., and Bystryn, J.-C., Anti-trichohyalin antibodies in canine alopecia areata, in Advances in Veterinary Dermatology, Kwochka, K.W., Willemse, T., and von Tscharner, C., Eds., Butterworth-Heinemann, Boston, 355. 30. Scott, D.W., Large Animal Dermatology, W.B. Saunders, Philadelphia, 1988, 393. 31. Tobin, D.J., Alhaidari, Z., and Olivry, T., Equine alopecia areata autoantibodies target multiple hair follicle antigens and may alter hair growth in passive transfer studies, Exp. Dermatol., 7, 289, 1998. 32. Scott, D.W. and Guard, C.L.G., Alopecia areata in a cow, Agric. Pract., 9, 16, 1988. 33. Paradis, M., Fecteau, G., and Scott, G.W., Alopecia areata in a cow, Can. Vet. J., 29, 727, 1988. 34. Eichberg, J.W. and DeVillez, R.L., Alopecia totalis in a chimpanzee, J. Med. Primatol., 13, 81, 1984. 35. Tobin, D.J. et al., A natural canine homologue of alopecia areata in humans, Br. J. Dermatol., in press. 36. Messenger, A.G. and Bleehen, S.S., Alopecia areata: light and electron microscopic pathology of the regrowing white hair, Br. J. Dermatol., 110, 155, 1984. 37. Gilhar, A. et al., Melanocyte-associated T cell epitopes can function as autoantigens for transfer of alopecia areata to human scalp explants on Prkdc(scid) mice, J. Invest. Dermatol., 117, 1357, 2001. 38. Kim, I.H. et al., Quantitative image analysis of hair follicles in alopecia areata, Acta Derm. Venereol., 79, 214, 1999. 39. Bystryn, J.-C., Orentreich, N., Stengel, F., Direct immunofluorescence studies in alopecia areata and male pattern alopecia, J. Invest. Dermatol., 73, 317, 1979. 40. Tobin, D.J. et al., Hair follicle structures targeted by antibodies in alopecia areata, Arch. Dermatol., 133, 57, 1997. 41. Tobin, D.J. et al., Antibodies to hair follicles in alopecia areata, J. Invest. Dermatol., 102, 721, 1994. 42. Tobin, D.J. and Bystryn, J-C., Alopecia areata is associated with antibodies to hair follicle-specific antigens located predominantly in the proliferative region of hair follicles, in Hair Research for the Next Millennium, Neste, D.J.J. and Randall, V.A., Eds., Elsevier Science, Amsterdam, 1996, p. 237. 43. Gollnick, H. and Orfanos, C.E., Alopecia areata: pathogenesis and clinical picture, in Hair and Hair Diseases, Orfanos, C.E. and Happle, R., Eds., Springer, Berlin, 1990 p. 529. 44. Guin, J.D, Kumar, V., and Petersen, B.H., Immunofluorescence findings in rapid whitening of scalp hair, Arch. Dermatol., 117, 576, 1981. 45. Madani, S. and Shapiro, J., Alopecia areata update, J. Am. Acad. Dermatol., 42, 549, 2000. 46. Shapiro, J., Alopecia areata. Update on therapy, Dermatol. Clin., 11, 35, 1993. 47. Perret, C., Weisner-Menzel, L., and Happle, R., Immunohistochemical analysis of T-cell subsets in the peribulbar and intrabulbar infiltrates in alopecia areata, Acta Derm. Venereol., 64, 26, 1984. 48. Tobin, D.J. and Bystryn, J-C., Immunology of alopecia areata, in Hair and Hair Disorders: Research, Pathology and Management, Camacho, F.M., Randall, V.A., and Price, V., Eds., Martin Dunitz, London, 2000, p. 187. 49. McElwee, K.J., Pickett, P., and Oliver, R.F., The DEBR rat, alopecia areata and autoantibodies to the hair follicle, Br. J. Dermatol., 134, 55, 1996. 50. O’Guin, W.M., Sun, T.T., and Manabe, M., Interaction of trichohyalin with intermediate filaments: three immunologically defined stages of trichohyalin maturation, J. Invest. Dermatol., 98, 24, 1992. 51. Gilhar, A. et al., Failure of passive transfer of serum from patients with alopecia areata and alopecia universalis to inhibit hair growth in transplants of human scalp skin grafted on to nude mice, Br. J. Dermatol., 126, 166, 1992.



CHAPTER

32

Experimental Chimeric SCID Mouse/Human Skin Model of Alopecia Areata: Induction by Transfer of Cellular Immunity Richard S. Kalish and Amos Gilhar

CONTENTS I. History ................................................................................................................................483 II. Laboratory Animals............................................................................................................484 A. Human Skin Graft/SCID Mouse as an Experimental System for Studying Inflammatory Conditions ...........................................................................................484 B. Characteristics of SCID Mice ...................................................................................484 C. Immunodeficient Mice as Recipients for Human Scalp Grafting ............................484 III. Disease Induction...............................................................................................................485 A. The Role of T Cells...................................................................................................485 B. The Role of Pretransfer Activation with Follicular Antigen ....................................485 C. The Roles of CD4+ and CD8+ T-Cell Subsets ..........................................................486 D. The Role of Melanocyte-Derived Antigen................................................................486 IV. The Disease Assessment ....................................................................................................487 A. Clinical Manifestation ...............................................................................................487 B. Histopathological and Immunopathological Examination........................................489 V. Lessons Learned.................................................................................................................489 References ......................................................................................................................................489

I. HISTORY Much clinical evidence suggests that alopecia areata (AA) is an autoimmune disease. AA responds to immunosuppressive doses of systemic steroids [1], as well as immunotherapy with contact sensitizers [2]. The condition also has an association with known autoimmune processes such as autoimmune thyroiditis [3] and vitiligo [4]. As is the case with other autoimmune diseases, AA has associations with HLA, specifically DQB1 03 [5,6], HLA-B18 [7], and possibly HLA-A2 [8]. The LOD score for HLA-DR is 2.34 and that for DQB1 is 2.41, with a significant association with DQB1 302 [9]. Alopecia totalis/universalis has an association with DQB1 0301 [10,11]. This

0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

483


484


association is not found for patchy AA, suggesting a genetic difference between these two conditions. However, both patient groups have a positive association with HLA-DQ3 (DQB1 03). Hair loss in AA is associated with a perifollicular lymphocytic infiltrate made up primarily of CD4+ cells, along with a CD8+ intrafollicular infiltrate [12]. Evidence for immune activation includes expression of both HLA-DR and ICAM-1 on the follicular epithelium [13–15]. The role of antibodies in AA is uncertain. Circulating autoantibodies to follicular structures are found in patients with AA [16]. Similar antibodies are also reported in normal controls, and there is no consistent pattern of reactivity of antibodies to hair follicle structures. It is not possible to transfer AA by injection of patient IgG into human skin explants on nude mice [17].

II. LABORATORY ANIMALS A. Human Skin Graft/SCID Mouse as an Experimental System for Studying Inflammatory Conditions Severe combined immunodeficiency (SCID) mice are a useful model for the study of human inflammatory diseases [18,19]. SCID mice can accept grafts of both human skin and human leukocytes. Human immune reactions can be demonstrated in human skin grafted to SCID mice, and human skin graft/SCID mouse systems have been used to model inflammation [20], leukocyte homing [21,22], delayed hypersensitivity [12,23,24], skin allograft rejection [25,26], and autoimmune diseases of the skin. Human skin graft/SCID mouse systems have been extensively used in the study of psoriasis [27–33]. B. Characteristics of SCID Mice SCID mice are deficient in the ability to rearrange both T-cell receptor genes and immunoglobulin genes [34]. This results in a lack of T cells, B cells, and antibodies [35]. The mice exhibit lymphopenia, with almost empty splenic follicles and lymph nodes, as well as absent thymic cortex [36]. The function of antigen-presenting cells is normal [37]. The mutation responsible for the SCID phenotype involves a DNA-dependent protein kinase (Prkdc) that inhibits DNA recombination [38,39]. With age, SCID mice exhibit a low number of rearranged B and T cells (“leakiness”) [40,41]. Natural killer (NK) function is normal or elevated in SCID mice [42], and NK cells can mediate rejection of hematopoietic grafts, as well as clearance of infiltrating lymphocytes in skin grafts. SCID mice will not reject xenografts of human skin; however, engraftment of human bone marrow and lymphocytes are more complex [43]. Issues include the lack of human colonystimulating factors in the mouse, as well as the presence of NK activity. Several approaches have been taken to reduce NK activity, including development of NOD-SCID mice, and beige-SCID mice [44–46]. The choice of SCID mouse type and NK activity must be considered in data interpretation and experimental design. C. Immunodeficient Mice as Recipients for Human Scalp Grafting Bald (lesional) AA scalp grafted to immunodeficient nude mice will regrow hair [47,48]. It is presumed that hair regrowth results from removal of the hair follicle from the host immune system. Hair also regrows when AA lesional scalp is grafted to SCID mice. Regrowth of hair is associated with loss of CD8+ T cells from the infiltrate, along with a decrease in infiltrating CD4+ cells, particularly at the hair bulb [49]. Grafting of normal human hair to SCID mice initially results in a dystrophic catagen, with subsequent regrowth of normal anagen follicles [50]. Human hair will grow continually on a SCID mouse for at least 1 year [51].


INDUCTION BY TRANSFER OF CELLULAR IMMUNITY

485

NUMBER OF HAIRS PER GRAFT (STD)

NOT INJECTED

PBMC

SCALP T-CELLS

SCALP TC-HOM

0

DAY 68 Figure 32.1

2

4

DAY 75

6

8

DAY 82

The grafts were injected intradermally with T cells as indicated on day 40. Number of hairs per graft (N = 18 grafts). On day 82 the difference between Scalp TC-Hom and other treatments was statistically significant (ANOVA, p < 0.001). TC-HOM, T cell cultured with hair follicle homogenate. (From Gilhar, A. et al., J. Clin. Invest., 101, 62, 1998, with permission.)

III. DISEASE INDUCTION A. The Role of T Cells We have developed a system for transfer of AA to human scalp grafts on SCID mice by injection of lesional T cells [52]. Lesional (bald) scalp is grafted to CB-17 Prkdcscid (SCID) mice (Charles River, UK). Two-mm punch biopsies are used for grafting, and the grafts are inserted through an incision in the skin into the subcutaneous tissue over the lateral thoracic cage of each mouse, and covered with a standard band-aid dressing. Additional scalp biopsies are obtained for isolation of lymphocytes by collagenase treatment and mincing. These lymphocytes are cultured with irradiated autologous peripheral blood monocytes as antigen presenting cells, and IL-2. Homogenized hair follicles are added as a source of potential autoantigen. Forty days after grafting, the hair starts to regrow and the cultured autologous T cells are injected into the grafts, inducing loss of hair, along with morphological changes of AA. The number of hairs per graft on days 75 and 82 was significantly (ANOVA, p < 0.05) lower in grafts injected with T cells incubated with hair follicle homogenate than in control groups (Figure 32.1). Grafts injected with these T cells also had a significant reduction in hairs per graft on day 82 relative to day 68 (ANOVA, p < 0.05). B. The Role of Pretransfer Activation with Follicular Antigen Activation of lesional T cells to induce hair loss is specific for T cells cultured with hair follicle autoantigen. Control lesional and peripheral-blood T-cell cultures activated with PHA, or IL-2 do not induce hair loss. Lesional T cells cultured with homogenate of nonfollicular scalp also failed to induce hair loss [53]. The requirement for hair follicle homogenate to active lesional T cells


486


suggests that the pathogenic T cells are specific for a follicular autoantigens. Inability to induce hair loss with activated peripheral blood T cells may reflect a low proportion of pathogenic T cells in the peripheral blood. The data suggest that the proportion of pathogenic T cells in lesional skin is also very low, and requires antigen specific stimulation to obtain sufficient amplification of cell numbers to transfer hair loss. These data suggest that AA is an autoimmune condition mediated by T cells activated by a follicular antigen. C. The Roles of CD4+ and CD8+ T-Cell Subsets We next addressed the role of CD4+ and CD8+ T cells in the immunopathogenesis of AA, using the human scalp graft/SCID mouse transfer model [54]. Lesional scalp T cells were cultured with hair follicle homogenate, autologous antigen-presenting cells, and IL-2 as described above. Every 5 days the T cells were restimulated with feeders, antigen, and IL-2 for a total of culture of 30 days. After 30 days of culture, the T cells were separated into CD4+ and CD8+ cells by selection with magnetic beads. These T cells were injected into autologous lesional scalp grafts on SCID mice. Three treatment groups of transferred T cells included: CD4+ cells, CD8+ cells, and mixed CD4+ and CD8+ T cells. Experiments were repeated using both negative and positive selection of CD4+ and CD8+ T cells. In five experiments, the injection of unseparated T cells or mixed CD4+ and CD8+ T cells induced reproducible significant hair loss (ANOVA, p < 0.01). It was not possible to induce hair loss with injection of separated CD4+ or CD8+ T cells. This indicates that optimal hair loss requires cooperation between CD4+ and CD8+ T cells. Since the T cells were activated in vitro prior to injection, it may be expected that CD8+ effector cells would induce hair loss without the need for CD4+ help. The finding that both CD4+ and CD8+ T cells are required for optimal hair loss suggests that both T-cell subsets have a role in the induction of pathology. CD4+ T cells are generally more effective at cytokine production. Interferon-g produced by CD4+ cells may facilitate the effects of CD8+ T cells by inducing HLA-A, B, and C, as well as ICAM-1 (CD54) on follicular epithelium. There is additional support for an effector role for CD8+ T cells in the pathogenesis of AA. The intrafollicular T-cell infiltrate is predominantly composed of CD8+ T cells [55]. Intrafollicular T cells are cytotoxic and possess both the Fas/Fas ligand and granzyme B cytotoxic mechanisms [56]. In contrast the perifollicular infiltrate is composed primarily of CD4+ T cells. Depletion of either CD8+ T cells [57] or CD4+ T cells [58] can reverse AA in the Dundee experimental bald rat, indicating a synergy or cooperation between CD8+ and CD4+ T cells. One of the features of AA is aberrant expression of HLA-A, B, and C on the follicular epithelium of the hair bulb [59]. Paus et al. [60] have hypothesized that this expression of class I MHC allows an autoaggressive response by CD8+ T cells. The authors suggested that the CD8+ cells induce MHC class II on affected hair follicles, resulting in a second wave of CD4+ cells. We have previously reported CD4+ autoreactive T cells in the AA lesions [61]. D. The Role of Melanocyte-Derived Antigen It has been hypothesized that the autoantigen of AA is melanocyte derived [60]. The initial basis for this is the clinical observation that with disease activity, pigmented hairs are lost preferentially to nonpigmented (e.g., white) hairs. Furthermore, with regrowth there is a tendency for the initial regrowing hairs to be white. Melanocytes are a significant component of the hair bulb, which is the site of immunological attack. There is also an association of AA with vitiligo [62,63], and melanocytes of the hair bulb show both histological and ultrastructural abnormalities in AA [64]. Mice immunized against melanocyte-associated (e.g., melanoma) antigens can exhibit alopecia [65]. Since CD8+ T cells have a role in AA, it is logical to search for melanocyte antigens recognized by CD8+ T cells in association with HLA-A, B, and C molecules. Many such T-cell epitopes have been



487

identified in the search for melanoma-associated antigens, with particular attention paid to peptides presented by HLA-A2, which is present in approximately half the population [66]. HLA-A2 may also be associated with AA [67]. These melanocyte-associated peptides are derived from melanosomeassociated proteins such as gp100, MART-1/Melan-A, melanocortin 1 receptor (MC1R), or tyrosinase, and are present in both normal melanocytes and melanomas. MAGE antigens tend to be preferentially expressed on melanoma cells, and are less likely to function as autoantigens for AA. The following peptides were commercially synthesized (Chiron Technologies, Raleigh, NC) with free amino- and carboxylic-acid termini: Gp100/G9-154, KTWGQYWQV [68–70]; Gp100/G9-209, ITDQVPFSV [68–70]; Gp100/G9-280, YLEPGPVTA [68–70]; MC1R 291, AIIDPLIYA [71]; MC1R 244, TILLGIFFL [71]; MC1R 283, FLALIICNA [71]; MART-1 (amino acid sequence No. 27–35), AAGIGILTV [72,73]; and tyrosinase, AFLPWHRFL [74]. The goal of this study was to test the hypothesis that melanocyte-associated antigens can function as autoantigens to induce hair loss in AA. For this purpose, six HLA-A2 positive patients with AA were selected for studies of HLA-A2-restricted, melanocyte peptide epitopes. Patients exhibited either alopecia totalis, or severe AA. Severe AA is defined as large areas of alopecia with small residual areas of hair. These patients would be categorized as S4 (76% to 99% hair loss) by the AA investigational assessment guidelines [75]. Scalp biopsies were grafted to CB-17 Prkdcscid (SCID) mice (Charles River, UK) as described above, and additional scalp biopsies were obtained as a source of autologous lesional T cells. Scalp T cells were cultured with IL-2, autologous antigen-presenting cells, and either hair follicle homogenate (positive control) or melanocyte T-cell epitopes. The cells were then injected into autologous scalp explants on SCID mice, and the number of hairs per graft was measured. Melanoma peptide-activated T cells were able to significantly (ANOVA, p < 0.001) reduce the numbers of hair regrowing [76]. T cells incubated with follicular homogenate as a source of autoantigen resulted in a similar significant reduction in hair numbers. For all six donors, no hairs were present at day 90 in any of the three grafts per donor from grafts injected with T cells activated by either melanocyte peptides or hair follicle homogenate. There was no reduction of hair in grafts injected with control T cells cultured with IL-2 and antigen-presenting cells alone. As was observed following activation of T cells with the follicular homogentate, melanocyte peptide-activated T cells induced follicular expression of ICAM-1, HLA-DR, and HLA-A, B, and C. This demonstrates that melanocyte peptide epitopes are capable of activating T cells to induce hair loss in AA. Biopsies were taken from the mice in the above experiment after 90 days and analyzed both by histology and immunohistochemistry. Noninjected grafts showed growth of normal terminal anagen hairs. In contrast, injected grafts showed dystrophic hair follicles with a dense infiltrate of CD4+ T cells, as well as an intrafollicular infiltrate of CD8+ T cells. In contrast to noninjected grafts, the follicular epithelium of the injected grafts expressed HLA-A, B, and C; HLA-DR; and ICAM-1. These are known features of active AA. The ability of melanocyte-associated peptides to activate T cells to transfer AA indicates that melanocyte peptides are capable of functioning as autoantigens in AA. Multiple peptides were recognized in this small panel of six HLA-A2 positive patients, suggesting that there is a wide array of melanocyte autoantigen peptides. This work demonstrates the power of the human skin graft/SCID mouse system not only to delineate mechanisms of pathogenesis, but to identify autoantigens.

IV. THE DISEASE ASSESSMENT A. Clinical Manifestation Clinical assessment for the hair loss phenotype takes place by counting the number of hairs regrown per human scalp skin transplanted onto the SCID mice as described in Section III.


488

Figure 32.2


Histology of alopecia areata scalp explants prior to grafting, and on day 82. (A) Alopecia areata biopsy prior to grafting (¥20), (B) Noninjected graft day 82, horizontal section with mouse skin on upper part. Cross-section demonstrates ten human terminal hairs in anagen (¥20). (C) Graft injected with scalp T cells, with normal anagen hair follicles (¥32). (D) Graft injected with scalp T cells cultured with follicular homogenate. Hair follicles are surrounded by a dense lymphocytic infiltration (¥20). Mouse skin present at upper left and right. (From Gilhar, A. et al., J. Clin. Invest., 101, 62, 1998, with permission.)



489

B. Histopathological and Immunopathological Examination The histopathological examination of hair-loss, human scalp skin grafted onto SCID mice reveals evidence of AA in autologous, follicular antigen-activated, lesional T-cell–injected grafts: a dense infiltrate of mononuclear cells and follicular dystrophy with matrix and hair bulb degeneration (Figure 32.2). Few anagen follicles were present but only at higher levels of the dermis, and no terminal hairs were noted. In contrast, grafts injected with PBMC or scalp T cells cultured without hair follicle homogenate revealed normal terminal hairs without follicular damage (Figure 32.2). Other features of AA that are reproduced include expression of ICAM-1, HLA-DR, and HLA-A, B, and C by follicular epithelium.

V. LESSONS LEARNED First, we learned that immunodeficient mice, particularly SCID mice, are suitable recipients for human scalp transplantation. This suitability will provide available tools for other experimental inflammatory skin diseases, such as passive transfer of autoantibodies in autoimmune blistering skin diseases and psoriasis. Second, the ability to transfer of AA hair loss by injection of autologous lesional T cells into scalp grafts on SCID mice highly indicates that T cells are responsible for the induction of hair loss in this model. Furthermore, we learned that transfer of hair loss requires activation with autoantigen, specifically follicular antigen. In addition, we found that induction of hair loss requires cooperation between CD4+ and CD8+ T cells. Lastly, we learned that melanocyteassociated antigens in peptide form can serve as autoantigens for activation of lesional T cells to transfer AA. Thus, this model provides investigators with a tool not only in determining the immune cell types responsible for AA, but also the antigenic epitopes of the autoreactive T cells.

REFERENCES 1. Kiesch, N. et al., Pulse steroid therapy for children’s severe alopecia areata? Dermatology, 194, 395, 1997. 2. Micali, G. et al., Treatment of alopecia areata with squaric acid dibutylester, Int. J. Dermatol., 35, 52, 1996. 3. Milgraum, S.S. et al., Alopecia areata, endocrine function, and autoantibodies in patients 16 years of age or younger, J. Am. Acad. Dermatol., 17, 57, 1987. 4. Shellow, W.V., Edwards, J.E., and Koo, J.Y., Profile of alopecia areata: a questionnaire analysis of patient and family, Int. J. Dermatol., 31, 186, 1992. 5. Welsh, E.A. et al., Human leukocyte antigen-DQB1 03 alleles are associated with alopecia areata, J. Invest. Dermatol., 103, 758, 1994. 6. Morling, N. et al., DNA polymorphism of HLA class II genes in alopecia areata, Dis. Markers, 9, 35, 1991. 7. Hacham-Zadeh, S. et al., HLA and alopecia areata in Jerusalem, Tissue Antigens, 18, 71, 1981. 8. Hordinsky, M.K. et al., Familial alopecia areata. HLA antigens and autoantibody formation in an American family, Arch. Dermatol., 120, 464, 1984. 9. de Andrade, M. et al., Alopecia areata in families: association with the HLA locus, J. Invest. Dermatol. Symp. Proc., 4, 220, 1999. 10. Colombe, B.W. et al., HLA class II antigen associations help to define two types of alopecia areata, J. Amer. Acad. Dermatol., 33, 757, 1995. 11. Colombe, B.W., Lou, C.D., and Price, V.H., The genetic basis of alopecia areata: HLA associations with patchy alopecia areata versus alopecia totalis and alopecia universalis, J. Invest. Dermatol. Symp. Proc., 4, 216, 1999. 12. Todes-Taylor, N. et al., T cell subpopulations in alopecia areata, J. Am. Acad. Dermatol., 11, 216, 1984.


490


13. Messenger, A.G. and Bleehen, S.S., Expression of HLA-DR by anagen hair follicles in alopecia areata, J. Invest. Dermatol., 85, 569, 1985. 14. Khoury, E.L., Price, V.H., and Greenspan, J.S., HLA-DR expression by hair follicle keratinocytes in alopecia areata: evidence that it is secondary to the lymphoid infiltration, J. Invest. Dermatol., 90, 193, 1988. 15. Mcdonagh, A.J. et al., HLA and ICAM-1 expression in alopecia areata in vivo and in vitro: the role of cytokines, Br. J. Dermatol., 129, 250, 1993. 16. Tobin, D.J. et al., Hair follicle structures targeted by antibodies in patients with alopecia areata, Arch. Dermatol., 133, 57, 1997. 17. Gilhar, A. et al., Failure of passive transfer of serum from patients with alopecia areata and alopecia universalis to inhibit hair growth in transplants of human scalp skin grafted on to nude mice, Br. J. Dermatol., 126, 166, 1992. 18. Bosma, M.J. and Carroll, A.M., The SCID mouse mutant: definition, characterization, and potential uses, Annu. Rev. Immunol., 9, 323, 1991. 19. Boehncke, W.H., The SCID-hu xenogeneic transplantation model: complex but telling, Arch. Dermatol. Res., 291, 367, 1999. 20. Taub, D.D. et al., Chemokine-induced human lymphocyte infiltration and engraftment in huPBL-SCID mice, Methods Enzymol., 287, 265, 1997. 21. Kunstfeld, R. et al., HECA-452+ T cells migrate through superficial vascular plexus but not through deep vascular plexus endothelium, J. Invest. Dermatol., 108, 343, 1997. 22. Blades, M.C. et al., Stromal cell-derived factor 1 (CXCL12) induces human cell migration into human lymph nodes transplanted into SCID mice, J. Immunol., 168, 4308, 2002. 23. Tsicopoulos, A. et al., Tuberculin-induced delayed-type hypersensitivity reaction in a model of huPBMC-SCID mice grafted with autologous skin, Am. J. Pathol., 152, 1681, 1998. 24. Rosenblatt-Velin, N. et al., Transformed and nontransformed human T lymphocytes migrate to skin in a chimeric human skin/SCID mouse model, J. Invest. Dermatol., 109, 744, 1997. 25. Briscoe, D.M. et al., The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID mouse model of skin rejection, Transplantation, 67, 1590, 1999. 26. Rayner, D., Nelson, R., and Murray, A.G., Noncytolytic human lymphocytes injure dermal microvessels in the huPBL-SCID skin graft model, Hum. Immunol., 62, 598, 2001. 27. Gilhar, A. et al., T-lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice, J. Invest. Dermatol., 109, 283, 1997. 28. Boehncke, W.H. et al., Pulling the trigger on psoriasis, Nature, 379, 777, 1996. 29. Boehncke, W.H. et al., Induction of psoriasiform inflammation by a bacterial superantigen in the SCID-hu xenogeneic transplantation model, J. Cutan. Pathol., 24, 1, 1997. 30. Wrone-Smith, T. and Nickoloff, B.J., Dermal injection of immunocytes induces psoriasis, J. Clin. Invest., 98, 1878, 1996. 31. Nickoloff, B.J., Is psoriasis a T-cell disease? Controversies in experimental dermatology, Exp. Dermatol., 9, 359, 2000. 32. Nickoloff, B.J. et al., Characterization of a T cell line bearing natural killer receptors and capable of creating psoriasis in a SCID mouse model system, J. Dermatol. Sci., 24, 212, 2000. 33. Gilhar, A. et al., Psoriasis is mediated by a cutaneous defect triggered by activated immunocytes: induction of psoriasis by cells with NK receptors, J. Invest. Dermatol., 119, 384, 2002. 34. Lieber, M.R. et al., The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination, Cell, 55, 7, 1988. 35. Bosma, G.C., Custer, R.P., and Bosma, M.J., A severe combined immunodeficiency mutation in the mouse, Nature, 301, 527, 1983. 36. Custer, R.P., Bosma, G.C., and Bosma, M.J., Severe combined immunodeficiency (SCID) in the mouse. Pathology, reconstitution, neoplasms, Am. J. Pathol., 120, 464, 1985. 37. Czitrom, A.A. et al., The function of antigen-presenting cells in mice with severe combined immunodeficiency, J. Immunol., 134, 2276, 1985. 38. Kirchgessner, C.U. et al., DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect, Science, 267, 1178, 1995. 39. Blunt, T. et al., Identification of a nonsense mutation in the corboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse, Proc. Natl. Acad. Sci. U. S. A., 93, 10285, 1996.



491

40. Carroll, A.M. et al., T cell leakiness in scid mice, Curr. Top. Microbiol. Immunol., 152, 117, 1989. 41. Carroll, A.M., Hardy, R.R., and Bosma, M.J., Occurrence of mature B (IgM+, B220+) and T (CD3+) lymphocytes in scid mice, J. Immunol., 143, 1087, 1989. 42. Dorshkind, K. et al., Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid), J. Immunol., 134, 3798, 1985. 43. Boehncke, W.H., The SCID-hu xenogeneic transplantation model: complex but telling, Arch. Dermatol. Res., 291, 367, 1999. 44. Shultz, L.D. et al., Multiple defects in innate and adoptive immunologic function in NOD/LtSz-scid mice, J. Immunol., 154, 180, 1995. 45. Takizawa, Y. et al., New immunodeficient (nude-scid, beige-scid) mice as excellent recipients of human skin grafts containing intraepidermal neoplasms, Arch. Dermatol. Res., 289, 213, 1997. 46. Greiner, D.L., Hesselton, R.A., and Shultz, L.D., SCID mouse models of human stem cell engraftment, Stem Cells, 16, 166, 1998. 47. Gilhar, A. and Krueger, G.G., Hair growth in scalp grafts from patients with alopecia areata and alopecia universalis grafted onto nude mice, Arch. Dermatol., 123, 44, 1987. 48. Gilhar, A. et al., Failure of passive transfer of serum from patients with alopecia areata and alopecia universalis to inhibit hair growth in transplants of human scalp skin grafted on to nude mice, Br. J. Dermatol., 126, 166, 1992. 49. Tsuboi, H. et al., Characterization of infiltrating T cells in human scalp explants from alopecia areata to SCID nude mice: possible role of the disappearance of CD8+ T lymphocytes in the process of hair regrowth, J. Dermatol., 26, 797, 1999. 50. Hashimoto, T. et al., Histologic and cell kinetic studies of hair loss and subsequent recovery process of human scalp hair follicles grafted onto severe combined immunodeficient mice, J. Invest. Dermatol., 115, 200, 2000. 51. Kyoizumi, S. et al., Radiation sensitivity of human hair follicles in SCID-hu mice, Radiat. Res., 149, 11, 1998. 52. Gilhar, A. et al., Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice, J. Clin. Invest., 101, 62, 1998. 53. Gilhar, A. et al., Alopecia areata is a T-lymphocyte mediated autoimmune disease: lesional human T-lymphocytes transfer alopecia areata to human skin grafts on SCID mice, J. Investig. Dermatol. Symp. Proc., 4, 207, 1999. 54. Gilhar, A. et al., Alopecia areata is mediated by cooperation between CD4+ and CD8+ T-lymphocytes: Transfer to human scalp explants on Prkdcscid mice, Arch. Dermatol., 138, 916, 2002. 55. Todes-Taylor, N. et al., T cell subpopulations in alopecia areata, J. Am. Acad. Dermatol., 11, 216, 1984. 56. Bodemer, C. et al., Role of cytotoxic T cells in chronic alopecia areata, J. Invest. Dermatol., 114, 112, 2000. 57. McElwee, K.J., Spiers, E.M., and Oliver, R.F., In vivo depletion of CD8+ T-cells restores hair growth in the DEBR model for alopecia areata, Br. J. Dermatol., 135, 211, 1996. 58. McElwee, K.J., Spiers, E.M., and Oliver, R.F., Partial restoration of hair growth in the DEBR model for alopecia areata after in vivo depletion of CD4+ T cells, Br. J. Dermatol., 140, 432, 1999. 59. Brocker, E.B et al., Abnormal expression of class I and class II major histocompatibility antigens in alopecia areata: modulation by topical immunotherapy, J. Invest. Dermatol., 88, 564, 1987. 60. Paus, R., Slominski, A., and Czarnetzki, B.M., Is alopecia areata an autoimmune-response against melanogenesis-related proteins, exposed by abnormal MHC class I expression in the anagen hair bulb? Yale J. Biol. Med., 66, 541, 1994. 61. Kalish, R.S., Johnson, K.L., and Hordinsky, M.K., Autoreactive T-cells are variably enriched relative to peripheral blood in the scalp lesions of alopecia areata, Arch. Dermatol., 128, 1072, 1992. 62. Shong, Y.K. and Kim, J. A., Vitiligo in autoimmune thyroid disease, Thyroidology, 3, 89, 1991. 63. Shellow, W.V., Edwards, J. E., and Koo, J. Y., Profile of alopecia areata: a questionnaire analysis of patient and family, Int. J. Dermatol., 31, 186, 1992. 64. Tobin, D.J., Fenton, D.A., and Kendall, M.D., Ultrastructural observations on the hair bulb melanocytes and melanosomes in acute alopecia areata, J. Invest. Dermatol., 94, 803, 1990. 65. Becker, J.C. et al., Lymphocyte-mediated alopecia in C57Bl/6 mice following successful immunotherapy for melanoma, J. Invest. Dermatol., 107, 627, 1996.


492


66. UCLA Tissue Typing Laboratory, Histocompatibility Testing 1980, Tissue Typing Laboratory, University of California-Los Angeles, 1980 (antigen frequencies). 67. Hordinsky, M.K. et al., Familial alopecia areata. HLA antigens and autoantibody formation in an American family, Arch. Dermatol., 120, 464, 1984. 68. Parkhurst, M.R. et al., Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A 0201-binding residues, J. Immunol., 157, 2539, 1996. 69. Salgaller, M.L. et al., Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides, Cancer Res., 56, 4749, 1996. 70. Bakker, A.B. et al., Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line, Int. J. Cancer, 62, 97, 1995. 71. Salazar-Onfray, F. et al., Synthetic peptides derived from the melanocyte-stimulating hormone receptor MC1R can stimulate HLA-A2-restricted cytotoxic T lymphocytes that recognize naturally processed peptides on human melanoma cells, Cancer Res., 57, 4348, 1997. 72. Bettinotti, M.P. et al., Stringent allele/epitope requirements for MART-1/Melan A immunodominance: implications for peptide-based immunotherapy, J. Immunol., 161, 877, 1998. 73. Rivoltini, L. et al., Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1, J. Immunol., 154, 2257, 1995. 74. Kang, X. et al., Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumorinfiltrating lymphocytes, J. Immunol., 155, 1343, 1995. 75. Olsen, E. et al., Alopecia areata investigational assessment guidelines, J. Am. Acad. Dermatol., 40, 242, 1999. 76. Gilhar, A. et al., Melanocyte-associated T cell epitopes can function as autoantigens for transfer of alopecia areata to human scalp explants on Prkdc(scid) mice, J. Invest. Dermatol., 117, 1357, 2001.


SECTION

I

Scleroderma



CHAPTER

33

Spontaneous Mouse Models of Systemic Scleroderma Paul J. Christner and Sergio A. Jimenez

CONTENTS I. History ................................................................................................................................495 II. Laboratory Animals............................................................................................................496 III. Course and Assessment of Disease ...................................................................................496 A. Tight Skin 1 (Tsk1) Mouse........................................................................................496 1. Cutaneous Alterations..........................................................................................496 2. Internal Organ Involvement.................................................................................497 3. Humoral Immune Abnormalities.........................................................................499 4. Cellular Immune Abnormalities ..........................................................................501 5. Abnormalities in Connective Tissue Metabolism ...............................................502 6. The Molecular Defect in Tsk1/+ Mice................................................................503 7. A Mutation in the Fibrillin-1 Gene: the Genetic Defect in Tsk1 Mice .............504 B. The Tsk2 — A Novel Mutation Resembling SSc.....................................................507 1. Immunology.........................................................................................................507 2. Genetics................................................................................................................507 3. Biochemical Studies ............................................................................................509 C. Transgenic Animal Models Exhibiting Some Features of SSc ................................510 IV. Conclusion..........................................................................................................................511 References ......................................................................................................................................512

I. HISTORY Animal models of systemic connective tissue diseases have provided valuable insights into the causative mechanisms and the pathogenesis of these diseases, and have allowed the testing of potentially useful therapeutic interventions. Several spontaneous and induced animal models of systemic sclerosis or scleroderma (SSc) have been described. However, spontaneous murine animal models for the disease have been extensively studied because of the large number of inbred mouse strains available and the large body of genetic information for this species. Although these animal models do not reproduce all the clinical and pathological manifestations of human SSc, they display 0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

495


496


some of the most important pathological alterations of this disorder and the prudent interpretation of the results obtained from their study has provided substantial and valuable information about the pathogenesis of SSc. Here, two spontaneous murine models of SSc will be reviewed. In particular, the review will focus on the pathological phenotype as the result of a genomic mutation that is transmitted genetically as a stable trait, emphasizing the similarities and differences with the human disease. In addition, other models that result from either genetic alterations induced by gene transfer in transgenic animals or by gene inactivation in knockout mice will be described. Further study of these murine models is likely to allow identification of the genes that participate in the pathogenesis of SSc, and may help to elucidate the alterations in the molecular pathway(s) that regulate a variety of physiological processes leading to tissue fibrosis, the hallmark of this disease. These animal models will also be of substantial value to test potential therapeutic agents that may eventually be employed for the treatment of SSc.

II. LABORATORY ANIMALS There are two animal models of SSc that are transmitted genetically in a typical Mendelian dominant pattern. These are the tight skin 1 (Tsk1) and tight skin 2 (Tsk2) mice. It is currently believed that each of these phenotypes is the result of a mutation in a single gene. Genetically transmitted disease models have the advantage that their phenotype is stable and the causative gene has been (Tsk1) or is in the process of being identified (Tsk2). Therefore, each molecular event in the pathway leading from the genetic mutation to the disease phenotype is likely to be elucidated. Identification of the molecular alterations in these murine models may provide valuable clues to understanding the molecular pathogenesis of SSc.

III. COURSE AND ASSESSMENT OF DISEASE A. Tight Skin 1 (Tsk1) Mouse The Tsk mouse is a spontaneous dominant mutation that occurred in the inbred B10.D2 (58N)/Sn strain. Recently, a different mutation causing a tight skin phenotype has been described. Therefore, the original Tsk mutation is now referred to as Tsk1. The Tsk1 mutation was identified at the Jackson Laboratories by Helen Bunker and was reported in detail by Green et al. [1]. The most striking feature of these mice is the presence of thickened and tight skin which is firmly bound to the subcutaneous and deep muscular tissues. The Tsk1 mutation is lethal and homozygous embryos (Tsk1/Tsk1) degenerate and die in utero at 8 to 10 days of gestation. The heterozygous animals (Tsk1/+) display cutaneous and visceral alterations that closely resemble those present in patients with SSc, as well as biochemical and molecular abnormalities that mimic the fibrotic connective tissue alterations characteristic of the disease [2,3]. 1. Cutaneous Alterations Skin tightness in Tsk1/+ mice is readily detected at 7 days of age and is manifested by difficulty in gathering a fold of skin in the interscapular region [1–3]. At age 2 months, the animals exhibit a hunched posture with a pronounced hump and prominent skin thickening (Figure 33.1). Studies by Menton and Hess [4] and Menton et al. [5] confirmed the initial report of dermal thickening in Tsk1/+ mice and demonstrated decreased pliability and increased skin stiffness. Increased skin thickness was also found by Osborn et al. [6] and Jimenez et al. [2]. Light microscopy studies of Tsk1/+ mice skin showed dermal thickening and replacement of adipose tissue by collagen (Figure 33.2). Electron microscopy [4] revealed changes in the connective tissue architecture of


SPONTANEOUS MOUSE MODELS OF SYSTEMIC SCLERODERMA

Figure 33.1

497

Photograph of a Tsk1/+ mouse that carries one mutated and one normal Fbn1 gene. Note the hunched posture indicative of the excessive accumulation of connective tissue in the interscapular region.

the dermis, including irregular spatial organization of collagen fibrils that were of small diameter and tightly packed. Furthermore, abundant deposition of fine microfibrillar material in the deep dermis was noted (Figure 33.3). The thickened peritendinous fascia resulted in atrophy of tail and ankle tendons. Extensive collagen replacement of the subcutaneous fatty tissue was observed in the dorsal, lateral, and ventral thoracic and abdominal regions, and in the forelimbs and hindlimbs. Prominent changes were also noted around the mammary glands, the brown fat of the scapular regions, and the ventral side of the sternum. Connective tissue surrounding the kidneys, adrenal glands, and pancreas was also substantially increased [2,3]. 2. Internal Organ Involvement The most prominent visceral changes in Tsk1/+ mice occur in the lungs and heart [1]. Lung abnormalities are characterized by marked distention that is present at birth. Histologically, the alterations in the lung resemble human emphysema, with little fibrosis (Figure 33.4). Szapiel et al. [7] noted inflammatory cell accumulation in the interstitium and alveolar spaces. These alveolar spaces were dilated with thin, disrupted walls and subpleural cysts and bullae. Rossi et al. [8] found increased numbers of neutrophils in bronchoalveolar lavage fluids of Tsk1/+ lungs in the absence of infection. The T- and B-cell levels were normal in the lungs of these mice. O’Donnell et al. [9] investigated the elastin and collagen matrices in the Tsk1/+ mouse lung by scanning electron microscopy and found significant elastin destruction but no collagen destruction. Keil et al. [10] examined by scanning electron microscopy the lungs of Tsk1/+ in comparison to those of pallid and beige mutants and C57BL/6 (controls) at 1, 12, and 24 months of age. The parenchyma of Tsk1/+ mice lungs appeared distorted at all ages with enlargement of alveolar ducts and sacs and alveoli with a large number of pores. These changes increased with age. Only the Tsk1/+ mice had increased numbers of alveolar pores throughout life compared to the controls. Although the other mutant mice studied also displayed alterations in lung architecture compared to controls, the authors concluded that the pathogenetic mechanism leading to emphysema was unique to the Tsk1/+ mouse. Gardi et al. [11] examined collagen synthesis in Tsk1/+ lungs from birth to 12 months. They reported no differences at birth. However, by 2 months the collagen synthetic rate was markedly increased in Tsk1/+ lungs compared to controls as assessed by prolylhydroxylase activity and radiolabeled proline incorporation into collagen. At 6 and 12 months the increase was only slightly higher than the increase seen at 2 months, observations which the authors attributed to ongoing


498

Figure 33.2


Light microscopy of Masson’s trichrome-stained skin sections from 10-day-old normal (A) and Tsk1/+ (B) mice. Note the marked increase in dense, blue-staining connective tissue in the skin from Tsk1/+ mice compared with normal mice (original magnification ¥100).

parenchymal destruction. They also reported a relative increase in type I collagen in Tsk1/+ mice lungs over time. The cardiac enlargement reported by Green et al. [1] was confirmed by Osborn and Bauer [12]. Myocardial hypertrophy in these mice did not appear to be due to increased blood pressure or to valvular/arterial lesions. Increased cardiac weight and electron-microscopic evidence of moderately increased myocardial collagen deposits were found. No inflammatory cells were observed histologically. Osborn et al. [13] performed biochemical analysis of the collagen content of 1- and 12month-old Tsk1/+ hearts compared to normal controls. They reported that type I collagen was markedly increased with a proportional decrease in types III and V collagen in Tsk1/+ myocardium. Bashey et al. [14] reported that hearts from Tsk1/+ mice had up to two-fold greater protein and collagen biosynthesis and 2.5-fold greater type VI collagen content compared to controls. Gardi et al. [15] investigated cardiac collagen synthesis and content during the development of right ventricular hypertrophy (RVH) in Tsk1/+ mice. At 3 months both parameters were markedly increased. At 8 months the synthetic rate had returned to control values but the content was still elevated and there was no change in collagen types compared to controls. At 16 months the collagen synthesis and content in the Tsk1/+ hearts was comparable to control values; however, there was a shift to more type I collagen in the Tsk1/+ mouse hearts compared to controls. Martorana et al. [16] reported that RVH first began to develop between 8 and 16 months and progressed up to 24 months of age in the Tsk1/+ mouse. No pulmonary vascular changes were detected in the Tsk1/+ mice. Chapman and Eghbali [17] examined collagen gene expression in Tsk1/+ myocardium and reported elevated collagen types I, III, and IV mRNA levels. Using immunofluorescence light microscopy and



A

499

B

D C

E Figure 33.3

F

Ultrastructural study of normal and Tsk1/+ mice skin. Normal mouse (A, C, and E); Tsk1/+ mouse (B, D, and F). Low-power views (5000¥) showing basal keratinocytes, the dermo-epidermal junction as represented by the lamina densa (open arrows), and the upper dermis (A and B). In both mice, the dermis is dominated by collagen bundles. Microfibrillar bundles and elastic fibers are indicated by closed arrows. Larger microfibrillar bundles with abundant microfibrils are present, but little elastin (B). Elastic fibers and microfibrillar bundles in high-magnification views appear less well defined in Tsk1/+ skin (D and F) than in normal skin (C and E). In particular, the striation pattern of microfibrils (arrowheads) seen with the normal animals (C and E) was less apparent in the Tsk1/+ mice skin. (From Kielty, C.M. et al., J. Cell Biol., 140, 1159, 1998, with permission.)

monospecific antibodies, they reported the presence of collagen type I fibers that were thicker and denser in perivascular areas of the Tsk1/+ heart compared to the normal heart. No abnormal accumulation of type III collagen was observed. 3. Humoral Immune Abnormalities Mononuclear inflammatory cell infiltration of affected organs is not seen in the Tsk1/+ mouse. However, certain immunological abnormalities are present including the presence of antinuclear antibodies (ANA), which have been detected in about 50% of Tsk1/+ mice at 8 months of age. Prior to this age all animals tested were negative for ANA. The serum autoantibodies did not recognize dsDNA, SS-A/Ro, SS-B/La, Sm, RNP, or Scl-70 specificities when tested by immunodiffusion. However, antibodies that recognize the Scl-70 marker antigen (topoisomerase 1) were demonstrated in supernatants from hybridomas established from Tsk1/+ mice splenocytes [18]. These antibodies have been extensively characterized by Muryoi et al. [19] and Shibata et al. [20]. Shibata et al. [21] demonstrated the presence of autoantibodies to the nuclear protein RNA polymerase I, which is considered to be, like the presence of Scl-70 autoantibodies, a specific marker for SSc. They showed that the Tsk1/+ mouse developed autoantibodies directed against the 190-kDa


500


A

B Figure 33.4

Light photomicrograph of a lung section from normal (A) and Tsk1/+ (B) mice. The sections were stained with hematoxylin and eosin. Note the marked distortion of lung parenchyma with enlarged and fragmented alveoli in the section from the Tsk1/+ mouse (original magnification ¥200).

subunit. Autoantibodies to this same subunit are present in SSc patients. Shibata et al. [21] further determined that the autoantibodies against RNA polymerase I were encoded by the VH J558 gene family. Muryoi et al. [20] have reported the presence of serum anticellular antibodies in all Tsk1/+ mice studied and in none of the controls. The authors then produced a large panel of hybridomas in order to determine the frequency and specificity of clones producing antibodies with anticellular and antinuclear activity. They found that the frequency of hybridomas producing either type of autoantibody was higher in Tsk1/+ mice than in C57BL/6-pa/pa mice. However, the significance of these findings is not clear, because the frequency of clones producing anticellular antibodies in these two strains (Tsk1/+ and C57BL/6-pa/pa) was found to be lower than that in other strains. By testing the specificity of anticellular autoantibodies to other self- and foreign antigens by radioimmunoassays, they concluded that only a small fraction of Tsk1/+ mouse anticellular antibodies were natural antibodies. In order to understand the molecular basis of the generation of the autoantibody repertoire in the Tsk1/+ mouse, Muryoi et al. [20] and Kasturi et al. [22] investigated the expression of V gene families and VH/VK pairing in Tsk/+ mouse autoantibodies. They found that in the case of Vk gene families, Vk4, Vk22, Vk23, and Vk28 were overrepresented, and that VHJ558 and VGAM were slightly overrepresented in Tsk1/+ mice, whereas VHQ52 and VH7183 were



501

underrepresented. The pairing of VH and Vk gene families was random. This work was confirmed by Kasturi et al. [23] who later reported that 14 of 18 autoantibodies in Tsk1/+ mice shared a conserved heptapeptide sequence motif, YNEKFKG, in the second complementarity-determining region of heavy chains. They concluded that the usage of germ-line genes from diverse J558 genes bearing a common motif to encode autoantibodies suggests a regulatory role for this motif. Overall these studies indicated that the utilization of V gene families by Tsk1/+ mice for the production of anticellular antibodies differs from other murine strains. In other work, Muryoi et al. [24] reported that the B-cell clones producing autoantibodies in the Tsk1/+ mouse and in SSc patients share an interspecies cross-reactive idiotope, suggesting that the human and mouse repertoires are conserved during phylogeny, and are activated during the development of SSc. However, the significance of these extensive findings is still not clear, because the role of autoantibodies in the pathogenesis of SSc and in the Tsk1/+ mouse is not yet known. 4. Cellular Immune Abnormalities Alterations in cellular immunity have been examined in Tsk1/+ mice [25]. Responses to the Tand B-cell mitogens, concanavalin A and lipopolysaccharide, were found to be normal. The production of IL-2 was also found to be in the range of normal strains. However, a low, autologous mixed lymphocyte response was observed, as is typical of autoimmune strains of mice [26] and of human SSc [27]. The role of cellular immunity in the pathogenesis of tissue fibrosis in Tsk1/+ mice was examined by Phelps et al. [28]. These authors showed that infusion of bone marrow cells or T and B lymphocytes from Tsk1/+ mice into pallid mice led to tissue fibrosis, cellular infiltration, autoantibody production, and increased transcription of the a1(I) collagen gene. Several other studies have presented data indicating that participation of the immune system is required to cause the cutaneous fibrosis observed in the Tsk1/+ mice. Ong et al. reported the involvement of CD4+ T cells and that IL-4 was necessary for the development of fibrosis [29,30]. They showed that neutralizing antibodies to IL-4 prevented the development of dermal fibrosis in mice and suggested that Th2 cells and/or factors elaborated by a certain T-cell subset may play a key role in regulating dermal collagen content. They also reported that cutaneous fibrosis did not occur in Tsk1/+ mice with a null mutation for IL-4, and suggested that a specific usage of a portion of the T-cell repertoire, specifically the Vbeta8.2 gene segment, could prevent the development of fibrosis in Tsk 1/+ mice [29]. Kodera et al. [31] showed that embryos carrying the lethal Tsk1/Tsk1 genotype could be rescued by disrupting either one (+/-) or both (-/-) of the IL-4 genes. The IL4 deficient mice failed to develop cutaneous hyperplasia although they exhibited pulmonary emphysema. They also reported that IL-4 could stimulate the levels of TGF-b mRNA in fibroblasts and that the levels of TGF-b in the lungs of Tsk1/+, IL-4 (-/-) mice were lower than those in the lungs of Tsk1/+, IL-4(+/+) animals. McGaha et al. [32] reported that crossing the Tsk1/+ mouse to an IL-4 a receptor–deficient mouse prevented cutaneous fibrosis in the F1 generation, but did not prevent the emphysematous changes in the lungs. Tsuji-Yamada et al. [33] reported that injection of a plasmid that encodes for IL-12 into Tsk1/+ mice markedly decreased skin thickness, serum levels of antinuclear antibodies, and IL-4 production by spleen cells. The authors concluded that the expression of IL-12 prevented collagen accumulation and suppressed autoimmunity via improvement of the Th1/Th2 lymphocyte balance. In other studies, Wallace et al. [34] reported that CD4+ T cells were involved in the pathogenesis of skin fibrosis in Tsk1/+ mice. By breeding Tsk1/+ to CD4-deficient mutant mice, they showed that in the Tsk1/+ mice lacking CD4+ T cells, there was a marked reduction in skin fibrosis as well as decreased cellularity and only mild collagen disorganization compared to controls. In similar experiments with CD8-deficient Tsk1/+ mice they reported no change in fibrosis but significantly reduced levels of serum anti-topoisomerase 1 antibodies compared to CD4-deficient Tsk1/+ mice. Employing the same crossings, Hatakeyama et al. [35] reported a good correlation between the


502


levels of anti–topoisomerase 1 antibodies and the histological and biochemical alterations of the Tsk1 phenotype. Although the studies above suggest the involvement of inflammatory cells in the development of cutaneous fibrosis, other laboratories have reported contradictory results. Siracusa et al. [36], employing a cross between Tsk1/+ and Rag mice, reported that the tight skin phenotype of Tsk1/+ mice is not dependent on the presence of mature T or B lymphocytes. These results were confirmed by Dodig et al. [37]. In their investigations Tsk1/+ mice were mated to BALB/cByJSmn-Prkdcskid mice. Mice homozygous for the Prkcdskid mutation (skid/skid) lack mature T and B lymphocytes. Phenotypically tight F1 mice were back crossed to BALB/cByJSmn-Prkdcskid to produce N2 mice which were Tsk1/+, Prkcdskid/Prkcdskid. These tight skin mice were genotyped to ensure that they were homozygous for the skid mutation and assessed for antibody levels by ELISA. The results showed that skid/skid mice (T- and B-cell deficient) developed the tight skin phenotype. Kasturi et al. [38] and Saito et al. [39] also reported that the presence of mature B cells was not necessary for the development of cutaneous fibrosis in Tsk1/+ mice and that the fibrosis was independent of the presence of autoantibodies. They crossed Tsk1/+ mice with three immunocompromised mice strains: the JH-/-, which lacks mature B cells; the RAG2-/-, which lacks mature T and B cells; and the vit/vit, which is hyporesponsive in contact hypersensitivity. They found that mice which were JH-/-, Tsk1/+ still developed cutaneous fibrosis. The authors were unsuccessful in producing any Tsk1/+, RAG2-/- mice and could not, therefore, determine the role of T-cells in the development of cutaneous fibrosis. The authors did not describe the results obtained with Tsk1/+, vit/vit mice. Most recently, Oble and Teh [40] also reported that Tsk1/+ mice with a-receptor–deficient T cells showed no difference in skin thickness nor any obvious changes in architecture of the dermis. They concluded that the presence of CD4+ cells was not necessary for expression of the tight skin phenotype. Therefore, the putative role of inflammatory cells or their products in the pathogenesis of tissue fibrosis in Tsk1/+ mice has not been conclusively resolved to date. It has been suggested that mast cells may be important initiators of cutaneous fibrosis in SSc [41]. Walker et al. [42] examined this hypothesis in the Tsk1/+ model and found a twofold increase in mast cell numbers in Tsk1/+ skin compared to normal mouse skin up to 6 months of age. In these younger Tsk1/+ animals, the majority of mast cells were degranulated. By 15 months of age, the number of skin mast cells was comparable in Tsk1/+ and control groups and significantly reduced in both compared to younger animals. These observations suggest that mast cells may play an as yet undefined role in the progressive accumulation of connective tissue in the Tsk/+ mice. The increase in mast cells and their degranulation are similar to those seen in SSc [43], and Walker et al. [42] also showed that inhibition of mast cell degranulation by the administration of disodium cromoglycate significantly reduced the width of the subcutaneous fibrous layer in Tsk1/+ mice suggesting that mast cells and their products were key participants in the development of tissue fibrosis. Subsequent studies on the role of mast cells in tissue fibrosis have been controversial. Everett et al. [44] reported that mast cells were clearly not necessary for the development of fibrosis in the Tsk1/+ mouse; however, the presence of mast cells in a late stage of the fibrotic process correlated with a more pronounced fibrosis. They concluded that the Tsk1/+ skin lesions are a pleiotropic manifestation in which mast cells are involved/recruited by some as yet uncharacterized mechanism. 5. Abnormalities in Connective Tissue Metabolism The histological evidence suggesting an increase in skin collagen in the Tsk1/+ mouse has been confirmed by extensive biochemical studies [2,45]. The average total collagen content of skin from Tsk1/+ mice was 2.5-fold greater than that of normal mice skin. Since there is evidence that shifts in collagen type are associated with certain pathological conditions, this possibility was examined in the Tsk1/+ mutant. These studies failed to show qualitative differences in the collagens found in skin of Tsk1/+ and normal mice. These results are similar to those reported in biochemical



503

studies of affected skin from SSc patients showing increased collagen content but no alteration in the relative proportions of the various collagen types. Glycosaminoglycans (GAG) are the other major connective tissue component of skin. Ross et al. [46] and Dorner et al. [47] examined the GAG content of Tsk1/+ skin and found significant increases in total hexosamine and uronic acid for a given skin surface area. The mechanisms responsible for the excessive accumulation of extracellular matrix in Tsk1/+ skin were further examined in an organ culture system [2]. The results of these studies indicated that excessive biosynthesis was the primary mechanism of collagen accumulation in the Tsk1/+ tissues. No qualitative differences in the products synthesized by Tsk1/+ and normal skin organ cultures were found upon electrophoretic analysis [2,3]. Studies by Uitto [48] and Jimenez et al. [49] of collagen biosynthesis in organ cultures of SSc skin have shown similar findings. Further in vitro studies of collagen biosynthesis and its regulation employed dermal fibroblast cultures established from Tsk1/+ and normal littermate control mice [45]. Collagen synthesis as assessed by incorporation of 14C-proline and production of 14C-hydroxyproline was two-fold greater in the Tsk1/+ fibroblast cultures. All of the increase was in the highly soluble fraction secreted into the culture medium. These findings are similar to results of collagen and protein biosynthesis studies in cultures of SSc dermal fibroblasts [50–53]. Characterization of the newly synthesized proteins from Tsk1/+ and control fibroblast cultures showed quantitative but not qualitative differences [45]. This is similar to the findings of Uitto et al. [54] with SSc and normal human fibroblasts. An increase in fibronectin synthesis is also seen in cultures of Tsk1/+ fibroblasts similar to the increased fibronectin reported in SSc skin by Cooper et al. [55]. A study of the expression of three collagen genes employing hybridizations with a1(I), a2(I), and a1(III) procollagen cDNA showed a five-fold increase in steady-state mRNA levels for the corresponding transcripts in Tsk1/+ fibroblasts [45]. In other studies, elevated type VI collagen mRNA levels were also found in cultured Tsk1/+ fibroblasts [2]. These results are similar to the findings of parallel increases in types I and III procollagens and type VI collagen mRNAs in SSc fibroblasts [53,56]. Pablos et al. [57] used in situ RNA hybridization in skin sections from Tsk1/+ and control mice to examine the expression of TGF-b, procollagens a1(I) and a1(III), and collagen a2(VI). They found that all genes were under temporospatial regulation and exhibited characteristic patterns of expression during postnatal growth and development. TGF-b, was expressed only during rapid post-natal growth of the skin in parallel with high expression of a1(I), a1(III) and a2(VI) collagen genes. Fibroblasts expressing types I and III collagens were increased in Tsk1/+ fibrotic lesions. An abnormal pattern of collagen a2(VI) gene expression was observed only at later stages of fibrosis, suggesting a noncoordinate regulation of type VI collagen. They concluded that the fibrosis observed in Tsk1/+ mice was the result of a subpopulation of fibroblasts overexpressing type I and III collagens and that this overexpression did not appear to be mediated by TGF-b. Pablos et al. [58] examined Tsk1/+ and normal skin tissue sections stained with proliferating cell nuclear antigen as an indicator for cellular proliferation and found no significant differences between each group. They also found no difference in apoptosis using in situ end-labeling of fragmented DNA and nuclear staining with propidium iodide. Thus, neither process could account for the fibrotic phenotype seen in the Tsk1/+ mice and it was concluded that transcriptional activation of collagen genes was responsible for the increased accumulation of collagen. Sgonc et al. [59] later confirmed that apoptosis was not involved in the development of the Tsk1/+ phenotype. 6. The Molecular Defect in Tsk1/+ Mice To elucidate the mechanisms by which the Tsk1 mutation modulated the coordinate expression of several genes of the large family of extracellular matrix proteins, the cis acting elements responsible for increased transcription of the a1(I) procollagen gene in Tsk1/+ fibroblasts were examined. Transient transfections of a series of human a1(I) procollagen promoter reporter gene


504


constructs into Tsk1/+ and normal fibroblast cultures were performed [60]. The results showed that the transcription of the a(1) procollagen gene was increased by more than threefold in the fibroblasts derived from Tsk1/+ mice. The experiments also showed that the up-regulation of the a1(I) procollagen gene was due to the lack of the strong inhibitory influence of the regulatory sequence contained in the promoter between -675 and -804 bp. This lack of inhibition was shown to be caused by lower binding activity of nuclear extracts from fibroblasts of Tsk1/+ mice to oligonucleotides spanning the mapped regulatory sequence. Additionally, it was shown that Tsk1/+ nuclear extracts displayed decreased binding to a consensus AP-1 sequence. Philips et al. [60] postulated that a strong negative regulatory sequence contained within the -675 bp to -804 bp region of the a1(I) procollagen gene promoter binds AP-1 transcription factor and mediates inhibition of gene transcription in normal murine fibroblasts. The Tsk1/+ fibroblasts lack this inhibitory control, due to lower available amounts and/or decreased binding activity to this inhibitory sequence. Therefore, they display increased a1(I) procollagen gene expression. Other studies employing reporter transgenes harboring upstream fragments of the 5' flanking region of the mouse a2(I) procollagen gene introduced into Tsk1/+ mice confirmed the transcriptional activation of collagen genes in Tsk1/+ mice [61]. These studies demonstrated that the reporter transgenes displayed higher expression starting at age 1 week and their expression was further stimulated by TGF-b in cultured cells. An important observation was that the transcriptional activation of the mouse transgene involved also an upstream enhancer and suggested the participation of fibroblast-specific pathways. 7. A Mutation in the Fibrillin-1 Gene: the Genetic Defect in Tsk1 Mice Initial genetic studies employing visible markers mapped the Tsk1 mutation to mouse chromosome 2 [1]. More recently, Siracusa et al. [62,63] localized the gene to a 3.5-cM region closely linked to the b2 macroglobulin gene. These observations were confirmed by Everett et al. [64]. Subsequently, Siracusa et al. [65] identified the Tsk1 mutation as a large in-frame duplication of exons 17 through 40 inserted between exons 40 and 41 within the fibrillin-1 gene (Figure 33.5). As a result of the large duplication, numerous important domains in the encoded mutant protein are duplicated, resulting in the addition of 18 Ca++-binding EGF domains and one fibrillin, one RGD integrin, and two TGF-b–binding domains. These results were confirmed by Bona et al. [66], who, in addition, found four amino acid differences between the two fibrillin-1 gene copies in the Tsk1/+ mouse. These sequence differences, however, are not likely to have functional impact [66]. The studies of Siracusa et al. [65] showed that Tsk1/+ mice express both the normal 11kb fibrillin1 mRNA transcript and a mutant 14kb transcript encoded by the mutated gene. Dermal fibroblasts from Tsk1/+ mice synthesize and secrete both the normal 350 kD and the mutant 450-kD fibrillin1 molecule in approximately equal amounts (Figure 33.6). This mixture of fibrillin-1 molecules is incorporated into the ECM. Siracusa et al. [65] hypothesized that the abnormal fibrillin-1 would change the homeostasis in the ECM owing to the greater number of EGF binding domains and the duplication of the TGF-b–binding domain in the mutant fibrillin-1 molecule. It would be expected that greater amounts of these growth factors would be recruited to the ECM resulting in the production of excessive amounts of collagen. Saito et al. [67] provided support for this hypothesis showing that the mutant fibrillin-1 binds greater amounts of TGF-b than the normal protein. Immunohistochemical and ultrastructural analyses of normal and Tsk1/+ mouse skin indicated differences in the gross organization and distribution of the microfibrillar arrays. The beaded microfibrillar structures in Tsk1/+ were abnormal, displaying diffuse interbead segments with longer periodicity which tended to aggregate [68] (Figure 33.7). Transmission electron microscopy indicated that the mass of the microfibrils in the skin of Tsk1/+ mice was greater than in normal skin. These data showed that the mutant fibrillin-1 from Tsk1/+ mice becomes incorporated into a discrete population of beaded microfibrils with altered molecular organization. However, Gayraud et al. [69], using Tsk1/Tsk1 dermal fibroblasts in co-culture with a human cell line that cannot assemble fibrillin1, concluded that the mutant fibrillin-1 microfibrils co-polymerize with wild-type fibrillin-1 and that



A

B

C

D

505

E

Fbn1

mutant Fbn1 LTBP1

LTBP2

Ltbp3

Figure 33.5

Schematic representation of protein motifs encoded by normal and mutant Fbn1 transcripts. Regions A to E indicate the five domains of the Fbn1 protein. “Fbn1” indicates the wild-type mouse Fbn1 protein and “mutant Fbn1” indicates the Tsk1-specific protein. The dashed lines indicate the region duplicated in the Tsk-specific transcript, and the arrow indicates the single junction resulting from the duplication. The human “LTBP1,” human “LTBP2,” and mouse “Ltbp3” proteins represent the three related TGF-b–binding proteins. Symbols represent the corresponding structural motifs: (patterned rectangles) cysteine-rich EGF-like repeats; (rectangles) EGF-CB repeats; (circles) Fib motif; (circles) Fib-like motif; (ovals) TGF-bp repeats; (black shaded and patterned ovals) TGFbp-like repeats; (asterisks) RGD domain; (thick bars) proline-rich region; (thin bars) proline/glycinerich region; (very thin lines) amino- and carboxy-terminal amino acids. (From Siracusa, L.D. et al., Genome Res., 6, 300, 1996, with permission.)

+/+ TSK/+

+/+ TSK/+

+/+

TSK/+

TSK-Fib

Fib

FN

Figure 33.6

SDS-PAGE analysis of newly synthesized radiolabeled fibrillin-1 immunoprecipitated from medium of normal and Tsk1/+ dermal fibroblast cultures. Tsk1/+ cells synthesized and secreted both normal (Fib, ~330 kDa) and Tsk1 fibrillin-1 (Tsk-Fib, ~450 kDa) in comparable amounts. Lanes 1, 3, and 5, medium from normal mice cells; lanes 2, 4, and 6, medium from Tsk1/+ mice cells. Lanes 1 and 2, crude medium; lanes 3 and 4, fibrillin-1 immunoprecipitated with a fibrillin-1–specific antiserum; lanes 5 and 6, immunoprecipitation controls using normal rabbit serum. FN, fibronectin. (From Kielty, C.M. et al., J. Cell Biol., 140, 1159, 1998, with permission.)


506


the co-polymerization rescues the abnormal morphology of the Tsk1/Tsk1 fibrillin-1 aggregates. These authors believe that lung and other connective tissue abnormalities of Tsk1/+ mice are due to co-polymerization of mutant and wild-type fibrillin-1 molecules into functionally deficient microfibrils. They postulate that in contrast with other fibrillin-1 mutations which cause the Marfan syndrome and other related syndromes with prominent vascular alterations, the vascular complications are not present in the Tsk1/+ mice because the level of functional microfibrils does not decrease below the critical threshold required to cause structural alterations of the large blood vessel walls.

Figure 33.7

Rotary shadowing analysis of normal and Tsk1/+ skin microfibrils. Normal skin microfibrils (A and C); Tsk1/+ skin microfibrils (B and D). (a) Normal mice skin microfibrils exhibited well-organized packing and regular diameter. (B) Tsk1/+ mice skin microfibrils. Some appeared normal in morphology and periodicity, whereas others appeared as periodic rows of beads with indistinct interbeads and repeat distances longer than normal (arrowheads). (C) Normal mice skin microfibrils after incubation for 10 minutes in 5 mM EDTA. (D) Tsk/+ mice skin microfibrils after incubation for 10 minutes in 5 mM EDTA. The apparently normal microfibrils responded to EDTA as control mice microfibrils, but the abnormal microfibrils remained morphologically distinct (arrowheads). (From Kielty, C.M. et al., J. Cell Biol., 140, 1159, 1998, with permission.)



507

The identification of fibrillin-1 as the mutated gene in the Tsk1/+ mice stimulated a large number of studies to examine whether alterations in fibrillin-1 were also present in SSc. Some of these studies focused on the Choctaw Indians, an ethnic group in which there is an extremely high incidence of SSc displaying a strong component of heritability. Studies in this population reported that there is genetic linkage to fibrillin-1 in affected individuals [70]. Tan et al. [71] reported an association of a single nucleotide polymorphism with SSc in the Choctaw Indian and extended this observation to Japanese populations. It should be noted, however, that this polymorphism is found in the nontranslated region of the gene and, therefore, it does not affect or modify the primary structure or the function of the protein. Studies were also undertaken to determine whether patients with SSc developed antibodies to fibrillin-1. Several reports have described the presence of antifibrillin-1 antibodies in sera from patients with SSc [72–74] and localized forms of scleroderma [75]. Similar to these observations, Murai et al. [76] reported that Tsk1/+ mice displayed high titer of antibodies (IgG) recognizing the carboxy-terminal region of fibrillin-1 molecules. B. The Tsk2 — A Novel Mutation Resembling SSc A second mutant mouse characterized by a tight skin phenotype was reported in 1986 [77]. The mutation appeared in the offspring of a male from the 101/H mouse strain as a result of administration of the mutagenic agent, ethylnitrosourea. This mutation has been called Tsk2 and has been localized to mouse chromosome 1. Like the Tsk1 mutation, the Tsk2 mutation is inherited as an autosomal dominant trait and appears to be lethal in utero; therefore, only heterozygous Tsk2/+ animals survive. Studies to characterize the Tsk2/+ mice have shown that these animals indeed develop a tight skin phenotype that becomes apparent at 3 to 4 weeks of age [78]. Histological examination of skin samples showed marked thickening of the dermis and excessive deposition of thick collagen fibers, which extended deeply into the subdermal adipose tissue and occasionally surrounded the fascicles of the panniculus carnosus (Figures 33.8A and B). Numerous mononuclear cells were present in the lower dermis and in the adipose tissue septa (Figures 33.8C and D). Furthermore, in contrast to normal skin, the dermal–adipose junction was not distinct in Tsk2/+ skin samples, because of accumulation of abundant connective tissue and the presence of mononuclear cells infiltrating the intercellular spaces of the adipose tissue (Figures 33.8E and F). 1. Immunology Immunological studies were performed by Wooley et al. [79] on the T cells infiltrating the dermis to determine whether Tsk2/+ mice exhibited a T-cell receptor bias. RT-PCR reactions using RNA extracted from the skin and lymph nodes were performed with 18 of the 21 Vb types. These experiments showed that mRNA extracted from involved skin exhibited a restricted pattern with positive Vb signals corresponding to eight T-cell subtypes (Vb1, 6, 8.1, 8.2, 10, 11, 16, and 18). Band intensity analysis revealed that three Vb subtypes (8.1, 11, and 18) predominated, and this pattern was consistent among the four skin samples from four different Tsk2/+ mice tested. The authors postulated that a restricted T-cell population participated in the inflammatory cell infiltrate seen in these mice. However, Sgonc et al. [59,80] reported that this inflammatory infiltrate did not lead to endothelial cell apoptosis and, therefore, the role of inflammatory cells in the pathogenesis of the Tsk2/+ phenotype is not clear. 2. Genetics Christner et al. [78] localized the Tsk2 gene to a 15 cM region on chromosome 1 flanked by the molecular markers D1Mit4 and D1Mit5. In a subsequent study, the authors described a highresolution linkage map of the gene further delimiting Tsk2 to a 1.5-cM interval between the molecular markers D1Mit233 and D1Mit213 [81], as shown in Figure 33.9. The approximate


508

Figure 33.8


Light photomicrographs of skin sections from 7- to 8-month-old normal (A) and Tsk2/+ (B) mice, stained with Masson’s trichrome. Note the marked increase in dense, blue-staining collagen fibers in the skin from Tsk2/+ mice compared with normal mice. Skin sections from normal (C) and Tsk2/+ (D) mice, stained with Verhoeff-Van Gieson stain. Note the densely packed connective tissue in the dermis of Tsk2/+ mice, extending into the subdermal adipose tissue. Also note the presence of mononuclear inflammatory cells in the lower dermis and in the adipose tissue septa. Higher magnification of the dermal–adipose junction of normal (E) and Tsk2/+ (F) mice, stained with hematoxylin and eosin. Note the abundant collagen in the dermis–adipose boundary in the skin from Tsk2/+ mice and the presence of mononuclear cells in the subdermal tissue and infiltrating the adipocyte septa. (Original magnification ¥100 in A and B, ¥200 in C and D, ¥400 in E and F.) (From Christner, P.J. et al., Arthritis Rheum., 38, 1791, 1995, with permission.)

position of the Tsk2 gene was reported at 28 cM by aligning the results of this study to the chromosome 1 consensus map at the molecular markers D1Mit175 and D1Mit236. This region is syntenic with the region of human chromosome 2, which contains the type III procollagen gene, and the authors mentioned that type III collagen gene may be considered a candidate gene for Tsk2 [81].



Figure 33.9

509

A molecular genetic linkage map of the proximal portion of mouse chromosome 1. Selected mapped loci are listed on the side of the chromosome. The map on the right is a partial version of a consensus map. The map on the left was generated from various crosses. The maps have been aligned at the D1Mit175 and D1Mit236 loci. Genes mapped in the human genome are underlined, and their map positions on human chromosomes are shown on the far right. (From Christner, P.J. et al., Mammalian Genome, 7, 610, 1996, with permission.)

3. Biochemical Studies The downstream effects of the Tsk2 mutation have been investigated by Christner et al. [78,82]. Histological examination confirmed that the dermis of Tsk2/+ mice was significantly thicker and biochemical analysis showed that Tsk2/+ mouse skin had 50% more collagen than the normal control mouse skin. Using Tsk2/+ cultured dermal fibroblasts, the authors showed that collagen synthesis was approximately 100% higher in Tsk2/+ cells compared to normal controls. Steadystate mRNA levels for a1(I) and a1(III) collagens were 50% and 100% higher, respectively (Figure 33.10). Transient transfection experiments with COL1A1 promoter constructs demonstrated that the elevated levels of a1(I) collagen mRNA in Tsk2/+ cells were due largely to increased transcriptional activity of the corresponding gene. Electrophoretic mobility shift assays performed with a probe encompassing the relevant COL1A1 promoter region revealed increased DNA-protein binding activities in nuclear extracts prepared from Tsk2/+ fibroblasts compared with normal fibroblasts. Competition experiments using consensus Sp1 and NF-1 oligonucleotides and supershift experiments using anti-Sp1 and anti–NF-1 antibodies indicated that at least two transcription factors, Sp1 and NF-1, or their homologs, are involved in the up-regulated transcriptional activity of COL1A1 promoter in Tsk2/+ fibroblasts. Subsequent experiments were performed with promoter deletion constructs of the mouse type III collagen gene. Transient transfection experiments with the shortest promoter construct (from -96 to +16 bp) yielded 25-times-higher CAT activity than that observed in normal fibroblasts [83]. Electrophoretic mobility shift assays showed that again Sp1 and NF-1 or their homologues were involved in the up-regulated transcriptional activity of the type III collagen gene promoter in Tsk2/+ fibroblasts. Additional experiments with type VI collagen have shown that the steady-state levels of mRNA for this collagen are increased 3.5-fold in Tsk2/+ dermal fibroblasts compared to controls [83]. The Tsk2 mutation, which was originally caused by exposure to the toxic agent ethylnitrosourea, is particularly interesting because of the awareness that several human cutaneous fibrotic diseases resembling SSc appear to result from exposure to chemical substances such as in the case of the


510


toxic oil syndrome and the L-tryptophan–induced eosinophilia-myalgia syndrome. Studies to identify the mechanisms responsible for the connective tissue abnormalities in the Tsk2 mutation may, therefore, provide valuable information regarding the role of environmental exposures in the pathogenesis of SSc and other chemically induced fibrotic diseases. C. Transgenic Animal Models Exhibiting Some Features of SSc In addition to the animal models that display some features of SSc as a result of a genetically transmitted trait, there are also other models that have been obtained by genetic manipulations induced either by the introduction of genes by gene transfer in transgenic animals or by the abolishment of a gene, by gene inactivation in knockout animals. Following the identification of a mutation in the fibrillin-1 gene in Tsk1 mice, several laboratories initiated studies to establish transgenic mice harboring the mutation. Saito et al. [84] generated transgenic mice expressing the Tsk1 mutated fibrillin-1 in order to determine whether the mutation was responsible for the tight skin phenotype. With the same goal in mind these authors also injected normal mice after birth with a plasmid-bearing mutated fibrillin-1. Their results demonstrated that the transgenic mice carrying the Tsk1 mutant fibrillin-1 transgene developed permanent cutaneous hyperplasia. Those mice injected with the mutant fibrillin-1 plasmid after birth developed transient hyperplasia. The transgenic mice also produced antitopoisomerase I and antifibrillin-1 antibodies as do Tsk1/+ mice and SSc patients. However, in contrast to Tsk1/+ mice, neither the transgenic nor the plasmid injected mice developed lung emphysema. The results suggest that the cutaneous hyperplasia (tight skin) of the Tsk1/+ mouse is caused by the tandem duplication in the gene encoding fibrillin-1, but that the lung emphysema may be due to another cause. They also suggest that the level and duration of expression of the mutated fibrillin-1 protein may determine the severity of the fibrosis. Several other models with relevance to various aspects of SSc pathogenesis have been described. Sato et al. [85] investigated the role of CD19, a cell-surface protein that regulates intrinsic and antigen receptor-induced B-lymphocyte signaling thresholds. They produced CD19 transgenic mice that overexpress CD19. Mice with this transgene developed spontaneous autoantibodies in a genetic background not associated with autoimmunity. Subsequently, Sato et al. attempted to quantify the amount of excess CD19 expression required to induce autoantibody production. They found that even a 15% increase in CD19 production resulted in production of antibodies such as antispindle pole, rheumatoid factor, anti-ssDNA, anti-dsDNA, and anti-histone in C57BL/6-CD19 transgenic mice. They found remarkably similar changes in CD19 expression on the surface of B cells from patients with SSc. CD19 density on peripheral blood B cells from SSc patients was 20% higher compared to controls, whereas CD20, CD22, and CD40 were unchanged. They concluded that modest changes in the expression of CD19 may shift the balance between tolerance and immunity toward autoimmunity and that alterations in cell-surface signaling molecules may be important in the production of autoantibodies in patients with SSc. Ong et al. [29] have used IL4 and Stat6 knockout mice to understand the downstream events resulting from the fibrillin-1 duplication and leading to the development of tight skin in the Tsk1/+ mouse. They reported that Tsk1/+ mice carrying a null mutation for either IL4 or Stat6 failed to develop tissue fibrosis. They concluded that development of the CD4+ T cell determines whether fibrosis will occur. CD4+ T cells can develop into either T-helper 1 (Th1) or T-helper 2 (Th2) cells based on the immunizing conditions. When the CD4+ T cell develops into a Th1 cell no fibrosis occurs, whereas when it develops into a Th2 cell fibrosis does occur. By modulating the amount of IL-4 or Stat6, the ratio of Th1/Th2 can be changed. They further determined that alteration of the T-cell receptor (TCR) repertoire in Tsk1/+ mice could also prevent dermal fibrosis. By introducing TCR transgenes into Tsk1/+ mice, they were able to show that exclusive usage of the



511

Vbeta8.2 gene segment by T cells prevented the development of fibrosis-causing Th2 cells. They suggested that the restricted TCR usage prevents the generation of a very small subset of antigenspecific CD4+ T cells that cause skin fibrosis or that the exclusive usage of Vb8.2 by CD4+ T cells leads to an alteration of cytokines (e.g., absence of IL-4) preventing the development of Th2 cells. Liu et al. [86] obtained transgenic mice to study the role of collagen degradation on fibrosis. These mice carried a transgene with a mutation in the a1 collagen gene sequence that encodes the specific site for mammalian collagenase cleavage and therefore the resultant protein was resistant to digestion. These mice died in utero presumably because of excessive accumulation of collagenase-resistant mutant collagen coded by the transgene. However, when the same mutation was produced by gene targeting, the mice survived, probably because the production of mutant collagen was less in these mice than in the transgenic mice, although with increasing age they developed marked dermal fibrosis similar to that seen in SSc patients. In addition to type I collagen, type V collagen has been implicated in the molecular pathway resulting from the fibrillin-1 mutation in the Tsk1/+ mouse. In order to gain an understanding of the events downstream of the fibrillin-1 mutation, Phelps et al. [25] bred Tsk1/+ mice to type V collagen-deficient mice. These collagen type V knockout mice exhibit skeletal abnormalities, skin fragility, and alterations in the collagen fiber organization. The F1 mice did not develop cutaneous hyperplasia and did not produce autoantibodies. The diameter of the collagen fibrils in the dermis was comparable to controls. The results indicated that genetic complementation from the cross with a collagen V–defective mouse could reverse the phenotypic changes displayed by Tsk1/+ mice.

IV. CONCLUSION This review of the spontaneous mouse models for scleroderma described in the literature demonstrates that there are at least two models in which to study various aspects of this complex disorder. Each model has its strengths in mimicking certain aspects of the disease — inflammatory, vascular, immunologic, or fibrotic — as well as important differences as summarized in Table 33.1. It is apparent that the prudent interpretation of results obtained with each of these models can contribute to our knowledge of the mechanisms underlying this presently incurable disorder. The most extensively studied model from the histologic, immunologic, and biochemical viewpoint is the Tsk1 mutation. However, the Tsk1/+ mouse has several deficiencies as a model for human SSc including the absence of inflammatory, vascular, gastrointestinal, and joint involvement. These deficiencies are offset by the fact that the genetic mutation, a tandem duplication in fibrillin-1, which causes the Tsk1/+ phenotype has been identified. The mechanisms by which the mutated fibrillin-1 leads to tight skin and tissue fibrosis are now being actively investigated in several laboratories. With the likely identification of the gene for Tsk2, there will be two different mutations located on two separate chromosomes, and therefore involving at least two separate genes, which lead to a similar cutaneous fibrotic phenotype. It will then be possible to determine whether there are molecular events shared by both pathways and which are necessary to cause tissue fibrosis. Furthermore, the existence of transgenic animal models of SSc will allow the application of the techniques of cellular and molecular biology to address relevant questions related to the pathogenesis of SSc.


512

Table 33.1


Comparison of Animal Models and Human Scleroderma Feature

Clinical features Skin thickening Arthritis Visceral involvement Gastrointestinal Vascular Pulmonary Renal Cardiac Genetic transmission Immunological changes Mononuclear cell infiltrate IL-2 production TGF-b abnormalities ANA Scl-70 Anticentromere Anti–ds-DNA Antifibrillin Biochemical changes Increased tissue collagen Collagen phenotype changes Increased collagen biosynthesis Abnormal collagen regulation Increased procollagen gene expression Increased procollagen gene transcription Increased GAG Increased fibronectin

Tsk1/+

Tsk2/+

Human SSc

+ -

+ -

+ +

Emphysema ? + +

? ? ? ? ? +

+ + Fibrosis + + Rare

Normal + + + +

+(? cells) ? ? ? ? ? ? ?

+(T cells) increased + + + + +

+ + + + + + +

+ + + + + ? ?

+ + + + + + +

REFERENCES 1. Green, M.C., Sweet, H.O., and Bunker, L.E., Tight-skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton, Am. J. Pathol., 82, 493, 1976. 2. Jimenez, S.A., Millan, A., and Bashey, R.I., Scleroderma-like alterations in collagen metabolism occurring in the Tsk (tight skin) mouse, Arthritis Rheum., 27, 180, 1984. 3. Jimenez, S.A. et al., The tight skin (Tsk) mouse as an experimental model of scleroderma, in CRC Handbook of Animal Models for Rheumatic Diseases, Greenwald, R.A. and Diamond, H.S., Eds., CRC Press, Boca Raton, FL, 1988, p. 169. 4. Menton, D.N. and Hess, R.A., The ultrastructure of collagen in the dermis of tight-skin (Tsk) mutant mice, J. Invest. Dermatol., 7, 139, 1980. 5. Menton, D.N. et al., The structure and tensile properties of the skin of tight-skin (Tsk) mutant mice, J. Invest. Dermatol., 70, 4, 1978. 6. Osborn, T.G. et al., Tight-skin mouse: physical and chemical properties of the skin, J. Rheumatol., 10, 793, 1983. 7. Szapiel, S.V. et al., Hereditary emphysema in the tight-skin (Tsk/+) mouse, Am. Rev. Respir. Dis., 123, 680, 1981. 8. Rossi, G.A. et al., Hereditary emphysema in the tight skin mouse, Am. Rev. Respir. Dis., 129, 850, 1984. 9. O’Donnell, M.D. et al., Ultrastructure of lung elastin and collagen in mouse models of spontaneous emphysema, Matrix Biol., 18, 357, 1999. 10. Keil, M. et al., A scanning electron microscopic investigation of genetic emphysema in tight-skin, pallid, and beige mice, three different C57 BL/6J mutants, Lab. Invest., 74, 353, 1996.



513

11. Gardi, C. et al., Lung collagen synthesis and deposition in tight-skin mice with genetic emphysema, Exp. Mol. Pathol., 56, 163, 1992. 12. Osborn, T.G. and Bauer, N.E., Physical and biochemical manifestations of cardiomyopathy in the tight-skin mouse, Arthritis Rheum., 27, 75s, 1984. 13. Osborn, T.G. et al., Collagenous abnormalities in the heart of the tight-skin mouse, J. Mol. Cell Cardiol., 19, 581, 1987. 14. Bashey, R.I. et al., Increased collagen synthesis and increased content of type VI collagen in myocardium of tight skin mice, Cardiovasc. Res., 27, 1061, 1993. 15. Gardi, C. et al., Cardiac collagen changes during the development of right ventricular hypertrophy in tight-skin mice with emphysema, Exp. Mol. Pathol., 60, 100, 1994. 16. Martorana, P.A. et al., Tsk mice with genetic emphysema. Right ventricular hypertrophy occurs without hypertrophy of muscular pulmonary arteries or muscularization of arterioles, Am. Rev. Respir. Dis., 142, 333, 142. 17. Chapman, D. and Eghbali, M., Expression of fibrillar types I and III and basement membrane collagen type IV genes in myocardium of tight skin mouse, Cardiovasc. Res., 24, 578, 1990. 18. Bocchieri, M.H. et al., Evidence for autoimmunity in the tight skin (Tsk/+) mouse model of progressive systemic sclerosis, Arthritis Rheum., 34, 599, 1991. 19. Muryoi, T. et al., Self reactive repertoire of tight skin mouse: immunochemical and molecular characterization of anti-topoisomerase I autoantibodies, Autoimmunity, 9, 109, 1991. 20. Muryoi, T. et al., Self-reactive repertoire of tight skin (Tsk/+) mouse: immunochemical and molecular characterization of anti-cellular autoantibodies, Cell Immunol., 144, 43, 1992. 21. Shibata, S. et al., Immunochemical and molecular characterization of anti-RNA polymerase I autoantibodies produced by tight skin mouse, J. Clin. Invest., 92, 984, 1993. 22. Kasturi, K.N. et al., Tight skin mouse autoantibody repertoire: analysis of VH and VK gene usage, Mol. Immunol., 30, 969, 1993. 23. Kasturi, K.N., Yio, X.Y., and Bona, C.A., Molecular characterization of J558 genes encoding tightskin mouse autoantibodies: identical heavy-chain variable genes code for antibodies with different specificities, Proc. Natl. Acad. Sci. U. S. A., 91, 8067, 1994. 24. Muryoi, T. et al., Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes, J. Exp. Med., 175, 1103, 1992. 25. Phelps, R.G. et al., Effect of targeted mutation in collagen V a2 gene on development of cutaneous hyperplasia in tight skin mice, Mol. Med., 4, 356, 1998. 26. Hom, J.T. and Talal, N., Decreased syngeneic mixed lymphocyte response (SMLR) in genetically susceptible autoimmune mice, Scand. J. Immunol., 15, 5, 1982. 27. Morse, J.H. and Bodi, B.S., Autologous and allogeneic mixed lymphocyte reaction in progressive systemic sclerosis, Arthritis Rheum., 25, 390, 1982. 28. Phelps, R.G. et al., Induction of skin fibrosis and autoantibodies by infusion of immunocompetent cells from tight skin mice into C57BL/6 Pa/Pa mice, J. Autoimmunity, 6, 701, 1993. 29. Ong, C.J. et al., A role of T helper 2 cells in mediating skin fibrosis in tight-skin mice, Cell Immunol., 196, 60, 1999. 30. Ong, C. et al., Anti-IL-4 treatment prevents dermal collagen deposition in the tight-skin mouse model of scleroderma, Eur. J. Immunol., 28, 2619, 1998. 31. Kodera, T. et al., Disrupting the IL-4 gene rescues mice homozygous for the tight-skin mutation from embryonic death and diminishes TGF-b production by fibroblasts, Proc. Natl. Acad. Sci. U. S. A., 99, 3800, 2002. 32. McGaha, T. et al., Lack of skin fibrosis in tight skin (Tsk) mice with targeted mutation in the interleukin-4Ra and transforming growth 3 factor-b genes, J. Invest. Dermatol., 116, 136, 2001. 33. Tsuji-Yamada, J. et al., Effect of IL-12 encoding plasmid administration on tight-skin mouse, Biochem. Biophys. Res. Commun., 280, 707, 2001. 34. Wallace, V.A. et al., A role for CD4+ T-cells in the pathogenesis of skin fibrosis in tight skin mice, Eur. J. Immunol., 24, 1463, 1994. 35. Hatakeyama, A. et al., Correlation between the concentration of serum anti-topoisomerase I autoantibodies and histological and biochemical alterations in the skin of tight skin mice, Cell Immunol., 167, 135, 1996.


514


36. Siracusa, L.D. et al., The mouse tight skin (Tsk) phenotype is not dependent on the presence of mature T and B lymphocytes, Mamm. Genome, 9, 907, 1998. 37. Dodig, T.D. et al., Development of the tight-skin phenotype in immune-deficient mice, Arthritis Rheum., 44, 723, 2001. 38. Kasturi, K.N. et al., B-cell deficiency does not abrogate development of cutaneous hyperplasia in mice inheriting the defective fibrillin-1 gene, J. Autoimmunity, 10, 505, 1997. 39. Saito, S., Kasturi, K., and Bona, C., Genetic and immunologic features associated with sclerodermalike syndrome of Tsk mice, Curr. Rheumatol. Rep. 1, 34, 1999. 40. Oble, D.A. and Teh, H.S., Tight skin mouse subcutaneous hypertrophy can occur in the absence of a/b T cell receptor-bearing lymphocytes, J. Rheumatol., 28, 1851, 2001. 41. Hawkins, R.A. et al., Increased dermal mast cell population in progressive systemic sclerosis: A link in chronic fibrosis? Ann. Intern. Med. 102, 182, 1985. 42. Walker, M. et al., Mast cells and their degranulation in the Tsk mouse model of scleroderma, Proc. Soc. Exp. Biol. Med., 180, 323, 1985. 43. Pearson, M.E. et al., Immunologic dysfunction in scleroderma: Evidence for increased mast cell releaseability and HLA-Dr positivity in the dermis, Arthritis Rheum., 31, 672, 1988. 44. Everett, E.T. et al., The role of mast cells in the development of skin fibrosis in tight-skin mutant mice, Comp. Biochem. Physiol. A Physiol., 110, 159, 1995. 45. Jimenez, S.A. et al., Increased collagen biosynthesis and increased expression of type I and type III procollagen gene in tight skin (Tsk) mouse fibroblasts, J. Biol. Chem., 261, 657, 1986. 46. Ross, S.C. et al., Glycosaminoglycan content in skin of the tight-skin mouse, Arthritis Rheum., 26, 653, 1983. 47. Dorner, R.W., Osborn, T.G., and Ross, S.C., Glycosaminoglycan composition of tight skin and control mouse skins, J. Rheumatol., 14, 295, 1987. 48. Uitto, J., Collagen biosynthesis in human skin: a review with emphasis on scleroderma, Ann. Clin. Res., 1971;3, 250, 1971. 49. Jimenez, S.A., Yankowski, R., and Frontino, P.M., Biosynthetic heterogenetiy of sclerodermatous skin in organ cultures, J. Mol. Med., 2, 423, 1977. 50. LeRoy, E.C., Increased collagen synthesis by scleroderma skin fibroblasts in vitro: a possible defect in the regulation or activation of the scleroderma fibroblasts, J. Clin. Invest., 54, 880, 1974. 51. Buckingham, R.B. et al., Increased collagen accumulation in dermal fibroblast cultures from patients with progressive systemic sclerosis (scleroderma), J. Lab. Clin. Med., 92, 5, 1978. 52. Jimenez, S.A. and Bashey, R.I., Collagen synthesis by scleroderma fibroblasts in culture, Arthritis Rheum., 20, 902, 1977. 53. Jimenez, S.A. et al., Coordinate increase in the expression of type I and type III collagen genes in progressive systemic sclerosis fibroblasts, Biochem. J., 237, 837, 1986. 54. Uitto, J., Bauer, E.A., and Eisen, A.Z., Scleroderma: Increased biosynthesis of triple-helical type I and III procollagens associated with unaltered expression of collagenase by skin fibroblasts in culture, J. Clin. Invest., 64, 921, 1979. 55. Cooper, S.M. et al., Increase in fibronectin in the deep dermis of involved skin in progressive systemic sclerosis, Arthritis Rheum., 22, 983, 1979. 56. Peltonen, J. et al., Increased expression of type VI collagen genes in progressive systemic sclerosis lesions in situ, Arthritis Rheum., 33, 1829, 1990. 57. Pablos, J.L. et al., Transforming growth factor-b1 and collagen gene expression during postnatal skin development and fibrosis in the tight-skin mouse, Lab. Invest., 72, 670, 1995. 58. Pablos, J.L. et al., Apoptosis and proliferation of fibroblasts during postnatal skin development and scleroderma in the tight-skin mouse, J. Histochem. Cytochem., 45, 711, 1997. 59. Sgonc, R. et al., Lack of endothelial cell apoptosis in the dermis of tight skin 1 and tight skin 2 mice, Arthritis Rheum., 42, 581, 1999. 60. Philips, N., Bashey, R.I., and Jimenez, S.A., Increased a1(I) procollagen gene expression in tight skin (Tsk) mice myocardial fibroblasts is due to a reduced interaction of a negative regulatory sequence with AP-1 transcription factor, J. Biol. Chem., 270, 9313, 1995. 61. Denton, C.P. et al., Activation of a fibroblast-specific enhancer of the proa2(I) collagen gene in tightskin mice, Arthritis Rheum., 44, 712, 2001.



515

62. Siracusa, L.D. et al., The tight skin (Tsk) mutation in the mouse, a model for human fibrotic diseases, is tightly linked to the b2-microglobulin (B2m) gene on chromosome 2, Genomics, 17, 748, 1993. 63. Goldstein, C. et al., Of mice and Marfan: genetic linkage analyses of the fibrillin genes, Fbn1 and Fbn2, in the mouse genome, Mamm. Genome, 5, 696, 1994. 64. Everett, E.T. et al., The tight-skin (Tsk) mutation is closely linked to b2m on mouse chromosome 2, Mamm. Genome, 5, 55, 1994. 65. Siracusa, L.D. et al., A tandem duplication within the fibrillin-1 gene is associated with the mouse tight skin mutation, Genome Res., 6, 300, 1996. 66. Bona, C.A. et al., Structure of the mutant fibrillin-1 gene in the tight skin (Tsk) mouse, DNA Res., 4, 267, 1997. 67. Saito, S. et al., Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (Tsk) mice, Mol. Immunol., 36, 169, 1999. 68. Kielty, C.M. et al., The tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils, J. Cell Biol., 140, 1159, 1998. 69. Gayraud, B. et al., New insights into the assembly of extracellular microfibrils from the analysis of the fibrillin-1 mutation in the tight skin mouse, J. Cell Biol., 150, 667, 2000. 70. Tan, F.K. and Arnett, F.C., Genetic factors in the etiology of systemic sclerosis and Raynaud phenomenon, Curr. Opin. Rheumatol., 12, 511, 2000. 71. Tan, F.K. et al., Association of fibrillin-1 single-nucleotide polymorphism haplotypes with systemic sclerosis in Choctaw and Japanese populations, Arthritis Rheum., 44, 893, 2001. 72. Tan, F.K. et al., Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin-1, in patients with scleroderma and other connective tissue diseases, J. Immunol., 163, 1066, 1999. 73. Tan, F.K. et al., Autoantibodies to fibrillin-1 in systemic sclerosis: ethnic differences in antigen recognition and lack of correlation with specific clinical features or HLA alleles, Arthritis Rheum., 43, 2464, 2000. 74. Pandey, J.P. et al., Anti-fibrillin-1 autoantibodies in systemic sclerosis are GM and KM allotyperestricted, Exp. Clin. Immunogenet., 18, 123, 2001. 75. Arnett, F.C. et al., Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin-1, in patients with localized scleroderma, Arthritis Rheum., 42, 2656, 1999. 76. Murai, C. et al., Spontaneous occurrence of anti-fibrillin-1 autoantibodies in tight-skin mice, Autoimmunity, 28, 151, 1998. 77. Peters, J. and Ball, S.T., Tight skin-2 (Tsk-2), Mouse News Lett., 74, 91, 1986. 78. Christner, P.J. et al., The tight skin 2 mouse. An animal model of scleroderma displaying cutaneous fibrosis and mononuclear cell infiltration, Arthritis Rheum., 38, 1791, 1995. 79. Wooley, P.H. et al., T-cells infiltrating the skin of Tsk2 scleroderma-like mice exhibit T cell receptor bias, Autoimmunity, 27, 91, 1998. 80. Sgonc, R., The vascular perspective of systemic sclerosis: of chickens, mice and men, Int. Arch. Allergy Immunol., 120, 169, 1999. 81. Christner, P.J. et al., A high-resolution linkage map of the tight skin 2 (Tsk2) locus: A mouse model for scleroderma (SSc) and other cutaneous fibrotic diseases, Mamm. Genome, 7, 610, 1996. 82. Christner, P.J. et al., Transcriptional activation of the a1(I) procollagen gene and up-regulation of a1(I) and a1(III) procollagen messenger RNA in dermal fibroblasts from tight skin 2 mice, Arthritis Rheum., 41, 2132, 1998. 83. Christner, P.J. et al., Upregulated expression of collagen type III and VI genes in the Tsk2 mouse model, Arthritis Rheum., 41, S279, 1998. 84. Saito, S. et al., Induction of skin fibrosis in mice expressing a mutated fibrillin-1 gene, Mol. Med., 6, 825, 2000. 85. Sato, S. et al., Quantitative genetic variation in CD19 expression correlates with autoimmunity, J. Immunol., 165, 6635, 2000. 86. Liu, X. et al., A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling, J. Cell Biol., 130, 227, 1995.



CHAPTER

34

Experimental Mouse Model of Scleroderma/Graft versus Host Disease: Induction by Transfer of Cellular Immmunity Anita C. Gilliam

CONTENTS I. History ................................................................................................................................518 A. GVHD as a Model for Scleroderma .........................................................................518 B. The Diversity of GVHD ............................................................................................519 C. Major Issues in Characterizing a Mouse Model of Scleroderma.............................520 1. General Issues......................................................................................................520 2. The Reproducibility of Major Features of Human Scleroderma in Mouse Scl GVHD ...............................................................................................520 a. Vascular Injury...............................................................................................520 b. Mast Cell Degranulation................................................................................520 c. Up-regulation of Tissue Chemokines............................................................521 d. Tissue Influx of Inflammatory Cells and Their Activation...........................521 e. Up-regulation of Cytokines ...........................................................................522 f. Up-regulation of Collagen Synthesis Leading to Tissue Fibrosis ................522 g. Collagen Degradation ....................................................................................522 II. Animals ..............................................................................................................................522 A. Generating Scl GVHD...............................................................................................522 1. Strains of Mice ....................................................................................................522 2. Sex and Age.........................................................................................................522 B. Inherent Susceptibility to Fibrosis ............................................................................523 III. Disease Induction...............................................................................................................523 A. Mice Preparation........................................................................................................523 B. Irradiation of Recipient Mice ....................................................................................523 C. Bone Marrow and Spleen Cell Collection from Donor Mice ..................................523 1. Bone Marrow Cells (Source of Hematopoietic Cells for Engraftment).............523 2. Spleen Cells (Source of Mature T Cells to Generate GVHD)...........................524 D. Transplantation...........................................................................................................524 E. Maintenance of Transplanted Recipient Mice ..........................................................524 F. Experimental Design .................................................................................................524 0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

517


518


1. Controls for Scl GVHD.......................................................................................524 2. Controls for Engraftment.....................................................................................525 IV. Course of Disease ..............................................................................................................525 V. Assessment of Disease.......................................................................................................525 A. Skin in Scl GVHD.....................................................................................................525 1. Caliper Measurements .........................................................................................525 2. Histopathological and Morphometric Analysis...................................................525 a. Routine Histology..........................................................................................526 b. Image Analysis ..............................................................................................526 3. Ultrasonographic Evaluation ...............................................................................526 4. Cutaneous Immune Cell Infiltrates......................................................................526 a. Quantification of Immunostaining by Image Analysis .................................526 b. Flow Cytometric Analysis of Cutaneous Immune Cell Infiltrates ...............526 B. Lung in Scl GVHD....................................................................................................527 VI. Expert Experience ..............................................................................................................527 A. Skin ............................................................................................................................527 1. Skin Thickening and Collagen Synthesis............................................................527 2. Keratinocyte Injury ..............................................................................................527 3. Mast Cell Degranulation......................................................................................527 4. Chemokines and Cytokines .................................................................................527 5. Cutaneous Inflammatory Cells ............................................................................528 B. Lungs..........................................................................................................................528 C. Spleen.........................................................................................................................528 D. Liver ...........................................................................................................................529 E. Kidney........................................................................................................................530 VII. Therapeutic Responses.......................................................................................................530 A. Antibodies to TGF-b .................................................................................................530 B. Latency-Associated Peptide.......................................................................................530 C. Halofuginone..............................................................................................................530 D. Nedocromil Sodium...................................................................................................530 VIII. Conclusion..........................................................................................................................531 References ......................................................................................................................................531

I. HISTORY A. GVHD as a Model for Scleroderma Chronic graft versus host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT) that has autoimmune features resembling lupus erythematosus (lichenoid GVHD) or scleroderma (sclerodermatous GVHD, Scl GVHD). Henry Claman et al. [1–9] first recognized mouse Scl GVHD as a possible model for scleroderma in the 1980s. Also in the 1980s, two examples of mouse Scl GVHD were described [10–12]. The B10.D2>BALB/c transplantation pair is the best described and most commonly used. Since that early series of publications, there has been a revival of interest in the model because of a new concept in the possible etiology of scleroderma: microchimerism of persistent fetal cells in postpartum women [13–16]. An attractive, but still controversial, hypothesis is that a graft versus host (or host versus graft?)-like reaction due to microchimerism may help explain the predominant occurrence of scleroderma in women in their postchildbearing years. Maternal microchimerism (retention of maternal cells in the fetus) has also been proposed as a possible explanation of autoimmunity in men and children [17].


INDUCTION BY TRANSFER OF CELLULAR IMMMUNITY

519

As Claman et al. suggested in the 1980s [1–9], mouse Scl GVHD is a very useful model for scleroderma because it is possible to study the immunology and progression of early disease, and because interventions can be tested in the model. Since Claman’s initial work on the murine model, Levi Schaefer et al. [18–22] have used mouse Scl GVHD (the B10.D2>BALB/c transplantation pair) to study the role of mast cells in fibrosis. Mast cells release a wide variety of mediators of inflammation (histamine, proteases, cytokines) and when added to fibroblast cultures can stimulate both fibrogenic and fibrolytic changes [23]. Our understanding of the role that mast cells play in scleroderma is still incomplete, however. Levi-Schaffer et al. [20] and Pines and Nagler [24] tested interventions with halofuginone, an alkaloid inhibitor of collagen synthesis in mouse Scl GVHD (B10.D2>BALB/c). Chen et al. [25] induced long-term survival in this model with rapamycin, a macrolide used to suppress organ transplant rejection. Others have shown hepatic and small bowel inflammation in mouse Scl GVHD (B10.D2>BALB/c) [26] that is ameliorated by feeding recipient mice with proteins extracted from BALB/c splenocytes to induce oral tolerance [27,28]. Nagler et al. [28] demonstrated altered serum interleukin 10 (IL-10), interferon-gamma (IFN-g) and tumor necrosis factor alpha (TNF-a) levels, suggesting that the decrease in gut inflammation was due to a cytokine shift from proinflammatory to anti-inflammatory pattern. The B10.D2>BALB/c Scl GVHD mice also have biliary cirrhosis, similar to that in human primary biliary cirrhosis. Biliary cirrhosis has also been demonstrated in some individuals with CREST syndrome [29]. Howell et al. [30] characterized the hepatic GVHD in a series of articles in which they demonstrated increased ICAM-1 and Iad on bile duct epithelium, injury to bile ducts associated with increased T cells, an increased CD4/CD8 T-cell ratio, and increased Mac-1 positive macrophages. Hepatic GVHD lesions and elevated serum IgE were suppressed by anti-CD4 but not anti-CD8 T-cell antibodies in the mice with Scl GVHD [31]. A skewed T-cell receptor repertoire by two-color immunofluorescence on spleen and liver lymphocytes suggested a polyclonal response to multiple host non-MHC antigens [32]. Parotid gland dysfunction has also been documented in these mice (B10.D2>BALB/c) [33]. We have focused on the cutaneous immunology of early mouse Scl GVHD (B10.D2>BALB/c) because of the ready accessibility of skin and the extensive published data on and reagents for skin biology. We characterized the early cutaneous inflammatory cells and showed that monocyte/macrophages making TGF-b1, a potent fibrogenic cytokine, predominate over lymphocytes in skin [34]. Up-regulation of mRNA for chemokines MCP-1 (macrophage chemotactic factor); RANTES (regulated upon activation, normal T cell expressed and secreted), and MIP-1a (macrophage inhibitory factor) occurs early in cutaneous disease, and precedes cutaneous inflammatory cell influx [35]. We have also shown that latency-associated peptide (LAP), the naturally occurring pre-propeptide inhibitor of TGF-b, and antibodies to TGF-b both inhibit skin thickening in the mouse model, confirming the critical role of TGF-b in fibrosis [34–37]. B. The Diversity of GVHD GVHD results when immunologically competent cells with disparate histocompatibility antigens are transplanted into an immunologically compromised or suppressed individual that cannot reject the grafted cells. The donor cells then attack recipient host cells, tissues, organs, and immune system. Typically in human allogeneic BMT or stem cell transplantation, donor cells are matched for at least four to six major histocompatibility antigens. No attempt is made to match for minor antigens, which are less well characterized and for which reagents are not readily available. Therefore, it is no surprise that manifestations of GVHD can be quite different among individuals and from one animal model to another because they are dependent mainly on the unique genetic backgrounds of the individuals transplanted. Different mouse-strain pair combinations allow us to generate acute or chronic, mild nonfatal or devastating fatal disease; disease mainly with vasculitis


520


or with fibrosis; or disease directed to specific organs (skin, lungs, kidneys, liver, etc.). These mouse models are invaluable tools to dissect out the pathogenesis of epithelial injury, vasculitis, or tissue fibrosis because disease can be characterized from early to late time points, and interventions can be tested within fairly short periods of time (weeks to months). Most chronic human GVHD is of the classic lichenoid, scaling variant (lupus-like), with prominent cytotoxicity to epithelial cells producing “dermatitis, hepatitis, and enteritis” [38], whereas Scl GVHD develops in only a small percentage of humans with chronic GVHD, that is, 3.6% in one study [39]. Similarly, Scl GVHD can be generated in only a few mouse strain pairs that are transplanted across minor histocompatibility loci. These models most closely resemble human sibling allogeneic BMT, in which donor and recipient bone marrow are matched for major HLA loci, but may be disparate at minor histocompatibility loci. The B10.D2>BALB/c model (B10.D2 H-2d bone marrow and spleen cells transplanted to lethally irradiated BALB/c H-2d mice) is the most commonly used strain pair. Another strain-pair model utilizes C57Bl/6 (H-2b) bone marrow and spleen cells transplanted to LPJ (H-2b) mice (C57Bl/6>LPJ) [40]. In both of these models, syngeneic bone marrow/spleen transplanted mice serve as controls. In contrast to classic GVHD, transplanted animals with Scl GVHD do not develop skin dermatitis or diarrhea clinically. Histologically, minimal cytotoxic effects are seen in skin epidermis in Scl GVHD. The main features of mouse Scl GVHD that resemble scleroderma are skin thickening and lung fibrosis. C. Major Issues in Characterizing a Mouse Model of Scleroderma 1. General Issues Scleroderma is a complex disease involving production of autoantibodies to various cellular antigens (components of nucleoli, centromeres, small nuclear ribonucleoproteins, and RNase molecules), vascular injury, and skin and visceral fibrosis. No one animal model recapitulates all features of human scleroderma. Just as there are subsets of patients with different types of disease (systemic sclerosis, CREST), and with different autoantibody patterns associated with different ethnic groups [41], the mouse (Tight Skin, Tsk; bleomycin-induced sclerosis; and Scl GVHD) and UCD chicken scleroderma models may each reproduce some but not all features of human scleroderma. Scl GVHD most closely resembles explosive rapidly progressive scleroderma that evolves over months rather than years. This form of scleroderma may be more amenable to treatment, which could interrupt the disease process before significant irreversible tissue injury has occurred. 2. The Reproducibility of Major Features of Human Scleroderma in Mouse Scl GVHD a. Vascular Injury In human scleroderma, reduplicated basement membrane and concentric sclerosis with perivascular fibrosis are seen in deep dermal vessels. In mouse Scl GVHD, no vascular injury is seen in routine hematoxylin and eosin (H and E) sections of skin in mice with Scl GVHD, much like human sclerodermatous GVHD. In contrast, vascular injury is prominent in the UC Davis chicken model for scleroderma. b.

Mast Cell Degranulation

Claman et al. [9,42] were the first to describe degranulated “ghost” mast cells in scleroderma. In mouse Scl GVHD, mast cell degranulation is also seen, whereas most dermal mast cells are intact in syngeneic transplanted control mice.



c.

521

Up-regulation of Tissue Chemokines

RANTES, MCP-1, MIP1a, and MIP1b are increased in skin [43,44]. In mouse Scl GVHD, mRNA for cutaneous RANTES, MCP-1, and MIP1a are increased preceding development of skin fibrosis [35]. d. Tissue Influx of Inflammatory Cells and Their Activation T Cells — Chronic autoimmune diseases are thought to be antigen driven, and the focus to date has been on T cells in scleroderma, presumably specific for unknown cellular antigens [45–48]. Evidence for this includes skewing of T-cell receptor repertoires with oligoclonal proliferation of T-cell subsets in individuals with systemic sclerosis [49], and in individuals with CREST and primary biliary cirrhosis [29]. In general, scleroderma is said to be a predominantly Th2 process, but conflicting reports are found in the literature with respect to up-regulation of Th1 versus Th2 cytokines. There is a higher percentage of activated CD3+ DR+ T cells in peripheral blood of systemic sclerosis patients compared to matched controls, with detectable IFN-g mRNA-positive cells [50]. Under basal conditions, PBMC of a majority of the scleroderma patients showed both Th1 and Th2 cell activation. In mouse Scl GVHD, T cells in liver and spleen have an altered Tcell receptor repertoire skewed toward Vb2 and Vb3 [51], and both cutaneous T cells and monocyte/macrophages express Iad, consistent with an activated phenotype [35]. Th1 versus Th2 cytokine predominance is not yet established in the model. Monocyte/Macrophages — CD14+ monocytes predominate over T cells in scleroderma skin in early disease [52]. There is chronic activation of pulmonary T cells and macrophages [46], presumably brought there by increased chemokines. Parallels are seen in other rheumatic diseases. Activated macrophages, often located strategically near the pannus, and increased monocyte/macrophage chemokines are present in affected joints in rheumatoid arthritis. Clinical improvement in rat adjuvant–induced arthritis can be linked to down-regulation of the monocyte/macrophage system [53,54]. In mouse Scl GVHD, we observed that TGF-b producing monocyte/macrophages predominate over other immune cells (T cells, NK cells) in Scl GVHD skin before fibrosis begins, an important finding in understanding the pathophysiology of Scl GVHD and scleroderma, and in targeting a cell type for immunomodulatory therapies. T cells and monocytes are derived from CD34+ bone marrow precursors. Monocytes pass through several intermediate stages in the bone marrow (monoblasts and promonocytes). Then they enter the circulation as monocytes for approximately 5 days after which they home to various tissues and organs via mechanisms that are still poorly understood, but are thought to be similar to those of lymphocytes. In tissue, they differentiate into mature resident tissue macrophages or dendritic cells, a final stage thought in the past to be irreversible. The systemic and local triggers for differentiation of monocytes are not as well understood as those for lymphocytes. They include cytokines, T-cell interactions, matrix molecule interactions, and microorganisms. Several markers of differentiation and activation can be used to characterize monocyte/macrophages. They include up-regulation of surface markers (class II MHC, CD40, scavenger receptors, CD11b, LFA-1, and CD44); chemokine secretion (MCP-1, MIP-1a), and cytokine production (up-regulated TGF-b, IL-1b , TNF-a, GMCSF). During inflammation, macrophages can immigrate to the draining lymph nodes in response to cutaneous MCP-1 carried there via lymphatics [55]. There, they may play a role in presentation of antigens from inflamed skin, resembling dendritic cells. In mouse Scl GVHD, we have shown up-regulation of macrophage activation markers (CD11b, scavenger receptor molecules, and Iad) in early cutaneous Scl GVHD, suggesting a critical role of monocyte/macrophages in disease [35]. Like T cells, macrophages may contribute to the chemokine and cytokine environment that favors fibrosis, and our data suggest that they may also be important in antigen presentation.


522


e. Up-regulation of Cytokines Up-regulation of a critical fibrogenic cytokine, TGF-b, has been demonstrated in skin and lung of individuals with scleroderma [56]. TGF-b can induce increased collagen mRNA and protein synthesis in normal human fibroblasts in vitro. The inability of TGF-b to up-regulate collagen production in cultured scleroderma fibroblasts is thought to reflect a refractory state in which collagen synthesis by scleroderma fibroblasts is no longer under regulatory control of TGF-b. Connective tissue growth factor (CTGF) is also up-regulated in scleroderma, and may be an important inducer of fibroblast collagen synthesis that is downstream from TGF-b [57,58]. In mouse Scl GVHD, up-regulated TGF-b1 mRNA and protein and CTGF mRNA are present in mouse Scl GVHD skin by day 7 post-BMT, preceding collagen synthesis [34,35,36]. f.

Up-regulation of Collagen Synthesis Leading to Tissue Fibrosis

Type I collagen mRNA and protein synthesis are up-regulated in skin of individuals with scleroderma [59]. In mouse Scl GVHD, we have shown increased synthesis of cutaneous type I collagen, documented by RNase protection assays, RT/PCR, and by immunostaining of frozen skin sections in Scl GVHD [34,35,36]. g. Collagen Degradation This area of research is underdeveloped compared to the study of the immune cells, fibroblasts, and collagen synthesis in scleroderma. It is thought that extracellular matrix deposition is balanced in vivo by degradation, and one theory of scleroderma pathogenesis is that it may be a disease of “imbalance,” where collagen synthesis exceeds degradation. Clearly, some scleroderma patients have had complete resolution of their disease, and it is well known that scleroderma in a single patient is difficult to study over time because disease can wax and wane. Increased levels of tissue inhibitors of metalloproteinases (TIMPS) are documented in scleroderma serum, suggesting decreased collagen degradation [60]. In mouse Scl GVHD, no studies have been performed to date on collagen degradation in the murine model, but it provides the ideal system for those studies.

II. ANIMALS A. Generating Scl GVHD 1. Strains of Mice The two mouse strain pairs used to generate Scl GVHD are B10.D2>BALB/c and C57Bl/6>LPJ. We have the most experience with the B10.D2>BALB/c model and will concentrate on this strain pair in our discussion. The strain designations are BALB/cJ (H-2d, Mls2a, Mls3a) and B10.D2 (nSNJ H-2d, Mls-2a, Mls3a). 2. Sex and Age Male or female mice at 6 to 8 weeks of age are utilized as donors and recipients. We generally transplant female mice because when male mice from different litters are placed together, the fighting that ensues can lead to unwanted injuries with scarring of the skin. We have transplanted bone marrow and spleen from male mice into female mice in order to track donor cells by PCR analysis of Y chromosome sequences [35].



523

B. Inherent Susceptibility to Fibrosis Although it has not been studied in a methodical manner, some individuals are known to be more susceptible to fibrosis than others. For instance, keloid formation tends to be familial and is common in black individuals. There is a correlation between the MHC antigens and ethnically associated scleroderma, most dramatically illustrated in the Choctow American Indians [41]. In mice, the C57Bl/6 strain is more likely than the BALB/c strain to develop lung and skin fibrosis after intratracheal or intradermal administration of bleomycin [61,62]. C57Bl/6 mice are also more likely to develop lung fibrosis when exposed to asbestos fibers than 129 mice [63]. Decreased responsiveness in vitro to peptide growth factor PDGFa and to TNFa of 129 fibroblasts compared to C57BL/6 fibroblasts was suggested as a possible explanation of the fibrosis-resistant phenotype. In addition, C57Bl/6 mice are prone to lung fibrosis after ionizing radiation to the thorax (these doses are much higher than those employed for BMT) [64]. TGF-b that localized to alveolar macrophages by immunostaining was seen in fibrotic lungs of the irradiated mice but not in lungs of control unirradiated C57Bl/6 mice in those experiments. The fibrosis-prone mouse strains such as C57Bl/6 may reflect an inherent strain difference in responses to growth factors or cytokines, leading to tissue injury. Cytokine promoter polymorphisms are implicated in inflammatory disorders such as subacute systemic lupus erythematosus [65] and are another possible explanation for strain differences in fibrosis. Therefore, there are clearly strain differences in susceptibility to fibrosis in mice, but the genetic components of fibrosis are complex and multiple, and are not well characterized at this time.

III. DISEASE INDUCTION A. Mice Preparation Donor and recipient mice (Jackson Laboratory, Bar Harbor, ME) are approximately 6 to 8 weeks old, and have been allowed to adapt for approximately 1 week after shipping. They are maintained from time of arrival in Microisolator cages (Lab Products, Seaford, DE), three to five mice per cage. B. Irradiation of Recipient Mice Recipient mice are irradiated in groups of five to eight with 700 cGy using a Gammacel 137Cs source. BALB/c mice are sensitive to ionizing radiation and increased mortality unrelated to GVHD occurs at higher doses. C. Bone Marrow and Spleen Cell Collection from Donor Mice We use a standard protocol for preparation of transplantation inoculum [66]. Donor mice are sedated with ketamine, and injected intraperitoneally with 100 ml of heparin (10,000 units/ml, Sigma, St. Louis, MO). They are then sacrificed by cervical dislocation, and dipped in iodine solution and then in 75-percent ethanol. 1. Bone Marrow Cells (Source of Hematopoietic Cells for Engraftment) Using ethanol-cleaned surgical instruments, skin is stripped away from the lower extremity starting at the Achilles tendon with an incision. The femur is disarticulated from the pelvic socket with heavy scissors releasing the leg. The soft tissue is then stripped from femur and tibia with


524


fine scissors, and the fibula is discarded. Using heavy scissors, the ends of each bone are clipped and discarded. Three cleaned bones from each leg (femur, knee joint, and tibia) are then placed in a sterile Petri dish with media containing heparin (RPMI 1640 [BioWhittaker, Frederick, MD] with 20 U/ml heparin [Fisher Scientific, Pittsburgh, PA]). Bone marrow is flushed from the bone marrow canal into a 50-ml falcon tube using 27-gauge needles and 5-ml syringes preloaded with media/heparin. Bone marrow cells are resuspended in a final volume of 50 ml of media/heparin, washed two times in media/heparin and counted for total numbers and viability, using trypan blue exclusion. Bone marrow from four to five mice yields approximately 50 to 150 ¥ 106 bone marrow cells for transplantation. Injecting donor animals with 150 mg 5-fluorouracil (5-FU) 2 days before collection of bone marrow can increase yield. The bone marrow cells appear to be more fragile after 5-FU mobilization, however, and must be handled carefully. 2. Spleen Cells (Source of Mature T Cells to Generate GVHD) At the same time that leg bones are collected, spleen is removed, and minced with a sharp scalpel in a sterile Petri dish with media/heparin. Spleen pieces are pressed through an autoclaved stainless steel wire mesh screen over a 50-ml falcon tube to disaggregate cells. The mesh is washed in media/heparin, and spleen cells are collected into 50-ml media/heparin and centrifuged cells are then washed two times in media/heparin and counted for total numbers and viability. D. Transplantation Bone marrow and spleen cells are combined to ultimately deliver a ratio of 2:1 bone marrow (2 ¥ 106) and spleen (1 ¥ 106) cells in a volume of 0.2 ml per mouse. This is a standard dose for generating GVHD [67]. In a pilot experiment with a small number of animals, we found no difference in cutaneous GVHD when we increased the ratio of spleen/bone marrow cells in the inoculum. The bone marrow/spleen preparation is kept at room temperature. If some clotting occurs, the preparation is drawn up repeatedly in a 5-cc syringe with an 18-gauge needle to disaggregate the clots before use. Transplantation must occur within 6 hours of fresh bone marrow/spleen collection in order to have viable transplanted cells. It is also critical to deliver the entire 0.2 ml inoculum. Animals receiving less than the full transplantation inoculum may develop GVHD at a slower rate, producing more variability in skin thickening at given time points after transplantation. Deaths during the transplantation procedure itself are usually due to emboli (clotted bone marrow/spleen preparations or air emboli) during tail vein injections. Deaths at early time points (1 to 2 weeks after transplantation) are most likely due to failure of engraftment. E. Maintenance of Transplanted Recipient Mice Transplanted animals are maintained in Microisolator cages and given autoclaved acidified water (pH 2.5 with acetic acid) and autoclaved chow. Antibiotics are not used in drinking water. F.

Experimental Design

1. Controls for Scl GVHD In addition to transplantation of BALB/c mice with B10.D2 bone marrow/spleen cells (experimental group), transplantation with syngeneic BALB/c bone marrow/spleen cells is always performed (control group).



525

2. Controls for Engraftment Irradiated but not transplanted mice are included in each experiment to confirm that the irradiation dose was lethal. These mice typically die between 7 and 10 days. Engraftment can be evaluated by examination of spleen in sacrificed animals. Numerous colonies should be present in spleens of engrafted animals within 4 to 5 days after transplantation.

IV. COURSE OF DISEASE BALB/c mice transplanted with B10.D2 bone marrow and spleen cells show minimal systemic effects of Scl GVHD at any time point. Both experimental and control groups have a brief period of lethargy following total body irradiation and BMT. The experimental group of mice (B10.D2>BALB/c) do not develop dermatitis, enteritis, or dramatic weight loss in contrast to animals with classic acute GVHD, in which death often occurs within 3 to 7 weeks post-BMT. In classic GVHD, death is most likely due to profound immunosuppression and to loss of barrier function in the gastrointestinal tract and skin, which increases susceptibility to infection. Mice with Scl GVHD fail to gain weight as rapidly as syngeneic BMT control mice or normal mice but mortality is low compared with classic GVHD. In fact, we have followed transplanted animals with Scl GVHD and syngeneic BMT controls to up to 75 days after transplantation, and have noted little morbidity associated with fibrosing disease. In one study, approximately 50% of mice with Scl GVHD (B10.D2>BALB/c) were alive at day 60 post-BMT [25]. The course of their skin manifestation is described next.

V. ASSESSMENT OF DISEASE A. Skin in Scl GVHD When animals are sacrificed for evaluation after day 14 to 21 post-BMT, experimental mice have thicker back skin subjectively than control mice by visual examination. 1. Caliper Measurements Although caliper measurements are routinely used to evaluate mouse ear thickness in contact dermatitis protocols, we have not found caliper measurements of skin to be as reliable as measurements by image analysis of histopathological sections of skin. 2. Histopathological and Morphometric Analysis Skin samples (1-cm strips) are fixed in 10% buffered formalin (Surgipath Medical Industries, Richmond, IL) on filter paper to prevent curling. Quantification of skin thickening is best performed by histological analysis of paraffin sections stained with H and E (Surgipath). We use image analysis of multiple 10¥ views of skin sections, typically six to ten per mouse skin collected from the back, utilizing the Optimas 6.1 program (Bothell, WA). Any image analysis program allowing morphometric analysis can be used. Depending on the quality of histological sections, these results are quite reliable and significantly different in experimental versus control mice by day 21 after BMT. We use area of dermis (from bottom of epidermis to dermis–fat interface) on a 10¥ view of skin as an “integrated” skin thickness. This method minimizes the small variations of dermal skin thickness from one area to another if the thickness is measured directly.


526


a. Routine Histology All the tissue at one time point from one experiment is processed at the same time to obtain consistent cutting and staining conditions. Caveat: If paraffin sections are floated too long on the warming water bath in the histology laboratory, they may spread excessively and lead to unreliable measurements of skin thickness. Numerous holes in the tissue are a clue to this problem that can be solved by recutting sections with careful attention to spreading artifact. Correct orientation of sections is essential; tangentially oriented sections of skin are not measurable. b.

Image Analysis

Images of skin for an experiment are captured at the same time with the same image analysis threshold settings to minimize variability from one microscope slide to another. Then images can be analyzed separately at a later time. 3. Ultrasonographic Evaluation We have also used ultrasonography to evaluate skin thickness on sedated mice with comparable results [34]. DermaScan (Cortex Technology, Madsund, Denmark) makes a 20-MHz instrument designed for evaluation of human skin, and a small probe can be purchased that is appropriate for mouse skin. The advantage to ultrasound is that sequential evaluation of skin thickness is possible without sacrificing the animals. However, ultrasonography is less sensitive than histological measurements that we now use exclusively. 4. Cutaneous Immune Cell Infiltrates We have evaluated density of skin inflammatory infiltrates by immunostaining for T cells and monocyte/macrophages, and have quantified immune cell composition by flow cytometric analysis of single cell suspensions prepared from a 1 ¥ 1–cm piece of back skin. a. Quantification of Immunostaining by Image Analysis This is performed on slides stained with specific antibodies (Abs) and corresponding isotype Abs. Isotype control Ab staining is always tested on the same slide as specific Ab staining, and subtracted in the analysis. Dermal area is calculated in arbitrary square units by outlining the dermis on a 10¥ view for each microscopic image. The same threshold settings are used on the set of slides stained with the same Ab. Density of positive immune cell staining within the outlined areas is plotted as percent of positive area. A minimum of six measurements is taken from two or more skin sections from each animal, and the variation among animals is expressed as standard error. b.

Flow Cytometric Analysis of Cutaneous Immune Cell Infiltrates

A standard method for preparation of cutaneous immune cells for flow cytometric analysis is utilized [68]. Small pieces of depilated skin are digested in RPMI containing 10 mM HEPES (Irvine Scientific, Santa Anna, CA), 0.01% DNase (Sigma), 0.27% collagenase (Sigma), and 1000 units of hyaluronidase (Sigma) at 37∞C for 2 hours. The digested skin is filtered through 100-mm nylon mesh to generate a single-cell suspension of skin cells that contain resident cells (keratinocytes, dendritic cells, fibroblasts, endothelial cells, and perivascular cells such as mast cells) and infiltrating cells (lymphocytes, monocytes, and NK cells). Approximately 4 ¥ 106 cells are typically obtained from a 1 ¥ 2–cm2 piece of skin for control mice, and 8 ¥ 106 cells for mice with Scl GVHD at day 21 post-BMT. Prior to specific antibody staining, all isolated skin cells are blocked with purified



527

mouse IgG (1 mg per 106 cells, Sigma) for 5 minutes on ice. For cytokine and chemokine staining, permeabilization buffer containing 1% saponin is used as washing and staining buffers. Specific and isotype-matched antibodies are then directly applied (1 mg per 106 cells). Prior to fixation with 1% paraformaldehyde in PBS, all samples are washed twice in PBS supplemented with 1% BSA, 1% FCS, and 0.05% sodium azide. Sample data are acquired on a Becton Dickenson FACScan (Franklin Lakes, NJ) and analyzed using Cell Quest software. B. Lung in Scl GVHD Mice with Scl GVHD have increased lung fibrosis by image analysis of routine H and E sections, quantified as a decrease in free alveolar space on 10¥ sections of lung. VI. EXPERT EXPERIENCE In our laboratory, we have characterized the following findings in the mouse Scl GVHD model regarding the skin, lung, and other organ systems. A. Skin 1. Skin Thickening and Collagen Synthesis By histological examination, skin collected from back is approximately 30 to 40% thicker in mice with Scl GVHD than controls (Figures 34.1A and B) by day 21 post-BMT. Routine H and E-stained sections and Masson’s trichrome-stained sections for collagen show increased density of cutaneous collagen. RNase protection assays and semiquantitative RT/PCR analysis of total RNA prepared from back skin show up-regulation of proa1(I) collagen mRNA [34,35]. Increased collagen protein can be documented by immunostaining for type I collagen [35,36]. 2. Keratinocyte Injury Very rare apoptotic cells are found in epidermis at any time point in Scl GVHD (Figures 34.1A and B), consistent with mild GVHD directed to epithelium, whereas the skin fibrosis predominates. This is in dramatic contrast to classic GVHD, where numerous apoptotic keratinocytes are present in epithelium, and their numbers are predictive of severe cutaneous and visceral GVHD [69]. Mucosal epithelia are very sensitive indicators of early or mild GVHD. Mild cytotoxic changes are demonstrated in tongue epithelium (Figures 34.1E and F) where infiltrating lymphocytes and occasional apoptotic keratinocytes are found in experimental (Figure 34.1F) but not in control mice (Figure 34.1E). The lack of significant epithelial injury is consistent with the absence of a scaling dermatitis or diarrhea in Scl GVHD, and may help to explain the longevity of Scl GVHD mice that have little compromise in skin or gut barrier function. 3. Mast Cell Degranulation Increased numbers of degranulated mast cells are seen in Scl GVHD mice compared to controls (Figures 34.1C, D) [70]. 4. Chemokines and Cytokines Up-regulation of mRNA for chemokines RANTES, MCP-1, and MIP-1a is seen in skin at day 21 post-BMT, preceding the influx of cutaneous mononuclear cells [35]. Increased chemokines for


528

Figure 34.1


(A, B) Representative hematoxylin and eosin–stained sections of skin of mice with Scl GVHD (B) compared with syngeneic BMT control mice (A). There is increased skin thickness in B, and an influx of mononuclear cells that is usually in the deep dermis, where fibrosis is first apparent in early scleroderma. Minimal epidermal injury is seen in Scl GVHD. (C, D) Increased mast cell degranulation is seen in routine hematoxylin and eosin-stained sections in skin of Scl GVHD (D) compared with controls (C). (E, F) Mild cytotoxic graft versus host disease is seen in tongue of mice with Scl GHVD (F) compared with controls (E). Apoptotic cells associated with lymphocytes in epidermis (satellitosis) are rare. Slightly increased numbers of lymphocytes tag basal keratinocytes, indicating mild GVHD.

monocyte/macrophages (MCP-1 and MIP-1a) are particularly interesting in view of the predominance of CD11b+ monocyte/macrophages over CD3+ cells in the dermal infiltrates by immunostaining and flow cytometric analysis. 5. Cutaneous Inflammatory Cells We analyzed the composition of dermal immune cells by several methods: immunostaining, flow cytometry, and magnetic bead separation of CD11b+ cells and CD3+ cells. By flow cytometry, the cutaneous immune cells in skin by day 21 post-BMT are predominantly monocyte/macrophages (20 to 22% of total cells in dermal suspensions), whereas the percentage of T cells is less (15%). NK cells are a very minor percentage of total dermal cells (8%) [35]. B. Lungs BALB/c mice transplanted with B10.D2 bone marrow and spleen cells also develop lung fibrosis, measured by image analysis of histological sections as a decrease in free alveolar space (Figures 34.2A and B). Our focus has been on the skin fibrosis; immune cells, chemokines, and cytokines have not yet been characterized in the lungs in this model. C. Spleen The spleen shows histological changes of bone marrow engraftment at times following BMT, with no apparent differences in experimental and control mice (Figures 34.2C and D). Figures 34.2E and F show histology of skin and spleen in a mouse that failed to engraft.



Figure 34.2

529

(A, B) Increased numbers of inflammatory cells and decreased free alveolar space are seen in lungs of mice with Scl GVHD (B) compared with controls (A). (C, D) Spleens of mice with Scl GVHD are comparable to controls by histology of routine hematoxylin and eosin-stained sections. (E, F) Routine hematoxylin and eosin-stained sections of skin (E) and spleen (F) of a moribund mouse that failed to engraft. Skin shows early necrosis with focal areas of epidermal loss, and loss of the subcutaneous fat layer, typically found in cachexia. Spleen histology is abnormal with focal areas of cell death and early necrosis. (G, H) Periportal inflammation is seen in liver of mice with Scl GVHD (H) but not in controls (G) by day 21 post-BMT, consistent with early biliary cirrhosis.

D. Liver Some periportal inflammation is present in liver in Scl GVHD by day 21 (Figures 34.2G and H), consistent with the biliary cirrhosis documented by others. We have not examined liver at later time points. In some early pilot experiments, we observed binding of IgG antibodies in Scl GVHD


530


mouse serum to bile duct epithelium by immunofluorescence on frozen sections of normal mouse liver. Not all mice had these antibodies, however, and they were very low in titer. E. Kidney No inflammation is present in kidneys of mice with Scl GVHD or controls (not shown).

VII. THERAPEUTIC RESPONSES A. Antibodies to TGF-b Since elevated fibrogenic TGF-b mRNA and protein are seen in skin of mice with Scl GVHD, we tested inhibition of Scl GVHD with in vivo anti-TGF-b antibodies. When 150 mg of polyclonal rabbit antimouse TGF-b antibodies (Sigma) are administered by tail vein injection to mice with Scl GVHD at days 1 and 6 (total 300 mg), influx of inflammatory cells into dermis, skin thickening, and lung fibrosis do not occur [34]. The dose was selected based on published studies in other systems in which blocking of fibrosis in vivo by TGF-b antibodies was effective. B. Latency-Associated Peptide Latency-associated peptide (LAP) is the naturally occurring peptide inhibitor of TGF-b that is co-synthesized with TGF-b as a larger precursor protein. LAP is cleaved, dimerizes, and reassociates noncovalently with dimerized TGF-b to inactivate it. This complex of TGF-b and LAP is designated the small latent TGF-b complex. A large latent TGF-b complex (LAP-TGF-b-LTBP) is bound to extracellular matrix via TGF-b binding protein (LTBP) that is encoded on a different chromosome and is synthesized separately from LAP and TGF-b. Together, these forms of LAP-bound TGF-b regulate the release of active TGF-b into tissue. It is thought that a large reservoir of latent TGFb in tissue provides a means to rapidly release active TGF-b following injury [71,72]. Therefore, we also tested the ability of human recombinant LAP (specific for the TGF-b1 isoform, homology to mouse LAP approximately 86%, R&D, Systems Inc., Minneapolis, MN) to prevent skin thickening. When 2 ng of LAP was delivered by tail vein injection at days 1 and 6 post-BMT, skin thickening was completely inhibited and lung fibrosis was partially inhibited [35,36]. Collagen synthesis was decreased, documented by immunostaining with an antibody to newly synthesized mouse type I collagen. In contrast to inhibition with antibody, influx of cutaneous cells and their activation were not blocked by LAP, suggesting that LAP may affect a step further downstream in the TGF-b pathway of fibrosis [36]. TGF-b is a pleiotropic cytokine with many effects on immune cells, and we speculate that the chemokine functions of TGF-b may have been affected by the antibody intervention. However, we have not tested this hypothesis in our model. C. Halofuginone Daily intraperitoneal injections of halofuginone (1 mg/mouse), an alkaloid inhibitor of collagen synthesis, prevented skin thickening and loss of subdermal fat when given for 52 days starting 3 days before transplantation. Skin type I collagen synthesis is decreased [20]. Halofuginone is thought to act directly to inhibit collagen synthesis. D. Nedocromil Sodium Mast cell degranulation is seen in early human scleroderma and mouse Scl GVHD. A mast cell stabilizer, nedocromil sodium, alleviated skin fibrosis when applied topically to mice with Scl



531

GVHD at 5 mg/day from days 3 to 15 post-BMT. Peritoneal and cutaneous mast cell numbers were normalized by this treatment [21].

VIII. CONCLUSION In conclusion, the mouse Scl GVHD model is a useful model for studying human scleroderma because of the following factors: 1. Microchimerism and a chronic GVHD-like reaction may be important in the pathophysiology of scleroderma. 2. Skin and lung fibrosis with inflammatory infiltrates are seen in Scl GVHD that resemble early inflammatory scleroderma. 3. Increased T-cell and monocyte/macrophage chemokines (RANTES, MCP-1, and MIP-1a) and cytokines (TGF-b1) are seen in mouse Scl GVHD that resemble chemokine and cytokine changes in human scleroderma. Therefore, immunomodulatory agents can be selectively targeted to specific cell types, chemokines, or cytokines early when therapy may be more effective. 4. The model allows sequential assessment in developing disease that occurs over a relatively short period of time. The effects of interventions can be readily evaluated in this mouse model in which the immune system can be manipulated. At present, very few transgenic or knockout mice are available on the B10.D2 or BALB/c background. As they become available, the effects of welldefined genetic manipulations in the mice can be studied.

REFERENCES 1. Jaffee, B.D. and Claman, H.N., Chronic graft-versus-host disease (GVHD) as a model for scleroderma. I. Description of model systems, Cell. Immunol., 77, 1, 1983. 2. Claman, H.N. et al., Chronic graft-versus-host disease as a model for scleroderma. II. Mast cell depletion with deposition of immunoglobulins in the skin and fibrosis, Cell. Immunol., 94, 73, 1985. 3. Claman, H.N., Mast cell depletion in murine chronic graft-versus-host disease, J. Invest. Dermatol., 84, 246, 1985. 4. Claman, H.N. et al., Mast cell “disappearance” in chronic murine graft-vs-host disease (GVHD)ultrastructural demonstration of “phantom mast cells,” J. Immunol., 137, 2009, 1986. 5. Choi, K.L., Giorno, R., and Claman, H.N., Cutaneous mast cell depletion and recovery in murine graft-vs-host disease, J. Immunol., 138, 4093, 1987. 6. Choi, K.L. and Claman, H.N., Mast cells in murine graft-versus-host disease: a model of immunologically induced fibrosis, Immunol. Ser., 46, 641, 1989. 7. Claman, H.N., Graft-versus-host disease and animal models for scleroderma, Curr. Opin. Rheumatol., 2, 929, 1990. 8. Claman, H.N., Mast cells and fibrosis. The relevance to scleroderma, Rheum. Dis. Clin. North Am., 16, 141, 1990. 9. Claman, H.N., On scleroderma. Mast cells, endothelial cells, and fibroblasts, JAMA, 262, 1206, 1989. 10. Bangert, J. et al., A comparison of two histologic patterns of murine graft-vs-host skin disease (GVHSD), J. Invest. Dermatol., 80, 329, 1983. 11. Charley, M.R. et al., Murine graft-versus-host skin disease: a chronologic and quantitative analysis of two histologic patterns, J. Invest. Dermatol., 81, 412, 1983. 12. Charley, M.R. and Deng, J.S., Murine models of graft-versus-host disease with features of scleroderma, Immunol. Ser., 46, 625, 1989. 13. Nelson, J.L. et al., Microchimerism and HLA-compatible relationships of pregnancy in scleroderma [see comments], Lancet, 351, 559, 1998. 14. Nelson, J.L., Microchimerism and the pathogenesis of systemic sclerosis, Curr. Opin. Rheumatol., 10, 564, 1998.


532


15. Nelson, J.L., Microchimerism and autoimmune disease, N. Engl. J. Med., 338, 1224, 1998 (letter; comment). 16. Johnson, K.L. et al., Fetal cell microchimerism in tissue from multiple sites in women with systemic sclerosis, Arthritis Rheum., 44, 1848, 2001. 17. Maloney, S. et al., Microchimerism of maternal origin persists into adult life, J. Clin. Invest., 104, 41, 1999. 18. Levi-Schaffer, F. et al., Effect of coculture of rodent mast cells with murine chronic graft-versus-host disease (cGVHD)-derived fibroblasts, J. Allergy Clin. Immunol., 89, 501, 1992. 19. Levi-Schaffer, F. and Rubinchik, E., Mast cell/fibroblast interactions, Clin. Exp. Allergy, 24, 1016, 1994. 20. Levi-Schaffer, F. et al., Inhibition of collagen synthesis and changes in skin morphology in murine graft-versus-host disease and tight skin mice: effect of halofuginone, J. Invest. Dermatol., 106, 84, 1996. 21. Levi-Schaffer, F. et al., Nedocromil sodium ameliorates skin manifestations in a murine model of chronic graft-versus-host disease, Bone Marrow Transplant., 19, 823, 1997. 22. Levi-Schaffer, F. et al., Regulation of the functional activity of mast cells and fibroblasts by mononuclear cells in murine and human chronic graft-vs.-host disease, Exp. Hematol., 25, 238, 1997. 23. Rubinchik, E. and Levi-Schaffer, F., Mast cells and fibroblasts: two interacting cells, Int. J. Clin. Lab. Res. 24, 139, 1994. 24. Pines, M. and Nagler, A., Halofuginone: a novel antifibrotic therapy, Gen. Pharmacol., 30, 445, 1998. 25. Chen, B.J., Morris, R.E., and Chao, N.J., Graft-versus-host disease prevention by rapamycin: cellular mechanisms, Biol. Blood Marrow Transplant., 6, 529, 2000. 26. Vierling, J.M. et al., Hepatic lesions in murine chronic graft-versus-host disease to minor histocompatibility antigens. A reproducible model of nonsuppurative destructive cholangitis, Transplantation, 48, 717, 1989. 27. Ilan, Y. et al., Induction of oral tolerance in splenocyte recipients toward pretransplant antigens ameliorates chronic graft versus host disease in a murine model, Blood, 95, 3613, 2000. 28. Nagler, A. et al., Oral tolerization ameliorates liver disorders associated with chronic graft versus host disease in mice, Hepatology, 31, 641, 2000. 29. Mayo, M.J. et al., Association of clonally expanded T cells with the syndrome of primary biliary cirrhosis and limited scleroderma, Hepatology, 29, 1635, 1999. 30. Howell, C.D. et al., Liver T cell subsets and adhesion molecules in murine graft-versus-host disease, Bone Marrow Transplant., 16, 139, 1995. 31. Li, J., Helm, K., and Howell, C.D., Contributions of donor CD4 and CD8 cells to liver injury during murine graft-versus-host disease, Transplantation, 62, 1621, 1996. 32. Howell, C.D. et al., Biased liver T cell receptor V beta repertoire in a murine graft-versus-host disease model, J. Immunol., 155, 2350, 1995. 33. Levy, S. et al., Parotid salivary gland dysfunction in chronic graft-versus-host disease (cGVHD): a longitudinal study in a mouse model, Bone Marrow Transplant., 25, 1073, 2000. 34. McCormick, L.L. et al., Anti-TGF-beta treatment prevents skin and lung fibrosis in murine sclerodermatous graft-versus-host disease: a model for human scleroderma, J. Immunol., 163, 5693, 1999. 35. Zhang, Y. et al., Murine sclerodermatous graft-versus-host disease, a model for human scleroderma: cutaneous cytokines, chemokines, and immune cell activation, J. Immunol., 168, 3088, 2002. 36. Zhang, Y., McCormick, L.L., and Gilliam, A.C., Latency-associated peptide prevents skin fibrosis in sclerodermatous graft-versus-host disease, a model for human scleroderma., J.. Invest. Dermatol., in press. 37. Zhang, Y. and Gilliam, A.C., Animal models for scleroderma: an update, Curr. Rheumatol. Rep., 4, 150, 2002. 38. Gilliam, A.C. and Murphy, G.F., Cellular pathology of cutaneous graft-versus-host disease, in Graft versus Host Disease, 2nd ed., Burakoff, S.J. et al., Eds., Marcel Dekker, Inc., New York, 1997, p. 291. 39. Chosidow, O. et al., Sclerodermatous chronic graft-versus-host disease. Analysis of seven cases, J. Am. Acad. Dermatol., 26, 49, 1992. 40. Charley, M.R., Gilliam, J.N., and Sontheimer, R.D., Ultraviolet B exposure converts murine lichenoid graft-vs-host disease (GVHSD) into sclerotic GVHSD, J. Invest. Dermatol., 80, 328, 1983.



533

41. Johnson, R.W., Tew, M.B., and Arnett, F.C., The genetics of systemic sclerosis, Curr. Rheumatol. Rep., 4, 99, 2002. 42. Seibold, J.R., Giorno, R.C., and Claman, H.N., Dermal mast cell degranulation in systemic sclerosis, Arthritis Rheum., 33, 1702, 1990. 43. Distler, O. et al., Expression of RANTES in biopsies of skin and upper gastrointestinal tract from patients with systemic sclerosis, Rheumatol. Int., 19, 39, 1999. 44. Hasegawa, M., Sato, S., and Takehara, K., Augmented production of chemokines (monocyte chemotactic protein-1 (MCP- 1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis, Clin. Exp. Immunol., 117, 159, 1999. 45. White, B., Immune abnormalities in systemic sclerosis, Clin. Dermatol., 12, 349, 1994. 46. Luzina, I.G. et al., Gene expression in bronchoalveolar lavage cells from scleroderma patients, Am. J. Respir. Cell Mol. Biol., 26, 549, 2002. 47. Rose, N.R. and Leskovsek, N., Scleroderma: immunopathogenesis and treatment, Immunol. Today, 19, 499, 1998. 48. White, B., Immunopathogenesis of systemic sclerosis, Rheum. Dis. Clin. North Am., 22, 695, 1996. 49. Yurovsky, V.V., The repertoire of T-cell receptors in systemic sclerosis, Crit. Rev. Immunol., 15, 155, 1995. 50. Valentini, G. et al., Peripheral blood T lymphocytes from systemic sclerosis patients show both Th1 and Th2 activation, J. Clin. Immunol., 21, 210, 2001. 51. Chen, W. and Howell, C.D., Oligoclonal expansion of T cell receptor V beta 2 and 3 cells in the livers of mice with graft-versus-host disease, Hepatology, 35, 23, 2002. 52. Kraling, B.M., Maul, G.G., and Jimenez, S.A., Mononuclear cellular infiltrates in clinically involved skin from patients with systemic sclerosis of recent onset predominantly consist of monocytes/macrophages, Pathobiology, 63, 48, 1995. 53. Szekanecz, Z. et al., Chemokines in rheumatoid arthritis, Springer Semin. Immunopathol., 20, 115, 1998. 54. Szekanecz, Z. et al., Temporal expression of inflammatory cytokines and chemokines in rat adjuvantinduced arthritis, Arthritis Rheum., 43, 1266, 2000. 55. Palframan, R.T. et al., Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues, J. Exp. Med., 194, 1361, 2001 (see comments). 56. Peltonen, J. et al., Evaluation of transforming growth factor b and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization, J. Invest. Dermatol., 94, 365, 1990. 57. Shi-wen, X. et al., Autocrine overexpression of CTGF maintains fibrosis: RDA analysis of fibrosis genes in systemic sclerosis, Exp. Cell Res., 259, 213, 2000. 58. Denton, C.P. and Abraham, D.J., Transforming growth factor-beta and connective tissue growth factor: key cytokines in scleroderma pathogenesis, Curr. Opin. Rheumatol., 13, 505, 2001. 59. LeRoy, E.C., Increased collagen synthesis by scleroderma skin fibroblasts in vitro, J. Clin. Invest., 54, 880, 1974. 60. Young-Min, S.A. et al., Serum TIMP-1, TIMP-2, and MMP-1 in patients with systemic sclerosis, primary Raynaud’s phenomenon, and in normal controls, Ann. Rheum. Dis., 60, 846, 2001. 61. Schrier, D.J., Kunkel, R.G., and Phan, S.H., The role of strain variation in murine bleomycin-induced pulmonary fibrosis, Am. Rev. Respir. Dis., 127, 63, 1983. 62. Hoyt, D.G. and Lazo, J.S., Alterations in pulmonary mRNA encoding procollagens, fibronectin and transforming growth factor-beta precede bleomycin-induced pulmonary fibrosis in mice, J. Pharmacol. Exp. Ther., 246, 765, 1988. 63. Brass, D.M., Tsai, S.Y., and Brody, A.R., Primary lung fibroblasts from the 129 mouse strain exhibit reduced growth factor responsiveness in vitro, Exp. Lung Res., 27, 639, 2001. 64. Rube, C.E. et al., Dose-dependent induction of transforming growth factor beta (TGF-beta) in the lung tissue of fibrosis-prone mice after thoracic irradiation, Int. J. Radiat. Oncol. Biol. Physics, 47, 1033, 2000. 65. Werth, V.P. et al., Association of a promoter polymorphism of tumor necrosis factor-alpha with subacute cutaneous lupus erythematosus and distinct photoregulation of transcription, J. Invest. Dermatol., 115, 726, 2000.


534


66. Korngold, R. and Sprent, J., Graft-versus-host disease in experimental allogeneic bone marrow transplantation, Proc. Soc. Exp. Biol. Med., 197, 12, 1991. 67. Korngold, R., Pathophysiology of graft-versus-host disease directed to minor histocompatibility antigens, Bone Marrow Transplant., 7 (Suppl. 1), 38, 1991. 68. Hammerberg, C., Duraiswamy, N., and Cooper, K.D., Temporal correlation between UV radiation locally-inducible tolerance and the sequential appearance of dermal, then epidermal, class II MHC+CD11b+ monocytic/macrophagic cells, J. Invest. Dermatol., 107, 755, 1996. 69. Murphy, G.F. et al., Characterization of target injury of murine acute graft-versus-host disease directed to multiple minor histocompatibility antigens elicited by either CD4+ or CD8+ effector cells, Am. J. Pathol., 138, 983, 1991. 70. McCormick, L.L. et al., Activated macrophages express scavenger receptors in skin of mice with sclerodermatous graft versus host disease, a model for scleroderma, J. Invest. Dermatol., 117, 443, 2001. 71. Letterio, J.J. and Roberts, A.B., Regulation of immune responses by TGF-beta, Annual Rev. Immunol., 16, 137, 1998. 72. Roberts, A.B., Molecular and cell biology of TGF-b, Minor Electrolyte Metab., 24, 111, 1998.


CHAPTER

35

Experimental Mouse Model of Scleroderma: Induction by Bleomycin Toshiyuki Yamamoto

CONTENTS I. II. III. IV. V.

History ................................................................................................................................535 Animals ..............................................................................................................................536 Disease Induction...............................................................................................................537 Course of Disease ..............................................................................................................538 Assessment of Disease.......................................................................................................538 A. Clinical Manifestation ...............................................................................................538 B. Histopathological Examination .................................................................................538 C. Immunopathological Data .........................................................................................539 D. Biochemical Data ......................................................................................................539 E. Serum Cytokine .........................................................................................................539 VI. Therapeutic Responses.......................................................................................................541 VII. Expert Experience ..............................................................................................................542 VIII. Conclusion..........................................................................................................................542 References ......................................................................................................................................543

I. HISTORY Scleroderma is a connective tissue disease which shows fibrosis of the skin [1,2]. Although the pathogenesis of scleroderma is not fully elucidated as yet, it is characterized by excessive accumulation of extracellular matrix (ECM) in the skin and various internal organs, vascular injury, and immunological abnormalities [3]. In early stages of scleroderma, activated fibroblasts in the affected areas produce high amounts of collagen [4–6]. Histological analysis of the initial stage of scleroderma reveals perivascular infiltrates of mononuclear cells in the dermis, which is associated with increased collagen synthesis in the surrounding fibroblasts [7,8]. A number of studies have demonstrated the crucial role of several fibrogenic cytokines released from immunocytes infiltrating in the affected sites for initiating and/or leading to the sequential events of fibrosis [9–12].

0-8493-1391-0/04/$0.00+$1.50 © 2004 by CRC Press LLC

535


536


Although animal models are useful to provide clues and therapeutic interventions for various human diseases, animal models that exhibit al aspects of systemic sclerosis (SSc) are not currently available. Bleomycin is produced by Streptomyces verticullis, and is a frequently used antitumor antibiotic for various kinds of cancers [13]. Lung fibrosis is a well-known side effect of bleomycin. In addition, cutaneous changes including fibrosis, hyperpigmentation, alopecia, gangrene, edema, Raynaud’s phenomenon, and “flagellate” erythema (scratch dermatitis) have been described [14–17]. Scleroderma is also reported to be developed in malignancy-bearing patients after bleomycin therapy [18–20]. Bleomycin-induced pulmonary fibrosis, an established rodent model that has been extensively investigated [21–26], resembles human lung fibrosis histologically and biochemically. Rodents develop pulmonary fibrosis when given bleomycin by intratracheal or intravenous administration or by 1-week continuous infusion [27,28]. In these animals, the amounts of lung ECM were found to be increased [29–31]. In vitro, bleomycin up-regulates collagen mRNA expression in human lung or dermal fibroblasts [32,33]. Bleomycin was also shown to induce alveolar [34,35] as well as peripheral [36] macrophages to produce growth stimulatory factors for fibroblasts. Recent in vitro findings suggest that lung fibroblasts show chemotactic activity for neutrophils and monocytes in response to bleomycin [37]. Previously, Mountz et al. [38] reported that rats injected repeatedly with sublethal doses of bleomycin over a 58-week period developed severe dermal fibrosis similar to those found in human scleroderma, with structural abnormalities of collagen fibers. We have recently established a mice model for scleroderma by repeated local treatment of bleomycin [39–43]. Daily injections of bleomycin induced dermal sclerosis, but not fibrosis, in Balb/c mice after 4 weeks. Histological examination demonstrated thickened collagen bundles, deposition of homogenous materials in the bleomycin-injected skin with cellular infiltrates, which mimicked the histological features of human scleroderma.

II. ANIMALS Mice were usually used at 4 to 6 weeks old. Dermal sclerosis can be induced after 4 weeks of injections of bleomycin in various mice strains, although there is some variation among strains in the intensity of dermal sclerosis [40]. There is no difference of the intensity of dermal sclerosis between males and females. In particular, C3H/He, DBA2, B10.D2, and B10.A mice developed intense dermal sclerosis characterized by deposition of homogenous materials in the dermis and thickened collagen bundles. In A/J, C3H/He, B10.A, and B10.D2 mice, dermal thickness showed a more than 2.5-fold increase, as compared with phosphate buffered saline (PBS) treatment. On the contrary, dermal thickness was not strongly induced in C57BL/6J strain. Recent findings suggest that mast cells are important initiators of SSc [44], since mast cells are increased in number in the lesional skin of early stage scleroderma [45,46]. Mast cells produce a number of cytokines, growth factors, and mediators that are capable of activating fibroblasts or endothelial cells [47]. Furthermore, mast cells abundantly produce chemokines, including RANTES, macrophage inflammatory protein-1a (MIP-1a), and monocyte chemoattractant protein1 (MCP-1) [48]. In particular, MCP-1 is shown to up-regulate type I collagen gene expression in cultured fibroblasts [49], suggesting an important involvement in fibrosis. In tight skin (Tsk) mice, mast cells are abundant in the thickened dermis and exhibit prominent degranulation [50]. A decrease in fibrosis associated with inhibition of mast cell degranulation by cromolyn and ketotifen was also reported [50,51]. In this model, mast cells were increased in number in parallel with the induction of dermal sclerosis. Also, a marked degranulation was found in particular at the early phase, with elevated plasma histamine levels [39]. On the contrary, bleomycin could induce dermal sclerosis even in genetically mast cell-deficient WBB6F1-W/WV mice similarly to control littermates [40].


EXPERIMENTAL MOUSE MODEL OF SCLERODERMA: INDUCTION BY BLEOMYCIN

537

Although transforming growth factor-b (TGF-b), a key fibrogenic cytokine, is produced by mast cells, macophages are the main source of TGF-b. TGF-b can be detected immunohistologically on the infiltrating cells in both WBB6F1+/+ and WBB6F1-W/WV mice [40]. We thus speculate that mast cells may be associated but not necessary for the induction of dermal sclerosis. Mast cells may be not the sole pathway to the induction of dermal sclerosis. Mononuclear cell infiltrates in the skin is one of the most characteristic histological features in early scleroderma [52], which is suggested to secret cytokines stimulating ECM production. Moreover, infiltrating T lymphocytes, predominately CD4+, are also the major lymphocytes seen in the involved skin of scleroderma. As in human SSc, T cells, macrophages and mast cells are present in increased numbers or an activated state in involved tissues in animal models of SSc. In vivo T-cell depletion with anti-T-cell antibodies has been shown to either reduce or completely abrogate bleomycin-induced pulmonary fibrosis [53,54]. On the other hand, results of athymic nude mice lacking functional T lymphocytes have been controversial. One study showed histologically similar fibrosis in nude, euthymic mice after bleomycin treatment [55], whereas another study showed that bleomycin was not fibrogenic in nude mice [56]. On the contrary, a recent study demonstrated that bleomycin-induced lung fibrosis occurred in C57BL/6 severe combined immunodeficient (SCID) and (C57BL/6¥CB.17)F1 SCID mice comparable to that seen in wild-type mice, suggesting that initial induction of lung fibrosis is lymphocyte independent [57]. Our results using SCID mice confirmed that dermal sclerosis can be induced even in SCID mice comparable to control mice, suggesting that bleomycin-induced scleroderma is independent of T cells [42]. Furthermore, we have confirmed that dermal sclerosis can be induced also in nude mice (T.Y., unpublished data, 2002). These results suggest that dermal sclerosis can be inducible without the involvement of immunocytes. It is shown that bleomycin directly affects on lung fibroblasts to up-regulate collagen synthesis in vitro [32]. We also indicated that bleomycin up-regulates mRNA expressions of type I collagen and fibronectin, as well as fibrogenic cytokines in cultured normal skin fibroblasts [33]. In situ hybridization showed that cultured fibroblasts incubated with bleomycin expressed a1(I) collagen transcripts more intensely, as compared with untreated fibroblasts (Figure 35.1). Bleomycin exposure to rat lung fibroblast cultures results in elevated TGF-b mRNA synthesis, TGF-b mRNA steady-state levels, and TGF-b protein [58]. Increased TGF-b mRNA transcription is followed by TGF-b mRNA accumulation and TGF-b protein, which is followed by increased procollagen gene transcription [58]. A recent study showed that TGF-b is a mediator of the fibrotic effect of bleomycin at the transcriptional level and that the TGF-b response element is required for bleomycin stimulation of the pro a1(I) collagen promoter [59]. Taken together, these results show that induction of dermal sclerosis in this model can be mediated by fibrogenic cytokines released from various immunocytes, or by direct effect of bleomycin increasing ECM production, which is in part mediated by TGF-b. III. DISEASE INDUCTION Bleomycin is dissolved into PBS, and sterilized by filtration. Dermal sclerosis was induced by subcutaneous injections of 100 ml of bleomycin into the shaved back skins with 27-gauge needles every day for about 4 weeks. The concentrations of bleomycin used in the injection were ranging from 100 mg/ml to 1 mg/ml. Histopathological examination revealed definite dermal sclerosis characterized by deposition of homogenous materials in the thickened dermis with cellular infiltrates. Masson trichrome stain showed dense deposition of collagen in the thickened dermis. Dermal thickness was gradually increased, and significantly increased up to two-fold when the sclerosis was developed. Furthermore, in some strains, epidermal thickness was also induced as well [41].


538


B

A

C

Figure 35.1

In situ expression of a1(I) collagen in cultured human skin fibroblasts. (A) Fibroblasts were hybridized with control sense probe and (B) antisense probe. (C) Fibroblasts stimulated with bleomycin for 24 hours were hybridized with antisense probe.

IV. COURSE OF DISEASE Dermal sclerosis is gradually induced and histological sclerosis was observed after 4 weeks of treatment with bleomycin. Other than skin, lung fibrosis with thickened alveolar walls with cellular infiltrates was also observed early on. However, the kidney, liver, and heart were not involved. Cutaneous changes were relatively localized to around the injected site skin, and sclerotic changes were not induced in the remote region, such as fingers or abdominal skin. The induced sclerotic changes remained at least 6 weeks, when untreated.

V. ASSESSMENT OF DISEASE A. Clinical Manifestation Clinically, dermal sclerosis at the site of injections is not significant by cross inspection. B. Histopathological Examination Histological examination showed the changes with thickened and homogenous collagen bundles and cellular infiltrates in the dermis in mice treated with bleomycin, compared with those treated with PBS (Figure 35.2A and B). Mononuclear cell infiltration was observed in the dermis, which mainly consisted of CD4+ T cells and macrophages (Figure 35.2C). Thickening of vascular walls



539

was also found in the lower dermis (Figure 35.2D). Toluidine blue stain demonstrated increased numbers of mast cells around sclerotic lesions. In particular, degranulation was prominent an early phase (1 to 2 weeks) (Figure 35.2E). Myofibroblasts have features that intermediate between those of the fibroblasts and smooth muscle cells. They are observed in the wound healing [60,61] or tissue fibrotic process, including liver cirrhosis [62,63], kidney [64], lung [65], or skin [66]. It is shown that scleroderma skin expresses a-smooth muscle actin (a-SMA), suggesting myofibroblastic phenotype [67]. Myofibroblasts are shown to persist in scleroderma fibroblasts cultures [67]. TGF-b and platelet-derived growth factor (PDGF), fibrogenic cytokines important in fibrosis process, induce the expression of a-SMA by fibroblasts [68,69]. In our model, a-SMA–positive myofibroblast-like cells were observed in the dermis at 1 week, and gradually increased in parallel with the induction of dermal sclerosis [43]. Mononuclear infiltration in the lung was also found, and alveolar wall thickening was observed in the sclerotic stages (Figure 35.2F). Expression of type I collagen was enhanced in the sclerotic skin following bleomycin treatment (Figure 35.2G). C. Immunopathological Data TGF-b plays an important role in the fibrotic process [70]. TGF-b, which is found abundantly in platelets and released from activated macrophages or lymphocytes, is a strong chemoattractant for fibroblasts [71]. TGF-b increases the synthesis of collagen type I and type III or fibronectin by many cell types in vitro [72–74]. In addition, TGF-b modulates cell-matrix adhesion protein receptors [75,76]. TGF-b also regulates the production of proteins that can modify the ECM by proteolytic action, such as plasminogen activator, an inhibitor of plasminogen, or procollagenase [77–79]. TGF-b is capable of stimulating its own synthesis by fibroblasts through autoinduction [80]. Thus, maintenance of increased TGF-b production may lead to the progressive deposition of ECM, resulting in fibrosis. TGF-b induces rapid fibrosis and angiogenesis when injected subcutaneously into newborn mice [81]. Thus, multiple actions of TGF-b are supportive of the notion that TGF-b plays a key role in the pathogenesis of scleroderma. Bleomycin induces alveolar macrophages to secrete TGF-b [82]. In this model, immunohistological analysis showed that TGF-b was detected on the infiltrating cells, which were predominantly composed of macrophages, as well as fibroblasts at the sclerotic stage following bleomycin treatment. TGF-b1 and TGF-b2 mRNA expression were also detected in the lesional skin. In addition, recent findings show that scleroderma fibroblasts express elevated levels of TGFb receptor type I and type II mRNA, which correlate with elevated a2(I) collagen mRNA levels, suggesting that activation in scleroderma fibroblasts is, in part, due to an autocrine TGF-b loop [83]. We have recently observed increased immunohistological localization of TGF-b receptor I and II in sclerotic fibroblasts in the bleomycin-treated skin (T.Y., unpublished data, 2002). D. Biochemical Data Hydroxyproline content in the skin was significantly increased as compared with control PBStreated skin in parallel with the development of dermal sclerosis. SDS-PAGE showed up-regulation of type I collagen in the sclerotic skin. Northern blot analysis revealed up-regulation of type I collagen mRNA expression in the sclerotic skin after bleomycin treatment [39]. Of interest, autoantibody was detected in the serum after repeated bleomycin treatment [39]. E. Serum Cytokine Recent hypotheses have indicated that an imbalance exists between the type 1 and type 2 cytokine response in the pathogenesis of scleroderma. Type 2 cytokines include interleukin (IL)-4, IL-5, IL-10, IL-13 and MCP-1. A recent report shows that most CD4+ T-cell clones generated from


540


A

C

F

Figure 35.2

B

D

E

G

Histopathological evaluation of dermal sclerosis induced by bleomycin. Mice were treated with (A) PBS or (B) bleomycin for 4 weeks. Note marked dermal sclerosis with thickened and homogeneous collagen bundles in the dermis. (C) Cellular infiltrates of macrophages stained for BM-8. (D) Thickened vascular walls in the lower dermis. (E) Marked degranulation of mast cells shown by toluidine blue stain. (F) Lung sections showing thickened alveolar walls with cellular infiltrates. (G) Immunohistological expression of type I collagen in bleomycin-treated mice.

scleroderma skin biopsies exhibited type 2 cytokine profiles [84]. IL-4, which is produced by activated memory T-cells and mast cells, is known to promote fibroblast proliferation, collagen gene expression, and collagen synthesis [85–87]. Increased IL-4 and IL-6 levels are detected in the sera of SSc patients [88]. Serum in the majority of SSc patients showed elevated levels of CD30 [84], which is expressed on activated type 2 cells. In our model, serum IL-4, IL-6, and tumor necrosis factor-a (TNF-a) levels were significantly elevated following bleomycin treatment.



541

VI. THERAPEUTIC RESPONSES As mentioned above, TGF-b is suggested to play a crucial role in fibrosis. Indeed, TGF-b mRNA is elevated in the lesional skin of SSc and other fibrotic conditions [89–91], and also shown to co-localize with type I collagen in scleroderma skin lesions [89]. Recent studies have demonstrated that blockade of TGF-b by either antibody against TGF-b or soluble TGF-b receptor inhibits the development of tissue fibrosis in experimental animal models [92–96]. Bleomycin up-regulates TGF-b1 mRNA expression in cultured rat lung [32] or human skin fibroblasts [33]. This bleomycin sensitivity requires the activation of a specific TGF-b promoter-binding protein [59]. We observed increased expression of TGF-b at protein and mRNA levels in the lesional skin following bleomycin treatment, and further demonstrated that systemic administration of anti-TGF-b antibody, which cross-reacts with TGF-b1 and -b2, in combination with local bleomycin treatment suppressed the development of scleroderma [92]. However, on account of the problem of half-life, repeated injections of TGF-b antibody were required. Gene therapy targeting TGF-b signaling may be expected. Interferons (IFNs), in particular IFN-g, cause potent inhibition of collagen production, which correlates with reduction in the corresponding steady-state mRNA levels, in cultured skin fibroblasts [97–101]. In vitro study showed that IFN-g decreased TGF-b-induced a-SMA expression in palatal fibroblasts, as well as alteration of morphology [102]. In this model, systemic administration of IFN-g reduced dermal sclerosis even after the onset of scleroderma [103]. We speculate that IFNg was effective because it was administered just after the final treatment of bleomycin. On the other hand, IFN-a did not suppress the dermal sclerosis induced by bleomycin, which is consistent with the in vitro effect [98] and clinical efficacy [104]. IFN-g is a powerful type 1 inducer of cellular immunity that may indirectly contribute to the improvement of the imbalance of the type 2 shift. A recent report has shown that IFN-g inhibits the TGF-b–induced phosphorylation of Smad3 and the accumulation of Smad3 in the nucleus, whereas it induces the expression of Smad7, which prevents the interaction of Smad3 with the TGF-b receptor [105]. Bleomycin is known to generate reactive oxygen species (ROS), such as superoxide and hydroxyl radicals. ROS can cause several abnormalities such as endothelial cell damage or enhanced platelet activation, leading to up-regulation of expression of adhesion molecules or secretion of inflammatory or fibrogenic cytokines including PDGF and TGF-b. Other effects of oxygen radicals include the stimulation of skin fibroblast proliferation at low concentrations [106], and the production of increased amounts of collagen [107]. A recent study has demonstrated that several of the autoantigens targeted by scleroderma autoantibodies are fragmented in the presence of ROS and specific metals such as iron or copper [108]. They suggest that tissue ischemia generates ROS, which induces the fragmentation of specific autoantigens. These findings support the notion that scleroderma is characterized by ischemia-reperfusion injury, overproduction of ROS is commonly found in scleroderma patients with active disease [109], and scleroderma is occasionally associated with a variety of environmental toxins or organic solvents. Therefore, a reduction of free radical formation may contribute to the decrease of collagen content by inhibition of proline hydroxylation, which leads to the improvement of scleroderma. We observed the inhibitory effect of lecithinized superoxide dismutase (SOD), which shows high tissue accumulation and long half-life in blood, on bleomycin-induced scleroderma [110]. On the contrary, postonset administration of SOD could not attenuate the dermal sclerosis. Recent studies demonstrate that halofuginone, an alkaloid originally isolated from the plant Dichroa febrifuga, suppressed avian skin collagen synthesis in vivo [111]. In vitro, halofuginone attenuates collagen synthesis, as well as collagen gene expression in avian and murine skin fibroblasts [112]. Halofuginone specifically inhibits a1(I) collagen gene expression without affecting the synthesis of other types of collagen such as types II and III [112,113]. Halofuginone prevents skin fibroblasts in murine models of scleroderma, the chronic GVHD and the Tsk mouse [114].


542


On the contrary, we could not confirm the efficacy of this agent on bleomycin-induced scleroderma in our system [115].

VII. EXPERT EXPERIENCE Both intradermal as well as subcutaneous bleomycin injections can induce dermal sclerosis, because mouse skin is very thin. Besides daily injections, alternate day administration can also induce dermal sclerosis.

VIII. CONCLUSION We described here the histologic, immunologic, and biochemical characteristics of an experimental mouse model for scleroderma induced by bleomycin. Table 35.1 summarizes various aspects in this model. Skin sclerosis was induced only at the injected site skin in this model. In addition, gastrointestinal, cardiac, hepatic and renal involvement were also absent, although lung fibrosis was observed. The possible mechanisms of bleomycin-induced scleroderma are speculated in Figure 35.3. The induction of dermal sclerosis is, in part, mediated by fibrogenic cytokines, that is, TGF-b, derived from immunocytes. On the other hand, this experimental model may be specific for bleomycin, because of its direct effect on ECM synthesis in fibroblasts that modulate immune cell behavior by conditioning the local cellular and cytokine microenvironment [116]. To explore the pathogenesis of this model may enhance our understanding of the pathogenesis of human scleroderma, although this model may represent a specific subtype of scleroderma compulsorily induced by bleomycin. Animal models for scleroderma present promising tools for future studies of cellular and molecular mechanisms of this disease, and for the evaluation of new therapeutic interventions such as gene therapy.

Table 35.1 Characterization of Bleomycin-Induced Scleroderma Feature Scleroderma Sclerodactyly Epidermal proliferation Dermal sclerosis Dermal thickness Thickened collagen fibers Collagen deposition Thickened vascular wall Mononuclear cell infiltrate Mast cell increase Visceral involvement Lung Increased tissue collagen Increased procollagen gene expression Myofibrotic change Autoantibody

Bleomycin-Induced Scleroderma + + + + + + + + + + + + +



543

Bleomycin

T cell

Th1/2

Mast cell

Macrophage Endothelial cell

B cell

adhesion molecule

bFGF

TGF-β autoantibody

bFGF

TGF-β

IL-1 TNF-α MCP-1

TGF-β

Fibroblast altered gene expression oxidant damage

Integrin TGF-β CTGF PDGF Extracellular matrix

Proliferation Figure 35.3

Schematic design of the role of bleomycin in the pathogenesis of cutaneous fibrosis/sclerosis.

REFERENCES 1. Krieg, T. and Meurer, M., Systemic scleroderma. Clinical and pathophysiological aspects, J. Am. Acad. Dermatol., 18, 457, 1988. 2. LeRoy, E.C. et al., Scleroderma (systemic sclerosis): classification, subsets and pathogenesis, J. Rheumatol., 15, 202, 1988. 3. Jimenez, S.A. and Christner, P., Animal models of systemic sclerosis, Clin. Dermatol., 12, 425, 1994. 4. Bostein, G.R., Sherer, G.K., and LeRoy, E.C., Fibroblast selection in scleroderma. An alternative model of fibrosis, Arthritis Rheum., 25, 189, 1982. 5. Krieg, T. et al., Collagen synthesis by scleroderma fibroblasts, Ann. N. Y. Acad. Sci., 460, 375, 1986. 6. Mauch, C. et al., Control of fibrosis in systemic scleroderma, J. Invest. Dermatol., 100, 92, 1993. 7. Fleischmajer, R., Perlish, J.S., and Reeves, J.R.T., Cellular infiltrates in scleroderma skin, Arthritis Rheum., 20, 975, 1977. 8. Scharffetter, K., Lankat-Buttgereit, B., and Krieg, T., Localization of collagen mRNA in normal and scleroderma skin by in-situ hybridization, J. Clin. Invest.,Eur. J. Clin. Invest., 18, 9, 1988. 9. Fagundus, D.M. and LeRoy, E.C., Cytokines and systemic sclerosis, Clin. Dermatol., 12, 407, 1994. 10. Postlethwaite, A.E., Connective tissue metabolism including cytokine in scleroderma, Curr. Opin. Rheumatol., 7, 535, 1995. 11. Kovacs, E.J. and DiPietro, L.A., Fibrogenic cytokines and connective tissue production, FASEB J., 8, 854, 1994.


544


12. LeRoy, E.C. et al., A strategy for determining the pathogenesis of systemic sclerosis. Is transforming growth factor the answer? Arthritis Rheum., 32, 817, 1989. 13. Umezawa, H. et al., New antibiotics, bleomycin A and B, J. Antibiot., 19, 200, 1966. 14. Cohen, I.S. et al., Cutaneous toxicity of bleomycin in therapy, Arch. Dermatol., 107, 553, 1973. 15. Nixon, D.W., Pirozzi, D., and York, R.M., Dermatologic changes after systemic cancer therapy, Cutis, 27, 181, 1981. 16. Yamamoto, T., Yokozek,i H., and Nishioka, K., Dermal sclerosis in the lesional skin of ‘flagellate’ erythema (scratch dermatitis) induced by bleomycin, Dermatology, 297, 399, 1998. 17. Surville-Barland, J. et al., Bleomycin-induced digital gangrene, Eur. J. Dermatol., 8, 221, 1998. 18. Finch, W.R. et al., Bleomycin-induced scleroderma, J. Rheumatol., 7, 651, 1980. 19. Kerr, L.D. and Spiera, H., Scleroderma in association with the use of bleomycin: a report of 3 cases, J. Rheumatol., 19, 294, 1992. 20. Passiu, G. et al., Bleomycin-induced scleroderma: report of a case with a chronic course rather than the typical acute/subacute self-limiting form, Clin. Rheumatol., 18, 422, 1999. 21. Snider, G.L. et al., , E.C.Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin, Am. Rev. Respir. Dis., 117, 289, 1978. 22. Zia, S., Hyde, D.M., and Giri, S.N., Development of a bleomycin hamster model of subchronic lung fibrosis, Pathology, 24, 155, 1992. 23. Thrall, R.S. et al., Bleomycin-induced pulmonary fibrosis in the rat: inhibition with indomethacin, Am. J. Pathol., 95, 117, 1979. 24. Aso, Y., Yoneda, K., and Kikkawa, Y., Morphological and biological study of pulmonary changes induced by bleomycin in mice, Lab. Invest., 35, 558, 1976. 25. Chandler, D.B., Hyde, D.M., and Giri, S.N., Morphometric estimates of infiltrative cellular changes during the development of bleomycin-induced pulmonary fibrosis in hamsters, Am. J. Pathol., 112, 170, 1983. 26. Bowden, D.H., Unraveling pulmonary fibrosis, the bleomycin model, Lab. Invest., 50, 487, 1984. 27. Harrison, J.H. and Lazo, J.S., High dose continuous infusion of bleomycin in mice: A new model for drug-induced pulmonary fibrosis, J. Pharmacol. Exp. Ther., 243, 1185, 1987. 28. Lindenschmidt, R.C. et al., Intratracheal versus intravenous administration of bleomycin in mice: acute effects, Toxicol. Appl. Pharmacol., 85, 69, 1986. 29. Kelley, J. et al., Bleomycin selectively elevates mRNA levels for procollagen and fibronectin following acute lung injury, Biochem. Biophys. Res. Commun., 131, 836, 1985. 30. Westergren-Thorsson, G. et al., Altered expression of small proteoglycans, collagen, and transforming growth factor-b1 in developing bleomycin-induced pulmonary fibrosis in rats, J. Clin. Invest., 92, 632, 1993. 31. Raghow, R. et al., Profiles steady state levels of messenger RNAs coding for type I procollagen, elastin, and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis, J. Clin. Invest., 76, 1733, 1985. 32. Clark, J.C., Starcher, B.C., and Uitto, J., Bleomycin-induced synthesis of type I procollagen by human lung skin fibroblasts in culture, Biochim. Biophys. Acta, 631, 359, 1980. 33. Yamamoto, T., Eckes, B., and Krieg, T., Bleomycin increases steady-state levels of type I collagen, fibronectin and decorin gene expression in human skin fibroblasts, Arch. Dermatol. Res., 292, 556, 2000. 34. Denholm, E.M. and Phan, S.H., The effects of bleomycin on alveolar macrophage growth factor secretion, Am. J. Pathol., 134, 355, 1989. 35. Kovacs, E.J. and Kelley, J., Secretion of macrophage-derived growth factor during acute lung injury by bleomycin, J. Leukoc. Biol., 37, 1, 1985. 36. Yamamoto, T., Katayama, I., and Nishioka, K., Fibroblast proliferation by bleomycin stimulated peripheral blood mononuclear cell factors, J. Rheumatol., 26, 609, 1999. 37. Takamizawa, A. et al., Bleomycin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity, J. Immunol., 162, 6200, 1999. 38. Mountz, J.D. et al., Bleomycin-induced cutaneous toxicity in the rat: analysis of histopathology and ultrastructure compared with progressive systemic sclerosis (scleroderma), Br. J. Dermatol., 108, 679, 1983.



545

39. Yamamoto, T. et al., Animal model of sclerotic skin. I. Local injections of bleomycin induce sclerotic skin mimicking scleroderma, J. Invest. Dermatol., 112, 456, 1999. 40. Yamamoto, T. et al., Animal model of sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell deficient WBB6F1-W/WV mice, J. Rheumatol., 26, 2628, 1999. 41. Yamamoto, T. et al., Animal model of sclerotic skin. III: Histopathological comparison of bleomycininduced scleroderma in various mice strains, Arch. Dermatol. Res., 292, 535, 2000. 42. Yamamoto, T. and Nishioka, K., Animal model of sclerotic skin. IV, Induction of dermal sclerosis by bleomycin is T cell independent, J. Invest. Dermatol., 117, 999, 2001. 43. Yamamoto, T. and Nishioka, K., Animal model of sclerotic skin. V, Increased expression of a-smooth muscle actin in fibroblastic cells in bleomycin-induced scleroderma, Clin. Immunol., 102, 77, 2002. 44. Gruber, B.L., Mast cells: accessory cells which potentiate fibrosis, Intern. Rev. Immunol. 12, 259, 1995. 45. Hawkins, R.A. et al., Increased dermal mast cell populations in progressive systemic sclerosis: a link in chronic fibrosis? Ann. Intern. Med., 102, 1182, 1985. 46. Nishioka, K. et al., Mast cell numbers in diffuse scleroderma. Arch. Dermatol., 123, 205, 1987. 47. Galli, S.J., New concepts about the mast cell, N. Engl. J. Med., 328, 1657, 1989. 48. Selvan, R.S., Butterfield, J.H., and Krangel, M.S., Expression of multiple chemokine genes by human mast cells, J. Biol. Chem., 269, 13893, 1994. 49. Gharaee-Kermani, M., Denholm, E.M., and Phan, S.H., Costimulation of fibroblast collagen and transforming growth factor b1 gene expression by monocyte chemoattractant protein-1 via specific receptors, J. Biol. Chem., 271, 17779, 1996. 50. Walker, M., Harley, R., and LeRoy,, E.C., Inhibition of fibrosis in Tsk mice by blocking mast cell degranulation, J. Rheumatol., 14, 299, 1987. 51. Walker, M., Harley, R., and LeRoy,, E.C., Ketotifen prevents skin fibrosis in the tight skin mouse, J. Rheumatol., 17, 57, 1990. 52. Fleischmajer, R., Perlish, J.S., and Reeves, I.R.T., Cellular infiltrates in scleroderma skin, Arthritis Rheum., 20, 975, 1977. 53. Thrall, R.S. et al., The effect of T cell depletion on the development of bleomycin induced pulmonary fibrosis in the rat, Am. Rev. Respir. Dis., 121, 99, 1980. 54. Piguet, P.F. et al., Tumor necrosis factor/cachectin plays a key role in bleomycin induced pneumopathy and fibrosis, J. Exp. Med., 170, 655, 1989. 55. Szapiel, S.V. et al., Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse, Am. Rev. Respir. Dis., 120, 893, 1979. 56. Schrier, D.J., Phan, S.H., and McGarry, B.M., The effects of nude (nu/nu) mutation on bleomycin induced pulmonary fibrosis. A biochemical evaluation, Am. Rev. Respir. Dis., 127, 614, 1983. 57. Lake-Bullock, M.H.V. et al., T cell independence of bleomycin-induced pulmonary fibrosis, J. Leukoc. Biol., 65, 187, 1999. 58. Breen, E. et al., Bleomycin regulation of transforming growth factor-b mRNA in rat lung fibroblasts, Am. J. Respir. Cell Mol. Biol., 6, 146, 1992. 59. King, S.L. et al., Bleomycin stimulates proa1(I) collagen promoter through transforming growth factor-b response element by intracellular and extracellular signaling, J. Biol. Chem., 269, 13156, 1994. 60. Skalli, O. et al., Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins, Lab. Invest., 60, 275, 1989. 61. Darby, I., Skalli, O., and Gabbiani, G., Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing, Lab. Invest., 63, 21, 1992. 62. Bhatal, P.S., The presence of modified fibroblasts in cirrhotic livers in man, Pathology, 4, 139, 1972. 63. Rudolph, R., McLure, W.J., and Woodward, M., Contractile fibroblasts in chronic alcoholic cirrhosis, Gastroenterology, 76, 704, 1979. 64. Nagle, R.B. et al., Induction of smooth muscle characteristics in renal interstitial fibroblasts during obstructive nephropathy, Lab. Invest., 29, 422, 1973. 65. Adler, K.B. et al., Actin-containing cells in human pulmonary fibrosis, Am. J. Pathol., 102, 427, 1981. 66. Sappino, A.-P. et al., Smooth muscle differentiation in scleroderma fibroblastic cells, Am. J. Pathol., 137, 585, 1990. 67. Kirk, T.Z. et al., Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1, J. Biol. Chem., 270, 3423, 1995.


546


68. Serini, G. and Gabbiani, G., Modulation of alpha-smooth muscle actin expression in fibroblasts by transforming growth factor-b isoforms: an in vivo and in vitro study, Wound Repair Regen., 4, 278, 1996. 69. Bostrom, H. et al., PDGF-A signaling is a critical event in lung alveolar myofibroblasts development and alveogenesis, Cell, 85, 863, 1996. 70. Kovacs, E.J., Fibrogenic cytokines, The role of immune mediators in the development of scar tissue, Immunol. Today, 12, 17, 1991. 71. Roberts, A.B. and Sporn, M.B., The transforming growth factor bs, in Handbook of experimental pharmacology. Peptide growth factors and their receptors. Vol 95, Sporn, M.B. and Roberts, A.B., Eds., Springer-Verlag, New York, 1990, p. 419. 72. Varga, J., Rosenbloom, J., and Jimenez, S.A., Transforming growth factor b (TGFb) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts, Biochem. J., 247, 597, 1987. 73. Raghow, J. et al., Transforming growth factor-b increases steady state levels of type I procollagen and fibronectin messenger RNAs posttranscriptionally in cultured human dermal fibroblasts, J. Clin. Invest., 79, 1285, 1987. 74. Ignotz, R.A. and Massague, J., Transforming growth factor-b stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix, J. Biol. Chem., 261, 4337, 1988. 75. Heino, J. et al., Regulation of cell adhesion receptors by transforming growth factor-beta, J. Biol. Chem., 264, 380, 1989. 76. Ignotz, R.A., Heino, J., and Massague, J., Regulation of cell adhesion receptors by transforming growth factor-beta, J. Biol. Chem., 264, 389, 1989. 77. Laiho, M. et al., Enhanced production and extracellular deposition of the endothelial-type plasminogen activator in cultured human lung fibroblasts by transforming growth factor beta, J. Cell Biol., 103, 2403, 1986. 78. Laiho, M., Saksela, O., and Keski-Oja, J., Transforming growth factor-beta induction of type-1 plasminogen activator inhibitor. Pericellular deposition and sensitivity to exogenous urokinase, J. Biol. Chem., 262, 17467, 1987. 79. Edwards, D.R. et al., Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor, EMBO J., 6, 1899, 1987. 80. Obberghen-Schilling, E.V. et al., Transforming growth factor b1 positively regulates its own expression in normal and transformed cells, J. Biol. Chem., 263, 7741, 1988. 81. Roberts, A.B. et al., Transforming growth factor type b: rapid induction of fibroblasts and angiogenesis in vivo and stimulation of collagen formation in vitro, Proc. Natl. Acad. Sci. U. S. A., 83, 4167, 1986. 82. Denholm, E.M. and Rollins, S.M., Expression and secretion of transforming growth factor-b by bleomycin-stimulated rat alveolar macrophages, Am. J. Physiol., 264, 36, 1993. 83. Kawakami, T. et al., Increased expression of TGF-b receptors by scleroderma fibroblasts, Evidence for contribution of autocrine TGF-b signaling to scleroderma phenotype, J. Invest. Dermatol., 110, 47, 1998. 84. Mavilia, C. et al., Type 2 helper T-cell predominance and high CD30 expression in systemic sclerosis, Am. J. Pathol., 151, 1751, 1997. 85. Postlethwaite, A.E. et al., Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4, J. Clin. Invest., 90, 1479, 1992. 86. Gillery, P. et al., Interleukin-4 stimulates collagen gene expression in human fibroblast monolayer cultures. Potential role in fibrosis, FEBS Lett., 302, 231, 1992. 87. Sempowski, G.D. et al., Subsets of murine lung fibroblasts express membrane-bound and soluble IL-4 receptors. Role of IL-4 in enhancing fibroblast proliferation and collagen synthesis, J. Immunol., 152, 3606, 1994. 88. Needlemann, B.W., Wigley, F.M., and Stair, R.W., Interleukin-1, interleukin-2, interleukin-4, interleukin-6, tumor necrosis factor a, and interferon-g levels in sera from patients with scleroderma, Arthritis Rheum., 28, 775, 1985. 89. Kulozik, M. et al., Co-localization of transforming growth factor b2 with a1(I) procollagen mRNA in tissue sections of patients with systemic sclerosis, J. Clin. Invest., 86, 917, 1990. 90. Peltonen, J. et al., Evaluation of transforming growth factor b and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization, J. Invest. Dermatol., 94, 365, 1990.



547

91. Gruschwitz, M. et al., Transcription and expression of transforming growth factor type beta in the skin of progressive systemic sclerosis: a mediator of fibrosis? J. Invest. Dermatol., 94, 197, 1990. 92. Yamamoto, T. et al., Anti-sclerotic effect of anti-transforming growth factor-b antibody in bleomycininduced scleroderma, Clin. Immunol., 92, 6, 1999. 93. McCormick, L.L. et al., Anti-TGF-b treatment prevents skin and lung fibrosis in murine sclerodermatous graft-versus host disease: a model for human scleroderma, J. Immunol., 163, 5693, 1999. 94. Giri, S.N., Hyde, D.M., and Hollinger, M.A., Effect of antibody to transforming growth factor b on bleomycin induced accumulation of lung collagen in mice, Thorax, 48, 959, 1993. 95. Denis, M., Neutralization of transforming growth factor-b1 in a mouse model of immune-induced lung fibrosis, Immunology, 82, 584, 1994. 96. Wang, Q. et al., Reduction of bleomycin induced lung fibrosis by transforming growth factor b soluble receptor in hamsters, Thorax, 54, 805, 1999. 97. Duncan, M.R. and Berman, B., g-interferon is the lymphokine and b interferon the monokine responsible for inhibition of fibroblast collagen production and late but not early fibroblast proliferation, J. Exp. Med., 162, 516, 1985. 98. Jimenez, S.A., Freundlich, B., and Rosenbloom, J., Selective inhibition of human diploid fibroblast collagen by interferons, J. Clin. Invest., 74, 1112, 1984. 99. Duncan, M.R. and Berman, B., Persistence of a reduced-collagen-producing phenotype in cultured scleroderma fibroblasts after short term exposure to interferons, J. Clin. Invest., 79, 1318, 1987. 100. Rosenbloom, J. et al., Inhibition of excessive scleroderma fibroblast collagen production by recombinant gamma-interferon. Association with a coordinate decrease in types I and III procollagen messenger RNA levels, Arthritis Rheum., 29, 851, 1986. 101. Kahari, V.-M. et al., Interferon-alpha and interferon-gamma reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture, Biochim. Biophys. Acta, 968, 45, 1988. 102. Yokozeki, M. et al., Interferon-g inhibits the myofibroblastic phenotype of rat palatal fibroblasts induced by transforming growth factor-b1 in vitro, FEBS Lett., 442, 61, 1999. 103. Yamamoto, T. et al., Effect of interferon-g on experimental scleroderma induced by bleomycin, Arch. Dermatol. Res., 292, 362, 2000. 104. Black, C.M. et al., Interferon-a does not improve outcome at one year in patients with diffuse cutaneous scleroderma. Results of a randomized, double-blind, placebo-controlled trial, Arthritis Rheum., 42, 299, 1999. 105. Ulloa, L., Doody, J., and Massagué, J., Inhibition of transforming growth factor-b/SMAD signaling by the interferon-g/STAT pathway, Nature, 397, 710, 1999. 106. Murrell, G.A.C., Francis, M.J.O., and Bromley, L., Modulation of fibroblast proliferation by oxygen free radicals, Biochem. J., 265, 659, 1990. 107. Chojkier, M. et al., Stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. A role for lipid peroxidation, J. Biol. Chem., 264, 16957, 1989. 108. Casciola-Rosen, L., Wigley, F., and Rosen, A., Scleroderma autoantigens are uniquely fragmented by metal-catalyzed oxidation reactions: implications for pathogenesis, J. Exp. Med., 185, 71, 1997. 109. Sambo, P. et al., Monocytes of patients with systemic sclerosis (scleroderma) spontaneously release in vitro increased amounts of superoxide anion, J. Invest. Dermatol., 112, 78, 1999. 110. Yamamoto, T. et al., Effect of superoxide dismutase on bleomycin-induced dermal sclerosis: Implications for the treatment of systemic sclerosis, J. Invest. Dermatol., 113, 843, 1999. 111. Granot, I. et al., Increased skin tearing in broilers and reduced collagen synthesis in skin in vivo and in vitro in response to the coccidiostat halofuginone, Poult. Sci., 70, 1559, 1991. 112. Granot, I. et al., Halofuginone: an inhibitor of collagen type I synthesis, Biochim. Biophys. Acta, 1156, 107, 1993. 113. Choi, E.T. et al., Halofuginone, a specific collagen type I inhibitor, reduces anastomotic intima hyperplasia, Arch. Surg., 130, 257, 1995. 114. Levi-Schaffer, F. et al., Inhibition of collagen synthesis and changes in skin morphology in murine graft-versus-host disease and tight skin mice: effect of halofuginone, J. Invest. Dermatol., 106, 84, 1996. 115. Yamamoto, T. and Nishioka, K., Analysis of the effect of halofuginone on bleomycin-induced scleroderma, Rheumatology, 41, 259, 2002. 116. Buckley, C.D. et al., Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation, Trends Immunol., 22, 199, 2001.


1391__Index.fm Page 549 Tuesday, November 18, 2003 6:01 PM

Index A Active induction method, EAE model, 172–175 Adaptive immune system canine, 80–85 (See also Canine immune system) human, 44–66 (See also specific components) antigen processing and presentation, 50–52 B1 cells, 44–45 B lymphocytes (B cells), 44 chemokines, 65–66 cytokines, 53–65 growth factors, 65–66 immunoglobulins (Igs), 45–48 Langerhans cells, 52–53 major histocompatibility complex (MHC), 48–50 minor histocompatibility a (H) antigens, 50 Adhesion-molecule knock-out (KO) mice, 134 Adhesion molecules. See also ICAMs; Selectins; VCAMs laminin and mucous membrane pemphigoid, 251–258 mouse, 125–126 in psoriasis-like skin lesions, 342–343 Adhesion molecule very late antigen, 39–40 Adoptive transfer method, 179–185 approach, 180–183 cell preparation, 181–183 EAE illustration, 184–185 intraperitoneal, 181 intravenous, 181 local, 181 strength and limitation, 183–184 Adrenal steroids, in murine atopic dermatitis, 378–379 Albinism Griscelli syndrome, 7 ocular type 1, 7 oculocutaneous type 2, 7 Allergen avoidance, in atopic dermatitis, 363 Allergen-specific immunotherapy, in atopic dermatitis, 362

Allergy, food. See Food hypersensitivity Alopecia areata canine spontaneous, 469–481 animals (breed predilection), 470–471 assessment, 471–478 clinical manifestation, 471–472 disease course, 471 epidemiology, 471 expert experience, 478 histopathology, 472–473 history, 469–470 immunogenetics, 478 immunopathology, 473–477 lessons learned, 478–479 therapeutic responses, 478 vs. human, 478–479 Dundee experimental bald rat (DEBR) model, 451–467 animals, 452–453 assessment, 456–460 clinical manifestation, 456–457 disease course, 453–455 disease susceptibility, 453 genetics, 455–456 histopathology, 457–458 history, 451–452 immunopathology, 459–460 immunophenotyping, 458–459 lessons learned, 463–465 sexual dichotomy, 453 therapeutic responses, 461–463 feline, 470 genetics, 433–434 history, 429–434, 451–452 human as autoimmune disease, 430–433 acquired immunity, 433–433 innate immunity, 431–432 human vs. canine, 478–479 immune privilege collapse model of, 161–162 juvenile, 442 murine models for, 434 acquired (cellular) immunity in, 437–440 549


550


animals used in, 434 C3H mice, 435 cell transfer and depletion studies, 437 genetics in, 441–442 innate (humoral) system in, 436–437 longitudinal graft study, 438–440 melanin in C3H/HeJ model, 441 in pharmaceutical tests, 441 rationale for, 434–442 sexual dichotomy in, 441 skin graft induction, 437 species used in, 434 vs. papular atrichia, 442 murine SCID graft induction, 483–492 animals, 484 assessment, 487–489 clinical manifestation, 487–488 disease induction, 485–487 histopathology, 489 history, 483–484 immunopathology, 489 lessons learned, 489 in nonhuman mammals, 470 totalis, 429 trophoneurotic hypothesis of, 430 universalis, 429 Ancestral haplotypes, 80 Anterior chamber-associated immune deviation (ACAID), 144, 146–147. See also under Immune privilege Antibodies. See Immunoglobulins Antidepressants, in atopic dermatitis, 362 Anti-epiligrin cicatricial pemphigoid, 252. See also Mucous membrane pemphigoid Antigen-presenting cells (APCs). See also Langerhans cells in alopecia areata, 439–440 ocular, 146–147 Antigen processing and presentation, 50–52 Antigens adhesion molecule very late, 39–40 minor histocompatibility (H), 50 Antihistamines, in atopic dermatitis, 361 Antimicrobial peptides (AMPs), 35 Antinuclear antibodies (ANAs), in scleroderma, 499–501 Apoptosis, 55 Astemizole, in atopic dermatitis, 362 Asthma, 58 atopic dermatitis and, 371–385 Athymic mice. See Nude mice Atopic dermatitis asthma and, 371–385 canine spontaneous, 353–369 animals, 354–355

assessment, 356–361 clinical manifestation, 356–359 disease course, 355–356 epidemiology, 355 expert experience, 363 histopathology, 359 history, 353–354 immunogenetics, 361 immunopathology, 360–361 lessons learned, 363 therapeutic response, 361–363 murine epicutaneous induction, 417–426 animals, 418–419 assessment, 421–422 clinical manifestation, 421 disease course, 421 disease induction, 419–421 histopathology, 422 history, 417–418 immunogenetics, 422 immunopathology, 422 lessons learned, 423–435 therapeutic responses, 422–423 food allergy–induced, 399–415 animals, 401 assessment, 401–407 clinical manifestation, 401–402 disease course, 410 disease induction, 401 expert experience, 408–409 histopathology, 402–403 history-410, 400 immunopathology, 403–407 lessons learned, 409–412 therapeutic responses, 407–408 vs. human disease, 411–412 spontaneous in NC/Nga mice, 375–386 transgenic induction, 387–398 Azathioprine in canine pemphigus foliaceus, 317 in canine pemphigus vulgaris, 269

B B1 cells, 44–45 B2 cells, 129–130 B6SIL ¥ BALB/c mice, 388 B10.D2>BALBc mice, 517–531 B10-D2-NSN mice, pemphigus vulgaris in, 275–283 B10-D2-OSN mice, pemphigus vulgaris in, 275–283 B19RIII mouse, EAE in, 174


INDEX

BALB/cBy mice, 388 BALB/c mice anti-laminin 5 antibody passive transfer, 251–258 bullous pemphigoid in, 170–172, 201–221 pemphigus vulgaris in, 275–283 Basement membrane zone, 9, 19–30 components, 20–26 integrins, 24–25 laminins, 24 noninflammation-related, 25 type VII collagen, 20–24 type XVIII collagen, 25 overview, 19 structures, 20 BAY X 1005, in psoriasis, 337 B cells (B lymphocytes), 44 canine, 83 mouse, 128 rat, 97–98 marginal zone, 97–98 migratory, 97 Beagles, breed predilections, 354 Beige mice, 332 Beta chemokines, 124–125 Beuceron dogs, breed predilections, 354 Bleomycin-induced murine scleroderma, 535–547 Boston terrier, breed predilections, 354 Bovine alopecia areata, 470 BP180 antibody, 170–172 BP230 antibody, 170 Breed predilections, dogs alopecia areata, 470–471 atopic dermatitis, 354–355 border collies, 242 cocker spaniel, 310 cocker spaniels, 242 collie, 264, 310 dachshunds, 242 Doberman pinscher, 310 German shepherd, 242, 264 pemphigus foliaceus, 310 pemphigus vulgaris, 264 poodles, 242 Siberian husky, 242 Brittany spaniels, C3 deficiency in, 83 Bullous pemphigoid canine spontaneous, 201–209 disease scoring, 172 human epidemiology, 202 IgG passive transfer model, 170–172 murine experimental IgG passive transfer, 213–223 animals, 214 anti-BP antibodies in, 217–218

551

clinical manifestation, 216 complement in, 218 disease course, 216 disease induction, 214–216 histopathology, 216 history, 213–214 immunopathology, 216–217 lessons learned, 217–221 mast cells in, 218–219 neutrophils and, 219 proteolytic enzymes in, 220 therapeutic potential, 220–221 vs. human, 220 murine passive IgG transfer model, 213–223 animals, 214 anti-BP antibodies in, 217–218 clinical manifestation, 216 complement in, 218 disease course, 216 disease induction, 214–216 histopathology, 216 history, 213–214 immunopathology, 216–217 lessons learned, 217–221 mast cells in, 218–219 neutrophils and, 219 proteolytic enzymes in, 220 therapeutic potential, 220–221 vs. human, 220 spontaneous in companion animals, 201–211 age of onset, 209 animals affected, 202 assessment, 203–208 clinical manifestation, 203–204 disease course, 202–203 epidemiology, 202 expert experience, 208 histopathology, 204 history, 201–202 immunogenetics, 208 immunological data, 204–205 lessons learned, 209 therapeutic responses, 208

C C2 complement protein, 80 C3-activating pathway, 42–43 C3 complement protein, 81 canine, 83 rat, 99 C3 convertases, 43 C3 deficiency, canine, 83 C3H/HeJ (H2K) mice, 399–415


552


C3H/HeJ mouse, hair loss in, 161 C3H mice, 435 C4 complement protein canine, 80–81 human, 80 C5-deficient mice, pemphigus vulgaris in, 275–283 C5-sufficient (B10-D2-NSN) mice, pemphigus vulgaris in, 275–283 C57B1/6LPJ mice, 517–531 C57BI 6 (H2s) mouse, EAE in, 174 C57Bl/6-bal/bal mice, 292–294 C57BL/61J mice, bullous pemphigoid in, 214 Cadherin antigen Dsg4, 296–300 Cadherins, 7–8, 126, 289 in psoriasis-like skin lesions, 342 Cancavalin A, in scleroderma, 501 Canine C4 complement protein, 80–81 Canine immune system, 79–89 antibodies (Igs), 81–82 chemokines, 85 complement system, 80–81 cytokines, 84–85 dendritic cells, 84 lymphocytes, 83–84 major histocompatibility complex (MHC), 80 mast cells, 81 neutrophils, 82–83 Canine spontaneous alopecia areata animals (breed predilection), 470–471 assessment, 471–478 disease course, 471 epidemiology, 471 expert experience, 478 history, 469–470 lessons learned, 478–479 therapeutic responses, 478 vs. human, 478–479 Canine spontaneous atopic dermatitis, 353–369 animals, 354–355 assessment, 356–361 disease course, 355–356 epidemiology, 355 expert experience, 363 history, 353–354 lessons learned, 363 therapeutic response, 361–363 Canine spontaneous bullous pemphigoid, 201–209. See also Bullous pemphigoid in companion animals vs. human, 202 Canine spontaneous epidermylosis bullosa acquisita, 227–236 animals, 228 assessment, 229–234 disease course, 228–229

epidemiology, 228 expert experience, 234 history, 227–228 lessons learned, 234–236 therapeutic responses, 234 vs. human, 235–236 Canine spontaneous mucous membrane pemphigoid, 211–250 animals, 242 assessment, 243–246 disease course, 242–243 epidemiology, 242 expert experience, 247 history, 241–242 lessons learned, 247–248 therapeutic responses, 246–247 vs. human, 247–248 Canine spontaneous pemphigus foliaceus, 309–319 animals, 310 assessment, 311–317 drug-induced, 313 expert experience, 317 history, 309 lessons learned, 318 panepidermal pustular, 314 therapeutic response, 317 Canine spontaneous pemphigus vulgaris animals, 264 assessment, 265–266 disease course, 264 epidemiology, 264 expert experience, 269–270 history, 263–264 lessons learned, 270–271 therapeutic responses, 269 vs. human, 270–271 Cantharidin, 289 Cardiotropin-1, 59, 61 Cataract, 147–148 Catenins, 289 Cathedicins, 35 Cats. See also Feline entries bullous pemphigoid in, 202, 203, 205, 208 CB6 ¥ BALB/c mice, 388 CC chemokines, 66–69 CD3 + 1 helper T cells, 83 CD4+/CD8+ cells, in food-induced murine atopic dermatitis, 404 CD4+ cells mouse, 129 in murine scleroderma, 510–511 rat, 101 in scleroderma, 501–502 CD4 cytokines, 83 CD8 cytokines, 83


INDEX

CD11/CD18 complex, 37 CD14 receptors, 37 CD-15R antigen, 98 CD18, in alopecia areata, 439 CD19, in murine scleroderma, 510 CD25 cells, 101 CD34+ hemotopoietic stem cells, 39, 40 CD44var.10 receptors, 439 CD45RG cells, 101 CD84/NKG21 group, 38 Cells B (See B cells) B1, 44–45 CD34+ hemotopoietic stem, 39 dendritic, 52–53 (See also Dendritic cells) canine, 84 Langerhans, 5, 52–53 mast, 40–41 natural killer (NK), 38–39 T (See T cells) Ceramidase, 381 Ceramide, in murine atopic dermatitis, 381 Chemokine receptors, 57 Chemokines canine, 83–85 in humans, 66–69 MDC, 377 TARC, 85, 377 Chlamydia trachomatis, 148 Chlorambucil, in canine pemphigus vulgaris, 269 Chymase inhibitor, in murine atopic dermatitis, 380 Cicatricial pemphigoid. See Mucous membrane pemphigoid Ciliary neurotrophic factor, 59, 61 Class switching, 48 CLIP peptides, 51 Clobetasole propionate, in murine atopic dermatitis, 378 Cocker spaniel, breed predilections, 310, 354 Collagen types of, 9 type VII, 20–24 type VIII, 25 Collagen abnormalities, in graft versus host disease, 522 Collie, breed predilections, 264, 310 Columbia University rat strains, 93 Common cytokine receptor gamma chain, 57–58 Complement system canine, 80–81 human, 41–44 mouse, 126 Connective tissue abnormalities, in murine scleroderma, 502–503

553

Constitutively expressed transgenic techniques, 188–189, 190–191 Contact-sensitizing agents, in alopecia areata, rat, 461 Corticosteroids in atopic dermatitis, 361–362 in canine pemphigus foliaceus, 317 in canine pemphigus vulgaris, 269 in food-induced murine atopic dermatitis, 408 in mucous membrane pemphigoid, 246 in murine atopic dermatitis, 378, 383–384, 394 in murine mucous membrane pemphigoid, 257 in pemphigus vulgaris, 288–289 in rat alopecia areata, 461 Cows, alopecia areata in, 470 Crystallins, 147–148 CT-1, 59, 61 Cutaneous adverse food reactions. See Food hypersensitivity CXC chemokines, 66–69, 124–125 Cyclophosphamide, in canine pemphigus vulgaris, 269 Cyclosporin A in alopecia areata, rat, 462 in atopic dermatitis, 361 in psoriasis, 337 Cytokine knock-out (KO) mice, 134 Cytokine receptor modules (CRMs), 59 Cytokines. See also Growth factors and individual types canine, 83–85 in humans, 38, 53–65 interferons, 54 interleukins, 55–64 (See also IL entries) tumor necrosis factor (TNF), 54–55 mouse, 123–124 in murine atopic dermatitis, 379–380 rat, 103–105

D Dachshund, breed predilections, 354, 470 Dalmation, breed predilections, 354 DBA/2NCr mice, anti-laminin 5 antibody passive transfer, 251–258 Decay-accelerating factor (DAF), 99 Defensins, 122–123 Dehydroepiandrosterone (DHEA), in murine atopic dermatitis, 378–379 Delayed-type hypersensitivity, 145 Dendritic cells canine, 84 migration to lymph nodes, 103 mouse, 129


554


rat, 96–97, 102–103 Dermatitis atopic (See Atopic dermatitis) Malassezia, 355, 363 staphylococcal, 363 Desmocollin, 8 Desmoglein 3 (dsg3), 4–5. See Dsg3 Desmogleins, 8 Dexamethasone, in psoriasis, 337 Dichroa febrifuga, in collagen disorders, 541–542 Diet manipulation, in murine atopic dermatitis, 380–381 Diphencyprone, in rat alopecia areata, 461–462 DLA genes, 80 DNCB, in rat alopecia areata, 461–462 Doberman pinscher, breed predilections, 310, 354, 470 Dogs. See also Canine entries breed predilections atopic dermatitis, 354–355 border collies, 242 cocker spaniels, 242 collie, 264 dachshunds, 242 German shepherd, 242, 264 Great Danes, 228 pemphigus foliaceus, 310 pemphigus vulgaris, 264 poodles, 242 Siberian husky, 242 Dolphin, MHC molecules in, 80 DPCP, in alopecia areata, rat, 461–462 Drug-induced canine spontaneous pemphigus foliaceus, 313 Dsg3 mice, 292–294 Dsg 4 cadherin antigen, 296–300 Dundee experimental bald rat (DEBR) model, 161 alopecia areata, 451–467 (See also Rat spontaneous alopecia areata) history, 451–452

E EGF-R, integrins and, 347–348 English bulldog, breed predilections, 354 English setter, breed predilections, 354 Eosinophils canine, 83 in humans, 39–40 Eotaxin, 377 Epidermis, 4–9 dermis, 8–10 desmoglein 3 (dsg3), 4–5 keratinocytes, 7–8 Langerhans cells, 5.102, 52–53, 130

melanocytes, 5–7 murine vs. human, 297 Epidermylosis bullosa acquisita, 242 canine spontaneous, 227–236 animals, 228 assessment, 229–234 clinical manifestation, 229–230 disease course, 228–229 epidemiology, 228 expert experience, 234 histopathology, 230–231 history, 227–228 immunogenetics, 233–234 immunopathology, 231–232 lessons learned, 234–236 therapeutic responses, 234 vs. human, 235–236 Epiligrin, 252 Epithelial basement membrane zone. See Basement membrane zone Epithelium, Langerhans cells, 149 Equine bullous pemphigoid, 202, 204, 205, 208 E-selectin, 10 Experimental autoimmune encephalomyelitis (EAE) active induction model, 172–175 adoptive transfer SCID mouse example, 184–185 Experimental autoimmune uveitis, 148 Experimental methods. See also specific methods and models active induction, 170–175 adoptive transfer, 179–185 passive transfer, 170–172 strength and limitations, 170, 183–184, 190–193 transgenic techniques, 187–195

F FA+AD mice, 402–403 Familial systemic lupus erythematosus, 80 Fas-FasL pathway, in alopecia areata, 440 FasL molecules, 145–146 Fatty acid supplements in atopic dermatitis, 362 in murine atopic dermatitis, 383–384 Fcg receptors, 83 Fc receptors, 37 mouse, 128 Feline alopecia areata, 470 Feline bullous pemphigoid, 202, 203, 205, 208 Feline pemphigus foliaceus, 310, 312 Feline pullous pemphigoid, 202 Fibrillin-1 gene, in murine scleroderma, 504–507 Fibroblast growth factor, 124 Fibroblasts, rat, 96–97


INDEX

555

FK506 (tacrolimus) in murine atopic dermatitis, 379, 383–384 in rat alopecia areata, 462 Fluocinolone acetonide, in food-induced murine atopic dermatitis, 408 Food allergy–induced murine atopic dermatitis, 399–415. See also under Murine atopic dermatitis Food hypersensitivity, atopic dermatitis and, 356 Fox terrier, breed predilections, 354

G Gamma chemokines, 124–125 GATA factors, 129 G-CSF, mouse, 124 Genes DLA, 80 resistance, 132–133 German shepherd, breed predilections, 264, 354, 470 German short-haired pointer, breed predilections, 354 Glucocorticosteroids. See Corticosteroids and specific drugs GM-CSF, 59 canine, 86 mouse, 124 Golden retriever, breed predilections, 354 Gp130 proteins, 59–61 Graft versus host disease, 41 human chronic, 519–520 as scleroderma model, 517–534 (See also under Murine scleroderma) Granulocyte-colony stimulating factor. See G-CSF Granulocyte-macrophage colony-stimulating factor. See GM-CSF Granulocytes, canine, 83 Great Danes, breed predilections, 228 Griscelli syndrome, 7 Growth factors. See also Cytokines and specific factors in humans leukemia inhibitory factor (LIF), 65 platelet-derived growth factor (PDGF), 65 stem cell factor, 65 transforming growth factor, 65 mouse, 124

H H1 blockers, in atopic dermatitis, 362 Hair follicle, immune privilege of, 155–165. See also under Immune privilege

Hair follicle-specific IgGs, 430–433 Halofuginone in graft versus host disease, 530 in scleroderma, 541–542 H antigens, 50 Heparin, low-dose, in canine pemphigus vulgaris, 269 Heterohybridomas, canine, 82 HLA-G, 146 Horses. See also Equine entries alopecia areata in, 470 bullous pemphigoid in, 202, 204, 205, 208 MHC molecules in, 80 HSV keratitis, 148 Human defensins, 35 Human immune system, 33–78. See also under Immune system adaptive, 44–66 (See also Adaptive immunity and specific components) antigen processing and presentation, 50–52 B1 cells, 44–45 B lymphocytes (B cells), 44 chemokines, 65–66 cytokines, 53–65 growth factors, 65–66 immunoglobulins (Igs), 45–48 Langerhans cells, 52–53 major histocompatibility complex (MHC), 48–50 minor histocompatibility a (H) antigens, 50 innate, 35–40 (See also Innate immunity and specific components) complement, 41–44 eosinophils, 39–40 mast cells and basophils, 40–41 natural killer (NK) cells, 38–39 neutrophils, 37–38 phagocytes, 36–37 Human neutrophil peptides, 35 Hypersensitivity, food. See Food hypersensitivity Hypodermis, 10

I ICAM-1, 10 ICAMs, mouse, 127–128 IFN-a, 54 IFN-b, 54 IgA, 47 canine, 81 mouse, 128 IgD, 46 IgE, 47 canine, 82


556


in food-induced murine atopic dermatitis, 403–405 in murine atopic dermatitis, 376–377, 382–384 IgG, 46–47 canine, 81–82 hair follicle-specific, 430–433 mouse, 128 rat, 98–99 IgG4, in pemphigus foliaceus, 325–326. See also Murine passive transfer pemphigus foliaceus Ig-like killer immunoglobulin receptors, 38 IgM, 46 canine, 81 mouse, 128 rat, 99 IL-1, 55–57 rat, 103 IL-1b, 52–53 IL-2, 57–58 in alopecia areata, 430 rat, 103 IL-3, 59 IL-4, 57–58 in murine atopic dermatitis, 379, 423 rat, 103–104 IL-4-Tg mouse, 388 IL-5, 59 in food-induced murine atopic dermatitis, 407 in murine atopic dermatitis, 379, 423 IL6 mouse, 104 rat, 104 IL-6, 59–61 IL-7, 58 IL-8, 61 IL-9, 58 IL-10, 61–62 in psoriasis, 337 rat, 104 IL-12, 62–63 in murine atopic dermatitis, 379–380 rat, 104–105 IL-13, 63–64, 86 canine, 86 in food-induced murine atopic dermatitis, 407 IL-14, 63–64 IL-16, 64 IL-17, 64 IL-18, 56–57 in murine atopic dermatitis, 383 IL-21, 58 IL-25, 64 IL-27, 63 IL-28, 64

IL-29, 64 Immune cell knock-out (KO) mice, 135 Immune privilege anagen hair follicle as site, 157 concept, 156 hair follicle, 149–150, 155–165 (See also Alopecia areata) alopecia areata and, 161–162 in mouse, 157–161 physiological function of, 161 potential functions of, 160–161 ocular, 143–154 anatomical and structural factors, 144–145 anterior-chamber-associated immune deviation (ACAID), 146–147 anti-inflammatory and immunosuppressive factors, 145–146 ocular diseases and, 147–149 vs. hair follicle, 160 potential mechanisms, 156 Immune privilege collapse model, of alopecia areata, 161–162 Immune system canine, 79–89 antibodies (Igs), 81–82 chemokines, 84–85 complement system, 80–81 cytokines, 84–85 dendritic cells, 84 lymphocytes, 83–84 major histocompatibility complex, 80 mast cells, 82 neutrophils, 82 human, 33–78 mouse, 119–140 innate, 120–129 rat, 91–117 cells, 103–104 complement system, 99 cytokines and chemokines, 103–105 immunoglobulins (antibodies), 98–99 major histocompatibility complex (MHC), 94–95 organs, 95–98 Immunoglobulins (Igs), 45–48. See also Ig entries anti-lens, 147–148 canine, 81–82 class switching, 48 gene regulation, 48 mouse, 128 rat, 98–99 Immunosuppressants, murine hair cycle and, 159–160 Inducibly expressed transgenic techniques, 189–190, 192


INDEX

557

INF-g, 54 alopecia areata and, 430 in murine atopic dermatitis, 380 INF-g-inducing factor (IL-18), 56–57 Inflammation-related adhesion molecules. See Eselectin; ICAM; Selectins; VCAMs Inflammatory bowel disease, 436–437 Innate immune system. See also specific components human, 35–40 complement, 41–44 eosinophils, 39–40 mast cells and basophils, 40–41 natural killer (NK) cells, 38–39 neutrophils, 37–38 phagocytes, 36–37 ocular, 145 (See also Immune privilege) Integrins, 34–35, 102 EGF-R and, 347–348 hyperproliferation/inflammation and, 345–347 mouse, 126 in psoriasis-like skin lesions, 335–350 suprabasal as marker, 348 suprabasal expression, 347 Interferons. See also INF entries canine, 86 mouse, 128 in murine atopic dermatitis, 377, 423–424 in scleroderma, 541 Interleukins, 55–64. See also IL entries canine, 86 in food-induced murine atopic dermatitis, 407 mouse, 124, 129 in murine atopic dermatitis, 423 rat, 103–104 in scleroderma, 501 International Society for Animal Genetics, 80 In vivo lymphocyte depletion, in alopecia areata, in rat, 462–463 Irish setter, breed predilections, 354

Keratitis, HSV, 148 KIR receptors, 38 Knock-out (KO) mice adhesion-molecule, 134 cytokine, 134 immune cell, 135 RAG, 134

L Labrador retriever, breed predilections, 354 Labrit dogs, breed predilections, 354 Laminins, 24 Langerhans cells, 5 absence from cornea, 149 human, 52–53 mouse, 130 rat, 102 Latency-associated peptide (LAP), in graft versus host disease, 530 Lectin-like receptors, 38 Leishmania major, 132 Leukemia inhibitory factor (LIF), in humans, 59, 60–61, 65 Leukocyte adhesion deficiency, 37 Leukotriene inhibitors, in atopic dermatitis, 362 Lhasa apso, breed predilections, 354 Loratidine, in atopic dermatitis, 362 Lupus erythematosus familial systemic, 80 vesicular cutaneoous, 242 Lymph nodes, dendritic cell migration, 103 Lymphocyte depletion, in rat alopecia areata, 462–463 Lymphocytes. See B cells; T cells Lymphosarcoma, canine thymus, 264

M J Jackson Laboratory mice, 133, 135, 388 Janus kinases, 123 Japanese Nishiki Nezumi mice, 372 Juvenile alopecia areata, 442

K KC proteins, 289 Keratinocytes, 7–8 Keratins, 8 in alopecia areata, 436

Macrophage-derived chemokine (MDC), 377 Macrophages. See also Monocyte/macrophages human vs. rat, 101–102 rat, 101–102 Magyar miszla (dog), breed predilections, 470 Major basic protein (MBP), 40 Major histocompatibility complex (MHC). See also MHC entries canine, 80 in humans, 48–50 mouse, 130 rat, 94–95 Malassezia dermatitis, 355, 363 Mannose-binding lectin pathway, 43


558


Mannose receptors, 36–37 Mast cell degranulation, 520 Mast cells canine, 81 ghost, 520 human, 40–41 phenotypes, 41 mouse, 127–128 rat, 100 M-CSF, mouse, 124 Melanin, in alopecia areata, 441 Melanocytes, 5–7 Membrane attack complex (MAC), 126 Membrane cofactor proteins (MCPs), 99 Methylprednisolone. See also Corticosteroids in pemphigus vulgaris, 289 MHC ancestral haplotypes, 80 MHC molecules class I, 80 hair loss and, 162 murine hair cycle and, 157–159 class II, 80 human antigen processing and presentation, 50–51 class I, 50–51 class II, 49, 51 peptide binding, 49–50 MHC tetramers, 51–52 Miniature schnauzer, breed predilections, 354 Minor histocompatibility (H) antigens, 50 Misoprostol, in atopic dermatitis, 362 Mixed breed dogs, disease predilections, 354, 470 Monocyte/macrophages in graft versus host disease, 521 rat, 101–102 Monocytes, human vs. rat, 101–102 Mouse, laboratory. See also Murine entries 129xBL/6 Dsg3null, 292–294 B6SIL ¥ BALB/c, 388 B10.D2>BALBc, 517–531 B10-D2-OSN, 275–283 B19RIII, 174 BALB/c, 214, 251–258, 275–283 bullous pemphigoid in, 170–172 BALB/cBy, 388 beige, 332 C3H, 435 C3H/HeJ, 161 C3H/HeJ (H2K), 399–415 C5-deficient, 275–283 C5-sufficient (B10-D2-NSN), 275–283 C57B1/6LPJ, 517–531 C57BI 6 (H2s), 174 C57Bl/6-bal/bal, 292–294 C57BL/61J, 214

CB6 ¥ BALB/c, 388 CBA/CaHN-Bikxxid/J, 433 DBA/2NCr, 251–258 dendritic cells, 84 Dsg3, 292–294 FA+AD, 402–403 hair follicle immune privilege in, 157–161 IL-4-Tg, 388 Jackson Laboratory, 275–283, 388 NC/Nga, atopic dermatitis and asthma, 371–385 Nishiki Nezumi, 372 PL/J (H2u), 174 SCID (See SCID mice) EAE adoptive transfer, 184–185 Scl GVHD, 517–531 SJL/H2, 174 Stat6 knockout, 510–511 tsk1, 496–507 tsk1+ ¥ Rag, 502 tsk2, 507–510 vs. rat as model, 92 Mouse models, 130–135 disease susceptibility, 132–133 knock-out (KO) mice, 134–135 (See also Knockout (KO) mice) naturally occurring mutations, 133–135 nomenclature of inbred strains, 131 nude mice, 134 SCID mice, 133–134 use of inbred strains, 131–132 Mucous membrane pemphigoid, 149 canine spontaneous, 211–250 animals, 242 assessment, 243–246 clinical manifestation, 243 disease course, 242–243 epidemiology, 242 expert experience, 247 histopathology, 243–244 history, 241–242 immunogenetics, 246 immunopathology, 244–246 lessons learned, 247–248 therapeutic responses, 246–247 vs. human, 247–248 murine anti-laminin antibody passive transfer model, 251–259 animals, 253 clinical manifestation, 255 disease course, 255–257 disease induction, 253–255 histopathology, 255–256 history, 252 immunopathology, 256–257 lessons learned, 257–258


INDEX

therapeutic response, 257 "Multiple hit" hypothesis, of pemphigus vulgaris, 291 Murine alopecia areata, 434 acquired (cellular) immunity in, 437–440 C3H mice, 435 cell transfer and depletion studies, 437 genetics in, 441–442 innate (humoral) system in, 436–437 juvenile, 442 longitudinal graft study, 438–440 melanin in C3H/HeJ model, 441 in pharmaceutical tests, 441 rationale for, 434–442 SCID graft induction, 483–492 animals, 484 assessment, 487–489 disease induction, 485–487 history, 483–484 lessons learned, 489 sexual dichotomy in, 441 skin graft induction, 437 species used in, 434 vs. papular atrichia, 442 Murine atopic dermatitis epicutaneous induction, 417–426 animals, 418–419 assessment, 421–422 disease course, 421 disease induction, 419–421 lessons learned, 423–435 therapeutic responses, 422–423 food allergy–induced, 399–415 animals, 401 assessment, 401–407 disease course, 410 expert experience, 408–409 history-410, 400 lessons learned, 409–412 therapeutic responses, 407–408 vs. human disease, 411–412 NC/Nga mice, 371–385 assessment, 374–378 disease course, 374 epidemiology, 372–374 expert experience, 381–382 history, 372 skin barrier abnormalities, 381 therapeutic responses, 378–381 vs. human disease, 382–384 oral allergen induction, 399–415 spontaneous in NC/Nga mice, 375–386 transgenic induction, 387–398 animals, 388 assessment, 390–394

559

clinical manifestation, 390 disease course, 389–390 disease induction, 388–389 expert experience, 394–395 histopathology, 391–392 history, 387–388 immunogenetics, 394 immunopathology, 391–393 lessons learned, 395–396 therapeutic responses, 394 Murine bullous pemphigoid, anti-BP180 passive transfer model, 213–223 animals, 214 assessment of disease, 216–217 disease course, 216 disease induction, 214–216 history, 213–214 lessons learned, 217–221 relevance to human disease, 220 Murine experimental pemphigus vulgaris, with desmoglein-targeting antibodies, 275–283 disease course and assessment, 279–280 disease induction, 277–278 history, 275–276 lessons learned, 281–282 therapeutic response, 280–281 vs. human, 282 Murine immune system adaptive, 129–130 B cells, 129–130 Langerhans cells, 130 major histocompatibility complex (MHC), 130 T cells, 129 innate, 120–129 adhesion molecules, 125–126 B-1 cells and Igs, 128 cells, 126–129 chemokines, 124–125 complement, 126 cytokines, 123–124 defensins, 122–123 gamma/delta T cells, 128 interferon-producing cells, 128 intraepithelial lymphocytes, 129 mast cells, 127–128 natural killer (NK) cells, 127 NF-kB receptor family, 122–123 Toll-like receptors, 120–121 Murine mucous membrane pemphigoid, antilaminin antibody passive transfer model animals, 253 disease disease course and assessment, 255–257 disease induction, 253–255


560


history, 252 lessons learned, 257–258 Murine pemphigus foliaceus, passive transfer, 321–328 animals, 322 assessment, 323–324 disease induction, 322–323 history, 321–322 immunogenetics, 324 lessons learned, 324–326 therapeutic potential, 326 Murine pemphigus vulgaris, with nondesmoglein 1 and 2 antibodies, 285–305 animals, 292–294 disease induction, 294–296 history and rationale, 286–291 lessons learned, 296–297, 296–300, 298–300 "multiple-hit" hypothesis, 291 Nicolskiy's sign, 295 Murine psoriasis SCID/human skin graft, 331–339 animals, 334 assessment, 335–337 clinical manifestation, 335 disease induction, 334–335 histopathology, 335–336 immunogenetics, 336–337 immunopathology, 336 lessons learned, 337 therapeutic response, 337 transgenic expression of integrin, 341–350, 345–348 assessment, 345 clinical manifestation, 345 disease induction, 344–345 histopathology, 345 history, 341–342 immunopathology, 345 rationale, 342–343 Murine scleroderma bleomycin-induced, 535–547 animals, 536–537 assessment, 538–540 disease course, 538 disease induction, 537 history, 535–536 therapeutic responses, 541–542 connective tissue abnormalities, 502–503 cutaneous manifestations, 496–497 graft versus host disease model, 517–534 animals, 522–523 assessment, 525–527 disease course, 525 disease induction, 523–525 diversity of disease, 519–520

expert experience, 527–530 history, 518–522 reproducibility, 520–522 therapeutic responses, 530–531 histopathology, 509–510 immunopathology, 499–502 internal organ involvement, 497–499 molecular defects in, 503–504 spontaneous, 495–515 history, 495–496 tsk1 mouse, 496–507 tsk2 mutation, 507–510 in transgenic models, 510–511 vs. human, 512 Murine vs. human epidermis, 297 Mycoptes musculinus, 373

N NAChRa9 receptor, 287–288 Natural killer (NK) cells human, 38–39 mouse, 127 ocular immune privilege and, 146 rat, 100–101 NC/Nga mouse, atopic dermatitis and asthma, 371–385 Nedocromil sodium, in graft versus host disease, 530 Neshiki nezumi (Japanese mouse), 372 Neurotropin, 124 Neurotropin-1/B-cell stimulating factor (NNT-BSF2, 60 Neutrophils canine, 82–83 human, 37–38 NF-kB proteins, 122 Niacinimide/tetracycline, in mucous membrane pemphigoid, 246 Nicolskiy's sign, 295, 323 NK cell receptor protein, 38 Nude mice, 134, 332

O Occular immune privilege, 143–154. See also under Immune privilege Ocular albinism type 1, 7 Oculocutaneous albinism type 2, 7 Omega 3/omega 6 fatty acids, in atopic dermatitis, 362 Onchocerciasis, 148–149


INDEX

561

Onchocercia volvulus, 148–149 Oncostatin M, 59, 60–61

P P19 protein, 63 Panepidermal pustular canine spontaneous pemphigus foliaceus, 314 Papular atrichia, 442 Parasitic infections, 47–48 of eye, 148–149 Passive transfer method, 170–172 Pathogen-associated molecular pathways (PAMPs), 121 PDGFa, in graft versus host disease, 523 Pemphigoid bullous (See Bullous pemphigoid) cicatrical as term, 149 cicatricial (See Mucous membrane pemphigoid) mucous membrane (See Mucous membrane pemphigoid) mucous membrane as term, 149 Pemphigus foliaceus, 287 canine spontaneous, 309–319 animals, 310 assessment, 311–317 clinical manifestation, 311 disease course, 310–311 drug-induced, 313 epidemiology, 310 expert experience, 317 histopathology, 311–312 history, 309 immunopathology, 314–317 lessons learned, 318 panepidermal pustular, 314 therapeutic response, 317 vs. human, 318 feline, 310, 312 murine passive transfer, 321–328 animals, 322 assessment, 323–324 disease course, 323 disease induction, 322–323 history, 321–322 immunogenetics, 324 lessons learned, 324–326 Nicolskiy's sign in, 323 therapeutic potential, 326 Pemphigus vulgaris canine spontaneous, 263–273 animals, 264 assessment, 265–266 clinical manifestation, 265–266

disease course, 264 epidemiology, 264 expert experience, 269–270 histopathology, 266–267 history, 263–264 immunopathology, 268–269 lessons learned, 270–271 therapeutic responses, 269 vs. human, 270–271 murine with desmoglein-targeting antibodies, 275–283 animals, 275 clinical manifestation, 279 disease course and assessment, 279–280 disease induction, 277–278 histopathology, 279–280 history, 275–276 immunopathology, 280 lessons learned, 281–282 therapeutic response, 280–281 vs. human, 282 murine with nondesmoglein 1 and 2 antibodies, 285–305 animals, 292–294 disease induction, 294–296 gross pathology, 295 histopathology, 297–298 history and rationale, 286–291 identification of cadherin Dsg4 antigen, 296–297, 298–300 interpretation of mechanism, 296 lessons learned, 296–300 "multiple-hit" hypothesis, 291 Nicolskiy's sign, 295 spontaneous canine, variants, 265–266 Peptide growth factor, in graft versus host disease, 523 Peptides antimicrobial (AMPs), 35 CLIP, 51 human neutrophil, 35 Periateriolar lymphatic sheath (PALS), 96 Persimmon leaf extract, in murine atopic dermatitis, 380–381 Phagocytes, 36–37 Phagocytosis, in humans, 36–37 CD14 receptors and, 37 Fc receptors and, 37 mannose and CR3 receptors in, 36 scavenger receptors and, 36 Toll-like receptors in, 36–37 Pharmaceutical product testing, murine models in, 441 Phosphodiesterase inhibitors, in atopic dermatitis, 362


562


Pigs bullous pemphigoid in, 202, 203, 206, 298 MHC molecules in, 80 Plakoglobin, 289 Plasminogen activator (PA), in pemphigus vulgaris, 280 Platelet-derived growth factor (PDGF), 65 PL/J (H2u) mouse, EAE in, 174 Poodle, breed predilections, 354, 470 Prednisolone in canine pemphigus foliaceus, 317 in mucous membrane pemphigoid, 246 Programmed cell death. See Apoptosis Proteins. See also specific types gp130, 59–61 KC, 289 major basic, 40 membrane-bound complement, 41–43 membrane cofactor (MCPs), 99 NF-kB, 122 NK cell receptor, 38 p19, 63 P-selectin, 10 Psoriasis history, 331–333 immunogenetics, 336–337 murine models chimeric SCID/human cellular immunity transfer, 331–339 transgenic expression of integrin, 341–350 susceptibility loci, 337 Pug dogs, breed predilections, 354

R RAG knock-out (KO) mice, 134 Rat albino, 92 Columbia University strains, 93 Dundee experimental bald (DEBR), 161 as laboratory model, 92–93 non-Wistar strains, 93 vs. mouse as model, 92–93 Wistar crosses, 93 Wistar Institute strains, 93 Rat immune system cells, 103–104 dendritic cells, 102–103 lymphocytes, 101 mast cells, 100 monocyte/macrophages, 101–102 natural killer (NK) cells, 100–101 complement system, 99 cytokines and chemokines, 103–105

immunoglobulins (antibodies), 98–99 major histocompatibility complex (MHC), 94–95 organs, 95–98 spleen, 96–98 thymus, 95–96 Rat Resource and Research Center, 93 Rat spontaneous alopecia areata animals, 452–453 assessment, 456–460 disease course, 453–455 genetics, 455–456 history, 451–452 lessons learned, 463–465 sexual dichotomy, 453 therapeutic responses, 461–463 Rattus norwegicus (Norway rat), 91 Rattus rattus (house rat), 91 Reactive oxygen species, bleomycin and, 541 Receptors. See also specific types CD3 complement, 83 CD14, 37 CD44var.10, 439 chemokine, 57 complement, 43–44 cytokine, 123 Fc, 37, 128 Fcg, 83 Ig-like killer immunoglobulin, 38 IL-1, 56 lectin-like, 38 nAChRa9, 287–288 natural killer (NK), 337 scavenger, 36–37 T-cell, 53 Toll-like, 36–37, 120–121 Resistance genes, 132–133 Respiratory bursts, 37–38

S SADBE, in rat alopecia areata, 461–462 Scavenger receptors, 36–37 Schnauzer, miniature, breed predilections, 354 SCID mice, 133–134 3EAE adoptive transfer, 184–185 alopecia areata, 483–489 (See also under Murine alopecia areata) human skin grafting onto, 253 human skin psoriasis induction model, 331–339 "leaky" phenotype, 334 psoriasis transfer model, 331–339 Scleroderma, murine. See also Murine scleroderma bleomycin-induced, 535–547 spontaneous, 495–515


INDEX

563

animals, 496 history, 495–496 tsk1 mouse, 496–507 tsk2 mutation, 507–510 in transgenic models, 510–511 vs. human, 512 Scl GVHD mice, 517–531 Scottish terrier, breed predilections, 354 Sealyham terrier, breed predilections, 354 Selectins alopecia areata and, 430 E, 10 mouse, 126, 127–128 P, 10 in psoriasis-like skin lesions, 342–343 Sexual dichotomy atopic dermatitides, 372 canine pemphigus foliaceus, 310 graft versus host disease, 522 mucous membrane pemphigoid, 242 Signature sequences, 57 SJL/H2 mouse, EAE in, 174 Skin components, 4–10 (See also specific components) basement membrane zone, 9 dermis, 9–10 epidermis, 4–9 hypodermis, 10 function, 3–4 Smoothe™ (fluocinolone acetonide), in food-induced murine atopic dermatitis, 408 Spleen in ocular immune privilege, 147–148 rat, 96–98 Spontaneous canine pemphigus vulgaris, variants, 265–266 Staphylococcal dermatitis, 363 STAT6 deficiency, in murine atopic dermatitis, 383 Stat6 knockout mice, 510–511 STAT protein, 60 STATs, 123 Stem cell factor, 65 Syndecan-2, 126 Systemic lupus erythematosus, familial, 80

T TACK chemokines, 342 Tacrolimus (FK506) in murine atopic dermatitis, 379, 383–384 in rat alopecia areata, 462 TAP molecules, 159

TARC (thymus and cytokine-regulated chemokine), 85, 342, 377 T-cell receptors, 53 T cells (T lymphocytes), 53 in alopecia areata, 439–440 canine, 83–84 gamma-delta, 128 in graft versus host disease, 521 mouse, 129 in murine atopic dermatitis, 423 rat, 101 Terfenadine, in atopic dermatitis, 362 Tet-off system, 189, 192 Tet-off/transcriptional silencer system, 190, 192 Tet-on system, 188–190, 192 TGFa, in graft versus host disease, 523 TGF-b in graft versus host disease, 530 in murine atopic dermatitis, 380 in scleroderma, 541 TGF-b1, murine hair cycle and, 159–160 Th2 cytokine, in murine atopic dermatitis, 395 Thallium acetate, alopecia areata and, 430 Thymus. See also T cells rat, 95–96 Thymus and cytokine-regulated chemokine (TARC). See TARC TLR1, 121 TLR2, 121 TLR3, 121 TLR4, 121 TLR5, 121 TLR6, 121 TLR7, 121 TLR9, 121 T lymphocytes. See T cells TNF-a, in alopecia areata, 439 TNF receptor, 123 Toll-like receptors. See also TLR entries in humans, 36–37 in mice, 120–121 Trachoma, 148 Transforming growth factor (TGF), 65 Transgenic techniques, 187–195 constitutively expressed, 188–189 inducibly expressed, 189–190 strength and limitations, 190–193 Tretinoic acid, in psoriasis, 337 Triamcinolone, in atopic dermatitis, 362 Tricyclic antidepressants, in atopic dermatitis, 362 Trophoneurotic hypothesis, of alopecia areata, 430 Tsk1 mouse, 496–507 Tsk1+ ¥ Rag mice, 502 Tsk2 mouse, 507–510


564


Tumor necrosis factor (TNF), 54–55. See also TNF entries canine, 86 mouse, 124 rat, 105

U

W West Highland white terrier, breed predilections, 354 White terrier, breed predilections, 354 Wire-haired fox terrier, breed predilections, 354 Wistar Institute, 92 Wistar Institute rat strains, 93

Uveitis, experimental autoimmune, 148

Y V Vascular cell adhesion molecules (VCAMs), 40 VCAM-1, 10 VCAMs, mouse, 127–128 Vesicular cutaneoous lupus erythematosus, 242

Yorkshire terrier, breed predilections, 354 Yucatan minipigs, bullous pemphigoid in, 202, 203

Animal Models of Human Inflammatory Skin Diseases

New Animal Models of Human Neurological Diseases

Animal Models for Retinal Diseases

Sauer’s Manual of Skin Diseases

Skin Diseases of Exotic Pets

Animal Models of T-Cell Mediated Skin Diseases - Ernst Schering Research Foundation Workshop 50

Medicine - Inflammatory Diseases of the Brain

Diagnosis Aging Skin Diseases

Serological Diagnosis of Certain Human, Animal and Plant Diseases

The Book of Human Skin

Animal Models of Movement Disorders

Animal Models of Cognitive Impairment

Animal Encounters (Human-Animal Studies)

Pelvic Inflammatory Disease (Deadly Diseases and Epidemics)

Animal Models of Human Cognitive Aging (Aging Medicine)

Gene Therapy for Autoimmune and Inflammatory Diseases

Inflammatory Pancreatic Diseases, An Update: Digestive Diseases 2004

Skin Diseases of the Dog and Cat

Skin Diseases of the Dog and Cat

Sauer's Manual of Skin Diseases, 10th Edition

Osteoporosis Research: Animal Models

Epidemiology, Causes and Prevention of Skin Diseases

Sauer's manual of skin diseases 8th edition

Sauer's Manual of Skin Diseases, 10th Edition

Control of Human Parasitic Diseases

Atlas of Common Skin Diseases In Australia

Animal Models in Toxicology

Control of Human Parasitic Diseases

Polyomaviruses and Human Diseases

Human Diseases and Conditions

Animal and Human Calorimetry

Animal Models of Human Inflammatory Skin Diseases

New Animal Models of Human Neurological Diseases

Animal Models for Retinal Diseases

Sauer’s Manual of Skin Diseases

Skin Diseases of Exotic Pets

Animal Models of T-Cell Mediated Skin Diseases - Ernst Schering Research Foundation Workshop 50

Medicine - Inflammatory Diseases of the Brain

Diagnosis Aging Skin Diseases

Serological Diagnosis of Certain Human, Animal and Plant Diseases

The Book of Human Skin

Animal Models of Movement Disorders

Animal Models of Cognitive Impairment

Animal Encounters (Human-Animal Studies)

Pelvic Inflammatory Disease (Deadly Diseases and Epidemics)

Animal Models of Human Cognitive Aging (Aging Medicine)

Gene Therapy for Autoimmune and Inflammatory Diseases

Inflammatory Pancreatic Diseases, An Update: Digestive Diseases 2004

Skin Diseases of the Dog and Cat

Skin Diseases of the Dog and Cat

Sauer's Manual of Skin Diseases, 10th Edition

Osteoporosis Research: Animal Models

Epidemiology, Causes and Prevention of Skin Diseases

Sauer's manual of skin diseases 8th edition

Sauer's Manual of Skin Diseases, 10th Edition

Control of Human Parasitic Diseases

Atlas of Common Skin Diseases In Australia

Animal Models in Toxicology

Control of Human Parasitic Diseases

Polyomaviruses and Human Diseases

Human Diseases and Conditions

Animal and Human Calorimetry

Recommend Documents