The Mouse in Biomedical Research, 2nd Edition Volume IV Immunology
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THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION Volume IV Immunology EDITED
BY
James G. Fox
Muriel T. Davisson
Fred W. Quimby
Division of Comparative Medicine, MIT Cambridge, MA
The Jackson Laboratory Bar Harbor, ME
Laboratory Animal Research Center The Rockefeller University New York, NY
Stephen W. Barthold
Christian E. Newcomer
Abigail L. Smith
Center for Comparative Medicine Schools of Medicine and Veterinary Medicine University of California Davis, CA
Research Animal Resources and Department of Molecular and Comparative Pathobiology Johns Hopkins University Baltimore, MD
School of Veterinary Medicine University of Pennsylvania Philadelphia, PA
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Table of Contents Volume I History, Wild Mice, and Genetics List of Reviewers List of Contributors Foreword Preface
1.
Building a Better Mouse: One Hundred Years of Genetics and Biology
x xi xiii xv
10. 1
Herbert C. Morse III
Mouse Embryology: Research Techniques and a Comparison of Embryonic Development between Mouse and Man
165
Matthew H. Kaufman 2.
Systematics of the genus Mus
13
Priscilla K. Tucker
11.
Gamete and Embryo Manipulation
211
K.C. Kent Lloyd 3.
The Secret World of Wild Mice
25
Grant R. Singleton and Charles J. Krebs 12. 4.
Breeding Systems: Considerations, Genetic Fundamentals, Genetic Background, and Strain Types
Mouse Strain and Genetic Nomenclature: an Abbreviated Guide
225
Martin Hrabé de Angelis, Dian Michel, Sibylle Wagner, Sonja Becker, and Johannes Beckers 53
Melissa L. Berry and Carol Cutler Linder 5.
Chemical Mutagenesis in Mice
13.
Gene-Specific Mutagenesis
261
K.C. Kent Lloyd 79
Janan T. Eppig
14.
Gene Transfer Studies Using Mouse Models
267
Robert G. Pergolizzi and Ronald G. Crystal 6.
The Mouse Genome
99
Mark D. Adams 7.
Gene Mapping
15.
Mouse and Human Pluripotent Stem Cells
281
Leslie F. Lock
115
Muriel T. Davisson 16. 8.
Genetic Monitoring
135
Cytogenetics Muriel T. Davisson and Mary Ann Handel
289
Lucia F. Jorge-Nebert, Sandrine Derkenne, and Daniel W. Nebert
Richard R. Fox, Michael V. Wiles, and Petko M. Petkov 9.
Drugs and the Mouse: Pharmacology, Pharmacogenetics, and Pharmacogenomics
145 Index
321
v
vi
TA B L E O F C O N T E N T S
Volume II Diseases
10.
Retroelements in the Mouse
269
Herbert C. Morse III List of Reviewers List of Contributors Foreword Preface
x xi xiv xv
Viral Diseases
11.
Sendai Virus and Pneumonia Virus of Mice (PVM)
281
David G. Brownstein
12.
DNA Viruses
Cardioviruses: Encephalomyocarditis Virus and Theiler’s Murine Encephalomyelitis Virus
311
Howard L. Lipton, A.S. Manoj Kumar, and Shannon Hertzler 1.
Murine Cytomegalovirus and Other Herpesviruses
1 Bacterial Diseases
Geoffrey R. Shellam, Alec J. Redwood, Lee M. Smith, and Shelley Gorman 13. 2.
Mouse Adenoviruses
325
Roger G. Rank
49
Katherine R. Spindler, Martin L. Moore, and Angela N. Cauthen
Chlamydial Diseases
14.
Clostridial Species
349
Kimberly S. Waggie 3.
Mousepox
67
R. Mark L. Buller and Frank Fenner
4.
Parvoviruses
15.
Enterobacteriaceae, Pseudomonas aeruginosa, and Streptobacillus moniliformis
365
Hilda Holcombe and David B. Schauer
93
Robert O. Jacoby and Lisa Ball-Goodrich 16. 5.
Polyoma Viruses
Aerobic Gram-Positive Organisms
389
Cynthia Besch-Williford and Craig L. Franklin
105
Thomas L. Benjamin 17.
RNA Viruses
Helicobacter Infections in Mice
407
James G. Fox and Mark T. Whary 6.
Mouse Hepatitis Virus
141 18.
Stephen W. Barthold and Abigail L. Smith
Mycoplasma pulmonis, Other Murine Mycoplasmas, and Cilia-Associated Respiratory Bacillus
437
Trenton R. Schoeb 7.
Lymphocytic Choriomeningitis Virus
179
Stephen W. Barthold and Abigail L. Smith
19.
Pasteurellaceae
469
Werner Nicklas 8.
Lactate Dehydrogenase-Elevating Virus
215 Mycotic and Parasitic Diseases
Jean-Paul Coutelier and Margo A. Brinton
9.
Reoviridae Richard L. Ward, Monica M. McNeal, Mary B. Farone, and Anthony L. Farone
235
20.
Fungal Diseases in Laboratory Mice Virginia L. Godfrey
507
vii
TA B L E O F C O N T E N T S
21.
Protozoa
517
3.
Katherine Wasson
22.
Helminth Parasites of Laboratory Mice
Reproductive Biology of the Laboratory Mouse
91
Kathleen R. Pritchett and Robert Taft
551
4.
Kathleen R. Pritchett
Endocrinology: Bone as a Target Tissue for Hormonal Regulation
123
Krista M. Delahunty and Wesley G. Beamer 23.
Arthropods
565 5.
David G. Baker
Hematology of the Laboratory Mouse
133
Nancy E. Everds Miscellaneous Diseases 6. 24.
The Tumor Pathology of Genetically Engineered Mice: A New Approach to Molecular Pathology 581
Spontaneous Diseases in Commonly Used Mouse Strains
171
Fred W. Quimby and Richard H. Luong
Robert D. Cardiff, Robert J. Munn, and Jose J. Galvez
25.
Clinical Chemistry of the Laboratory Mouse
Management, Techniques, and Husbandry
7. 623
Gnotobiotics
217
Richard J. Rahija
Cory Brayton 8. 26.
Zoonoses and Other Human Health Hazards
Management and Design: Breeding Facilities
235
William J. White
719
Christian E. Newcomer and James G. Fox 9. Index
747
Design and Management of Research Facilities for Mice
271
Neil S. Lipman
Volume III Normative Biology, Husbandry, and Models List of Reviewers List of Contributors Foreword Preface
10.
Nutrition
321
Graham Tobin, Karla A. Stevens, and Robert J. Russell x xi xiv xv
Normative Biology
11.
Health Delivery and Quality Assurance Programs for Mice
385
Diane J. Gaertner, Glen Otto, and Margaret Batchelder
12.
Environmental and Equipment Monitoring
409
J. David Small and Rick Deitrich 1.
Gross Anatomy
1
Vladimír Komárek
2.
Mouse Physiology Robert F. Hoyt, Jr., James V. Hawkins, Mark B. St. Claire, and Mary B. Kennett
13.
23
Biomethodology and Surgical Techniques
437
Alison M. Hayward, Laura B. Lemke, Erin C. Bridgeford, Elizabeth J. Theve, Courtnye N. Jackson, Terrie L. Cunliffe-Beamer, and Robert P. Marini
viii 14.
TA B L E O F C O N T E N T S
In-Vivo Whole-Body Imaging of the Laboratory Mouse 489 Simon R. Cherry Use of Mice in Biomedical Research
Foreword Preface
xiii xv
Overview
1
Fred W. Quimby and David D. Chaplin 15.
Behavioral Testing
513
Douglas Wahlsten and John C. Crabbe
16.
Cardiovascular Disease: Mouse Models of Atherosclerosis
1.
The Molecular Basis of Lymphoid Architecture in the Mouse
57
Carola G. Vinuesa and Matthew C. Cook 535
Nobuyo Maeda, Raymond C. Givens, and Robert L. Reddick
2.
The Biology of Toll-Like Receptors in Mice
109
Osamu Takeuchi and Shizuo Akira 17.
Convulsive Disorders
565 3.
Mariana T. Todorova and Thomas N. Seyfried
Genomic Organization of the Mouse Major Histocompatibility Complex
119
Attila Kumánovics 18.
Eye Research
595
Richard S. Smith, Patsy M. Nishina, John P. Sundberg, Johann Zwaan, and Simon W.M. John
4.
Some Biological Features of Dendritic Cells in the Mouse
135
Kang Liu, Anna Charalambous, and Ralph M. Steinman 19.
Genetic Analysis of Rodent Obesity and Diabetes
617
Sally Chiu, Janis S. Fisler, and Craig H. Warden 5. 20.
Mouse Models in Aging Research
637
Kevin Flurkey, Joanne M. Currer, and D.E. Harrison
21.
Mouse Models of Inherited Human Neurodegenerative Disease 673
Mouse Skin Ectodermal Organs
155
Maria D. Iglesias-Ussel, Ziqiang Li, and Matthew D. Scharff
6.
Karl Herrup
22.
Mouse Models Revealed the Mechanisms for Somatic Hypermutation and Class Switch Recombination of Immunoglobulin Genes
Mouse Natural Killer Cells: Function and Activation
169
Francesco Colucci 691 7.
Maksim V. Plikus, John P. Sundberg, and Cheng-Ming Chuong
Cytokine-Activated JAK-STAT Signaling in the Mouse Immune System 179 Bin Liu and Ke Shuai
23.
Quality Control Testing of Biologics
731 8.
William R. Shek
Signal Transduction Events Regulating Integrin Function and T Cell Migration in the Mouse
195
Lakshmi R. Nagarajan and Yoji Shimizu Index
759 9.
Volume IV Immunology List of Reviewers List of Contributors
x xi
Mouse Models of Negative Selection Troy A. Baldwin, Timothy K. Starr, and Kristin A. Hogquist
207
ix
TA B L E O F C O N T E N T S
10.
Peripheral Tolerance of T Cells in the Mouse
223
14.
Vigo Heissmeyer, Bogdan Tanasa, and Anjana Rao
Mouse Models to Study the Pathogenesis of Allergic Asthma
291
Chad E. Green, Nicholas J. Kenyon, Scott I. Simon, and Fu-Tong Liu 11.
The Genetics of Mouse Models of Systemic Lupus
243
Srividya Subramanian and Edward K. Wakeland 15. 12.
Inhibitory Receptors and Autoimmunity in the Mouse 261
The Mouse Trap: How Well Do Mice Model Human Immunology?
303
Christopher C.W. Hughes and Javier Mestas
Menna R. Clatworthy and Kenneth G.C. Smith Index 13.
Mouse Models of Immunodeficiency B. Anne Croy, James P. Di Santo, Marcus Manz, and Richard B. Bankert
275
313
List of Reviewers for Chapters in this Volume Altmann, Danny M. Bankert, Richard Eckhardt, Laurel Fischer-Lindahl, Kristen Fu, Yang-Xin Kinashi, Tatsuo Kulski, Jerzy Latour, Sylvain Lee, James J. McGaha, Tracy Nimmerjahn, Falk Pernis, Alessandra B. Schultz, Leonard D. Skokos, Dimitris
x
Imperial College, London, UK State University of NY, Buffalo, NY Hunter College of CUNY, New York City, NY University of Texas Southwestern Medical Center, Dallas, TX University of Chicago, Dept of Pathology, Chicago, IL Kansai Medical School, Japan Murdoch University, Perth, Western Australia Hôpital Necker-Enfants Malades, Paris, France Mayo Clinic College of Medicine, Rochester, MN The Rockefeller University, New York, NY The Rockefeller University, New York, NY Columbia University, New York, NY The Jackson Laboratory, Bar Harbor, ME The Rockefeller University, New York, NY
Contributors Shizuo Akira Department of Host Defense Research Institute for Microbial Diseases Osaka University Osaka, Japan
B. Anne Croy Department of Anatomy and Cell Biology Faculty of Health Sciences Kingston Ontario, Canada
Troy A. Baldwin Center for Immunology University of Minnesota Minneapolis, MN 55455
James P. Di Santo Unite des Cytokines et Developpement Lymphoide Institut Pasteur Département d’Immunologie Paris, France
Richard B. Bankert Department of Microbiology and Immunology State University of New York Buffalo, NY 14260 David D. Chaplin Department of Microbiology University of Alabama at Birmingham Birmingham, AL 35294 Anna Charalambous Laboratory of Cellular Physiology and Immunology The Rockefeller University New York, NY 10021 Menna R. Clatworthy Cambridge Institute for Medical Research Department of Medicine University of Cambridge Cambridge, United Kingdom Francesco Colucci The Babraham Institute Babraham Research Campus Cambridge, United Kingdom Matthew C. Cook Department of Immunology The Canberra Hospital Woden, ACT, Australia
Chad E. Green Department of Biomedical Engineering School of Engineering University of California at Davis Davis, CA 95616 Vigo Heissmeyer National Research Center for Environment and Health-GSF Institute of Molecular Immunology Marchioninistr Munich, Germany Kristin A. Hogquist Center for Immunology University of Minnesota Minneapolis, MN 55455 Christopher C.W. Hughes Department of Molecular Biology & Biochemistry University of California at Irvine Irvine, CA 92697 Maria D. Iglesias-Ussel Department of Cell Biology Albert Einstein College of Medicine New York, NY 10461 Nicholas J. Kenyon Division of Pulmonary and Critical Care Medicine Department of Internal Medicine University of California at Davis School of Medicine Davis, CA 95616 xi
xii Attila Kumánovics Department of Pathology Division of Immunology and Cell Biology University of Utah School of Medicine Salt Lake City, UT 84132 Ziqiang Li Department of Cell Biology Albert Einstein College of Medicine New York, NY 10461 Bin Liu Division of Hematology/Oncology University of California Los Angeles Los Angeles, CA 90095 Fu-Tong Liu Department of Dermatology University of California at Davis Davis, CA 95616 Kang Liu Laboratory of Cellular Physiology and Immunology The Rockefeller University New York, NY 10021 Marcus Manz Institute for Research in Biomedicine (IRB) Bellinzona, Switzerland Javier Mestas Center for Immunology University of California at Irvine Irvine, CA 92697 Lakshmi R. Nagarajan Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, MN 55455 Fred W. Quimby Laboratory Animal Research Center The Rockefeller University New York, NY 10021 Anjana Rao Department of Pathology Harvard Medical School and the CBR Institute for Biomedical Research Boston, MA 02115 Matthew D. Scharff Department of Cell Biology Albert Einstein College of Medicine Bronx, New York, NY 10461
CONTRIBUTORS
Yoji Shimizu Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, MN 55455 Ke Shuai Division of Hematology/Oncology University of California Los Angeles Los Angeles, CA 90095 Scott I. Simon Department of Biomedical Engineering School of Engineering University of California at Davis Davis, CA 95616 Kenneth G.C. Smith Cambridge Institute for Medical Research Department of Medicine University of Cambridge Cambridge, United Kingdom Timothy K. Starr Center for Immunology University of Minnesota Minneapolis, MN 55455 Ralph M. Steinman Laboratory of Cellular Physiology and Immunology The Rockefeller University New York, NY 10021 Srividya Subramanian Center for Immunology University of Texas Southwestern Medical Center Dallas, TX 75390 Osamu Takeuchi Department of Host Defense Research Institute for Microbial Diseases Osaka University Osaka, Japan Bogdan Tanasa Department of Pathology Harvard Medical School and the CBR Institute for Biomedical Research Boston, MA 02115 Carola G. Vinuesa Division of Immunology and Genetics John Curtin School of Medical Research Canberra City, ACT, Australia Edward K. Wakeland Center for Immunology University of Texas Southwestern Medical Center Dallas, TX 75390
Foreword for Volume IV
More than 30 years ago, Rolf Zinkernagel and I made the chance discovery that virus-specific “killer” T cells only destroyed virus-infected target cells that shared at least one H2 haplotype. This very clear result depended on the use of a few, standard inbred mouse strains—CBA/H (H2k), BALBc/J (H2d), and C57BL/6J (H2b). Like any young scientists, we simply took it for granted that these mice were available for us to use in our experiments and didn’t think much about where or why they were developed. Years later, at a meeting that celebrated “50 years of H2” held at The Jackson Laboratory in Bar Harbor, Maine, Jan Klein and others told us some of the story1. In trying to study cancer, George Snell discovered that the tumor cells he was transplanting were being rapidly rejected. He quickly realized that it wasn’t something specific to the tumor that the recipients were recognizing, but the identity of the donor mouse strain. Together with serological observations made by Peter Gorer in London, this began the story of what we now know as the major histocompatibility complex (MHC). Klein traced for us how C.C. Little, the founder of The Jackson Laboratories, had used founder stock inbred to select ornamental mouse strains by Abbie Lathrop, a commercial breeder in Vermont. Lathrop is, in a sense, the mother of mouse transplantation genetics. Genetics is now, and has long been, the most powerful analytical tool available to biologists. Without the further development of recombinant and mutant mice that mapped and defined the MHC class I genes, the observation that Rolf and I made would not have gone very far. As our discovery and the way we interpreted our results led to a Nobel Prize, we owe an enormous debt to George Snell, Peter Gorer, Don Bailey, Chella David, Don Shreffler, Hugh McDevitt, Jack Stimpfling, Igor Egorov, and a host of other geneticists who were trying to understand transplantation and the so-called immune response (Ir) genes.
1. David, C.S. Ed H-2 Antigens: Genes, Molecules and Function NATO AASI Series, Plenum Press, New York, 1987
In short, we were able to exploit an extraordinarily elegant genetic system to answer the linked questions, “What do killer T cells see, and how do they see it?” Some similar observations had been made in humans, but the interpretation of these results was quite wrong and the area was immensely confused. Without mouse transplantation genetics and the ready, open availability of those inbred mouse strains, our understanding of T cell recognition would have been delayed for decades. I think it must have hurt just a little when these two, young, naive nonentities came along and told the mouse genetics community the nature of what it was they had been investigating for all those years. That is, though, how science works. The breakthrough discoveries are often made by chance, and come from the most unlikely direction. As late as 1987, some of the geneticists still had difficulty acknowledging that the primary function of the transplantation antigens was to serve as targets for “surveillance of self.” The development of transgenic “knock-out” and “knock-in” mice is now, of course, central to almost every in vivo investigation in mammalian cell biology. Although cancer biologists had largely lost interest in mouse experiments by the mid1980s—and the experimental immunologists had few friends when they argued that it was necessary to build bigger animal facilities—that changed dramatically in the 1990s and we soon found it hard to hold on to whatever space we had. Now, making the relevant mutant mouse is a prerequisite for any serious study of a potential cancer gene. Any honest commentator on the laboratory mouse must acknowledge that these genetic analyses are essential to contemporary biomedical research. Of course, there are many opponents who—because they are hostile to molecular genetics, animal experimentation, or both—use emotionally charged arguments to convince the public and the politicians that this is not the case. We have a constant battle on our hands as we try to ensure that there is no interruption in that rapid progress through transgenic mouse studies that has been our experience over the past 15 or so years.
xiii
xiv Of course, the rules have changed over the past 5 to 10 years and if the more regressive elements in society succeeded in shutting down animal experiments tomorrow in the United States and Europe, we would simply see an exacerbation of the progressive move of such research to emerging Asian centers of excellence. As science and technology are tightly tied to economic development, this could be disastrous for the so-called “knowledge” economies of the West.
FOREWORD
PETER C. DOHERTY, DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY, UNIVERSITY OF MELBOURNE, VICTORIA 3010, AUSTRALIA
[email protected] and, DEPARTMENT OF IMMUNOLOGY, ST. JUDE CHILDREN’S RESEARCH HOSPITAL, MEMPHIS, TN 38105
[email protected]
Preface The American College of Laboratory Animal Medicine (ACLAM) was formed in 1957 in response to the need for specialists in laboratory animal medicine. The college has promoted high standards for laboratory animal medicine by providing a structured framework to achieve certification for professional competency and by stressing the need for scientific inquiry and exchange via progressive continuing education programs. The first edition of “The Mouse in Biomedical Research” consisting of four volumes, and published in 19811983 was a part of the College’s effort to fulfill those goals. It is one of a series of comprehensive texts on laboratory animals developed by ACLAM over the past three decades: “The Biology of the Laboratory Rabbit” was published in 1974, “The Biology of The Guinea Pig” in 1976 and a two-volume work “Biology of The Laboratory Rat” in 1979 and 1980. Also, in 1979 the College published a two-volume text on “Spontaneous Animal Models of Human Disease”. In 1984 the first edition of “Laboratory Animal Medicine” appeared in print followed by “Laboratory Hamsters” in 1987. The second edition of The Biology of the Laboratory Rabbit was published in 1994. A two-volume treatise on “Nonhuman Primates in Biomedical Research” was published in 1995 and 1998. A text “Anesthesia and Analgesia in Laboratory Animals” was published in 1997 followed by the second edition of “Laboratory Animal Medicine” in 2002. Most recently, the second edition of “The Laboratory Rat” was published in 2005. The estimated annual use of 100 million-plus mice worldwide attests to the importance of the mouse in experimental research. The introduction of genetically engineered mice has only increased the usefulness of the mouse model in biomedical research. In no other species of animal has such a wealth of experimental data been utilized for scientific pursuits. Knowledge of the mouse that has been accumulated is, for the most part, scattered throughout a multitude of journals, monographs and symposia. It has been 25 years since the publication of the first edition of the “Mouse in Biomedical Research”. The intent of this second edition is to build upon the framework of the first edition, rather than simply to update and duplicate the earlier effort. The intended purpose of this text is to assemble established scientific data emphasizing recent information on the biology and use of the laboratory mouse. Separation of the material into multiple volumes was essential because of the number of
subject areas covered. The four volumes consist of 80 chapters coauthored by 167 scientists. The information in Volume 1 serves as a primer for scientists new to the field of mouse research. It provides information about the history, basic biology and genomics of the laboratory mouse (Mus musculus), as well as basic information on maintenance and use of mouse stocks. Mouse origins and relationships are covered in chapters on history, evolutionary taxonomy and wild mice. Genetics and genomics of the mouse are covered in chapters on genetic nomenclature, gene mapping, cytogenetics and the molecular organization of the mouse genome. Maintenance of laboratory mice is described in chapters on breeding systems for various types of strains and stocks and genetic monitoring. Use of the mouse as a model system for basic biomedical research is described in chapters on chemical mutagenesis, gene trapping, gene therapy, pharmacogenetics and embryo manipulation. Volume 2 entitled Diseases departs from the first edition of the same title by discussing specific disease-causing microorganisms, whereas the first edition discussed infectious diseases affecting specific organs and tissues. This volume consists of 26 chapters subdivided into RNA viruses and DNA viruses, as well as bacterial, mycotic and parasitic infections. These chapters not only provide updates on pathogenesis, epidemiology and prevention of previously recognized murine pathogens, but also include chapters on newly recognized disease-causing organisms: mouse parvovirus, cilia-associated respiratory bacilli and Helicobacter spp. A separate category, consisting of 3 chapters, discusses zoonoses, tumor pathology of genetically engineered mice and spontaneous diseases in commonly used mouse strains. Volume 3 encompasses 23 chapters whose contents provide a broad overview on the laboratory mouse’s normative biology, husbandry and its use as a model in biomedical research. This consists of chapters on behavior, physiology, reproductive physiology, anatomy, endocrinology, hematology and clinical chemistry. Other chapters cover management, as well as nutrition, gnotobiotics and disease surveillance. Individual chapters describe the mouse as a model for the study of aging, eye research, neurodegenerative diseases, convulsive disorders, diabetes and cardiovascular and skin diseases. Chapters on imaging, surgical and other research techniques and the use of the mouse in assays of biological products also are included.
xv
xvi Volume 4 is a completely new addition to this series, dedicated to mouse immunology. It is based on the vast body of knowledge which has made the mouse the model of choice when studying immunity in human beings. Arguably more is known about the immune system in mice than any other species except human. In large part this is due to the power of genetic engineering to delineate molecular mechanisms. This volume includes an overview of mouse immunology, including both the innate and adaptive immune systems, followed by 15 chapters (mini-reviews), each dealing with a specific area of immunology. The overview addresses broad concepts concerning molecular and cellular immunology and cites both current references and the appropriate chapter, for more detailed information, from the mini-reviews which follow. The 15 chapters illustrate the power of genetic engineering in dissecting each component of the immune response from the development of lymphoid tissues to signal transduction pathways in activated cells. Individual chapters address: The Genomic Organization of the MHC, Toll-like Receptors, The Molecular Basis of Lymphoid Architecture, The Biology of Dendritic Cells, Somatic Hypermutation and Class Switching, Natural Killer Cell Function and Activation, Cytokine Mediated Signaling, Signal Transduction Events Regulating Integrin Function and T-Cell Migration, Central Tolerance in T-Cells, Peripheral Tolerance in T-cells, Inhibitory Receptors and Autoimmunity. The volume also includes the use of mice in studies of Systemic Autoimmunity, Immunodeficiency, Allergic Airway Inflammation and the Differences Between Mouse and Human Immunology. This treatise was conceived with the intent to offer information suitable to a wide cross section of the scientific community. It is hoped that the four volumes will serve as a standard reference source for scientists using mice in biomedical research. Students embarking on scientific careers also will benefit from the broad coverage of material presented in compendium format. Certainly, specialists in laboratory animal
P R E FA C E
science will benefit from these volumes; technicians in both animal care and research will find topics on surgical techniques, management and environmental monitoring of particular value. The editors wish to extend special appreciation to the contributors to these volumes. Authors were selected because of knowledge and expertise in their respective fields. Each individual contributed his or her time, expertise and considerable effort to compile this resource treatise. In addition, the contributors and editors of this book, as with all volumes of the ACLAM series texts, have donated publication royalties to the American College of Laboratory Animal Medicine for the purpose of continuing education in laboratory animal science and comparative medicine. This book could not have been completed without the full support and resources of the editors’ parent institutions which allowed us the time and freedom to assemble this text. A special thanks is also extended to the numerous reviewers of the edited work whose suggestions helped the authors and editors present the material in a meaningful and concise manner. We also thank the editorial staff of Elsevier for their assistance. Finally, we especially acknowledge with deep appreciation the editorial assistance of Lucille Wilhelm, whose dedication and tireless commitment, as well as good humor, throughout this project were of immeasurable benefit to the editors in the completion of this text.
JAMES G. FOX STEPHEN W. BARTHOLD MURIEL T. DAVISSON CHRISTIAN E. NEWCOMER FRED W. QUIMBY ABIGAIL L. SMITH
Overview Overview of Immunology in the Mouse: Molecular and Cellular Immunology Fred W. Quimby and David D. Chaplin
I. II.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Receptors and Ligands of the Innate Immune System . . . . . . . . . . . . . 1. Toll-Like Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. TLR1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. TLR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. TLR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. TLR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f. TLR5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. TLR6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . h. TLR7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i. TLR8 and TLR10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . j. TLR9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . k. TLR11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Fc Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Poly-Ig Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. The Neonatal FcR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. FcγRIIB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. Activating FcγRI and FcγRIII . . . . . . . . . . . . . . . . . . . . . . . . . . f. FcγRIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g. FcεRI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . h. Other FcRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Complement and Complement Receptors . . . . . . . . . . . . . . . . . . . . a. Overview of the Complement System . . . . . . . . . . . . . . . . . . . b. Types of Complement Receptors . . . . . . . . . . . . . . . . . . . . . . . i. CR1 and CR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii. CR3 and CR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii. Receptors for Anaphylatoxins . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 5 5 6 6 7 7 7 8 8 8 10 10 11 11 Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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4. C-Type Lectin Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Type I Receptor Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Type II CLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. Collectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. Type V Family Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. NK Cell Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Ly49 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. CD94/NKG2 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Major Histocompatibility Complex . . . . . . . . . . . . . . . . . . . . . . . . 1. Classic Class I Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Classic Class II Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Nonclassic Class I and Class II Molecules . . . . . . . . . . . . . . . . . . . 4. Molecules Associated with Antigen Processing . . . . . . . . . . . . . . . a. TAP Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. H2-DM and H2-DO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. The CD1 Family of Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Antigen Receptors and Coreceptors in Adaptive Immunity . . . . . . . . . 1. Antibodies and the B-Cell Receptor (BCR) . . . . . . . . . . . . . . . . . . a. Primary Antibody Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Secondary, Tertiary, and Quaternary Structure . . . . . . . . . . . . . c. Antibody Classes and Subclasses . . . . . . . . . . . . . . . . . . . . . . . d. The BCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. BCR Coreceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The T-Cell Receptor (TCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. The Antigen-Binding Complex . . . . . . . . . . . . . . . . . . . . . . . . . b. The Signal-Transducing Complex . . . . . . . . . . . . . . . . . . . . . . c. T Cell Coreceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Costimulatory Receptors and Ligands in Adaptive Immunity . . . . . . . 1. The B7 Family and Its Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . a. B7-1 and B7-2 and Their Receptors . . . . . . . . . . . . . . . . . . . . . b. ICOS and ICOS-L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. PD-1 and Its Ligands PD-L1 and PD-L2 . . . . . . . . . . . . . . . . . 2. CD40 and CD40 Ligand (CD154) . . . . . . . . . . . . . . . . . . . . . . . . . . E. Cytokines, Chemokines, and Their Receptors . . . . . . . . . . . . . . . . . . . . III. Cellular Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cells of the Innate Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cells That Provide Anatomical Barriers . . . . . . . . . . . . . . . . . . . . . a. Physical Epithelial Barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Secreted Protective Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Phagocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Monocytes and Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . c. Eosinophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Mast Cells and Basophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. NK Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cells of the Adaptive Immune System . . . . . . . . . . . . . . . . . . . . . . . . . 1. T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Early T Cell Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Gene Rearrangements in γδ T Cells . . . . . . . . . . . . . . . . . . . . . c. Thymic Selection of CD4+ and CD8+ T Cells . . . . . . . . . . . . . d. Differentiation of T Cells into the Type 1 and Type 2 Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. Differentiation of Th17 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . f. Regulatory T Cells and T Cell Tolerance . . . . . . . . . . . . . . . . . g. NKT Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. B Cells and Plasma Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. B Cell Development before Encounter with Antigen . . . . . . . . b. B Cell Development after Encounter with Antigen: The Germinal Center, Affinity Maturation, and Differentiation of Plasma Cells . . . . . . . . . . . . . . . . . . . . . . . . . c. Receptor Editing and B Cell Tolerance . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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OVERVIEW
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INTRODUCTION
As in man, the immune system of the mouse can be divided into innate and adaptive components. The innate immune response is evolutionally primitive with functions such as phagocytosis present in protozoa and sponges. Cells have receptors capable of recognizing structural motifs shared by families of infectious agents, and these cells are capable of making an immediate response. The adaptive immune response first appeared in jawed fish, agnathia, and, in contrast to the innate system, requires time after foreign agent recognition to develop effector mechanisms. It incorporates fine specificity for molecular sequences unique to individual infectious agents as well as the capacity for immunological memory. Because the development of immunological specificity also introduced the chance for self-recognition, cells of the adaptive response have developed a sophisticated mechanism for induction of tolerance to “self.” At many levels there is cross-talk between innate and adaptive immune responses. The cells of the innate system recruit cells of the adaptive responses to sites of inflammation, they process and present antigens to cells of the adaptive system, and at many levels they modify adaptive immune responses. In this volume a general overview of molecular and cellular immunology in the mouse is first presented. It is followed by 15 chapters, each of which is a mini-review concentrating on a specific immunological concept, cell type, or effector process. These chapters are meant to illustrate how mutant mice have been used to better understand immune mechanisms in mice and man. Chapter 15, entitled “The Mouse Trap: How Well do Mice Model Human Immunology?” details differences discovered between the species. This volume does not provide a section on techniques used in immunological research. A complete and up-to-date listing of these techniques may be obtained through reading Current Protocols in Immunology by Coligan et al. (2006). In addition, the modifications in engineering and husbandry needed to maintain immunodeficient mice are not addressed in this volume; that subject is discussed of chapter 3 in Volume 3, entitled “Management and Design: Research Facilities for Mice.”
II. A. 1.
MOLECULAR IMMUNOLOGY
Receptors and Ligands of the Innate Immune System Toll-Like Receptors
a. INTRODUCTION In the mouse nine Toll-like receptors (TLRs) have been recognized. Those receptors are conserved throughout evolution from flies to humans and are composed of an extracellular peptide containing leucine-rich repeat (LRR)
3 motifs, a transmembrane region and a cytoplasmic Toll/interleukin (IL)-1 receptor homology domain (TIR). Through the LRR motif TLRs bind pathogen-associated molecular patterns (PAMPs), which are molecular configurations common to groups of bacteria, fungi, or viruses. Intracellular triggering is initiated through the TIR. Ligand binding and signaling may require homodimerization or heterodimerization between two TLRs. The basic structure is shown in Fig. 1. TLRs are found on the cells of the innate immune system (e.g., monocytes, macrophages, neutrophils, mast cells, and dendritic cells [DCs]), where they signal the cell to induce phagocytosis and activate the transcription factors nuclear factor (NF)-κB and adaptor protein (AP)-1), leading to cytokine secretion and the initiation of inflammation. The fusion event between the phagosome and lysosome, resulting in the acquisition of hydrolase enzymes necessary for bacterial degradation, is greatly accelerated by TLR signaling (Blander and Medzhitov 2004). On DCs, TLRs play an important role preparing molecules derived from pathogens for antigen presentation to cells of the adaptive immune response. This is accomplished by the upregulation of both major histocompatability complex (MHC) class I and class II on the surface of DCs as well as the upregulation of costimulatory molecules necessary for triggering responses by B and T cells (West et al. 2004). TLRs can also be found on B cells, fibroblasts, intestinal epithelial cells, and vascular endothelial cells. On vascular endothelium bacterial products elicit upregulation of adhesion molecules contributing to the recruitment of leukocytes (Vasselon and Detmers 2002). All TLRs have at least one signaling pathway–dependent adaptor molecule called myeloid differentiation factor 88 (MyD88). This feature is also shared with IL-1 and IL-18 receptor signaling (Scanga et al. 2004). Additional signaling pathway adaptor proteins vary according to the TLR activated. Readers desiring more information on murine TLRs should refer to chapter 2 in this volume, entitled “The Biology of Toll-Like Receptors in Mice.” b. TLR1 TLR1 shares amino acid sequence homology with TLR2 and TLR6 and these form a subfamily of receptors, which also share in ligand recognition. Expression of TLR-1 alone fails to impart recognition but coexpression with TLR-2 results in activation in response to triacyl-lipopeptide (Takeuchi et al. 2002). The heterodimer utilizes both the signal adapter molecule MyD88 and MyD88 adaptor-like (MAL) protein to initiate intracellular signaling (O’Neill et al. 2003). c. TLR2 TLR2 recognizes PAMPs derived from a broad range of infectious agents including bacterial lipoprotein, lipoteichoic acid, and glycosylphosphatidylinositol (GPI) anchors. In TLR2 knockout mice these substances fail to activate macrophages, rendering the mice highly susceptible to agents such as Staphylococcus aureus and Streptococcus pneumoniae, to name a few. TLR2 heterodimers with TLR1 recognize bacterial lipopeptides, whereas heterodimers with TLR6 recognize mycoplasmal lipopeptides (O’Neill et al. 2003).
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A TLR Structure
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B Specificities of TLRs TLR
Ligand
Microbial Source
TLR2
Lipoproteins Peptidoglycan Zymosan LPS GPI anchor Lipoarabinomannan Phosphatidylinositol dimannoside
Bacteria Gram-positive bacteria Fungi Leptospira Trypansosomes Mycobacteria Mycobacteria
TLR3
Double-standard RNA
Viruses
TLR7/8
Single-standard RNA
Viruses
TLR4
LPS HSP60
Gram-negative bacteria Chlamydia
TLR5
Flagellin
Various bacteria
TLR9
CpG DNA
Bacteria, protozoans
Leucine-Rish Repeat Motifs Cysteine-Rich Flanking Motif
TIR Domain
C TLR Signal Transduction LPS-Binding Protein
TIR Domain
LPS Death Domain TLR4 CD14 MD2
Active AP-1
MyD88
TRAF6 (Inactive)
IRAK
Inactive NF-IκB MAP Kinase Cascade
P TRAF6 (Active)
Gene Transcription; Inflammatory Response
Jun– P
IκB
IκB Kinase Cascade Active NF-IκB
NF-IκB IκB
P
IκB
P
Fig. 1 Mammalian TLRs. A typical TLR contains conserved extracellular and cytoplasmic domains. Different TLRs contain different numbers and arrangements of the extracellular leucine-rich and cysteine-rich motifs. Different TLRs are involved in responses to different microbial products. Some of the specificities listed may require heterodimerization with other TLRs (e.g., TLR2 with TLR6). The signaling pathway triggered by TLRs that results in the generation of the NF-κB transcription factor is shown. Intracellular adapter proteins other than MyD88 may also be involved in some TLR signaling pathways. In addition to NF-κB activation, TLRs are also linked to AP-1 activation. Reproduced from Abbas, A.K., and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
OVERVIEW
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d. TLR3 TLR3 binds to and is activated by viral doublestranded RNA or the synthetic polyinosinic:polycytidylic acid. Homodimers also appear to be activated by lipopolysaccharides (LPSs). Intracellular signaling leads to the production of type I interferons (IFNs) and inflammatory cytokines. The TIR domain-containing adapter inducing interferon TRIF (which is also involved with TLR2, 4, and 7) prevents activation of the IFNβ promoter (O’Neill et al. 2003). TRIF has a receptor-interacting domain similar to MyD88 and may inactivate MyD88. A mutation in TRIF called Lps2 severely impairs responses mediated through TLR3 and TLR4 (Hoebe et al. 2003). Mice lacking both TRIF and MyD88 cannot be activated by LPSs. Thus, like MAL, which is used by TLR2, TRIF may collaborate with MyD88 to produce different signaling outcomes. e. TLR4 TLR4 binds to and is activated by LPSs. TLR4 knockout mice have macrophages unresponsive to LPSs, and the mice are highly resistant to LPS-induced shock. The extracellular domain of TLR4 associates with MD-2, which enhances the response to LPS. Although intracellular signaling may be mediated by MyD88, TLR4 can also activate NF-κB without MyD88 by utilizing the TIR domain containing adaptor protein, MAL (also called TIRAP). MAL, in addition to mimicking MyD88 has unique functions and is not required for activation of the transcription factor, IFN regulatory factor 3 (IRF3) which regulates IFN-β production and other IFN-dependent genes (O’Neill et al. 2003). TLR4 has been shown to protect mice against Pasteurella pneumotropica (Hart et al. 2003). f. TLR5 TLR5 recognizes bacterial flagellin through the conserved amino- and carboxyl-terminal regions shared among many Gram-negative bacteria (Takeda et al. 2003). TLR expression–deficient mice are more susceptive to infection by Salmonella typhimurium. g. TLR6 TLR6, after heterodimerizing to TLR2, recognizes diacyl lipopeptides. Expressed alone, TLR6 fails to respond to lipopeptide. The heterodimer uses both MyD88 and MAL signal adaptor molecules. h. TLR7 TLR7 forms a subfamily with TLR8 and TLR9 based on genomic structure and sequence similarities. Murine TLR7 has been shown to bind single-stranded (ss) RNA and activate the transcription factor NF-κB through MyD88. Using guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus (HIV), mouse DCs and macrophages were shown to be activated by and secrete IFN-α and proinflammatory and regulatory cytokines (Heil et al. 2004). Similarly stimulation of murine TLR7 by influenza ssRNA demonstrated that TLR7 sense endosomal ssRNA and produce large amounts of IFN-α in response to it (Diebold et al. 2004). i. TLR8 AND TLR10 Whereas mice express TLR8, they do not appear to have any specific phenotype. Unlike human TLR8, mouse TLR8 is not activated by ssRNA (Heil et al. 2004). There is a missense mutation in the mouse TLR10 gene that prevents its expression.
j. TLR9 Unmethylated CpG motifs, which are infrequent in the human genome, are common in bacterial and viral DNA and are known to bind and activate murine TLR9 (Takeda et al. 2003). Like TLR7, TLR9 is located in the endosome and endosomal reticulum in which nucleotides are transported. Mice with a mutated TLR9 were susceptible to murine cytomegalovirus (MCMV) infection. Normal mice recognize MCMV DNA and induce IL-12- and IFN-promoting natural killer (NK) cell clearance of the virus (Krug et al. 2004). k. TLR11 Mice express a functional TLR11 on macrophages, liver, kidney, and bladder epithelial cells. Although a natural ligand for this receptor is not known, knockout mice are highly susceptible to infection of the kidneys by uropathogenic bacteria, suggesting an important role for TLR11 in preventing infection in those organs (Zhang et al. 2004). 2.
Fc Receptors
a. OVERVIEW Many cells have membrane glycoproteins called Fc receptors (FcRs) due to their affinity to bind the Fc portion of the mouse immunoglobulins. They act as a bridge between innate and adaptive immunity by transporting antibodies across cell membranes and across the placenta, by activating or inhibiting the functions of lymphocytes, by modulating inflammation including phagocytosis and the proliferation of monocytes, and by triggering effector functions such as antibody-dependent cellular cytotoxicity (ADCC). Recent information suggests that FcRs play a role in maintaining peripheral tolerance (Fukuyama et al. 2005; Samsom et al. 2005). FcRs always have a transmembrane ligand-binding protein composed of an extracellular domain, a transmembrane domain, and a cytoplasmic domain, this protein is called the α chain α, and structurally it belongs to the immunoglobulin (Ig) superfamily of proteins because the genes are derive from a primordial gene encoding basic domain structure. Several of the FcRs have transmembrane domains that physically associate with other accessory polypeptide chains via a charged amino acid residue found in the transmembrane domain. Together with accessory polypeptides, these FcRs engage in signal transduction. Depending on the receptor, the accessory polypeptides may be composed of two γ chains or combinations of γ and β chains (Goldsby et al. 2003). More on this subject is presented in chapter 12. b. POLY-IG RECEPTOR Mouse poly-Ig receptor (pIgR) is a single glycoprotein of 120 kDa capable of binding IgA and IgM (Asano et al. 1998). The pIgR is expressed on the basolateral surfaces of mucosal cells and the glandular epithelia of mammary, salivary and lacrimal glands, where it binds polymeric immunoglobulins via their J chains and transports them into mucus or glandular secretions. In mice pIgR is also expressed in the liver and is involved in IgA transport into bile. Mice differ from humans in that most of their proximal intestinal secretory IgA comes from hepatobiliary transfer (Johansen and Brandtzaeg 2004).
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Approximately 80% of mouse plasma cells are located in the mucosal lamina propria of the gut and respiratory tracts and most of them secrete IgA with J chains. Mice differ from humans in that J chain mRNA is not expressed until the antigen-driven stages of B cell development. The J chain is an inherent part of polymeric immunoglobulins and allows for the pIgR-mediated vesicular trafficking to the luminal side of the mucosae. The extracellular domain of pIgR is cleaved and remains attached as secretory component (SC) to the secreted IgA or IgM after release into the lumen. Covalently bound SC confers extra stability to the immunoglobulin (Fig. 2). The abundance of IgA-secreting plasma cells in the gut has been attributed to a number of factors, however, in the mouse, a class switch to IgA in the lamina propria is probably restricted to the T-independent B1 cell, derived from the mouse peritoneal cavity (Johansen and Brandtzaeg 2004). This appears to differ from the situation humans in whom ligation of CD40 on B cells with CD154 (CD40 ligand [CD40L]) on T helper (Th) cells is a prerequisite for class switch. Epithelial cell expression of pIgR may be seen as early as 20 weeks of gestation in humans but does not appear until weaning in mice, suggesting that it is dependent on exogenous stimuli for induction. pIgR expression in the mouse gut is both constitutive and induced by proinflammatory and immunoregulatory cytokines; however, pIgR expression in the female genital tract, lacrimal glands, and mammary glands is regulated by steroid hormones using the glucocorticoid response element in humans and an androgen response element in the upstream promoter of the murine gene. pIgR knockouts accumulate IgA in serum and intestinal lamina propria but thrive under conventional conditions, implicating a critical role for intraepithelial lymphocytes in the homeostatic control of intestinal microflora (Yamazaki et al. 2005).
Lamina Propria
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A mouse pIgR that is nearly identical to epithelial cell pIgR has been reported on a mouse B cell lymphoma; however, this receptor binds IgM with higher affinity than IgA, making it similar to human pIgR but different from mouse epithelial cell pIgR (Phillips-Quagliata et al. 2000). c. THE NEONATAL FCR The neonatal FcR (FcRn) is a distant member of the MHC class I protein family that diverged in evolution to bind the hinge region of IgG Fc. The α chain has three immunoglobulin-like loop structures, and the functional molecule is dependent on dimerization with β2-microglobulin. Mouse FcRn has a broad range of binding specificities for IgGs in contrast to the stringent binding specificity of human FcRn. These differences in IgG binding specificity are due to residues close to the interaction site and a shorter α1 helix in the human molecule (Zhou et al. 2005). The FcRns are responsible for transfer and protection of IgG from mother to fetus (across placenta) or to neonate. In the neonate the FcRn is expressed in the gut and, more weakly, in adult tissues including vascular endothelium (in which protection occurs). Intracellular FcRn resides primarily in endosomal vesicles where it binds and recycles endocytosed IgG destined for degradation. This latter protective function may explain why murine IgG has a longer half life (t1/2 6–8 days) compared with that of non-IgG classes (t1/2 1–2 days) (Roopenian et al. 2003). d. FCγRIIB FcγRIIB (CD32) is a 40-kDa protein composed of an α chain with two disulfide bond loops in the extracellular domain and transmembrane and cytoplasmic domains. The cytoplasmic domain contains one immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence. Phosphorylation of the ITIM leads to recruitment of the src homology (SH) 2 containing inositol polyphosphate phosphatase (SHIP) and the hydrolysis of phosphatidylinositol 3-kinase (PI3K) products. FcγRIIB binds immune complexes (ICs) containing IgG1 only.
Mucosal Epithelial Cell
Lumen
Poly-Ig Receptor with Bound IgA J Chain Secreted IgA IgA-Producing Plasma Cell Dimeric IgA
Endocytosed Complex of IgA and Poly-Ig Receptor
Proteolytic Cleavage
Fig. 2 Transport of IgA through epithelial cells. IgA is produced by plasma cells in the lamina propria of mucosal tissue and binds to the poly-Ig receptor at the base of an epithelial cell. The complex is actively transported through the epithelial cell, and the bound IgA is released into the lumen by proteolytic cleavage. The process of active transport through the cell is called transcytosis. Reproduced from Abbas and A.K., Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
OVERVIEW
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IMMUNOLOGY
IN
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Because this is the most common isotype in mice, this inhibitory receptor probably prevents incidental activation of circulating myeloid cells by ICs and the systemic inflammation that ensues. FcγRIIB is found on B cells, monocytes, macrophages, activated neutrophils, and mast cells. There are two isoforms of FcγRIIB, one on B cells, which prevents internalization of ICs, and another on myeloid cells, which initiates phagocytosis. Through its ITIM, this receptor can inhibit the activating signals for other FcRs as well as for the B cell receptor (BCR) and T cell receptor (TCR). The FcγRIIB is required to maintain tolerance in mice. Mice lacking FcγRIIB spontaneously develop autoimmunity. Lupusprone strains such as NZM, BXSB, and B6 Fcgr2b−/−, when given bone marrow cells transfected to express FcγRIIB, stop developing autoimmune disease (McGaha et al. 2005). FcγRIIB knockout mice failed to develop mucosal tolerance to ovalbumin, and CD4+ ovalbumin-responding T cells failed to differentiate into IgE class–restricted regulatory T (Treg) cells. The receptor was found to play a critical role in modulating antigen presentation by DCs (Samsom et al. 2005). Disruption of FcγRIIB leads to enhanced inflammation, phagocytosis, and ADCC. Anaphylatoxin (C5a), intravenous immunoglobulin, and IFNγ each modulate inflammation by acting on FcγRs. Both FcγRIIB and the activatory receptor FcγRIII are present on antigenpresenting cells (APCs) and ICs containing IgG can bind either receptor. The ratio of these opposing signaling receptors is critical for setting thresholds for inflammatory activity. As myeloid cells leave the bone marrow they contain mostly FcγRIIB with little FcγRIII. Within an inflammatory environment, C5a, IFN-γ, and tumor necrosis factor (TNF) reverse the ratio of FcRs favoring FcγRIII, whereas intravenous immunoglobulin up-regulates FcγRIIB, making it more difficult for ICs to induce FcγRIII (Ravetch 2002; Shushakova et al. 2002). Mice lack FcγRIIA, which is found in man; however, transgenic mice expressing the human FcγRIIA receptor were able to functionally remove human IgG1-coated erythrocytes from the circulation (van Royen-Kerkhof et al. 2005). e. ACTIVATING FCγRI AND FCγRIII FcγRI (CD64) and FcγRIII (CD16) are multichain receptors, each having a ligand binding α chain associated with one or two accessory signal-transducing subunits containing immunoreceptor tyrosine-based activating motif (ITAM) sequences. These γ chains are transmembrane polypeptides with a cytoplasmic tail containing ITAM in their cytoplasmic tail. Activation responses are dependent on the sequential activation of members of the src and syk kinase families. Whereas α chains are unique to either FcγRI or FcγRIII, both receptors use a common γ chain. FcγRI binds ICs containing IgG1 and IgG2b (Rivera and Casadevall 2005). In addition mouse FcγRI is a high-affinity FcR capable of binding monomeric IgG. FcγRI is expressed on macrophages, monocytes, and neutrophils, whereas FcγRIII is expressed on macrophages, activated monocytes, NK cells, pre-B cells, γδ T cells and serosal and mucosal mast cells. Mice, with disruption of the
7 IgE heavy chain gene and incapable of making IgE, do make IgG and develop anaphylaxis. However, disruption of the FcγRIII gene abolishes IgG IC-induced mast cell degranulation, IgG-induced passive cutaneous anaphylaxis (PCA), and the Arthus reactions (Daeron 1997). Likewise, mice with a disrupted FcγRIII gene failed to mount ADCC against NK-resistant IgG-coated target cells. Abolishing the common γ chain also abolishes ADCC by NK cells and the Arthus reaction. FcγRI mediates ADCC by macrophages and monocytes and triggers superoxide production and secretion of inflammatory cytokines. f. FCγRIV FcγRIV is unique to the mouse; it has an α chain encoded by a gene on chromosome 1 adjacent to genes for FcγRIIB and FcγRIII. The α chain has two disulfide loops in the extracellular domain, a transmembrane domain containing a charged amino acid residue and a cytoplasmic domain. The transmembrane domain is physically associated with the common γ chain. FcγRIV is expressed on myeloid cells including monocytes, splenic and bone marrow DCs, macrophages, and neutrophils; it is not expressed on lymphocytes. Expression is upregulated by IFN-γ and LPSs and downregulated by transforming growth factor (TGF)-β and IL-4. FcγRIV selectively binds IgG2a and IgG2b with affinity intermediate between that for high-affinity FcγRI and low-affinity FcγRIIB and FcγRIII. ICs containing these IgG subclasses induce calcium influx characteristic of FcR signaling. Whereas IgG2a can activate complement via the classic pathway, it mediates its in vivo effects by preferentially binding activator FcγRIV (over the inhibitory FcγRIIB). Thus, IgG2a functions as a highly effective opsonin (Nimmerjahn et al. 2005). g. FCεRI FcεRI is a high-affinity IgE binding receptor composed of a single α chain with two disulfide loops in the extracellular domain and transmembrane and cytoplasmic domains. The transmembrane domain is associated with two γ chains and a β chain, each containing ITAM motifs. The α chain is encoded by a single gene located on chromosome 1 near the gene for the α subunit of FcγRIII. FcεRI is expressed on mast cells and basophils. Within 15 seconds after FcεRI aggregation by antigen on mast cells, the γ and β subunits become tyrosine phosphorylated. As with other activatory FcRs, phosphorylation of ITAMs correlates with the activation of several sets of tyrosine kinases including the src, lyn, and syk family kinases (Daeron 1997). Activation of FcεRI leads to mast cell release of inflammatory mediators including granular and lipid mediators and cytokines. FcεRI mediates local and systemic anaphylaxis. Disruption of the FcεRI gene abolishes PCA in mice. When monomeric IgE binds the receptor, it upregulates expression of FcεRI and enhances survival. When monovalent hapten binds receptor IgE, it prevents receptor upregulation and aggregation but allows receptor internalization, degranulation, and IL-6 production. Receptor aggregation is induced by multivalent antigen and leads to ITAM phosphorylation (Kitaura et al. 2004). IgE class-restricted tolerance may be induced in mice by
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neonatal treatment with monoclonal IgE; it is unique to the IgE class of immunoglobulins and involves both failure to develop FcεR+ cells and the production of Treg cells (Chen et al. 1984). Recently, FcεRI has been documented on mouse sensory neurons; its significance is unknown (Andoh and Kuraishi 2004). More on this receptor is found in chapter 14 in this volume, entitled “Mouse Models to Study the Pathogenesis of Allergic Asthma.” h. OTHER FCRS It appears that there is no true homolog of human FcαRI (Monteiro and Van De Winkel 2003), although recently an FcR for IgM was described on murine oligodendrocytes and myelin which may be an Fcα/µR (Nakahara et al. 2003). The transferrin receptor (TfR) has been shown to serve as an IgA receptor on mouse eosinophils. Two isoforms are made by the mouse. Binding by IgA blocks transferrin-induced synthesis of reactive oxygen species and eosinophil-derived neurotoxin (Decot et al. 2005). FREB is a unique FcR in that it lacks a transmembrane domain and is expressed intracellularly in germinal center B cells of mouse and man. It is a homolog of FcγRI found on mouse B cells and contains three immunoglobulin-like domains and a C terminus containing a proline-rich stalk region followed by a leucine-rich amphipathic α helix. It is thought to play a role in regulating clonal expansion or differentiation of B cells during the germinal center reaction (Wilson and Colonna 2005). 3.
Complement and Complement Receptors
a. OVERVIEW OF THE COMPLEMENT SYSTEM The complement system provides a vital link between innate and adaptive immunity. It is evolutionarily highly conserved and is composed of approximately 20 serum glycoproteins that circulate in an inactive form. Various foreign substances may interact with a complement component, initiating proteolysis and subsequently generating a proteolytic product with enzymatic activity. This product then participates in an enzymatic cascade with tremendous amplification. The biologically active cleavage products of complement proteins bind covalently to microbes, antibodies of certain isotypes, and tissues in which complement is activated. Regulatory proteins, found on normal host cells, minimize complement-mediated damage to the host. Four pathways lead to complement activation: the classical pathway, in which mouse IgM and IgG2b bound to antigen initiates the cascade; the alternative pathway in which activation is induced by a microbial cell surface without antibody; the lectin pathway which is activated by a plasma lectin that binds mannose residues on microbial cells; and the pentraxin pathway in which C-reactive protein, serum amyloid P component, and PTX3 bind to C1q and activate the classic pathway. The latter appears to be an important mechanism for the removal of apoptotic cells. Regardless of the pathway activated, the central event in each cascade is the proteolysis of complement protein 3 to produce biologically active split products with one product, C3b,
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becoming covalently attached to microbial cell surfaces or antibody bound to antigen. A second product, C3a, binds to a specific receptor found on inflammatory cells (C3aR), inducing a series of biological activities from opsonization and phagocytosis to degranulation of intracytoplasmic granules. In the classic pathway IgM or IgG2b bound to antigen exposes a binding site for C1q on the Fc region of the µ or γ heavy chain. Each heavy chain has a single C1q binding site and each C1q molecule must bind two or more Ig heavy chains to be activated. Once activated, C1q leads to the enzymatic activation of C1r, which cleaves and activates C1s. Activated C1s cleave and activate C4 to generate C4b (with C4a being released as an anaphylatoxin). The attachment of C4b to the cell surface next to cell-bound C1s leads to C2 activation. C2 binds to C4b and is cleaved by C1s to generate C2b. This C4b2b complex acts as a C3 convertase. Once C3b is bound to the surface it can bind factor B and generate more C3 convertase. Through this mechanism thousands of C3b molecules can be deposited on the surface where complement is activated. The C4b2b3b complex acts as a C5 convertase needed to generate the late stage of the cascade. The alternative pathway of activation is based on the phenomenon of continual low level plasma cleavage of C3 to C3b. Some C3b reacts with amino or hydroxyl groups on cell surfaces or with polysaccharides to form amide or ester bonds. If these bonds are not generated, C3b becomes inactivated in the fluid phase. However, once the bonds are formed, the C3b can bind Factor B and then Factor B is cleaved by Factor D to generate the Bb fragment. This C3bBb complex is also a C3 convertase and cleaves more C3 molecules, resulting in tremendous amplification. C3Bb complexes on mammalian cells are rapidly degraded by regulatory proteins not found on microbial cells. On microbial cells some of the C3b generated by C3 convertase binds to the complex, producing the C5 convertase, C3bBb3b (see Fig. 3). In both the lectin and pentraxin pathways, complexes formed activate C1 and the classic pathway. Plasma mannose binding lectin binds mannose residues on microbial polysaccharides, and because it is structurally similar to C1q, it triggers the activation of the C1r-C1s complex. In the late steps of complement activation C5 convertases cleave C5 into C5a and C5b. The C5a split product is a biologically active anaphylatoxin (like C3a and C4a). C5b remains bound to the complex and binds both C6 and C7. The resulting C5b, 6, and 7 complex inserts into the lipid bilayer of cell membranes and is a C8 receptor. The resulting complex of C5b, 6, 7, and 8 (C5b-8) attracts C9, which polymerizes at the complex site and forms pores in plasma membranes. The C5b-9 complex is known as the membrane attack complex (MAC) and the pores allow for the exchange of ions across the membrane, leading to cell death (see Fig. 4). The C9 serum protein is homologous to perforin, which is found in cytotoxic T lymphocytes and NK cells. Readers interesting in more detail concerning the regulation of the complement cascade in mice
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Late Steps of Complement Activation Fig. 3 The early steps of complement activation by the alternative and classical pathways. The alternate pathway (A) is activated by C3b binding to various activating surfaces, such as microbial cell walls, and the classical pathway (B) is initiated by C1 binding to antigen-antibody complexes. The C3b that is generated by the action of the C3 convertase binds to the microbial cell surface or the antibody and becomes a component of the enzyme that cleaves C5 (C5 convertase) and initiates the late steps of complement activation. The late steps of both pathways are the same (not shown here), and complement activated by both pathways serves the same functions. The lectin pathway (not shown) activates C1 in the absence of antibody, and the remaining steps are the same as in the classical pathway. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
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Membrane Attack Complex (MAC) Fig. 4 Late steps of complement activation and formation of the MAC. Schematic view of the cell surface events leading to formation of the MAC is shown. Cell-associated C5 convertase cleaves C5 and generates C5b, which becomes bound to the convertase. C6 and C7 bind sequentially, and the C5b, 6, 7 complex becomes directly inserted into the lipid bilayer of the plasma membrane, followed by stable insertion of C8. Up to 15 C9 molecules may then polymerize around the complex to form the MAC, which creates pores in the membrane and induces cell lysis. C5a released on proteolysis of C5 stimulates inflammation. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
and differences between the complement system in mice and man are encouraged to read chapter 6, entitled “Clinical Chemistry of the Laboratory Mouse” in Volume 3. Those interested in a more detailed explanation of the pathways may consult several recent reviews (Abbas and Lichtman 2005; Goldsby et al. 2003; Lambris et al. 1999). b. TYPES OF COMPLEMENT RECEPTORS Complement receptors are varied: structurally, in cell distribution, and in function. They are capable of binding C3b and its degradation products, C3dg, C3d, and iC3b, and C4b and the anaphylatoxins, C3a, C5a, and C5a desArg. C3b is converted to the inactive iC3b and subsequently to C3dg by fluid-phase Factor I and Factor H. C3dg can be further cleaved to C3d by serum proteases; however, the biological functions of C3d and C3dg appear to be identical (Rickert 2005). C3a desArg and C5a desArg are generated when serum carboxypeptidase N rapidly removes the C-terminal arginyl residue of C3a or C5a, respectively. I. CR1 AND CR2 Complement 1 and complement 2 receptors (CR1 and CR2) are both encoded by the same gene, Cr2, by alternative splicing. Both CR1 and CR2 are single-chain transmembrane glycoproteins with a large extracellular domain. The CR1 (CD35) receptor is generated by the addition of six short consensus repeat units (SCRs) to the amino-terminal
end of the CR2 (CD21) protein. Thus, the extracellular domain of CR1 has 22 SCRs whereas CR2 has 15–16 SCRs. In mice, but not man, CR1 serves as a receptor for C4b and C3b in addition to C3d and iC3b. CR2 binds C3d and iC3b. In mice, both CR1 and CR2 are expressed on mature B cells and follicular DCs (FDCs). CR1 is also expressed on monocytes, neutrophils, and eosinophils. Unlike humans, mouse CR1 is not expressed on erythrocytes or platelets and thus is not an immune adherence receptor (Haas et al. 2002; Molina et al. 1996). CR1 on phagocytes causes internalization of particles opsonized with C3b or C4b. The receptor also transduces signals that activate microbicidal mechanisms, especially if there is simultaneous engagement of FcγRI and FcγRIII receptors. CR1 and CR2, present on B cells and FDCs, are critical regulators of both thymic-independent (TI) and thymic-dependent (TD) antibody responses. In mice CR1 and CR2 play a unique role in promoting IgG3 antibody responses (isotype responsive toTI-2 antigens); this is due to their ability to localize bloodborne antigen-C3d complexes to marginal zone B cells of the spleen and cause immediate isotype switch (Haas et al. 2002; Zandvoort and Timens 2002). Mice lacking CR1 and CR2 have normal serum levels of total IgM and the various IgG isotypes and have no evidence of
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altered B or T cell development or germinal center formation. However, their humoral responses to TD antigens, especially secondary responses, are markedly impaired. CR2 is noncovalently associated with other molecules to form a multimolecular signal transduction complex on the B cell surface. One molecule in this complex, CD19, is a glycoprotein belonging to the immunoglobulin superfamily and is involved with B cell activation and proliferation. When CR2 interacts with C3d-coated surface IgM-bound antigens, CD19 acts as the signaling component initiating intracellular events that amplify and facilitate B cell activation (Molina et al. 1996). In addition, expression of CR1 and CR2 on FDCs is essential for the development of long-term memory B- cells (Barrington et al. 2001). Finally, the Cr2 gene is a candidate for the Sle1c lupus susceptibility locus (Boackle et al. 2001); also see the chapter 11 in this volume, entitled “The Genetics of Mouse Models of Systemic Lupus”. Altered expression of CR2 has been associated with systemic lupus erythematosus in multiple mouse strains and in man. A mutation in Cr2 of mice leads to a polymorphism in the SCR1–2 site near the N terminus of the extracellular domain. The mutation introduced a unique glycosylation site, which molecular modeling, based on the crystal structure of CR2-C3d, predicts will interfere with ligand binding and receptor dimerization. This mutation is seen in many lupus-prone strains. CR2 is also required to develop autoimmunity to murine cardiac myosin (Hannan et al. 2002). II. CR3 AND CR4 Complement receptors 3 and 4 (CR3 and CR4) are integrin molecules found on the surface of mononuclear phagocytes, neutrophils, and NK cells. CR3 (CD11b CD18) is composed of a unique 165-kDa α chain (CD11b) also called Mac-1 and a common 95-kDa β chain (CD18). The two chains are noncovalently linked and bind iC3b, a degradation product of C3b. The β chain is identical in two closely related molecules, leukocyte function-associated-antigen-1 (LFA-1) and p150, 95. The CR3 receptor promotes phagocytosis of iC3b-coated microbes and also acts as an adhesion molecule by binding intercellular adhesion molecule (ICAM)-1 on endothelial cells, promoting stable attachment of leukocytes to endothelium without complement (see Fig. 5). CR4 is composed of a unique 150-kDa α chain (CD11c) and the 95-kDa common β chain (CD18). It is found on the surface of phagocytes and especially DCs where it binds iC3b and induces phagocytosis. Recent reports suggest that both CR3 and CR4 play a role in the removal of iC3b opsonized apoptotic cells. Here, complement may be activated by the lectin or pentraxin pathways. Uptake of apoptotic cells by phagocytes has an immunomodulatory effect, which favors T cell tolerance (Pittoni and Valesini 2002). III. RECEPTORS FOR ANAPHYLATOXINS Receptors for anaphylatoxins include the C3aR and the C5aR, both of which are members of the rhodopsin-type receptor superfamily and have seven transmembrane domains. In this respect, they are similar to other chemoattractant receptors, for example, the receptors for N-formyl peptides, leukotriene B4, and various α- and
11 β-chemokines. The coding sequence for all these receptors except C5aR is contained on a single exon. In the case of C5aR, the initiating methionine codon is separated from the rest of the coding sequence by a single large intron, similar to the human sequence (Gerard et al. 1992). Both receptors couple to pertussis toxin-sensitive and -insensitive G proteins. Mouse C3aR is composed of 477 amino acids and shares 65% sequence identity with human C3aR. The large second extracellular loop is the most divergent, containing only 44% identity (Hollmann et al. 1998). In man, C3aR is a chemoattractant receptor that mediates directed chemotaxis of eosinophils and mast cells. It also mediates histamine release from mast cells and IL-3-treated basophils, smooth muscle contraction, and modulation of cellular and humoral immune responses. In mice, C3aR is found on bronchial epithelium, endothelium, and smooth muscle in the lung where binding to the C3a ligand leads to hyperresponsiveness to aerosolized methacholine. C3aR is also present in parasympathetic ganglia cells in the airway, and it can modulate the effects of muscarinic cholinergic receptors. C3aR knockout mice have protection from airway reduction, reduced levels of IL-4, reduced antigen- specific IgE and IgG, and reduced eosinophil infiltration in the ovalbumin challenge model (Gerard and Gerard 2002) and Aspergillus fumigatus extract model of allergic asthma (Baelder et al. 2005). Paradoxically, C3aR knockouts are more susceptible to intravenous injection with LPSs. This finding suggests that although most C3aR functions are proinflammatory in nature, their responses to LPSs may be anti-inflammatory, suppressing LPS-induced secretion of TNFα, IL-1β, and IL-6 from peripheral blood mononuclear cells and B cells (Kildsgaard et al. 2000). Presentation of ovalbumin to APCs in the skin lacking C3aR resulted in more IL-4 and IL-5 secretion by T cells. C3a inhibited the ability of splenocytes to secrete Th2 cytokines (Kawamoto et al. 2004). C3aR also appears to play a significant role in murine autoimmune diseases. Receptor blockade of C3aR in MRLlpr mice prolonged survival and reduced the number of neutrophils, monocytes, and apoptotic cells in the kidneys of this lupus-prone strain (Bao, Osawe, Haas, et al. 2005). Boos et al. (2004) found that transgenic mice overexpressing C3a under the glial fibrillary acid protein promoter expressed much more C3a in the brain, which was associated with massive meningeal and perivascular infiltration of macrophages and CD4 lymphocytes in a model of experimental autoimmune encephalitis (EAE). They also showed that C3aR knockouts had a significantly attenuated course of EAE with greatly reduced macrophage and lymphocyte infiltration into the spinal cord. The C5aR of mice, as in man, contains a single N-linked glycosylation site near the amino terminus and a general acidic property for the first extracellular region. Overall mouse C5aR shares 65% sequence homology with human C5aR with the most divergence appearing in the extracellular positions of the molecule. Despite these differences, mouse C5aR binds human C5a
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Phagocyte Oxidase Killing of Phagocytosed Microbes by ROIs and NO Fig. 5 Phagocytosis and intracellular destruction of microbes. Microbes may be ingested by different membrane receptors of phagocytes; some directly bind microbes, and others bind opsonized microbes. (Note that the Mac-1 integrin binds microbes opsonized with complement proteins [not shown].) The microbes are internalized into phagosomes, which fuse with lysosomes to form phagolysosomes, where the microbes are killed by reactive oxygen and nitrogen intermediates. iNOS, inducible nitric oxide synthase; NO, nitric oxide; ROIs, reactive oxygen intermediates. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
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with high affinity and mediates mast cell vascular permeability and smooth muscle contraction (Gerard et al. 1992). This result is consistent with the fact that only the amino terminus is useful for ligand affinity chromatography (Gerard and Gerard 1994). In man, C5aR is found on mast cells, basophils, granulocytes, mononuclear phagocytes, platelets, and endothelial cells, and ligation of the receptor is responsible for mast cell and basophil degranulation. In mice, C5aR induces IL-6 and TNF-α in response to sepsis and suppresses neutrophil function. In contrast C5aR protects mice from Pseudomonas pneumonia when it activates lung macrophages essential for bacterial phagocytosis. A deficit of C5a was associated with decreased bronchial responsiveness to antigenic challenge, and at least part of this effect is mediated through IL-12 release that induces a Th1 helper response from monocytes and macrophages. In the absence of C5, a more vigorous Th2 response ensues (Gerard and Gerard 2002). Furthermore airway hyperresponsiveness was substantially improved after C5aR blockade in an Aspergillus extract model. In addition C5aR blockade was associated with reduced lymphocyte numbers in bronchoalveolar lavage fluid (Baelder et al. 2005). C5aR was shown to control the migration of neutrophils into the intraperitoneal cavity where they served as killer cells for iC3b-antibody opsonized tumor cells (Allendorf et al. 2005). C5aR also controlled the development and progression of lupus nephritis in MRLlpr mice (Bao, Osawe, Puri, et al. 2005; Wenderfer et al. 2005). An orphan receptor, C5L2, which binds both C5a and C5a desArg, has been reported in mice. This enigmatic serpentine receptor is coexpressed with the C5aR on many cells including neutrophils. C5L2 is uncoupled from G proteins and may modulate the activity of C5a by acting as a decoy receptor and limiting the proinflammatory activities of C5a. Recently the rat homolog of this receptor was found to be a noradrenalininduced anti-inflammatory receptor on brain astrocytes (Gavrilyuk et al. 2005). 4.
C-Type Lectin Receptors
a. INTRODUCTION Antigen uptake by cells for processing and presentation is mediated in part by C-type lectin receptors (CLRs). The CLRs contain one or more carbohydrate recognition domains (CRD) used by the CLRs to bind carbohydrates found on pathogens and host cells. Host cell carbohydrate binding is involved in cell adhesion, glycoprotein regulation, tissue repair, and healing. There are four families of CLRs based on differences in molecular structure: type I (mannose receptors); type II receptors; collectins; and NK cell receptors (type V). b. TYPE 1 RECEPTOR FAMILY Type 1 receptors include the macrophage mannose receptor (CD206) and the Endo 180 receptor. These transmembrane receptors have an N-terminal cysteine-rich domain followed by a fibronectin type II domain and a series of eight tandemly arranged CRDs, all in the extracellular portion of the molecule. They also contain a short
13 cytoplasmic tail that confers the ability to rapidly recycle between the cell surface and the intracellular compartment (McGreal et al. 2004). CD206 displays a calcium-dependent lectin activity toward terminal mannose, fucose, and N-acetylglucosamine residues. Among the eight CRDs only CRD4 and 5 show true affinity for monosaccharides. CD206 has been shown to be a pattern recognition receptor for a broad range of bacteria including Klebsiella pneumoniae. The CRDs of CD206 also bind a range of self-ligands including many mediators of inflammation. In this respect they are thought to play a major role in homeostasis and the resolution of inflammation. Anti-inflammatory mediators such as IL-4, IL-10, and IL-13 upregulate CD206 expression. CD206 is expressed on macrophages, DCs, and lymphatic and hepatic endothelium (Fig. 5) (McGreal et al. 2004). Although CD206-positive DCs are not found in T cell areas of lymphoid organs, and thus may not target ligands for antigen presentation to T cells, DCs expressing DEC-205 (CD205) are abundant in T cell areas. DEC-205 is structurally similar to CD206 except that it has 10 CRDs on the extracellular segment. After ligand binding, CD206 recycles quickly through cells via early endosomes, and by contrast ligand-bound DEC205 localized both to early endosomes and MHC class II late endosomes and lysosomes. The EDE sequence on the cytoplasmic domain of DEC-205 enables the receptor to target MHC II compartments. In addition, ligands for DEC-205 are processed via the exogenous pathway to MHC class I in a “transporters for antigenic peptides” (TAP) dependent manner (Steinman et al. 2003). More on the role of CLRs in DC biology is presented in chapter 4 in this volume, entitled “Some Biological Features of Dendritic Cells in the Mouse.” Endo 180, expressed on fibroblasts, macrophages, and certain endothelial cells, has been shown to bind mannose, fructose and N-acetylglucosamine via its CRD2 domain. In addition, the fibronectin type II (FNII) domain has a novel receptor for collagen. Mice with a targeted mutation of FNII exhibited defective collagen uptake and reduced migration of fibroblast and a defect in chemotactic activity to urokinase-type plasminogen activator. Whereas the pathogen recognition capacity of Endo 180 is largely unknown, this receptor appears to be involved in tissue remodeling and repair. c. TYPE II CLRS Type II CLRs include the specific intracellular adhesion molecule-3 grabbing non-integrin (SIGN) family including mDC-SIGN, mSIGNR1, mSIGNR2, mSIGNR3, and mSIGNR4 as well as Dectin-1, Dectin-2 and Langerin. They are all type II CLRs containing a single CRD. Genes for the SIGN CLRs are located on adjacent regions of chromosome 8 in close proximity to the CD23 gene. mSIGNR1, mSIGNR3, and mSIGNR4 are transmembrane receptors whereas mSIGNR2 lacks the transmembrane domain and is a soluble receptor. The CRD is found on the N terminus of the extracellular domain. All mouse SIGNs, except mSIGNR4, have a highly conserved EPN sequence within the CRD, which recognizes
14 mannose-containing structures. mSIGNR4 has a QPN motif instead and probably recognizes another ligand specificity, possibly galactose. Within the cytoplasmic tail several internalization motifs are found. All membrane-bound SIGNs have a triacidic cluster motif, whereas mDC-SIGN also has a di-leucine motif and mSIGNR1 and R3 have a tyrosine-based motif. These various cytoplasmic tail motifs are important for antigen internalization (Koppel et al. 2005). These CLRs tend to form tetramers by interactions between neck domains. mDC-SlGN is abundant on DCs in tissues throughout the body. There are also low levels of expression on B cells but not on T cells. mSIGNR1 mRNA is expressed in high levels in liver and lymph nodes and in moderate levels in spleen. No mSIGNR1 is found on DCs, B cells, or T cells. In lymph node, mSIGNR1 is expressed on medullary and subcapsular macrophages, whereas in the spleen it is expressed on the marginal zone macrophages (MZMs). mSIGNR1 is also expressed on liver sinusoidal endothelial cells and peritoneal macrophages. mSIGNR2 mRNA is present in testes and in LPS-stimulated B cells, mSIGNR3 RNA has been detected in spleen and lymph node, and mSlGNR4 mRNA is detected in testes and spleen. Little is known about the function of mSIGNR2, mSIGNR3, or mSIGNR4. The CRDs of mSIGNR1 and R3 bind mannose-, fucose-, and N-acetylglucosamine-terminating oligosaccharides. Fucose recognition was demonstrated by binding Lewis antigens. The binding specificity of mDC-SIGN is unknown. mSIGNR1 can interact with ICAM-2 expressed widely on leukocytes and especially on lymphocytes and may serve to bring mSIGNR1 cells in contact with lymphocytes. In addition, mouse L-selectin may bind circulating leukocytes using the same interaction. Likewise mSIGNR1 on MZMs may interact with lymphocytes migrating through the marginal sinus of the spleen, attracting them to the white pulp. Here MZMs are among the first cells to see blood-borne antigens including carbohydrates on Candida albicans, Mycobacterium tuberculosis, Escherichia coli, and Streptococcus typhimurium, leading to their internalization, degradation, and shedding, where they are taken up by marginal zone B cells after opsonization by complement. Dectin-2 is a type II receptor with a classical calciumdependent CRD. It appears to bind mannose residues although a ligand is also present on CD4+ CD25+ T cells. Dectin-2 is expressed on macrophages, DCs, and Langerhans cells. Langerin (CD207) is a type II CLR with binding specificity for mannose, fucose, and N-acetylglucosamine. It is expressed on Langerhans cells and subpopulations of DCs. Langerin appears to form trimers by interactions between the neck domains. After ligand binding, Langerin is rapidly internalized but does not traffic to MHC II compartments. Langerin induces the formation of Birbeck granules (Cambi et al. 2005; McGreal et al. 2004). d. COLLECTINS Collectins comprise a family of CLRs that are not transmembrane proteins but rather are soluble proteins that tend to form trimers or oligomers. They include the lung
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surfactant proteins SP-A and SP-D and mannose binding protein. SP-A and SP-D are secreted at the luminal surface of pulmonary epithelial cells and form oligomers necessary for regulating surfactant phospholipid homeostasis and reducing surface tension within alveoli, allowing less pressure to maintain them open; they also have broad specificities for pathogen-associated molecular patterns. SP-A and SP-D interact with allergens and immune cells and modulate cytokine and chemokine profiles during the hypersensitivity response (Madan et al. 2005). Mannose-binding protein is a plasma protein with a single CRD, but its ability to form oligomers correlates with complement activation (Cambi et al. 2005). e. TYPE V FAMILY RECEPTORS Dectin-1 is a unique type V CLR because it is one of the only CLRs that recognizes carbohydrate ligands in the absence of calcium, and its carbohydrate-binding motif is not the classic CRD. The gene for Dectin-1 is located in the NK gene complex. This CLR is a 28-kDa transmembrane receptor that contains an ITAM motif in its cytoplasmic tail. It is expressed on leukocytes targeted for β-glucan activity: monocytes, macrophages, DCs, and Langerhans cells but not NK cells. Dectin-1 expression is upregulated by IL-4 and IL-13 and is downregulated by IL-10 and glucocorticoids. Dectin-1 has specificity for β-1,3 and β-1,6 linked glucans found in fungi, plant cell walls, and some bacteria. It appears to be primarily involved with protection against pathogenic yeast infections where it mediates phagocytic uptake, killing, and the release of TNF-α and macrophage inflammatory protein (MIP)-2 by macrophages. Dectin-1 accomplishes this by cooperating with the TLR-2 signaling pathway through the activation of its ITAM (McGreal et al. 2004). 5.
NK Cell Receptors
a. INTRODUCTION NK cells were originally described for their ability to kill tumor target cells without the necessity of prior sensitization. They also are effectors against viruses, parasites, and intracellular bacteria. After antigen recognition they secrete chemokines and cytokines, which regulate the adaptive immune response. Upon contact with antigen on cells, both stimulatory and inhibitory receptors on the NK cells are activated. The balance between these opposing signals will either favor NK cell cytotoxicity or tolerance to self. A more complete discussion on NK cells and their biology is presented in chapter 6 in this volume, entitled “Mouse Natural Killer Cells: Function and Activation.” b. LY49 RECEPTORS Ly49 receptors belong to the family of lectin-like type II transmembrane receptors. They are expressed as disulfide-bonded homodimers on NK cells and some T-cell subsets. Ligands for these receptors include MHC class II molecules and MCMV. After ligand engagement, the receptors may either stimulate or inhibit further NK responses. Inhibitory receptors have ITIM motifs in their cytoplasmic domain, which, upon ligand binding, become tyrosine phosphorylated and recruit intracellular phosphatases such as Src homology
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domain-2–containing protein tyrosine phosphatase-1 (SHP-1). SHP-1 is responsible for dephosphorylating downstream signaling proteins in the activation pathway. By contrast, stimulatory Ly49 receptors have short cytoplasmic tails lacking signal-transducing elements. They associate with and signal through adaptor molecules such as DNAX-activation protein-12 (DAP-12). More than 24 genes or pseudo genes have been found for Ly49 receptors. The C57BL/6 strain has 11 active Ly49 genes and 5 pseudogenes. Of these Ly49A, Ly49B, Ly49C, Ly49E, Ly49F, Ly49G, Ly49I, Ly49J, and Ly49Q are inhibitory and only two, Ly49D and Ly49H, are stimulatory. The ligand for Ly49D is H-2Dd, Dr, and Dspz, and the ligand for Ly49H is involved with resistance to MCMV. In the 129 strain MCMV protein m157 binds inhibitory receptor Ly49I, since this strain does not express Ly49H (Backstrom et al. 2004; Kane et al. 2004). c. CD94/NKG2 RECEPTORS CD94/NKG2 receptors are CRLs and together with Ly49 they stimulate or inhibit NK cell activation. NKG2 belongs to the type V lectin-like transmembrane receptor family; NKG2A and NKG2B have two ITIM motifs in their cytoplasm domain. The NKG2 molecule forms a heterodimer with CD94 that is required for cell surface expression and recognition of the nonclassic MHC class I molecule, Qalb. Qalb displays peptides derived from the signal sequences of classical MHC class I molecules. NK cells indirectly monitor the expression of classical MHC class I by having CD94/NKG2 monitor the expression of nonclassic MHC class I molecules. When CD94 forms heterodimers with NKG2C and E they form stimulatory receptors that recognize Qalb. However, the binding affinity of NKG2C/E is much lower than that of NKG2A/B, thus favoring inhibition. CD94/NKG2A only binds Qalb loaded with the peptide, Qdm, and replacement of this defined peptide results in loss of NK cell inhibition (Backstrom et al. 2004). NKG2C/E contains ITAM motifs in its adaptor molecule. NKG2D is unique among CD94/NKG2 family members in that it forms homodimers, only shares about 20% amino acid homology with other members, and does not bind Qa-1b. Two charged amino acid residues in the transmembrane domain associate with the adaptor molecules DAP-10 or DAP-12. DAP-10 contains an YxxM motif whereas DAP-12 contains an ITAM motif. The adaptors are selectively expressed in different cell types and mediate signaling via different pathways. Mouse NKG2D-short (S) can associate with either DAP-10 or DAP-12; however, NKG2D-long (L) associates only with DAP-10. Naive NK cells express only NKG2D-L but activated NK cells can express both isoforms. Activated CD8+ T cells express both isoforms but mediate effects through DAP-10 only. Ligands for NKG2D include two proteins distantly related to MHC class 1 molecules, having MHC class 1–like α1 and α2 domains but no α3 domain. RAE1α, β, γ, δ, and ε are GPIlinked proteins, whereas H60 is a transmembrane protein with
a short cytoplasmic tail. RAE1 family members are expressed during embryonic days 9–14 and not in adults. These proteins have been induced by MCMV infection. H60 is not expressed in C57BL/6 mice but is found in thymocytes and lymphoblasts of BALB/c mice. The mouse UL16 binding protein–like transcript 1 (MULT1) possesses MHC class 1–like α1 and α2 domains and has a long cytoplasmic domain. MULT1 is expressed widely in mice (Backstrom et al. 2004). Figure 6 is a diagram of the role of activating and inhibitory receptors of NK cells.
B.
The Major Histocompatibility Complex
The major histocompatibility complex (MHC), also called H-2 in the mouse, encodes a large number of genes found on chromosome 17. The first genes studied in this region encoded proteins that served as the basis for allograft rejection, so called histo-incompatibility, which provided the name for this complex. Within the MHC there are three classes, clusters of closely linked genes, named class I, II, and III. Class III genes encoded several proteins of the complement system as well as genes that aid in the regulation of the immune system. MHC class I and II genes encode highly polymorphic molecules that play a role in antigen presentation to BCRs and TCRs, in addition to their role in graft rejection. Haplotypes of the MHC complex are determined by the combination of alleles of the class I (K, D, L, Q, T, and M), class II (I-Aa, I-Ab, I-Ea, and I-Eb), and class III (S region) genes. Table 1 lists the allelic designations for the MHC for inbred and recombinant strains. Readers should refer to chapter 3 in this volume, entitled “Genomic Organization of the Mouse Major Histocompatability Complex,” for further information. 1.
Classic Class I Molecules
Classic class I molecules are composed of a 45-kDa α chain noncovalently linked to a 12-kDa β2-microglobulin molecule and are expressed on a wide variety of cells. The α chain is encoded by highly polymorphic genes found within the K and D/L regions of the mouse MHC. The α chain has three extracellular domains, each containing 90 amino acids, a transmembrane domain, and a cytoplasmic anchor segment. The transmembrane domain contains about 25 hydrophobic amino acids on the extracellular side of the membrane, followed by a short segment of charged (hydrophilic) residues. β2-Microglobulin does not have a transmembrane segment and is noncovalently linked to the α3 domain, with which it shares sequence homology. Crystallization studies have shown there are two pairs of interacting domains in the extracellular fragment: a membrane-distal pair made up of the α chain α1 and α2 domains and a membrane-proximal pair composed of the α3 domain and β2-microglobulin. The α1 and α2 domains form a platform of eight antiparallel β strands bordered by two
16
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Inhibitory Receptor Engaged Removal of Phosphates and Inhibition
Activating Signals
P P PTK PTP
P
Inhibitory Receptor
Activating Receptor Ligand for NK Cell
Self Class I MHC–Self Peptide Complex
NK Cell Not Activated; No Cell Killing
Normal Autologous Cell
B
Inhibitory Receptor Not Engaged
Virus Inhibits Class I MHC Expression
NK Cell Activated; Killing of Infected Cell
Virus-Infected Cell (Class I MHC Negative) Fig. 6 Activating and inhibitory receptors of NK cells. Activating receptors of NK cells recognize ligands on target cells and activate protein tyrosine kinase (PTK), whose activity is inhibited by inhibitory receptors that recognize class 1 MHC molecules and activate protein tyrosine phosphatase (PTP). As a result, NK cells do not efficiently kill class 1 MHC–expressing targets. If a virus infection inhibits class 1 MHC expression on infected cells, the NK cell inhibitory receptor is not engaged, and the activating receptor functions unopposed to trigger responses of NK cells, such as cytolysin secretion. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
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TABLE 1
ALLELIC DESIGNATIONS OF THE H2 COMPLEX OF SOME INBRED, CONGENIC, AND RECOMBINANT STRAINS MHC (H2) Loci Haplotype
Strains
K
I-Ab
I-Aa
I-Eb
I-Ea*
S
D
L
Qa2
T18
Qa1
a b
A/J, AXB-5/Pgn, BXA-4/Pgn CXBK 129P2/J, C57L/J, BXSB/MpJ, C57BL/10J, C57BL/6J BALB/cJ, DBA/2J, AKXD13/Ty BIO.M–H2f/nMob, A.CAH2fH2-T18d/Sn WB/ReJ+/+, WC/ReJ+/+ MRL/MpJ, AKR/J, C58/5, CBA/J, RF/J NOD/LtJ, NOR/LtJ, NOcCB1/Lt R III/J, R III/DmMob ALS/LtJ, NON/LtJ P/J, C3H.NB-H2PH2-T18c/Sn DBA/1J, FVB/NJ, SWR/J B10.R III-H2r (71NS)/nMob SJL/J, CXJ-15/SlkJ, SWXJ-8/Bm PL/J, B10.PL-H2u H2-T18a (73NS)/Sn SM/J, NXSM-I/Ei, NXSM-L/Ei NZM64/J, NZ0/HEllJ, NZW/LacJ
k b
k b
k b
k b
k b
d b
d b
d b
a a
a b
a b
d
d
d
d
d
d
d
d
b
d
b
f
f
f
f
f
f
f
f
b
d
b
j k
j k
j k
j k
j k
j k
b k
b k
a b
b b
b b
d k b p q r s u
g7 k nb1 p q r s u
g7 k nb1 p q r s u
— k k p q r s u
— k k p q r s u
— k — p q r s u
b q b p q r s d
— q — p q r s d
— a — b a b a a
— a — e b b b a
— a — a b b b a
v u
v u
v u
v u
v u
v z
v z
v z
a b
b b
b b
d f j k g7 m nb1 p q r s u v z
*Strains that are I-Ea null: 129, ASW/Sn, BALB.B, C57BL/6, C57BL/10, DBA/1, NOD/LtJ, NOR/LtJ, SJL/J. Adapted from the JAX MICE Catalog 2000, The Jackson Laboratory, Bar Harbor, ME.
α-helical regions to form a deep groove known as the peptidebinding cleft. The cleft is displayed on the top surface of the MHC molecule and can bind a peptide of 8–10 amino acids with interactions between conserved residues and the bound peptide, resulting in a closed groove; these structural features are shared between mouse and man (Fig. 7). The α3 domain is highly conserved among class I molecules and together with β2-microglobulin it forms an immunoglobulin fold structure that interacts with the CD8 membrane molecule on T cells (see section II.C.2.c.). β2-Microglobulin also interacts with a peptide in the class I α chain, and this interaction is essential for proper fold conformation and delivery of the class I molecule and peptide to the cell surface (Abbas and Lichtman 2005; Goldsby et al. 2003). 2.
Classic Class II Molecules
Classic class II molecules contain a 33-kDa α chain and a 28-kDa β chain, which are noncovalently linked. Genes encoding these classic class II molecules are located in the IA or IE regions of the MHC. Inbred strains of the H2 haplotypes b, s, f, and g do not express E class II molecules. Each membrane-bound chain has two extracellular domains, α1 and α2, on the α chain and β1 and β2 on the β chain. The membrane-proximal α2β2 domains have an immunoglobulin fold structure, whereas the
membrane-distal α1β1 form the antigen-binding cleft (Fig. 8). Crystallographic studies show marked similarities between the antigen-binding cleft of classic class I and classic class II molecules. Both have a floor of β pleated sheets and sides of antiparallel α helices; however, the class II cleft does not have residues that interact with the ends of the bound peptide, and thus class II molecules have a open-ended groove. CD4 binding sites are found on opposite sides of the class II molecule in the α2β2 domains (Abbas and Lichtman 2005; Goldsby et al. 2003). 3.
Nonclassic Class I and Class II Molecules
Nonclassic class I molecules (also called class 1b) lack one or more features of the classic molecules and thus either have limited tissue distributions, low polymorphism, and unknown function or lack the ability to present antigen to CD8 T cells. Class Ib molecules are encoded by genes in the H2-Q, T, and M regions. Molecules derived from the Q region have either unknown function or, like Qa2, can protect from NKmediated lysis and play a role in defense against tumors or in resistance to parasites. Molecules derived from T region genes may be found on leukemia cells or, like Qa1, regulate NK cells (see chapter 6 and section II.A.5.). Molecules of the H2-T10 and T22 gene pair form a modified class I fold but together with
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Class I MHC Peptide-Binding Cleft Peptide
α1
α2 α1
N
α2
N α3
α3 β2m
β2Microglobulin Transmembrane Region
C
Disulfide Bond lg Domain C Fig. 7 Structure of a class 1 MHC molecule. The schematic diagram (left) illustrates the different regions of the MHC molecules (not drawn to scale). Class 1 molecules are composed of a polymorphic α chain noncovalently attached to the nonpolymorphic β2-microglobulin (β2m). The α chain is glycosylated; carbohydrate residues are not shown. The ribbon diagram (right) shows the structure of the extracellular portion of the HLA-B27 molecule (human class I) with a bound peptide, resolved by x-ray crystallography. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
β2-microglobulin can bind to γδ T cells. M region proteins are thought to participate in pheromone receptor function. Nonclassic class II molecules encoded in the H2-DM and DO regions are involved with peptide loading into classic class II molecules and are referred to as accessory molecules (Braud et al. 1999; Goldsby et al. 2003; Jensen et al. 2004; Ploss et al. 2003; Rodgers and Cook 2005). 4.
Molecules Associated with Antigen Processing
Two different pathways are used to eliminate intracellular and extracellular antigens; they are the cytosolic and endocytic pathways, respectively. The cytosolic pathway involves degradation of ubiquitin-protein conjugates within a multifunctional protease complex called the proteasome. The proteasome is composed of three subunits with peptidase activity, low molecular mass polypeptides LMP2, LMP7, and LMP10, which generate peptides that preferentially bind MHC class I molecules. Peptides generated from the proteasome are transported to the rough
endoplasmic reticulum (RER) by a transporter called transporter associated with antigen processing (TAP). Class I molecules are assembled in the RER with the assistance of molecular chaperones that facilitate folding. Calnexin is a chaperone that associates with free class 1 α chain to promote folding. As β2-microglobulin binds the α chain, calnexin is released, and the chaperone calreticulin and tapasin (TAP-associated protein) become involved. Tapasin brings the TAP transporter next to class I and allows the peptide to be captured in the cleft. Unbound peptides are rapidly degraded. Once peptide is bound, the complex is transported to the plasma membrane through the Golgi. It is known that each type of class I MHC binds a unique set of peptides; however, regardless of class I type (K, D, or L), all bound peptides are 8–10 amino acids long and contain similar amino acid residues at defined positions, called anchor residues. All peptides that bind class I contain a carboxyl-terminal anchor that is hydrophobic (leucine or isoleucine) and makes contact with class I in the groove region (Goldsby et al. 2003; Kloetzel and Ossendorp 2004; Lehner and Cresswell 2004).
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Class I MHC Peptide-Binding Cleft Peptide β1
α1
NN
β1
α1
β2
α2
α2
β2
Transmembrane Region Disulfide Bond lg Domain C
C
Fig. 8 Structure of a class I MHC molecule. The schematic diagram (left) illustrates the different regions of the MHC molecule (not drawn to scale). Class II molecules are composed of a polymorphic α chain non-covalently attached to a polymorphic β chain. Both chains are glycosylated; carbohydrate residues are not shown. The ribbon diagram (right) shows the structure of the extracellular portion of the HLA-DR1 (human class II) molecule with a bound peptide, resolved by x-ray crystallography. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
Antigens external to the cell are internalized by phagocytosis (macrophages) or endocytosis (receptor-mediated endocytosis or pinocytosis). B cells use their membrane-bound antibody as a receptor for receptor-mediated endocytosis. Internalized antigen moves through three increasingly acidic compartments—the early endosome, the late endosome (endolysosome), and the lysosome—in which they encounter hydrolytic enzymes and are degraded into oligopeptides of 13–18 residues. While MHC class II molecules are being assembled in the RER, their binding cleft associates with a preassembled trimer of a protein known as invariant chain (CD74). This trimer interacts with the binding grooves of three class II molecules, prevents endogenously derived peptides from binding, aids in the folding of α and β chains (so class II can exit the RER), and routs the complex to the endocytic-processing pathway via the trans-Golgi network. As the complex moves through the endosomes, the invariant chain is degraded, leaving only a small fragment, called class II-associated invariant chain peptide (CLIP), occupying the peptide groove. A nonclassic,
nonpolymorphic class II MHC molecule, H2-DM, catalyzes the exchange of CLIP for antigenic peptide. Another molecule, H2-DO, inhibits the activity of DM. Once peptide is bound, the peptide-MHC class II complex is transported to the plasma membrane. Unlike peptide binding class I molecules, class II peptides have no anchor residues; however, they have an internal sequence of 7–10 residues that provide contact points with class II. Pathways leading to antigen presentation by MHC class I and class II molecules are illustrated in Fig. 9. a. TAP PROTEINS TAP1 and TAP2 are encoded within the class II region of the mouse MHC adjacent to genes for LMP2 and LMP7. Together they form membrane-spanning heterodimers with each protein having multiple transmembrane segments. Each protein has a domain projecting into the lumen of the RER and an ATP-binding domain that projects into the cytosol. Peptides generated in the cytosol by the proteasome are translocated into the RER by TAP via a process involving the hydrolysis of ATP (Lehner and Cresswell 2004).
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Peptide-MHC Association
CD4+ T Cell
Endocytosis of Extracellular Protein
Invariant Chain (Ii)
ER
Proteasome
Class I MHC Pathway
Class II MHC
Peptides in Cytosol
Cytosilic Protein
W.
TAP
Class I MHC
CD8+ CTL ER
Class I MHC Pathway
Fig. 9 Pathways of antigen processing and presentation. In the class II MHC pathway (top panel), extracellular protein antigens are endocytosed into vesicles, where the antigens are processed and the peptides bind to class II MHC molecules. In the class I MHC pathway (bottom panel), protein antigens in the cytosol are processed by proteasomes, and peptides are transported into the endoplasmic reticulum (ER), where they bind to class I MHC molecules. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
b. H2-DM AND H2-DO DM and DO are heterodimers of α and β chains encoded by genes in the mouse MHC class II region adjacent to genes for TAP and LMP. H2-DM is not expressed on the cell membrane but exclusively in the endosomal compartment and is induced by IFN-γ. The exchange of CLIP for antigenic peptide is impaired in the presence of H2-DO. DO binds DM and reduces its efficacy. DO is expressed only in B cells and the thymus and is not induced by IFN-γ. Because higher acidity weakens the association of DO with DM, it is possible that in the B cell there is preferential selection of class II peptides from lysosomal compartments (Bryant and Ploegh 2004; Goldsby et al. 2003). 5.
The CD1 Family of Molecules
The CD1 genes encoded a family of nonpolymorphic cell surface glycoproteins that are highly conserved in mammals. CD1 proteins are structurally related to the MHC class I gene family but diverged from class I in the structure of their antigen-binding domains, allowing them to present lipids and
glycolipids to T cells. The CD1 locus is not linked to MHC and, in fact, it is found on chromosome 3. CD1d, the only member found in mice, is composed of a heavy chain containing extracellular, transmembrane, and cytoplasmic tail domains. It associates with β2-microglobulin to form a heterodimer that colocalizes with lysosomal associated membrane protein-1 in lysosomal vesicles. In addition to deep penetration into the endosomal system, the CD1d cytoplasmic tail binds the AP-3 adaptor complex, which mediates sorting of cargo proteins in late endosomes and lysosomes. This allows CD1d to sample more intracellular compartments and acquire self-lipids for presentation on APCs (Dascher and Brenner 2003). CD1d is the antigen-presenting molecule that restricts natural killer T (NKT) cells. Mouse NKT cells are defined as a population of T cells that express an invariant TCR α chain (Vα14/Jα281) in association with Vβ2, -7, or -8 and express the NK1.1 antigen (NKR-P1C), a cell surface C-type lectin. Phenotypically NK1.1+ T cells are either CD4+CD8− or CD4−CD8− and represent a major fraction of the mature T cells in thymus and liver and about 5% of splenic T cells. They are rare in lymph nodes
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(Skold and Behar 2003). By using CD1d tetramers loaded with the synthetic glycolipid α-galactosyl-ceramide (α-GalCer), murine NKT cells have been characterized into three populations based on their binding of the tetramer and expression of NK1.1 (MacDonald 2002). Injection of α-GalCer into mice leads to activation of many immune cell types and is dependent on the presence of both CD1d and invariant NKT (iNKT) cells. It is known that under these conditions iNKT cells activate NK cells to produce IFN-γ. This form of immunological stimulation has been shown to enhance host resistance to a wide variety of pathogens including bacteria, yeast, viruses, and protozoans (Skold and Behar 2003) and stimulates tumor rejection and suppresses autoimmunity (Vincent et al. 2003).
C.
Antigen Receptors and Coreceptors in Adaptive Immunity
1.
Antibodies and the B-Cell Receptor (BCR)
a. PRIMARY ANTIBODY STRUCTURE Antibodies are the antigen-binding glycoproteins found in plasma and certain secretions; the membrane-bound versions are the antigen-binding portions of the BCR. All antibody molecules have, as a common structural feature, four peptide chains. The basic structure consists of two identical heavy chains (H) of 50 kDa and two identical light chains (L) of 25 kDa molecule mass. One light chain is bound to a heavy chain by a disulfide bond as well as salt linkages, hydrophobic bonds, and hydrogen bonds to form a H-L heterodimer. The two H chains are bound to each other using similar bonds including at least one interchain disulfide bond. The N-terminal 110 amino acids of both the L chains and H chains are highly variable between antibody molecules and constitute the variable (V) region, which is the antigen binding site. These V regions are called complementarity-determining regions (CDRs) and will be discussed in more detail later in this section. Beyond the V region, both H and L chains of an antibody class have a constant sequence. Enzymatic cleavage of the molecules using papain leads to two heterodimers, each consisting of an L chain bonded to the N-terminal half of the H chain (called the Fab fragment) and a homodimer consisting of the carboxyl-terminal half of each H chain bonded to one another (called the Fc fragment) as depicted in Fig. 10. The Fab fragment is responsible for antigen binding whereas the Fc fragment imparts many other functions to the antibody, such as complement fixation, FcR binding, and transcytosis. Sequencing L chains elucidated the N-terminal V region and a carboxyl-terminal C region. The C region has two basic sequences that correspond to the κ and λ light chain types. Of all mouse antibodies, 95% are of a single κ-chain type. The remaining 5% that are λ chains consist of three subtypes based on their amino acid sequence (Goldsby et al. 2003). Sequencing of H chains elucidated an N-terminal V region followed by one of five difference constant regions known as µ,
21 δ, γ, ε, and α. Each of these five heavy chain types is known as an isotype. Whereas the δ, γ, and α chains have approximately 330 amino acids in their constant regions, µ and ε chains have 440 amino acids. These H-chain isotypes determine the class of antibody molecule: IgM (µ), IgG /(γ), IgA (α), IgD (δ), and IgE (ε). Further variations in amino acid sequence of the IgG isotype determine subisotypic specificities; mice have four subisotypes: IgG1, IgG2a, IgG2b, and IgG3. Certain inbred strains do not have IgG2a but rather have IgG2c. In addition, genetic loci that are allelic within a species determine amino acid sequences on both the light and heavy chains; these specificities among different inbred strains of mice are known as allotypes. A full discussion of isotype and allotype specificities in inbred strains is found in Bankert and Mazzaferro (1999). The N-terminal variable regions of light and heavy chains are folded and bound into globular units through the formation of covalent and noncovalent linkages to be described later under secondary and tertiary structure. Antigen-binding sites were found to be complementary to the structure of the portion of antigen bound (epitope), and thus they are known as CDRs. There are three heavy and three light chain CDRs in each N-terminal variable region domain, and the great diversity of antigen-binding sites is due to variations in the length and amino acid sequence of the six CDRs in each Fab fragment. X-ray diffraction studies of antigen-antibody complexes have shown that all six CDRs bind the antigenic epitope but that the VH domain contributes more to antigen binding than the VL domain. In some instances, antigen binding causes further conformational changes in both the antigen and antibody, which lead to more effective epitope binding. b. SECONDARY, TERTIARY, AND QUATERNARY STRUCTURE The secondary structure of antibodies is formed by folding of the polypeptide chains into antiparallel β-pleated sheets. Both heavy and light chains are composed of multiple homologous sequences of 110 amino acid residues, termed domains, which have intrachain disulfide bonds forming loops of approximately 60 amino acid residues. Light chains have one N-terminal variable domain (VL) and one constant domain (CL), whereas heavy chains, depending on the class of antibody, have an N-terminal variable domain (VH) followed by three or four constant domains (CH1–CH4). Each domain is folded into a sandwich of two β-pleated sheets, stabilized by hydrogen bonds, which connect NH groups of one strand with carbonyl groups of the other strand. Hydrophobic amino acids orientate toward the center of the sandwich whereas hydrophilic groups are oriented outward. This characteristic compact structure is known as the “immunoglobulin fold” and is characteristic of all members of the immunoglobulin superfamily of proteins, which include FcRs, the T-cell receptor, MHC class I and II molecules, T-cell accessory proteins such as CD2, CD4, and CD8 and various adhesion molecules. The tertiary structure is formed by these chains then folding into compact globular domains, which are connected to each other by the continuation of the polypeptide chains (both heavy
22
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B N N
Heavy Chain
N
N
VH
N
VL
Heavy Chain
VL
Cγ1
N Cγ1
CL
Light Chain
CL
N
VH
N
C
C
Pepsin
Pepsin Papain
Papain
Cγ2
Cγ2
Cγ3
Cγ3
C
N
Light Chain
C
C
C
N N
N
F(ab')2
N
Fab
N
N C C C
C
N
N
Fab N Papain Products
C
C C
C
Papain Products FC Papain Fragments C C Fig. 10 Proteolytic fragments of an IgG molecule. IgG molecules are cleaved by the enzymes papain (A) and pepsin (B) at the sites indicated by arrows. Papain digestion allows separation of two antigen-binding regions (the Fab fragments) from the portion of the IgG molecules that binds to complement and Fc receptors (the Fc fragment). Pepsin generates a single bivalent antigen-binding fragment, F(ab’)2. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
and light) that fall outside of the β-pleated sheets. The globular domains of adjacent heavy and light chains interact to form the quaternary structure, and this structure, situated at the N-terminal of nonidentical chains (VH-VL), forms the antigen-binding site. Similar noncovalent interactions occur between identical domains (CH2-CH2) (Goldsby et al. 2003). c. ANTIBODY CLASSES AND SUBCLASSES Mice have five major classes of immunoglobulins that differ in both structure and function. The most abundant class in serum is IgG, and
mice make four subclasses IgG1, IgG2a, IgG2b, and IgG3, all of which circulate as 150-kDa glycoproteins. These subclasses do not necessarily correlate exactly with the human counterparts. The four subclasses are distinguished by amino acid sequences encoded in germline CH genes. In addition, their structures are affected by the size of the hinge region, although all four subclasses have three disulfide bonds between heavy chains. These characteristics also affect the biological function of the subclasses. Unlike humans, the four mouse IgG
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subclasses have a complex banding pattern on electrophoresis. IgG1 has a serum half-life of 4 days and mean serum concentration of 6.5mg/mL, is cytophilic for mast cells, and causes a homologous PCA reaction. IgG2a has a serum half-life of 5 days and a mean concentration of 4.2 mg/mL, can cross the placenta by FcRn transcytosis, is cytophilic for macrophages, and causes a heterologous PCA reaction. IgG2a is preferentially expressed in the host response to viral infections (Greenspan and Cooper 1992). IgG2b has a short half-life (t1/2 = 2 days), mean serum concentration of 1.2 mg/mL, crosses the placenta, activates complement by the classical pathway but is not cytophilic. IgG3 has a long half-life (t1/2 = 4 days) but is present in very low serum concentrations (100–200 µg/mL). Unlike the other IgG subclasses, IgG3 predominates in the humoral response to bacterial polysaccharides (TI type 2). The binding characteristics of IgG3 antibodies against N-acetylglucosamine residues on group A streptococci are markedly different from those of variable domain-identical IgG1 and IgG2b antibodies and involve noncovalent interactions between Fc fragments of adjacent molecules, leading to oligomerization and cooperativity in binding multivalent antigens. IgG3 can also cross the placenta (Greenspan and Cooper 1992). Cytokines involved in a subclass switch to IgG2a, IgG2b, and IgG3 are elaborated by Th1 responses (IFN-γ), whereas those for IgG1 are elaborated by Th2 responses (IL-4). TGF-β induces a class switch to IgG2b (Nimmerjahn and Ravetch 2005) (also see chapter 5 in this volume, entitled “Mouse Models Revealed the Mechanisms for Somatic Hypermutation and Class Switch Recombination of Immunoglobulin Genes”). Binding of the various IgG subclasses to FcRs is discussed in section II.A.2. IgM accounts for approximately 5% of serum immunoglobulin in outbred mice with average concentrations of ~1 mg/mL. B cell surface IgM exists as a monomer with the heavy chain bound by a single disulfide bond at cysteine position 575. Secreted IgM exists primarily as a pentamer of five monomeric units held together by disulfide bonds between heavy chains. The pentamer is arranged with the antigenbinding Fab subunits facing outside (peripheral). Monomeric subunits are larger than typical IgG monomers because the heavy chain contains an extra, fourth, constant region domain. They generally have higher carbohydrate contents as well. In the pentamer, a J chain is disulfide bonded to the penultimate half-cysteine residue of the heavy chains. These J chains ensure that the five subunits are held in a closed ring, aid in transcytosis, and are highly conserved among species (Matsuuchi et al. 1986). In mice, IgM appears as a single class with no subclasses and mutation of cysteine at position 575 is known to inactivate complement fixation (Bankert and Mazzaferro 1999). IgA is a class found in secretions (saliva, bile, colostrums, and tears) and serum. In serum it may exist as either a monomer or dimer, whereas the secreted form is typically a dimer with two monomeric units linked by both a J chain and a secretory
23 component (SC). The J chain is similar to that described for IgM. The SC is derived from the PIgR (see section II.A.2.b.) responsible for transporting IgA across the epithelial cells into mucosal or glandular secretions. The SC is bound to mouse IgA by both disulfide and noncovalent bonds. Mice have two subclasses of IgA, one with the normal disulfide linked structure (as seen in the NZB strain) and another in which the light chains are linked to each other by disulfide bonds but not to the heavy chains (as seen in BALB/c mice) (Bankert and Mazzaferro 1999). IgE, also known as reaginic antibody, exists in serum as a fourchain monomer of 185–200 kDa. Like IgM, the IgE monomers contain a fourth constant region domain. Serum concentrations of IgE are reported to be <10 µg/mL. IgE is cytophilic for mast cells, where it binds the FcεRI receptor (see section II.A.2.g.) and is able to mediate the release of granular and lipid mediators of inflammation, including the PCA reaction. The IgE class switch is induced by IL-4 as part of a Th2 response. IgD exists primarily as a monomer bound to the surface of B cells. Levels of secreted IgD in mouse serum are generally low (<10 µg/1mL), but relatively high levels can be achieved after repeated immunization (White and Gray 2000) or in IgM knockout mice (Lutz et al. 1998). Mouse δ chains are unusual in that they contain two constant domains only, Cδ1 and Cδ3; however, because of the large amount of carbohydrate, the IgD heavy chain has a molecular mass of 70 kDa. Mouse CD4+ T cells have a receptor that binds the Fd and Fc regions of the δ chain in a calcium-dependent fashion. Increased secondary antibody responses appear to be mediated through T cell IgD receptors and membrane IgD that enhances T-B cell interactions (Preud’homme et al. 2000). A full discussion of mouse antibodies and their quantization is found in chapter 6 in Volume 3 of this series. d. THE BCR The ligand (antigen)-binding moiety of the BCR is membrane-anchored immunoglobulin (mIg). All five classes of mouse Igs can be expressed as mIgs, and they differ from the secreted protein by being expressed as monomers with a transmembrane domain and a very short cytoplasmic tail (3–28 amino acids). Because this cytoplasmic tail is too short to associate with intracytoplasmic signaling molecules, the antigen-binding moiety is associated with a disulfide-linked heterodimer called Ig-α/Ig-β. Both the Ig-α and Ig-β chains belong to the immunoglobulin superfamily of proteins and express a single extracellular Ig-like domain. The α chain has a 61-amino acid cytoplasmic tail, whereas the tail of the β chain contains 48 amino acids. These tails each contain 18 residue ITAM motifs (Fig. 11). After cross-linking of mIg by antigen, the tyrosines within ITAMs are phosphorylated by receptor-associated protein tyrosine kinases (PTKs). Once phosphorylated, the ITAMs provide docking sites for Syk kinases (Fyn, Blk, and Lyn), which activate the second messengers inositol 1,4,5-triphosphate and diacylglycerol, causing release of calcium from intracytoplasmic stores. The Syk kinases also activate the small G proteins, Ras and Rac, which allow activated transcription factors to translocate to the nucleus where they stimulate or inhibit the transcription of specific genes (Goldsby et al. 2003).
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IgM
C
C
Extracellular Space
Igβ
Igα
Plasma embrane Membrane Cytoplasm
Immunoreceptor Tyrosine-Based Activation Motif (ITAM) Fig. 11 B cell antigen receptor complex. Membrane IgM (and IgD) on the surface of mature B cells is associated with the invariant Igα and Igβ molecules, which contain ITAMs in their cytoplasmic tails that mediate signaling functions. Note the similarity to the TCR complex. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
e. BCR CORECEPTORS BCR coreceptors include the proteins CD19, CR2 (CD21), and TAPA (CD81). CR2 is a receptor for complement component C3d and has been previously discussed (see section II.A.3.b.i.). CD19 has three extracellular domains and a long cytoplasmic tail containing six tyrosine residues that may be phosphorylated by PTKs after BCR crosslinking. When antigen coated with C3d is simultaneously bound by mIg and CR2, this cross linkage allows CD19 to interact with the Ig-α/Ig-β complex (Poe et al. 2001; Rickert 2005). This mechanism amplifies the signal transmitted through the BCR and is a major pathway for TI-2 antigenic stimulation of IgG3 in mice (Fig. 12). 2.
The T-cell Receptor (TCR)
a. THE ANTIGEN-BINDING COMPLEX Like the BCR, the TCR is a multi-protein complex consisting of a ligand (antigen)-binding
motif and signal-transducing complex called CD3. The antigenbinding component of the TCR is made up of heterodimers linked by interchain disulfide bonds. T cells may have heterodimers of α and β chains or heterodimers of γδ chains. In mice most T cells express αβ heterodimers. The domain structure of those heterodimers is similar to that seen in immunoglobulins. Each chain has two domains containing intrachain disulfide bonds that span 60–75 amino acids. The N-terminal domain has marked sequence variation, whereas the remainder of the chain is constant. The TCR variable domains have three hypervariable regions equivalent to CDRs of immunoglobulin L and H chains. Following the constant region domain each chain has an extracellular connecting sequence containing the interchain disulfide bond. Each chain has a transmembrane component with highly charged amino acid residues and a short cytoplasmic tail. The charged amino acids of the transmembrane moiety interact with the chains of the CD3 signaling complex. Although γδ
OVERVIEW
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Microbe
Complement Activation
Recognition by B Cells
Bound C3d
IgM
CR2 CD19 Igα
Igβ CD81
Signals from Ig and CR2 Complex
P13-Kinase
B Cell Activation Fig. 12 Role of complement in B cell activation. B cells express a complex of the CR2 complement receptor, CD19, and CD81. Microbial antigens that have bound the complement fragment C3d can simultaneously engage both the CR2 molecule and the membrane Ig on the surface of a B cell. This leads to the initiation of signaling cascades from both the BCR complex and the CR2 complex, because of which the response to C3d-antigen complexes is greatly enhanced compared with the response to antigen alone. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
heterodimeric receptors are very similar to αβ receptors certain structural features, especially dealing with the orientation of the V and C regions, differ. Whereas αβ receptors recognize antigenic peptides in conjunction with MHC class I or class II antigens, no such restriction is seen with γδ receptors (Goldsby et al. 2003). b. THE SIGNAL-TRANSDUCING COMPLEX CD3 is a complex of five polypeptide chains that associate to form three dimers: a γε heterodimer, a δε heterodimer, and either a homodimer of ζ chains (ζζ) or a ζ and η chain heterodimer (ζη). Most TCRs have the ζ homodimeric chains. It requires the combination of all three dimeric molecules (γε , δε, and ζχ or ζη) to impart the signal-transducing function on the TCR. The γ, δ, and ε chains of CD3 are all members of the immunoglobulin superfamily and have a single extracellular domain containing aspartic acid residues, followed by a transmembrane region and
a cytoplasmic tail containing a single ITAM. In contrast, ζ chains have a very short external region, a transmembrane region containing a negatively charged aspartic acid residue (that interacts with positively charged amino acids in the TCR αβ chains), and a cytoplasmic tail containing three ITAM motifs. This latter feature is shared with the η chain (Fig. 13). c. T CELL CORECEPTORS Antigen is presented to the αβ TCR combined with either MHC class I or MHC class II molecules. Two molecules found on T cells are capable of binding to conserved regions of the MHC molecules: CD4 recognizes MHC class II molecules, whereas CD8 recognizes MHC class I molecules. Mature T cells display only one of these CD molecules, which help define the function of the T cell. Because CD4 and CD8 both bind MHC molecules and play a role in signal transduction, they are considered coreceptors.
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TCR α
β
Extracellular Space CD3 ε
CD3 γ
ε
δ
Plasma Membrane ζ
ζ Immunoreceptor Tyrosine-Base Activation Motif (ITAM)
Cytoplasm
Disulfide Bond Fig. 13 Components of the TCR complex. The TCR complex of MHC-restricted T cells consist of the α β TCR noncovalently linked to the CD3 and ζ proteins. One possible stoichiometric combination is shown, but this may vary. The associations of these proteins are medicated by charged residues in their transmembrane regions, which are not shown. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
I. CD 8 CD8 takes the form of either a disulfide-linked αβ heterodimer or a αα homodimer. Both α and β chains are 30to 38-kDa glycoproteins consisting of a single extracellular immunoglobulin-like domain, a hydrophobic transmembrane region, and a cytoplasmic tail that can be phosphorylated (Fig. 14). CD8 binds class I molecules by contacting β2-microglobulin (see section II.B.). Because MHC class I combines with peptides derived from proteasomal processing of ubiquitin-conjugated intracellular proteins, T cells displaying CD8 are best suited for recognition and activation by antigens arising from within host cells such as components of viruses. Thus, CD8-positive T cells exhibit antiviral effector mechanisms such as cellular cytotoxicity and secretion of antiviral cytokines. They are commonly called cytotoxic lymphocytes (CTLs) (Goldsby et al. 2003). II. CD4 CD4 is a 55-kDa monomeric glycoprotein containing four extracellular immunoglobulin-like domains, a hydrophobic transmembrane region, and a long cytoplasmic tail, which contains three serine residues that can be phosphorylated. The N-terminal domain of CD4 contacts the hydrophobic pocket formed by the α2 and β2 domains of the MHC class II heterodimer (Fig. 14). Because MHC class II molecules combine with peptides processed through the exogenous pathway, T cells exhibiting CD4 are best suited for recognition and activation by antigens arising from outside the cell and processed by endolysosomal degradation. CD4-positive T cells
are called helper cells (Th) and may be further divided into Th1 or Th2 cells, depending on cytokines secreted after antigenic activation (Fig. 15). The cells play an essential role in most B cell responses and help determine the subclass of antibody to be secreted (see chapter 5) as well as promote macrophage activation and delayed type hypersensitivity (Goldsby et al. 2003).
D.
Costimulatory Receptors and Ligands in Adaptive Immunity
1.
The B7 Family and Its Receptors
Whereas binding of antigen to the TCR and activation of signal transduction via the CD3 complex generate the initial signal, by itself it is insufficient to activate naïve T cells. Members of the B7 family of ligands, B7-1 (CD80) and B7-2 (CD86) bind to receptors CD28 and CTL-associated antigen 4 (CTLA-4) and provide the second signal. Both B7-1 and B7-2 bind the CD28 receptor promoting T cell activation and homeostasis of regulatory CD4+CD25+ T cells (Treg cells) (Fig. 16). CTLA-4 binding by the same ligands, albeit with higher affinity than CD28, results in T cell inhibition (Greenwald et al. 2005). a. B7-1 AND B7-2 AND THEIR RECEPTORS B7-1 and B7-2 are members of the immunoglobulin superfamily and are
OVERVIEW
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Class II MHC
IN
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α1 CD4 N
β1 Peptide
V
α2 H CD4 V
β2
H V H
Class I MHC α1
Peptide
C
α2
CD8 CD8 N
β2m
N
CD8α1 α1
α3
α2
CD8α2
C
C
Fig. 14 Structure of CD4 and CD8. The models of CD4 and CD8 binding to class II MHC and class I MHC molecules, respectively, are based on the structures defined by x-ray crystallography. Both CD4 and CD8 bind to nonpolymorphic regions of MHC molecules (CD4 to the class II β2 domain and CD8 to the class I α3 domain) away from the peptide-binding clefts. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
transmembrane proteins with two extracellular immunoglobulin-like domains including an N-terminal V-like domain and a proximal C-like domain. Although their extracellular domains are very similar, their cytosolic domains are different and do not appear to transduce signals in the cells that express them. B7-1 and B7-2 are expressed on APCs, including macrophages,
DCs, and B cells. Whereas they may be expressed constitutively on DCs, both molecules are induced by inflammatory cytokines and CD40-CD40L interactions. B7-1 is never expressed constitutively on DCs. Both molecules are induced late after activation of APCs, and B7-2 can be expressed constitutively and induced early after APC activation.
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Costimulator (e.g., B7) CD28
Activation of Naive CD4+ T Cell
Naive CD4+ T Cell Dendritic Cell
Clonal Expansion and Differentiation
TH1 Cell
Property
TH2 Cell
TH1 Subset
TH2 Subset
Cytokines Produced
+++
− +++
IL-10
− +/−
IL-3, GM-CSF
++
++
IL-12R β Chain
++
−
IL-18R
++
−
CCR4
+/−
++
CXCR3, CCR5
++
+/−
Ligands for E- and P-Selection
++
+/−
Antibody Isotyped Stimulated
IgG2a (mouse)
IgGE, IgG1 (mouse)/IgG4 (humans)
+++
−
IFN-γ IL-4, IL-5, IL-13
Cytokine Receptor Expression
Chemoline Receptor Expression
Macrophage Activation
Fig. 15 Properties of Th1 and Th2 subsets of CD4+ helper T cells. Naive CD4+ T cells may differentiate into distinct subsets, such as Th1 and Th2 cells, in response to antigen, costimulators, and cytokines. Reproduced from Abbas, A.K., and Lichtman, A.H. (2005). Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
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All of the receptors of the CD28 family are expressed on T cells. They are transmembrane proteins with a single extracellular immunoglobulin V-like domain and a cytosolic tail containing tyrosine residues. Both CD28 and CTLA-4 are expressed as disulfide-linked homodimers, and their V domains have an MYPPPY motif, which is essential for B7 binding. After binding B7, the tyrosine residues on CD28 and CTLA-4 become phosphorylated and serve as docking sites for PI3K and Grb-2. CTLA-4 also has ITIM motifs on its cytoplasmic tail and recruits SHP-2, activating the inhibitory signaling pathway. CD28 is constitutively expressed on both CD4 and CD8 T cells, whereas CTLA-4 is inducible (Abbas and Lichtman 2005). b. ICOS AND ICOS-L Inducible costimulator (ICOS) ligand (ICOS-L) is structurally similar to B7-1 and B7-2 and its expression can be induced on APCs, B cells, and T cells and on fibroblasts, endothelial cells, and some epithelial cells. The gene is found on mouse chromosome 10. ICOS-L is the ligand for ICOS which is an inducible, transmembrane, homodimer with a structure similar to CD28. ICOS is expressed on T cells only after TCR antigen binding, and its induction is enhanced by CD28 signals. The ligand-binding motif of ICOS is FDPPPF and after binding, its cytoplasmic tail tyrosine residues phosphorylate, providing a docking site for PI3K. ICOS signaling promotes expression of IL-10 and IL-4 but not IL-2. ICOS appears more important for effector T cell responses such as the Th2 response. It does not affect the maturation status of B cells in mice (Greenwald et al. 2005). c. PD-1 AND ITS LIGANDS PD-L1 AND PD-L2 Programmed death1 (PD-1) is an Ig family member related to CD28 and CTLA-4 but lacks the membrane-proximal cysteine that allows for homodimerization, and thus it is expressed as a monomer. The cytoplasmic domain contains an ITIM motif and an immunoreceptor tyrosine-based switch (ITSM) motif: PD-1 is expressed during thymic development on CD4−CD8− (doublenegative) T cells, and it may be induced on peripheral CD4+ and CD8+ T cells, B cells, and monocytes. NKT cells express low levels. PD-L2 has greater affinity for PD-1 than PD-L1 and is more broadly expressed on tissues including endothelium, placenta, and many tumors in addition to B cells, T cells, DCs, and macrophages. PD-L1 may regulate self-reactive T or B cells in peripheral tissues. Upon binding to PD-1, phosphorylation of the tyrosine on the ITSM motif leads to inhibitory signals (Greenwald et al. 2005). 2.
CD40 and CD40 Ligand (CD154)
CD40 is a 50-kDa membrane receptor of the TNF receptor (TNFR) superfamily, which serves as a costimulatory receptor for B cell activation by T cells and selection of B cells to become long-term memory cells. In addition, CD40 is found on macrophages and DCs, where it functions to enhance
OVERVIEW
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Antigen Presentation to Helper T Cell
IN
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Activation of T Cell; Expression of CD40 Ligand, Cytokine Secretion
CD40
Activation of B Cell by Cytokines and CD40 Ligation
CD40 Ligand
B Cell Proliferation and Differentiation
CD40 Ligand
T Cell
B Cell
B7-1, CD28 B7-2
Cytokines
Cytokines
Fig. 16 Mechanisms of helper T cell–mediated B cell activation. B cells display processed peptides derived from endocytosed protein antigens and express the costimulators B7-1 and B7-2. Helper T cells recognize the antigen (in the form of peptide-MHC complexes) and the costimulators and are stimulated to express CD40 ligand and to secrete cytokines. CD40 ligand then binds to CD40 on the B cells and initiates B cell proliferation and differentiation. Cytokines bind to cytokine receptors on the B cells and also stimulate B cell responses. Reproduced from Abbas, A.K. and Lichtman, A.H. (2005) Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
antigen presentation. Like other members of TNFR family, CD40 utilizes distinct overlapping sets of cytoplasmic adapter proteins, TNFR-associated factors (TRAFs), to deliver signals to cell. At least four distinct TRAFs, which bind to distinct structural motifs in the CD40 cytoplasmic tail, have been identified. TRAFs are normally found unbound in the cytoplasm of nonactivated B-cells; upon binding CD154, CD40 aggregates into discrete microdomains in the plasma membrane (lipid rafts) and recruits TRAF molecules from the cytoplasm. Although TRAFs have no known enzyme activity, they interact with downstream signaling molecules and can lead to the activation of c-Jun NH2-terminal kinase (JNK), the stress-activated protein kinase p38, and NF-κB (Bishop and Hostager 2003). CD154 is a T cell membrane homotrimer that is structurally homologous to TNF and Fas ligand. Its expression is induced on Th cells after activation by antigen and costimulators. After Th activation, the T cells encounter B cells with processed antigenic peptides associated with MHC class II on the B cell surface. This allows the two cells to form a T-B conjugate. Further ligation of CD40 with CD154 brings the cell surfaces into contact and allows B cell activation and directional release of Th cell cytokines, which promote class switch and differentiation of B cells into plasma cells or long-term memory cells (Abbas and Lichtman 2005; Goldsby et al. 2003). There is some evidence that the abnormal expression of CD40 on T cells and CD154 on B cells is associated with the
development of autoimmune disease in mice (Bishop and Hostager 2003).
E.
Cytokines, Chemokines, and Their Receptors
Cytokines are proteins made by various cells, which may have autocrine, paracrine, or endocrine activities. The cytokines involved with immunity in mice belong to the interleukins, the TGF-β superfamily, the TNF superfamily or the IFNs. Important members of each of these classes of cytokines and their receptors are discussed in detail in chapter 6 in Volume 3 of this series; Table 2 summarizes these cytokines. Chemokines, along with adhesion molecules, are involved with leukocyte migration and mediate the relocation of leukocytes from sites of hematopoiesis to sites of immune defense or inflammation. Chemokines induce signaling via G protein–coupled cell surface receptors. All but two mouse chemokines are secreted proteins, and all can be classified structurally on their arrangement of two NH2-terminal cysteine residues, which either are located adjacent to each other (C-C) or are separated by a single amino acid (CXC). Minor subfamilies based on modifications of this structure also exist. Chemokine receptors are classified on the basis of whether they recognize one of the two structural types of cytokines. Chemokines and their receptors in mice have been discussed in detail in volume 3 of this series (see chapter 6) and are summarized in Table 3.
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TABLE 2
CYTOKINES AND THEIR RECEPTORS Cytokine
Sources
Activities
Receptor
IL-1 exists as IL-1α, IL-1β secreted as procytokines and cleaved to active 17-kDa molecules; also called lymphocyte activation factor
M, mast, dendritic, T, B, NK, and nonimmune cells
Modulate inflammation; also effects on bone remodeling, insulin secretion, fever regulation, and neuronal development
IL-1RI binds both IL-1α and IL-1β and contains extracellular Ig domains and a cytoplasmic TLR/IL-1 signaling domain
IL-2,15–20 kDa; also called T cell growth factor
Activated Th cells
Stimulates T cells to induce IL2R and modulates the activities B and NK cells
IL-3, 30 kDa dimer; known as hematopoietic cell growth factor (HCGF) and mast cell growth factor (MCGF)
Activated T cells, mast cells eosinophils
Stimulates colony formation for N, E, B, M, mast, megakaryocytes, and erythroid cells; promotes mast cell and DC differentiation
IL-2R is multisubunit heterotrimer; signal-transducing γ chain shared with IL-4R, IL-7R, IL-15R; found on T, B, and NK cells IL-3R is a heterodimer found on most hematopoietic cells
IL-4, 15–19 kDa; also known as Mast cells, T cells, basophils, B cell stimulating factor-1 (BSF-1) bone marrow stromal cells
Many effects on T, B, M, myeloid cells, erythroid cells fibroblasts and endothelial cells; induces class switch to IgG1 and IgE; induces differentiation of Th2 cells; induces expression of vascular cell adhesion molecule, IL-5, IL-6, and eotaxin-1 and 2 Induces IgA and IgG receptors on eosinophils; induces class switch to IgA, IgG1, and IgE in B2 cells and IgM production from B1 cells
IL-4R has two subunits and is widely expressed on tissues
IL-5, 45 kDa; also called eosinophil differentiation factor (EDF) and eosinophil colony stimulating factor (E-CSF)
T cells
1IL-6, 25 kDa; also known as B cell stimulatory factor-2 (BSF-2), hybridoma/plasmacytoma growth factor (HPGF), and endothelial hepatocyte stimulating factor (HSF)
T, B, M, and bone marrow stromal cells and fibroblasts
Broad pleiotropic effects on host defense, acute-phase responses, immune responses, and hematopoiesis. Induces monocyte chemotaxic protein-1
IL-6R heterodimer widely expressed
IL-7, 20–28 kDa; also called pre-B cell growth factor and lymphopoietin-1 (LP-1)
Bone marrow + thymic stromal cells
Promotes thymopoiesis and differentiation of pro-B cells into pre-B cells
IL-10, 35–40 kDa; also called cytokine synthesis inhibitory factor (CS1F)
Activated CD4 and CD8 T cells
IL-12, a heterodimer with p35 and p40 subunits; also called NK cell stimulatory factor (NKSF)
M, DC after activation of TLR
Inhibits IFN-γ and GM-CSF from Th1 cells and induces CD8 T cell chemotaxis; promotes IgA class switch and inhibits T cell apoptosis; enhances histamine release from mast cells and inhibits M1P-1α, M1P-1β, IL-1β, and TNF-α by neutrophils Induces differentiation of the Th1 subset and induces IFN-γ by Th1 and NK cells
IL-7R is a heterodimer sharing the common γ chain with IL-2R; expressed on spleen, thymus, fetal liver developing T and B cells and bone marrow macrophages IL-10R is a heterodimer distributed on lymphoid and myeloid cells
IL-13, 17 kDa
Activated Th2 cells, mast cells, and NK cells
IL-15, 14 kDa
DCs, M
IFN-γ, 40 kDa homodimer
Th1 cells, NK cells, CTLs
Involved with inflammation, mucus production, tissue remodeling and fibrosis; induces IgE class switch; involved with allergy Stimulates T cell proliferation and the development and activation of NK cells Induces macrophages to secrete TNF-α, express MHC class II, and produce antimicrobial activities and class switch to IgG2A; it inhibits Th2 differentiation and promotes CTL differentiation from CD8 precursors
IL-5R is a heterodimer with signal-transducing β unit shared with IL-3R
IL-12R is a heterodimer expressed on Th1 and NK cells IL-13R is composed of IL-4Rα and IL-13Rα1 chains and is widely expressed IL-15R is a heterotrimer sharing the common γ chain with IL-2R IFN-γR is a heterodimer with the IFN-γRα chain constitutively expressed on many cells
OVERVIEW
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TABLE 2
CYTOKINES AND THEIR RECEPTORS—cont’d Cytokine
Sources
Activities
Receptor
TGF-β, 25 kDa; also called differentiation inhibiting factor; three forms exist: TGF-β1, β2, β3
Many nucleated cells and platelets
TNF-a, 52 kDa; also known as cachectin; 17-kDa fragment circulates as a homotrimer
M, macrophages, T cells, fibroblasts
TGF-β1 is stimulatory for cells of mesenchymal origin and inhibitory for cells of epithelial or neuroectodermal origin; it suppresses Treg cells, inhibits B-cell proliferation, but promotes isotype switch to IgA; it mediates oral tolerance Strong mediator of inflammation and immune function; regulates growth and differentiation and is cytotoxic to many transformed cells
TNF-β, 25 kDa; also known as lymphotoxin (LT)
Activated T + B cells
There are three TGF-β receptors: type 1 (53 kDa) type 2 (70 kDa), type 3 (250 kDa); types 1 and 2 act as heterodimers to mediate antiproliferative activities, and type 3 cannot mediate signal transduction TNFRI is widespread and mediates the effects of TNF-α and TNF-β; the receptor can shed its extracellular domain and circulate TNFRI
Mediates inflammation and immune function; affects healing
T-thymic derived lymphocyte, B-bone marrow derived lymphocyte, M-monocyte, Ma-macrophage, NK-natural killer cell, DC-dendritic cell, mega-megakaryocyte, Baso-basophil, N-neutraphil, E-eosinophil
III.
CELLULAR IMMUNOLOGY
A protective immune response includes contributions from many cellular elements. Some of these cellular elements serve a barrier function to exclude potentially pathogenic microbes from delicate tissues. Other cellular elements contribute
directly to the recognition and elimination of pathogens that have penetrated these physical defenses. In this portion of the overview, we will review the major cellular elements that contribute to intact host immunity and define their major mechanisms of activation, interactions, and effector mechanisms.
TABLE 3
FUNCTION OF MURINE CHEMOKINES Receptor
Chemokine
Function
CCR1 CCR2 CCR3 CCR4 CCR5
M1P-1α, RANTES MCP-1, MCP-3, CCL8, MCP-2 CCL11 (eotaxin), CCL24 eotaxin-2 CCL17 (TARC) M1P-1α, M1P-1β, CCL5, (RANTES), CXCL11 (eotaxin-1) CCL6 (exodus-1) CCL19, (M1P-3β), CCL21
Antiviral responses Macrophage migration, especially to brain, IgE class switch Th2 reactions and eosinophil chemotaxis Skin homing of memory T cells, Th2 migration Apoptosis of CD1d NKT cells, Th1 responses, and macrophage migration
CCR6 CCR7 CCR8 CCR8 CCR9 CCR10 CXR2 CXR2 CXCR3 CXCR4
CCL1 (TCA-3) CCL20 CCL25 CCL27 CXCL1 (KC), CXCL3 (M1P-2) CXCL6 (LIX) CXCL9 (M1G), CXCL10 (1P-10), CXCL11 (I-TAC) CXCL12 (SDF-1α)
CXCR5 CXCR5
CXCL14 (KS1) CXCL13 (BCA-1)
Development of CD4+ Treg and memory cell formation Maturation of DCs, which are programmed for Th1 induction, migration of skin DCs into lymphatics, lymphocyte homing via hepatitis E virus Th2 responses and recruitment of T cells and DCs to inflamed skin Dendritic cell homing Intestinal homing for effector T cells, thymocyte migration Skin homing for memory T cells Induces the NF-κB signal pathway and controls migration and degranulation of neutrophils Activates NF-κB and induces proinflammatory cytokines Chemotaxic for lymphocytes and NK cells, controls activated T cell responses; controls intestinal epithelial cell turnover, T cell allograft rejection, and angiogenesis Germinal center development, CTL recruitment into tumors, inhibits chemokine-induced myelosuppression, involved with T and B cell lymphopoiesis, homing of DCs to spleen and migration of sensing neuron progenitors B cell and monocyte chemotaxis, APC development B cell follicle interactions, recruitment of B cells, induction of XCL1, XCL2
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A. 1.
Cells of the Innate Immune System
Cells That Provide Anatomical Barriers
a. PHYSICAL EPITHELIAL BARRIERS A critical, but often overlooked, component of the host defense system is the physical barrier established by the skin and the mucosal epithelia (Cario et al. 2002; Jane et al. 2005). In the epidermis, the cornified layer of dehydrated keratinocytes provides the initial barrier that prevents the entry of water-soluble toxins and microbes. Cornification depends importantly on the induction by homeobox genes of the keratinocyte transglutaminase that catalyzes dense cross-linking of the intracellular cytokeratin (Candi et al. 2005; Mack et al. 2005). Below the cornified layer, the live keratinocytes represent a second barrier that depends on the desmosomes that form tight intercellular junctions (Yin et al. 2005) and the hemidesmosomes that anchor the basal layer of epidermal keratinocytes to the basement membrane (Andriani et al. 2003). Similar barrier functions are expressed by epithelial cells in the mucosal tissues, preventing entry of pathogenic microbes that are introduced as these mucosal surfaces contact the environment in which the rodent lives (Cario et al. 2002). These barriers provide protection unless the microbes express proteases that digest the extracellular matrix or the tight junction components themselves (Andrian et al. 2004). b. SECRETED PROTECTIVE PROTEINS The mucosal epithelia characteristically produce a barrier layer of mucus that floats over the watery secretory fluid that bathes the cilia on the apical surface of the epithelium (Cohn 2006). This mucous blanket serves to trap particulate antigens that deposit on mucosal surfaces and prevents their penetration to the epithelial cell layer. In the airways, the coordinated movement of the epithelial cilia propels the mucous layer toward and up the trachea where it ultimately is deposited into the hypopharynx and swallowed (Drannik et al. 2004). The differentiation of epithelial cells into mucus-producing goblet cells is enhanced by the activation of epithelial growth factor receptors and by the action of IL-13 (Tyner et al. 2006). Thus, mucus clearance of microbes can be regulated by immunomodulatory molecules acting through enhancement of this physical barrier. In addition to the barrier function of the skin and mucosal epithelia, these anatomic barriers also produce secreted products that are directly toxic to many forms of microbes. For example, epidermal keratinocytes produce many different kinds of secreted antibacterial peptides, including cathelicidin (Braff et al. 2005). This peptide is expressed constitutively by polymorphonuclear leukocytes but is also expressed by keratinocytes that have been activated by TLR ligation. Phagocytic cells and epithelial cells also express other antimicrobial peptides such as the defensins (Oppenheim et al. 2003). The defensins can be grouped into two families (α-defensins and β-defensins) on the basis of the arrangement of their three intramolecular disulfide bonds. In addition to these characteristic three intramolecular disulfide bonds, the defensins have
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three antiparallel β strands resembling the structures of many chemokines (Hoover et al. 2000). The α-defensins are largely stored preformed in the granules of neutrophils and some macrophages, but they also are found in the granules of Paneth cells at the base of the villi in the gastrointestinal mucosa (Ayabe et al. 2000). After their secretion in response to bacterial products, they are activated by cellular metalloproteinases, providing an extra level of regulation controlling their function (Wilson et al. 1999). The β-defensins represent a large gene family encoded by more than 20 genes. Despite their apparent redundancy, targeting of the gene encoding the mouse homolog of human β-defensin-1 (HBD1) results in delayed ability to clear experimental Haemophilus influenzae infection in the lung (Moser et al. 2002). In addition to their direct antibacterial activities, both the defensins and cathelicidin have additional actions that permit them to recruit other portions of both the innate and the adaptive immune systems. This recruiting is based on the ability of the antibacterial peptides to bind to and activate many G protein–coupled receptors, particularly selected chemokine receptors. For example, many β-defensins can bind to the chemokine receptor CCR6 (Yang et al. 1999), contributing to the recruitment of DCs and resting memory and cytotoxic T cells. The receptors for the α-defensins have not been defined, but because α-defensins induce chemotaxis of immature DCs and subsets of CD8+ T lymphocytes in a pertussis toxin–sensitive fashion, they are assumed to interact with G protein–coupled receptors (Yang et al. 2000). Perhaps through their ability to mobilize recruitment of DCs, both α- and β-defensins show prominent adjuvant activity for both Th1 and Th2 type immune responses (Tani et al. 2000). 2.
The Phagocytes
An intact immune response includes contributions from many subsets of leukocytes. These subsets were originally defined based on the morphology of the cells as assessed using conventional histological stains. In the past few decades, the subsets have been more precisely defined by examination of their cell surface phenotypes as defined by monoclonal antibody binding to registered differentiation antigens. Many of these differentiation antigens have been precisely defined by molecular cloning, but some remain defined only as serological specificities. Based on their serological reactivity, each antigen is assigned a cluster of differentiation (CD) number. There are currently more than 350 defined CD antigens. International workshops that regulate these CD designations have focused on the molecules expressed on human cells. Mouse homologs have been designated on the basis of structural and occasionally functional homology to the human molecules. Updated and annotated listings of the defined CD antigens are published at http://www.sciencegateway.org/resources/prow/default.htm/. The circulating leukocytes all derive from pluripotent hematopoietic stem cells that also give rise to the erythroid lineage.
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In the mouse, hematopoiesis begins in a poorly defined anterior region of the embryo and then moves to the yolk sac where the expansion and differentiation of blood precursors form blood islands (Orkin and Zon 2002). The hematopoietic stem cells (HSCs) that are present in these blood islands constitute a population of self-renewing precursor cells that sustain the formation of all the required blood elements throughout the life of the animal. In addition to the blood elements, HSCs also give rise to vascular endothelial cells and perhaps some additional cell lineages (Ogawa et al. 2001). Shortly after the liver forms from the gut endoderm, HSCs migrate to this richly vascular organ, and hematopoiesis occurs in this tissue for most of gestation (Wolber et al. 2002). Near the end of normal gestation, the major site of hematopoiesis moves to the spleen and then to the bone marrow (Tsai et al. 1994). In mice, detectable hematopoiesis continues in the spleen throughout most of the life of the animal, although the major site of hematopoiesis is normally the bone marrow (Godin and Cumano 2005). In the bone marrow, the pluripotent HSCs differentiate under the influence of defined growth factors into lymphoid stem cells and myeloid stem cells (Richards et al. 2003). The myeloid stem cell population gives rise to all of the granulocyte cells, to megakaryocytes and platelets, and to erythrocytes (Fig. 17). It has generally been assumed that once this differentiation step has occurred, then commitment to the lymphoid or the myeloid lineage is absolute; however, recent studies have suggested that intermediate lineages of partially committed stem cells with both lymphoid and myeloid, but not erythroid and megakaryocytic, potential exist (Adolfsson et al. 2005). The myeloid stem cell then differentiates further under the influence of lineage-specific growth factors to form lineagespecific CFUs that behave as lineage-specific stem cells. The granulocyte lineage includes neutrophils, monocytes, macrophages, eosinophils, basophils, and mast cells, as well as megakaryocytes and platelets. For each of these end lineages, a defined CFU serves as the progenitor for these terminally differentiated cells. Thus, the intermediate precursor for neutrophils and monocytes is the CFU-GM, and the precursor for eosinophils is the CFU-Eo. a. NEUTROPHILS The granulocytic cells are all marked by their prominent expression of cytoplasmic granules that contain effector molecules stored preformed to permit these cells to express their various effector functions quickly after they encounter an activating stimulus. Neutrophils are actively phagocytic cells that produce large quantities of reactive oxygen species which are toxic to bacterial pathogens and other microbes (Moraes et al. 2006). They also produce proteolytic enzymes that demonstrate some direct microbial cytotoxicity and that also appear to participate in inflammatory cell recruitment, tissue remodeling, and repair after injury (Parks 2003). In mice, neutrophils represent 10–20% of total peripheral leukocytes, substantially lower than the values seen in humans. Although there are no defined cell surface markers that are absolutely specific for neutrophils, their combined expression
33 of CD11b (the receptor for the inactivated form of complement component C3, iC3b, expressed on neutrophils and monocytes as well as some additional cell lineages) and the Gr-1 antigen (a 21- to 25-kDa GPI-linked protein also know as Ly-6G/Ly-6C, expressed on neutrophils, eosinophils, some populations of macrophages, and plasmacytoid DCs) allows their definitive detection by flow cytometry or immunostaining in tissue sections (Goni et al. 2002). Neutrophils differentiate in the bone marrow and require signals delivered via their cell surface granulocyte colony-stimulating factor (CSF) receptors for their trafficking from the marrow into the circulation (Semerad et al. 2002). They are recruited from the circulation to sites of tissue damage by acute inflammatory chemokines such as CXCL1 (KC) and CXCL2 (MIP-2), reaching a peak accumulation within 6–12 hours after initiation of the recruitment signal. They are relatively short-lived, with most surviving less than 2 days after their release from the bone marrow. The existence of strains of mice with either abnormal differentiation of neutrophils or neutrophils that are deficient in specific reactive oxygen effector pathways demonstrates the essential role of these cells in antibacterial host defense (Schaper et al. 2003). Neutrophils are also recognized to produce chemokines and cytokines, suggesting that they may play important immunoregulatory roles in addition to their roles in direct antimicrobial host defense (Kasama et al. 2005). b. MONOCYTES AND MACROPHAGES Like neutrophils, monocytes and macrophages are highly phagocytic for microbes and particulate antigens. They are detected by their distinctive morphology characterized by large cell size (11–14 µm in diameter) with indented nuclei, surface expression of CD11b and F4/80, and expression of nonspecific esterase (Henkel et al. 1999). Their expression of the well-defined complement and Fc receptors emphasizes their role in clearance of opsonized microbes and particles that have been marked by specific antibodies and complement. Novel receptors for complement and other opsonic signals are still being discovered, including a recently identified complement receptor of the immunoglobulin gene superfamily whose expression appears limited to macrophages (Helmy et al. 2006). This receptor appears essential for clearance of opsonized bacteria. Monocytes and macrophages are recruited more slowly to sites of inflammation than neutrophils, appearing several hours after the peak of neutrophil influx. They persist at sites of acute and chronic inflammation for long periods, apparently contributing importantly both to the clearance of the initiating pathogen or inflammatory stimulus and also to the ultimate tissue repair. Macrophages are particularly prominent in granulomatous processes. Activated macrophages produce large amounts of nitric oxide (via the inducible nitric oxide synthase), an important antimicrobial agent, and large amounts of cytokines such as IL-12, IL-23, and IFN-γ, giving them important regulatory roles in adaptive as well as innate responses (Hume 2006; Langrish et al. 2004). c. EOSINOPHILS Eosinophils can be readily recognized because they contain prominent cytoplasmic granules containing toxic
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B Lymphocyte T Lymphocyte
Lymphoid Stem Cell
Plasma Cell
NK-T Cell NK Cell
Pluripotent Hematopoietic Stem Cell
Neutrophil
Dendritic Cell Langerhans Cell
CFU-GM Monocyte
Myeloid Stem Cell
Macrophage
Eosinophil CFU-Eo Basophil BMCP
Mast Cell Megakaryocyte
CFU-Meg Erythrocyte CFU-E Fig. 17 Leukocytes that support normal host defense. Pluripotent hematopoietic stem cells differentiate in bone marrow into lymphoid and myeloid stem cells. Lymphoid stem cells give rise to B cell, T cell, NK cell, and NKT cell lineages. Myeloid stem cells give rise to a second level of lineage-specific colony-forming unit (CFU) cells that ultimately lead to the production of neutrophils, monocytes, DCs, macrophages, eosinophils, basophils, mast cells, megakaryocytes, and erythrocytes. Modified from Chaplin (2003).
molecules and enzymes that are particularly active against helminths and other parasites. Whereas eosinophils belong clearly to the granulocyte lineage and are capable of ingesting complement-coated ICs and particles with their surface complement and Fc receptors (Lopez et al. 1981), they are substantially less phagocytic than neutrophils and macrophages and should probably not be considered professional phagocytes (Rabinovitch 1995). Eosinophils differentiate from bone marrow precursors that are specified by expression of the transcription factors GATA-1, PU.1, and C/EBP (CCAAT enhancer-binding protein) (McNagny and Graf 2002; Nerlov and Graf 1998; Nerlov et al. 1998) and that depend for their terminal expansion and release from the marrow on IL-3, IL-5, and granulocyte-macrophage (GM)-CSF (Rothenberg and Hogan 2006). IL-5 also supports extended survival in peripheral tissues, permitting these potent granulocytes to emerge as prominent components of allergic inflammation driven by Th2 cells (Ochiai et al. 1997). Interestingly, in established allergic responses, eosinophils can themselves release IL-5 that acts in an autocrine fashion to sustain the eosinophil’s survival and
consolidate the allergic character of the inflammatory response (Huang et al. 2005). Until recently, eosinophils were thought of as cells that acted protectively in the host response against many types of parasites and that could cause substantial tissue injury because of their release of toxic granule components in the course of allergic responses (Kariyawasam and Robinson 2006). Eosinophils are now becoming recognized as multifunctional leukocytes that contribute to the initiation of a variety of inflammatory responses and that participate as regulators of both innate and adaptive immunity (Rothenberg and Hogan 2006). Eosinophils are recruited to sites of tissue inflammation by the chemokines CCL11 (eotaxin) and CCL24 (eotaxin-2) acting on the chemokine receptor CCR3 on the eosinophil itself. In addition to their role in inflammatory reactions, eosinophils are components of normal homeostatic responses, including normal function of the thymus, in which at certain times of development eosinophils are present in numbers equal to those of thymic DCs (Throsby et al. 2000). In addition, by virtue of their secretion of an array of cytokines (IL-2, IL-4, IL-5, IL-6,
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IL-10, IL-12, and TNF), eosinophils can also promote CD4+ T cell proliferation and differentiation contributing to the polarization of helper cell responses towards the Th1 or Th2 phenotype (Lacy and Moqbel 2000; MacKenzie et al. 2001). In addition, eosinophils are able to regulate mast cell function, presumably by their release of the highly charged granule protein, major basic protein, together with eosinophil peroxidase and eosinophil cationic protein. Together, these eosinophil granule proteins induce mast cell release of histamine, TNF, chemokines, and prostaglandins (Piliponsky et al. 2002; Zheutlin et al. 1984). 3.
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Mast Cells and Basophils
Mast cells and basophils are morphologically similar cells that have long been thought to represent distinct lineages. Recent studies have, however, identified bipotent progenitors in the mouse spleen that can give rise to both basophils and mast cells (Arinobu et al. 2005). These cells, designated basophil/mast cell and progenitors, express c-Kit (CD117, the receptor for stem cell factor [SCF]) and require SCF for their growth and survival both in vitro and in vivo. In this same study, progenitor cells committed to the basophil lineage were also detected in mouse bone marrow, whereas progenitor cells committed to the mast cell lineage were detected in the gastrointestinal mucosa, suggesting that the microenvironment in which the progenitor cell resides provides differentiating signals that govern the phenotype of the progenitor and its lineage potential. Mast cells and basophils each contain proteoglycans in their granules. These proteoglycans are responsible for the metachromatic staining characteristics of the granules with dyes such as toluidine blue. In addition, both cell lineages express biologically significant levels of the high-affinity receptor for IgE (FcεRI) and after antigen challenge of sensitized animals, both cell types can release histamine and a variety of other mediators including proteases, chemokines, cytokines, and arachidonic acid metabolites, all of which act to induce immediate hypersensitivity reactions. Basophils are generally found circulating in the blood, although they can emigrate into the lungs and other tissues under conditions of antigen-induced tissue inflammation (Luccioli et al. 2002). Mast cells are normally found in peripheral tissues. They exist in two subtypes, designated mucosal mast cells and connective tissue mast cells. These two subtypes differ in their expression of inflammatory mediators. Triggering of the high-affinity IgE receptor on mouse mucosal mast cells results in release of preformed histamine, chondroitin sulfate E, and mast cell proteases 1 and 2 (Onah and Nawa 2004; Pemberton et al. 2003). In contrast, triggering of murine connective tissue mast cells induces release of both histamine and serotonin, heparin, and mast cell proteases-3, -4, and -5, as well as a mast cell carboxypeptidase and two tryptases (Tchougounova et al. 2003). Activation of both types of mast cells leads to release of cytokines, including IL-4, IL-5, IL-6, and TNF, as well as production of arachidonic acid metabolites, primarily
leukotriene C4 and prostaglandin D2. Although the mechanisms are not fully understood, these mediators participate in an essential fashion in the killing and clearance of nematodes and other parasites. Recent studies have identified additional actions of mast cells in host defense, particularly in antibacterial host responses. The participation of mast cells in the response to bacterial pathogens was first suggested by the observation of mast cell phagocytosis of E. coli and other enterobacteria by virtue of interactions with the mannose-binding subunit on the bacterial fimbriae, FimH (Malaviya et al. 1994). Subsequent studies showed that mice deficient in mast cells because of a mutation in c-Kit (strain WBB6F1-W/Wv) showed more than 20-fold reduced ability to clear experimental E. coli infections, largely because of the absent local production of TNF that was usually generated by mast cells in response to the microbes (Malaviya et al. 1996). The specific role of mast cell–derived TNF in the host response to these bacteria has not been fully defined, but the ability of mast cell–derived TNF to recruit neutrophils via a macrophage inflammatory protein (MCP)1-dependent mechanism may be physiologically important (Wang and Thorlacius 2005). In addition, mast cell-derived TNF has been found to promote recruitment of DCs and to enhance antigen presentation (Suto et al. 2006). Lastly, the production of TNF after mast cell activation by bacterial pathogens results in the hypertrophy of draining lymph nodes and increased accumulation of T lymphocytes in these nodes, enhancing their ability to generate a robust CD4+ T cell response (McLachlan et al. 2003). Thus, both through IgEdependent and IgE-independent mechanisms, mast cells contribute importantly to innate and adaptive host defenses. More details on the actions of mast cells and eosinophils with a focus on allergic airway disease is found in chapter 14. 4.
DCs
DCs are MHC class II–expressing bone marrow–derived leukocytes that are distributed through all tissues in the body and that serve as potent APCs (Adams et al. 2005). As specified by their name, most have typical dendritic morphology. Studies in both humans and mice have demonstrated that DCs exist in distinct subsets, each expressing distinctive functional characteristics (Shortman and Liu 2002). DCs can arise from both common lymphoid and common myeloid progenitor cells through a Fms-like tyrosine kinase 3+ (Flt3+) precursor cell (Fig. 18) (Jackson et al. 2002; Karsunky et al. 2003). Many studies have shown that these progenitors can differentiate into multiple DC subtypes. There is not, however, general agreement on the characteristics of each of these cell lineages, or even on which cell types should be considered to be DCs. We will briefly describe the most commonly accepted subsets of DCs, but caution the reader that this is a rapidly developing field and that as new DC surface markers are defined, it is likely that new subsets of DC will be defined.
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Plasmacytoid DC
Lymphoid Stem Cell
CD4−CD8+ DC
Pluripotent Hematopoietic Stem Cell
Flt3+ DC Precursor
CD4−CD8− DC
CD4+CD8− DC
Myeloid Stem Cell
Langerhans cell
Interdigitating DC Fig. 18 Differentiation of DCs at rest. DC precursors can differentiate from both lymphoid and myeloid stem cells. After a signal from Flt3-ligand, the Flt3+ DC precursor proliferates, and prepares to give rise to all the major subsets of DCs.
In the epidermis, the major DC population consists of Langerhans cells, characterized by their high surface expression of MHC class II and Langerin, a type II transmembrane C-type lectin with mannose-binding specificity (Valladeau et al. 2000). These cells also contain characteristic Birbeck granules, specialized components of the endosomal recycling compartment (Mc Dermott et al. 2002), and express TLRs 2, 4, and 9 (Mitsui et al. 2004). In the skin, these cells express highly phagocytic and endocytic behavior. When they internalize a foreign antigen, they become activated, often in a TLR-dependent fashion. The activated cells upregulate expression of the chemokine receptor CCR7, rendering them sensitive to CCR7 ligands including CCL21 and CCL19 (Ohl et al. 2004) and directing them to nearby lymphatic vessels and the draining lymph nodes (Yoshino et al. 2003). In the lymph node, the Langerhans cells either transfer their antigen to or differentiate into interstitial or “interdigitating” DCs that express costimulatory molecules and are active as APCs for T cells.
Interstitial DCs are located in all of the body tissues including the dermis. They are often identified by their high expression of the myeloid cell antigen CD11c. Some DCs express a homodimer of the α chain of CD8 and manifest both high phagocytic activity for dying cells and effective antigen presentation to cytolytic T cells (Iyoda et al. 2002). CD8+ DCs that present antigen to CTLs perform the process termed “cross-presentation.” APCs usually present exogenous antigens via the MHC class II pathway, stimulating CD4+ αβ T cells, and present endogenous antigens (from digested self-proteins or peptides from intracellular pathogens) via the MHC class I pathway. It is clear, however, that some antigens from cells outside the APC can be taken up exogenously and processed in a fashion that they somehow enter the MHC class I processing pathway and are ultimately presented via class I proteins. This allows the immune system to generate cytotoxic T cells to viral antigens that do not infect the APC themselves and to maintain peripheral tolerance (see section III.B.1.e.) to antigens that are
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not expressed endogenously in the DC. Experiments in both mice and humans demonstrate that DCs can perform the important process of cross-presentation (den Haan et al. 2000). The molecular pathways by which the endosomal pathway and the class I presentation pathway intersect remain to be defined. Other populations of DCs are CD11c+CD11b+CD4+CD8−, are also highly phagocytic/endocytic, and present exogenous antigens via the conventional MHC class-restricted pathway. These cells, together with a population of CD4−CD8− CD11c+ CD11b+CD205− DCs are largely distributed in the spleen marginal zone in naïve animals, migrating to the T cell zones of the spleen white pulp after stimulation with microbial products. One more population of murine DCs is CD4−CD8−CD11c+ CD11b+. This latter population is moderately positive for expression of the surface marker DEC205 (CD205) and localizes in higher numbers in lymph nodes, with little representation in the spleen. The ways in which the DCs are activated, including, importantly, the repertoire of their TLRs that are activated by interaction with the antigen, ultimately determine the nature of the T cell response that is generated, with DCs activated via TLR9 inducing a Th1 or Tc1 response, and cells activated by TLRs that elicit production of IL-4 induce the production of Th2 and Tc2 response (d’Ostiani et al. 2000). DCs that present antigens via both the class I–restricted and the class II–restricted pathways localize in a lymphotoxindependent fashion within the T cell zones of secondary lymphoid tissues and there contribute signals that support recruitment of T cells to these lymphoid compartments (Wu et al. 1999). In addition to the above classes of DCs that are designated “myeloid” because of their expression of CD11c, there is an additional class of DC present in the secondary lymphoid tissues and in small numbers in some peripheral tissue that is distinguished by its large size and round morphology similar to that of plasma cells. These plasmacytoid DCs are CD11cloB220+Gr-1+class IIlo and can be either CD4+ or CD4− and either CD8+ or CD8− (Blasius and Colonna 2006). These cells also express multiple TLRs, including the TLR7 and TLR9 that drive their activation by viruses and other intracellular microbes in response to ssDNA and CpG DNA, respectively. When activated in this fashion, they produce very large quantities of the type I interferons (IFNα and IFNβ) and are thought to play a pivotal role linking the innate and the adaptive immune systems (Fuchsberger et al. 2005). Additional information regarding the biology of DCs is found in chapter 4 in this volume. 5.
NK Cells
NK cells are lymphocytes derived from lymphoid stem cells that arise in the bone marrow (Fig. 17). This lineage was initially characterized by its ability to kill a broad range of tumor cells without prior sensitization to the tumor antigens and was initially thought to function primarily in antitumor host defense
37 (Oldham 1983). NK cells are now recognized to participate in many aspects of both innate and adaptive immunity. An important area that should be addressed in future research is the potential role of NK cells during pregnancy. NK cells accumulate in high numbers at the maternal-fetal interface in the placenta (Moffett-King 2002), and the decidua of mice genetically deficient in NK cells is abnormal in structure and function (Greenwood et al. 2000). Because the basic mechanisms of NK cell target cell recognition and the receptors used in that recognition have already been described (see section II.A.5.), we will focus here on the requirements for NK cell development and differentiation. NK cells have the morphology of large granular lymphocytes (Becknell and Caligiuri 2005). They can be distinguished phenotypically from the other classes of lymphocytes by their lack of B lymphocyte and T lymphocyte antigen receptors (surface immunoglobulin or the TCR). Because their development does not require rearrangement of antigen receptor genes, they are present in substantial numbers in scid and RAG-1- or RAG-2deficient mice (Dorshkind et al. 1985; Mombaerts et al. 1992). Spleen cells from athymic nu/nu mice were just as active natural killers as spleen cells from wild-type mice, demonstrating that the thymus is not required for NK cell development (Kiessling et al. 1975). Thus, NK cells clearly follow a developmental pathway distinct from that of either the B cell or T cell lineage. A critical role for the bone marrow microenvironment in the development of NK cells was suggested by the observations that congenitally osteopetrotic mice with the mi/mi mutation had NK cells with grossly defective natural killing activity (Seaman et al. 1979). Additionally, mice in which the marrow compartment had been damaged by treatment with boneseeking isotopes such as 89Sr also showed NK cells with impaired function. More recent studies in mice with disturbed development of secondary lymphoid tissues because of a deficiency of the membrane lymphotoxin heterotrimer (LTα1β2) showed congenital absence of both NK cells and NKT cells (Iizuka et al. 1999). Reciprocal bone marrow transplantation studies using membrane lymphotoxin-deficient mice and wild-type mice suggested that close interactions between NK cell precursors that expressed membrane lymphotoxin and lymphotoxin-responsive stromal cells was required for normal development of both NK and NKT cells (Iizuka et al. 1999; Wu et al. 2001). In vitro systems for induction of NK cells from bone marrow progenitors have been developed on the basis of marrow culture with a cocktail of cytokines that are prevalent in the marrow itself. Culture of hematopoietic progenitor cells with bone marrow stromal cells plus SCF (c-Kit ligand), IL-7, Flt-3 ligand, and IL-15 supports the development of functional NK cells with full expression of the NK-specific Ly49 receptors (Roth et al. 2000; Williams et al. 1999; Yu et al. 1998). Culture of committed NK cell bone marrow progenitors with IL-15 alone supports the final maturation of these committed cells to
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functional NK cells (Waldmann and Tagaya 1999). Together these and other studies suggest that common lymphoid progenitor cells give rise to T, B, and NK cells (Kondo et al. 1997). Common lymphoid progenitors, then, differentiate to bipotential T cell/NK cell progenitors that no longer have the potential to generate cells of the B lineage. Development of T/NK progenitors requires the expression of IL-15 and the IL-15 receptor α chain (Kennedy et al. 2000). Consistent with the important role of IL-15 in NK cell development, mice deficient in Janus-family tyrosine kinase-3 (Jak3) or signal transducer and activator of transcription (STAT) 5 show major defects in NK cell development (Imada et al. 1998; Park et al. 1995). In addition to STAT5, the Ets1 and Id2 transcription factors are required for NK cell progenitor development, although they are not required for B cell and T cell development (Barton et al. 1998; Yokota et al. 1999). The final maturation of NK cell progenitors to mature, fully functional NK cells involves the upregulation of NK cell receptors (CD94 and Ly49) and the differentiation antigens NK1.1 and DX-5. All of these depend on further stimulation with IL-15 and interactions with bone marrow stromal cells (Yokoyama et al. 2004). Additional information on the function and activation of NK cells is found in chapter 6.
B. Cells of the Adaptive Immune System 1.
T Cells
a. EARLY T CELL DEVELOPMENT This discussion will focus primarily on the subset of T cells that recognize antigen using the αβ TCR. A second pathway of T cell development produces cells with an antigen receptor consisting of a VγJγ paired with a VδDδJδ forming a γδ T cell. The αβ TCR has evolved to recognize primarily peptide antigens presented as a complex with class I or class II MHC proteins. As discussed in section II.C.2., the antigen-binding portion of these receptors is composed of a heterodimer consisting of one α chain and one β chain. Individual T cells express a single functional α chain and a single functional β chain that combine to form an antigen-binding receptor with a single antigenic specificity. Development of a repertoire of T cells that can recognize and respond to the vast universe of pathogenic microbes requires a vast number of cells encoding a correspondingly vast number of discrete TCRs. The genes encoding functional TCR are produced by somatic joining of variable (both α and β chains), diversity (β chain only), and joining (both α and β chains) gene elements to generate mature VαJα and VβDβJβ exons. The somatic joining process occurs by site-specific DNA recombination mediated by the lymphoid cell–specific recombinase-activating gene 1 (RAG1) and RAG2 proteins interacting with moderately conserved recombination signal sequences (RSSs) that flank each of the V, D, and J gene elements. Each V gene element is composed of an upstream leader exon followed by a short (20–250 base pair
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[bp]) intron and the variable gene segment. The 3’ end of each V element is followed by an RSS that consists of a conserved heptamer and nonamer with a 23-bp long spacer. There are approximately 65 Vβ gene segments on mouse chromosome 6. Downstream of the Vβ gene cluster lie two gene clusters consisting of a Dβ gene, followed by Jβ gene elements (~6 in the upstream cluster and ~7 in the downstream cluster) and a Cβ gene. The two Cβ gene elements are almost identical in sequence, but there is considerable sequence variability in the sequences of the Dβ and Jβ segments. The D segments are each bounded on their 5’ end by an RSS with a 12-bp spacer, and at their 3’ end by an RSS with a 23-bp spacer. The Jβ gene elements are bounded on their 5’ ends by a 12-bp spacer and on their 3’ ends by mRNA splice sites permitting RNA splicing to the downstream Cβ loci (Schatz 2004). The receptor gene recombinase enzyme has specificity to join gene elements bounded by one RSS with a 23-bp spacer and another gene element with a 12-bp spacer, assuring the proper orientation of Vβ, Dβ, and Jβ gene elements. There are approximately 100 Vα gene segments on chromosome 14 in the mouse germline. A few hundred thousand base pairs downstream are ~60 Jα gene segments, each flanked on their 5’ side by an RSS with a 12-bp spacer. The 3’ boundary of the Jα gene segments are RNA splice sites that permit splicing from a rearranged VαJα to the downstream Cα constant regions. The assembly of a functional rearranged VαJα occurs when the recombinase enzyme binds to one RSS containing a 23-bp spacer and one RSS containing a 12-bp spacer (Schatz 2004). The receptor recombinase is a complex enzyme that must recognize two appropriate RSSs in either the D and J gene clusters or the V and D gene clusters for the β chain, cut the DNA duplex immediately adjacent to the two RSS, and then catalyze the joining of the cut ends to yield a recombined receptor gene. The joining process represents a type of DNA repair and is catalyzed by a collection of DNA repair enzymes that include the DNA-dependent protein kinase (Gellert 2002), Ku80 and Ku86 (Errami et al. 1996; Liang and Jasin 1996), DNA ligase IV (Grawunder et al. 1998), and Artemis, a DNA binding factor that is mutated in some subjects with severe combined immunodeficiency (Grawunder et al. 1998). V-D-J rearrangement in the TCR β chain locus and V-J rearrangement in the TCR α chain locus produce sequence diversity in the CDR3 regions of the α and β chains of the receptor. Variability in the CDR1 and CDR2 is the result of sequence variability in the body of the different V region genes. The extent of sequence variability of the CDR3 is manyfold higher than that seen in the CDR1 and CDR2. Crystal structures of the TCR associated with a class II MHC molecule containing the antigenic peptide show that the TCR CDR1 and CDR2 are involved primarily in contacting the α helices of the MHC class II proteins, with CDR3 being primarily responsible for interacting with the MHC-bound peptides. Adding potential complications to the recombination process is the fact that the TCR Vδ gene sequences are scattered among
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the Vα gene elements, and the Dδ and Jδ elements are just upstream of the Jα segments on chromosome 14. Thus, the TCR δ chain gene elements are embedded within the TCR α chain gene elements. This means that regulated expression of the rearrangement and expression of the δ chain genes must be distinct from that of the α chain gene elements. The genes encoding the TCR γ chain are located on their own on mouse chromosome 13. b. GENE REARRANGEMENTS IN γδ T CELLS Activation of γδ TCR gene rearrangement occurs in sequence, beginning at day 12 of mouse gestation. At this time, developing T cells in the thymus attempt to rearrange their TCR δ genes, first rearranging the DδJδ segments on both chromosomes. This is then followed by attempted rearrangement of a Vδ to a DδJδ element. If that produces an in-frame, functional δ chain, then rearrangement on the other chromosome is stopped, and rearrangement of the γ chain gene begins (Haas et al. 1993). If a functional TCR γ chain is also formed, then the cell leaves the thymus to continue as a γδ T cell. In adult mice, rearrangement of γ and δ chains can also occur in an extrathymic location. In fact, the majority of γδ T cells in adult mice are thought to arise in an extrathymic compartment, with these cells largely populating the gastrointestinal tract. γδ T cells are also prominent in the epidermis where they are described as “dendritic epidermal T cells,” but these cells appear to derive largely from the thymus (Hara et al. 2000). αβ T cells develop in the thymus, with β chain rearrangement beginning also around embryonic day 13. As soon as a functional β chain is formed, it pairs with a monomorphic surrogate α chain called pre-Tα. Expression of a pre-Tαβ chain heteromer extinguishes further β chain rearrangement (Aifantis et al. 2006). This process is called allelic exclusion and serves to prevent two functional TCR β chain genes from being expressed in the same cell. After assembly of a functional TCR β chain, rearrangement of the TCR α chain gene segments begins. Allelic exclusion of α chains seems less complete than that for β chains. Thus, it is possible to detect peripheral T cells with dual specificity because of their ability to pair either of two functional α chains with the single functional β chain. Not well defined is the mechanism by which a developing thymic T cell decides whether to commit to becoming a γδ or an αβ T cell. Initial studies were interpreted to support a sequential model in which T cell precursors first attempted to rearrange down the γδ lineage and if that failed then to attempt rearrangement by the αβ lineage. Alternatively, differentiation may be via a separate lineage model. From analysis of the sequences of a large number of αβ T receptors, it is clear that most chromosomes show lack of rearrangements of their δ chains (Winoto and Baltimore 1989). Additional evidence supporting the separate lineage model comes from analysis of mice that carry transgenic functional γ and δ TCR genes. In the sequential model, the expression of a functional γδ receptor should lead to heavily reduced production of cells expressing αβ TCR. However, in these mice, normal numbers of αβ T cells were found (Dent et al. 1990).
39 c. THYMIC SELECTION OF CD4+ AND CD8+ T CELLS For T cells carrying the αβ TCR, assembly of a functional antigen receptor occurs exclusively in the thymus as does evaluation of whether the receptor lacks self-reactivity, is likely to recognize antigens in the context of self-MHC, and should be allowed to enter the peripheral pool of circulating T cells (Fig. 19). The thymus is a complex lymphoid organ that is located in the anterior mediastinum, at the base of the neck and in front of the heart (Miller 2002). In certain strains of mice, there is also a smaller cervical thymus found in the neck (Terszowski et al. 2006). In terms of its function in the support of T cell development and testing whether the newly produced T cells have appropriate antigen specificity, the thymus has three major compartments. The first, the subcapsular zone, is the compartment in which bone marrow–derived T cell precursors first localize, becoming definitive prothymocytes, and begin to differentiate. These cells enter the thymus as CD3−CD4− CD8− cells with germline configuration of their TCR genes. In the subcapsular zone, they begin to rearrange their TCR β chains. If β chain rearrangement is unsuccessful, they are eliminated by apoptosis. If they produce β chains that can pair with pre-Tα, they proceed to migrate into the cortex where the α gene elements rearrange in an attempt to form a functional, mature αβ TCR. With expression of an αβ TCR heterodimer, the cells upregulate the TCR-associated CD3 complex and express on their surfaces CD4 and CD8. By virtue of their dual expression of CD4 and CD8, these cells are designated as double positive. In the cortex, the cells are tested to determine whether their newly assembled TCR has sufficient affinity for self-MHC molecules to permit them to recognize antigen-MHC complexes. This process of “positive selection” depends on close interactions between the developing lymphocyte and the specialized cortical epithelial cells (Cosgrove et al. 1992). Positive selection of CD4+ T cells depends on cortical epithelial expression of class II MHC molecules, whereas positive selection of CD8+ T cells depends on cortical epithelial class I MHC expression (Cosgrove et al. 1992). If the lymphocyte fails this positive selection, it loses survival signals, undergoes apoptosis, and is cleared by phagocytosis by thymic cortical macrophages. At this point, the T cell has declared whether it sees antigen in the context of a class I MHC molecule or a class II MHC molecule. If the cell’s TCR shows appropriate affinity for antigen in the context of class I MHC, the cell extinguishes expression of CD4 and remains TCR+CD3+CD8+. If its TCR recognizes antigen in the context of class II MHC, then the cell extinguishes expression of CD8 and remains TCR+CD3+CD4+. The positively selected cells next traffic to the thymic medulla where they are tested for dangerous autoreactivity in a process called negative selection. This process, like the cortical positive selection, is dependent on the presence of specialized epithelial cells in the medulla. Trafficking of positively selected cells from the cortex to the medulla is driven by upregulation of CCR7 on the positively selected cells (Kurobe et al. 2006; Kwan and Killeen 2004) and by CCL19 and/or CCL21 expression in
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Excessive Affinity for Self Peptide + MHC
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Apoptosis γδ T Cell CD3+ CD4−CD8− γ δ TCR+
Fig. 19 Differentiation and maturation of T cells in the thymus. Hematopoietic stem cells committed to the T cell lineage move from the bone marrow to the thymic subcapsular zone. There they begin rearrangement of the TCR genes. Once a productive TCR β chain has been produced, they move to the thymic cortex where α chain rearrangement occurs, and the cells express both the CD4 and the CD8 surface proteins. These DP cells are positively selected on cortical epithelial cells for their abilities to recognize self-MHC proteins. Selected cells move to the thymic medulla where they are negatively selected on thymic epithelial cells to remove cells with excessive affinity for self-antigens presented in MHC proteins. Cells emerge from positive selection single positive for either CD4 or CD8 expression and then are exported to the periphery. Cells that fail positive or negative selection are removed by apoptosis. A small fraction of cells rearrange their TCR γ and δ chains rather than their α and β chains. Modified from Chaplin (2003).
medullary stromal cells (Ueno et al. 2004). Once in the medulla, the developing T cells interact with the specialized medullary epithelial cells. These cells manifest exceptionally promiscuous expression of self-proteins that are normally limited in their expression to peripheral tissues (Derbinski et al. 2001). This promiscuous expression is driven by the AIRE protein, a transcription factor designated the autoimmune regulator (Anderson et al. 2002). Positively selected T cells that show signs of autoreactivity when they interact with medullary epithelial cells that promiscuously present self-antigens are induced to undergo apoptosis before they can escape into the circulation. This apoptotic response is independent of the Fas death receptor but is dependent on the proapoptotic protein Bim (Villunger et al. 2004). In addition to macrophages, eosinophils appear to play an important function in the selection process, particularly in the induction of apoptosis in class I–restricted autoreactive cells (Throsby et al. 2000). The stochastic process that leads to the assembly of αβ TCR is inefficient, frequently generating cells that are either autoreactive or that cannot recognize self-MHC sufficiently to support antigen recognition. In fact, fewer than 5% of developing T cells that are generated in the thymus survive the positive and negative selection process (Sprent and Kishimoto 2002).
More information on central T cell tolerance is found in chapter 9, entitled “Mouse Models of Negative Selection.” d. DIFFERENTIATION OF T CELLS INTO THE TYPE 1 AND TYPE 2 PHENOTYPE Seminal studies by Mosmann and Coffman demonstrated in the mid-1980s that Th cells can be divided into two major classes based on their expression of selective profiles of cytokines and other secreted mediators (Mosmann et al. 1986). Cells designated Th1 (or type 1 Th cells) showed potent activity supporting cellular immune responses and produced large amounts of IL-2, IFN-γ, IL-3, and GM-CSF. Cells designated Th2 (or type 2 Th cells) secreted very low levels of IL-2 and IFN-γ and high levels of IL-4 (originally designated BSF-1) and IL-9 (originally designated T cell growth factor distinct from IL-2). Over the years since this original description of these two helper cell phenotypes, it became apparent that most CD4+ T cell–dependent immunological reactions developed a predominantly Th1 or Th2 quality. It also became clear that Th2 responses often were also associated with cellular production of IL-5 and IL-13, whereas Th1 responses often included production of lymphotoxin or expression of the membrane lymphotoxin α1β2 heterotrimer (O’Garra and Arai 2000). The effector cytokine profiles associated with the type 1 and the type 2 responses suggested that Th1 cells were most closely
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associated with cell-mediated immune responses, with responses to intracellular pathogens, and with many types of organ-specific autoimmune disorders (e.g., rheumatoid arthritis and multiple sclerosis). In contrast, Th2 cells appeared most closely associated with the host response to helminth infestations, humoral immune responses (especially responses supported by IL-4, such as IgG1 and IgE responses), allergic reactions, and atopy. It also has become clear that the major determinants of whether naïve CD4+ T cells differentiate towards a Th1 or a Th2 response are characteristics of the cytokine milieu in the site where the cell receives its antigen stimulus via an APC. Antigen stimulation in an environment dominated by IL-4 (produced by mast cells, eosinophils, NKT cells, or previously activated and polarized Th2 cells) leads to polarization of the responding cell toward Th2 cytokine production. In contrast, activation of the naïve cell in an environment rich in IL-12 or IL-18 (produced by activated macrophages, by DCs that have been stimulated through any of several types of TLRs (especially TLR4 and TLR9) or by other phagocytic cells that have ingested intracellular bacteria leads to polarization of the responding cell toward Th1 cytokine production. In particular, antigen challenges or deliberate immunization that activates TLR9 expressed by DCs induces expression of IL-12 and induction of a Th1 type response, whereas antigen challenge or deliberate immunization that activates tissue mast cells or NKT cells can lead to substantial production of IL-4 and consequently establishment of a Th2 response (Mowen and Glimcher 2004). Portions of the intracellular signaling pathways leading to type 1 or type 2 polarization are becoming well known. It is now clear that signals leading to Th1 polarization are delivered through the T-bet transcription factor (Szabo et al. 2000). In fact, mice deficient in T-bet develop pathological changes in their lungs similar to those seen in human subjects with allergic asthma, suggesting that in the absence of T-bet, the effector Th response diverts dominantly toward Th2 (Finotto et al. 2002). The Th2 lineage, in contrast, depends on expression of the transcription factor GATA-3 for its development and maintenance (Zheng and Flavell 1997). GATA-3 upregulates IL-4 and IL-5 gene expression in CD4+ T cells and downmodulates IFN-γ expression in Th1 cells (Ferber et al. 1999; Ouyang et al. 1998; Ranganath et al. 1998). The identification of these intracellular signaling pathways in the expression of type 1 and type 2 responses may identify important new targets for immunomodulatory drug therapy. An awareness of the consequences of polarized cytokine production has prompted investigation of the possibility of polarization in other experimental systems. It is now clear that some CD8+ T cell responses can be polarized in a type 1 or type 2 fashion, although it is not so clear what environmental factors control the polarization process. Polarized CD8+ T cell responses are designated Tc1 and Tc2 to designate whether they are substantially associated with responses to intracellular microbes or to extracellular parasites. Tc1 cells showed
41 variable cytotoxic activity, depending on the tissue source they were derived from, but expressed large amounts of IL-2, IFNγ, and IL-6. Tc2 cells, in contrast, always showed high cytotoxic function and secreted large amounts of IL-4 and IL-5 and moderate amounts of IFN-γ (Kemp et al. 2005). In addition to CD8+ T cells, some subsets of DCs are closely associated with expression of a type 1 or a type 2 cytokine response and therefore have been designated DC1 and DC2 (Hochrein et al. 2001; O’Keeffe et al. 2003). e. DIFFERENTIATION OF TH17 CELLS Skewing of the effector Th response toward production of Th1 cells is governed by the production of IL-12 at sites of Th cell activation (Berenson et al. 2004). IL-12 is a heterodimer of IL-12 p35 and IL-12 p40 subunits covalently joined by a disulfide bond and signals by binding to the dimeric IL-12 receptor. This receptor is constituted of IL-12Rβ1 and IL-12Rβ2 subunits (Trinchieri 2003). IL-12 is now recognized as belonging to a small family of cytokines that share structural features, including, in addition to IL-12, the highly related cytokine IL-23 and the more distantly related cytokines IL-27, cardiothrophin-like cytokine (CCL) paired with a soluble form of the ciliary neutrotrophic factor receptor (CNTFR), and CCL paired with a CNTFR-like chain designated cytokine-like factor-1 (CLF-1). The most closely related family member, IL-23, consists of an IL-23 p19 subunit covalently linked to the IL-12 p40 subunit. The IL-23 heterodimer signals by interacting with a dimeric receptor that is composed of the IL-23 receptor chain associated with the IL-12Rβ1 subunit (Trinchieri et al. 2003). Thus, IL-12 p40 paired with the p35 subunit represents IL-12 and signals through the IL-12Rβ1β2 heteroreceptor, and IL-12 p40 paired with the p19 subunit represents IL-23 and signals through the IL-12Rβ1IL-23 heteroreceptor. Because both the IL-12 and the IL-23 ligand and receptor pairs signal through STAT1-, STAT3-, and STAT4-dependent pathways, they were originally thought to be functionally redundant, both contributing to the development of Th1 responses and to a host of inflammatory autoimmune processes. However, recent studies have shown that mice deficient in IL-12 p40 (deficient in both IL-12 and IL-23) show reduced severity of brain inflammation in EAE, as did mice deficient in the IL-23 p19 subunit (deficient in IL-23, but with wild-type expression of IL-12). In contrast, mice deficient in the IL-12 p35 subunit (IL-12 deficient, but IL-23 expressing) showed dramatically exacerbated disease (Cua et al. 2003). These findings suggested that IL-12 was not the primary mediator of EAE pathogenesis and placed emphasis on IL-23 as a key regulatory signal in autoimmunity. Similar findings have now been obtained in models of inflammatory bowel disease and several inflammatory arthritis models, suggesting the generality of these findings for autoimmune pathogenesis (Murphy et al. 2003; Yen et al. 2006). Identification of IL-23 as a major signal that determines the outcome of CD4+ T cell inflammatory responses has directed intense interest on the role of IL-17–producing CD4+ T cells as autoimmune effector cells. IL-23 promotes the development of
42 IL-17–producing effector cells (Aggarwal et al. 2003), and it is now clear that under appropriate conditions, CD4+ T cells are induced that express IL-17 in the absence of either IL-4 or IFN-γ. These cells have been designated Th17 (Park et al. 2005). They secrete both IL-17 and a related isoform of unknown function designated IL-17F. IL-17 secreted by Th17 cells can activate a broad range of host proinflammatory mediators including IL-1, IL-6, TNF, nitric oxide, metalloproteinases, and chemokines (Iwakura and Ishigame 2006). It is now recognized that Th17 cells are important regulators of the host response against extracellular bacteria and that they cause many of the pathological features of a broad range of autoimmune disorders and may participate importantly in allograft rejection. Recent studies by several groups have addressed the developmental pathway leading from the naïve CD4+ T cell to a mature Th17 cell. Based on studies performed both in vitro and in vivo in mice, there are now convincing data that Th17 cells represent a lineage that is fully distinct from the Th1 and Th2 lineages and that they are produced directly from naïve CD4+ T cells (Harrington et al. 2005; Park et al. 2005). Although IL-23 is clearly identified as a cytokine that can upregulate expression of IL-17 by these cells, differentiation of Th17 cells from the naïve precursors is directed by TGF-β and IL-6 (Bettelli et al. 2006; Mangan et al. 2006; Veldhoen et al. 2006). Of interest, CD4+ T cells that express suppressive activity (Treg cells, see below) appear to participate in the induction of Th17 cells in vivo (Veldhoen et al. 2006). This finding is particularly intriguing because regulatory T cells can secrete TGF-β, and some can express TGF-β on their surfaces. DCs and stromal cells can also express TGF-β, so the cellular source of this Th17-polarizing cytokine remains to be defined. Additional information on cytokine-activated intracellular signaling in lymphocytes may be found in chapter 7, entitled “Cytokine-Activated JAK-STAT Signaling in the Mouse Immune System.” f. REGULATORY T CELLS AND T CELL TOLERANCE The adaptive immune response plays a major role in the host defense mechanisms of mice and other mammals. Central to the sensitivity and specificity of the murine adaptive host response is this system’s use of stochastically rearranging genes that encode antigen-specific receptors. A perhaps inevitable consequence of such a rearranging gene system is that receptors with self-reactivity arise with appreciable frequency. These selfreactive receptors represent a significant immunological threat to the animal, and it is essential that reliable mechanisms be in place to prevent adverse outcomes when the cells that produce these antigen-binding molecules encounter self-antigens. One form of self-tolerance is provided by negative selection against self-reactive cells. This type of selection usually takes place in the central lymphoid tissues near the time that the lymphocyte first acquires its antigenic specificity. Because this mechanism for eliminating self-reactive cells is expressed in a central lymphoid tissue, the immune tolerance it provides is referred to as central tolerance. The selection against self-reactive
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T cells that occurs in the thymic medulla (discussed above) represents an important form of central tolerance; however, even if the central mechanisms to remove self-reactive cells are working well, autoreactive T cells still escape from the thymus into the peripheral circulation and tissues with detectable frequency. It is, therefore, essential to develop robust mechanisms to maintain tolerance in the periphery. One way in which self-tolerance is supported in the periphery is that the immune system generally requires the convergence of both immunological signals from the non-self-antigen and danger signals that upregulate the expression of molecules which provide T cell costimulatory functions (Greenwald et al. 2005). Thus, self-tissues that do not elicit the expression of costimulatory signals usually do not provide a sufficiently strong activating signal to induce a response from potentially autoreactive cells. In fact, T cells that receive immunological signals through their antigen receptors (referred to as “signal 1”) without receiving costimulatory signals (referred to as “signal 2”) often are rendered anergic, becoming resistant to activation by the antigen on subsequent exposures even when accompanied by costimulatory signals (Powell 2006). In addition to pathways leading to cell anergy, the immune system has evolved an important additional mechanism to prevent the activation of antigen-specific T cells. This mechanism suppresses the activation of effector antigen-specific T cells by the actions of the several populations of regulatory T cells (Treg cells). The best characterized type of Treg, also called the “naturally arising regulatory T cell” (Yamaguchi and Sakaguchi 2006), is defined by its surface expression of CD4 and CD25 (the IL-2 receptor )IL-2R) α chain that contributes to the production of the high-affinity IL-2R) (Shevach 2002). This is in marked contrast to conventional CD4+ T cells that express very low or undetectable levels of CD25 until they have been activated by antigen plus costimulatory signals (Greene and Leonard 1986). The constitutive expression of CD25 appears to be central to the differentiation and function of the naturally arising Treg. This characteristic is manifested by the fact that mice deficient in IL-2 or either the CD25 component (α chain) or the CD122 component (β chain) of the high-affinity IL-2R manifest a fulminant lymphoproliferative condition with autoimmune features (Sadlack et al. 1993; Suzuki et al. 1995; Willerford et al. 1995). It appears that IL-2 is not required for the development of CD25+ T cells in the thymus (Fontenot et al. 2005). IL-2 is, however, required for the activation of these cells for them to express their effector function (Thornton et al. 2004). In addition to their signature expression of CD25, naturally arising Treg cells are also distinguished by their expression of Foxp3, the gene encoding the Foxp3 transcription factor, which plays a key role in establishing the differentiation and supporting the functions of these cells (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003). The molecular targets of Foxp3 are not yet well defined, but the importance of this factor is underscored by the finding that spontaneous (Brunkow et al. 2001)
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or targeted mutation (Fontenot et al. 2003) of the Foxp3 locus results in a fatal autoimmune syndrome with deficiency of CD4+CD25+ Treg cells. Thus, it is clear that the Foxp3 transcription factor plays a central role in either the induction or survival of the CD4+CD25+ Treg lineage. It remains the most specific molecular marker for this cell lineage. Foxp3+CD4+CD25+ Treg cells arise in the thymus as a mature cell population that expresses its full phenotype before any encounter with its specific antigen. In mice, these cells exit the thymus beginning 3 days after birth and ultimately represent 5–10% of the repertoire of circulating CD4+ T cells in healthy adult animals (Shevach 2002). Thymectomy of mice before day 3 after birth results in a broad range of autoimmune manifestations in multiple tissues, indicating the essential function of the thymus in the formation of this form of Treg (Sakaguchi et al. 1995). Although parallel studies have not been performed in mice, interesting studies in humans suggest a role for Hassall’s corpuscles, unique thymic epithelial cell structures, in the development of Treg cells. Stromal cells in Hassall’s corpuscles express thymic stromal lymphopoietin that activates thymic CD11c+ DCs to express high levels of costimulatory molecules (including CD80 and CD86). These activated thymic DCs then signal the differentiation of CD4+CD25− cells to express surface CD25 and intracellular Foxp3, assuming the full CD4+CD25+ Treg phenotype (Watanabe et al. 2005). In addition to these naturally arising Treg cells, Treg cells can also be generated after antigen exposure in the peripheral tissues (Buckner and Ziegler 2004). Some, but not all, of these peripherally induced Treg cells upregulate expression of Foxp3 when they are activated by antigen (Chen et al. 2003; Vieira et al. 2004; Walker et al. 2003). For some peripherally activated Treg cells, IL-10 is an important effector cytokine (Vieira et al. 2004). In addition to CD4-expressing Treg cells, CD8+ T cells also can express regulatory functions (Keino et al. 2006; Maile et al. 2006). These cells are activated by antigen, express TGF-β, upregulate genes involved in resistance to apoptosis, and appear to use CD103 to facilitate their entry to sites of tissue inflammation (Keino et al. 2006). In addition to TGF-β, other effector cytokines with a Tc2 character, including IL-4, IL-5, and IL-10, may be central to the inhibitory function of these cells. An interesting feature of these cells is that they express low levels of surface CD8 (Maile et al. 2006), suggesting that the avidity of the interaction between these cells and the antigen-bearing cells may be particularly important in their regulation. Perhaps as a consequence of their low avidity, these CD8+ Treg cells seem unable to suppress the function of memory T cells, restricting their function to naïve T cells. Additional information on peripheral T cell tolerance is found in chapter 10, entitled “Peripheral Tolerance of T Cells in the Mouse.” g. NKT CELLS NKT cells belong to a cell lineage that constitutively expresses surface markers of both NK cells (in
C57BL/6 mice, the NK cell antigens NK1.1 and DX-5) and T cells (expressing CD3 and sometimes CD4 and/or CD8). They also bear rearranged TCR genes; however, unlike conventional αβ or γδ T cells, most NKT cells express a specific TCR α chain, Vα14/Jα28 or Vα14/Jα18 (Taniguchi et al. 2003). The TCR formed by the combination of this “invariant” TCR α chain with any of a number of TCR β chains shows binding reactivity with a class of glycolipids antigens that are modeled by the synthetic antigen α-GalCer presented on the nonclassic class I MHC protein CD1d (Bendelac et al. 1995). Antigens with glycolipid character in the same class as α-GalCer can be generated from many self-tissues including cancers and from a variety of microbes (Wu et al. 2006), suggesting that NKT cells may play immunoregulatory roles in antibacterial host defense and tumor surveillance. A signature quality of NKT cells is their ability to produce large quantities of IL-4 and/or IFN-γ within a few hours after stimulation (Leite-De-Moraes et al. 1998). Perhaps because they exist in substantial numbers (up to several percent of peripheral circulating lymphocytes) with broad specificity of one class of antigen, there is no need for clonal expansion before their delivery of an effector response. In this regard, they behave like effectors of the innate immune system. Recent studies have identified important roles for NKT cells in host defense against tumors through an effect on IL-12–dependent antitumor mechanisms (Cui et al. 1997; Liu et al. 2005). NKT cells appear to play a central effector role in certain allergic responses as well, being required for the induction of allergeninduced airway inflammation and hyperreactivity in mice (Akbari et al. 2003) and in human asthmatic patients (Akbari et al. 2006). Interestingly, the transcription factor T-bet, the T-box transcription factor required for differentiation of Th1 cells, is also required for the differentiation of mature and functional NK and NKT cells (Townsend et al. 2004). 2.
B Cells and Plasma Cells
B cells constitute approximately 15% of the peripheral blood leukocytes. They are defined by their ability to produce Igs that represent the primary soluble antigen-binding molecules of the adaptive immune response. The structures of the Ig classes and subclasses were described earlier in this chapter (see section II.C.1.). Like the antigen-binding receptors on T cells, the BCRs are the product of pairing of two chains to yield a complete antigen-binding unit. For B cells, the two chains are designated heavy and light chains on the basis of their molecular weights. The genes encoding both chains are generated by somatic joining of variable (both heavy and light chains), diversity (heavy chain only), and joining (both heavy and light chains) gene elements to generate exons encoding the mature, antigen-binding portions of the heavy (VHDHJH) and light (VLJL) chain genes. Unlike the TCR, each Ig unit is composed of two heavy and light chain pairs (Fig. 10), and in the case of the secreted forms of IgA and IgM, larger multimers consisting
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of 2 and 5 units are assembled. The assembly of two pairs of heavy and light chains into single Ig molecules creates antigenbinding molecules that are bivalent for antigen binding. This increases the avidity of the interaction between antigen and antibody and permits the formation of lattice-like antigen-antibody aggregates, or ICs, that enhance the clearance of the antigen, and increase the binding of the antigen-antibody complex to Fc and other receptors. a. B CELL DEVELOPMENT BEFORE ENCOUNTER WITH ANTIGEN Assembly of a functional Ig gene from V (heavy and light chain), D (heavy chain only), and J (heavy and light chains) elements occurs via many of the same mechanisms as are used to assemble functional TCR genes, although in some regards, the program of gene rearrangement is more complex as outlined in the following. The enzyme complex that performs the gene rearrangement process appears to be the same as that involved in TCR gene assembly. The gene elements that encode the heavy chain locus are situated on mouse chromosome 12. There are two types of light chains, designated κ and λ. The genes encoding the κ chains are located on mouse chromosome 6 and the genes encoding the λ chains are located on mouse chromosome 22. Like the TCR V gene segments, each heavy and light chain V gene element is composed of two exons, an upstream leader sequence exon followed by a short intron and an approximately 300-bp long V sequence exon. The 3’ ends of the V gene exons, both ends of the D gene elements, and the 5’ ends of the J elements are bounded by heptamer/nonamer RSSs (Fig. 20). As for rearrangement of the TCR gene elements, the recombinase only permits recombination between RSSs that differ in their spacer lengths, so that an RSS with a 23-bp spacer can recombine with an RSS containing a 12-bp spacer.
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In the Ig heavy chain locus, the DH gene elements are flanked on both sides with RSS containing 12-bp spacers, and both VH and JH elements are flanked by RSSs containing 23-bp spacers. A JH segment is, therefore, unable to recombine with a VH segment until after it has recombined with a DH element. Rearrangement of the mouse heavy and light chain genes occurs in the bone marrow in cells that arise from the common lymphoid progenitors and that are committed to the B cell lineage. Gene rearrangement is an ordered process. The first step is, rearrangement of a DH element to a JH element occurring on both chromosomes. After DHJH rearrangement has occurred on both chromosomes, then a first attempt at VH gene rearrangement occurs. If rearrangement is successful, the process results in a recombined heavy chain VDJ exon or, as described below, a light chain VJ exon that is now located upstream of constant region exons that are responsible for the unique characteristics of the particular heavy chain isotype (µ, δ, γ, α, or ε) or the light chain isoform (κ or λ). Thus, after successful rearrangement of the first heavy chain locus, a functional heavy chain gene is formed that directs the synthesis of an intact µ heavy chain. This chain pairs in the cell with two chains, λ5 and Vpre-B, that together make up the surrogate light chain. Assembly of the rearranged µ heavy chain with the surrogate light chain creates a stable protein that can be expressed on the cell surface as the pre-BCR and that then signals within the cell to terminate further heavy chain rearrangement. This results in allelic exclusion (the expression of a heavy chain expressed from only one of the diploid chromosomes) and also induces the start of ordered light chain rearrangement. The variable portions of Ig light chains consist only of a V gene element joined to a J gene element. Attempts at successful
Orientation of V(D)J Gene Segments and Recombination Signal Sequences Jκ
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Fig. 20 Recombination signal sequences. Conserved heptamer (7-bp) and nonamer (9-bp) sequences, separated by 12-bp or 23-bp spacers, are located adjacent to V and J exons (for κ and λ loci) or to V, D, and J exons (in the H chain locus). The V(D)J recombinase recognizes these recombination signal sequences and brings the exons together. Reproduced from Abbas, A.K., and Lichtman, A.H. (2005). Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
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light chain rearrangement occur first at the κ locus, involving joining of a Vκ to a Jκ element. If the first rearrangement produces a functional light chain, then this chain pairs with the heavy chain to form an intact immunoglobulin that can be expressed on the membrane of the B cell and that signals for further rearrangement of light chains to be suppressed. If the first rearrangement does not yield a functional light chain, then rearrangement proceeds at the κ locus on the other chromosome. If a functional light chain does not emerge from this attempt, then rearrangement is activated at the λ light chain locus. With a functional λ rearrangement, then a functional Ig is produced, and B cell differentiation can proceed. If λ rearrangement is not successful, then it is thought that the cell is removed via an apoptotic mechanism. In functional B cells, perhaps largely because attempted rearrangement of κ genes precedes the attempted rearrangement of λ chains, cells using κ as the light chain element represent close to 95% of the total B cells in the circulation and spleen of mice. Diversity of B cell antigen receptors is supported by several mechanisms. The first is based on the combinatorial association of V, D, and J gene elements in the heavy chains and V and J gene elements in the light chains. In addition, there is substantial diversity generated at the junctions between these rearranged gene segments caused as a result of the molecular mechanisms of gene rearrangement. Cleavage of the germline DNA sequences by the RAG-1/RAG-2-containing recombinase enzyme complex generates DNA that terminates in hairpin loops formed by linkage of the two strands of the DNA duplex. Joining of gene elements that are terminated by these hairpin ends requires nucleolytic opening of the hairpins, a process that can occur asymmetrically, forming what are termed P (palindromic) nucleotides. In addition to sequence variability created by the action of the hairpin-cleaving endonuclease, the ends of the DNA fragments can be altered by nucleotide deletion (via an exonuclease) or addition. Nucleotide addition is mediated by the action of the enzyme terminal deoxynucleotidyl transferase (TdT), and occurs more commonly in Ig heavy chain genes than in light chain genes. The nucleotides that are added by TdT are called N-region nucleotides, because they represent nongermline encoded nucleotides. P nucleotides and N nucleotides are common in TCR genes as well, underscoring the similarity in the molecular mechanisms that generate functional TCR genes and functional Ig genes. In the TCR, N-region nucleotides are more common in β and δ chain genes than in α and γ chain genes. Altogether, the ability to create diversity in antigen-binding sites by combinatorial association of heavy and light chains, by variable joining of different V, D, and J elements, and by junctional sequence variability with P and N nucleotides results in a staggering potential repertoire of Ig and TCR antigenic specificities. These mechanisms have the potential to generate more than 1012 to 1016 different antigen-binding Ig and TCR molecules. The process of B cell maturation from a committed stem cell with its Ig gene elements still in their germline configuration
45 through to a fully formed mature B cell occurs completely in the bone marrow in the absence of the antigens that the B cell will ultimately recognize (Fig. 21). This differentiation is dependent on interactions between the developing B cell and bone marrow stromal cells that produce IL-7. Once the precursor cell is committed to the B cell lineage, it can be recognized by its expression of CD45 (specifically its B220 isoform), CD19, and low to moderate levels of MHC class II (Fig. 21). Once heavy chain rearrangement has occurred and the cell expresses the pre-B cell receptor on its cell surface, it is designated a pre-B cell. After successful light chain rearrangement and the expression of an intact IgM molecule on the cell surface, the cell is classified as an immature B cell. Further differentiation of the cell to the mature B cell stage is accompanied by coexpression on the cell surface of both IgM and IgD. The ability of the same cell to express both IgM and IgD forms of antibody recognizing the same antigen at the same time is the consequence of the genomic organization of the heavy chain constant region exons as a gene cluster on chromosome 12. The exons encoding the constant region of the δ heavy chain are located immediately downstream of the exon encoding the constant region of the µ heavy chain. These two isotypes are then expressed by alternative RNA splicing of the same rearranged VHDHJH exon to either the µ exons or the δ exons. In the early phases of B cell development, before encounter with antigen, mRNA splicing of the µ and δ constant region exons results in inclusion of transmembrane exons that encode a short intracellular peptide which provides an endoplasmic reticulum stop transfer signal, anchoring the heavy chain and its associated light chains in the membrane (YamawakiKataoka et al. 1982). When the heavy chains are anchored in the B lymphocyte membrane, they associate with the Igα:Igβ heterodimer to form an intact signaling complex (see section II.C.1.d. and Fig. 11) that informs the cell when it encounters its specific multivalent antigen. After activation by antigen, alternative mRNA splicing replaces the transmembrane and intracytoplasmic portions of the heavy chain constant region with an exon that encodes an Ig tail piece that permits efficient secretion (Calame et al. 1980). Before activation by antigen, very few heavy chain transcripts are spliced to the secreted exons, resulting in most or all of the synthesized IgM and IgD being placed on the cell membrane and very little or none being secreted (Goldsby et al. 2003). When B cells transition to the mature stage, they are released from the bone marrow in an SDF-1- and CXCR4-dependent process (Ma et al. 1998). Mature B cells travel via the blood to localize in the secondary lymphoid tissues where the tissue organization favors encounter with antigen and, if necessary, with other leukocytes. In the periphery, B cells manifest a variety of different phenotypic and functional characteristics that divide them into discrete subsets (Kearney 2004). The largest B cell subset is designated B-2. B cells in this subset circulate relatively rapidly from one lymphoid tissue compartment to
46
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Fig. 21 B cell differentiation and development. B cells differentiate in the bone marrow from stem cells to become mature surface IgM- and IgD-expressing cells. This occurs in the absence of antigens. In peripheral lymphoid tissues, the B cell can then mature further under the influence of antigen and T cell help to undergo isotype switching and affinity maturation by somatic mutation. The factors controlling the final differentiation from an antibody-secreting B cell to a plasma cell are not well characterized. Correlations are shown between the stage of cell differentiation and the expression of key molecules in the cell (TdT, RAG1/RAG2, and cytoplasmic µ) and on the cell surface (class II MHC, CD19, CD21, CD45, and surface Ig). Modified from Chaplin (2003).
another and localize primarily in the B cell follicles in spleen, lymph nodes, and Peyer’s patches. These cells are primarily involved in responses that depend on help from T cells and that lead to the production of high-affinity antibodies recognizing protein antigens (Baumgarth et al. 2000). A special subset of B-2 cells localizes in the marginal zone (MZ) of the spleen (Martin et al. 2001). The B-1 subset is self-renewing, exists in two forms based on expression or lack of expression of CD5, and is found primarily in the peritoneal and pleural cavities of mice (Stall et al. 1996). Localization to these compartments appears to depend on the expression of the chemokine CXCL13 (BLC), although the cellular sources of this chemokine that drive this localization remain undefined (Ansel et al. 2002). The B-1 and MZ B cell populations share some functional characteristics, responding early after encounter with antigen, generally acting independent of T cell help, and having a preference for responding to carbohydrate and other T cell–independent antigens (Martin et al. 2001). b. B CELL DEVELOPMENT AFTER ENCOUNTER WITH ANTIGEN: THE GERMINAL CENTER, AFFINITY MATURATION, AND DIFFERENTIATION OF PLASMA CELLS The initial activation of
mature B cells by antigen is thought to occur in different microenvironments, depending on the subset of B cell involved. For example, activation of B-1 cells is thought to occur at the sites of their greatest prevalence in the peritoneal or pleural cavities (Martin et al. 2001). The mechanisms by which B-1 cells are activated remain incompletely defined. B-1 cells appear to produce IgM antibodies in a constitutive fashion, and, under at least some circumstances, cross-linking of surface IgM results in cellular apoptosis (Tsubata et al. 1994). Surface expression of CD5 on the B-1 cell appears to contribute to this apoptotic response after BCR cross-linking, since mice deficient in CD5 show enhancement of antibody production by B-1 cells after BCR cross-linking (Bikah et al. 1996). In the case of B-2 cells, the outcome of the encounter with antigen can be importantly modulated by interactions with regulatory and helper T cells. These interactions are fostered by the organized structures of secondary lymphoid tissues (Fu and Chaplin 1999). When a T cell–dependent antigen penetrates past the physical barriers of the skin or mucous membranes, it is taken up by a phagocytic cell or DC and transported to a nearby secondary lymphoid tissue where it can be presented to antigen-specific
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CD4+ T cells as peptide fragments in a class II MHC protein. Alternatively it can be taken up in its native, unprocessed form by binding to the antigen-receptor on the surface of B cells (Chaplin 2003). In the secondary lymphoid organs, T cells and B cells are segregated from each other into discrete T cells zones and B cell areas containing B cell follicles (Fig. 22). These discrete zones are established and maintained by the expression of the chemokines CCL19 and CCL21 in stromal cells in the areas that become the T cell zones and CXCL13 in stromal cells in the areas destined to become B cell follicles (Ansel et al. 2000; Ngo et al. 1999). Within the B cell follicles, there are clusters of FDCs, cells with many dendritic processes, that carry large quantities of Fc and complement receptors and that appear not to be derived from bone marrow precursors (Le Hir et al. 1996). In the lymph nodes, the uptake of antigen activates a program of B cell and T cell migration that first brings antigen-specific B and T cells into physical contact with each other near the boundary between the B cell and the T cell zones (Garside et al. 1998). This contact is thought to deliver T cell
Antigen
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Afferent Lymphatic Vessel
Artery
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Fig. 22 Schematic diagram of a lymph node. This diagram illustrates the T cell–rich and B cell–rich zones, including B cell follicles and the routes of entry of lymphocytes and antigen (shown captured by a DC). Reproduced from Abbas, A.K., and Lichtman, A.H. (2005). Cellular and Molecular Immunology, 5th ed. Elsevier Saunders, Philadelphia.
47 help to the B cell. The ability of a T cell to provide help is dependent on its expression of the CD40L and its interaction with CD40 on the B cell (Renshaw et al. 1994). Under the influence of T cell–derived cytokines, the CD40-CD40L interaction induces B cells to undergo isotype switching, the process by which the functional rearranged VHDHJH exon is moved to a position upstream of constant region exons that encode either Ig γ, α, or ε heavy chains. This movement permits the functionally rearranged VDJ exon to be used to produce antibodies of the IgG, IgA, or IgE class while preserving the same antigenbinding capability (Kinoshita and Honjo 2000). Many studies over the past decade have demonstrated that class switching is the result of DNA recombination between switch regions, sequences located upstream of the first µ constant region exon and upstream of the first exons of each of the γ, α, and ε heavy chain exons (Manis et al. 2002). This recombination results in deletion of the DNA sequences between the upstream and the downstream switch region. The molecular mechanisms that govern class switching are just currently being determined. Recombination is clearly mediated in part by the RNA-editing enzyme called activation-induced cytidine deaminase (AID) and also involves enzymes involved in general DNA double strand break repair (Dudley et al. 2005). After contact with the T cell, the activated B cell migrates back into the B cell follicle where it participates in the formation of a germinal center, a structure induced by antigen challenge and characterized by rapid proliferation of B cells that are marked by surface glycoproteins that bind peanut agglutinin. There are conflicting data regarding whether class switch recombination occurs before or after the activated B cell enters the germinal center (Przylepa et al. 1998). It is, however, clear that in the germinal center, the expressed variable region exons of the activated B cells can be altered by somatic hypermutation. This process, also dependent on the AID enzyme (Muramatsu et al. 2000), introduces mutations in an apparently stochastic fashion that frequently alter the affinity of the encoded antibody for its immunizing antigen. If the mutation results in a reduced affinity for the antigen, the B cell loses important BCR-mediated growth and survival signals and dies an apoptotic death (Guzman-Rojas et al. 2002). If, however, the mutations result in increased affinity for antigen, then the cell producing that antibody has a proliferative advantage in response to the antigen, permitting it to grow to dominate the pool of responding cells and leading to an enhanced affinity of the overall antibody response to the T cell-dependent antigen. Somatic mutation and selection for and clonal expansion of high-affinity B cells all occur in the germinal center (Przylepa et al. 1998). More information on class switching and somatic hypermutation can be found in chapter 5. B cells that have completed isotype switching and somatic mutation exit the germinal center and differentiate further into antigen-secreting lymphocytes, plasma cells, and memory cells. Activation of antibody secretion involves both enhanced expression of Ig heavy and light chain genes and also alternative
48 splicing of the heavy chain mRNA to change the secreted Ig tail exon for the transmembrane and intracytoplasmic exons (Calame et al. 1980). The signals that govern the transition from an antibody-producing B cell to a fully differentiated plasma cell remain only partly defined but depend on expression of the transcription factor Blimp-1 (Sciammas and Davis 2005). The signals that result in formation of long-lived memory cells remain incompletely defined (McHeyzerWilliams and McHeyzer-Williams 2005). Memory cells usually express isotype-switched Ig and somatic hypermutations, suggesting that acquisition of B cell memory follows and may be dependent on passage through the germinal center (Baumgarth 2000). Finally, the release of B cells and plasma cells from the secondary lymphoid tissues to allow both B cell recirculation to other tissues and plasma cell migration to the bone marrow is regulated by their expression of the sphingosine-1-phosphate receptor 1 (Matloubian et al. 2004). More information on the architecture of primary and secondary lymphoid tissues is found in chapter 1 of this volume, entitled “The Molecular Basis of Lymphoid Architecture in the Mouse.” c. RECEPTOR EDITING AND B CELL TOLERANCE The process of somatic rearrangement of BCR gene elements to yield antigenbinding heavy and light chain variable exons provides the exceptional level of Ig diversity necessary to provide antibodies with specificity for the universe of microbial pathogens and toxins. As for the TCR, it also results with appreciable frequency in the formation of BCR with reactivity against self-structures. The mechanisms used to eliminate autoreactivity in the B cell pool appear to depend largely on the avidity of the BCR for the autoantigen. When a B cell expresses a BCR with high avidity for a membrane bound autoantigen, it undergoes apoptotic clonal deletion, whereas if a B cell bearing the same BCR encounters an autoantigen that interacts with low avidity, the B cell becomes anergic (Goodnow et al. 1995). Experiments using mice carrying transgenic Igs with autoreactive specificity showed that many B cells underwent either apoptotic negative selection or the induction of anergy (Nossal 1991). However, a small portion of the B cells did not undergo clonal deletion. These cells, rather, escaped deletion by activating a secondary V-D-J and/or V-J recombination event that lead to expression of BCRs with new antigenic specificity (Tiegs et al. 1993). This process, which was dependent on induction of expression of RAG-1 and RAG-2, has been designated receptor editing. This mechanism provides a second chance for an immature or nearly mature B cell to develop a functional immunoglobulin with antigenic specificity that is not autoreactive. Studies using both transgenic and wild-type mice have now shown that receptor editing may be the predominant mechanism maintaining B cell tolerance, with clonal deletion and induction of anergy only operating when receptor editing has not been successful (Halverson et al. 2004; Retter and Nemazee 1998). In studies designed to probe the molecular mechanisms of receptor editing, it has become clear that normal, functional,
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naïve B cells transduce a tonic low level signal that helps to maintain the cell’s survival and suppress RAG-1/RAG-2 expression and receptor editing (Kraus et al. 2004). Specific signals appear to underlie the activation of receptor editing. Although much of the signaling cascade remains to be defined, it is now clear that signals involving BLNK, Btk, RelA/p65, and E2A all contribute to activating the receptor editing pathway (Pelanda and Torres 2006). It will be important to understand better the ways in which tonic activity of cellular BCR suppresses receptor editing and to determine if this contributes to the mechanism underlying the all too frequent development of autoreactive B cells in both tissue specific and systemic autoimmune diseases. More information concerning the molecular basis of autoimmunity may be found in chapters 11 and 12.
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Chapter 1 The Molecular Basis of Lymphoid Architecture in the Mouse Carola G. Vinuesa and Matthew C. Cook
I. II.
III.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primary Lymphoid Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Bone Marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Bone Marrow Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Cellular Organization of the Bone Marrow . . . . . . . . . . . . . . . . . . . C. Thymus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Thymus Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. T Cell Compartments within the Thymus . . . . . . . . . . . . . . . . . . . . 3. Early Thymus Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Signals Guiding Epithelium-Thymocyte Interactions in Early Thymus Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. T Cell-Epithelium Interactions during Thymocyte Selection . . . . . Secondary Lymphoid Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Secondary Lymphoid Organs Are Crucial for Immune Responses . . . . B. Overview of Secondary Lymphoid Organs . . . . . . . . . . . . . . . . . . . . . . C. Molecular Regulation of Secondary Lymphoid Organ Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Overview of Lymph Node and Mucosal Lymphoid Organ Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Immediate Tumor Necrosis Factor Family . . . . . . . . . . . . . . . . a. Receptors and Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Signaling Events Downstream of TNFRSF . . . . . . . . . . . . . . . . . 3. Ligation of LTβR during Organogenesis . . . . . . . . . . . . . . . . . . . . . 4. Lymphoid Tissue–Inducing Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Induction of LTα1β2 on Lymphoid Tissue–Inducing Cells . . . . . . . 6. Signaling Mutations Affecting Early Lymphoid Organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Tumor Necrosis Factor Immediate Family, Chemokine Transcription, and Lymphocyte Recruitment . . . . . . . . . . . . . . . . . . D. Spleen Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Microenvironments in Secondary Lymphoid Organs . . . . . . . . . . . . . . . . . . A. Normal B Cell Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Lymphatic Drainage to the Secondary Lymphoid Organs . . . . . . . . 2. Entry into and Exit from Lymph Nodes and Peyer’s Patches . . . . . 3. Entry into and Exit from the Spleen . . . . . . . . . . . . . . . . . . . . . . . . . B. Microdomains in Secondary Lymphoid Organs . . . . . . . . . . . . . . . . . . 1. B Cell Zones in Secondary Lymphoid Organs . . . . . . . . . . . . . . . . . a. Follicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Chemokine Regulation of B Cell Migration to Follicles . . . . . . c. Marginal Zone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. B-1 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. T Cell Zones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Homeostasis and Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. B Cell Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Competition for Entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. B Cell Extrinsic Signal: BAFF and B Cell Homeostasis . . . . . . . . . 3. B Cell Intrinsic Signals: B Cell Receptor Signaling and B Cell Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. B Cell Receptor Signaling Thresholds . . . . . . . . . . . . . . . . . . . . b. NF-κB Activation via the B Cell Receptor . . . . . . . . . . . . . . . . . c. Negative and Positive Selection of B Cells . . . . . . . . . . . . . . . . . d. Integration of BAFF, B Cell Receptor, and Chemokine Signals: Survival versus Location . . . . . . . . . . . . . . B. Differentiation of Peripheral B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Transitional B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Marginal Zone versus Follicular B Cells . . . . . . . . . . . . . . . . . . . . . 3. B-1 Cell Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Immune Response Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overview of Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Structural and Molecular Architecture of Immune Responses . . . . . . . 1. T Cell Activation and Differentiation . . . . . . . . . . . . . . . . . . . . . . . . a. Dendritic Cell-T Cell Interactions, Formation of Immunological Synapses, and T Cell Priming . . . . . . . . . . . . . . b. Effector T Cell Subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. T Cell Memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Effector B Cell Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. B Cell Responses in Secondary Lymphoid Microenvironments: T Cell–B Cell Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Extrafollicular B Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . a. Kinetics and Architecture of the Extrafollicular B Cell Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Molecular Drivers of Extrafollicular Responses . . . . . . . . . . . . . 2. Molecular Regulation of Germinal Centers . . . . . . . . . . . . . . . . . . . a. Overview of the Kinetics and Architecture of Germinal Center Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Molecular Signals That Establish the Stromal Support for Germinal Centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Molecular Signals during Germinal Center B Cell Differentiation after Immunization . . . . . . . . . . . . . . . . . . . . . . . d. Cell-Intrinsic Regulation of Germinal Center B Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. Molecular Regulation of Somatic Hypermutation . . . . . . . . . . . . f. Selection of Germinal Center B Cells . . . . . . . . . . . . . . . . . . . . . g. Terminal Differentiation of Centrocytes and Involution of Germinal Centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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76 76 76 76 77 78 78 78 78 78 81 82 82 82 82 82 83 83 84 84 85 85 85 85 86 87 87 88 88 88 89 90 91 91 92 92 93 93 93 93 94 94 94 95 96 96
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1. THE
MOLECULAR
I.
BASIS
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ARCHITECTURE
INTRODUCTION
The mammalian immune system uses numerous strategies to fulfill its principal function of defense against pathogens. At the most fundamental level, each strategy involves an antigen recognition event followed by an effector event. Receptors on cells, as well as circulating host defense molecules, recognize antigens on pathogens, and then through sophisticated processes that frequently involve the cooperation of numerous cell types, the pathogen is eliminated or is at least compartmentalized to prevent ongoing tissue damage. The nature of the receptors used by the immune system for antigen recognition changed dramatically during the evolution of jawed vertebrates from agnathia; compared with lower organisms, the diversity of the repertoire in all jawed vertebrates is enormous. This development came about when cells of the immune system (B and T lymphocytes) acquired the capacity to randomly rearrange gene segments that encode their antigen receptors and marked a transition from an immune system comprising a relatively small number of receptors that recognized common molecular motifs present on pathogens but not on host tissue to a system in which virtually any antigenic structure could be recognized in a specific manner. Massive stochastic diversification of the receptor repertoire has two crucial implications. First, any antigen-specific lymphocyte is a rare cell within a vast repertoire. Second, receptors emerge that are specific for host molecules. Numerous strategies appear to have evolved concomitantly to meet these challenges. Mechanisms that deal with the risk of self-reactivity, in large part, take place during the development of lymphocytes in the primary lymphoid organs. The ontogeny of lymphocytes in the bone marrow and thymus has been resolved to fine detail, and it is clear that the molecular events regulating receptor diversification and selection according to the likelihood of self-reactivity are coregulated. Equally complex is the sophisticated networks of communication between different components of the immune system that are triggered by infection or immunization. Effective immune responses depend on cooperation between rare antigen-specific cells, and these take place within secondary lymphoid organs. Given the rarity of cells specific for any particular antigen, it is not surprising that the organization and structure of these organs is highly sophisticated and carefully regulated. The sophistication and diversity of defense strategies has been driven by the selective pressure exerted by co-evolving pathogens, whose survival depends on evading host defense mechanisms. Once activated, antigen-specific cells undergo clonal expansion to generate sufficient cells to mount an effective immune response. During and after this phase of expansion, differentiation occurs, resulting in cells that are better able to carry out effector functions for silencing or eliminating pathogens. In most cases, this phase of differentiation takes place in secondary lymphoid organs as well, but the structures that form to
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generate an effector population are usually transient, and they dissipate once the threat has been eliminated. In this chapter we outline the current state of knowledge of the molecular events that regulate the development and maintenance of each of these environments.
II.
PRIMARY LYMPHOID ORGANS A.
Overview
Both T and B cells differentiate from hematopoietic precursors and then undergo receptor rearrangement, resulting in the establishment of a diverse repertoire of lymphocytes capable of meeting most of the antigenic challenges posed by the normal environment of the host. Receptor diversification is a stochastic process that is largely independent of antigen, although signals delivered through the antigen receptor play an important role in regulating lymphocyte development and shaping the repertoire. The antigen-independent phase of lymphocyte development takes place in primary lymphoid organs. Under normal circumstances, mature cells from the recirculating population only account for a minority of the cellular constituents of primary lymphoid organs, and immune responses to antigen do not occur here. There is some variation in the location of primary lymphoid organs between species. For example, B cells are produced in the bursa of Fabricius in chickens (Cooper et al. 1966), in Peyer’s patches (PPs) in sheep (Reynaud et al. 1991), and in the kidney and pancreas in zebra fish (Danilova and Steiner 2002). In adult mice, as in adult humans, B cells develop in the bone marrow (Osmond and Everett 1964), and T cells develop in the thymus (Miller 1961). Ontogeny partially replicates phylogeny: For a brief period, pools of hematopoietic cells accumulate in an ill-defined region along the anterior of the early mouse embryo. Next, blood islands that give rise to all hematopoietic lineages are found in the yolk sac (Moore and Metcalf 1970), and then hematopoiesis moves to the liver, which develops as an outpouch of the gut endoderm (Owen et al. 1974). Late in gestation, hematopoiesis takes place in the spleen and continues here until 2–4 weeks of age, overlapping with the shift to the bone marrow, where it continues throughout adult life (Hardy and Hayakawa 2001).
B.
Bone Marrow
In the mouse, the bone marrow is the principal hematopoietic organ, giving rise to all blood lineages. It is located in the medullary spaces of the long bones, and the spongy medullary bone of the vertebral bodies, sternum, and ribs. Blood cells differentiate from pluripotent precursors, and hematopoiesis
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continues throughout life. Thus, the bone marrow is a dynamic environment, with substantial cellular turnover, and hosts a remarkable spectrum of cellular differentiation. Clearly, only a subset of the total bone marrow activity is captured by the term primary lymphoid organ, as it refers specifically to the production and differentiation of B cells. The bone marrow also harbors a substantial component of the long-lived plasma cell population generated after contact with antigen in the secondary lymphoid organs (see “Normal B Cell Migration”) (Manz et al. 2005). In this respect it also functions as part of the secondary lymphoid organ system. Like all lymphoid tissues, the bone marrow can undergo hypertrophy during times of intense activity. However, the location of the bone marrow within bony cavities imposes an absolute physical restraint on the extent of hypertrophy. Nevertheless, there is considerable reserve for expansion, since up to 80% of the bone marrow in adult mice comprises adipose tissue, which can be replaced by hematopoietic elements (Prockop 1997). When demand for new cells in the periphery exceeds the productive capacity of the bone marrow, hematopoiesis also takes place in extramedullary sites, usually recapitulating ontogeny by occurring in liver and spleen. Extramedullary hematopoiesis can also be observed when the bone marrow cavity is encroached upon, either by the bone itself (e.g., osteopetrosis), or by nonhematopoietic infiltration (e.g., malignancy). During B cell development, several key events take place. First, the B cell population expands dramatically from a small number of precursors into a very large population of immature B cells, which emigrate to the periphery to compete for entry in the recirculating repertoire of naïve mature B cells (Hardy and Hayakawa 2001). Second, within each developing B cell, genes encoding the variable regions of immunoglobulin heavy-chain (IgH) and light-chain (IgL) molecules undergo stochastic rearrangements to diversify the repertoire of available receptors, resulting in an estimated 1011 specificities (Davis and Bjorkman 1988). Third, the repertoire of B cells is modified to eliminate or silence those lymphocytes that express antigen receptors (surface Ig) that bind self-antigen with high affinity. Several mechanisms operate to prevent self-reactivity, referred to collectively as self-tolerance. 1.
Bone Marrow Structure
As there is no lymphatic supply or drainage from the bone marrow, cellular traffic enters and leaves exclusively via the bloodstream. Arteries perfuse the bone marrow and empty directly into venous sinuses, a loose and interconnected network of blood-filled spaces. The bone marrow is organized centripetally, with the earliest precursors located closest to the bone, and the most mature elements adjacent to the medullary cavity (Lord et al. 1975). Hematopoietic cell precursors are supported by a stroma of cellular and noncellular elements. Once mature, cellular elements dislodge from the cellular marrow to enter the circulation via the venous system.
2.
G.
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COOK
Cellular Organization of the Bone Marrow
Early experiments in which lethally irradiated mice were reconstituted with limiting numbers of bone marrow-derived cells established the presence of pluripotent hematopoietic precursors (hematopoietic stem cells [HSCs]) that give rise to all blood cells in the periphery, including lymphocytes (Wu et al. 1968). Furthermore, passage of these cells through more than one animal established that these HSCs were self-renewing. Once lineage markers became available, HSCs were enumerated and found to represent between 0.1 and 0.05% of the bone marrow cellular content (Spangrude et al. 1988). Clonal analysis of these cells demonstrated that as few as 10 HSCs can give rise to all hematopoietic elements in vivo (Smith et al. 1991). Stromal cultures have recapitulated several phases of early B cell ontogeny in vitro. HSCs differentiate from mesoderm via a still poorly understood process that depends on Wnt, Hedgehog, and Notch signaling pathways. Once generated, Wnt signaling via the canonical β-catenin pathway (see “Thymus”) is crucial for HSC renewal (Reya et al. 2003; Staal and Clevers 2005). B lineage cells account for approximately 30% of the nucleated cells in mouse adult bone marrow (Picker and Siegelman 1999). HSCs differentiate to common lymphoid precursors (interleukin [IL]-7Rα+, c-kitlo), which account for about 1 in 3000 bone marrow cells, and can give rise to B, T, and natural killer (NK) cells (Kondo et al. 1997). Lymphoid commitment depends on expression of Ikaros and Aiolos (Georgopoulos et al. 1994; Wang et al. 1998). Several key B cell lineage genes, including Rag1, TdT, λ5, and VpreB are regulated by Ikaros. Commitment to B lineage differentiation is signaled by initiation of rearrangement of the variable (V), diversity (D), and joining (J) segments of DNA that encode the IgH genes [V(D)J rearrangement] and intranuclear expression of terminal deoxynucleotide transferase, which inserts nontemplated nucleotides at VH segment joints. B lineage commitment depends on expression of the transcription factors E2A, EBF, and low level expression of PU.1 (Bain et al. 1994; DeKoter and Singh 2000). Expression of high levels of PU.1 leads to myeloid commitment. E2A is under the negative control of Notch1, which regulates T and B commitment; Notch1 inhibits E2A expression, biasing lymphoid development toward T and NK cells, blocking B cell development. Expression of EBF and E2A leads to upregulation of Igα/β, VpreB, λ5 and RAG1/2 (O’Riordan and Grosschedl 1999). Subsequent phases of B cell ontogeny are punctuated by events in IgH and IgL rearrangement. A detailed description of V(D)J recombination is beyond the scope of this chapter (for recent reviews, see Gellert 2002; Jung and Alt 2004), but an outline sufficient to understand the architectural arrangement of B cell development in the bone marrow is provided here. D-J rearrangement takes place in the earliest precursors (pro-B cells), followed by V-D rearrangement to complete assembly of a µ heavy-chain gene. This associates with a surrogate invariant
1. THE
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light chain (λ5 and Vpre-B) enabling expression of a modified surface Ig on the pre-BI cell. After successful expression of the pre-B receptor, pre-B cells enter the cell cycle and undergo considerable expansion as large pre-BII cells. During this phase, V-J recombination of IgL genes takes place, culminating in assembly and expression of mature IgM on the surface of immature B cells. V(D)J recombination is dependent on the activity of the recombinase-activating genes (rag-1 and rag-2), whose expression is tightly regulated during each phase of Ig gene rearrangement. The majority of mature lymphocytes express only one species of antigen-specific receptor (one lymphocyte–one receptor rule; Nossal and Lederberg 1958). In B cells, this is accounted for by the sequential rearrangement of each IgH and IgL allele and the cessation of V(D)J rearrangement upon expression of a functional Ig molecule, so-called allelic exclusion (Mostoslavsky et al. 2004). However, in addition to the assembly of a functional receptor, comprising a heterotetramer of two heavy and two light chains and the capacity to associate with the signaling apparatus proximal to the inner cell membrane, a second constraint is imposed on developing B cells, which is to minimize self-reactive cells from entering the periphery. These twin constraints, quality control of the diversification process and maintenance of self-tolerance, are reflected in the dynamics of cellular proliferation, cell death by apoptosis, and V(D)J rearrangement during B cell development in the bone marrow. A detailed account of the mechanisms of self-tolerance that operate during B cell development in the bone marrow is also beyond the scope of this chapter (reviewed in Ait-Azzouzene et al. 2004; Hardy and Hayakawa 2001). In brief, once an intact IgM is expressed on the earliest immature B cells, ligation with antigen triggers reactivation of rag-1/2, and another round of V(D)J recombination takes place, which has the potential to alter the specificity of the receptor expressed by that B cell and rescue it from deletion. This process is referred to as receptor editing (Casellas et al. 2001; Nemazee 2000). However, if late immature B cells bind antigen with high avidity (e.g., to cell surface–bound antigen), then they undergo apoptosis. Lowavidity binding to antigen (e.g., to soluble antigen) can result in unresponsive and short-lived cells that are referred to as anergic B cells (Goodnow et al. 1988). Studies tracking B cell ontogeny in situ, based on the expression of markers of differentiation have confirmed that the architectural arrangement of B lymphopoiesis is the same as that for other hematopoietic lineages. The earliest precursors (pro-B cells) are located close to the endosteum in association with reticulum/stromal cells, and the most mature precursors (immature B cells) are found adjacent to the venous sinusoids (Hermans et al. 1989; Jacobsen and Osmond 1990). Intermediate phases are characterized by substantial proliferation and cell death. Cell death is triggered by either failure to assemble an effective Ig molecule or high-avidity ligation with antigen. Thus, apoptosis peaks at the pro-B/pre-B transition and again in immature B cells (Deenen et al. 1990), probably accounting
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for the population of macrophages containing large amounts of apoptotic debris that are located in regions of B cell lymphopoiesis in the bone marrow (Osmond and Everett 1962). Numerous B cell intrinsic defects have been identified that arrest B cell ontogeny in the bone marrow. These include defects affecting the assembly of the surrogate receptor or the mature receptor or the B cell signaling apparatus, defects of V(D)J recombination, and defects of B cell receptor (BCR) signaling (summarized in Table 1-1). In addition, a number of defects have been identified in transcription factors that operate B cell intrinsically to disrupt B cell differentiation. The sophisticated spatiotemporal organization of B cell precursors within the bone marrow, which are established in microenvironments separate from those of other lineages, implies specific interactions between cells at different stages of ontogeny and the bone marrow stroma. It seems likely that chemokine gradients exist from the endosteum to the medullary cavity, although these are not well characterized. On the other hand, adhesion molecules that regulate hematopoietic–stromal cell interactions once B lineage differentiation is underway have been identified and include hyaluronate and VCAM-1
TABLE 1-1
B CELL-INTRINSIC DEFECTS THAT BLOCK DEVELOPMENT IN THE BONE MARROW Defect Transcription factors Ikaros E2A Early B cell factor Pax-5 SOX-4 Oct-2 OBF-1 Inhibitors of B cell development PU.1 Notch-1 Defects of the antigen receptor λ5/Vpre-B µ Hc Defects of V(D)J recombination RAG DNA-PKcs Artemis Defects of BCR signaling Ig-β PI3K Btk BLNK Syk Lyn Frizzled9
B Cell Arrest
Reference
No lymphocytes Pro-B Pro-B Pro-B Early pre-B Immature/B-1 cells Immature/B-1 cells
(Georgopoulos et al. 1994) (Bain et al. 1994) (Bain et al. 1994) (Urbanek et al. 1994) (Schilham et al. 1996) (Corcoran et al. 1993) (Kim et al. 1996)
No B cells No B cells
(Scott et al. 1994) (Wilson et al. 2001)
Pro-B Pro-B
(Kitamura et al. 1992) (Kitamura et al. 1991)
Pro-B Pro-B Pro-B
(Mombaerts et al. 1992) (Blunt et al. 1995) (Bassing et al. 2002)
Pro-B Pro-B Pro-B Pro-B Pro-B Immature B Pre-B
(Gong and Nussenzweig 1996) (Fruman et al. 1999) (Middendorp et al. 2002) (Xu et al. 2000) (Turner et al. 1995) (Hibbs et al. 1995) (Ranheim et al. 2004)
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(vascular cell adhesion molecule) on stroma, which ligate CD44 and VLA-4, respectively, on B cells (Miyake et al. 1991). The stroma also produces growth factors essential for pro/pre-B cell survival and expansion, including IL-3, IL-7, IL-11, granulocyte colony-stimulating factor, granulocyte–macrophage colony-stimulating factor, c-kit, and insulin-like growth factor-1 (Chaplin 2003). IL-7 is particularly important for survival and proliferation of pro- and pre-B cells (Morrissey et al. 1991; Peschon et al. 1994). Deficiency of the α chain of the IL-7 receptor (IL-7R) (which is unique to the IL-7R) results in a severe defect in T and B cell development. By contrast, deficiency of the IL-7R common γ chain causes a much milder defect. This is because thymic stromal-derived lymphopoietin (TSLP) also ligates a receptor that contains IL-7Rα (Ray et al. 1996). The centripetal organization of B cell ontogeny, from endosteum to marrow lumen, is likely to depend on chemokine expression and differential expression of adhesion molecules, although the fine details of this arrangement are not well understood. One chemokine known to be expressed by bone marrow stroma and in endothelium of bone marrow vessels is CXCL12, which is crucial for the recruitment and maintenance of HSCs and B cell progenitors, which express CXCR4 (Wright et al. 2002). CXCR4 appears to be necessary to maintain both B lineage and granulocyte lineage precursors in the marrow during normal development (Ma et al. 1999). CXCR4 is downregulated when B cells reach maturity, enabling egress from the bone marrow and migration to the periphery (Nie et al. 2004).
C.
Thymus
Unlike all other hematopoietic lineages that develop in the bone marrow from early life, T cells develop outside of the bone marrow in the thymus (Miller 1961). The key events during T cell development are similar to those described for B cell development in the bone marrow. The T cell repertoire is diversified by stochastic V(D)J recombination of T cell receptor (TCR) genes, and selection takes place to minimize the emergence of self-reactive T cells into the peripheral compartment (Gellert 2002; Jung and Alt 2004). Diversification of the T cell repertoire by V(D)J recombination shows close parallels with diversification of the B cell repertoire in the bone marrow. First, the TCRβ chain locus undergoes recombination, and the nascent receptor is expressed with an invariant surrogate light chain (pre-TCRα), analogous to recombination and quality control of IgH gene recombination in B cells. Successful expression of the pre-TCR leads to thymocyte expansion and activation of TCRα chain locus recombination, analogous to IgL recombination in B cells, resulting in expression of a mature TCR on CD4+ CD8+ (double-positive [DP]) T cells. The most significant difference between T and B cell development hinges on the way T cells interact with antigen. Unlike B cells, which bind intact free antigen via surface Ig, T cells bind to antigenic peptides presented by major histocompatibility
G.
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complex (MHC) class I and class II molecules on the surface of other cells (Doherty and Zinkernagel 1975). In the thymus, MHC molecules are expressed by hematopoietic dendritic cells (DCs) and thymic epithelial cells (TECs). During T cell development the repertoire is modified from a population of cells that expresses a random array of TCRs into one that at once shows a preference for the MHC molecules of the host (positive selection) but not for self-antigens presented in the context of these self-MHC molecules (negative selection). The details of how the repertoire is so modified have been one of the two key conundrums of thymus biology and remain incompletely resolved. The details of positive and negative selection are well beyond the scope of this chapter (for a recent review, see Starr et al. 2003). The second conundrum arises from the observation that, in the periphery, each mature T cell exhibits a preference for binding to antigenic peptides in the context of either MHC class I or class II, determined by their expression of either CD8+ or CD4+, which stabilize interactions between TCR and the respective antigen/MHC complexes. It remains unclear whether differentiation of DP thymocytes into either CD4+ or CD8+ single-positive (SP) T cells in the thymus is determined by the affinity of a T cell affinity for antigens presented by either MHC class I or II (instruction model) or whether differentiation into CD4+ or CD8+ cells occurs stochastically irrespective of the affinity of its TCR for class I or class II, and selection follows (stochastic model) (reviewed in Germain 2002). It has been shown recently that the zinc finger transcription factor Th-POK (T-helper-inducing POZ/Kruppel-like factor) is a master regulator of CD4/CD8 lineage commitment: Th-POK overexpression drives commitment toward the CD4 lineage whereas a point mutation in this transcription factor redirects class II–restricted thymocytes toward the CD8 lineage (He et al. 2005). 1.
Thymus Structure
The thymus is a bilobed, solid organ located in the superior mediastinum, adjacent to the pericardium. The thymus is surrounded by a thin connective tissue capsule, which invaginates as septae that carry arteries, veins, efferent lymphatics, and nerves and divide the thymic parenchyma into lobes. There are no afferent lymphatics entering the thymus and, indeed, the importance of efferent lymphatics for egress of newly formed T cells remains uncertain (Kato 1988). From the outside in, the thymic parenchyma is divided into the subcapsular zone, which contains the earliest T cell precursors, the lymphocyte-dense cortex, and the epithelium-rich medulla (Figure 1-1A). Exchange of cellular and soluble components between the circulation and the thymus takes place at the corticomedullary junction, where arteries leave the septae, and venous sinuses form (Kendall 1989). Non-T cell hematopoietic elements, which represent a small but significant fraction of the nucleated cellular content of the thymus, are located in the perivascular spaces of the corticomedullary junction.
1. THE
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Cortex
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ARCHITECTURE
Medulla
A
White pulp
Follicle T zone MZ
Central arteriole
B Medulla Germinal center
Paracortex (T zone) Follicle (B zone)
C
Subcapsular sinus
Fig. 1-1 Histology of thymus (A), spleen (B), and lymph node (C), showing the principal microenvironments. (Thymus, 20×, spleen and lymph node 10× magnification; hematoxylin and eosin.) (See color insert in the back of the book.)
In the mature thymus, several types of epithelium can be identified. First, the subcapsular perivascular epithelium, which separates the cellular components from the vascularized mesenchymal stroma (capsule). The subcapsular epithelium is not in intimate contact with developing lymphocytes, and its function remains uncertain. By contrast, cortical epithelial cells (CECs) and medullary epithelial cells (MECs) are located in close contact with T cells in the cortex and corticomedullary junction/medulla, respectively (Chaplin 2003). Both CECs and
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MECs express high levels of MHC class I and II molecules. The interactions between these epithelia and T cells are crucial for positive and negative selection (see later). CECs have a dendritic morphology, providing extensive cell membrane contact with thymocytes; each cortical epithelial cell is thought to make contact with approximately 200 T cells (de Waal Malefijt et al. 1986). This interaction contributes significantly to thymocyte positive selection. MECs make fewer contacts with thymocytes and are important for negative selection of T cells. Finally, epithelial cells forming keratinized concentric epithelial structures (Hassall’s corpuscles) are found within the thymic medulla. The function of these structure is uncertain, but they might contribute to removal of apoptotic cells (Douek and Altmann 2000). 2.
Red pulp
IN
T Cell Compartments within the Thymus
T cell precursors are derived from HSCs in the bone marrow, which emigrate to the thymus. Although these precursors exhibit evidence of differentiation from HSCs, they have no discernible T cell differentiation markers. Furthermore, adoptive transfer of the earliest thymocyte precursors can give rise to B cells, NK cells, DCs, and T cells (Antica et al. 1994; Shortman and Wu 1996). Thus, whether or not prothymocytes are committed to T cell differentiation has been the subject of controversy. This issue appears to have been resolved by studies of the Notch1 signaling pathway, which is crucial for the T versus B cell fate decision (see earlier discussion) (Pui et al. 1999; Radtke et al. 1999). Notch1-deficient mice show a severe T cell development defect, with B cells colonizing the thymus. Conversely, constitutive expression of Notch1 results in B cell deficiency with emergence of DP T cells in the bone marrow. Hematopoietic progenitors of T cells from fetal liver express Notch1, but this does not transmit a signal until they enter the thymus, where they bind to Delta-like 1 (a Notch1 ligand) expressed by TECs (Harman et al. 2003). Thus, it is only after entering the thymus that prothymocytes become committed to T cell differentiation. After entering the thymus near the corticomedullary junction (CMJ), the early progenitors proliferate and migrate to the subcapsular region and then move inward again toward the medulla as they mature further. Subsets of thymocytes have been defined according to their stage in ontogeny based on expression of CD4 and CD8: Development proceeds from double negative (DN) to DP and then to SP. DN cells are further categorized into four subsets (DN1–DN4) based on expression of CD44 and CD25 (Godfrey and Zlotnik 1993). Each of these subsets occupies different microenvironments within the thymus (Lind et al. 2001). Thus, T cell differentiation is compartmentalized as thymocytes follow a pathway of orderly migration: from the CMJ to the subcapsular region, where cells have completed TCRβ gene rearrangement, express pre-TCRs, and are DN, and then from the cortex to the CMJ, where cells have also rearranged TCRα genes, thus express intact TCRs, and are DP.
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TABLE 1-2
CHEMOKINES AND CHEMOKINE RECEPTORS Receptor
Ligand
CCR1
CCL3 (MIP-1a), CCL5 (RANTES), CCL7 (MARC), CCL8 (MCP-2), CCL9 (MCP-2) CCL2 (MCP-1), CCL7 (MARC), CCL8 (MCP-2), CCL12 (MCP-5) CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL11 (eotaxin) CCL17 (TARC), CCL22 (ABCD-1) CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL8 (MCP-2), CCL11 (eotaxin) CCL20 (LARC) CCL19 (ELC), CCL21 (SLC) CCL1 (TCA-3) CCL25 (TECK) CCL27 (CTACK), CCL28 (MEC) CXCL6, CXCL8 (IL-8) CXCL9 (Mig), CXCL10 (CRG-2), CXCL11 (I-TAC) CXCL12 (SDF-1) CXCL13 (BLC) XCL1 (lymphotactin) XCL1 (lymphotactin)
CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CCR9 CCR10 CXCR2 CXCR3 CXCR4 CXCR5 XCR1 XCL2
Positive selection is accompanied by migration to the medulla and downregulation of either CD4 or CD8 to become SP T cells. Changes in expression of chemokine receptors are likely to orchestrate the movement of thymocytes through their microenvironments (Chaffin et al. 1990; Kim et al. 1998) (Table 1-2). There is differential expression of several chemokine receptors by thymocytes at different stages of development, whereas their ligands exhibit different but significantly overlapping patterns of expression, suggesting that changes in receptor expression during ontogeny and integration of more than one chemokine stimulus regulate thymocyte positioning. At present, there are some conflicting experimental findings, so only an approximation of chemokine-guided thymocyte migration is possible at this time. Cortical migration of DN thymocytes (from the CMJ) depends on CXCR4 expression. CXCR4-deficient thymocyte development is arrested at DN1 (Plotkin et al. 2003). CCR7deficient thymocyte development is arrested during the transition from DN1 to DN2, causing a general reduction in thymic cellularity and an accumulation of DN cells in the CMJ (Misslitz et al. 2004). After migration to the cortex, ligation of pre-TCRs on cortical thymocytes leads to induction of CCR9, and this remains upregulated during positive selection of DP thymocytes. CCR9 binds to CCL25 in medulla and cortex. (Norment et al. 2000; Wurbel et al. 2000). During positive selection, migration across the corticomedullary junction is accompanied by expression of CCR4, CCR7, and CCR8 and responsiveness to the medullary chemokines CCL22 (MDC), CCL17 (TARC), CCL19, and CCL1 (Campbell et al. 1999; Kwan and Killeen, 2004). CCR7 expression by most mature
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populations of CD4+ and CD8+ SP T cells prepares them for recruitment to the T zones of secondary lymphoid organs (see “T Cell Zones”). Abrogation of CCR7 signaling or ligation impairs but does not prevent migration of mature T cells to the periphery (Ueno et al. 2002). Similarly, premature overexpression of CCR7 on DP thymocytes does not result in their premature egress from the thymus (Kwan and Killeen 2004). In other words, the key signals that control migration out of the thymus remain to be determined. 3.
Early Thymus Development
Unlike bone marrow and secondary lymphoid organs, in which the stroma is of mesenchymal origin, much of the thymic stroma is of epithelial origin. Thymic epithelium is derived from foregut endoderm, specifically from evaginations of the lateral third pharyngeal pouches. Thymic ectoderm is derived from corresponding evaginations of the third branchial clefts. Back-to-back association of ectoderm and endoderm form the pharyngeal membrane, which descends in the neck and is surrounded by mesenchyme derived from the cranial neural crest to form the thymic anlagen on embryonic (E) days 10–11 (Le Lievre and Le Douarin 1975) (Figure 1-2). Several transcription factors have been found to be crucial for the development of pharyngeal organs (thymus, thyroid, parathyroids, and ultimobranchial bodies). Inactivation of the Hoxa3 gene, which is expressed in both the endoderm of the third and fourth pharyngeal pouches and neural crest mesenchyme, results in aparathyroidism athymia, and persistence of the ultimobranchial bodies (Manley and Capecchi 1995). Disruption of Pbx-1 impairs patterning of the caudal pharyngeal pouch, resulting in reduced proliferation of the thymic epithelium (Manley et al. 2004). A network of transcription factors—Pax1-Eya1-Six1—known to act cell autonomously during the development of the eye in Drosophila, has also been shown to be essential for thymus development. Inactivation of Pax9 results in early failure of thymus, parathyroid, and ultimobranchial body formation, with failure of the thymic primordium to invaginate into the mesenchyme (Peters et al. 1998), whereas Pax1 mutants have hypoplastic parathyroids and thymus and perturbed thymocyte maturation (Wallin et al. 1996). Disruption of the mammalian homolog of Drosophila absent eyes-1 (Eya1), which is expressed in the third branchial arch, pouch, and pharyngeal arch, as well as in cranial sensory placodes, results in athymia (Xu et al. 2002). Increased cell death of surface ectoderm of the pharyngeal region in Eya1−/− mice indicates that Eya1 is necessary for ectodermal cell survival. In Eya1 mutants, Six1 (sine oculis-related homeobox-1 homolog) expression in pharyngeal arch mesenchyme, pouch endoderm, and surface ectoderm is markedly reduced, and Gcm2 (glial cells missing homolog 2) expression in the third pouch endoderm is undetectable. Subsequent studies have demonstrated that Six1 mutants also exhibit marked thymic hypoplasia (Laclef et al. 2003).
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Endoderm
Mesoderm
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Hoxa3 Pax1, 9 Eya1 Six1
Hoxa3 Six1 Eya1
Notch1 Thymocyte Precursors (fetal liver)
Thymic anlage
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FGFR Epithelium FoxN1
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Wnt Fz
Thymocyte
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B LTα1β2LTβR
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Thymocyte AIRE MEC MHC + Self-antigen TCR
Self-reactive T cell
B Fig. 1-2 Summary of the cellular interactions that take place during thymic organogenesis. A: Mesoderm and endoderm derived from the neural crest and pharyngeal pouches, respectively, meet thymocyte precursors in the thymic anlage. Thymic epithelial differentiation is dependent on Hoxa3, Pax, Six1, and Eya1 transcription factors. Thymocyte differentiation depends on ligation of Notch1 within the thymus. B: Two-way interactions between thymic mesenchyme and epithelium results in early differentiation of epithelial cells, which can then support differentiation of early thymocytes, via signals including ligation of Frizzled (Fz), which in turn provide signals that direct further TEC differentiation. C: Signals delivered to MECs via LTβR lead to upregulation of AIRE and expression of self-antigens, which modify the mature T cell repertoire by negative selection.
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Hoxa3, Pax1 and Pax9, Eya, and Six1 are expressed in the pharyngeal pouch endoderm and Hoxa3, Eya1, and Six1 are expressed in the neural crest mesenchyme (Figure 1-2). A model has been proposed in which this axis controls positioning of the thymic rudiment. Gcm2 is expressed in a discrete domain of the pouch endoderm, which may mark the parathyroid-thymus boundary (Blackburn and Manley 2004). A parallel thymus selector determining thymus identity is either yet to be found or could be FoxN1 (see later). An alternative explanation is that the endoderm differentiates into thymic epithelium by default. The mesenchymal component of the stroma is derived from the cephalic neural crest via the pharyngeal arch, which covers the epithelial rudiment on E12, then migrates into the rudiment and surrounds the epithelium by E13. Hematopoietic elements are recruited from outside the thymus rudiment (Le Douarin and Jotereau 1975). Thymocyte progenitors enter from the yolk sac and para-aortic foci before the gland is properly vascularized on E11.5 (Amagai et al. 1995). Although most thymic patterning appears to be determined cell autonomously, positioning and outgrowth of the thymic rudiment are likely to depend on cross-talk between mesenchyme and endodermderived TEC progenitors. In turn, stromal differentiation is necessary to support thymocyte development, and developing T cells provide positive feedback for TEC differentiation. The early migration of mesenchyme (derived from the pharyngeal pouch) establishes a network of epithelium, mesenchyme, and thymocytes (Auerbach 1960). The mesenchyme appears to be instrumental in directing early thymic development, since extirpation of avian neural crest mesenchyme precludes normal thymic development (Bockman and Kirby 1984), and fetal thymic organ cultures lacking mesenchyme fail to support thymocyte development (Anderson et al. 1993; Suniara et al. 2000). However, understanding of the precise role of the three-way cross-talk between mesenchyme, epithelium, and thymocytes is incomplete. Recent evidence indicates that although the mesenchyme is necessary for early TEC survival and proliferation, it does not promote TEC differentiation into CECs and MECs. The origin of TECs remains controversial. One model suggests that cortical TECs are derived from ectoderm and MECs are derived from pharyngeal pouch endoderm (Cordier and Haumont 1980). However, more recent studies suggest that the ectoderm undergoes apoptosis within the thymic anlagen, and all TECs are derived from endoderm (Blackburn and Manley 2004; Gordon et al. 2004). The earliest identifiable TEC precursor expresses the winged-helix transcription factor FoxN1 (winged-helix-nude [Whn]) (Blackburn et al. 1996). The TEC precursors are identified by the surface markers Mts20 and Mts24, and keratin types 5 and 8 (K5+8+), although these are not specific for the TEC precursors and also occur on other structures derived from the pharyngeal pouch. Although Mts 20+Mts24+ cells have been shown to differentiate into K5+8− MECs and K5–8+ cortical TECs (Bennett et al. 2002; Gill et al. 2002; Klug et al. 1998), it remains unclear whether endoderm
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IL7Rα
IL7Rα
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IL-7
IL-7
I
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Jak3 TRAF6
Jak3
LTα1β2
NIK
LTβR
LTα1β 2
LTβR
NF-κB2
LNIC
CXCR5 CCR7
RORγt Id2
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CXCR5 CCR7 CXCL13 CCL19 CCL21
PPIC RORγt Id2
II LTα1β2
LTβR
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MOC
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CXCR5 CCR7
LTα
1 β2
β LTα 1 2
VCAM-1 ICAM-1
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α4β2
CXCR5
NonB cell
CXCR5
B cell B cell
NonB cell LT
αβ 1 2
β2 α1
LT
LT
FDC
αR
LT αR
IV TNFR1
LTα1β2
LTβR
TNFR1
FDC
Fig. 1-3 Cellular interactions during formation of lymph nodes and PPs. Schematic overview of key molecular signals that guide organogenesis of lymph nodes and PPs. These interactions take place in separate secondary lymphoid organs. I, LTICs and PP-inducing cells colocalize with MOCs under the influence of chemokines, including CXCL13, CCL19, and CCL21. LTα1β2 is induced on LTICs by signals through IL-7Rα and RANK and on PPICs via IL-7Rα signals. II, Ligation of LTβR on MOCs induces chemokine secretion, as well as upregulation of adhesion molecules. Chemokine signals delivered to PPICs lead to upregulation of adhesion molecules. III, B cells are recruited and upregulate LTα1β2 and CXCR5. Ligation of LTβR on stromal cells results in a positive feedback loop that augments CXCL13 secretion, which induces further LTα1β2 upregulation. IV, Ligation of LTβR on immature follicular dendritic cells (FDCs) and ligation of TNFR1 promotes FDC maturation that permits patterning of B cell zones in the spleen. In the lymph node (box), induction of FDC maturation requires similar signals, although these appear to be delivered by T and NK cells.
gives rise to two separate MEC and cortical TEC stem cells or whether the same stem cell can differentiate down either pathway. Irrespective of this uncertainty, the medulla appears to develop as a series of clonally derived islets of MEC progenitors, first detected morphologically around E14, that persist until E16.5 and develop independently of the surrounding cortical TECs (Rodewald et al. 2001).
Signals from the mesenchyme are important for early TEC survival and proliferation. The mesenchymal rudiment releases fibroblast growth factor (FGF)-7 and -10, which ligate FGFR2-IIIb on thymic epithelium to stimulate proliferation in the early embryo (Jenkinson et al. 2003), although these mesenchymal signals do not appear to be necessary for epithelial differentiation (into cortical TECs and MECs) after E12
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(Jenkinson et al. 2003). The earliest phase of TEC differentiation appears to be independent of thymocytes as well (Klug et al. 2002). However, once cortical TECs appear, their differentiation is under the influence of differentiating thymocytes (Klug et al. 1998). Signals from DN thymocytes (DN2 and/or DN3) induce the three-dimensional organization of TEC (Hollander et al. 1995a, 1995b). DN3 T cells may be necessary for inducing normal downregulation of IL-7 in cortical TECs (Zamisch et al. 2005). A positive feedback loop appears to operate, since TECs provide trophic support for early thymocyte precursors once they begin to differentiate. One key signal is IL-7, which is expressed by the earliest TEC rudiments, and IL-7R is present on early thymocyte precursors that emerge from the fetal liver (Mebius et al. 2001). 4. Signals Guiding Epithelium-Thymocyte Interactions in Early Thymus Development
Several lines of evidence point to a crucial role for activation of the β-catenin pathway via the wnt/fz/lrp axis in the mesenchymal-epithelial interactions during early thymic development (van de Wetering et al. 2002) (Figure 1-3). Identification of the defect responsible for the nude mouse phenotype (a spontaneous mutant strain of mice that is athymic and has abnormal hair follicle development) has provided important insights into thymus development. Nude mice carry a mutation of the FoxN1 transcription factor gene, which results in involution of the pharyngeal pouch ectoderm on E11.5, curtailing thymic epithelial development, and, as a consequence, the thymic remnant cannot support T lymphopoiesis (Nehls et al. 1994). FoxN1 acts cell autonomously to generate all TECs (Blackburn et al. 1996) and is also crucial for allowing the thymic rudiment to attract prothymocytes. In the absence of FoxN1, prothymocytes accumulate caudally to the thymic rudiment in the vicinity of the parathyroid, apparently as a consequence of defective production of CCL25 (TECK) and CXCL12 (SDF-1) by the thymic anlagen (Bleul et al. 1998). Normally, Wnt glycoproteins in thymic epithelium regulate expression of FoxN1 (Balciunaite et al. 2002). The mammalian genome encodes 19 Wnt (mammalian analogs of Drosophila gene wingless and homologs of mammalian gene Int1) molecules, which are cysteine-rich, secreted glycoproteins. When Wnt molecules ligate their receptors, complexes of frizzled (Fz) family members and low-density lipoprotein receptor–related proteins lead to activation of β-catenin. This pathway has been thoroughly investigated in Drosophila development and in colon carcinogenesis. In unstimulated cells, cytoplasmic β-catenin is complexed with adenomatous polyposis coli, glycogen synthase kinase 3β (GSK3β) and axin, where it is rapidly phosphorylated, making it a substrate for β-TrCP within an E3 ubiquitin ligase complex (Aberle et al. 1997). Consequently, β-catenin is subject to rapid proteosomal degradation. Ligation of Fz/Lrp prevents GSK3β activation by Dishevelled (Noordermeer et al. 1994), inhibiting β-catenin phosphorylation,
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and instead of undergoing proteosomal degradation, β-catenin translocates to the nucleus where it associates with nuclear transcription factors T cell factor (TCF) and lymphoid enhancing factor (LEF) (Pongracz et al. 2003). The link between Wnt signaling and activation of TCF/LEF provided the crucial clue to the significance of this pathway for thymic development, since TCF and LEF were identified through screens for T cell–specific transcription factors (van de Wetering et al. 1991; Waterman et al. 1991). Subsequent studies revealed that several different Wnt molecules are released from thymic epithelium and that several types of Fz are expressed on thymocyte precursors. However, the possibility that Wnt is also released from prothymocytes to induce FoxN1 in the epithelial rudiment cannot be discounted. Other aspects of the transcriptional program regulated by β-catenin/TCF/LEF are beginning to be elucidated. Gene expression analysis suggests that the β-catenin pathway regulates the expression of adhesion molecules that maintain immature thymocytes within their correct thymic microenvironment (Staal et al. 2004). Taken together with evidence from HSCs and B cells, a picture emerges of Wnt/β-catenin pathways as regulating precursor proliferation and location but not differentiation. 5.
T Cell-Epithelium Interactions during Thymocyte Selection
T cells remain in intimate contact with TECs throughout the thymus. However, a detailed understanding of the nature of the trophic support provided to T cells remains to be determined in detail, although IL-7 is crucial. Consequently, at present generation of SP T cells in vitro can only be achieved with thymic explants. Positive selection is thought to be mediated by interactions between DP thymocytes and cortical epithelium (Anderson et al. 1994). MECs mediate the final stages of T cell maturation and contribute to negative selection (Bonomo and Matzinger 1993), although less efficiently than hematopoietic DCs (Burkly et al. 1993; Hoffmann et al. 1992). Adoptive transfer of normal bone marrow into SCID mice restores normal MEC organization (Shores et al. 1991), suggesting a crucial role for thymocytes in inducing differentiation of MECs and maintaining the normal thymic structure necessary for negative selection. More recently, the molecular basis for this interaction has been refined. T cells expressing lymphotoxin (LT) α1β2 ligate the LTβ receptor (LTβR) on MECs, which signals via NIK (nuclear factor-κB [NF-κB]–inducing kinase) and RelB (NF-κB) (Burkly et al. 1995) (see “The Immediate Tumor Necrosis Factor Family” and “Signaling Mutations Affecting Early Lymphoid Organogenesis”). After thymic development, RelB is confined to the medulla (Carrasco et al. 1993). RelB−/− mice exhibit a disorganized thymic medulla and absence of 29+ MECs, and negative selection of thymocytes is compromised (Burkly et al. 1995). Deficiencies of LTβR and NIK exhibit a similar thymic phenotype (Boehm et al. 2003; Miyawaki et al. 1994).
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Recent studies have identified autoimmune regulator (Aire) as a master regulator of transcription that is expressed predominantly in the thymus, where it controls expression of a variety of ostensibly tissue-specific proteins, some of which are the target of autoimmune responses. Mutations of Aire in humans cause autoimmune polyendocrinopathy syndrome type I (1997; Nagamine et al. 1997). Engineered deficiency of Aire in mice results in a dramatic autoimmune phenotype, with inflammatory responses affecting multiple organs (Anderson et al. 2002). The distribution of RelB expression correlates with Aire expression (Zuklys et al. 2000). Furthermore, both LT and LTβR deficiencies result in failure of Aire induction and overt autoimmunity against self-antigens normally protected by Aire. Stimulation of LTβR by agonistic antibody leads to increased expression of Aire and tissue-restricted antigens in intact thymus (Chin et al. 2003). Thus, expression of Aire appears to be under the control of signaling via LTβR, which is triggered by LTα1β2 on T cells. As described in the section on “Secondary Lymphoid Organs,” this interaction is also crucial for the development of secondary lymphoid organs, and it therefore represents a key similarity between the lymphocytestromal interactions in the development of primary and secondary lymphoid organs.
III. A.
SECONDARY LYMPHOID ORGANS
Secondary Lymphoid Organs Are Crucial for Immune Responses
Stochastic diversification of the antigen receptor genes during lymphocyte development in the primary lymphoid organs results in a staggering array of antigen specificities. As a result, lymphocytes specific for any particular antigen represent only a tiny fraction of the repertoire. To generate an effective immune response, these rare antigen-specific clones proliferate, a process called clonal expansion. This takes place in secondary lymphoid organs (McMaster and Hudack 1935). Immune responses are perturbed or even abolished when specialized immune compartments are absent. The importance of afferent lymphatics for initiation of normal immune responses was suggested by early studies (Barker and Billingham 1968). These experiments can now be interpreted as demonstrating the role of migrating DCs for initiation of T cell activation. Other experiments have demonstrated that selective absence of the spleen results in a defective antibody response to encapsulated organisms (Amlot and Hayes 1985). In the absence of all secondary lymphoid organs, normal immune responses are delayed significantly, contact hypersensitivity responses are abolished (Karrer et al. 1997; Rennert et al. 2001) and allografts are tolerated indefinitely (Lakkis et al. 2000).
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Under some circumstances, clonal expansion is triggered by direct interactions between lymphocyte and antigen, but often proliferation of T and B cells depends on collaboration between more than one cell type. For example, antigen-specific T cells interact with antigen-presenting cells during T cell priming. Subsequently, antigen-specific T cells provide cognate help to antigen-specific B cells for B cell clonal expansion in response to protein antigens (see “Effector B Cell Populations”). Within secondary lymphoid organs, lymphocytes segregate into compartments. Nevertheless, most cells within the secondary lymphoid organs are constantly on the move. These two features deserve special emphasis because they help explain how rare encounters between antigen-specific cells occur. Maintenance of lymphoid structure in the face of a dynamic and constantly changing population explains why the signals that coordinate and maintain these structures are so complex. The level of sophistication of secondary lymphoid organs is highlighted by several additional and crucial features. First, the size of the secondary lymphoid organ population remains constant during periods of immune quiescence (see “Normal B Cell Migration”). The population can expand dramatically during an immune response but then returns to normal when the response is over. This occurs despite a constant influx of newly formed lymphocytes from the primary lymphoid organs and loss of cells either by attritional death or recruitment into effector populations. Second, during immune responses, new compartments form within the secondary lymphoid organs. If the immune response is ongoing, then these compartments persist, but if the antigen is eliminated, they dissipate, and the secondary lymphoid organs return to their quiescent organization. Finally, a stochastic process of receptor diversification carries a risk of generation of autoreactive lymphocytes, and if these remain in the repertoire and become activated, they can cause autoimmune disease. Mechanisms have evolved to prevent these self-reactive cells from being recruited into the immune response. Although many of these mechanisms operate in the primary lymphoid organs early in lymphocyte ontogeny, there is evidence that selection favors recruitment of non-self-reactive lymphocytes into the secondary lymphoid organs. Furthermore, because another round of BCR diversification takes place during B cell responses to antigen (somatic hypermutation; see “Effector B Cell Populations”), mechanisms to deal with newly formed but self-reactive B cells in secondary lymphoid organs are of considerable importance.
B.
Overview of Secondary Lymphoid Organs
The secondary lymphoid organs comprise the spleen, lymph nodes, and aggregates of lymphoid tissue associated with mucosal surfaces of the gut and respiratory and reproductive tracts, including PPs in the small intestine (see Figure 1-1). Peripheral lymph nodes are located in clusters throughout the body, usually in close association with bifurcations of the arteries.
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Their location is propitious for surveying antigen exposure of the skin and viscera. Lymph nodes develop as outpouches of lymphatic vessels (Eikelenboom et al. 1978; Wigle and Oliver 1999). As a result, cellular and soluble material enters and leaves lymph nodes via afferent and efferent lymphatics, respectively. The afferent lymphatics arise in parenchymal tissues and collect fluid that has extravasated from the systemic circulation into the interstitium. Foreign antigen can travel to the peripheral lymph nodes via the afferent lymphatics, either in free form or within DCs that phagocytose antigen in the nonlymphoid parenchyma and then migrate via the lymphatics to the lymph nodes (Romani et al. 2001). Peripheral lymph nodes also receive a vascular supply, and naïve lymphocytes enter lymph nodes from the arterial circulation. Lymphocytes exit lymph nodes via the efferent lymphatics moving centripetally, eventually draining via the thoracic duct into the superior vena cava to reenter the systemic circulation. The spleen lacks both afferent and efferent lymphatics. Thus, antigen and lymphocytes enter exclusively via the systemic circulation. Although the spleen is the largest of the secondary lymphoid organs, not all of it has the structure of a secondary lymphoid organ. The spleen is divided into white and red pulp. The white pulp contains the bulk of the lymphoid tissue and is organized similarly to the other secondary lymphoid organs. The red pulp contains blood sinuses lined by specialized macrophages. Several unique features of the splenic white pulp structure (compared with other secondary lymphoid organs) appear to reflect the crucial role the spleen plays in clearing the blood of pathogens and mounting rapid immune responses to virulent bacteria. Various organized lymphoid structures are observed on mucosal surfaces and are referred to collectively as mucosaassociated lymphoid tissue (MALT) (Newberry and Lorenz 2005). The best characterized of these are PPs. Isolated lymphoid follicles and cryptopatches also occur in the gastrointestinal tract, whereas nasal-associated lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT) occur in the respiratory tract. PPs are true secondary lymphoid organs, comprising T cells, B cells, and DCs. However, they do not receive afferent lymphatics. Lymphocytes enter via high endothelial venules and exit via efferent lymphatics, migrating to draining lymph nodes, and then via the thoracic duct to the systemic circulation. Antigen enters directly across the mucosal surface, traversing specialized epithelial cells (referred to collectively as follicleassociated epithelium [FAE]) that bridge the lymphoid aggregates and the lumen by micropinocytosis, endocytosis, or phagocytosis (Bockman and Cooper 1973; Mowat 2003; Owen and Jones 1974). The FAE overlying PPs contains M (microfold) cells (Wolf et al. 1981) identified by Ulex lectin (Clark et al. 1995). Similar cells have also been demonstrated overlying isolated lymphoid follicles (ILFs), cryptopatches, BALT, and NALT. M cells are distinguished from conventional mucosal epithelial cells by their capacity to selectively transport
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macromolecules, viruses and bacteria, and IgA-immune complexes (Neutra et al. 2001). They have an irregular brush border and decreased alkaline phosphatase activity and do not make IgA secretory piece. In PPs, the subepithelial dome lies beneath the FAE, and this is rich in CD11b− CD8α− CD11c+ DCs (Iwasaki and Kelsall 2000). Initiation of immune responses in the PPs depends on the presence of this population, which can migrate to the adjacent T zones of PPs or via efferent lymphatics to mesenteric lymph nodes. Cryptopatches (CPs) are small, loose collections of c-kit+ IL-7R+ Thy1+ cells that lack TCRs and BCRs, DCs, and VCAM-1+ stromal cells (Kanamori et al. 1996). There are virtually no T or B cells present. CPs occur at the bases of intestinal crypts throughout the large and small intestine. Their function remains uncertain. One line of evidence suggests that they are the site of origin of intraepithelial T cells that develop independently of the thymus (Saito et al. 1998). However, the demonstration that intraepithelial T cells are present in mice that lack CPs refutes this hypothesis, and sophisticated cell labeling studies indicate that intraepithelial αβ T cells arise in the thymus (Eberl and Littman 2004; Pabst et al. 2005). It now appears that c-kit+ IL-7R+ Thy1+ cells are adult counterparts of lymphoid tissue–inducing cells (see “Molecular Regulation of Secondary Lymphoid Organ Development”), in which case CPs may be precursors of isolated lymphoid follicles. ILFs are small aggregates of up to 300 cells that occur throughout the intestine (Hamada et al. 2002). B cells account for ~70% of their cellular content with CD4+ T cells, DCs, and IL-7R+ kit+ cells making up the rest. Like PPs, ILFs are covered by FAE, can support germinal center formation, and generate IgA-secreting plasma cells. However, their contribution to total mucosal IgA appears to be much less significant than that of PPs. In the respiratory tract, lymphoid follicles in the lower third of the bronchial tree are part of the systemic immune system, and they bear similarities to PPs and ILFs (Bienenstock and McDermott 2005). They comprise predominantly B cells and are covered by FAE. These aggregates also lack afferent lymphatics, and lymphocytes enter via high endothelial venules (HEVs) via interaction with VCAM-1, and L-selectin (Xu et al. 2003). In the upper third of the respiratory tract, lymphoid aggregates resembling PPs occur, and these are part of the mucosal immune system, since lymphocytes tend to recirculate among these mucosal aggregates (McDermott and Bienenstock 1979). The majority of BALT lymphocytes are B cells expressing IgA and IgM. Rodents also have NALT (Heritage et al. 1997; Kiyono and Fukuyama 2004), which may be analogous to the Waldeyer’s ring (bilateral tubule, palatine and lingual tonsils, and adenoid) that occurs in other mammals including humans but not in rodents. NALT consists of two cylindrical structures at the junction of the soft palate and pharyngeal duct (Heritage et al. 1997). They contain 1–2 × 106 cells, which are organized like PPs into follicles that support germinal center formation, and are covered by FAE. Although the majority of cells are B cells, T cells and DCs occur in parafollicular areas.
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The T cell population includes an unusual regulatory subset that is B220lo, CD3lo, CD4−, CD8−, and TLR (Toll-like receptor)-2+ (Rharbaoui et al. 2005), which is induced during tolerogenic immune responses and drains to cervical lymph nodes. The NALT generates IgA responses, including IgA-producing cells in draining cervical lymph nodes. Germinal centers within NALT are responsible for high-affinity IgA production (Shimoda et al. 2001).
C.
Molecular Regulation of Secondary Lymphoid Organ Development
1.
Overview of Lymph Node and Mucosal Lymphoid Organ Formation
The molecular signals governing embryogenesis of peripheral lymph nodes and PPs have been resolved in reasonable detail, and there appear to be sufficient similarities to consider them together, emphasizing differences where they exist (Cupedo and Mebius 2005; Mebius 2003; Nishikawa et al. 2003). Based on the available evidence, organogenesis is a four-stage process. First, non-T, non-B hematopoietic cells referred to as lymphoid tissue–inducing cells (LTICs) (or specifically, lymph node–inducing cells [LNICs] and PP-inducing cells [PPICs]), migrate to lymph node and PP anlagen where mesenchymal organizer cells (MOCs) are present (Honda et al. 2001). B cells and γδ T cells are recruited at this stage, but no cellular organization into microdomains is apparent, and studies using lymphocyte-deficient mice indicate that the early patterning of lymph nodes and PPs proceeds independently of T and B cells (Hashi et al. 2001). Second, interactions between LTα1β2+ LTICs and LTβR+ MOCs lead to LTα1β2-dependent, NFκB–dependent induction of chemokines that create an environment permissive for follicle formation. Next, B cells upregulate CXCR5, which leads to their upregulation of LTα1β2. Once this maturation event has taken place, B cells, possibly T cells, and LTICs all contribute to a positive feedback loop, resulting in CXCL13 secretion that maintains organization of discrete B- and T cell zones. These phases are elaborated on later and summarized in Figure 1-3. What determines the exact location and number of secondary lymphoid organs remains uncertain. PPs are distributed evenly along the antimesenteric mucosal surfaces of the small intestine, and the number of PPs is remarkably similar in different animals from the same species. Similarly, the location of peripheral lymph nodes is very consistent at sites of venous bifurcation, which correlates to sites where lymph and venous sacs are in close opposition during embryogenesis. This finding suggests that the earliest event in embryogenesis might be a signal emanating from the mesenchyme (as described in the thymus). Consistent with this interpretation, PPICs are distributed diffusely through the intestine until they receive a localizing signal. For both PP and lymph node formation, the key initiating events appear to be induction of LTα1β2 on
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LTICs, and their recruitment to lymphoid anlagen where they deliver a LTα1β2 signal to the mesenchyme. Intestinal CPs depend on similar signals (Taylor et al. 2004). The major constituent of CPs appear to be adult counterparts of LTICs. CPs probably give rise to isolated lymphoid follicles, where the most abundant population is B cells. Recruitment of B cells induces isolated lymphoid follicle formation in LTα/LTβRdependent fashion (Hamada et al. 2002). By contrast, the molecular signals governing formation of BALT and NALT are not well understood. Neither is dependent on LTα, IL-7Rα, NIK, or RORγ (retinoic acid orphan receptor-γ), and they develop in the absence of other secondary lymphoid tissues, although their micro-organization is abnormal in the absence of these signals (Fukuyama et al. 2002; Harmsen et al. 2002). 2.
The Immediate Tumor Necrosis Factor Family
a. RECEPTORS AND LIGANDS The tumor necrosis factor (TNF) superfamily of surface receptors comprises a large group of highly conserved molecules that play important roles in host defense and cellular homeostasis through regulation of apoptosis. TNF superfamily members and their ligands are also key regulators of lymphoid architecture, from the earliest phase of lymphoid organogenesis through to the regulation of lymphoid patterning, coordination of the transient compartments that develop during immune responses, and even in organizing the so-called tertiary lymphoid structures that develop and sustain certain forms of chronic inflammation in nonlymphoid parenchyma. These molecular signals will be discussed in turn. Because interactions between LTα1β2 on LTICs and LTβR on MOCs are crucial for understanding lymphoid organogenesis, a brief outline of these ligand pairs follows. Unlike most cytokines, many TNF family members are expressed in a membrane-bound state, where they form trimers via conserved aromatic residues, which bind to ligand trimers (Locksley et al. 2001). Because heterotrimers can form between different family members, there is extensive flexibility in the system. A subset of this superfamily, the so-called TNF immediate family (TNF, LTα, LTβ, and LIGHT [homologous to LTs, inducible expression, competes with herpes simplex virus glycoprotein D for HVEM (herpes simplex virus entry mediator), a receptor expressed on T lymphocytes]), has been defined based on their close structural similarity, their propensity to assemble into heterotrimers, and their overlapping patterns of receptor-ligand interactions (Schneider et al. 2004) (Figure 1-4). There are two TNF receptors, type I TNF receptor (TNFR1, p55) and type II TNF receptor (TNFR2, p75), which are expressed ubiquitously on cell surfaces and also exist in circulating soluble forms that compete with transmembrane receptors for ligand binding and inhibit cell-to-cell interactions. Expression of transmembrane TNF is limited mainly to macrophages, NK cells, and activated T and B lymphocytes. Transmembrane TNF and LTα both exist as homotrimers
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The immediate TNF superfamily of molecules, their ligands, and their principal signaling pathways. See text for details.
(TNF3 and LTα3) that bind TNFR1 and TNFR2 with similar affinity, although transmembrane TNF appears to preferentially ligate TNFR2 (Grell et al. 1995). LTα is expressed by activated T, B, and NK cells. TNF and LTα also exist as circulating (soluble) cytokines, generated when membrane-bound molecules are cleaved by a specific metalloproteinase (TACE) (Moss et al. 1997). LTβ does not exist as a free or transmembrane ligand but associates with LTα to form two different membrane-anchored heterotrimers: LTα1β2 (the predominant form) and LTα2β1. LTα2β1 binds to TNFR1 or TNFR2, although the biological significance of this interaction remains uncertain. By contrast, LTα1β2 shows no affinity for either TNFR1 or TNFR2, but binds to a distinct cellular receptor designated LTβR (Crowe et al. 1994). Significantly, LTβR is not expressed by lymphoid cells. LIGHT (TNF super family [SF] 14) also binds to LTβR, as well as to HVEM.
More recently, RANKL (receptor activator of NF-κB ligand; also known as TRANCE [TNF-related activation-induced cytokine]) was identified as an inducible TNFSF member, which is crucial for lymph node organogenesis, as well as osteoclast differentiation (Anderson et al. 1997; Dougall et al. 1999; Kong et al. 1999b; Wong et al. 1997). RANKL binds to trimeric RANK (receptor Activator of NF-κB), which is widely expressed and inducible, and the decoy receptor osteoprotegerin, which exists as both a soluble secreted molecule and membrane bound on bone marrow stroma, B cells, DCs, and follicular dendritic cells (FDCs) (Yun et al. 1998). b. SIGNALING EVENTS DOWNSTREAM OF TNFRSF TNFRSF members signal to induce caspase-dependent cell death or activation via TRAFs (TNF receptor–associated factors) adaptor proteins and NF-κB. During lymphoid organogenesis, the latter
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pathway is crucial. NF-κB is one of the most important signaling pathways in the immune system and can be activated by many stimuli (Hayden and Ghosh 2004). These include activators of the innate immune system (TLRs), cytokines, and members of the TNFRSF. NF-κB has the potential to activate hundreds of genes that regulate different aspects of the immune response. The overlapping receptor ligand binding of the immediate TNF family, together with the pleiotropic actions of NF-κB might suggest that there is redundancy in the system. However, results of gene deletion studies argue to the contrary. A combination of tight temporal regulation of expression and limited cellular distribution of receptors and ligands, together with a complex subunit structure of the five NF-κB family members that associate with different receptor SF members, leads to precision and specificity. The NF-κB family consists of RelA (p65), RelB, c-Rel, NF-κB1 (p50 and its precursor p105), and NF-κB2 (p52 and its precursor p100). Normally, NF-κB is maintained in an inactive
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state by inhibitors of κB (IκBs), which neutralize a NF-κB nuclear localization signal. There are seven IκB family members: IκBα, IκBβ, Bcl3, IκBε, IκBγ, and the p100 and p105 precursor proteins. NF-κB activation is triggered by activation of the Iκ kinase (IKK) complex, comprising IKKα, IKKβ, and IKKγ (also known as NEMO [NF-κB essential modifier]), which targets IκBs to ubiquitin ligase machinery and degradation. Freed NF-κB then translocates to the nucleus to bind promoter and enhancer sequences in target genes (Hayden and Ghosh 2004). There are two pathways downstream of TNFRSF members that activate NF-κB (Dejardin et al. 2002; Senftleben et al. 2001). The classic pathway leads to an inflammatory response, with synthesis and secretion of chemokines and upregulation of adhesion molecules. The alternative pathway also regulates a suite of chemokines and adhesion molecules that appear to be crucial for lymphoid organogenesis (Figure 1-4). Each pathway is distinguished by different regulators of IκB degradation. In the classic pathway, which can be activated via both TNFR1
Notch2 Delta-like1,3,4 Jagged 1,2 RGP-Jk/CBF-1 Mastermind-like 1 Id2
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and LTβR, NF-κB/RelA heterodimers are maintained in an inactive state by IκBα. In the alternative pathway, which operates downstream of LTβR via NIK and BAFF-R (see “B Cell Extrinsic Signal: BAFF and B Cell Homeostasis”), p52/RelB heterodimers are retained in their inactive state by p100 until activated by IKKα. NIK acts both as an IKKα-activating kinase and as a docking protein that links IKKα to p100 (Xiao et al. 2004). Thus, TNFR1 is a much more potent proinflammatory stimulus than LTβR ligation, as the latter leads to a signal that integrates both pathways, and the p52/RelB pathway attenuates gene expression induced by NF-κB1/RelA (Dejardin et al. 2002). Clearly, the complexity of the effects of TNFRSF ligation is accentuated by these signaling cascades, since there is not a one-to-one relation between a particular ligand receptor pair and an intracellular signaling pathway. Indeed, LTβR can activate via both pathways (see “Signaling Mutations Affecting Early Lymphoid Organogenesis”). Furthermore, there is the potential for mutual cross-regulation of each signaling pathway by the induction of genes by one NF-κB heterodimer that inhibit the activation of the other heterodimer. Finally, further complexity is added by the semiredundant TRAF family of molecules, which provide the proximal link between TNFRSF members and downstream signaling pathways. 3.
Ligation of LTβR during Organogenesis
The importance of LTα in lymphoid organogenesis was first realized when LTα−/− mice were generated and found to have no lymph nodes or PPs and chaotic splenic white pulp in which segregation of T and B cells is absent (Banks et al. 1995; De Togni et al. 1994). Subsequently, it was shown that LTβR−/− mice also lack all peripheral lymph nodes, PPs, and aggregates of MALT (Futterer et al. 1998). It is now clear that interactions between LTα1β2 and LTβR are crucial for lymph node development as well as for formation of PPs and normal splenic white pulp architecture. Studies using reciprocal bone marrow chimeras have helped delineate the roles of hematopoietic and mesenchymal cells bearing these receptor–ligand pairs during organogenesis. When wild-type recipients are reconstituted with LTα−/−derived bone marrow, lymphocytes segregate normally within lymph nodes. When wild-type bone marrow is used to reconstitute irradiated LTα−/− recipients after embryogenesis is complete, development of PPs and lymph nodes is not rescued, although microarchitecture in the splenic white pulp is partially restored (Mariathasan et al. 1995; Matsumoto 1999). In LTα−/− mice, lymph node organogenesis is restored by the administration of agonistic anti-LTβR antibodies during gestation, which confirms the importance of temporal ligation of this receptor during lymph node development (a function normally performed by LTIC) (Browning and French 2002). Once the normal phase of lymph node development in embryogenesis has passed, restoration of LTβR ligation cannot restore lymph
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node formation. By contrast, ongoing LTα1β2 is required for maintenance of lymphoid patterning (see “B Cell Zones in Secondary Lymphoid Organs”). The conclusion from these and other studies is that a crucial interaction during lymph node organogenesis takes place between LTβR on mesenchymederived stroma and LTα1β2 on LTICs. Although these studies might be taken to indicate that LTα1β2 binding to LTβR is the key TNFRSF ligation event governing early lymphoid organogenesis, the phenotype of some mutants raise questions about this conclusion. Seventyfive percent of LTβ−/− mice have one prominent mesenteric lymph node, and a variable number have cervical lymph nodes, indicating an incomplete defect in lymphoid organogenesis (Koni et al. 1997). This finding raised the possibility that LTα1β2 is not the only ligand for LTβR during lymphoid development. LIGHT is a candidate to explain these findings, since it also binds LTβR. However, LIGHT−/− mice have normal lymphoid structures. In mice doubly deficient for LIGHT and LTβ, the incidence of mesenteric lymph nodes is reduced from 75% to 25%, indicating a contribution by LIGHT to mesenteric lymph node organogenesis (Scheu et al. 2002). By contrast, mice doubly deficient in TNFR1 and LTβ lack all lymph nodes (Koni and Flavell 1998), although LTβ/TNF double-deficient mice have mucosal lymph nodes (Kuprash 1999). Blockade of the ligand for LTβR with dimeric soluble LTβR-human IgG1 fusion proteins during gestation, results in near total abrogation of lymph node and PP development in the fetus (Rennert et al. 1996). Because complete blockade of all lymph node development is obtained by the addition of TNFR1-Ig (Rennert et al. 1998), it is possible that ligation of TNFR1 (by LTα3) also contributes to mucosal lymphogenesis. Taken together, these findings indicate that an as yet undiscovered LTβR and/or TNFR1 ligand contributes to lymph node organogenesis, but signaling via LTβR or RANK appears to be the dominant pathway during lymph node and PP organogenesis (RANK only contributes to lymph nodes, see later, with other receptors modifying development and regulating microarchitecture). 4.
Lymphoid Tissue–Inducing Cells
LTICs can be identified early during embryogenesis as hematopoietic cells that interact with mesenchymal cells in lymph node and PP anlagen during secondary lymphoid organ development. The precise ontogeny of these cells remains controversial. PPICs appear to arise from fetal liver, whereas there is evidence that LNICs arise from the thymus, but there are no functional data indicating that they arise from the fetal thymus (Eberl et al. 2004). LTICs express CD45 but lack T, B, and DC lineage markers (Mebius et al. 1997; Yoshida et al. 1999). They can be induced to form NK1.1 T cells and CD11c+ antigen-presenting cells in vitro. LTIC development appears to depend on the transcriptional regulators RORγ (Kurebayashi et al. 2000) and Id2
74 (Yokota et al. 1999). In the fetus, the RORγt isoform is expressed exclusively in LTICs. LTICs are absent from mice deficient in either of these factors, as are all peripheral lymph nodes, PPs, and CPs (Eberl et al. 2004). RORγt−/− mice also exhibit extensive thymocyte apoptosis, perhaps consistent with the idea that LTICs arise in the thymus (Sun et al. 2000). Although the precise function of Id2 and RORγt in LTICs remains to be determined, both are known to inhibit cell differentiation and proliferation, and RORγ has been shown to inhibit apoptosis by promoting bcl-xL expression and downregulating Fas ligand (Eberl and Littman 2003). LTIC are identified by the surface phenotype, CD45+ IL-7R LTα1β2+ CXCR5+ (see Figure 1-3). They are first detectable in the vicinity of the pharynx, intestine, and pericardium on E12.5 (Sun et al. 2000) and then in spleen, blood and lymph nodes, and PP anlagen later in fetal development. MOCs are identified by the surface phenotype, IL-7+ LTβR+ MAdCAM-1+ (mucosal addressin adhesion molecule-1) VCAM-1+ ICAM-1+ (intracellular adhesion molecule-1) CXCL13+ CCL19+, and are detected in clusters along the antimesenteric surface of the intestine on E15.5. PPICs infiltrate these locations during the next day (Adachi et al. 1997; Honda et al. 2001). MOCs are present within lymph node anlagen on E12.5–E13.5. Development of MOCs within the intestine is dependent on the winged helix transcription factor Foxl1, which is expressed specifically within VCAM-1+ ICAM-1+ cells in the mesenchyme close to the intestinal epithelium (Fukuda et al. 2003). Foxl1 deficiency results in a reduction of PPs, especially in the distal intestine, and dispersal of the VCAM-1+ MOCs, with fewer hematopoietic cells accumulating in the VCAM-1+ spots. Furthermore, Foxl1 deficiency results in a significant reduction in LTβR, CCL19, and CCL21 expression in the MOCs. Spleen and lymph node development is normal in Foxl1-deficient mice, and the nature of transcriptional regulation of MOCs in these locations remains unknown. Understanding of the earliest events governing the interaction between LTICs and MOCs remains incomplete. Considerable evidence points to a critical role for recruitment of LTICs to sites where lymph nodes and PPs will develop, although conclusive evidence that secondary lymphoid organ development is abrogated by the specific depletion of this population has not been obtained. LTIC recruitment depends on upregulation of LTα1β2 on LTICs and their migration to the earliest lymph nodes and PP anlagen. Overlapping stimuli delivered via chemokine receptors, IL-7Rα and RANKL, mediate these events (Cupedo and Mebius 2005). LTIC recruitment is at least partially dependent on CXCR5 ligation by stromal CXCL13, but mesenteric lymph nodes, PPs, and a variable number of peripheral lymph nodes develop in CXCR5−/− and CXCL13−/− mice (Ansel et al. 2000; Cupedo and Mebius 2005; Forster et al. 1996). This development is explained by the contribution of CCR7 ligation by CCL19 and CCL21 during recruitment of LTICs to peripheral lymph nodes and IL-7Rα ligation during LTIC recruitment to peripheral and
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mesenteric lymph nodes and PPs (Luther et al. 2003; Nishikawa et al. 2003). CXCL13, which is secreted by MOCs, activates α4β1 integrin on LTICs and VCAM-1 on MOCs (Finke et al. 2002; Honda et al. 2001). Blockade of this interaction partially disrupts normal PP induction, but additional adhesion events are probably necessary for LTICs to home effectively to lymph node and PP anlagen. α4β7 is also induced on LTICs and its ligand, MAdCAM-1, is expressed on MOCs, but disruption of this ligand pair does not affect PP formation (Wagner et al. 1996). 5.
Induction of LTα1β2 on Lymphoid Tissue–Inducing Cells
Early lymph node and PP development depends on delivery of LTα1β2 signals, which appears to be dependent on IL-7Rα and for lymph nodes on RANK (Luther et al. 2003). IL-7Rα contributes to receptors for two known ligands, IL-7 and TSLP. Several lines of evidence indicate that IL-7 is the key ligand for early lymphoid organogenesis. PP and some lymph node development is abrogated by administration of IL-7Rα antagonists between E12 and E14 (Yoshida et al. 1999, 2002). Deficiencies of Jak3 and common γ chain, which are necessary for IL-7 but not TSLP signaling, result in partial peripheral lymph node deficiency (but normal mesenteric lymph nodes). Furthermore, intraembryonic injection of IL-7 rescues lymph node development in TRAF6−/− mice (Yoshida et al. 2002). Systemic expression of IL-7 rather than expression within the PP anlagen appears to be responsible for LTα1β2 induction (Laky et al. 2000; Yoshida et al. 2002), and the level of LTα1β2 expression by LTICs is similar whether they are examined in peripheral blood or early mesenteric lymph nodes. The failure of IL-7 deficiency to completely abrogate PP and lymph node development (Nishikawa et al. 2003) is unlikely to reflect a role for other IL-7Rα ligands, since LTICs still express significant amounts of LTα1β2 in the absence of IL-7Rα (Luther et al. 2003). Instead, this reflects the contribution of RANK to LTα1β2 induction on LNICs (Kim et al. 2000). RANK, RANKL, and TRAF6 deficiencies all result in absent peripheral lymph nodes but normal PP formation (Dougall et al. 1999; Kong et al. 1999a). This defect is not corrected by transfer of wild-type bone marrow into RANKL−/− recipients. Interactions between RANK and RANKL regulates homing of LNICs to nascent lymph nodes (Kim et al. 2000). The number of LNICs in lymph node rudiments of RANKL−/− mice is reduced significantly, and they fail to form clusters within the lymph node anlagen (Kim et al. 2000). RANKL deficiency cannot be corrected with agonistic anti-LTβR antibody treatment, and the LTα−/− phenotype is not corrected with transgenic RANKL expression. Taken together, these findings suggest that ligation of RANKL and signaling via TRAF6 could play roles similar to that of IL-7Rα ligation of PPICs to induce LTα1β2 expression on LNICs (Nishikawa et al. 2003).
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Signaling Mutations Affecting Early Lymphoid Organogenesis
Signaling through LTβR activates both the classic and alternative NF-κB signaling pathways (Lo et al. 2006; Senftleben et al. 2001); thus, it is not surprising that mice deficient in either NF-κB1 (p50) or NF-κB2 (p52) have defects in the formation of inguinal lymph nodes (Lo et al. 2006; Weih and Caamano 2003). Absence of NF-kB1 results in a reduction of PPs, and PPs are completely absent in mice deficient in NF-κB2 (Paxian et al. 2002). Mice doubly deficient for p50 and p52 completely recapitulate the absence of lymph nodes observed in LTα-deficient mice (Lo et al. 2006). Retention of one p50 allele (nfκb1+/−nfκb2−/−) results in the retention of a single mesenteric lymph node, which is also observed in LTβ-deficient mice, whereas retention of one p52 allele (nfκb1−/−nfκb2+/−) restores most lymph nodes except the inguinal and brachial ones. These data together indicate that p50 and p52 both mediate nonredundant functions in LTβR-mediated lymph node organogenesis. Consistent with a role for the classic NF-κB signaling pathway involving RelA/p50 complexes, mice doubly deficient in RelA and TNFR1 lack all secondary lymphoid organs (Alcamo et al. 2002), although the relative contributions of RelA and TNFR1 have not been assessed. Mice with truncated c-Rel exhibit disordered B cell microdomains and impaired germinal center formation (Carrasco et al. 1998). Consistent with an essential role for the alternative NF-κB signaling pathway involving IKK/IKKα and NIK that yields RelB/p52 complexes in lymphoid organogenesis, IKKα-deficient mice lack PP (Matsushima et al. 2001; Senftleben et al. 2001) (IKKα deficiency is perinatal lethal and lymph node development has not been assessed), and the natural mutant alymphoplasia (aly) strain of mice with a mutated NIK, has no lymph nodes or PPs and has disordered splenic white pulp, a phenotype very similar to that of LTα−/− and LTβR−/− mice (Miyawaki et al. 1994) (see Table 1-5). NIK is normally activated after ligation of LTβR in MOCs. Interestingly, CP and isolated lymphoid follicle formation depends on LTβR signaling, but unlike in lymph node and PP formation, LTβR signaling seems to occur exclusively via the canonical NF-κB pathway in CP formation (Taylor and Williams 2005). Thus, CPs form in the absence of NIK. Isolated lymphoid follicle formation parallels lymph node development and is dependent on the alternative NF-κB pathway (Kanamori et al. 1996; Taylor et al. 2004). Development of most peripheral lymph nodes appears intact in 1-day-old RelB−/− mice, although lymphoid depletion and inflammation occur after 1 week of age (Weih et al. 1995). PPs are absent in RelB−/− mice (Yilmaz et al. 2003). The phenotype of TRAF mutants is somewhat confusing. TRAF2, 3, and 5 are all implicated in LTβR signaling, yet deletion of any one of these genes, or double TRAF2/5 deletion does not prevent lymph node organogenesis. On the other hand, deficiency of TRAF6, which binds the cytoplasmic tail of RANK but not LTβR results in lymph node deficiency and splenomegaly, but normal PP development and osteopetrosis
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(Akiyama et al. 2005; Naito et al. 1999). A similar phenotype is observed with RANK deficiency. This finding reinforces the view that RANK induces LTα1β2 on LNICs. 7. Tumor Necrosis Factor Immediate Family, Chemokine Transcription, and Lymphocyte Recruitment
The final phase of PP and lymph node organogenesis is patterning of lymphocytes, and this is determined by chemokine production. Chemokines are a family of small soluble molecules that are unified by the capacity to bind to seven-transmembrane— spanning receptors that are coupled with cytoplasmic heterotrimeric G-proteins (Table 1-2) (for reviews, see Luther and Cyster 2001; Zlotnik and Yoshie 2000). These interactions are complex, since chemokines have pleiotropic functions and they exhibit promiscuity in receptor binding. Furthermore, different combinations of G-protein subunits mediate linkage with different intracellular signaling cascades. Moreover, chemotaxis is not mediated exclusively by chemokine receptors, and the cross-talk between signaling activated by stimulation of nonchemokine receptors and chemokine receptors remains uncertain. Chemokines are important in lymphoid organogenesis from the earliest phase of development. Recruitment of LTICs depends on contributions from CXCR5 ligation by CXCL13 and CCR7 ligation by CCL21 and CCL19 (Luther et al. 2003). Deficiency of any one of these chemokine-ligand pairs leads to partial lymph node and PP deficiency. Enforced expression of CXCR5 on LTIC restores normal lymph node formation in CXCR5−/− mice (Ansel et al. 2000; Cyster 2003). During PP development, release of CXCL13 by mesenchymal cells activates α4 integrin on PPICs for binding to VCAM-1 on MOCs (Finke et al. 2002). At the same time, ligation of LTβR on MOCs enhances VCAM-1 and ICAM-1 expression (Yoshida et al. 2002). It remains possible that additional ligand pairs also contribute to LTIC recruitment. After entry into lymph node and PP anlagen, CXCL13 is upregulated in an LTα1β2-dependent manner. CXCL13 expression in the splenic stroma is severely reduced in the absence of LTα or LTβ and is also reduced in the absence of TNF or TNFR1 (Ngo et al. 1999). Ligation of LTβR signals via NIK to activate NF-κB p52/RelB and induce CCL19, CCL21, and CXCL13 (Dejardin et al. 2002). Initially, CXCL13 is induced by LTICs, and, subsequently, this permits recruitment of lymphocytes and patterning. B cells become responsive to CXCL13 and acquire LTα1β2, which establishes a positive feedback loop of further CXCL13 production (Cupedo and Mebius 2005).
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Spleen Development
The molecular control of splenic embryogenesis depends on several transcription factors that play no part in either lymph node or PP development. However, there is considerable overlap between development and organization of splenic
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white pulp and development and patterning of other secondary lymphoid organs. The spleen arises on E11 from the dorsal mesogastrium, a mesodermal precursor of adult mesentery. The NK homeobox family member Bapx1 (mouse homolog of Drosophila bagpipes) is an early developmental marker for splanchnic mesoderm, consistent with its putative role in visceral mesoderm specification. Deficiency of Bapx1 results in asplenia, with no sign of the splenic anlagen, as well as abnormal development of the ventromedial structure of the vertebral column and some of the craniofacial bones and gastroduodenal malformation (Akazawa et al. 2000; Lettice et al. 1999). Splenic development is specifically abrogated by deficiency of the orphan homeobox 11 gene (Hox11) (Roberts et al. 1994). Interestingly, the pancreas is normal even though it also arises from the dorsal mesogastrium, indicating that Hox11 only affects the mesodermal cells within the mesogastrium that are destined to form the spleen. Hox11 expression increases in the mesogastrium on E11 and appears to be necessary for survival of the splenic rudiment and positioning, since the splenic anlagen is present transiently in Hox11−/− mice (Dear et al. 1995). Soon after this, some splenic precursors undergo apoptosis, whereas others disperse to the region of the tail of the pancreas (Kanzler and Dear 2001). Capsulin is a basic helix-loop-helix transcription factor that appears to act on E12.5 after splenic specification to control morphogenetic expansion of the splenic anlagen via interactions with Hox11 and Bapx1 (Lu et al. 2000). In Bapx1−/− mice, Hox11 expression is abrogated and in capsulin−/− mice, neither Bapx1 nor Hox11 is expressed, suggesting a capsulin-Bapx1Hox11 axis for splenic patterning and development. Homozygous dominant hemimelia (Dh/+) mutants exhibit a range of defects, including skeletal abnormalities and severe splenic dysgenesis. Chimera embryo aggregate studies with Dh/Dh and +/+ cells restored spleens in some offspring, and the spleens contained Dh/Dh cells, suggesting that Dh provides a signal for normal spleen cell development (Suto et al. 1995). In addition to these transcriptional regulators that are unique to the spleen, LTβR is also crucial for splenic development. The white pulp is severely disrupted in LTα−/−, LTβ−/−, and LTβR−/− mice. Postnatal blockade of LTβR via expression of a LTβR-IgG1 transgene results in splenic white pulp disorganization, indicating that LTβR ligation is also necessary to maintain white pulp architecture once it has been established (Ettinger et al. 1996).
IV.
MICROENVIRONMENTS IN SECONDARY LYMPHOID ORGANS A.
Normal B Cell Migration
In contrast to nonlymphoid organs in which cells remain fixed in place by tight intercellular junctions, a large component of
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lymphoid organ parenchyma comprises cellular constituents (including lymphocytes and DCs) that are only temporary residents. Naïve lymphocytes recirculate continuously between different lymphoid organs. Within secondary lymphoid organs, naïve lymphocytes make transient interactions with each other and with the stromal components of the organs, moving randomly through the lymphoid parenchyma at speeds of 6–15 µm/min (Miller et al. 2002, 2003). Traffic through the secondary lymphoid organs and organization of different subsets of lymphocytes into microdomains within those organs enhances the efficiency of immune responses in two ways. First, the chances of rare antigenspecific cells meeting each other are increased. Second, antigen is concentrated within the secondary lymphoid organs. 1.
Lymphatic Drainage to the Secondary Lymphoid Organs
Lymphatics drain all organs except the central nervous system, the eye, bone marrow, and the spleen. They are richest in sites of high antigen exposure, such as the dermis and gastrointestinal submucosa. The murine lymphatics bud off from veins at E10.5 under the influence of the homeobox transcription factor Prox-1 (Miller et al. 2002, 2003; Wigle and Oliver 1999) and then the endothelium acquires a unique phenotype (VEGFR3+ LYVE-1+; Banerji et al. 1999; Karkkainen and Alitalo 2002). Afferent lymphatics drain into the convex surface of lymph nodes, emptying into the subcapsular sinus (see Figure 1-1C). The afferent lymphatics carry memory lymphocytes, DCs, extracellular molecules, and lymph. Immature DCs migrating from nonlymphoid parenchyma enter lymph nodes via the afferent lymphatics and are deposited in the paracortex in the vicinity of HEVs (Cavanagh and Von Andrian 2002; Gretz et al. 1997). The subcapsular sinus is lined by a reticular network that extends throughout the paracortex to the HEV (Marchesi and Gowans 1964). This network comprises numerous proteins, including collagens, fibronectin, and tenascin, which provide a scaffold for lymphocytes, and also appears to be a conduit for lymph and low-molecular-weight antigens (Miller et al. 2002). This reticular network is produced by radiation-resistant fibroblastic reticular cells (of which FDCs are a subset), and these cells also form conduits between the subcapsular sinus and the HEVs for the trafficking of small molecules (Gretz et al. 1996). Resident conduit-associated DCs have been shown to be capable of taking up and presenting soluble antigen circulating throughout the conduits (Sixt et al. 2005). However, larger free molecules are retained in the subcapsular sinus, where they may be trapped by macrophages and DCs located on the fibrous septae (Gretz et al. 2000). These antigens enter the lymphoid parenchyma most efficiently when transported by DCs via the afferent lymphatics, across the subcapsular sinus to the paracortex. 2.
Entry into and Exit from Lymph Nodes and Peyer’s Patches
Arteries enter the lymph node hilum. Naive lymphocytes and plasmacytoid DCs enter lymph nodes across the specialized
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cuboidal endothelia of the HEV in the paracortex (T cell zone) (Cella et al. 1999; Gowans and Knight 1964). Some memory and effector lymphocytes also enter lymph nodes from the bloodstream via HEVs, although the principal route is via the afferent lymphatics. Migration of lymphocytes through the HEV is a multistep process. First, interactions between lymphocytes and adhesion molecules expressed on HEV within peripheral and mesenteric lymph nodes and PPs cause lymphocytes to roll slowly along the endothelium. In mesenteric lymph nodes and PPs, rolling depends on α4β7 expressed by lymphocytes binding to MAdCAM-1 (Berlin et al. 1993), whose expression depends on ligation of TNFR1 and signaling involving NF-κB. In peripheral lymph nodes, L-selectin on naïve lymphocytes binds to peripheral node addressin on HEV (Gallatin et al. 1983). Rolling lymphocytes can then bind ICAM-1 and ICAM-2 via LFA-1 (lymphocyte function–associated antigen-1). However, L-selectin and LFA-1 are expressed by most leukocytes, and migration across HEV into lymph nodes is largely confined to lymphocytes. This specificity is determined by chemokines (Miyasaka and Tanaka 2004). Not all the chemokines that act within the HEV are produced there; chemokines produced elsewhere are selectively trafficked to HEVs from the subcapsular sinus along the fibroreticular cell conduits (Gretz et al. 1996). CCL21 is expressed by HEVs and CCL19 (not produced by endothelium) binds to the apical surface of HEVs and specifically enhances binding of T cells to ICAM-1. Similarly, CXCL12 ligates CXCR4 on lymphocytes to activate LFA-1 (αLβ2 integrin) and enhance binding to ICAM-1 (Campbell et al. 1998). A similar process operates for B cells, in which CXCL13 in HEVs binds to CXCR5 on B cells to enhance adhesion to HEVs (Okada et al. 2002). Chemokine responsiveness and induction of integrins depends on signaling downstream of the chemokine receptors, especially activation of Rac, which is a key modulator of actin polymerization and lamellipodia formation (EtienneManneville and Hall 2002). In T and B cells, activation of this pathway, and correct migration across the HEV, depends on DOCK2 (dedicator of cytokinesis-2) (Nombela-Arrieta et al. 2004). This also contributes to the specificity of lymphocyte recruitment into lymph nodes, since DOCK2 is not expressed in some nonlymphoid cell subsets such as neutrophils (Nishihara et al. 1999). Once lymphocytes enter lymphoid organs, their location is also determined by chemokines. Evidence suggests that correct localization of B and T cells within the lymphoid organ depends on balancing of signals from the chemoattractants CXCL13 and CCL21 concentrated in different microenvironments (Reif et al. 2002). On the other hand, convincing evidence for migration of cells in vivo along chemokine gradients is lacking. An alternative hypothesis is that chemokine molecules are located precisely on the extracellular matrix, with which lymphocytes make transient interactions. This would be consistent with recent real-time studies of lymphocyte
IN
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77
movement through lymphoid parenchyma, which shows cells taking a random, almost chaotic, walk (Miller et al. 2002). If lymphocytes are not activated by antigen, then their time within a secondary lymphoid organ is limited (e.g., about 30 hours for a B cell in a lymph node). Unstimulated lymphocytes migrate through a lymph node, draining via cortical sinusoids onto medullary sinuses and into efferent lymphatics (Soderstrom and Stenstrom 1969). Egress appears to depend on expression of S1P receptor 1 (S1P1) on lymphocytes (Matloubian et al. 2004). S1P1 is a receptor for sphingosine-1phosphate, which is abundant within the circulation (Cyster 2004). The signaling mechanism downstream of S1P1 that regulates lymphocyte egress remains unknown. 3.
Entry into and Exit from Spleen
Traffic into and through the spleen is different from that in other secondary lymphoid organs in several aspects. First, the spleen is divided into two compartments with different functions: red pulp, which comprises venous sinuses and contains macrophages that remove senescent red blood cells from the circulation; and white pulp, which is the lymphoid component, comprising follicles (B cell zones), T cell zones (also known as periarteriolar lymphoid sheaths), and marginal zones (see Figure 1-1B). The marginal zone (MZ) is located between the white and red pulp and contains specialized macrophages (metallophilic macrophages and MZ macrophages), B cells, and DCs (Veerman and van Ewijk 1975). A second difference stems from the absence of both afferent lymphatics and HEVs from the spleen, so antigen and lymphocytes can only enter via the arterial circulation. The white pulp is perfused by central arterioles located in T zones, which give off branches that open into the marginal sinuses. These are loose endothelial structures located between the white pulp and marginal zones (Nieuwenhuis and Ford 1976; Pabst 1988). Cells and antigens in the circulation enter the white pulp via the marginal sinus, migrating toward the T zone, through the follicles, draining via venous sinuses in the red pulp (cords of Billroth) to return to the systemic circulation (Grayson et al. 2001). Antigen also enters via the marginal sinus, where it comes into contact with macrophages and marginal zone B cells, before diffusing via a similar path through the T zone and into the red pulp. Marginal sinus endothelium expresses MAdCAM-1 (Kraal et al. 1995); however, the significance of this adhesion molecule for lymphocyte entry into the splenic white pulp is not clear, since neither MAdCAM-1 blockade with antibodies, nor blockade of its ligand, α4β7, affects B cell homing to the white pulp of the spleen (Kraal et al. 1995). Instead αLβ2 (LFA-1) and α4β1 integrins (Lo et al. 2003) and binding to marginal zone macrophages adjacent to the marginal sinus (Lyons and Parish 1995) seem to contribute to lymphocytes entering the splenic white pulp. Once inside, correct positioning within the splenic white pulp depends on chemokines within microdomains (Cyster and Goodnow 1995a).
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B. 1.
Microdomains in Secondary Lymphoid Organs
B Cell Zones in Secondary Lymphoid Organs
Even before contact with antigen, B cells differentiate and become organized into several peripheral compartments (Vinuesa and Cook 2001). In adults, the most populous is the follicular compartment, comprising naïve recirculating B cells (B-2 cells) that express low to intermediate levels of surface IgM and high levels of IgD. Unstimulated, follicular B cells appear to persist in the resting state for several months (Hao and Rajewsky 2001). These cells recirculate among the follicles of spleen and lymph nodes and are available for recruitment into immune responses by protein antigens. a. FOLLICLES Mature recirculating B cells segregate into lymphoid follicles within secondary lymphoid organs. Follicles also contain FDCs derived from local mesenchymal cells (Kapasi et al. 1993) that play an important role as an antigen depot, a unique population of T cells (follicular B helper T cells [TFH cells]), DCs of hematopoietic origin, and macrophages. After immunization follicles support the development of germinal centers, when they are referred to as secondary follicles. Follicle development depends on a recurring theme of lymphoid organogenesis in which hematopoietic cells, in this case B cells, are recruited and induce maturation of mesenchymal elements, in this case FDCs, within the nascent organ. LTβR and TNFR1 are expressed by FDCs, and both receptors need to be ligated to induce a mature FDC network and normal follicle formation. Bone marrow chimera studies have established that expression of TNF, LTα, and LTβ by hematopoietic cells induces FDC maturation and normal follicular architecture in LTα−/− and TNF−/− recipients (Le Hir et al. 1996; Tkachuk et al. 1998). Indeed, purified wild-type B cells are sufficient to restore FDC, follicles and germinal center reactions to LTα−/− recipients (Fu et al. 1998). LTα+ B cells induce FDC maturation, which establishes a positive feedback loop that consolidates follicle formation because FDCs secrete CXCL13, which acts both to recruit B cells to the follicle and makes B cells competent to deliver the LT signal (Ansel et al. 2000). Lymph nodes are present in both TNF−/− and TNFR1−/− mice, but mature FDC networks are absent and instead of follicles B cells form homogenous rims between the T cell zone and the red pulp (Korner et al. 1997; Le Hir et al. 1996; Pasparakis et al. 1996). Membrane TNF is not sufficient to maintain primary B cell follicles (Ruuls et al. 2001). When assessed by flow cytometry, both marginal zone and follicular B cell populations are present in normal numbers in TNF−/− mice, indicating that both B cell populations are located within this region (Korner et al. 2001; Liu and Banchereau 1997). Lymphoid architectural abnormalities such as those observed in TNF−/− and TNFR1−/− mice have also been observed in NFκB2/p52−/− mice (Caamano et al. 1998; Poljak et al. 1999).
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VINUESA
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COOK
Transfer of normal bone marrow into NF-κB2 /p52−/− recipients restores metallophilic macrophages but not FDC networks. A similar but incomplete defect is also observed in mice lacking Bcl-3, an I-κB related regulator (Table 1-3). In Bcl-3−/− mice, FDC networks are observed after immunization. It is then likely that TNFR1 signaling via NF-κB2/p52 is crucial for FDC development although it is possible that LTβR but not TNFR1 may signal via NF-κB2/p52. b. CHEMOKINE REGULATION OF B CELL MIGRATION TO FOLLIAlthough TNF and LTα must be expressed on B cells during development for normal follicles to form, when TNF-/and LTα-/- Β cells are injected into normal mice, they migrate normally into established follicles (Cook et al. 1998). This is because expression of TNF and LTα by B cells is necessary to establish follicles. Ligation of LTβR on FDCs and their immature stromal precursors by LTα1β2+ B cells during early follicle development induces CXCL13 production (Ansel et al. 2000; Ngo et al. 1999). This interaction appears to be crucial for the colonization of follicles by B cells; naive B cells localize within B cell follicles when CXCR5 on B cells binds to CXCL13 (Bowman et al. 2000; Gunn et al. 1998) (see Table 1-2). After this, ligation of TNFR1 is crucial to establish and maintain the mature B cell/FDC environment. CXCL13 expression is partially TNF dependent (Poljak et al. 1999); the similarities between TNF-/- (and TNFR1-/-) and NF-κB2/p52-/mice reflect the failure of FDCs in NF-κB2/p52-/- mice to express CXCL13. When naïve CXCR5-deficient B cells are transferred into normal recipients, they locate in the T zones, but fail to enter the follicles. A similar pattern of migration is observed after stimulation with antigen when B cells migrate to the outer T zones of spleen and lymph nodes (see “Immune Response Structures”). The splenic white pulp of CXCR5−/− mice resembles that of TNF−/− mice rather than that of LTβR−/− mice (Forster et al. 1996), although CXCL13 production is only marginally dependent on TNFR signaling. CLES
c. MARGINAL ZONE The MZ comprises a unique population of B cells and DCs, as well as two specialized macrophage populations: metallophilic macrophages and MZ macrophages (Kraal 1992). Although MZ B cells exhibit a relatively homogenous surface phenotype (IgM+++ IgD−/+ CD23− CD9+ CD21hi CD5− CD1d+ CD38hi CD9+), they are in fact a mixture of memory B cells, direct recruits from the bone marrow, and B cells that appear to enter from the recirculating population without participating in an immune response (i.e., without proceeding through germinal centers) (Martin and Kearney 2002; Vinuesa et al. 2003) (Figure 1-5). MZ B cells are larger, have less condensed chromatin, and express higher levels of costimulatory molecules (CD80 and CD86) than recirculating follicular B cells (MacLennan and Liu 1991; Spencer et al. 1998). These features suggest that MZ B cells are in a state of partial activation and are consistent with evidence that they can
1. THE
TABLE 1-3
SUMMARY OF THE MOLECULES RESPONSIBLE FOR SECONDARY LYMPHOID ORGAN FORMATION AND PATTERNING BASED ON RESULTS FROM GENETIC MANIPULATION OF MICE
MOLECULAR
Spleen GC
CP
ILF
NALT
References
TNF family members and ligands LTα LTβ LTβR RANK RANKL LIGHT LIGHT + LTβ TNF
− C − − − + − +
− One (75%) One − − + One (25%) +
− − − + + + − +
− Partial − + + + − Disrupted
− Partial − + + + − +
− − − + + + − Disrupted
− − Ectopic + + + − Ectopic
− ? ? ? ? ? ? ?
− ? − ? ? ? ? ?
Disrupted ? Disrupted ? ? ? ? ?
TNFR1 TNFR2 TNFR1 + LTβ TNF + LTβ NF-κB pathway TRAF6
+ + − −
+ + − +
+ + − −
Disrupted + − Disrupted
− + − −
Disrupted + − Disrupted
Ectopic + − −
? ? ? ?
? ? ? ?
+ ? ? ?
(De Togni et al. 1994) (Koni et al. 1997) (Futterer et al. 1998) (Dougall et al. 1999) (Kong et al. 1999b) (Scheu et al. 2002) (Scheu et al. 2002) (Pasparakis et al. 1996; Korner et al. 1997) (Le Hir et al. 1996) (Le Hir et al. 1996) (Koni and Flavell, 1998) (Koni and Flavell, 1998)
−
−
+
?
?
?
?
?
?
?
Aly/Aly NIK
− −
− −
− −
− −
− −
− −
− −
+ +
− −
? Disrupted
NF-κB1 (p50)
No I
+
Reduced
+
+
+
+
?
?
+
NF-κB2 (p52)
A, small I, Po − No I, B, small C − +
+
−
Disrupted
−
Disrupted
−
?
?
Disrupted
− +
− ?
− ?
− ?
− ?
− ?
? ?
? ?
? ?
One +
? Reduced
? Disrupted
? Disrupted
? −
? ?
? ?
? ?
? − +
? − +
− − +
? ? +
? Induced by immunization ? ? +
? ? +
? − +
? ? ?
? ? ?
Disrupted ? ?
(Lo et al. 2005) (Franzoso et al. 1997; Poljak et al. 1999) (Matsushima et al. 2001) (Senftleben et al. 2001) (Rudolph et al. 2000)
−
−
−
−
−
−
−
?
?
?
(Alcamo et al. 2002)
Reduced
Reduced
−
Disrupted
−
Disrupted
−
?
?
+
(Weih et al. 2001; Yilmaz et al. 2003)
p50 + p52 Nfkb1−/− nfkb2+/− Nfkb1+/− nfkb2−/− Bcl-3 IKKα IKKαAA IKKγ (NEMO) RelA† + TNFR1 RelB
(Naito et al. 1999; Akiyama et al. 2005) (Miyawaki et al. 1994) (Shinkura et al. 1999); (Yin et al. 2001) (Weih and Caamano, 2003); (Paxian et al. 2002) (Franzoso et al. 1998); (Poljak et al. 1999) (Franzoso et al. 1997) (Lo et al. 2005)
MOUSE
MZ
THE
FDC
IN
Follicle
ARCHITECTURE
PP
LY M P H O I D
MLN
OF
PLN*
BASIS
Deficiency
Continued
79
80
TABLE 1-3
SUMMARY OF THE MOLECULES RESPONSIBLE FOR SECONDARY LYMPHOID ORGAN FORMATION AND PATTERNING BASED ON RESULTS FROM GENETIC MANIPULATION OF MICE—cont’d Spleen FDC
MZ
GC
CP
ILF
NALT
References
c-Rel c-Rel∆CT/∆CT Chemokines CXCR5 CXCL13
+ ?
+ +
+ +
+ Disrupted
+ ?
+ Enlarged
Reduced Reduced
? ?
? ?
? ?
(Tumang et al. 1998) (Carrasco et al. 1998)
C, F, Po C, F
+ +
Variable Variable
Disrupted ?
− ?
Disrupted ?
− ?
? ?
? ?
? ?
CCR7
No I (variable) No A (rare)
+
?
?
?
?
?
−
?
?
(Forster et al. 1996) (Ansel et al. 2000; Cupedo and Mebius, 2005) (Luther et al. 2003)
+
?
?
?
?
?
?
?
?
(Luther et al. 2003)
No F, C, B, A −
+
?
?
?
?
?
?
?
?
(Luther et al. 2003)
−
?
?
?
?
?
?
?
?
(Luther et al. 2003)
+
+
+
+
+
+
?
?
?
(Fukuda et al. 2003)
− −
− −
Reduced distally − −
− −
− −
− −
− −
− −
− −
? −
(Eberl et al. 2004) (Yokota et al. 1999)
Partial Partial Small + fewer C Small + fewer I, A, C
+ + +
− Reduced Reduced
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
? ? Disrupted
+
−
?
?
?
?
?
−
?
(Park et al. 1995) (Nishikawa et al. 2003) (Nishikawa et al. 2003; Luther et al. 2003) (Luther et al. 2003; Yoshida et al. 2002)
(CCL19 + CCL21-ser (plt/plt) plt/plt + CXCL13 CXCL13 + IL-7Rα Transcription factors Foxl1 RORγt Id2 Cytokines and cytokine signaling Jak3 IL-7 IL-7Rα IL-2Rγc
*PLN, peripheral lymph nodes: A, axillary; B, brachial; C, cervical; F, facial; I, inguinal; Po, popliteal; MLN, mesenteric lymph nodes. †RelA−/− (and IKKβ−/−), embryonic lethal liver apoptosis, E15–16.
MATTHEW
Follicle
AND
PP
VINUESA
MLN
G.
PLN*
CAROLA
Deficiency
C. COOK
1. THE
MOLECULAR
BASIS
OF
LY M P H O I D
ARCHITECTURE
IN
THE
BCR
MOUSE
81
BAFF-R
PKC CARMA1 Bcl10
NIK
MALT1
NFAT
RelB NF- B2
RelA NF- B1 Classical AP-1
Alternative BAFF -R JNK Bim
Bcl2, A1, BclxL Pim-2
Fig. 1-6 Overview of B cell signaling to NF-κB. The classic pathway is activated by ligation of the B cell receptor and involves PKCθ, MALT1, Bcl10, and CARMA1. By contrast, the alternative pathway is activated by various signals, including ligation of the BAFF receptor and LTβR.
undergo rapid differentiation to plasma cells after ligation with antigen (Oliver et al. 1997). MZ B cells play a key role in immune responses to polysaccharides and may also play an important role in removal of senescent cells, apoptotic debris, and immune complexes. The marginal zone is absent in the neonatal period, only appearing at 2–3 weeks of age. Because MZ B cells are nonrecirculating (Gray et al. 1982), it follows that the signals that position B cells in the marginal zone are different from those that localize recirculating B cells to the follicles. Thus, CXCR5 does not regulate MZ location. Signals that maintain MZ B cells in their correct location need to be distinguished from signals that are necessary for MZ B cell differentiation (see “Marginal Zone versus Follicular B Cells”). Thus, in the absence of LTα or LTβR ligation, marginal zones are absent, but this is because MZ B cells are absent (Korner et al. 2001). Both MZ macrophages and metallophilic macrophages are also absent from these mice. By contrast, the MZ is not discernible in TNF−/− or TNFR1−/−, but MZ and follicular B cells are both present but located within the same microdomain (Korner et al. 2001), and the specialized macrophage populations are sparse and dispersed. αLβ2 and α4β1 integrin-mediated adhesion to ICAM-1 and VCAM-1 within the MZ stroma and S1P are crucial for retaining the MZ B cells in the MZ, and expression of these adhesion molecules on the stroma is LTα1β2 dependent (Cinamon et al. 2004; Karlsson et al. 2003; Lu and Cyster 2002). B cell intrinsic-defects that affect responsiveness to chemokines have also been shown to abolish the MZ. Mice lacking the tyrosine kinases Pyk-2, DOCK2, or Lsc, which all regulate cytoskeletal dynamics in response to chemokine receptor ligation, exhibit a selective MZ deficiency (Girkontaite et al. 2001; Guinamard et al. 2000). Furthermore, administration of pertussis toxin, which
blocks signaling through chemokine receptors (Cyster and Goodnow 1995a), has also been shown to abolish MZ B cell formation (Guinamard et al. 2000). Several models reveal that disorganization of the MZ involves both B cells and the specialized macrophage populations, raising the possibility that MZ macrophages (MZMs) and/or metallophilic macrophages contribute to localization of MZ B cells or vice versa. One possible mechanism is that MARCO on MZMs ligates an unknown receptor on MZ B cells (Karlsson et al. 2003) although this seems unlikely since MZMs have been depleted without affecting MZ B cells (Aichele et al. 2003). There is now evidence to support the alternative scenario, that is, that B cells are required for the maintenance of metallophilic macrophages and MZMs (Nolte et al. 2004) and that LT provided by both B and T cells is required for the maintenance of the marginal zone (Tumanov et al. 2003). d. B-1 CELLS B-1 cells constitute the major B cell population in neonates (Berland and Wortis 2002). B-1 cells, defined according to functional characteristics, have been subdivided into two sister populations on the basis of CD5 expression: CD5+ B-1a cells and CD5− B-1b cells. Both subsets can be distinguished from conventional (B-2) cells because they are CD11b+, IgMhi, IgDlo, and CD23− (Hayakawa and Hardy 2000; Martin and Kearney 2001). During early life, B-1 cells are soon overtaken by conventional B cells in most peripheral compartments and become largely confined to the serosal surfaces under the influence of CXCR4 (Ansel et al. 2002; Herzenberg et al. 1986). B-1 cells represent only a very small minority of the adult peripheral B cell repertoire, and most peripheral B-1 cells are located in the marginal zone of the spleen. B-1 cells are maintained by self-renewal rather than replenishment from the bone marrow (Hayakawa and Hardy 2000). Their function
82
CAROLA
and ontogeny have been controversial. They are responsible for production of natural IgM antibodies that bind antigen with low affinity (e.g., anti-blood group antibodies). It has been postulated that these cells are selected for defense against antigenic motifs prevalent in pathogens and are very efficient in mounting responses against polysaccharides. 2.
T Cell Zones
Migration and positioning of T cells and DCs in the T zone depends on expression of CCR7, a receptor for CCL19 (ELC) and CCL21 (SLC), which are both expressed in the T zones by radiation-resistant stroma, HEV, and DCs (Cyster 1999; Luther et al. 2000). Within the T zone, DCs contact up to 5000 T cells per hour (Miller et al. 2004). Like CXCL13, expression of CCL19 and CCL21 is dependent on ligation of LTβR on stromal cells by LTα1β2, and TNFR1 ligation by TNF. Interestingly, B cells expressing LTα1β2 are important for CCL21 induction in the spleen but not in lymph nodes, where the function appears to depend on LTICs (Mebius 2003; Ngo et al. 2001).
V.
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COOK
recirculating population is reconstituted from transplanted bone marrow after sublethal irradiation, the fraction of bone marrow emigrants that enter the recirculating population increases (Bazin et al. 1985). In the presence of constant input from the bone marrow, the half-life of follicular B cells has been estimated to be 4–6 weeks. However, when mature B cells are transferred into immunodeficient recipients, they survive for much longer than 6 weeks (Sprent et al. 1994). Similarly, when marrow output is suspended, the half-life of mature follicular B cells is approximately 4.5 months (Hao and Rajewsky 2001). Taken together, these findings indicate (1) that the number of recent bone marrow emigrants vastly exceeds the requirement to maintain the follicular population in steady state, (2) that selection must take place for bone marrow emigrants to become incorporated into the recirculating population, and (3) that there is competition between bone marrow emigrants and established follicular cells for places in the recirculating population. Homeostasis of many cellular populations depends on extrinsic signals (Raff 1992). For B cells, two signals appear to be crucial for maintenance in the unstimulated mature state. The first is delivered through the BCR and the second through a TNF family member, BAFF. BCR signals are also instrumental in determining peripheral differentiation of B cells into each of the three principal compartments: follicular, MZ, and B-1.
HOMEOSTASIS AND SELECTION 2.
The size of the recirculating population of naïve T and B cells remains constant over time in the adult animal. This is a remarkable feature, first because most of the populations of T and B cells in the periphery are continuously migrating from one secondary lymphoid organ to another, and, second, because the number of new lymphocytes emerging from the primary lymphoid organs exceeds those leaving the repertoire through apoptosis or recruitment into immune responses.
A. 1.
G.
B Cell Homeostasis
Competition for Entry
The naïve B cell population provides a reserve with which to replenish cells that become activated during immune responses, as well as a potential repertoire of new antigen specificities. This population is determined by the rate of entry of cells arriving from the bone marrow, and the life span of cells within the established repertoire. Temporary expansions of the pool induced by B cell proliferation in response to antigen are soon compensated for, and the size of the peripheral B cell compartment returns to normal. In mice, this remains constant at approximately 109 cells and appears to be largely the result of peripheral mechanisms (Gaudin et al. 2004). Estimates of immature B cell turnover indicate that there are sufficient bone marrow emigrants to replace the recirculating population every 4–5 days (Osmond 1991). When the
B Cell Extrinsic Signal: BAFF and B Cell Homeostasis
A/WySnJ is a spontaneous mutant strain of mice, first described in 1991, that has only 10% of the normal number of peripheral B cells but normal numbers of bone marrow precursors, T cells, and B-1 cells (Miller and Hayes 1991) due to a defect that compromises recruitment of newly formed B cells into the long-lived peripheral pool and survival of naïve B cells (Harless et al. 2001). A/WySnJ mice were found to have a retrotransposon integrated in the gene encoding a novel TNFR family member, BAFF (also known as B lymphocyte survival, BLyS) (Schiemann et al. 2001). Thus, BAFF emerged as a TNF family member that is crucial for survival of peripheral B cells. BAFF is expressed by T cells, monocytes/macrophages, neutrophils, and DCs, as well as by radiation-resistant (nonhematopoietic) cells (Gorelik et al. 2004). Production of BAFF can be induced by a variety of inflammatory stimuli, particularly interferon-γ (IFN-γ). In addition to BAFF receptor (BAFF-R), BAFF binds the ligands BCMA and TACI. BCMA is found only on B cells whereas TACI is also found on some T cell subsets (Laabi and Strasser 2000). Transgenic overexpression of BAFF results in increased numbers of transitional cells, follicular B cells, and MZ B cells (but not B-1 cells; see “B-1 Cell Selection”) (Moore et al. 1999). Conversely, blockade of BAFF with soluble BAFF-R or inactivation of the BAFF gene results in a striking reduction of all peripheral B cell pools except B-1 cells (Gross et al. 2001). BAFF-R is expressed by B cells after they reach the periphery. Continuous exposure and stimulation by BAFF is a prerequisite
1. THE
MOLECULAR
BASIS
OF
LY M P H O I D
ARCHITECTURE
for peripheral B cell survival. A/WySnJ heterozygotic B cells exhibit an intermediate but uniform life span, and, in mixed chimeras, mutant B cells are outcompeted by wild-type cells (Harless et al. 2001). Ligation of BAFF-R regulates survival of B cells by increasing expression of antiapoptotic Bcl-2 family members, including Bcl-2 itself, A1, and Bcl-xL via the NF-κB/RelB pathway (Do et al. 2000; Hsu et al. 2002) (see Figure 1-6). 3. B Cell Intrinsic Signals: B Cell Receptor Signaling and B Cell Homeostasis
a. B CELL RECEPTOR SIGNALING THRESHOLDS Many aspects of B cell biology are determined by signals through the BCR, including selection and survival within the recirculating follicular repertoire, location in secondary lymphoid tissue, tolerance of self-reactive B cells, and responsiveness to antigen. Ligation of the BCR activates many intracellular signaling pathways, which ultimately modulate survival versus apoptosis, cell cycle progression, and differentiation. The BCR consists of membrane Ig (mIg), which contains a short intracytoplasmic tail and a 25-amino acid transmembrane domain noncovalently linked to Igα and Igβ (CD79a and CD79b) (Shaw et al. 1990), which are crucial for interacting with the BCR signaling apparatus (Reth 1992). Both Igα and Igβ contain one immunoreceptor tyrosinebased activation motif (ITAM), which is crucial for BCR signal transduction via tyrosine kinase activation and calcium mobilization (Reth 1989). These are activated primarily by Srcrelated protein tyrosine kinases after BCR cross-linking by
IN
THE
MOUSE
antigen and provide crucial links with downstream protein tyrosine kinases. After BCR ligation, the BCR complex and Src-family kinases (Lyn, Fyn, Blk, and Lck) accumulate in glycolipid-enriched microdomains (lipid rafts), which are also rich in related kinases (Pierce 2002; Rodgers and Rose 1996). This culminates in the assembly of molecular complexes of enzymes including Bruton’s tyrosine kinase (Btk), phosphoinositide 3-kinase (PI3K), phospholipase C (PLC)-γ2, associated with nonenzymatic adaptors (BLNK [B cell linker protein] and BCAP [B cell cytoplasmic adaptor protein]). Many other surface molecules, including negative regulators of B cell activation are excluded from lipid rafts. Negative regulators of BCR signaling include CD22, CD72, and FcγRIIB1 (Table 1-4). The cytoplasmic tails of CD22, CD72, and FcγRIIB1 contain immunoreceptor tyrosine-based inhibition motifs, which increase the BCR activation threshold by recruiting phosphatases that downmodulate BCR signaling. CD22 and CD72 are constitutively associated with the BCR, whereas FcγRIIB1 associates with the BCR after ligation by Fc regions of Ig within circulating immune complexes. CD22 is ligated by α2–6-sialylated moieties on CD45RO, CD22 itself (Hennet et al. 1998) and possibly other unidentified ligands. CD72 binds the class IV semaphorin CD100, which is expressed on B cells and activated T cells (Kumanogoh et al. 2000; Shi et al. 2000) and CD5 (Van de Velde et al. 1991), which is present on B-1 cells and at high levels on T cells. CD22 and CD72 recruit SH2-containing protein tyrosine phosphatase (SHP)-1 and FcγRIIB1 recruits SH2-containing inositol polyphosphate 5-phosphatase. Activation of both phosphatases leads to reduced calcium mobilization in response to
TABLE 1-4
MODIFICATION OF BCR SIGNALING AND CONSEQUENCES FOR RECRUITMENT INTO PERIPHERAL COMPARTMENTS Mutation Enhanced BCR Signaling CD22 SHP-1 Lyn CD72 FcγRIIB Igα Aiolis Inhibited BCR signaling CD19 CD21 Igλ CD45 BCAP PI3K Btk BLNK PLCγ2 PKCβ PD-1
Follicle
MZ
B-1
Reference
Decreased Decreased Decreased Decreased
Decreased
Increased Increased Increased Increased Increased
(Samardzic et al. 2002) (Cyster and Goodnow 1995b) (Chan et al. 1997) (Pan et al. 1999) (Ravetch and Bolland 2001) (Kurosaki 1999) (Cariappa et al. 2001)
Reduced Reduced
(Tedder et al. 1997) (Cariappa et al. 2001) (Sun et al. 2002) (Byth et al. 1996) (Yamazaki et al. 2002) (Fruman et al. 1999) (Hardy et al. 1983) (Jumaa et al. 1999) (Wang et al. 2000) (Leitges et al. 1996) (Nishimura et al. 1998)
Increased
Decreased Decreased Decreased Decreased Decreased Decreased
Decreased Decreased Decreased Increased Increased Normal Normal Normal Increased Normal Normal Decreased Increased
83
Reduced
Reduced
Decreased
84 BCR ligation (Liu et al. 1998; Ono et al. 1997). The tyrosine kinase Lyn, which is activated after BCR ligation, phosphorylates CD22 and FcγRIIB1 (Chan et al. 1997; Smith et al. 1998). Although Lyn is also thought to phosphorylate CD79a/b ITAMs, studies with mutant mice suggest that its net effect is negative regulation. Mice deficient in either CD22, SHP-1, FcγRIIB1, or Lyn all exhibit a similar phenotype of B cell hyper-responsiveness (Bolland and Ravetch 2000; Nishizumi et al. 1995). Furthermore, the interaction between these negative regulators helps to explain why mice with heterozygous defects in Lyn, CD22, and SHP-1 exhibit a hyper-responsive phenotype similar to that of Lyn−/− mice (Cornall et al. 1998). Aiolos is another important negative B cell regulator, since Aiolos−/− mice exhibit an activated cell surface phenotype and undergo augmented BCR-mediated in vitro proliferative responses, even with limiting amounts of antigen (Wang et al. 1998). CD45 is a membrane-bound tyrosine phosphatase that provides an essential role in B cell activation: CD45−/− mice are defective in BCR stimulation and show impaired calcium mobilization and activation of multiple downstream signaling pathways. In the absence of CD45, CD40-mediated signals are not affected, and T-independent signals to viral antigens remain intact (Byth et al. 1996). CD19, CD21, CD81 (TAPA-1), leu13, and γ-glutamyl transpeptidase form a coreceptor complex that decreases the threshold of B cell activation. Consequently, deficiency of these molecules results in impairment of antibody responses. In addition, this coreceptor complex provides an important link with the innate immune response, because the C3d complement degradation product binds CD21 (Dempsey et al. 1996). This binding helps explain how complementcoated antigens enhance B cell responsiveness, since ligation of CD21 recruits CD19 into the BCR complex, which in turn enhances downstream calcium fluxes and phosphorylation of Src-family kinases. Most mice bearing mutations of positive coreceptors exhibit diminished B cell responses to immunization. However, the role of CD19 is complicated because CD19 also activates the CD22/SHP-1 inhibitory pathway, which then acts in a negative feedback loop to negatively regulate CD19 signaling (Fujimoto et al. 1999). Furthermore, CD19 function can be modulated by other coreceptors including CD38, an ectoenzyme that appears to enhance BCR signaling. Consequently, CD38−/− mice have reduced antibody responses to protein antigens (although the phenotype is less severe than that of CD19−/− mice) (Kitanaka et al. 1997; Wang et al. 1998). Mutations that modify the threshold of BCR signaling modulate several aspects of B cell function, including activation to produce antibodies and selection into peripheral B cell compartments (Table 1-4). b. NF-κB ACTIVATION VIA THE B CELL RECEPTOR B cell survival, activation, and differentiation into different peripheral compartments depends on activation of NF-κB. NF-κB plays a crucial role in enabling the immune system to adapt to
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environmental plasticity (Hayden and Ghosh 2004). Furthermore, because NF-κB can be activated via more than one pathway on both B and T cells (including BAFF-R, CD40, and TLRs as well as BCRs), diverse signals appear to converge on this crucial transcription factor. Once activated, NF-kB regulates expression of many cytokines and growth factors. As described in the section on “Primary Lymphoid Organs,” NF-κB activation through both the classic and alternative pathways plays a critical role in lymphoid organogenesis (see Figure 1-4) patterning of lymphoid organs, maintenance of B cells in the periphery, and B and T cell activation (Table 1-3). Downstream of the BCR, the link with proximal tyrosine kinases depends on PKCβ/CARMA1/Bcl10/MALT1 (Lin and Wang 2004), and a similar pathway operates downstream of the TCR, where the link with proximal tyrosine kinases is made by PKCθ. PKCβ deficiency results in a deficiency of follicular B cells (Su et al. 2002). The IKK complex is rapidly recruited to the proximal signaling complex after antigen receptor ligation of both T and B cells. CARMA1 is a scaffolding protein, which recruits Bcl10 and MALT1 into lipid rafts, resulting in IKK and NF-κB activation. In the absence of this pathway, calcium flux and AP-1 activation is preserved. CARMA1 deficiency or mutation abrogates NF-κB and c-Jun kinase activation after BCR ligation and results in a deficiency of follicular B cells and an absence of B-1 cells (Hara et al. 2003; Jun et al. 2003; Newton and Dixit 2003). Bcl10−/− mice have a reduction in follicular, MZ, and B-1 cells (Xue et al. 2003). By contrast, MALT1-deficient mice display normal follicles, but a complete absence of MZ and B-1 cells (Ruefli-Brasse et al. 2003; Ruland et al. 2003). c. NEGATIVE AND POSITIVE SELECTION OF B CELLS There is considerable circumstantial evidence to suggest that the naïve peripheral B cell repertoire is ligand selected. Analysis of the Ig VH loci expressed in mature B cells reveals that they differ from those expressed in B cell precursors, and there is also skewing of the light chain usage by peripheral B cells, consistent with the proposition that antigen specificity influences B cell selection (Freitas et al. 1991; Gu et al. 1991; Levine et al. 2000; Malynn et al. 1990). The evidence for negative selection of B cells that bind antigen with high affinity is beyond doubt, although most experimental evidence for clonal deletion of self-reactive B cells indicates that this takes place in the bone marrow. However, not all self-reactive B cell precursors undergo clonal deletion after contact with antigen. Binding to soluble self-antigen can render B cells anergic; these cells migrate to the periphery but have a shortened life span, are hyporesponsive to subsequent BCR ligation, and compete poorly for entry into the recirculating follicular population (Cook et al. 1997; Cyster et al. 1994; Goodnow et al. 1988). Changes in the BCR signaling threshold influence the efficiency of negative selection. When the BCR signaling threshold is increased, self-antigen selects self-reactive B cells to enter the follicles in preference to nontransgenic B cells
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(Cyster et al. 1996). By contrast, when the BCR signaling threshold is decreased, self-reactive B cells are deleted before they enter the follicles (Cyster and Goodnow 1995b). Although negative selection could account for the repertoire shift between immature and mature B cells, there is also considerable evidence to suggest that positive selection of B cells contributes to shape the peripheral B cell repertoire. Key experiments using genetically manipulated mice in which surface immunoglobulin expression is selectively extinguished on mature B cells have established that surface Ig is necessary for peripheral B cell survival (Kraus et al. 2004; Lam et al. 1997). Thus, the signal transmitted through the BCR is necessary for B cell survival. However, this BCR-mediated signal is transmitted by cells that remain antigen-naïve, in that they do not differentiate down effector pathways. This finding presents a paradox, which could be resolved by at least two explanations: First, BCR ligation with antigen transmits a signal below the threshold for activation and recruitment into the immune response, or, second, BCR signaling occurs in the absence of ligation with antigen due to receptor oligomerization (Wienands et al. 1996). The first explanation is supported by evidence that the V region of the BCR rather than the BCR per se is crucial for B cell competition and survival in the periphery (Rosado and Freitas 1998). Possible ligands that might provide this selection stimulus include environmental antigens, selfantigen, or Ig idiotypes. According to the second explanation, the BCR transmits a signal constitutively, and this tonic signal is modified when the BCR itself is ligated, which would cause disruption of the oligomerized receptor complex (Schamel and Reth 2000). A functional BCR would therefore generate a selection and survival signal in the absence of antigen. d. INTEGRATION OF BAFF, B CELL RECEPTOR, AND CHEMOKINE SIGNALS: SURVIVAL VERSUS LOCATION Irrespective of the ligand, BCR signaling and BAFF-R ligation represent the two crucial signals that determine recruitment and survival of peripheral B cells. The signaling pathways appear to operate via separate cascades (see Figure 1-6). BCR ligation results in calcium flux but also activation of several key transcriptional regulatory pathways. Like other TNFR family members, BAFF-R ligation results in NF-κB activation. Evidence is beginning to emerge to explain how these two key signals are integrated. (1) B cells that have received a negative selection signal via contact with self-antigen require more BAFF than unstimulated cells to survive (Lesley et al. 2004). BCR ligation leads to increased expression of proapoptotic Bim (Lesley et al. 2004). (2) A positive selection signal mediated via BCR upregulates BAFF-R expression. BAFF-R ligation leads to increased production of the prosurvival Ser/Thr kinase Pim-2 (Lesley et al. 2004). By contrast with the survival signals transmitted through the BCR and BAFF-R, signals through chemokine receptors and integrins appear to determine location but not survival. The observation that anergic B cells were excluded from follicles (Cyster et al. 1994) raised the possibility that the B cell survival
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was dependent on follicular location. However, it is now clear that exclusion from the follicle is determined by changes in responsiveness to chemokines based on the strength of BCR signal, whereas survival is determined by sensitivity to BAFF irrespective of location (Cook et al. 1997; Lesley et al. 2004).
B. 1.
Differentiation of Peripheral B Cells
Transitional B Cells
Recent bone marrow emigrants have not yet reached maturity and only do so within the peripheral lymphoid organs. Upon entry to the spleen, they are referred to as transitional B cells. These cells have the capacity to differentiate into either follicular or MZ B cells (Batten et al. 2000; Martin and Kearney 2000; Osmond 1993). Two stages of transitional B cell development have been identified (Allman et al. 2001; Loder et al. 1999; Pillai et al. 2004): T1 cells, identified by the phenotype, IgMhi CD23− B220+ AA4.1+, enter the follicles of all secondary lymphoid organs under the influence of CXCL13. They depend on BCR for survival but do not express BAFF-R (Hsu et al. 2002). T1 cells upregulate IgD and CD23 and CD21 and undergo further differentiation to become mature follicular B cells (IgMlo IgDhi, CD21int, and CD23+). This final differentiation step appears to require a BCR signal, as it is lost in the absence of Btk. The size of this follicular population is modified by mutations that affect the BCR signaling threshold. T2 cells, identified by the phenotype, IgMlo IgD+ B220+ CD21hi CD1dhi CD23hi AA4.1+, are located exclusively within the follicles of the spleen. This population depends on BAFF for survival and increases in mice that constitutively express high levels of BAFF (Batten et al. 2000). It has been suggested that T1 cells have the potential to become either naïve follicular or naïve MZ B cells, whereas T2 cells are precursors of the naïve subset of MZ B cells, but this suggestion remains to be proved. The factors that determine the MZ versus follicular B cell decision remain controversial and are discussed below. 2.
Marginal Zone versus Follicular B Cells
Differences between follicular and marginal zone B cells include their repertoire of receptor specificities, response to antigens (see “Homeostasis and Selection”), response to chemokines, and adhesion molecules. The last factor appears to be important for localizing MZ B cells (see earlier). However, it remains unclear whether B cells are selected into these respective populations according to their receptor specificity, which could determine their state of activation and adhesion molecule expression, or whether they differentiate independently of BCR specificity and then acquire the other phenotypic characteristics. These explanations are not mutually exclusive, especially since MZ B cells appear to arise from more than one type of precursor.
86 As mentioned earlier, transitional B cells have the potential to become either follicular or MZ B cells. Signaling via Notch family members influences this cell fate determination. Notch is known to affect other lineage decisions in the immune system, including T lineage commitment (see “Early Thymus Development”). Ligation of Notch2 is an absolute requirement for MZ B cell differentiation (Saito et al. 2003). Ligation of Notch2 by its ligands Delta-like 1, 3, and 4 or Jagged 1 and 2 causes proteolysis of the intracellular portion of Notch, which translocates to the nucleus and forms a transcriptional regulatory complex with RBP-Jκ/CBF1 (mammalian homolog of suppressor of Hairless) and Mastermind-like 1. Deficiencies of Notch2, RBP-Jκ/CBF1, Delta-like 1, and Mastermind-like 1, all result in absence of MZ B cells (Hozumi et al. 2004; Maillard et al. 2003). Conversely, mutations of MINT (Msx2interacting nuclear target), which is a negative regulator of Notch signaling, increase MZ B cell differentiation and reduce follicular B cells (Kuroda et al. 2003). The Id family of transcriptional regulators antagonize basic helix-loop-helix transcriptional regulators, especially E proteins (Benezra et al. 1990). T1 transitional cells in the spleen express high levels of Id2, which inactivates E2A function. Normally, E2A promotes differentiation into follicular B cells. Expression of Id2 antagonizes this signal, promoting MZ B cell differentiation. By contrast, in the absence of Id2, differentiation to MZ cells is almost completely inhibited (Becker-Herman et al. 2002). BAFF also appears to enhance the MZ B cell population (Schiemann et al. 2001) and DCs within the MZ have been suggested as the source of BAFF (Balazs et al. 2002). However, it is unclear whether BAFF acts to enhance transitional cell differentiation to MZ B cells or whether it simply promotes MZ B cell survival. Nevertheless, there is abundant, albeit circumstantial, evidence that the follicular versus MZ B cell decision is cell autonomous based on the specificity of the BCR. In both normal and transgenic mice, the MZ is enriched with self-reactive B cells (Lopes-Carvalho and Kearney 2004). Several strains of mice expressing BCR transgenes have been identified, in which B cells are preferentially selected to enter the MZ based on Ig heavy chain expression. Selection does not occur in the absence of Btk (signaling via the BCR), raising the possibility of positive selection (Martin and Kearney 2000; Vinuesa et al. 2001). In addition, the partially activated phenotype of MZ B cells suggests that they have already come into contact with antigen. Opposing these findings, in several models in which the threshold of BCR signaling has been modified, increasing the signal threshold appears to enhance recruitment to the MZ (Table 1-4). These modifications include defects directly downstream of the BCR that affect signal transduction via Btk, as well as defects in positive and negative regulators of the BCR signal (Pillai et al. 2005). Diversion into the MZ population based on the absence of BCR signaling is consistent with the surface phenotype of
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follicular and marginal zone B cells, and their responsiveness to antigen. BCR ligation results in IgM downregulation, and BCR sensitivity or the capacity to respond to ligation correlates with surface IgM expression. Recent bone marrow emigrants that are IgMhi are the most responsive, whereas anergic cells (IgMlo IgDhi) are least responsive. Similarly, MZ cells that are IgMhi are also very responsive to BCR ligation and readily differentiate into antibody-forming cells. Stimulation of B cells with antigen in vivo causes a reduction in the level of IgM expression in proportion to the intensity of the stimulus (Cook et al. 1997). Concordant with the idea that a weak signal leads to MZ selection is the idea that a stronger selection signal selects cells to the follicle. Downregulation of IgM on follicular B cells, therefore, may represent further evidence of a subthreshold BCR ligation during follicular selection. Again this must be a signal that is still below the threshold that initiates an immune response, because under these conditions, B cells move to the outer T zone (Liu et al. 1991). As noted earlier, MZ B cells are not derived exclusively from transitional B cells. At least in rats and humans, the MZ is enriched for memory B cells that have undergone somatic hypermutation (Dunn-Walters et al. 1995). During the course of a T-dependent immune response in mice, high-affinity B cells colonize the MZ (Shih et al. 2002). Furthermore, the MZ can also be populated by follicular B cells from lymph nodes, and these cellular immigrants share the functional characteristics of MZ B cells (Vinuesa et al. 2003). Thus, it is conceivable that each of these pathways of MZ selection will account for different subsets of this heterogeneous population (Figure 1-6). 3.
B-1 Cell Selection
For many years, it was thought that B-1 and conventional follicular B cells (B-2 cells) belong to separate lineages; however, recent evidence suggests that B-1 and B-2 cells may be selected after BCR ligation. BCR specificities appear to guide the development of B-1 or B-2 cells (Lam and Rajewsky 1999). Indeed, strong self-antigen binding appears to be a prerequisite for B-1 selection (Hayakawa et al. 1999). There is a close correlation between the BCR activation threshold and production of B-1 cells (Table 1-4). A decrease in the BCR activation threshold leads to an expansion of B-1 cells, whereas mutations that increase the B cell activation threshold abolish the B-1 population (Clarke and McCray 1993; Cyster and Goodnow 1995b; Rickert et al. 1995). B-1 cells are selectively absent from both Btk-mutant and CD19−/− mice, as well as from mice bearing other mutations that affect the Btk signaling pathway. Conversely, the population is increased in mice such as CD22−/−, CD72−/−, and Lyn−/−, in which the BCR signaling threshold is increased. Thus, the size of the B-1 compartment is a surrogate marker of the BCR activation threshold. These data are also consistent with the finding that treatment with cyclosporin A, which interferes with signaling downstream of the BCR, appears to block selection into the B-1 repertoire (Arnold et al. 2000).
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IMMUNE RESPONSE STRUCTURES A.
Overview of Immune Responses
After either infection or immunization, innate and adaptive components of the immune system are activated in a coordinated and complex fashion. This usually involves activation of individual cells, which liberate cytokines that have effector functions or enable communication between one cell and another or both. Activation is also accompanied by a change in the expression of surface molecules that influence patterns of migration, cell-cell interaction, and mutual stimulation. As antigen specific-lymphocytes are rare, proliferation is usually crucial for mounting an effective response. Furthermore, cells often differentiate, generating populations of cells such as plasma cells and memory cells that are better suited to deal with the pathogenic threat. All of these events take place in secondary lymphoid organs where new microenvironments dedicated to mounting an immune response form. Pathogens have evolved many different mechanisms to evade the immune response and vary tremendously in their mechanisms of virulence and means of damaging host cells. These include variations in the nature of antigens exposed on the surface of the pathogen, an ability to change these antigens at different stages in their life cycle, and resistance to effector mechanisms. In addition, there are many differences in the behaviors of pathogens within the host. Important variables include residence inside or outside of host cells, rate of replication, life span in the host, and capacity for direct cytotoxicity. Consequently, many different effector strategies have evolved to deal with different types of pathogens. For example, a rapid antibody response and complement deposition are important to facilitate phagocytosis of encapsulated bacteria, cytolytic CD8+ cells are essential to kill virus-infected cells, and IFN-γ–secreting CD4+ T cells are required to activate macrophage killing of intracellular mycobacteria. Immune responses are initiated at the sites of antigen entry. Of particular importance are the skin and mucosal surfaces because of the potential for exposure to pathogens at these sites. As noted, these tissues are rich in lymphatics, which drain into lymph nodes. The first events of an immune response take place in the nonlymphoid parenchyma, where DCs resident in infected tissues capture antigen. After this, DCs migrate to draining lymph nodes (or spleen if the infection is blood-borne) where they present processed antigen to T cells. The outcome of the DC-T cell interaction depends not only on the characteristics of the antigen but also on the type of dendritic cell, its stage of development, and its state of activation. The phenotype of DCs changes during their migration from the periphery to the secondary lymphoid organs, from a cell efficient at phagocytosis, to one efficient at antigen presentation to T cells (Cavanagh and Von Andrian 2002). This change is often triggered by recognition of molecular motifs on the
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pathogen that are not present on host cells via a discrete subset of highly conserved germline receptors called TLRs (Iwasaki and Medzhitov 2004). This recognition event provides complex signals that bias the subsequent immune response toward activation and inflammation. Upon entry into the secondary lymphoid organs, DCs migrate to the outer T zones under the influence of chemokines, where they become interdigitating dendritic cells (IDCs). Here, they come into contact with many T cells, which survey antigen/MHC complexes displayed on the IDC (Miller et al. 2003, 2004). Upon recognition of specific peptide bound by MHC class II molecules (so-called cognate interaction), CD4+ T cells form an intimate association with the DC (an immunological synapse, see later section), which permits establishment of a complex and specific communication between the IDC and the T cell to be established. Depending on the state of DC activation and differentiation, this can result in T cell priming (Inaba and Steinman 1984), thus setting in train a specific immune response or induction of T cell tolerance. After receiving priming signals from DCs, T cells differentiate into effector cells. The pathway of differentiation is determined by the nature of the priming signals (Kapsenberg 2003; Pulendran 2004). The two most important subsets are helper T cells and cytotoxic T cells. Most CD8+ T cells differentiate into cytotoxic T cells. CD4+ cells, by contrast, can differentiate into three types of effector helper T cells (Th): Th1, Th2, and TFH. Th1 cells are particularly effective at activating macrophages through provision of IFN-γ and TNF, which then acquire the capacity to kill intracellular bacteria (e.g., mycobacteria). Th2 cells are very effective at activating B cells and stimulating their differentiation to make antibody. TFH cells express CXCR5 and the highest levels of inducible T cell costimulator (ICOS) and operate on the subset of B cells that differentiate in germinal centers within lymphoid follicles (Breitfeld et al. 2000; Hutloff et al. 1999; Kim et al. 2001; Moser and Ebert 2003; Schaerli et al. 2000). This TFH subset is crucial for germinal center B cell selection and terminal differentiation into memory B cells or long-lived antibody-forming cells (Vinuesa et al. 2005b). To become properly activated and to differentiate into effector cells, B cells require two types of signals (Bretscher and Cohn 1970) (Figure 1-7). The first is delivered through the antigen receptor (surface Ig) after binding of intact antigen, either free or on the surface of another cell (e.g., on the pathogen itself or on the surface of a FDC). The second signal takes different forms. For so called T-independent (TI) antigens, the second signal is delivered through TLRs, for example, lipopolysaccharide present within the wall of gram-negative bacteria (Nagai et al. 2002). For T-dependent (TD) antigens, signal 2 is delivered when B cells make specific interactions with helper T cells, which deliver signals to the B cells via cell-to-cell contact and secretion of cytokines. Once B cells receive two activating signals; they proliferate in the outer T zone (Liu et al. 1991). Subsequent differentiation
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Short-lived Plasmablasts and plasma cells Spleen and LNs
Outer T zone
Germinal Center
DZ
Somatic Hypermutation
centroblasts
Affinity maturation
LZ centrocytes
High affinity Long-lived plasma cells
High affinity memory cells
Bone marrow
Fig. 1-7 Overview of B cell activation and differentiation in response to antigen. After B cell receptor ligation, B cells move to the outer T zones of secondary lymphoid organs. From here they can differentiate into plasmablasts, located in the bridging channels of the spleen and medullary cords of lymph nodes, or germinal center B cells, depending on the intensity of the B cell receptor stimulus, and the presence or absence of T cell help. Germinal centers give rise to longlived plasma cells, which reside in the bone marrow, and memory B cells.
depends on the nature of both the antigenic stimulus and the T cell help. B cells activated through TI mechanisms, which typically occurs during infections with encapsulated bacteria, differentiate exclusively outside follicles and give rise to shortlived plasma cells. By contrast, after stimulation with TD antigens (such as proteins), B cells can proceed down one of two differentiation pathways. In the first pathway, extrafollicular proliferation and differentiation give rise to short-lived plasma cells, in a response that is similar to that observed after contact with TI antigen (MacLennan et al. 2003). The second pathway takes place within follicles, where a small number of seeding cells give rise to germinal centers. Here, affinity maturation takes place, and B cell memory is established (MacLennan 1994). Whereas the majority of murine memory B cells migrate to the splenic marginal zones where they reside for prolonged periods of time, some memory T cells circulate in the blood (Figure 1-7). Affinity maturation is a crucial and remarkable event that helps explain why the germinal center environment and cellular dynamics are so complex (see later). During affinity maturation, somatic mutations are introduced stochastically into the germline of Ig variable region genes. Outside of malignant and immature B cells, acquisition of somatic mutations is confined to germinal center B cells. As a consequence of
somatic mutation, rare B cells come to express surface Ig that binds to the immunogen with even higher affinity than their precursors. However, these rare cells must be selected and allowed to proliferate preferentially if the overall affinity for antigen is to increase. In addition, there is also the potential for B cells to either lose affinity for antigen altogether, or to acquire a self-reactive receptor.
B.
Structural and Molecular Architecture of Immune Responses
1.
T Cell Activation and Differentiation
a. DENDRITIC CELL-T CELL INTERACTIONS, FORMATION OF IMMUNOLOGICAL SYNAPSES, AND T CELL PRIMING When DCs bind antigen in peripheral tissues, they mature and upregulate CCR7, and then under the influence of the CCR7 ligands CCL19 and CCL21, they enter T zones of draining lymph nodes and spleen. In lymph nodes, they locate around the HEVs (Bajenoff et al. 2003; Cavanagh and Von Andrian 2002; Gretz et al. 1997) (see “Normal B Cell Migration”). Naïve T cells also express CCR7 and enter the T zones of secondary lymphoid organs. Individual DCs form clusters with several
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antigen-specific T cells, and this interaction lasts for 24–48 hours (Bajenoff et al. 2003; Ingulli et al. 1997). Priming of naïve T cells by DCs is a critical event for the initiation of all TD immune responses. Priming is initiated when a sustained signal is delivered via the TCR and initiates chromatin remodeling, which is essential for cytokine synthesis (Avni et al. 2002; Lezzi et al. 1999). Because the T cell zone is full of T cells that are not specific for the peptide presented by any particular DC, the stimulus needs to be given precisely and specifically. Organ culture of explanted lymph nodes and intravital imaging of immune responses using two-photon microscopy have provided crucial insights into how this is achieved, documenting T cell movement in the lymph nodes and the stability of DC-T cell interactions that take place during T cell priming. Naïve T cells migrate randomly in the deep paracortex of lymph nodes so that each DC contacts 500–5000 DCs per hour (Bousso et al. 2002; Miller et al. 2002, 2003, 2004). Antigen-specific T cells “swarm” within specific microenvironments in the T zone, probably as an initial reaction to a particular DC, before they establish stable cognate interactions (Miller et al. 2002). Using anti-L-selectin antibody to transiently block entry of lymphocytes into draining popliteal lymph nodes to synchronize immune responses, three stages of the DC-T cell interaction have been identified (Mempel et al. 2004). There is an initial transient interaction that lasts 2–8 hours, followed by a stable interaction over the next 8–24 hours, and finally reversion to a transient interaction during the last 12 hours. TCR signaling takes place during the first stage, and IL-2 production is initiated during the second stage. Activated T cells transiently downregulate S1P1 (see “Entry into and Exit from the Spleen”), which leads to retention of activated T cells on lymphoid organs for up to the first four cell divisions, and this probably aids the process of T cell priming (Matloubian et al. 2004). Specificity of the interaction between T cells and DCs is achieved by the formation of an immunological synapse (named as an analogy of specific neurotransmission). Formation of the synapse depends on stabilization of T cell/DC complexes by adhesion molecules. After this, T cells undergo polarization, in which extensive membrane cytoskeletal reorganization results in a central cluster of TCRs, also known as a cSMAC (central supramolecular activation cluster), surrounded by a ring of adhesion molecules, particularly LFA-1, which binds ICAM-1 on the DC (Grakoui et al. 1999). As with B cells, two signals are necessary for effective activation, survival, and differentiation of T cells into effector populations. Signal one is delivered by the TCR after ligation by MHC/peptide complexes. The second signal, also referred to as costimulation, takes various forms. CD28 on T cells is one pathway through which the second signal is delivered. CD28 is a critical costimulatory molecule on T cells that binds B7-1 and B7-2 on antigen-presenting cells. Disruption of CD28 signals impairs T cell activation in vitro and in vivo (Schweitzer
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and Sharpe 1998). However, the view that S1 = TCR and S2 = CD28 is probably an oversimplification. A more complex picture is now emerging in which different costimulatory molecules play important and interdependent roles during T cell activation. Thus, while CD28 is probably at the top of the hierarchy of costimulatory molecules, in the absence of CD28, ICOS can still be upregulated although to a lower degree (Beier et al. 2000; McAdam et al. 2000) and can provide sufficient costimulation to restore effector T cell responses (Suh et al. 2004). ICOS-ICOS ligand interactions also acquire a prominent role during T cell costimulation by endothelial cells (Khayyamian et al. 2002). Of importance, engagement of CD40 on DCs by CD40L on T cells upregulates expression of B7 molecules on antigen-presenting cells (Foy et al. 1996). In addition, both CD4 and CD8 coreceptors, which bind to nonpolymorphic regions of MHC class II and I molecules, respectively, and determine the specificity of CD4+ T cells for class II/peptide and CD8+ T cells for MHC class I/peptide complexes, also contribute to the primary activation signal by increasing the sensitivity of TCR responsiveness to antigen (Ledbetter et al. 1990). Finally, interactions between adhesion molecules on T cells and DCs (LFA-1 with ICAM-1 or ICAM-2; CD2 with LFA-3 or CD59) also increase the overall avidity of the DC–T cell interaction (Shaw and Dustin 1997). The avidity of the interaction determines the extent of proliferation, outgrowth of T cells with the highest affinity for the peptide/MHC ligand complex, and effector cell differentiation. b. EFFECTOR T CELL SUBSETS In vitro, CD4+ T cell activation in the presence of IL-4 or IL-12 results in polarization toward Th2 and Th1 cells, respectively (Mosmann and Coffman 1989). The transcriptional program that leads to establishment of each of these subsets is influenced critically by T-bet and Gata-3, respectively (Mowen and Glimcher 2004), which are master regulators of cytokine production. Gata-3 and T-bet act through the induction of chromatin remodeling and DNA methylation. Th2 cells predominantly secrete IL-4, IL-5, IL-6, IL-10, and IL-13, which induce a B cell class switch recombination (CSR) to IgG1. By contrast, Th1 cells predominantly secrete IL-2, IFN-γ, and TNF and induce B cell switching to IgG2a. Th1 and Th2 cells reciprocally regulate each other: IL-4 negatively regulates Th1 responses whereas IFN-γ is a potent suppressor of IL-4 secretion by Th2 cells (Seder and Paul 1994). Th1 and Th2 cells can be identified by different chemokine receptors—CXCR3 on Th1 cells and CCR4 on Th2 cells (Bonecchi et al. 1998). However, this means of identification is not completely reliable, because under different activating conditions, the pattern of chemokine receptor expression can change. In vivo, the distinction between Th1 and Th2 cells on the basis of cytokine and chemokine expression is far less perfect. Furthermore, the idea that Th1 cells favor cytotoxicity and Th2 cells favor antibody responses is an impoverished one, and cannot account for the complexity of effector mechanisms that
90 have evolved to deal with different pathogens. Other criteria such as expression of CD62L and sialylated P-selectin ligand (sPSGL-1) in immunized mice are better predictors of the different abilities of effector T cells to stimulate either B cell or delayed-type hypersensitivity (DTH) responses (Campbell et al. 2001). Such differences in the pattern of adhesion molecule expression by effector subsets also help explain how they migrate to particular microenvironments during immune responses to different types of pathogens. For example, sPSGL-1 expression on T cells is induced by IL-12 (Xie et al. 1999) and mediates T cell migration from CD62P-expressing blood vessels into inflamed tissues (Austrup et al. 1997). CD62L+ sPSGL-1+ cells are potent stimulators of DTH reactions and secrete large amounts of IFN-γ. Also, CD62L+ sPSGL-1+ effector T cells capable of IL-4 production are typically found in the lungs during leishmanial infection. These IL-4–secreting effector T cells also lead to macrophage and granulocyte activation and parasite clearance. By contrast, CD62L− sPSGL-1− T cells are found in the lymph nodes after injections of antigen and cholera toxin provide effective help for B cell antibody responses. These T cells do not induce effective DTH reactions, do not express CD40L, and do not produce significant amounts of either IFN-γ or IL-4 (Campbell et al. 2001). It is thus unclear whether this cellular subset corresponds to the T cells important for providing the initial stimulus for B cell activation in the outer T zone (which would be equivalent to in vitro-generated Th2 cells) or whether they represent the TFH cell subset that predominantly provide help to germinal center B cells. TFH are emerging as a discrete T cell subset with cytokine, chemokine receptor, and gene expression signature different from Th1 or Th2 cells (Vinuesa et al. 2005b). They express CXCR5 (important for follicular localization), Bcl-6, CD200, CD84, PD-1, IL-21, CXCL13, and high levels of ICOS. ICOS and ICOS ligand (B7h)-deficient mice have impaired TD-antibody responses and germinal center formation. IL-21 has been shown to drive both Bcl-6 and Blimp-1 expression and IL-21 receptor-deficient mice exhibit CSR defects (Ozaki et al. 2004; Pene et al. 2004). The ontogeny of TFH cells is still unknown, but a CD3− CD4+ accessory cell that expresses Ox40L and CD30L and localizes to follicular cell-T cell boundaries and within follicles appears to play a role in directing TFH cell differentiation (Gaspal et al. 2005; Kim et al. 2003). The fact that TFH cells do not express Gata-3 or T-bet suggests that they differentiate separately from Th1 and Th2 cells. In mice deficient for SAP, a T cell adaptor molecule for the Slam-family of receptors including CD84 and Ly9, germinal center formation is severely impaired (Crotty et al. 2003; Engel et al. 2003; Hron et al. 2004). SAP is known to transmit signals to Fyn, leading to NF-κB activation (Davidson et al. 2004). This suggests the TCR/SAP/Fyn signaling pathway may also be critical for TFH differentiation. Recently, a novel ubiquitin ligase, Roquin, has been identified as a negative regulator of self-reactive TFH differentiation. Mice bearing a point mutation in this gene develop spontaneous germinal center formation and systemic autoimmunity (Vinuesa et al. 2005a).
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Effector CD8+ T cells are important for killing virus-infected cells. Perforin and granzyme are the main mediators of cytolysis. CD8+ effectors are potent producers of IFN-γ but lose the capacity to produce IL-2 and require help from IL-2–secreting CD4+ cells to sustain proliferation (Matloubian et al. 1994). After differentiation in the secondary lymphoid organs, CD8+ cells migrate to nonlymphoid parenchyma (where infected cells are located). This migration depends on downregulation of CCR7 and CD62L and upregulation of another suite of receptors expressed in the HEVs of the destination organs, such as α4β7 integrins within mucosal tissues. c. T CELL MEMORY During an immune response, most effector T cells die after the peak of proliferation due to upregulation of Fas (Lenardo et al. 1999). This process maintains homeostasis of the T cell population. Nevertheless, a small population of antigen-experienced CD4+ and CD8+ T cells remain for long periods of time after immunization. It is not clear what determines whether activated T cells go on to become memory cells. It has been shown that IL-7R+ effector CD8+ T cells eventually become memory T cells. These IL-7R+ CD8+ T cells also coexpress CD8αα. CD8αα plays a role in thymic selection of self-reactive CD8+ T cells and can interact with the nonclassic MHC-molecule TL to modulate TCR. CD8αα T cells are enriched in the intraepithelial lymphocyte pool (reviewed in Gangadharan and Cheroutre 2004), but it remains unclear whether gut CD8αα T cells are bona fide memory T cells. It has also been shown that CD8+ T cell differentiation into the memory pool requires CD4+ T cell help and CD40-CD40L interactions (Rocha and Tanchot 2004). Memory cells differ in phenotype, function, circulation patterns, survival requirements, and activation requirements from recently activated effector T cell subsets. Memory T cells can be easily reactivated upon antigen reencounter without the need for CD28 costimulation and do not produce a significant amount of cytokines in the absence of activation. Memory CD8+ and CD4+ subsets are heterogeneous in terms of circulation and homing patterns. Different subsets circulate through secondary lymphoid organs (CCR7+ and CD62L+) and nonlymphoid tissues (CCR7−). Liver and lung have been found to be the largest reservoirs of CD4+ memory T cells in mice after the effector phase of an immune reaction. These tissue-homing memory CD4+ cells are poor producers of IFN-γ and IL-2. Some memory CD8+ T cells retain perforin expression and thus cytotoxic capacity (Hamann et al. 1997). The signals necessary for maintenance of the population of long-lived memory cells is controversial. In general, maintenance of T cell memory does not require antigen or the presence of MHC, but although CD8+ cells can live for the lifetime of the host, CD4+ memory cells are not indefinitely maintained (Homann et al. 2001). Most memory cells are not in cell cycle, although a small proportion of CD8+ T cells are actively cycling, independent of MHC/peptide binding and probably driven by IL-15 (Zhang et al. 1998). By contrast, IL-15 does not enhance the proliferation of CD4+ memory T cells.
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Effector B Cell Populations
To mount an effective antibody response, B cells undergo terminal differentiation to plasma cells, which are the principal source of antibody. During differentiation, B cells can undergo CSR, so that the immunoglobulin molecules expressed and ultimately secreted by the plasma cells change from IgM and IgD on naïve cells to IgG, IgA, or IgE. Switching is not universal, and some plasma cells continue to secrete IgM. CSR to TD isotypes (all except IgG3) is absolutely dependent on CD40 ligation (Armitage et al. 1992), and the pattern of switching is determined by cytokines released from helper T cells. The molecular machinery that mediates DNA cleavage and recombination has been found to be similar to that necessary for somatic mutation (see later and review by Chaudhuri and Alt 2004). CSR takes place in both extrafollicular responses and germinal centers. CSR does not affect antigen specificity, but it does have important implications for effector responses. For example, IgM molecules associate into pentamers so that even low-affinity antibodies can result in a complex that binds antigen with high avidity. In addition, IgM and IgG1–3 fix complement efficiently. IgA associates with the secretory piece, rendering it resistant to proteolysis in the gut lumen. Perhaps of greatest significance is the ability of IgG to cross the placenta, providing the neonate with passive immunity. B cells differentiate to plasma cells via several pathways. First, naïve recirculating B cells can be activated in both T-dependent and T-independent pathways to generate a mostly short-lived population located in the extrafollicular regions of secondary lymphoid organs. This population is crucial for the early host response to pathogens. This population can also be generated very efficiently from MZ B cell precursors (Martin and Kearney 2002). By virtue of their location and partially activated phenotype, this population appears to be critical for rapid responses to blood-borne antigens. Long-lived plasma cells that bind antigen with high affinity are generated as a terminal differentiation pathway of germinal center B cells. After emerging from germinal centers in secondary lymphoid organs, these long-lived plasma cells are usually located in the bone marrow. Finally, there are memory B cells, which are the other product of germinal centers and are located in highest numbers in the marginal zone. Memory B cells express high-affinity receptors and can be stimulated to differentiate into plasma cells. A T-independent pathway of memory B cell formation derived from B1b cells has recently been reported (Alugupalli et al. 2004; Haas et al. 2005). C.
B Cell Response in Secondary Lymphoid
Microenvironments: T Cell–B Cell Interactions Naïve B cells recirculate between the follicles of secondary lymphoid organs due to expression of CXCR5 (see “Chemokine Regulation of B Cell Migration to Follicles”) (Figure 1-8).
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After binding to antigen in the circulation, or on the surface of FDCs within follicles, B cells migrate towards the follicular-T zone border by decreasing expression of CXCR5 and CCR7 (Cyster 2003). By contrast, primed T cells change chemokine receptor expression in the opposite direction, upregulating CXCR5 and downregulating CCR7. Consequently, activated T and B cells migrate toward each other for a rendezvous in the outer T zone at the follicular border (Luther and Cyster 2001). During TD responses, help from T cells is essential for B cell proliferation, CSR, and differentiation of B cells. After receiving T cell help B cells can differentiate extrafollicularly or enter the follicles to give rise to germinal center reactions. If B cells that have bound protein antigen do not receive T cell help (this can occur if the B cell binds self-antigen), B cells undergo apoptosis in the outer T zone. The conclusion is that location of antigen-stimulated B cells in the outer T zone is not only critical to maximize the chances of establishing cognate interactions with T cells, but it is also decisive in deciding between B cell immunity and tolerance (Goodnow 2005). Cognate T cell–B cell interactions require specific recognition of the peptide/MHC complex on B cells by TCRs as well as accessory signals in the form of cell-cell contact and cytokines. Cell-cell contact signals include adhesion molecules such as LFA-1 and ICAM-1, CD40, and the B7 family of molecules. Provision of CD40L signals by activated T cells is essential for TD antibody responses, since these are severely crippled in mice deficient in CD40 and CD40L. Activated B cells upregulate costimulatory molecules (B71, B7–2, and OX40L) (Bluestone 1995). The kinetics of B7.1 and B7.2 upregulation and their respective roles during the initiation of immune responses differ slightly. B7.2 is upregulated more rapidly than B7.1 after BCR ligation, but the latter is induced very efficiently by LPS (Hathcock et al. 1994). CD28−/− and mice doubly deficient for B7.1 and B7.2 have severe impairment of TD B cell responses with complete absence of germinal centers and CSR (Ferguson et al. 1996; Shahinian et al. 1993). B7.2 is absolutely required to respond to soluble antigen, but responses to antigen in adjuvant are normal in B7.2−/− mice. Adjuvant has been shown to upregulate B7.1, which is probably sufficient to drive antibody response to strong immunogens (Borriello et al. 1997). Another B7 family member, B7h (or ICOS ligand) is constitutively expressed on B cells and binds the CD28 homolog ICOS. B7h expression increases in the presence of LPS or TNF, and ICOS is expressed on T cells after TCR ligation. Many nonlymphoid tissues also express B7h, especially after exposure to bacterial endotoxin (Swallow et al. 1999). Ligation of ICOS by B7h induces CD40L expression on T cells. ICOS−/− mice exhibit a defect in CSR and germinal center formation in response to protein antigen. Cytokines secreted by helper T cells augment B cell CSR and proliferation. IL-4 and IL-13 mainly favor switching to IgG1, whereas IFN-γ is a potent inducer of IgG2a. ICOS expression is important for Th2 cytokine secretion since ICOS−/− T cells produce high levels of IFN-γ but significantly
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T zone Follicle Recirculation
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Differentiation TFH
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Unmutated Short-lived Low affinity
Mutated Long-lived High affinity
CXCL12 Bone marrow
Fig. 1-8 Overview of B and T lymphocyte chemokine–driven migration to the different lymphoid microenvironments during responses to antigen. After binding antigen, follicular B cells upregulate CCR7 and migrate toward the T zone where they encounter primed T cells that have upregulated CXCR5 while downregulating CCR7 and relocating to the T zone-follicle boundary. B cells can then move into the follicles or differentiate into short-lived plasma cells in extrafollicular foci at the T zone-red pulp junctions. Expression of CXCR4 by plasma cells (PC) leads their migration towards CXCL12-expressing stromal cells, which includes red pulp dendritic cells and bone marrow cells. Within follicles, proliferating B blasts are first induced to move toward the dark zone due to expression of CXCR4 in response to CXCL12 produced at this site and differentiate into centroblasts (CB). At this CB stage somatic hypermutation is targeted to Ig V region genes, and isotype switching is initiated. The differentiated progeny, centrocytes (CC), migrate toward the light zone as a consequence of upregulation of CXCR5, the receptor for CXCL13 secreted by FDCs. Centrocytes terminally differentiate into memory cells or long-lived plasma cells that generally migrate to the bone marrow, again due to increased responsiveness to CXCL12. (See color insert in the back of the book.)
lower levels of IL-4 and IL-10 than wild-type animals. Mice and humans with mutations in ICOS have very low levels of IgG1 and IgE (McAdam et al. 2001; Tafuri et al. 2001). T cell–B cell interactions involve mutual stimulation, but it remains unclear how B cell costimulation influences the outcome of T cell differentiation, although there is emerging evidence to suggest that B cell–mediated signals may be critical for directing follicular migration of T cells and subsequent differentiation into germinal TFH cells and at least a proportion of memory T cells. 1.
Extrafollicular B Cell Differentiation
a. KINETICS AND ARCHITECTURE OF THE EXTRAFOLLICULAR B CELL RESPONSE Extrafollicular responses are initiated when B blasts migrate to the junction zones of the spleen (between
the T zones and the red pulp) or lymph node medullary cords, where they differentiate into proliferating antibody-secreting plasmablasts. Extrafollicular responses play a critical role in host defense because they are responsible for early antibody production (Thorbecke and Keuning 1956). Initially the extrafollicular response produces IgM, but, later, CSR takes place and IgG is produced (Jacob et al. 1991). Survival and differentiation of extrafollicular plasmablasts into nondividing plasma cells is at least in part regulated by a subset of CD11chi DCs that colocalize with the plasmablasts in the junction zones of the spleen and medullary cords of lymph nodes (Vinuesa et al. 1999). Migration of plasmablasts to this location is influenced by chemokine expression (Figure 1-8). Plasmablasts downregulate expression of receptors for T zone chemokines such as CCL21 and CCL19 and express CXCR4,
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the receptor for the chemokine CXCL12. CXCL12 is expressed in the splenic red pulp medullary cords of lymph nodes and the bone marrow (Hargreaves et al. 2001), and in the absence of CXCL12 plasmablasts fail to migrate to the normal sites of antibody production. It is not clear whether CXCL12 is produced exclusively by CD11chi DCs or whether other stromal components of the splenic red pulp and medullary cords also contribute. Plasmablasts upregulate syndecan-1 (CD138) and downregulate surface Ig but contain high levels of cytoplasmic Ig. They undergo approximately five cell divisions (Sze et al. 2000) before coming out of cell cycle and terminally differentiating into nondividing plasma cells. Most extrafollicular plasma cells are short-lived and undergo apoptosis within 2–3 days. Nevertheless, a small proportion of up to 10% appear to survive for much longer in the spleen (Sze et al. 2000). There is evidence that IL-6 and BAFF produced by macrophages and DCs play an important role in sustaining growth and survival of plasmablasts in extrafollicular foci (Balazs et al. 2002). In addition, BAFF and its close relative APRIL can promote CSR, although in contrast with the effects of T cell–derived cytokines, no particular pattern of switching appears to be induced by either BAFF or APRIL (Litinskiy et al. 2002). b. MOLECULAR DRIVERS OF EXTRAFOLLICULAR RESPONSES Two transcriptional repressors, Blimp-1 (B lymphocyte–induced maturation protein-1) and Bcl-6, coordinate the transcriptional program of plasma cell and germinal center cell differentiation, respectively. Upregulation of Blimp-1 induces plasma cell differentiation. Several other transcription factors have been identified that are required for plasma cell differentiation. Expression of c-myc is repressed by Blimp-1, and this induces a switch from proliferation to differentiation and seems to be necessary but not sufficient for plasma cell differentiation (Lin et al. 2000). Blimp-1 also suppresses CIITA (class II transactivator) transcription. CIITA is a cofactor required for MHC class II transcription and is expressed at all stages of B cell development until plasma cell differentiation. As a consequence of MHC downregulation, plasma cells cannot make cognate interactions with T cells (Piskurich et al. 2000). XBP-1 (human X box binding protein-1) is induced after B cell activation and remains expressed at high levels in plasma cells. XBP-1 deficiency is embryo lethal, but it has been shown to be essential for plasma cell formation by blastocyst complementation (Reimold et al. 2001). Finally, differentiation of mature B cells to plasma cells is negatively regulated by BSAP (B cellspecific activator protein, also known as PAX-5), which is expressed in all stages of B cell development except plasma cells. BSAP overexpression in a mature B cell line reduced Blimp-1 expression and suppressed spontaneous appearance of plasma cells (Usui et al. 1997). Ligand pairs from the TNF family also regulate plasma cell differentiation. CD27 is expressed by B and T cells and binds CD70 to deliver a signal that appears to be important for
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terminal differentiation of B cells into plasma cells (Jacquot et al. 1997). By contrast, engagement of CD153 by CD30 inhibits plasma cell differentiation and expression of Blimp-1 (Cerutti et al. 2000). In addition, growth of plasma cells depends on the availability of IL-6, which is secreted by a variety of cells including DCs and B cells themselves. Overexpression of IL-6 is associated with plasma cell dyscrasia, polyclonal plasmacytosis, lymphadenopathy, extramedullary hematopoiesis, and mesangiocapilliary glomerulonephritis. On the other hand, in mice with IL-6 deficiency, pristane-induced plasmacytosis cannot be induced (Dedera et al. 1996), and these mice exhibit defects of both germinal center formation and CSR (Kopf et al. 1998). 2.
Molecular Regulation of Germinal Centers
a. OVERVIEW OF THE KINETICS AND ARCHITECTURE OF GERMINAL CENTER REACTIONS Germinal centers are typically, but not exclusively, the result of TD B cell responses (Garcia de Vinuesa et al. 2000). Germinal centers arise from a small number of precursors derived from B cells activated in the T zone, which undergo massive proliferation, giving rise to centroblasts that populate what is known as the dark zone (MacLennan 1994; Martinez-Valdez et al. 1996). Centroblast localization to the dark zone is due to expression of CXCR4 (Allen et al. 2004) (Figure 1-8). Centroblasts downregulate surface Ig expression and during proliferation, Ig genes acquire somatic point mutations (Berek et al. 1991; Feuillard et al. 1995). Centroblasts exit cell cycle to become centrocytes, which are located in close proximity to mature FDC networks in the germinal center light zone. Expression of CXCR5 by centrocytes has been shown to drive localization to the light zone (Allen et al. 2004) (Figure 1-8). Centrocytes upregulate somatically mutated Ig, and those that have either lost affinity for the immunizing antigen or have acquired affinity for a self-antigen are deleted, whereas rare centrocytes that have acquired IgV mutations that increase their affinity for antigen are selected for survival and differentiation. Selected centrocytes differentiate into longlived plasma cells (Slifka et al. 1995; Slifka et al. 1998), which migrate to the bone marrow or lamina propria of the gut, and memory B cells (Liu et al. 1988), which colonize the marginal zones. Some centrocytes remain in the germinal center and are recycled to become centroblasts again (Casamayor-Palleja et al. 1996). b. MOLECULAR SIGNALS THAT ESTABLISH THE STROMAL SUPPORT FOR GERMINAL CENTERS Radiation-resistant stroma provides the crucial source of chemokines that regulate development of lymphoid follicles (see “Follicles”). A subset of this stroma, FDCs, regulate B cell recruitment and also play a key role during selection of centrocytes in established germinal center reactions. FDCs trap antigen in the form of immune complexes by virtue of their expression of receptors for the Fc portion of Ig (FcγRs). However, even in the absence of
94 circulating immune complexes (demonstrated with transgenic mice that express membrane Ig but not secreted [circulating] Ig; Hannuma et al. 2000), germinal center reactions occur and affinity maturation is intact. This is probably because FDCs also express CD21 and CD35, which are receptors for complement degradation products (types 2 and 3 complement receptors [CR2 and CR3]). Because complement is activated on the surface of pathogens, complement degradation products also help localize antigen to FDC networks. This mechanism is crucial, since mice depleted of complement fail to form germinal centers (Klaus and Humphrey 1977). In addition, CD21 and CD35 are expressed by germinal center B cells, and coligation of BCR with antigen and CD21 with complement dramatically reduces the threshold of B cell activation (Dempsey et al. 1996). Predictably, germinal center formation and memory antibody responses are compromised in CD21/35−/− mice (Ahearn et al. 1996; Molina et al. 1996). CD21/35 expression on FDCs is necessary to maintain B cell memory, whereas ligation of CD21/35 on germinal center B cells promotes their survival during the germinal center reaction (Fischer et al. 1998). As described in the section on “Microenvironments in Secondary Lymphoid Organs,” interactions between hematopoietic (B cell) and mesenchymal (FDC) elements are critical during establishment of normal lymphoid follicles. Probably as a consequence of the absence of FDC-derived CXCL13, germinal centers do not form in the B cell areas of LTβR-, TNFRI-, LTα1β2-, and TNF-deficient mice after immunization with TD antigen. However, immunization of LTα−/−, LTβ−/− and LTβR−/− mice results in accumulation of germinal center B cells located ectopically in T zones adjacent to central arterioles that do not contain mature FDC networks. Although in LTα−/− mice germinal center B cell differentiation appears to take place and affinity maturation has also been reported (Matsumoto et al. 1996), this process is less efficient than in wild-type mice. It requires high doses of antigen, consistent with the view that FDCs normally function to provide an available depot of antigen to mediate efficient germinal center selection. c. MOLECULAR SIGNALS DURING GERMINAL CENTER B CELL DIFFERENTIATION AFTER IMMUNIZATION Signals delivered during the initiation of TD responses appear to be critical for germinal center formation. Germinal centers do not form after TD immunization of CD40L−/− mice or after administration of CD40-blocking antibody at the time of TD immunization (Foy et al. 1993; Han et al. 1995; Xu et al. 1994). CD40 ligation also appears to be important for maintenance of established germinal centers, since administration of CD40-blocking antibody after germinal center reactions are underway results in their dissolution (Han et al. 1995). On the other hand, CD40 ligation alone is not sufficient to induce germinal center B cell differentiation, because B cells stimulated with CD40L in vitro differentiate slowly and acquire an unphysiological phenotype.
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Blockade of CD86 (B7.2) ligation at the time of B cell activation abrogates germinal center formation but, unlike CD40 ligation, blockade of CD86 after germinal centers have formed has no effect (Han et al. 1995). Similarly, germinal centers fail to form in CD28−/− mice (Lane et al. 1994). This lack of formation probably reflects the importance of CD86-CD28 interactions during T cell priming rather than B cell activation. On the other hand, CD86 ligation may also activate T cells destined to migrate into the germinal center. Costimulation through OX40 and CD28 has been shown to upregulate CXCR5 on T cells, suggesting that this signal may be crucial during differentiation of T cells to migrate into B cell follicles and promote germinal center formation (Flynn et al. 1998). d. CELL-INTRINSIC REGULATION OF GERMINAL CENTER B CELL DIFFERENTIATION Bcl-6 is a transcriptional repressor that appears to be a master regulator of germinal center B cell differentiation (Dent et al. 1997; Ye et al. 1997). Bcl-6 acts within B cells to inhibit the extrafollicular pathway of B cell differentiation by suppressing Blimp-1 expression (Reljic et al. 2000; Shaffer et al. 2000). In secondary lymphoid organs of mice, Bcl-6 expression is confined to germinal center B cells, both centrocytes and centroblasts, and TFH cells (Cattoretti et al. 1995). In mice deficient in Bcl-6, germinal centers are absent even though the primary follicular structure is normal (Dent et al. 1997; Ye et al. 1997). Because Bcl-6−/− B cells proliferate normally in vitro and Bcl-6−/− mice make normal extrafollicular responses, Bcl-6 appears to be a specific regulator of germinal center differentiation. The B cell-specific transcription factor OBF-1 (also known as OCA-B or BOB-1) is a coactivator of the transcription factors Oct-1 and Oct-2, which bind to the Ig promoter. OBF-1−/− mice have a severe reduction in the number of recirculating follicular B cells. OBF-1 is upregulated in germinal center B cells under the influence of CD40 ligation and IL-4 (Qin et al. 1998). Germinal centers fail to form after immunization of OBF-1−/− mice with TD antigen. Even though CSR takes place in these mice, OBF-1 is also essential for expression of downstream Ig isotypes (Kim et al. 1996). Bone marrow transplantation studies show that this is due to B cell intrinsic effects, rather than to failure of FDC differentiation (Qin et al. 1998; Schubart et al. 2001). e. MOLECULAR REGULATION OF SOMATIC HYPERMUTATION The molecular machinery that mediates somatic mutation has remained a key mystery of immune function until recently. Although a detailed discussion of the way in which somatic hypermutation (SHM) is activated and sustained is beyond the scope of this chapter (see recent reviews by Honjo et al. 2004; Li et al. 2004), a brief outline is provided. SHM introduces point-missense mutations (and, more rarely, deletions or insertions) into immunoglobulin V genes and their flanking regions. Mutations are introduced downstream of the variable region promoter, being most concentrated in the V(D)J rearranged
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region at a density of about 10−3/base pair and a rate of one mutation per cell division. Germinal center–T cell triplets are the preferred targets of SHM, although how the mutator is targeted to these sites remains uncertain. Mutations are transcription dependent, and double-strand (ds) DNA breaks are noted in association with SHM. Activation-induced cytidine deaminase (AID) is essential for SHM and is specifically expressed in germinal center B cells in mice (Muramatsu et al. 2000). Initially, AID was thought to edit RNA, because of its homology with another cytidine deaminase, APOBEC1. However, the prevailing model is that AID binds to single-strand DNA and deaminates deoxycytidine to create deoxyuridine (dU). dU residues are removed by uracilDNA glycosylase, and then apyrimidic endonuclease generates DNA breaks. During class CSR, double-stranded (ds) DNA breaks are also dependent on the action of AID, but they are repaired by nonhomologous end-joining enzymes (DNAdependent protein kinase and the Ku70-Ku80 recombination complex). These are not involved in SHM (Casellas et al. 1998). Instead, error-prone polymerases, including Pol µ, Pol η, Pol ζ or Pol ι, which have been shown to be required for SHM, could account for the introduction of mutations once dsDNA breaks have occurred (Faili et al. 2002; Zeng et al. 2001). f. SELECTION OF GERMINAL CENTER B CELLS Overall improvement of the affinity of serum antibodies for antigen after immunization reflects affinity maturation of B cell responses and indicates that germinal center B cells that have acquired high affinity for antigen are selected. However, somatic mutation in germinal center B cells poses two risks. First, it can lead to emergence of self-reactive B cells and second, introduction of mutations or translocations may transform B cells and lead to malignancy (B cell lymphoma or leukemia). This is illustrated by the observation that the majority of B cell malignancies are derived from post–germinal center B cell precursors (Kuppers et al. 1999). Furthermore, in situations in which germinal center reactions are stimulated persistently, such as in sites of autoimmune inflammation, the incidence of malignant transformation is increased (Mackay and Rose 2001). Finally, in autoimmune disease, autoantibodies often bind self-antigen with high affinity and have been shown to contain somatic mutations, indicating that they have arisen from germinal center reactions. Experimental models relying on immunization with artificial antigens such as haptenated proteins have revealed that germinal centers undergo spontaneous involution approximately 3 weeks after immunization. This timing correlates with the disappearance of antigen held on FDCs. There also appears to be an affinity ceiling for antibody-antigen interactions, which might explain why germinal center reactions are self-limited (Batista and Neuberger 1998). Another possibility is that germinal centers involute when T cell help is no longer available. This would be consistent with the finding that massive germinal centers, which involute at the time when the first T cell selection
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signals are expected to induce centrocyte recycling to renew the centroblast pool can be induced by polysaccharide antigen (in the absence of T cell help) (Vinuesa et al. 2000). On the other hand, recent studies using real antigen (i.e., live virus) indicate that the germinal center life span may extend beyond 3 months and therefore provide a continuous source of high-affinity antibody-forming cells (Bachmann and Zinkernagel 1996). Recently a role for the Ets family transcription factor Spi-B in maintaining germinal center B cell survival has been identified. Spi-B is expressed exclusively in lymphoid cells and is critical for normal B cell development and function. Indeed, the expression of Spi-B increases as B cells mature, and a deficiency of Spi-B has a profound effect on B cell responses to protein antigen (Su et al. 1997). After immunization, Spi-B−/− mice form small germinal centers, which rapidly acquire excessive apoptotic bodies and involute prematurely. Consequently, memory responses are defective. The prevailing model is that germinal center B cells are programmed to undergo apoptosis by default and only survive if they receive T cell selection signals. This model is consistent with evidence that centrocytes downregulate apoptosis inhibitors such as Bcl-2 and Bcl-xL while upregulating proapoptotic molecules such as Fas and Bim (Yokoyama et al. 2002). This coordinated proapoptotic response is critical not only for maintaining B cell tolerance but also for selection of high-affinity memory B cells and long-lived plasma cells (Smith et al. 2000). In the presence of enforced expression of Bcl-2, low-affinity plasma cells accumulate in the bone marrow and overexpression of Bcl-xL results in accumulation of both low-affinity plasma cells and memory B cells (Takahashi et al. 1999). Fas also plays a role in the elimination of autoreactive germinal center B cells (Hoch et al. 2000) since mice with defective Fas/Fas ligand signaling accumulate somatically mutated self-reactive B cells. Nevertheless, there is evidence to suggest that in these mice, somatic hypermutation can occur in extrafollicular locations (William et al. 2002). Germinal center B cells express a pre-formed death-inducing signaling complex (DISC) formed by Fas, FLICE (caspase-8/FADD-like IL-1β–converting enzyme) and the FLICE inhibitor, cFLIPL (FADD-like IL-1β–converting enzyme-inhibitory protein) (Hennino et al. 2001). Centrocyte-positive selection requires two signals. Signal 1 is delivered when centrocytes bind the antigen that initiated the TD B cell response, displayed on the surface of FDCs. Signal 2 is delivered when centrocytes that compete successfully to bind antigen also receive help from TFH cells within the germinal center (MacLennan 1994). So far, the only well-demonstrated signal that is critical for centrocyte selection is CD40 ligation. This finding is consistent with in vitro studies of human centrocytes, which show that in the absence of CD40 ligation, centrocytes rapidly undergo apoptosis (Casamayor-Palleja et al. 1996), and evidence that centrocyte binding to self-antigen when T cell help is unavailable results in centrocyte death (Shokat and Goodnow 1995). ICOS-ICOS ligand interactions
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are also important because ICOS−/− T cells can enter the germinal centers but fail to provide help to B cells (Smith et al. 2003). g. TERMINAL DIFFERENTIATION OF CENTROCYTES AND INVOLUTION OF GERMINAL CENTERS Selected centrocytes can differentiate into memory B cells or long-lived plasmablast precursors, or they can recycle back into the centroblast pool to undergo further rounds of somatic hypermutation. The last pathway is crucial to sustain germinal center reactions; abrogation of recycling by anti-CD40 treatment or in the context of T-independent germinal centers causes them to dissipate quickly (Garcia de Vinuesa et al. 2000; Han et al. 1995; Vinuesa et al. 2000). The signals that determine the centrocyte terminal differentiation decision have not been fully elucidated. Blimp-1 is upregulated in a subset of germinal center B cells, which are probably destined to become high-affinity plasma cells rather than memory B cells. Plasma cells derived from germinal centers secrete mutated high-affinity antibody and migrate to the lamina propria of the gut or the bone marrow where they can survive for long periods without dividing (Slifka et al. 1998).
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Chapter 2 The Biology of Toll-Like Receptors in Mice Osamu Takeuchi and Shizuo Akira
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Toll-Like Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. TLR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. TLR2, TLR1, and TLR6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. TLR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. TLR5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. TLR7, TLR8, and TLR9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. TLR11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. The TLR Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MyD88-Dependent Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . B. TRIF-Dependent Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. TLR7 and TLR9 Signaling in pDCs . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Microorganisms that have invaded a host are initially deciphered by innate immune cells such as macrophages and dendritic cells (DCs). Upon pathogen recognition, these cells phagocytose invading microorganisms, process them, and present antigens to T cells. Furthermore, these cells increase the surface expression of costimulatory molecules, as well as produce
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cytokines, interferons (IFNs) and chemokines to evoke inflammation (Akira and Takeda, 2004). These actions result in the activation of antigen-specific T cells, leading to the generation of immunological memory. Therefore, innate immune cells play a central role in regulating immune responses in the initial stage of an infection. The adaptive immune system acquires an ability to react with various antigens by rearrangement of antigen receptors. In contrast, the innate system is characterized by
Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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the use of a limited number of germline-encoded receptors that recognize diverse pathogens invading the host. The innate system therefore targets a set of molecular structures that are absent from host cells, but unique to microorganisms and shared by various pathogens. By recognizing these pathogenspecific molecular patterns (PAMPs), the innate system is able to prevent autoimmune responses (Akira and Takeda, 2004).
II.
TOLL-LIKE RECEPTORS
The innate immune system is evolutionally conserved from fruit flies to vertebrates. In fact, Toll was originally identified as a molecule essential for determining dorsoventral polarity in the development of Drosophila melanogaster. Later, the role of Toll in Drosophila antifungal responses was uncovered by Hoffmann and his colleagues (Lemaitre et al. 1996). Moreover, studies in the field of Drosophila immunity have had a tremendous influence on advancing research into mammalian innate immunity. A mammalian homologue of Toll was discovered in 1997 by Janeway and Medzhitov (Medzhitov et al. 1997). They proposed that mammalian Toll was also involved in immune responses by inducing the production of proinflammatory cytokines and expression of costimulatory molecules. Subsequently, a family of Toll, named Toll-like receptors (TLRs), has been discovered via a database search (Rock et al. 1998). Thirteen mammalian TLRs have been reported to date, and 10 of them have been shown to recognize specific PAMPs. TLRs are expressed not only on macrophages, DCs, and B cells but also on fibroblasts, vascular endothelial cells, and intestinal epithelial cells. The TLR family proteins comprise extracellular leucine-rich repeat (LRR) motifs, a transmembrane region, and a cytoplasmic Toll/interleukin (IL)-1 receptor homology (TIR) domain (Akira and Takeda, 2004). The LRR motifs are responsible for ligand recognition, and the TIR domain is essential for triggering intracellular signaling pathways. The activation of TLR signaling pathways leads to the activation of transcription factors including nuclear factor-κB (NF-κB) and AP-1, and ultimately the expression of proinflammatory cytokine and chemokine genes is induced. Mouse genetics have greatly contributed to the identification of the microbial components recognized by each TLR and of the role of signaling molecules. In the following sections, we will describe functions of mouse TLRs and their signaling revealed by the analysis of mice lacking each TLR or molecules involved in the TLR signaling (Table 2-1).
A.
TLR4
Two strains of mice, C3H/HeJ and C57BL/10ScCr, were known to be hyporesponsive to lipopolysaccharide (LPS), a component of the outer cell membrane of Gram-negative bacteria.
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Beutler and his colleagues (Poltorak et al. 1998) performed positional cloning of the gene responsible for LPS recognition in these mice and identified TLR4. A missense point mutation of the TLR4 gene resulted in the replacement of a proline in the cytoplasmic domain of the TLR4 protein with histidine in C3H/HeJ mice. In C57BL/10ScCr mice, the Tlr4 locus was entirely deleted. TLR4-deficient (TLR4−/−) mice generated by gene targeting were also highly resistant to LPS-induced shock, and macrophages and B cells derived from these mice were unresponsive to LPS in terms of the activation of intracellular signaling molecules, production of cytokines, and proliferative responses (Hoshino et al. 1999). Moreover, C3H/HeJ mice were highly susceptible to infection by Salmonella and Escherichia coli, indicating that recognition of bacteria by TLR4 is critical to host defense (O’Brien et al. 1980). TLR4 interacts with a secreted protein, MD-2, at the extracellular portion (Shimazu, 1999). MD-2 was reported to bind directly to LPS, and expression of MD-2 greatly enhances cellular responses against LPS in the presence of TLR4. The response of MD-2−/− mice to LPS was severely impaired (Nagai et al. 2002. Interestingly, surface expression of TLR4 was abolished in MD-2−/− macrophages, suggesting that MD-2 is also important for the proper trafficking of TLR4 to the cell membrane. In addition to LPS, TLR4 recognizes several viral components such as the respiratory syncytial virus F protein and mouse mammary tumor virus envelope protein. Moreover, TLR4 is implicated in the recognition of endogenous products, such as heat-shock proteins, fibronectin fragments, and so on (Tsan and Gao 2004). However, it is possible that these ligand preparations were contaminated with LPS or other PAMPs, because in most studies recombinant proteins synthesized in E. coli were used. Further studies are required to confirm whether TLR4 is also responsible for endogenous ligands.
B.
TLR2, TLR1, and TLR6
Amino acid sequence alignment revealed that TLR1, TLR2, TLR6, and human TLR10 form a subfamily among TLRs. TLRs in this subfamily are not only similar in amino acid sequences but also in their ligands. TLR2 recognizes various PAMPs from bacteria, fungi, and parasites including bacterial lipoprotein, peptidoglycan (PGN), lipoteichoic acid, Saccharomyces cerevisiae zymosan, and glycosylphosphatidylinositol (GPI) anchors from Trypanosoma cruzi (Campos et al. 2001; Takeuchi et al. 1999a, 2000b; Underhill et al. 1999). Macrophages derived from TLR2−/− mice did not produce proinflammatory cytokines in response to TLR2 ligands, such as PGN and bacterial lipoproteins (Takeuchi, Hoshino, et al. 1999, 2000). Corresponding to the failure to detect these components through TLR2, TLR2−/− mice were susceptible to infections by various Gram-positive bacteria and fungi, including Staphylococcus aureus, group B Streptococcus, Borrelia burgdorferi, Chlamydia trachomatis, Streptococcus pneumoniae, and so on
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TABLE 2-1
THE PHENOTYPE OF MICE DEFICIENT IN TLR OR ITS SIGNALING MOLECULES Gene Mutated
Type of Mutation
Phenotype
Reference
TLR1 TLR2
Knockout (KO) KO
Defective response to triacyl lipoproteins Defective response to lipoproteins, PGN, LTA, zymosan, GPI anchors; highly susceptible to S. aureus infection
TLR3 TLR4
KO KO or natural mutation
TLR6 TLR7
KO KO
Defective response to dsRNA and poly(I:C) Defective response to LPS, Taxol, and envelope proteins from respiratory syncytial virus and MMTV Defective response to di-acyl lipoproteins Defective IFN response to ssRNA and RNA viruses
Takeuchi et al. 2002 Campos et al. 2004; Ozinsky et al. 2000; Schwandner et al. 1999; Takeuchi et al. 1999a, 2000a, 2000b; Underhill et al. 1999 Alexopoulou et al. 2001 Hoshino et al. 1999; Poltorak et al. 1998
TLR8 TLR9
KO KO
TLR11
KO
MyD88
KO
TRIF TIRAP
KO or point mutation KO
TRAM IRAK4 TRAF6
KO KO KO
TAK1
KO
TBK1
KO
IKK-i
KO
No phenotype in mice; a ssRNA detector in humans Defective response to CpG DNA and DNA viruses (HSV, MCMV) Defective response to uropathogenic bacteria, Toxoplasma Defective response to IL-1, TLR ligands except TLR3; impaired immune responses to various pathogens including S. aureus, M. tuberculosis, M. avium, L. monocytogenes, group B Streptococcus, LCMV, HSV-1, B. burgdorferi, C. albicans, and T. cruzi Defective response to TLR3 and TLR4 ligands Defective proinflammatory cytokine production to TLR2 and TLR4 ligands Defective TLR4 (LPS)-mediated responses Defective response to IL-1 and TLR ligands Osteopetrosis; defective response to IL-1 and TLR ligands Embryo lethal; TAK1-deficient B cells and MEFs show defective response to IL-1, TNF, and TLR ligands Embryo lethal; TBK1-deficient MEFs showed defective type I IFN production in response to TLR ligands Involved in type I IFN production in response to TLR ligands
(Darville et al. 2003; Echchannaoui et al. 2002; Takeuchi et al. 2000a; Wooten et al. 2002). On the other hand, recent reports have shown that TLR2 signaling suppresses immunity against Candida infection (Netea et al. 2004). Compared with ligands for other TLRs, TLR2 stimulation will activate cells to induce high amounts of IL-10. The population of regulatory T cells and IL-10 secretion were partially reduced in TLR2−/− mice, which may explain the improved resistance to Candida infections. Although the precise mechanism of this suppression is yet to be clarified, it is an interesting phenomenon that TLR signaling can work in a both stimulatory and inhibitory manner. TLR1 and TLR6 are highly homologous, and heterodimerization of TLR2 with TLR1 or TLR6 determines the ligand specificity (Takeuchi, Kawai et al. 1999). Although expression
Takeuchi et al. 2001 Diebold et al. 2004; Heil et al. 2004; Hemmi et al. 2002 Heil et al. 2004 Hemmi et al. 2000; Lund et al. 2003; Krug et al. 2004 Yarovinsky et al. 2005; Zhang et al. 2004 Adachi et al. 1998; Bellocchio et al. 2004; Bolz et al. 2004; Campos et al. 2004; Henneke et al. 2002; Kawai et al. 1999; Mansur et al. 2005; Seki et al. 2002; Takeuchi et al. 2000a; Zhou et al. 2005 Yamamoto, Sato, Hemmi, Hoshino et al. 2003 Horng et al. 2002; Yamamoto, Sato, Hemmi et al. 2002 Yamamoto, Sato Hemmi, Uemtasu et al. 2003 Suzuki et al. 2002 Lomaga et al. 1999; Naito et al. 1999 Sato et al. in press
Bonnard et al. 2000; Hemmi et al. 2004; McWhirter et al. 2004 Hemmi et al. 2004
of either TLR1 or TLR6 alone failed to confer any response, coexpression of TLR1 or TLR6 with TLR2 resulted in cell activation in response to triacyl- or diacyl-lipopeptide (Takeuchi et al. 2001, 2002). The cellular response to mycoplasmal diacyllipopeptide was abolished in TLR6−/− mice (Takeuchi et al. 2001). In contrast, TLR1−/− mice showed an impaired response to triacyl-lipopeptide, whereas the response to diacyl-lipopeptide was normal (Takeuchi et al. 2002). Overexpression studies have revealed that TLR1 and TLR6 each interacted with TLR2 in cells. These observations suggest that TLR1 and TLR6 recognize the difference in the lipid portion of lipoproteins through heterodimerization with TLR2. Although human TLR10 expresses as a receptor with LRR and a TIR domain, mouse TLR10 do not express because of a missense mutation of the gene (Hasan, 2005).
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C.
TLR3
Infection with RNA viruses leads to double-stranded RNA (dsRNA) generation in the cytoplasm of infected cells. Cells expressing TLR3 respond to viral dsRNA or its synthetic mimic, polyinosinic acid-polycytidylic acid [poly(I:C)], and activate an intracellular signaling pathway leading to upregulation of type I IFNs and proinflammatory cytokines (Alexopoulou et al. 2001). Myeloid DCs and lung fibroblasts derived from TLR3−/− mice were hyporesponsive to poly(I:C) stimulation in terms of IL-12p40 production (Yamamoto, Sato, Hemmi, Hoshino, et al. 2003). However, inoculation of poly(I:C) into TLR3−/− mice resulted in a normal production of serum IFN-α, indicating that other molecule(s) are also involved in the dsRNA-induced immune responses. In accordance with the impaired production of cytokines, TLR3−/− mice are more susceptible to murine cytomegalovirus (MCMV) infection than control mice (Tabeta et al. 2004). However, West Nile virus takes advantage of TLR3-mediated inflammation to invade the central nervous system (Wang et al. 2004). Therefore, TLR3−/− mice were more resistant to West Nile virus infection and showed impaired induction of cytokines. This suggests that because the TLR3-mediated viral recognition is important for the elimination of the virus, some viruses reciprocally target TLR3 to facilitate the infection. This is an example of the complex relationship between viruses and hosts.
D.
TLR5
TLR5 recognizes bacterial flagellin, a unique PAMP composed of protein (Hayashi et al., 2001. Flagellin is a protein component of the flagellum, a structure used by some bacteria to move through liquid medium. Chinese hamster ovary cells expressing human TLR5 can activate NF-κB in response to flagellin. Moreover, Salmonella typhimurium lacking flagellin failed to activate TLR5, indicating that TLR5 is critical for flagellin recognition. A stop codon within the open reading frame (ORF) of human TLR5 in many individuals renders them incapable of responding adequately to flagellated bacterium (Hawn et al. 2003). The MOLF/Ei mouse strain is shown to be susceptible to S. typhimurium infection, and one of the MOLF/Ei susceptibility loci was mapped to chromosome 1, which contains the mouse Tlr5 gene (Sebastiani et al. 2000). Furthermore, TLR5 expression was reduced in MOLF/Ei mice.
E.
TLR7, TLR8, and TLR9
TLR7 is closely related to TLR8 and shows significant homology with TLR9. Recent studies have revealed that ligands of these three TLRs are nucleotides and their derivatives. TLRs in this subfamily are localized in the endosome and the endoplasmic reticulum (ER), where nucleotides are incorporated
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and transported (Latz et al. 2004). TLR7 and TLR9 are highly expressed on plasmacytoid DCs (pDCs), which are known to produce huge amounts of IFN-α upon exposure to viruses (Iwasaki and Medzhitov, 2004; Liu 2005). TLR7 recognizes viral single-stranded RNAs (ssRNAs) and induces proinflammatory cytokines and type I IFNs (Diebold et al., 2004; Heil et al. 2004). TLR7-deficient pDCs failed to produce type I IFNs in response to influenza or vesicular stomatitis virus infection, or stimulation with GU-rich synthetic ssRNA (Diebold et al. 2004; Heil et al. 2004; Lund et al. 2004). Moreover, TLR7 is activated by small nucleotide derivatives, including imiquimod and resiquimod (R-848) in mice (Hemmi et al. 2002). These derivatives are small synthetic compounds that are approved for use as antiviral drugs because of their potency for inducing type I IFNs. TLR7−/− mice failed to respond to these compounds by not producing proinflammatory cytokines or type I IFNs (Hemmi et al. 2002). Expression of human TLR8 as well as human TLR7 in HEK293 cells conferred the responsiveness to ssRNAs to activate a NF-κB reporter gene (Heil et al. 2004). In contrast, the expression of mouse TLR7, but not TLR8, could restore ssRNA response in culture cells. Moreover, antigenpresenting cells from TLR8−/− mice normally produce proinflammatory cytokines in response to ssRNAs, suggesting that the role of TLR8 is different between humans and mice (Heil et al. 2004). TLR9 detects unmethylated DNA with a CpG motif (Hemmi et al., 2000). Although host genomic DNA has fewer CpG motifs and is largely methylated, bacterial or viral DNA is characterized by an abundance of CpG motifs and the lack of methylation (Hemmi et al. 2000). Furthermore, synthetic oligodeoxynucleotides containing an unmethylated CpG motif (CpG-DNA) show strong immunostimulatory activity. TLR9 was shown to be essential for CpG-DNA detection, since the immune response to CpG-DNA stimulation was abolished in TLR9−/− mice (Hemmi et al. 2000). In pDCs, TLR9 is responsible for the detection of DNA viruses, such as herpes simplex virus (HSV) types I and II, and production of type I IFNs (Krug et al. 2004; Lund et al. 2003). Moreover, mice deficient in TLR9 or those with a mutation in the Tlr9 locus were susceptible to MCMV infection (Tabeta et al. 2004). TLR9-mediated recognition of MCMV genomic DNA induces IL-12 and type I IFN production in DCs, which promotes viral clearance by natural killer cells (Krug et al. 2004). These observations indicate that TLR9 is critically involved in the immune response against DNA viruses.
F.
TLR11
Mouse TLR11 is expressed in macrophages and in liver, kidney, and bladder epithelial cells. TLR11 recognizes uropathogenic bacteria and profiling-like protein purified from Toxoplasma gondii (Yarovinsky et al. 2005; Zhang et al. 2004). TLR11−/− mice were highly susceptible to kidney infections by
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uropathogenic bacteria, and macrophages derived from TLR11−/− mice failed to produce proinflammatory cytokines in response to these bacteria. However, human TLR11 is likely to be nonfunctional, since the stop codons were observed in its ORF. This suggests that uropathogenic bacteria are recognized in humans in a TLR11-independent manner.
III.
THE TLR SIGNALING PATHWAY
Stimulation with ligands triggers activation of TLR-mediated intracellular signaling cascades. The signaling leads to the activation of transcription factors including NF-κB and IFN-regulatory factors, which mediate induction of various genes involved in host defense (Akira et al. 2004). The initial step of TLR signaling is recruitment of cytoplasmic adaptor molecules to the receptor (Akira et al. 2004). These adaptors possess a TIR domain and associate with the TIR domain of TLR/IL-1R with a homophilic interaction. So far, five TIRdomain containing adaptor molecules have been identified in mammals, including MyD88, TIR domain-containing adaptor inducing IFN-β (TRIF), TIR domain-containing adaptor protein (TIRAP)/MyD88 adaptor like (MAL), and TRIF-related adaptor molecule (TRAM)1 (Fitzgerald 2001, 2002; Horng 2001; Muzio 1997; Yamamoto, Sato, Hemmi, et al. 2002; Yamamoto, Sato, Hemmi, Hoshino, et al. 2003). Among these adaptor molecules, MyD88 and TRIF regulate the activation of two main signaling pathways leading to the production of proinflammatory cytokines and type I IFNs. Furthermore, cell type-specific recruitment of signaling machinery to the receptor is also important for the adaptive response to TLR ligands. In the following sections, we focus on mechanisms by which TLRs stimulate distinct signaling pathway leading to the adaptive responses.
A.
MyD88-Dependent Signaling Pathway
MyD88 is an adaptor molecule consisting of an N-terminal DD and a C-terminal TIR domain (Muzio, 1997). All IL-1R/ TLR family members, except for TLR3, share at least one common signaling pathway via MyD88 (Adachi et al. 1998; Takeuchi, Hoshino, et al. 2000). TIRAP bridges MyD88 to TLR2 or TLR4 (Horng et al. 2002; Yamamoto, Sato, Hemmi, et al. 2002). After ligand stimulation, TLR/IL-1R interacts with MyD88 via the TIR domains, and MyD88 subsequently recruits and associates with IL-1R-associated kinase (IRAK) proteins. Analysis of MyD88−/− mice revealed that MyD88 is essential for signaling through all the TLR/IL-1R family members, except for TLR3 (Adachi et al. 1998; Kawai et al. 1999; Takeuchi, Hoshino, et al. 2000). Cells derived from MyD88−/− mice failed to produce proinflammatory cytokines in response to various TLR ligands, such as bacterial lipoprotein, PGN,
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LPS, flagellin, ssRNA, and CpG-DNA, in addition to IL-1β and IL-18. MyD88−/− cells showed defective activation of NF-κB and mitogen-activated protein (MAP) kinases in response to TLR ligands, except for the TLR4 ligand (LPS) and TLR3 ligand [poly(I:C)]. When MyD88−/− cells were stimulated with LPS, delayed activation of NF-κB and MAP kinases was observed. Moreover, it was reported that TLR ligands promote bacterial phagocytosis in a MyD88-dependent manner. The analysis of MyD88−/− mice has greatly contributed to elucidation of the role of the TLR system in host defense against various pathogens. Because of the abrogation in TLR-mediated pathogen recognition, MyD88−/− mice showed impaired immune responses to challenges with various pathogens. These included bacteria, fungi, viruses, and parasites, such as S. aureus, Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, group B Streptococcus, Lymphocytic choriomeningitis virus (LCMV), HSV-1, B. burgdorferi, Candida albicans, and T. cruzi (Bellocchio et al. 2004; Bolz et al. 2004; Campos et al. 2004; Feng et al. 2003; Henneke et al. 2002; Mansur et al. 2005; Seki et al. 2002; Takeuchi et al. 2000a; Zhou et al. 2005). Altogether, these observations indicate that MyD88 is essential for host defense against various pathogens by activating innate immune responses. Of note, MyD88−/− mice are much more susceptible to challenges from various pathogens compared with mice-deficient in each TLR, suggesting that a pathogen is recognized by a combination of various TLRs. Activated IRAKs interact with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), a protein comprised of an N-terminal RING finger domain and a conserved C-terminal TRAF-domain (Akira et al. 2004). The RING finger domain is also found in a large family of E3 ubiquitin ligases, and TRAF6 was recently shown to function as an ubiquitin ligase, along with an E2 ligase complex consisting of Ubc13 and Uev1A/Mms2, to catalyze the formation of a polyubiquitin chain through lysine-63 (K63) of ubiquitin. Ubiquitin-dependent activation of transforming growth factor (TGF)-β-activated kinase 1 (TAK1) results in phosphorylation of Iκ kinase (IKK)-β and mitogen-activated protein kinase kinase 6 (MKK6), leading to NF-κB and MAP kinase activation. Ultimately, these transcription factors promote expression of proinflammatory cytokine genes. Mice lacking IRAK4 or TRAF6 were defective in the activation of the MyD88-signaling pathway (Lomaga et al. 1999; Naito et al., 1999; Suzuki et al. 2002). Inherited IRAK4 deficiency in humans was also reported (Picard et al. 2003). These patients were recurrently infected with Grampositive and Gram-negative bacteria. Cells taken from these patients were unresponsive to various TLR stimuli, indicating that IRAK4 is critical for the TLR signaling in both humans and mice. Recently, we generated conditional TAK1-deficient mice, because TAK1 straight knockout mice were terminal in an early embryonic stage. Analysis of B cells or fibroblasts lacking the TAK1 gene revealed that TAK1 is required for IL-1R/TLR-mediated cellular responses, by regulating activation of both NF-κB and MAP kinases (Sato et al., in press).
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Additionally, IFN regulatory factor (IRF) 5 was recently shown to regulate the production of proinflammatory cytokines in MyD88-dependent signaling (Takaoka et al. 2005). IRF5 is localized to a MyD88-containing complex and translocates into the nucleus to induce cytokine gene transcription in response to TLR stimulation (Takaoka et al. 2005). IRF5−/− DCs showed impaired cytokine production in response to stimulation with broad TLR stimuli.
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TBK1/IKK-i, kinases known to phosphorylate IRF3 to induce IFN-β gene induction. The transcription factors, IRF3 and IRF7 are essential for the expression of type I IFNs in response to viral infection and TLR stimulation (Honda et al. 2005). Mice deficient in either IRF-3 or IRF-7 are susceptible to infection with RNA and DNA viruses.
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TLR7 and TLR9 Signaling in pDCs
TRIF-Dependent Pathway
TRIF contains a TIR domain, a TRAF6 binding motif, and a C-terminal receptor interacting protein (RIP) homotypic interaction domain (Sato et al. 2003; Yamamoto, Sato, Mori, et al. 2002). A feature of the TRIF-dependent pathway is the production of type I IFNs. TRIF−/− mice or lps2 mutants obtained by mouse forward genetics were defective in TLR3- and TLR4-mediated proinflammatory cytokine production and type I IFN responses (Hoebe et al. 2003; Yamamoto, Sato, Hemmi, Hoshino, et al. 2003). Although LPS-mediated initial activation of NF-κB occurred in TRIF−/− cells, it was attenuated more rapidly than that in wild-type cells (Yamamoto, Sato, Hemmi, Hoshino, et al. 2003). LPS-induced signaling pathways were not activated in the absence of both MyD88 and TRIF, indicating that LPS signaling is entirely dependent on these two adaptors. Another TIR-containing adaptor, TRAM, bridges between TLR4 and TRIF (Yamamoto, Sato, Hemmi, Uemtasu, et al. 2003). TRIF can associate with TRAF6 through its TRAF6 binding motif, and also interact with RIP1 via the RIP homotypic interaction motif. Poly(I:C)-mediated NF-κB activation was abolished in RIPI−/− cells, indicating that RIPI is essential for TLR3-mediated NF-κB activation (Meylan et al. 2004). Two IKK-related kinases, inducible IκB kinase (IKK-i; also known as IKKε) and TRAF family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1; also known as T2K), play an essential role in TRIF-mediated type I IFN induction (Fitzgerald et al. 2003; Hemmi et al. 2004; Sharma et al. 2003). TRIF can directly associate with TBK1 (Sato et al. 2003). IKK-i and TBK1 phosphorylate IRF3, and IRF7 can activate the IFN-β promoter (Fitzgerald et al. 2003; Sharma et al. 2003). TBK1−/− mouse embryo fibroblasts (MEFs), but not IKK-i−/−, showed severely impaired induction of IFN-β and IFN-inducible genes in response to LPS, intracellular introduction of poly(I:C), and RNA virus infection (Hemmi et al. 2004; McWhirter et al. 2004). Activation of IRF3, but not NF-κB, in response to LPS and poly(I:C) was also diminished in TBK1−/− cells. IKK-i/TBK1 double-deficient MEFs failed to express any detectable levels of IFN-β and IFN-inducible genes in response to poly(I:C), indicating that both IKK-i and TBK1 contribute to the IFN pathway (Hemmi et al. 2004). Furthermore, these observations suggest that the signaling pathways triggered by TLR stimulation, as well as cytoplasmic viral recognition, converge at the level of
pDCs are known to be recruited to inflamed lymph nodes and produce large amounts of type I IFNs. pDCs highly express TLR7 and TLR9, and stimulation by their ligands induces IFN-α production. It was revealed that a complex composed of MyD88, IRAK4, IRAK1, TRAF6, and IRF7 was recruited to the receptors in response to ligand stimulation (Honda et al. 2004; Kawai et al. 2004). This complex is responsible for the activation of both NF-κB and IRF-7. Interestingly, IRAK1 is essential for the activation of IRF-7, but not NF-κB, suggesting that IRAK1 mediates the phosphorylation of IRF7 (Uematsu et al. 2005). The modification induces nuclear translocation of IRF7 and expression of IFN-α genes.
IV.
PERSPECTIVES
The application of mouse reverse genetics has greatly contributed to advances in the research field of innate immunity. However, the entire mechanism of innate immune recognition is not simply explained by the TLR system. For example, fibroblasts or conventional DCs still efficiently produce type I IFNs upon infection with DNA and RNA viruses. A recently identified family of molecules, comprising caspase recruitment domain (CARD) domains and a helicase domain (called retinoic acid inducible gene-I [RIG-I] and MDA-5), has been shown to recognize viral dsRNA in the cytoplasm (Andrejeva et al. 2004; Yoneyama et al. 2004). We have recently shown that RIG-I and MOAS are essential for type I IFN responses after RNA virus infection in various type of cells, except for pDCs (Kato et al. 2006). Cytoplasmic receptors have also been implicated in the recognition of bacteria that have invaded the cytoplasm of a cell. These include proteins classified as belonging to the nucleotide-binding oligomerization domain (NOD)-LRR family. This family comprises more than 20 different cytoplasmic proteins (Inohara et al. 2005). Among them, NOD1 and NOD2, proteins composed of CARDs, NOD, and LRR domains, recognize components of bacterial peptidoglycan, γ-D-glutamyl-meso diaminopimelic acid and muramyl dipeptide (Inohara et al. 2005). A missense point mutation in the human Nod2 gene is correlated with susceptibility to Crohn’s disease, an inflammatory bowel disease (Inohara et al. 2005).
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Although the mechanisms underlying involvement of NOD2 in the disease are still controversial, the relation between cytoplasmic pathogen detectors and inflammatory diseases are quite intriguing for future studies. In addition, the function of NOD-LRR proteins other than NOD1 and NOD2 is yet to be clarified. Taken together, numerous studies using mouse genetics revealed that the TLR system controls a coordinated and adaptive response depending on the pathogen. We hope that future studies will clarify the detailed mechanisms of cytoplasmic pathogen recognition and the intricate pathogen-host relationship.
V.
SUMMARY
TLRs are evolutionally conserved transmembrane receptors involved in innate immune recognition. To date, 13 TLRs have been identified either in mice or humans. TLRs detect PAMPs and trigger activation of signaling cascades, leading to the induction of genes involved in inflammation and further activation of adaptive immunity. Mouse reverse genetics have contributed greatly to increasing our understanding of microbial components recognized by each TLR and their signaling pathways. In this chapter, we have reviewed the functions of TLRs revealed by mouse reverse genetics.
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Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., et al. (2002). Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420, 324–329. Yamamoto, M., Sato, S., Hemmi, H., Uemtasu, S., Hoshino, K., Kaisho, T., et al. (2003). TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol 4, 1144–1150. Yamamoto, M., Sato, S., Mori, K., Hoshino, K., Takeuchi, O., Takeda, K., et al. (2002). Cutting edge: A novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-β promoter in the Toll-like receptor signaling. J Immunol 169, 6668–6672. Yarovinsky, F., Zhang, D., Andersen, J.F., Bannenberg, G.L., Serhan, C.N., Hayden, M.S., et al. (2005). TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 308, 1626–1629. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., et al. (2004). The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 5, 730–737. Zhang, D., Zhang. G., Hayden, M.S., Greenblatt, M.B., Bussey, C., Flavell, R.A., et al. (2004). A Toll-like receptor that prevents infection by uropathogenic bacteria. Science 303, 1522–1526. Zhou, S., Kurt-Jones, F.A., Mandell, L., Cerny, A., Chan, M., Golenbock, D.T., et al. (2005). MyD88 is critical for the development of innate and adaptive immunity during acute lymphocytic choriomeningitis virus infection. Eur J Immunol 35, 822–830.
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Chapter 3 Genomic Organization of the Mouse Major Histocompatibility Complex Attila Kumánovics
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. From Consensus Maps to Sequence-Based Maps . . . . . . . . . . . . . . . . . . . . . III. Comparative Map of the Mhc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Class II Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Non–Class II Genes in the Class II Region . . . . . . . . . . . . . . . . . . . . . . . C. The Class III Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Other Genes in the Class III Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The Class I Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. The H2-Q Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Genes between the H2-Q and -T Regions . . . . . . . . . . . . . . . . . . . . . . . . H. The H2-T Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. The H2-M Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. M1 and M10 Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. H2-M4, M5, and M6 Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. Telomeric End of the M Region or the Extended Class I Region . . . . . . M. Unity of the Class I, II, and III Regions/Shared Features of the Mhc Class I, II, and III Regions . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
The Mhc molecules together with the T cell and B cell receptors are the foundations of the adaptive immune system of vertebrates. The highly polymorphic MHC molecules are the major cause of histoincompatibility (hence the name major histocompatibility complex) when tissues are transplanted from THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
119 120 121 123 123 124 124 125 126 127 128 129 129 129 129 130 130 131
one individual into another within a species (allogenic transplantation). The Mhc is also associated with hundreds of diseases. For all these reasons, the Mhc is one of the mostinvestigated genomic regions in vertebrates. The transplantation antigens (Snell 1992) and the Mhc-restricted antigen presentation (Zinkernagel and Doherty 1997) were both discovered in mouse. The mouse Mhc, called H2, continues to serve as a reference for all other vertebrate Mhcs. Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
119
120 There are two classes of Mhc molecules. The class Ia or classic class I molecules are highly polymorphic, are expressed ubiquitously, and present antigens to the CD8+ cytotoxic T cells. Class I polypeptides are synthesized in the endoplasmatic reticulum (ER). The class I heavy chain is stabilized by β2-microglobulin, which is the shared light chain of all class Ia molecules, and by an 8–9-amino acid-long peptide usually derived from intracellular proteins. Only the class I heavy chains are encoded in the Mhc, whereas the nonpolymorphic β2-microglobulin is encoded outside of the Mhc. The nonclassic or class Ib molecules are defined by lack of one or more of the typical features of the class Ia molecules: limited tissue-specific expression, low polymorphism, unknown function, or function other than antigen presentation to CD8+ T cells. The class II molecules are heterodimers of α and β chains, both encoded in the Mhc. The mouse classic class II molecules, H2-A (or IA) and H2-E (or IE), are constitutively expressed on the surface of antigen-presenting cells, such as dendritic cells, B lymphocytes, macrophages, and thymic epithelium. Class II proteins are required for antigen presentation to T lymphocytes bearing the CD4 coreceptor (CD4+ T cells). The classic class II molecules are synthesized in the ER and targeted to the endocytic pathway, where they acquire peptides derived from proteins internalized from the extracellular space. The nonclassic class II molecules, H2-DM and H2-DO (H2-DM was also called H2-M; the DO a chain gene was also called H2-N, before its identity was clarified), serve as accessory molecules in the class II presentation pathway by promoting or modulating the peptide loading of the classic class II molecules. Large-scale sequencing and comparative sequence analyses transformed our understanding of the organization of the mammalian genome and, consequently, the organization of the Mhc. Sequencing of the mouse genome soon followed the sequencing of the human genome (Waterston et al. 2002), and we now have near complete genomic sequences of the Mhc from two inbred strains, 129/SvJ (haplotype H2bc; Kumánovics et al. 2003) and C57BL/6 (H2b; Waterston et al. 2002). There are plans to fund the sequencing of 15 more mouse strains (Pearson 2004). Sequence analysis of the complete Mhc allows us to draw definitive physical maps, which can serve as reference for all other maps, such as linkage, quantitative trait loci, and others. The map of H2 based on the two available sequences is presented here. Comparative analysis of H2 and Mhc sequences from other organisms, such as human, rat, cat, dog, and pig, makes it possible to go beyond the simple listing of genes from one end of the chromosome to the other. Comparative maps help the systematic evaluation of the various experimental animals and approaches; they also help us to reveal some of the evolutionary forces shaping the Mhc. This understanding allows us to make predictions about unsequenced mouse haplotypes and other organisms as well.
AT T I L A K U M Á N O V I C S
II.
FROM CONSENSUS MAPS TO SEQUENCE-BASED MAPS
Sequencing of the Mhc was preceded by more than 50 years of studies that established the basic organization and function of both the mouse and human Mhc. H2 is located on chromosome 17. The human Mhc, or HLA, is located on the short arm of chromosome 6, in the same centromere to telomere orientation as in mouse. Traditionally, the Mhc is divided into the class I, II, and III regions (Fig. 3-1A). The class I and class II regions encode the class I and II genes, respectively. The class III region is now best defined as the segment between the class I and II regions where the genes do not belong to a predominant class. The major organizational difference between mouse and human is the presence of class I genes (H2-K) centromeric to the class II region in mouse. The bordering segments that are in tight linkage with the Mhc have recently been designated as extended class I and class II regions (Stephens et al. 1999). The consensus maps of H2, assembled before the complete genomic sequence, represented a “virtual mouse” (e.g., the chromosome 17 reports published in Mammalian Genome; Forejt et al. 1999). These maps were an attempt to integrate information available from widely different sources and methods, and data were gathered from various mouse species and strains. Early physical maps used cosmid libraries derived from different strains to map smaller segments of H2. Similarly, the current complete human genome sequence represents only a “virtual human,” as the libraries used for sequencing were derived from several individuals. Now, the human Mhc is being resequenced from haplotype-specific libraries, and two haplotypes associated with autoimmune disease are already finished (Stewart et al. 2004). These complete sequences provide real, haplotype-specific physical maps together with polymorphism data. The Mouse Genome Project used a different approach, as it sequenced the inbred strain C57BL/6, H2 haplotype b (Waterston et al. 2002), therefore providing data for a real physical map. In addition, the mouse Mhc was also sequenced from strain 129, haplotype bc (Kumánovics et al. 2003), thus making the mouse and human data comparable. The characteristic feature of the Mhc is the extraordinary level of polymorphism. This polymorphism is the cause of the histoincompatibility that makes tissue and organ transplantation between unrelated individuals difficult. There are uncommonly high numbers of alleles, 50 or more, for some of the class I and II genes, and unlike most other cases, the alleles usually differ by multiple amino acids. Furthermore, the number of genes may also differ between haplotypes. Copy number polymorphism or genomic segmental polymorphism (Li et al. 2004; Snijders et al. 2005) is the process in which genes expand in a haplotype-specific manner to form subfamilies of closely related genes, varying in number. In this chapter
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MMU17
~500 kb
A
II
EII
I
III
K1/K2
D, Q1-10
T1-24
MICA/B HLA-B/C
HLA-E
EI
M1/M10
M4-6
M3 M2
H2 C4
Class II
HLA-A/G/F
HLA HLA-L
Class II
C4
Class II
Cen.
SLA C4
SLA6/7/8 MIC1/2
SLA-1-5, 9,11
Cen.
FLA B
Fig. 3-1 A, Traditional representation of the Mhc. The Mhc is divided into three regions, classes I, II, and III. The Mhc class I molecules are encoded in the class I region, and the class II molecules are encoded in the class II region. The class III region is located between the class I and II regions and contains the gene for the complement factor C4. B, Realistic representation of the Mhc from four species. Genes are divided into two groups: non-polymorphic genes and the polymorphic class I, class II, and C4. Dotted lines connect the orthologous positions in the class I regions. H2 is the mouse, HLA is the human, SLA is the pig, and FLA is the cat Mhc. Cen. shows the location of the centromere. (See color insert at the back of this book.)
I will concentrate on this latter type of polymorphism, as it has a profound influence on the map of the H2. To describe the genomic organization of the mouse, the diagram of genes is provided (Fig. 3-2) (Shiina et al. 2004). In addition to the linear representation of the gene content of the H2, results of the comparative sequence analysis of various mammalian Mhcs will also be discussed. The organizational rules derived from these studies help us to understand how the Mhcs from different individuals and organisms relate to each other.
III.
COMPARATIVE MAP OF THE MHC
The Mhc was sequenced or mapped from a number of mammalian species. There is a complete or close to complete Mhc sequence from human (MHC Sequencing Consortium 1999; Stewart et al. 2004), rhesus macaque (Daza-Vamenta et al. 2004; Kulski et al. 2004; Otting et al. 2005) chimpanzee (Anzai et al. 2003), rat (Hurt et al. 2004), cat (Beck et al. 2001, 2005; Yuhki and Beck 2003), and pig (Chardon et al. 1999, 2001;
Renard et al. 2001). In addition to these sequences, the Mhc was sequenced or mapped in some detail in cattle, horse, and dog (Debenham et al. 2005; Di Palma et al. 2002; Gustafson et al. 2003; Hess et al. 1999). Comparative analyses of these maps lead to the conclusion that the Mhc unit is present in all mammals. Moreover, the overall organization of the mammalian Mhc is similar to that in mouse and human (Fig. 3-1B). The class I, II, and III regions can be identified in all these species, although the location of the centromere in some species interrupts the linearity of the Mhc, and the “ends” of the Mhc vary among species. The class II region is separated from the class III region by the centromere in pig, cattle, and horse (Fig. 3-1B). In the domestic cat, the centromere splits a portion of the class I region from the rest of the Mhc. In human, the class I region can be extended until some 4 megabases (Mb) telomeric to the last class I gene (HLA-F) to include the class I–like hereditary hemochromatosis gene (HFE). Hereditary hemochromatosis in man is an HLA-linked disease, but the mouse Hfe gene is on chromosome 13 and therefore is not linked to H2. The conserved synteny is disrupted in mouse in the olfactory receptor region, which is located between the class I genes and the hemochromatosis gene (Albig et al. 1998; Amadou et al. 2003).
H 2 -M 3
Mouse
HLA-Z PSMB9 TAP1 PSMB8 TAP2
BTII
*
β β α
Mouse
HNRPA1P2 TSBP/C6orf10
BTNL2
MTCO3P1 DR / IE
β
ZN F57 MOG GABBR1
BRD2
β β β β α
H 2 -M 6 H 2 -M 4 H 2 -M 5
ββ α
H C G V II H C G V III-1 H L A -5 9 H C G IX -4 M IC D H L A -8 0 H L A -A H L A -2 1 H L A -7 0 H C G I V- 6 H C G II-7 H L A -1 6 H L A -5 4 M IC F H L A -G H L A -9 0 H L A -7 5 HCG4 MIC E H L A -F
β
DQ / IA
PPP1R11 T C T E X 5 ZNRD1 T C T E X 6 C6orf12 T C T E X 4
H L A -9 2
O
TRIM26 T R IM 1 5 T R IM 1 0 TRIM40 T R IM 3 1 RNF39
R PP21 TRIM39
α β
H 2 -M 1 0 .1 H 2 -M 1 0 .2 H 2 -M 1 0 .3 H 2 -M 1 0 .4 H 2 -M 1 0 .5 H 2 -M 7 .2 H2 - M 1 - lik e H 2 -M 9 H 2 -M 1 H 2 -M 7 H 2 -M 1 0 .6 H 2 -M 1 0 .7 H 2 -M 1 0 .8
DM
β β αβ β α
BTII
Human Telomeric end of conserved synteny
inversion
Fig. 3-2 Gene content of the human and mouse Mhc. The map starts from the centromeric end at the top and continues down to the telomeric end. The first segment corresponds to the class II and extended class II regions, the second segment corresponds to the class III regions, the third segment corresponds to the class I regions, and the fourth and fifth segments correspond to the extended class I region. Shared genes are shown as boxes crossing the middle line, whereas genes not shared are located above (human) or below (mouse) the line. Species-specific gene expansions are represented by various symbols crossing the middle line. The extended class I region encoding olfactory receptors resists simplifications, therefore the mouse and human region is shown separately. For all the details, see Amadou et al., 2003. (See color insert in the back of this book.) AT T I L A K U M Á N O V I C S
H 2 -M 2
Human
RAN P1 SUCLA2P TMPOP1 M IC C UBQLN1P H L A -3 0
α
H 2 - Tw 5 H 2 - Tw 4 H 2 - Tw 3 H 2 - Tw 2 H 2 - Tw 1
VARS2 G7C/C6orf27 NG23/C6orf26 MSH5 CLIC1 DDAH2 G6B/C6orf25 LYG6C LYG6D LYG6E G6F BAT5 LYG5C LYG5B CSK2B BAT4 G4/C6orf47 APOM BAT3 BAT2 AIF1 NCR3 LST1 LTB TNFA LTA NFKBIL1 ATP6V1G2 BAT1
O
αβ
H 2 -M 3 .3
Mouse G7e
RING1 HSD17B8 SLC39A7 RXRB COL11A2
HTATSF1P ZNF314P
KIFC1 LYPLA2P1 MYL8P DAXX ZNF297 TAPBP RGL2 KE2 BING4/C6orf11 BING5 B3GALT4 RPS18 VPS52
αβαβα α
H2-T11
β α
H2-T18
H2-K2 H2-K
DP
H 2 -M 3 .2
RDBP BF C2 ZBTB12 BAT8 Corf29 NEU1 G8/C6orf48 HSPA1B HSPA1A HSPA1L LSM2
TNXB CYP21A C4 STK19 DOM3Z SKIV2L
NOTCH4 GPSM3 PBX2 AGER RNF5 AGPAT1 EGFL8 PPT2 NG5/C6orf31 FKBPL CREBL1
Mouse
H2-T25
H 2 -T 2 4 H 2 -T 2 3 H2-T22
HCG27 PSORS1C3 PO U 5F1 TCF19 H C R/C6orf18 PSORS1C2 PSORS1C1 CDSN S T G/C6ORF15 HCG22 C6ORF205 C6ORF37OS DPCR1 VA R S2L GTF2H4 DDR1 IE R 3 FLO T1 TUBB MDC1 N RM KIA1949 DHX16 C6orf136 PTMAP1 C6orf134 MRPS18B PPR 1R 10 RBX1PS ABCF1 PRR3 G NL1 H L A -E
M IC B H L A -X M IC A H L A -1 7 H L A -B H L A -C
Human
H 2 -M 3 .1
Ubd
UBD
RPL15P4 PPIP9 Centromeric end of conserved synteny
122
H 2 -D H 2 -Q 1 H 2 -Q 2 H 2 -Q 3 H 2 -Q 4 H 2 -Q e 1 2 -Q 6 .2 H 2 -Q 7 HH 2 - Q o 1 H 2 -Q 6 H 2 -Q 9 H 2 -Q 1 0
Human class II class II pseudogene
butyrophylin-like non-class I/II non-class I/II pseudogene class I
class I pseudogene olfactory receptor
transcript
duplication and translocation orthologus class II and C4 expansions
paralogus class I expansion gene shared by mouse and human
mouse or human specific gene
MMU13
3 . G E N O M I C O R G A N I Z AT I O N O F T H E M O U S E M A J O R H I S T O C O M PAT I B I L I T Y C O M P L E X
The physical size of the Mhc can also vary significantly. In addition to variation in the number of genes, differences in genome-wide repeat content (short and long interspersed repeats and retroviral elements) can also strongly influence the size of the Mhc.
A.
The Class II Region
Class II molecules are present in all mammalian species investigated, but the number of genes varies. Five evolutionarily conserved groups of class II molecules are recognized: DM, DO, DP, DQ, and DR-like (following the human nomenclature). We find all five subgroups expressed in humans, but the DP-like genes are pseudogenes in mice, dogs, and cats. In cattle, goats, and sheep, we find ruminant-specific DYA and DIB genes instead of DP (Ballingall et al. 2001; Hess et al. 1999). In mole rats (Spalax ehrenbergi), however, the DP genes expanded to become the major class II locus, whereas there are no DR genes (Schöpfer et al. 1987). The domestic cat lacks all DQ genes, but an expanded, perhaps as compensation, number of DR genes (Yuhki and Beck 2003). The number of genes in each group also varies, and the variation is often haplotype specific. The human DRB genes are the best studied for these haplotype-specific expansions: Individual chromosomes contain one to five DRB genes in the most common haplotypes (Gongora et al. 1996; Satta et al. 1996). The mouse DQ-like genes are called H2-A (or IA), and the DR-like genes are called H2-E (or IE). In the strains investigated, there is only one A and one E a gene, but there are A and E b pseudogenes in addition to the active A and E b genes. There is one a and one b gene for DO, and one a and two active b genes for DM. This DMb duplication appears to be specific to mouse (Hermel et al. 1995). Unlike the classic class II genes, H2-DM and DO are mono- or oligomorphic. Four of the 11 inbred H2 haplotypes (b, s, f, and q) that were investigated and ~15% of wild haplotypes do not express E class II molecules (Tacchini-Cottier et al. 1995). In contrast to the significant number of H2 haplotypes in both inbred and wild mice that do not express E proteins, there are no reports of naturally A-deficient mice. Analysis of the wildderived haplotypes showed that the origin of the E-null mutations predates the speciation within Mus; therefore, the loss of H2-E protein expression may be advantageous and naturally selected. It is hypothesized that the E-null strains are resistant to infection by mouse mammary tumor virus (MMTV), as the superantigens need to bind to E molecules. Superantigens bind to class II molecules and T cell receptor molecules and thus activate certain subpopulations of T cells. Superantigens can be viral (e.g., MMTV) or bacterial, such as the staphylococcal enterotoxins, for example, staphylococcal enterotoxin B.
B.
123
Non–Class II Genes in the Class II Region
In addition to the class II genes, the class II region also contains a number of other genes. The functions of Tap1 (Abcb2), Tap2 (Abcb3), Psmb8 (Lmp7), and Psmb9 (Lmp2) are closely related to antigen presentation. Tap1 and Tap2 encode the peptide transporter that carries peptides from the cytoplasm into the ER, where the class I molecules are synthesized and assembled (see chapter 1 and the Overview). Psmb8 and Psmb9 are interferon-γ-inducible components of the proteasome, which is the main source of the antigenic peptides that are transported into the ER by Tap1/2. The main organizational difference between murine rodents and the other mammals investigated is that in rodents some of the class I genes (H2-K in mouse and RT1-A in rat) are in close linkage with the Tap1/Tap2 genes (Fig. 3-2). In rats, this linkage is thought to maximize the supply of appropriate peptides to the presenting molecules by promoting the coevolution of Tap transporters and class I molecules, because all the class Ia (or classic class I) molecules in rats are encoded in the RT1-A (or extended class II) region (Joly et al. 1998). In mice, only the H2-K class Ia gene is close to the Tap1/2 genes, but the H2-D locus is separated from the class II region by the entire class III region. In all the other mammals investigated, the class Ia genes are separated from the class II region encoded Tap1/2 genes by the class III region, as are the mouse H2-D (and L) genes. Therefore, a very close genetic linkage between the Tap and proteasome genes and the class Ia genes is probably not obligatory, although the linkage is still only 0.3 cM in mouse. It was long assumed that the presence of class I genes in the extended class II regions of mice and rats is the result of a rodent-specific translocation from the “real” class I region. Sequence analysis of the H2-K and H2-D/Q regions has contradicted this assumption (Kumánovics et al. 2002). The details will be discussed later under the class I region. The other genes in the class II region are without any obvious connection to antigen presentation or to the immune response. Ring3 or bromodomain 2 (Brd2) encodes a nuclear serine-threonine kinase with a bromodomain. Its function is unknown, but it is a homolog of fsh (female sterile homeotic) from Drosophila, and it may play a role in a cell cycle–responsive transcription (Denis et al. 2000; Guo et al. 2000; Tanaguchi et al. 1998; Thorpe et al. 1996). In humans, RING3 is also a probable susceptibility gene for common juvenile myoclonic epilepsy (Pal et al. 2003). Tbsp (or C6orf10 in human) is a testis-specific basic protein of unknown function (Stammers et al. 2000). There are six butyrophilin-like genes in the mouse class II region. Butyrophilins, first identified from milk fat, have no known function. They are related to B7-1 (CD80) and B7-2 (CD86), costimulatory molecules for T lymphocytes (see chapter 1 and the Overview), and to the myelin-oligodendrocyte glycoprotein (Mog), encoded at the telomeric end of H2 (Fig. 3-2). The number of class II region butyrophilin-like genes is also expanded in rats, which have eight, but not in man (Hurt et al. 2004).
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AT T I L A K U M Á N O V I C S
C.
The Class III Region
The class III region is the best-conserved part of the Mhc among mammals. The mouse, rat, pig, and human contain the same set of genes, which are similar in length (Hurt et al. 2004; Nunes et al. 1994; Peelman et al. 1996; Xie et al. 2003). The main difference among the species and various haplotypes is caused by the C4 expansions. C4 (complement component 4) plays an essential role in linking the recognition pathways of the complement system to the effectors of the humoral immune response (see the Overview) Complete or partial C4 deficiency leads to increased risk of infection and autoimmune diseases, such as lupus erythematosus (Blanchong et al. 2000). The class III region was first identified by mapping the immunologically detected Serum substance (Ss) or C4 into the H2 (hence the old name of the class III region: H2-S region). The serum C4 proteins are encoded by a complex genetic system (Blanchong et al. 2001). There are usually two C4-like genes in mice, the constitutively active C4, and Slp (sex limited protein), that is expressed only in adult males of some strains (e.g., B10.D2) (Beurskens et al. 1999). Insertion of a retroviral long-terminal repeat in the promoter region of Slp is the cause of the restricted expression (Stavenhagen and Robins 1988). In some strains, Slp is predominantly but not exclusively expressed in males (e.g., B10.WR), whereas in others, it is expressed in both sexes (e.g., PL/J). C4 and Slp are derived from the duplication of an ancestral C4 gene (Blanchong et al. 2000, 2001; Pattanakitsakul et al. 1990; Yang et al. 1999). The duplication unit also includes the neighboring Tnxb, Cyp21, Rp1, Dom3l, and Ski2w genes. There is a substantial C4 gene dosage variation among the various haplotypes. The H2w7 haplotype has three hybrid C4-Slp genes in addition to the C4 and Slp, leading to a high serum level of Slp in both female and male mice (Huang et al. 1991; Pattanakitsakul et al. 1990). Haplotypes w16 and w19 also contain more than two C4-like genes, but they were not investigated in detail. In H2d mice, however, Slp is a pseudogene due to a single nucleotide insertion, and Slp protein is not expressed. The gene dosage differences in the C4-like genes lead to C4 serum level differences, but gene dosage is not the only cause of serum level differences. The decreased serum level of C4 in H2k mice is caused by a short interspersed element repeat insertion into intron 13 of C4, and not by gene loss (Pattanakitsakul et al. 1992). Unequal meiotic recombination between homologous chromosomes of two different haplotypes can lead to further rearrangements, and, in some instances, to deleterious consequences. Meiotic recombination caused the lethal deletion of C4 and the adjacent steroid 21-hydroxylase (Cyp21) genes in the H2aw18 haplotype (Gotoh et al. 1988; Shiroishi et al. 1987). In addition to the gene dosage and expression-level differences, the C4 genes are also polymorphic (Falus et al. 1987; Miller et al. 1992). Ten polymorphic sites were found in the C4 molecules from the four haplotypes (b, d, w7 and FM) investigated,
and another 10 polymorphic sites among the Slp molecules between haplotypes w7 and FM (Blanchong et al. 2001). The serologically defined C4d.1/ C4d.2 polymorphism is the result of a single amino acid difference (Q1187R). This polymorphism was first described as an H2-associated antigen (first as Antigen G, later H-2.7). Unlike the other H2 antigens that are present on most cells, H-2.7 is present on erythrocytes only. The corresponding human C4 polymorphism (L1191R) results in the Chido/Rodgers (Ch1/Rg1) blood group antigenic determinants (Blanchong et al. 2001). The human C4 gene complex is similar to that of the mouse. There are haplotype-specific C4 expansions in human as well, but the duplication unit is different. Only the TNXB, CYP21, C4, and RP genes are amplified in human, whereas the DOM3L and SKI2W genes are not. As in mouse, the most common is the bimodular arrangement, with two C4-containing segments (Blanchong et al. 2000; Yang et al. 1999). Retroviral (HERV-K) insertions occurred in the human C4 genes (in intron 9) too; therefore, there are long C4 modules containing the retrovirus and short C4 modules without it. As in mouse, heterozygous combinations of mono-, bi- and trimodular arrangements can lead to further rearrangements by unequal crossing-overs during meiosis. Misalignment between a bimodular and a monomodular chromosome can cause deletion of the steroid 21-hydroxylase (CYP21) gene, leading to congenital adrenal hyperplasia, one of the most common genetic errors of metabolism in human (Yang et al. 1999). Deletion of TNXB leads to Ehlers-Danlos syndrome, a generalized connective tissue disorder (Burch et al. 1997; Mao et al. 2002). The human C4 exists in two isoforms: C4A and C4B. C4A and C4B have different substrate preferences, and Slp bears some functional resemblance to the human C4A (Beurskens et al. 1999; Blanchong et al. 2001). The plasma levels of C4A/C4B and C4/Slp are determined mainly by gene dosage (Blanchong et al. 2001). The human C4 genes are polymorphic, and about 30 allotypes are known (Blanchong et al. 2000; Mauff et al. 1984, 1990, 1998). Similarly to human and mouse, species (and conceivably haplotype)-specific C4 duplications occurred in rat too. In the BN/ssNHsd rat strain, C4 module duplication and translocation lead to a second C4 gene (C4-2) and Cyp21ψ and Stk19ψ pseudogenes between the butyrophilin gene cluster and Notch4 gene at the class II–class III border (Roos et al. 2005).
D.
Other Genes in the Class III Region
The class III region is the most gene-dense part of the human and mouse genome. There are 59 genes in this ~700-kilobase (kb)-long segment of human and mouse genome, not counting the duplicated genes of the C4 modules, and all except two are conserved between mouse and man (Fig. 3-2). 1c7 is probably a pseudogene in mouse, whereas it is intact in human and rat (Sivakamasundari et al. 2000). G7e is a retroviral envelope
3 . G E N O M I C O R G A N I Z AT I O N O F T H E M O U S E M A J O R H I S T O C O M PAT I B I L I T Y C O M P L E X
class II region or H2-K region (Dick et al. 2002; Gao et al. 2002; Grandea and Van Kaer 2001; Williams et al. 2002). The class Ia molecules are encoded in the K and D loci. The H2-K region contains two class I genes, K and K2; K2 is a pseudogene in all investigated strains. The D region shows more variation. The H2-D and -Q regions have been mapped previously with cosmids from different H2 haplotypes (b, d, k, p, and q), and now they are sequenced from b and bc haplotypes. All investigated haplotypes were found to be different (Litaker et al. 1996; Kumánovics et al. 2002; Stephan et al. 1986; Watts et al. 1989; Weiss et al. 1984, 1989) (Fig. 3-3). The two prototypical D region organizations in the commonly used laboratory strains are the “one gene” (D in haplotypes b, bc, and k), and the “five genes” (D, D2, D3, D4, and L in haplotypes d and q). H2-L, encoding a class Ia molecule present only in some of the haplotypes, is derived from H2-D by a segmental duplication, in which a portion of the neighboring Bat1 gene is part of the duplication unit and does not constitute a separate “H2-L” region (Rubocki et al. 1990; Wroblewski et al. 1994). D2, D3, and D4 genes were thought to originate from the Q region by unequal crossing-over (Rubocki et al. 1990; Stephan et al. 1986); however, based on the sequence analysis of the H2-Q region, this no longer seems to be likely (Kumánovics et al. 2002). They are probably the result of the complex interaction between the H2-K and -D regions. Sequence comparison of K and D genes from various haplotypes showed weak or no locus specificity (Pullen et al. 1992).
(env)–like gene, present only in mouse (Snoek et al. 1996). In addition to C4, two more complement components (C2 and Bf) are also encoded in the class III region. Other well-known immune genes of the class III region encode the cytokines lymphotoxins α and β and tumor necrosis factor α (Fu and Chaplin 1999; Marino et al. 1997). Intriguingly, about half of the 57 functional genes of the class III region can be associated with the immune response (Gruen and Weissman 2001; Kumánovics et al. 2003).
E.
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The Class I Region
The mouse class I genes are encoded in the H2-K, -D, -Q, -T, and -M regions (Fig. 3-2). The K region was already mentioned, as it is located centromeric to the class II region (Fig. 3-1) and is called the extended class II region in human. The D, Q, T, and M regions are telomeric to the class III region. The class I molecules present antigens to the CD8+ cytotoxic T cells and bind to natural killer (NK) cell receptors (see chapter 6 and the Overview). Many components of the class I antigen presentation pathway are encoded in the class II region: the two Tap proteins and the two interferon-γ-inducible proteasome components are located in the class II region (Fig. 3-2). Tapasin (or TAP binding protein, TAPBP), another accessory molecule for the class I antigen presentation, is encoded in the extended
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Fig. 3-3 Schematic representation of the class I gene content of the H2-D and -Q regions in five mouse haplotypes. Class I genes are represented with black boxes, whereas pseudogenes and gene-fragments are represented with gray boxes. Haplotypes b, bc, and p are based on sequence data, whereas the rest are based on mapping. The question marks represent missing data. In haplotype f, a large deletion is assumed that removed most of the Q region. Preliminary analysis (T. Takada, A. Kumánovics, K. Fischer Lindahl, unpublished) of the sequence from haplotype p (strain B10.P) shows an additional D-region class I gene, named Q11 before the sequence became available, which is equally similar to K and D genes.
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In other words, the H2-K from some strains looks more like the D genes from others and vice versa. Therefore, based solely on the sequence, we cannot tell which locus encodes a mouse class Ia gene. Gene conversion (exchange of short sequences) is the most commonly invoked process to explain this phenomenon. Rat is the only other species investigated that has class I genes in the extended class II region. As all the other mammals lack class I genes in this region, it was assumed that the mouse H2-K and rat RT1-A class I genes are the result of translocation from the main class I region. In mouse, the origin was localized in the H2-D/Q region, based on Southern blot cross-hybridization of probes made from various noncoding sequences (Weiss et al. 1984). Whether this class I “insertion” in the extended class II region happened independently in mouse and rat or before the speciation is debatable (Lambracht-Washington et al. 2000; Walter and Günther 2000). However, analysis of the genomic sequence of the H2-K and D/Q regions showed that the K region cannot be the result of a simple translocation from the H2-D/Q region but rather suggested an ongoing interaction between the K and D regions in which long genomic segments containing entire class I genes and not only short pieces, as suggested by the gene-conversion hypothesis, are exchanged (Kumánovics et al. 2002). The H2-K and RT1-A class I genes were suggested to be the ancestral remains of the Mhc class I genes found in teleost fish, which were lost from other mammalian species. In fish, the class I genes are bordered by TAPBP, RXRB, and COL11A2 genes, similarly to mouse and rat (Kulski et al. 2002).
F.
The H2-Q Region
The class Ib or nonclassic class I molecules are encoded in the H2-Q, -T, and -M regions. In the Q region, haplotype b is thought to represent the basic organization with genes from Q1 to Q10 (Watts et al. 1989) (Fig. 3-3). Haplotype bc (strain 129/SvJ), similar to haplotype b, contains Q1, 2, 3ψ, 4, 6, 7, 9, and 10 genes, but it also encodes two additional genes (Qo1 and Qe1) that are not present in H2b and are most similar to Q8/9d. In BALB/c mice, a putative deletion thought to generate a fusion gene between Q8 and Q9 (Matsuura et al. 1989), but Q8/9d is probably not a result of a fusion: It is just like Qe1bc and Qo1bc. A deletion is thought to have created a fusion gene between Q5 and Q9 in C3H (H2k) removing most of the Qa2 encoding genes (Watts et al. 1989). Assuming a haplotype b–like ancestral arrangement, an even larger deletion removed most of the Q region in haplotype f, leaving only Q10 behind (O’Neill et al. 1986). Detailed investigation of these putative deletions and fusion genes has to wait until more H2 haplotypes are sequenced. The Q region of haplotype p (B10.P) was partially mapped using cosmid clones (Litaker et al. 1996) and is now being sequenced (T. Takada, A. Kumánovics, and K. Fischer Lindahl, unpublished data).
Analysis of the class I sequences and the genomic region suggests that three sets of class I gene expansions generated the Q region class I genes: Q10 separated first; then Q1-like gene expanded to form Q1, Q2, and Q3. An ancestral Qa2-encoding gene expanded differentially in the various haplotypes to form Q4- to Q9-like genes. These expansions were further modified by insertions and deletions. This variation is not artificial and is restricted to the laboratory strains as it is present in wild-derived mice as well (Tine et al. 1990). Depending on the haplotype, the Q region encodes 5–10 class I genes, which fall into two groups. Some of the Q genes (Q1, Q2, Q4, and Q10) have a tissue-restricted expression and, although present in nearly all haplotypes, their function is unknown. The Qa2 antigen, detected on the surface of many cell types, is encoded by a variable number of genes; for example, Qa2 is encoded by Q6, Q7, Q8, and Q9 in haplotype b. The Qa2 expression level varies among haplotypes with the number of genes encoding Qa2. Qa2 has secreted and membrane-bound isoforms (Tabaczewski et al. 1994). Qa2 can protect cells from NK-mediated lysis and plays a role in resistance to Taenia crassiceps infection (Fragoso et al. 1998). Q genes can participate in the defense against tumors. Q9 expressed on the surface of tumor cells protects syngeneic hosts from melanoma outgrowth (Chiang et al. 2002; Chiang and Stroynowski 2004, 2005). Qa2-encoding genes were also identified as the Ped (preimplantation embryo development) locus, affecting the cleavage rate of preimplantation embryos (Goldbard et al. 1982; Wu et al. 1999). Having secreted and membrane-bound isoforms, a broad peptide-binding repertoire (He et al. 2001; Tabaczewski et al. 1997) similar to that of the class Ia proteins, a protective role against NK lysis, and a putative role in embryonic development are features shared between Qa2 and the human HLA-G (Le Bouteiller 1997). HLA-G and Qa2 might be analogous functionally, but they are not related phylogenetically (i.e., not orthologs). The corresponding segment of the human Mhc (Fig. 3-2) is also the result of a class I expansion, it contains two class I–like genes, MICA and MICB, and the class Ia genes HLA-B and HLA-C, in addition to several class I gene fragments (Guillaudeux et al. 1998). MIC genes are found in other mammals, such as pigs (Chardon et al. 2001), but not in rodents, leading to the conclusion that the rodent lineage lost the MIC-like genes. There are several class Ib genes in mouse that appear rodent specific, but according to phylogenetic analysis, these genes are older than the separation of rodent lineage, suggesting that some ancient class I–like genes were lost in some of the lineages and others were amplified: MIC genes are lost in rodents, and TL and M10 genes are lost in primates and human (see below). Moreover, there are MIC-like Mill genes (Mill1 and Mill2, encoded outside of the H2) in rodents that are missing in human (Watanabe et al. 2004). HLA-B and HLA-C are not orthologous to H2-D (or H2-L). The HLA-B/C region expansion derived from an already human-specific (in a human-mouse comparison) ancestor,
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whereas the H2-D/L and H2-Q duplications derived from mouse-specific ancestors (Kumánovics and Fischer Lindahl 2004). Phylogenetic analysis of human and mouse class I sequences clearly showed that the human genes and the mouse genes group according to species and not according to location. For example, HLA-B/C does not group together with H2-D/L or HLA-A/G/F with H2-M4/5/6. The same holds true for the comparison of mouse with pig (Renard et al. 2003). The corresponding segment in pigs contain three class Ib genes, a MIC gene, and a MIC pseudogene (Chardon et al. 2001) (Fig. 3-4). The corresponding region in rat between Bat1 and Pou5f1 is called RT1-C/E, and it also contains class I expansions. But even the closely related rat class I expansions are completely independent of the ones in mice. Sequence analysis of the region showed class I duplications mechanistically similar to the H2-Q duplications, but the RT1-C/E genes are not orthologous to the H2-D/Q genes. The H2-Q (and RT1-C/E) genes appear to be the youngest class Ib genes, as they are not shared even among rodents (Roos and Walter 2005).
G.
Genes between the H2-Q and -T Regions
The various regions in the mouse Mhc were first defined genetically using recombinant strains. Genetically, the H2-Q region extends until Abcf1 (Figs. 3-2 and 3-4), leaving two nonclass I genes for the T region (Prr3 and Gln1). On the basis of the gene structures and functions, it is more practical to discuss the many genes located between H2-Q10, the most
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telomeric class I gene in the H2-Q region, and H2-T24, the most centromeric class I gene in the H2-T region. The genes of the region are well conserved between the investigated mammals; mapping and sequencing showed that they are present in human, mouse, rat, pig, cat, and horse. This is in sharp contrast to the neighboring regions encoding Mhc class I proteins, which are not conserved. These conserved genes are often referred to as “framework genes” (Amadou 1999). Using these conserved genes as anchors or a framework, we can align the Mhc from all species (Fig. 3-4). The genomic segments located between H2-Q to -T in mice and HLA-C to -E in humans are reminiscent of the Mhc class III region. They are similar in size (the class III region is ~600 kb in both species), and they both encode ~30 non–class I or II genes, which are mainly unrelated to each other and conserved between species. Both the class III regions and the Q-T/C-E regions have about four to five times higher gene density than the genome average of 10 per Mb (Waterston et al. 2002; Xie et al. 2003). Thirty-one loci are known in human, and 26 of these are conserved between man and mouse. All of the conserved loci are predicted to be expressed genes, whereas the nonconserved loci are unconfirmed transcripts (e.g., HCG27), pseudogenes (e.g., PTMAP1), or putative noncoding regulatory RNA genes (e.g., PSORS1C1; Fig. 3-2). None of these 26 genes have an obvious functional connection to antigen presentation or even to the immune response in general (Shigenari et al. 2004; Shiina et al. 2004). The human region between HLA-C and VARS2L is thought to contain a psoriasis susceptibility gene (PSORS1; Nair et al. 2000; Oka et al. 1999), nonmelanoma
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Fig. 3-4 Schematic representation of the human, mouse, rat, and pig Mhc. The traditional regions of the mammalian and mouse Mhc are given on the top of the figure. Black blocks represent conserved non-polymorphic or framework genes. Colored symbols represent class I, II, C4, and olfactory receptor expansions. Orthologous gene expansions are shown where the genes in the same position of the species are more similar to each other than to other genes from the same family in the same species. Paralogous class I gene expansions have also occurred in the same position as defined by the framework, but the class I genes are more related to other class I genes from the same species than to class I genes in the same position in other species. The name of the class I expansions is shown below the double arrows; the number of genes in the expansions, including pseudogenes, is shown above the double arrows. A few other genes are named for orientation. In pig, the class II region is separated from the class III and class I regions by the centromere (Fig. 3-1).
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skin cancer, hypotrichosis (CDSN; Levy-Nissenbaum et al. 2003) and diffuse panbronchiolitis (Matsuzaka et al. 2002).
H.
The H2-T Region
Even though the H2-T region encodes the first class Ib antigen to be described (TL; Old and Boyse 1963), it is the last part of the mouse Mhc to be sequenced because of technical difficulties. The number of class I genes is ~20; this number differs greatly among haplotypes because of large-scale duplications (BALB/c 2× and A/J 3×; Brorson et al. 1989; Hammerling et al. 1985; Teitell et al. 1994). Duplications have generated multiple highly similar genes and gene fragments. Organization of the H2-T region class I genes was investigated using cosmid clones derived from H2d (BALB/c) and H2b (C57BL/10 and C57BL/6). In these mapping studies a single contiguous map covering the entire region could not be built. With use of BAC clones, the region was cloned and sequenced from C57BL/6 and 129/SvJ. But so far the sequencing failed to give a correct assembly. The C57BL/6 sequence assembly generated by the Mouse Genome Project contains too few genes; previous cloning and mapping studies on this strain identified more genes than what we can find in the present assembly. Some of the repeated units are probably merged in the current assembly. The 129/SvJ sequences also have a problem: There are large segments of near-identical sequences that cannot be arranged; therefore the exact number of genes and gene order cannot be determined. One thing is clear, however: There are multiple copies of T23/T11-, T22/T10-, T25-, and T18/T3-like genes, pseudogenes, and gene fragments. The large numbers of pseudogenes and gene fragments of various sizes suggest that the majority of the class I genes detected by hybridization in the T region are not functional. The two ends of the region, however, are well connected to the neighboring regions, and the sequence clearly shows that the most centromeric genes are H2-T24, 23, and 22, and the most telomeric ones are H2-T5, T4, T3, T2, and T1, ordering and orienting the earlier cosmid-based maps (Fig. 3-2). Only a few of the H2-T genes have been characterized functionally. As already mentioned, the majority of the class I genes are probably nonfunctional; therefore, we might know more about the T region class I genes than we thought. The TL (for thymus leukemia) antigen is found on intestinal epithelium/intraepithelial lymphocytes in all strains tested, whereas only some express it on thymocytes (Wu et al. 1991). Depending on the haplotype, it can be encoded by one (T3), two (T3 and T18), or three genes (in the A strain). The H2-T10, H2-T22 gene pair encodes heavy chains with a severely modified Mhc class I fold (Adams et al. 2005; Shin et al. 2005; Wingren et al. 2000) that still form heterodimers with β2-microglobulin, but do not require any ligand for binding to γδ T cells. About 0.4% of the γδ T cells from naïve animals recognize T10/T22 (Crowley et al. 2000). H2-T25 or “blastocyst MHC” is expressed in the placenta and in preimplantation
embryos at the blastocyst stage. Because of this expression pattern, it was proposed to be a functional analog of HLA-G (Sipes et al. 1996; Tajima et al. 2003). H2-T23 (and perhaps the very similar T11) encodes Qa1 (Hermel et al. 2004), the mouse functional equivalent of HLA-E (Rodgers and Cook 2005). Qa1 predominantly binds Qdm peptide, which is derived from the signal sequence of class Ia molecules. The Qa1/Qdm complex is the ligand for CD94/NKG2A or CD94/NKG2C inhibitory receptors expressed on some NK cells. Qa1-deficient mice develop exaggerated secondary CD4+ T cell responses after viral infection or immunization with foreign or self peptides. Qa1 and HLA-E both present class Ia leader peptides, but they are not orthologs. Leader peptide presentation by HLA-E and Qa1 was shown to be the result of convergent evolution (Yeager et al. 1997). Although the case for convergent evolution is strong, it should be mentioned that HLE-E and Qa1 map to the same location (Fig. 3-2) and that HLA-E could not be fitted into the series of class I duplications that formed the present day human class I region (Shiina et al. 1999). One hypothesis about the origin of the class Ib genes proposes that the class I genes derive from the class Ia genes by rapid gene duplication; hence they are young and shared between closely related species only (Hughes and Nei 1989; Nei et al. 1997). Such class Ib genes might be the H2-Q genes. However, phylogenetic analysis of TL-encoding genes from 11 Murinae species showed that TL is nearly as old (~100 million years) as the radiation of placental mammals (Davis et al. 2002). Therefore, the ancestors of species, which do not contain TL-like genes, have lost TL instead of rodents inventing it. A similar analysis was carried out with M3, another rodent-specific class Ib gene, which was also shown to be old. Similarly, an ancient origin was suggested for M1/M10 genes (see below under M region). In human, the corresponding region contains only one functional gene, HLA-E, and several pseudogenes and gene fragments, including a MIC (MICC) and a class I pseudogene (HLA-30; Stewart et al. 2004). In pig, this region does not exist: There are no class I genes between Gnl1 and Rpp21 (Fig. 3-4) (Shigenari et al. 2004). The corresponding region in rat is called RT1-N and contains a large number of class Ib genes (Fig. 3-4) (Hurt et al. 2004). Just like H2-T24 in the H2-T region, the orthologous RT1-T24-1 is the most centromeric class I gene. There are, however, three more T24-like genes in rat derived from the duplication of RT1-T24. This type relationship is true for all the other genes in the region: Rather than the simple one-to-one orthologous gene pairs that we see in the framework regions, we find orthologous gene families that undergo species-specific expansions (co-orthologs) such as the class II genes in a human-mouse comparison. Further examples from the RT1-N and H2-T region are RT1-V1, V2, P1, and P2 that encode genes similar to H2-T1, T3, and T18, respectively, and RT1-N2 and N3 are similar to H2-T9 and T10/T20, respectively. The rat ortholog of the mouse Qa1 (encoded by H2-T23) is called RT-BM1 and is also located in RT1-N.
3 . G E N O M I C O R G A N I Z AT I O N O F T H E M O U S E M A J O R H I S T O C O M PAT I B I L I T Y C O M P L E X
The T region is separated from the M region by two conserved non–class I genes, Rpp21 and Trim39. Rpp21 encodes a subunit of the pre-tRNA processing factor RNase P, and Trim39 encodes a protein of unknown function, which belongs to the tripartite motif (RING domain, B box, and coiled coil region) protein family (Meyer et al. 2003).
I.
The H2-M Region
The H2-M region encodes the best-characterized mouse class Ib molecule, H2-M3 (Fischer Lindahl et al. 1997). M3 presents N-formylated peptides derived from bacteria, thus playing an important role in defense against infection. It can also serve as a minor histocompatibility alloantigen by presenting N-formylated peptides from the mitochondrion. The region was named after M3, first described as Maternally transmitted antigen (Mta), as it presents mitochondrial peptides. The other M region genes are not as well characterized. The three functional H2-M1–like and the six H2-M10–like class I genes are expressed only in the vomeronasal organ (VNO; Ishii et al. 2003; Loconto et al. 2003). M2 and M5 also have open reading frames; M2 is expressed in the thymus (Brorson et al. 1989; Moore et al. 2004; Wang and Fischer Lindahl 1993). Unlike the H2-D, -Q, and -T regions, the M region appears to be stable among the haplotypes (Jones et al. 1995, 1999).
J.
M1 and M10 Genes
M region class I genes are found in three different locations. The first expansion occurred between Trim39 and Trim26, resulting in nine M1/M10 class Ib genes expressed in the VNO (Jones et al. 1999; Ishii et al. 2003; Loconto et al. 2003; Takada et al. 2003). The corresponding region in human contains only one class I pseudogene, HLA-92. In pig, the region contains seven class I genes, three of which are pseudogenes (Renard et al. 2001). As before, the pig and human class I genes found in this location are not related to the M1/M10 genes. Phylogenetic analysis of the M1/M10 genes suggests that they originated before the separation of human and rodent lineages; therefore, it is likely that these genes were lost by human ancestors (Kumánovics et al. 2003). M1 and M10 are proteins thought to participate in the pheromone receptor function in the VNO (Ishii et al. 2003; Loconto et al. 2003; Olson et al. 2005). Pheromones are water-soluble chemicals used by individuals of the same species to elicit social and reproductive behaviors. Pheromones are sensed primarily by the VNO in terrestrial vertebrates. In humans, the VNO is probably not functional. Almost all of the human VNO receptors are pseudogenes, as well as some key components of receptor signaling, such as the TRP2 ion channel. Presumably there is no need for M1/M10 proteins in humans if the VNO is not functional.
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In rat, the M1- and M10-like genes are found in the same position as in mouse (Fig. 3-4). Similarly to the case of the H2-T and RT1-N regions, the M1 and M10 genes expanded independently in the two species, but the orthology can be recognized between the mouse and rat M1/M10 genes (Hurt et al. 2004). In cat, this is the region (between Trim26 and Trim39) where the centromere disjointed the telomeric end of the class I region from the rest of the Mhc (Fig. 3-1) (Beck et al. 2005). The second group of M region class I genes are separated from the M1/M10 genes by nine conserved framework genes. Five of these genes encode proteins from the Trim family (Fig. 3-2). Among these, Trim10 protein (or Herf1) plays a role in terminal differentiation of erythroid cells. Rnf39 might play a role in an early phase of synaptic plasticity. Tctex6 encodes a zinc ribbon domain containing protein. Tctex5 (t-complex testis-expressed 5) or Ppp1R11 encodes the regulatory subunit 11 of protein phosphatase 1. Tctex4 encodes a β subunit of casein kinase II (Csnk2b), a ubiquitous multifunctional serinethreonine kinase (Reymond et al. 2001; Takada et al. 2003).
K.
H2-M4, M5, and M6 Genes
H2-M4, M5, and M6 form the second group of M region class I genes. M4 and M6 are pseudogenes, but M5 is potentially expressed. All three are orthologous between rat and mouse. Interestingly, the rat RT1-M4 and M6 genes are intact and not pseudogenes like their mouse orthologs (LambrachtWashington and Fischer Lindahl 2004). In rat, there are two M6 genes derived from a local duplication. The functions of these genes have not been investigated. In human, the region contains HLA-A, -G, and -F, in addition to a large number of class I, MIC, and other pseudogenes (Fig. 3-2). This is the largest class I expansion in human. HLA-A is a class Ia protein, whereas HLA-G and -F are both class Ib proteins. None of these class I genes nor any of the pseudogenes from this region are related to H2-M4, M5, or M6. HLA-G is indicated in embryonic development and therefore often is compared with the mouse Qa2 encoding genes, which also have a number of splice variants, including secreted forms (Comiskey et al. 2003; Tabaczewski et al. 1994). HLA-G interacts with the inhibitory leukocyte immunoglobulin-like receptors LIR-1 and LIR-2. The function of HLA-F remains unknown (Rodgers and Cook 2005).
L.
Telomeric End of the M Region or the Extended Class I Region
M3 and M2 class Ib genes are embedded in a region of olfactory receptor genes. The olfactory receptor (OR) genes are the largest gene family in mammals. There are 59 Mhc-linked OR loci in mouse and 25 in human. About 20% of the mouse genes, and ~50% of the human OR genes are pseudogenes.
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The OR genes can be grouped into subfamilies, and most of these OR subfamilies are conserved between man and mouse (Fig. 3-2) (Younger et al. 2001). Similarly to the class II genes, the OR genes underwent species-specific expansions, resulting in highly similar family members. These OR genes in mouse coduplicated with class I genes that resulted in three M3-like pseudogenes in addition to M3 (Amadou et al. 2003). Whereas M3 is well studied, the function of M2 is not known, but it is found in most strains and shows limited polymorphism (Moore et al. 2004). Its surface expression is independent of TAP, indicating a function different from that of class Ia proteins. Rats have M3 and M2 genes, and similar to mouse, the rat OR genes coduplicated with the M3, creating M3-like pseudogenes (Hurt et al. 2004). The region in human is called the extended class I region. In human, the genetic linkage, and therefore the extended class I region, extends ~4 Mb beyond the most telomeric class I gene, HLA-F, including the Mhc class I–like hereditary hemochromatosis gene, HFE (Albig et al. 1998). The conserved synteny between man and mouse ends in the OR region (Fig. 3-2) (Amadou et al. 2003). The mouse homologs of the human genes hs6M1-1 and hs6M1-15 as well as the mouse Hfe belong to a block of conserved synteny on mouse Chr 13 (Albig et al. 1998). The order of the OR genes and their transcriptional orientation are conserved between mouse and man. The only exception is the relative inversion of Olfr124 and Olfr125 with regard to hs6M1-28 and hs6M1-22. A single inversion in the mouse, marked by a double arrow on Fig. 3-2, would reestablish the order and transcriptional orientation. Intriguingly, no polymorphism was observed for the 10 OR genes located distal to this ~300-kb inversion, whereas the corresponding human genes do harbor polymorphic positions (Ehlers et al. 2000). The break of synteny marks the end of the physical linkage, and the inversion may mark the end of the tight genetic linkage with the Mhc. Mice prefer to mate with a partner with a dissimilar H2 haplotype. Both the human and mouse OR genes are polymorphic, but there is no association between the classic H2 haplotypes and OR alleles in the mouse strains studied (C57BL/6, 129/SvJ, DBA/2, and A/J). Therefore, the H2-dependent, olfaction-associated mate choice is unlikely to be mediated by the Mhc-linked OR genes (Amadou et al. 2003).
M.
Unity of the Class I, II, and III Regions/Shared
Features of the Mhc Class I, II, and III Regions Replacing the traditional tripartite Mhc depiction (Fig. 3-1A) with figures contrasting the conserved framework genes and nonconserved class I, II, and C4 genes (Figs. 3-1B and 3-4) helps illustrate the genomic structure of the Mhc. Before the genomic sequencing and comparative analysis, the class III region appeared to be somewhat of an outsider: it does not encode Mhc class I or class II genes or any other component of antigen presentation.
Some thought it should not be called Mhc (Klein and Sato 1998); others divided it to differentiate the telomeric half, in which several inflammatory genes reside (Gruen and Weissman 2001). In the light of the entire sequence of the Mhc, one can clearly see (Figs. 3-1B and 3-4) that the class III region is no different from the class II/extended class II or class I/extended class I regions. They all contain a large number of conserved framework genes, among which polymorphic immune genes expand in a speciesand haplotype-specific manner. The class II region is where we find class II gene expansions, the class I region is the place of the class I gene expansions, and the class III region is defined by the C4 expansion. The class I, II, and C4 genes are already linked in the phylogenetically oldest vertebrates with Mhc, in the cartilaginous fishes (Ohta et al. 2000). This linkage is not an absolute requirement, as it is broken in the teleost fishes, but it is largely maintained in mammals. The class I, II, and C4 gene expansions took place in different organisms in slightly different places as defined by the framework genes. For example, the K-region expressed 4 (Ke4) gene is considered a class I region gene in mouse and also in zebrafish, but it is usually described as an extended class II region gene in human. Similarly, many mammalian class III region genes (Ppt2, Pbx2, Ski2w, G9a, Csk2b, and G2) are present in the class I region of zebrafish (Sültmann et al. 2000). In mammals, the class II region contains the Tap1, Tap2, and proteasome (Psmb) genes, that are clearly part of the class I antigen presentation pathway. Even though the Tap genes can coevolve with the class Ia genes, as shown in rat (Joly et al. 1998), the Tap genes are formally part of the class II region. Tap and Psmb genes are located in the class I region of fishes. If we replace the old class I, II, and III region partition of the Mhc with a more realistic depiction, such as the ones presented here in Figs. 3-1 and 3-4, then these counterintuitive classifications disappear, and the various species and haplotypes can easily be compared.
IV.
SUMMARY
The sequence of the Mhc from mouse and human, together with a large amount of sequence and mapping information from several other species, allows us to draw simple rules about the organization (Fig. 3-4) and origin of this crucial part of the immune system: 1. The Mhc, as a genomic region maintained across species, is a mosaic of conserved and nonconserved genes. The segments that encode the class I, class II, and C4 genes are the least conserved parts, whereas segments that encode other genes are highly conserved among mammals. 2. The class I-, class II-, and C4-encoding segments are also the regions in which the various haplotypes within a species differ. 3. Two different types of segmental polymorphisms can be distinguished in the Mhc according to the ancestor of the expanding segment: a) In the class II and III, C4, and
3 . G E N O M I C O R G A N I Z AT I O N O F T H E M O U S E M A J O R H I S T O C O M PAT I B I L I T Y C O M P L E X
olfactory receptor regions, the orthologous relationship is largely maintained among the subgroups (e.g., DP, DR/IA, DQ/IE, DM, and DO); and b) the class I region is more complex; the level of conservation depends on the relationship of the compared species. Unlike in the class II regions, there are no universally conserved groups of class I genes among mammals. In other words, the expanding ancestral gene(s) can already be species specific.
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Chapter 4 Some Biological Features of Dendritic Cells in the Mouse Kang Liu, Anna Charalambous, and Ralph M. Steinman
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Some Properties That Have Been Used to Identify Mouse Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Generation of DCs from Proliferating Progenitors . . . . . . . . . . . . . . . . . . . IV. Distribution of DCs in Lymphoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . V. DCs in Nonlymphoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Life Span and Turnover of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Features of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Antigen Handling: Uptake, Processing, Presentation . . . . . . . . . . . . . . C. Maturation of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. DC Subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Migration of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Functions of DCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Innate Responses via Toll-Like Receptors and Interactions with Innate Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . B. Initiating Adaptive T Cell Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Controlling the Quality of the T Cell Response . . . . . . . . . . . . . . . . . . D. Initiating Different Types of Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . E. Interactions with Antibody-Forming B Cells . . . . . . . . . . . . . . . . . . . . . IX. Summary of Approaches to Analyzing DC Function in Mice . . . . . . . . . . . A. DCs Studied Ex Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. DCs Generated Ex Vivo from Proliferating Progenitors . . . . . . . . . . . . C. Direct Targeting of Antigens to DCs within Intact Lymphoid Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Creation of Improved Mice Carrying Human Immune Systems . . . . . . X. Appendix: Protocols for the Isolation of DCs . . . . . . . . . . . . . . . . . . . . . . . A. Isolating Spleen Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Isolating Bone Marrow Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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I.
LIU,
ANNA
INTRODUCTION
The discovery of dendritic cells (DCs) was the result of an effort to understand the initiation of immunity. The mouse as an experimental animal was critical because of a system, developed by Mishell and Dutton (1967), in which spleen cell suspensions could be stimulated to generate antibody responses in culture. At the time, this was the one system in which a primary immune response could be induced to an added antigen, sheep red blood cells. In contrast, other studies of immune responses were emphasizing the use of polyclonal mitogens, rather than specific antigens, or the use of cells that had already
CHARALAMBOUS,
AND
RALPH
M.
STEINMAN
been primed or immunized beforehand in vivo. Distinctive DCs were recognized in mouse spleen. Subsequent investigations revealed that these previously unknown cells could organize many layers of immune function, both initiating and restraining (tolerizing) antigen-specific immunity and recently, immune memory. In this chapter, we discuss the preparation of DCs from mice, as well as the tissues in which they are found and through which they migrate. Some methods that are currently used to study mouse DCs are provided in the Appendix. Then we outline some of the special features and functions of DCs, with emphasis on knowledge gained from genetically defined mice. The mouse strains cited in this review are listed in Table 4-1.
TABLE 4-1
GENETICALLY MODIFIED MOUSE STRAINS USEFUL FOR THE STUDY OF DENDRITIC CELLS Modifications that primarily affect DCs CD11c−DTR A diphtheria toxin receptor (DTR)/DT−based system that allows the inducible, short−term (1–2 days) ablation of DCs, e.g., DC−depleted mice fail to present cell−associated antigen to cross−prime cytotoxic T lymphocyte (CTL) precursors (Jung et al. 2002) and develop maximal memory T cell responses (Zammit et al. 2005). Bone marrow chimeras allow for repeated injection of DT and long−term ablation of DCs (Zammit et al. 2005). Langerin (CD207)−DTR mice
A DTR/DT−based system for inducible ablation of LCs up to 4 weeks without affecting the skin environment. The dermal DC compartment remains normal. In Langerin−DTR mice, contact hypersensitivity is decreased but not absent, indicating that dermal DCs also contribute (Bennett et al. 2005).
B6CD11c−Eα
Targeted expression of MHC class II gene (I−E) to DCs in vivo to show that thymic DCs are sufficient to negatively select I−E reactive CD4+ T cells (Brocker et al. 1997).
Langerin−/−
Disruption of the langerin/CD207 gene abolishes Birbeck granules without affecting LC function (Kissenpfennig, Ait−Yahia, et al. 2005).
Langerin−EGFP, Langerin−DTREGFP
LC motility and migration to lymph nodes are triggered by skin inflammation. Dermal DCs arrive in lymph nodes earlier than LCs and colonize distinct areas (Kissenpfennig, Henri, et al. 2005).
Antigen capture and presentation RIP−mOVA, RIP−YSS
Transgenic mice expressing ovalbumin (OVA) specifically in pancreatic β−islet cells. OVA−specific, OT−I, T cells only proliferate in the draining pancreatic lymph nodes, and the presentation of tissue−specific antigen depends on bone marrow–derived CD8α+ DCs (Belz et al. 2002; Kurts et al. 1996).
DEC−205−/− (or CD205−/−)
DEC−205 is a multilectin receptor on a subset of DCs. Antigens delivered to DEC−205 are endocytosed and processed efficiently for T cell priming (Bonifaz et al. 2002; Hawiger et al. 2001).
MR−/− (mannose receptor) (or CD206−/−)
MR, a multilectin receptor on macrophages and DCs, is homologous to DEC−205/CD205. It influences serum glycoprotein homeostasis; e.g., MR−/− mice are defective in clearing proteins bearing accessible mannose and N−acetylglucosamine residues and have elevated levels of eight different lysosomal hydrolases (Lee et al. 2002).
cx3cr1GFP/GFP
Transgenic mice with fractalkine receptor CX3CR1 replaced by green fluorescent protein. CX3CR1−dependent processes control host interactions of specialized DCs with commensal and pathogenic bacteria; e.g., lamina propria DCs depend on the CX3CR1 to form transepithelial dendrites, which enable the cells to directly sample luminal antigens. CX3CR1 also controls the clearance of entero−invasive pathogens by DCs (Niess et al. 2005).
Development, differentiation and homeostasis
Because none of these transcription factors is DC−restricted, these knockout mice often have defects in other cells, e.g., T cells and B cells.
Fms−like tyrosine kinase 3 ligand (flt3L)4−/−
Flt3L−/− mice have deficient hematopoiesis affecting DCs and NK cells (McKenna et al. 2000).
IRF−2−/−
IRF−2−/− mice have a selective loss of the CD8α− DC subset (Honda et al. 2004; Ichikawa et al. 2004).
IRF−4−/−
IRF−4−/− mice have a selective loss of the CD8α− DC subset (Suzuki et al. 2004).
IRF−8−/−
IRF−8 is essential for the development of mouse type I interferon−producing cells and CD8α+ DCs (Schiavoni et al. 2002). IRF−8 is involved in the maturation of CD8α− DCs (Aliberti et al. 2003).
4. SOME
BIOLOGICAL
FEATURES
OF
DENDRITIC
CELLS
IN
THE
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MOUSE
TABLE 4-1
GENETICALLY MODIFIED MOUSE STRAINS USEFUL FOR THE STUDY OF DENDRITIC CELLS—cont’d NF−κB (RelB−/−, p50−/−RelA−/−, p50−/−cRel−/−)
DC development and survival require distinct nuclear factor (NF)−κB subunits. DC development in doubly deficient p50−/−RelA−/− mice is significantly impaired. In contrast, DCs from p50−/−cRel−/− mice develop normally, but survival and IL−12 production are impaired (Ouaaz et al. 2002). RelB is important for normal thymic medullary structure and development of splenic CD8α− DCs (Wu et al. 1998).
TGF−β−/−, Id2−/−
Id2−/− mice lack LCs, and the splenic CD8α+ DC subset is markedly reduced. Mice deficient for TGF−β also lack LCs. In DCs, TGF−β induces Id2 expression, which represses B cell genes (Borkowski et al. 1996; Hacker et al. 2003).
Ikaros−/−
Ikaros−/− mice have reduced numbers of T cells and CD8α+ DCs (Wu et al. 1997).
LT−βR−/−
LT−βR is required for homeostasis of splenic DCs (Kabashima et al. 2005).
DC-T cell interaction CD11c−EYFP
Transgenic mice with DCs expressing enhanced yellow fluorescence protein at high levels permit examination of DCs with two−photon microscopy in live mice. Steady−state DCs form an extensive network in which the DCs do not move translationally but instead probe adjacent T cells with their processes. Early antigen−dependent T cell arrest on DCs is a shared feature of the tolerance and priming that is associated with T cell proliferation (Lindquist et al. 2004; Shakhar et al. 2005).
TSLP−/−
TSLP is required to mount a CD4+ T cell–mediated inflammatory response. It not only acts directly on naive CD4+ T cells to promote their proliferation in response to antigen but also exerts an effect indirectly through DCs to promote Th2 differentiation of CD4+ T cells (Al-Shami et al. 2005).
Signaling and survival MyD88−/−
MyD88−deficient mice have a profound defect in the activation of antigen−specific Th1 but not Th2 immune responses (Schnare et al. 2001). MyD88 is indispensable for inflammatory cytokine production in IL−1 receptor/TLR signaling. DCs from MyD88−deficient mice fail to produce inflammatory cytokines (TNF−α, IL−6, and IL−12) in response to IL−1 or other TLR ligands, such as pam−3−cys and CpG−DNA. However, they do mature in response to lipopolysaccharide (TLR4 ligand) or double−stranded RNA (TLR3 ligand) (Kaisho et al. 2001), which uses TRIF as a TLR adapter.
CD40−/−, CD40L−/−
Signaling through CD40 can replace the requirement for TH cells, indicating that T−cell “help,” at least for generation of CTLs by cross−priming, is mediated by signaling through CD40 on the antigen−presenting cell (Bennett et al. 1998). CD40 ligand−independent T helper cell activation requires the TNF family member, TRANCE (Bachmann et al. 1999). CD40 is critical for NKT cell–activated DC maturation (Fujii et al. 2004). Other than CD40L, there may be alternative ligands for CD40 such as Mycobacterium tuberculosis hsp70 (Lazarevic et al. 2003).
γ chain−/−, FcγRIIB−/−
Immune complexes can function to maintain tolerance through binding to inhibitory FcγRIIB on DCs, but immune complexes can also serve as potent immunogenic stimuli by selective engagement of activating FcγRs (Kalergis and Ravetch 2002).
TRAF6−/−
TRAF6 is an adapter molecule for signaling through IL−1 receptors, TLRs, and TNF receptors. TRAF6 is required for DC responses to some maturation stimuli. TRAF6−/− bone marrow chimeras lack the CD8α− DC subset (Kobayashi et al. 2003).
DC-B cell interaction SOCS1−/−
SOCS1 plays an essential role in normal DC functions and suppression of systemic autoimmunity. SOCS1−/− DCs accumulate in the thymus and spleen and produce high levels of BAFF/BLyS and APRIL, resulting in the aberrant expansion of B cells and autoreactive antibody formation in vivo (Hanada et al. 2003).
DC-innate lymphocyte interaction Jα18−/−, MyD88−/−, CD40−/−
NKT cells mediate DC maturation in a MyD88−independent but CD40−dependent manner (Fujii et al. 2003, 2004).
CXCR3−/−, CCR7−/−, IFN−γ−/−
Mature DCs recruit NK cells to lymph nodes in a CCR7− and CXCR3−dependent manner, and the NK cells provide IFN−γ for Th1 priming (Martin-Fontecha et al. 2004).
IL−15−/−
The IL−15/IL−15R interaction is critical in the early activation of antigen−presenting cells and plays an important role in the innate immune system (Ohteki et al. 2001). Continued
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TABLE 4-1
GENETICALLY MODIFIED MOUSE STRAINS USEFUL FOR THE STUDY OF DENDRITIC CELLS—cont’d DC migration CCR2−/−, CCR5−/−
The recruitment of new LCs is dependent on their expression of the CCR2 chemokine receptor and on the secretion of CCR2−binding chemokines by inflamed skin (Merad et al. 2002). Recruitment of the TNF/iNOS−producing (Tip)−DC subset in spleens of L. monocytogenes–infected mice is dependent on CCR2 and independent of CCR5 (Serbina et al. 2003; Zhong et al. 2004).
CCR6−/−
CCR6 mediates DC localization, lymphocyte homeostasis, and immune responses in mucosal tissue. In CCR6−/− mice, DCs expressing CD11c and CD11b are absent from the subepithelial dome region of Peyer’s patches (Cook et al. 2000).
CCR7−/−, plt/plt (CCL19 and CCL21−/−)
CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs (Forster et al. 1999). plt/plt mice lacking CCR7 ligands, CCL19 and CCL21, have defects in lymphocyte homing and DC localization (Gunn et al. 1999). CCR7 also governs skin DC migration under inflammatory and steady−state conditions (Ohl et al. 2004). CCR7 ligands, CCL19 and CCL21, induce a proinflammatory differentiation program in DCs (Marsland et al. 2005).
CCR8−/−
CCR7 and CCR8 pathways are used by monocyte−derived DCs during mobilization from skin to lymph nodes (Qu et al. 2004).
CXCR3−/−, IFNR−/−
Plasmacytoid DCs migrate to inflamed lymph nodes, produce INF−α, and help lymph node DCs to induce antiviral CTLs (Asselin-Paturel et al. 2005; Yoneyama et al. 2004, 2005)
II.
SOME PROPERTIES THAT HAVE BEEN USED TO IDENTIFY MOUSE DENDRITIC CELLS
Morphological and physical properties were helpful in the initial research on DCs, which occurred before the monoclonal antibody (mAb) revolution (reviewed in Steinman 2004). A useful feature was the low buoyant density of DCs, which permitted enrichment over density gradients, for example, in dense bovine serum albumin solutions (Steinman and Cohn 1974). Many of the DCs from mouse spleen were adherent cells, but, in contrast to macrophages and many other types of cultured cells, the DCs only had a transient capacity to adhere to plastic. The DCs could then be enriched by successive adherence and culture steps (Steinman et al. 1979). Before or after enrichment, DCs had an unusual cell shape, continually forming and retracting cell processes or dendrites in many different directions. Interestingly, these morphological features of DCs have now been observed in vivo in studies of living lymph nodes, using mice genetically modified so that DCs brightly express enhanced yellow fluorescent protein under the control of a CD11c promoter. With two-photon microscopy, one can observe the processes of DCs by constantly probing the environment in situ (Lindquist et al. 2004). In addition to their morphology, the cell surface composition of the newly discovered DCs proved to be distinct. Unlike other accessory cells such as macrophages and B cells, DCs were found to express high levels of major histocompatibility complex (MHC) products (Nussenzweig et al. 1981; Steinman et al. 1979), which are required for antigen presentation to T lymphocytes. At the time, the function of the MHC in presenting antigenic peptides was not known, but the MHC was known to
be responsible for encoding major antigens to stimulate transplant rejection. DCs were tested as stimulators of rejection in vitro in the mixed leukocyte reaction (MLR, originally known as the mixed lymphocyte reaction), in which MHC class II products on the cells from one mouse strain stimulate vigorous proliferation by T cells of another strain. Surprisingly DCs were unusually potent, whereas other class II–bearing cells were inactive, including MHC class II–bearing B lymphocytes and macrophages (Steinman and Witmer 1978; Steinman et al. 1980, 1983; Van Voorhis et al. 1983). Likewise, DCs were powerful accessory cells for inducing a T cell response to a so-called “nominal” antigen (Nussenzweig et al. 1980). These experiments were the basis for two new streams of research: DCs were a distinct type of leukocyte, and the initiation of T-cell mediated immunity could be achieved when DCs were presenting antigen. A series of DC-restricted mAbs was also made in the 1980s, including 33D1 (Nussenzweig et al. 1982), NLDC-145 (specific for the adsorptive endocytosis receptor, DEC205/ CD205; Kraal et al. 1986), and N418 (specific for the CD11c integrin; Metlay et al. 1990). These mAbs helped to establish the distinct functions of DCs within heterogeneous cell suspensions from mouse spleen, in which the DCs comprised only ~1% of the leukocytes. 33D1, for example, was used to selectively deplete DCs from mouse spleen, and this ablated the bulk of the MLR stimulating activity (Steinman et al. 1983) and the primary antibody response (Inaba et al. 1983). N418 was used to select DCs by fluorescence-activated cell sorting, and this process showed that the CD11c+ cells were the principal stimulators of the MLR (Crowley, Inaba, Steinman, 1990). 33D1 and N418 were also used in experiments in which DCs proved to be the major cells capturing soluble protein antigens that
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were administered intravenously to mice (Crowley, Inaba, Witmer-Pack, et al. 1990). Antibodies to DEC-205 and 33D1 now have a new role in studies of DC function in intact lymphoid organs. The approach is to introduce antigens into the antibodies by chemical conjugation or genetic engineering. The antibodies then target the antigen selectively and efficiently to DCs in situ. This permits an interrogation of DC function in situ under different conditions, either in the steady state without ostensible infection or inflammation and after administration of various DC stimuli (Bonifaz et al. 2002, 2004; Boscardin et al. 2006; Hawiger et al. 2001; Trumpfheller et al. 2006). Selective delivery of antigens within antibodies to these DC receptors increases the efficiency of antigen presentation to T cells more than 100-fold. Anti-CD11c antibodies also are frequently used to select DCs from different mouse tissues. The distinguishing feature of DCs is that they express very high levels of CD11c. Other cells, in particular some stimulated natural killer (NK) cells, can express CD11c to some extent. In contrast to DCs, NK cells are DX5/CD49b positive and express only low levels of CD80, CD86, and MHC class II products. In tissue sections of normal mice and in mice in which the CD11c promoter is used to express various markers such as MHC class II or green fluorescent proteins (Brocker 1997; Brocker et al. 1997; Kerksiek et al. 2005; Lindquist et al. 2004), the MHC class II-rich DCs are the main cell type that is identified as CD11c positive.
III.
GENERATION OF DCS
FROM PROLIFERATING PROGENITORS Cytokines that allow DC progenitors to expand in culture have been identified. The resulting increased availability of DCs has significantly stimulated research. The first system to be identified involved the application of granulocyte-macrophage colony-stimulating factor (GM-CSF) to blood (Inaba, Steinman, et al. 1992) and bone marrow cell suspension cultures (Inaba, Inaba, et al. 1992) (see Appendix). Some laboratories find it valuable to add interleukin (IL)-4 to these cultures. More recently, flt-3 ligand (flt-3L) has been used. flt-3L appears to be the major cytokine responsible for the production of DCs in vivo (D’Amico and Wu 2003; Maraskovsky et al. 2000; McKenna et al. 2000; Tussiwand et al. 2005), whereas GM-CSF may primarily be used to mobilize DCs during infection, the setting in which GM-CSF was first identified (reviewed in Metcalf 1985). To use flt-3L, the cytokine is given for at least 7 days, whereupon the numbers of DCs in various lymphoid tissues expands >10-fold. The DCs that are produced in GM-CSF– and flt3L–expanded cultures have some differences, particularly with regard to subsets of DCs. DCs generated with GM-CSF consist primarily of cells termed myeloid, DCs that resemble the CD8α− DC subset in mouse spleen (see below). flt-3L–expanded DCs, on the other hand, include both plasmacytoid and myeloid DCs
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(Gilliet et al. 2002), and the latter include cells with features of CD8α+ spleen DCs, such as enhanced presentation of ovalbumin antigen on MHC class I products (Naik et al. 2005). At this point, neither GM-CSF nor flt-3L is being used to generate cells with the properties of Langerhans cells (LCs), although a recent report indicates that the addition of estrogen to the GM-CSF cultures can increase the development of cells with properties of LCs (Mao et al. 2005). In contrast, LCs are readily identified in an important human cell culture system in which CD34+ progenitors are expanded with GM-CSF and tumor necrosis factor (TNF)-α (Caux et al. 1992). In all systems used for the expansion of DC progenitors, the progeny are primarily in an immature functional state, which means that the cells are able to capture antigens but require further stimuli to differentiate into cells that in turn initiate many pathways of lymphocyte differentiation (Inaba et al. 2000; Pierre et al. 1997; Turley et al. 2000). One stimulus for maturation is to disrupt the clusters of immature DCs and replate the cells in a fresh culture vessel, whereas other stimuli include ligands for Toll-like receptors, for example, lipopolysaccharide.
IV.
DISTRIBUTION OF DCS
IN LYMPHOID TISSUES DCs are abundant in lymphoid tissues. In the thymus, DCs are restricted to the medulla. The thymus is the primary lymphoid organ in which developing T cells rearrange T cell receptor genes to produce a diverse repertoire of clones, each with a single receptor. These T cells are then educated, undergoing positive selection for affinity to self-MHC products followed by elimination, tolerance, or negative selection against reactivity to self-antigens. DCs function in this central tolerance (Matzinger and Guerder 1989, Zal et al. 1994). When antigens are selectively expressed under the control of the mouse CD11c promoter, the thymic DCs function in negative rather than positive selection (Brocker et al. 1997). Recent data with human thymus reveal that thymic DCs, when matured by thymic stromal lymphopoietin, are also able to stimulate CD4+ CD25− single positive thymocytes to proliferate and differentiate into CD4+ CD25+ suppressor or regulatory T cells (Watanabe et al. 2005). This would mean that DCs play a critical role in the thymic development of natural suppressor T cells, which is a dominant or regulatory form of peripheral tolerance. In peripheral lymphoid tissues, DCs are most abundant in the T cell areas, that is, the periarterial sheaths of spleen, the deep cortex of lymph nodes, and the interfollicular zones of mucosaassociated lymphoid tissues such as Peyer’s patches and nasal-associated lymphoid tissue. As mentioned, DCs in the T cell area of living lymph nodes comprise a widely distributed network in which the DCs constantly probe adjacent T cells with their dendrites (Lindquist et al. 2004). There are additional important pools of DCs in lymphoid tissues, that is, DCs
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in the marginal zone of the spleen, the subcapsular sinus, and the outer cortex of lymph nodes and beneath the antigen-transporting epithelium of mucosa-associated lymphoid tissue. These DCs can function in antigen capture. In the B cell area, the major accessory cell is the follicular dendritic cell (FDC). The nomenclature may seem confusing, but at the time of the discovery of DCs, FDCs were known as “dendritic macrophages” and “dendritic reticular cells” (Nossal et al. 1968), and there was a possibility that these two new cell types—eventually to be called DCs and FDCs—were related (Chen, Adams, et al. 1978; Chen, Frank, et al. 1978). However, FDCs are nonhematopoietic cells, and they specialize in retention of intact antigens on their labyrinthine cell surface. The antigens are retained via Fc receptors or C3 receptors. DCs, in contrast, are bone marrow derived and specialize in the presentation of processed antigens. Nevertheless, DCs also are able to present intact antigens to B cells (Balazs et al. 2002; Wykes et al. 1998). Although this may take place primarily in extrafollicular sites in the so-called “plasmablast reaction” (Garcia De Vinuesa et al. 1999), it is possible that DCs enter B cell follicles under some conditions.
V.
DCS IN NONLYMPHOID TISSUES
The bone marrow is the source for DCs in several sites. The rat was used in many of the early studies of DCs in nonlymphoid tissues. For example, DCs were found in the interstitial spaces of many organs, such as heart and kidney but not brain (Hart and Fabre 1981). DCs were noted along mucosal surfaces such as the airway (Holt et al. 1987) and intestine (Maric et al. 1996). Using genetically modified mice, Niess et al. (2005) have demonstrated that in the ileum, DCs can extend processes through the epithelium, without disrupting the epithelial barrier, and that this requires CX3CR1 fractalkine receptors. DCs are also found in the circulation, both blood and afferent lymphatics (reviewed in Randolph et al. 2005). An important discovery was that bone marrow progenitors gave rise to LCs in the skin (Frelinger et al. 1979; Katz et al. 1979). Formally speaking, LCs were the first DCs to be visualized in 1868 by Paul Langerhans, although over the ensuing century, scientists considered LCs to be of neural or neural crest origin (reviewed in Rowden 1981; Stingl and Shevach 1991). This view changed with the discoveries that mouse LCs were bone marrow derived and that LCs in other species had many features that were more typical of leukocytes, including Fc receptors (Stingl et al. 1977), MHC class II antigens (Rowden et al. 1977), and antigen presentation to primed T cells (Stingl et al. 1978). Recent experiments in bone marrow chimeras and in parabiotic mice have demonstrated, surprisingly, that whereas LCs are hematopoietic cells, the bone marrow input is primarily used during inflammation and not in the steady state, in which LCs primarily undergo self-regeneration (Merad et al. 2002, 2004).
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Mouse LCs have been valuable for discovering the basic properties of DCs, particularly their differentiation from immature antigen-capturing cells to mature immunostimulatory ones (Inaba et al. 1986, Pure et al. 1990; Romani, Inaba, et al. 1989, Romani, Koide, et al. 1989; Schuler and Steinman 1985). LCs also provide a model to study DC migration from a tissue such as skin via lymphatics to the draining lymph node (Larsen et al. 1990; Macatonia et al. 1987). LCs in the epithelial layer of the skin can take up particles (Reis e Sousa et al. 1993) and contain distinct granules, known as Birbeck granules, which are a type of endocytic organelle (Romani et al. 2005). LCs express Fc receptors and MHC class II products in situ, but the expression of FcR drops dramatically in maturing cultured LCs (Romani, Inaba, et al. 1989; Schuler and Steinman 1985), whereas the MHC class II molecules redistribute from intracellular compartments to the cell surface (Pierre et al. 1997). Cells similar to LCs are found in other stratified squamous epithelia such as the vagina, cervix, anus, pharynx, and esophagus. Many other tissues of the mouse contain DCs in an immature state, including the dermis and the interstitial spaces of other organs. However, Birbeck granules and high levels of intracellular MHC class II products are not typically found in interstitial DCs in mouse peripheral tissues. LCs can act as potent inducers of T cell responses when they are allowed to mature in suspension culture (Schuler and Steinman 1985) or after migration from skin explants (Larsen et al. 1990). However, the roles of LCs in vivo are less clear and are coming under renewed scrutiny. For example, in herpes infection of the skin and vagina, LCs are not the major presenting cells for viral antigens in the draining lymph nodes (Allan et al. 2003; Zhao et al. 2003). It is possible that the infected LCs die and are presented by other DCs in the dermis or in the lymph node (Inaba et al. 1998). New genetic tools for studying LCs in mice have recently been generated. When the Langerin gene is ablated, the LCs lack Birbeck granules, but other abnormalities have yet to be defined (Kissenpfennig, Ait-Yahia, et al. 2005). Of some interest are mice that express a diphtheria toxin receptor under the control of the Langerin/CD207 promoter (Bennett et al. 2005) (where Langerin is a lectin that is abundant in LCs and is responsible for the formation of Birbeck granules; Valladeau et al. 2000). Administration of diphtheria toxin leads to the selective depletion of LCs for several weeks. Another tool is a CD207 promoter knockin mouse, which expresses enhanced green fluorescent protein (EGFP) only in LCs (Kissenpfennig, Henri, et al. 2005). These new mouse models make it possible to distinguish the behavior and functions of LCs from those of dermal DCs. LCs are for the most part sessile in the steady state, but application of a contact allergen induces increased LC motility and migration to lymph nodes. Dermal DCs also migrate to the lymph nodes during contact sensitization, but they do so earlier than LCs, and they colonize distinct areas after they arrive in the lymph nodes (Kissenpfennig, Henri, et al. 2005). Ablation of LCs does not ablate contact hypersensitivity, suggesting that dermal DCs contribute to T cell priming in this
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strong cell-mediated immune response (Bennett et al. 2005; Kissenpfennig, Henri, et al. 2005).
VI.
LIFE SPAN AND TURNOVER OF DCS
Irradiation eliminates many DCs in lymphoid organs, whereas reconstitution with bone marrow cells leads to their recovery in 1–2 weeks. Many DCs have a short life span, with a half-life of 1.5–3 days (Kamath et al. 2000). In contrast, a recent study with bromodeoxyuridine pulse labeling and with parabiotic mice has revealed that 5–10% of the DCs are in cell cycle and that the replacement of DCs from the blood is actually relatively slow (Kabashima et al. 2005). In other words, DCs in spleen, surprisingly, have some of the features recently documented for LCs, which regenerate locally and resist repopulation from the blood and marrow unless inflammation in the skin is induced (most likely through the production of CCR2-binding chemokines; Merad et al. 2002, 2004). Therefore, homeostasis of DCs in lymphoid organs is maintained through a balance of rapid local regeneration, death, and slow replenishment of precursors from blood. Current thinking is that when bone marrow–derived DC precursors arrive in lymphoid organs, they seed the tissue and wait for cues from their microenvironment (e.g., lymphotoxin β) to develop into DC subsets, at least some of which have the capacity to divide locally.
VII. FEATURES OF DCS A.
Morphology
No other blood cell exhibits the shape and motility that are the basis for the term dendritic cell (Romani et al. 2005; Steinman and Cohn 1973). In situ, as in the skin, airways, and lymphoid organs, DCs are stellate and motile. When isolated and spun onto slides, DCs display many fine dendrites. In the electron microscope, the processes are long (>10 µm) and thin, either spiny or sheet-like. When alive and viewed by phasecontrast microscopy, DCs extend large, delicate processes or veils in many directions from the cell body. These bend, retract, and re-extend in a nonpolarized fashion for a day or more. Actin cables are scarce. The shape and motility of DCs fit their function, which is to select antigen-specific T cells for the initiation of adaptive T cell immunity and for clonal selection for purposes of antigen-specific tolerance and memory.
B.
Antigen Handling: Uptake, Processing, Presentation
During the early years of DC research, antigen handling by DCs was mostly taken for granted. In other words, one would
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inject an antigen and assume that DCs would capture it and present it to the immune system. Although this is true to some extent, it is now evident that DCs possess multiple specializations for the capture, processing, and presentation of antigens. Studies with mouse and human DCs reveal the existence of multiple endocytic receptors (reviewed in Figdor et al. 2002; Mellman and Steinman 2001). These include Fcγ receptors for immune complexes and antibody-coated cells; several C-type lectin receptors that are often restricted to select subsets of DCs such as the macrophage mannose receptor/CD206, Langerin/CD207, DC-SIGN/CD209, and DEC-205/CD205; several potential receptors for the uptake of dying cells; and receptors for heat shock proteins such as CD91 (Basu et al. 2001) and LOX-1 (Delneste et al. 2002). By delivering antigen to DCs via these receptors, the efficiency of antigen presentation in vivo can be enhanced by 100-fold or more (Bonifaz et al. 2002; Hawiger et al. 2001; Liu et al. 2002). After uptake, DCs process the ingested antigens via both MHC class I and MHC class II pathways. This leads to recognition by CD8+ and CD4+ T cells, respectively. The processing of nonreplicating or exogenous antigens onto MHC class I molecules is called “cross-presentation” or the “exogenous” pathway of antigen presentation. To assess the contribution of DCs to this nontraditional pathway, Jung et al. (2002) generated mice in which the CD11c promoter was used to drive expression of the human diphtheria toxin receptor (mice are not normally sensitive to diphtheria toxin). This development permitted the ablation of DCs in lymphoid tissues for 1–2 days. The mice were unable to process several different antigens through the exogenous pathway, indicating that DCs were a major cell type for cross-presentation to CD8+ T cells in vivo (Jung et al. 2002). DCs also are a major site for the expression of CD1 molecules (CD1d in mice and CD1a, b, c, and d in humans). These molecules are used to present different forms of glycolipid. The mouse has been of major importance in delineating the immunological consequences of presentation of glycolipids on CD1d. The pivotal discovery was that natural killer T (NKT) cells bear invariant T cell receptors specific for CD1d-glycolipid complexes (Kawano et al. 1997). The glycolipids can be of endogenous (Zhou et al. 2004), microbial (Kinjo et al. 2005; Mattner et al. 2005), or synthetic (Gonzalez-Aseguinolaza et al. 2002; Schmieg et al. 2003, 2005) origin. When mouse DCs present the synthetic glycolipid α-galactosyl ceramide, the DCs undergo extensive maturation and become potent stimulators of combined CD4+ and CD8+ T cell–mediated immunity (Fujii et al. 2003, 2004; Hermans et al. 2003). However multiple doses of this glycolipid can lead to the development of DCs that produce IL-10 and have regulatory properties (Kojo et al. 2005). Antigen uptake and/or processing seems to be particularly efficient in DCs, because nanomolar concentrations of antigen can suffice. This is much less than the micromolar levels typically employed to load antigens into other antigenpresenting cells. Extensive studies by Mellman and colleagues (reviewed in Trombetta and Mellman 2005) have uncovered another special feature of DCs, which is their extensive regulation
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of the endocytic system during stimulus-dependent maturation (see next section). The uptake or endocytic activity of the DCs is dampened, the acidity and proteolytic activity of the deep endosomal lysosomal system are enhanced, and peptide MHC class II complexes actively form and move to the surface in discrete nonlysosomal organelles.
C.
Maturation of DCs
The development of DCs comprises two functionally distinct and phenotypically different stages. In most tissues, DCs are present in a so-called immature state, equipped to capture antigens but unable to carry out one of their classic functions, the initiation of a primary T cell response. These immature DCs can take up particles and microbes by phagocytosis, they can form large pinocytic vesicles in which extracellular fluid and solutes are sampled (“macropinocytosis”), and they express several receptors on their surface that mediate adsorptive endocytosis (e.g., C-type lectin receptors such as the macrophage mannose receptor and DEC-205 and Fc receptors). Immature DCs also express receptors that allow them to sense changes in the environment, including many Toll-like receptors (TLRs) for microbial ligands as well as receptors for different inflammatory cytokines. DCs then undergo extensive differentiation or maturation in response to different microbial and nonmicrobial stimuli. The differentiation program varies with the type of maturation stimulus. The stimuli can be microbial ligands for TLRs, TNF family members such as TNF and CD40 ligand (CD40L), heat shock proteins, different types of innate lymphocytes (NK, NKT, and γδ T cells), and immune complexes for Fc receptors. Genetically defined mice have been essential to uncover the intricate function of the Fcγ receptor (FcγR) family (Ravetch and Lanier 2000). FcγRs have activating and inhibitory forms, the latter being due to the presence of an immunoreceptor tyrosine-based inhibition motif that was first discovered in the mouse FcγRIIB (Muta et al. 1994). Mice that genetically lack the FcγRIIB undergo maturation when challenged with immune complexes, because the activating, immunoreceptor tyrosine-based activation motif–associated FcγRIIA is no longer subject to inhibition (Kalergis and Ravetch 2002). These observations have been extended in mice and humans through the identification of mAbs that selectively block inhibitory receptors (Dhodapkar et al. 2005; Samuelsson et al. 2001). Once DCs have captured antigen, the capacity to take up additional antigens rapidly declines if there is also a signal to mature (Garrett et al. 2000; Romani, Koide, et al. 1989). During maturation, antigen–MHC class II complexes are formed within deep endosomal and MHC class II–positive compartments (Inaba et al. 2000). The complexes move to the cell surface within distinct nonlysosomal vesicles, which also contain high levels of the CD86 costimulator (Chow et al. 2002; Turley et al. 2000). Functional maturation of DCs also includes
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major changes in cytokine and chemokine profiles (and their receptors) (Sallusto et al. 1999), many of which, such as IL-12 and type I interferons, are immune enhancing (Honda et al. 2005; Takaoka et al. 2005). In sum, maturing DCs increase the display of surface MHC-peptide complexes, produce large amounts of cytokines, and upregulate numerous cell surface costimulatory molecules such as CD40 and CD86. This maturation process endows the DCs with the molecular machinery for the initiation of immunity, but the specific maturation program and type of immune response is influenced by the nature of the maturation stimulus (described later). Transcriptional profiling with gene chips has shown the extensive nature of the differentiation of DCs during maturation. Expression of hundreds of genes changes by 4-fold or more after triggering of immature mouse DCs with a single TLR ligand. These changes are not coordinated, with some genes changing rapidly within hours and others taking much longer (Granucci et al. 2001). One of the features of the maturing DC is a high level of the cell surface costimulator, CD86 or B7-2 (Caux et al. 1994; Inaba et al. 1994). However, increased CD86 does not by itself dictate the development of T cell immunity. Fujii et al. (2004) showed that DCs maturing in response to NKT cells are able to present antigens on MHC class I and II, produce cytokines, and express high levels of CD86. Yet CD40 ligation was additionally vital to initiate immunity. Likewise Sporri and Reis e Sousa (2005) showed that DCs lacking the appropriate TLR could express high levels of CD86 when the cells were bystanders to other DCs that were directly responding to TLR ligation. However, bystander DCs were unable to initiate immunity. Thus, CD86 upregulation accompanies DC maturation in many instances but is by itself insufficient to explain the T cell immunizing properties of mature DCs.
D.
DC Subsets
DCs are composed of several subsets that differ significantly in their phenotype, localization, and function. In mouse lymphoid tissues, the subsets are often identified on the basis of CD4 and CD8 expression (reviewed in Shortman and Liu 2002). One functional difference involves receptors for antigen uptake. CD8+ DCs express high levels of DEC-205/CD205, Langerin/CD207, and CD36 molecules, whereas CD8− DCs express 33D1 (Crowley et al. 1989). DCs subsets are found in the thymus, spleen, lymph nodes, Peyer’s patches, and the liver, although the percentage of each subset and often cell surface markers vary from organ to organ. LCs and dermal DCs in the skin are a source of at least two other DC subsets in lymph nodes. LCs give rise to lymph node DCs that express Langerin and DEC-205 but low levels of CD8 (Henri et al. 2001). The expression of the CD45 isoform B220 was important in defining the mouse functional counterpart of human plasmacytoid DCs (Asselin-Paturel et al. 2001; Bjorck 2001;
4. SOME
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Nakano et al. 2001). These are found in all mouse lymphoid organs and are characterized by a capacity to produce large amounts of type I interferon in response to nucleic acids, including inactivated viruses (Asselin-Paturel et al. 2001). Plasmacytoid DCs are a major cell type expressing the TLR7 and TLR9 receptors for RNA and DNA. For a period of time, CD8+ and CD8− DC subsets were thought to have distinct origins from lymphoid and myeloid progenitors, respectively. More direct studies on this issue have indicated that each subset, as well as plasmacytoid DCs, can derive from either type of progenitor (Chicha et al. 2004; Manz et al. 2001; Traver et al. 2000). At this point, it should be mentioned that DC reconstitution assays utilize irradiated recipients, which might allow DC precursors to express homing and differentiation potentials that are not identical to normal physiology. With analyses of knockout mice, researchers have begun to identify transcription factors that play an important role in controlling the development of different DC subsets, for example, RelB, interferon regulatory factor (IRF)-2, and IRF-4 for CD8− DCs and IRF-8 and Ikaros for CD8+ DCs. Although these transcription factors are not DC specific, the findings indicate that DC subsets result from distinct transcriptional programs. DC subsets are also proving to have distinct functional properties, and the definition of these functions, particularly in vivo, represents a major frontier in DC biology. As mentioned, DC subsets differ in their ability to capture antigens, depending on their cell surface receptor expression. CD8+ splenic DCs specialize in capturing many sources of dying cells, including targets killed by NK cells; yet both CD8+ and CD8− splenic DCs phagocytose other particles such as injected latex beads (Iyoda et al. 2002). DC subsets also differentially express receptors for maturation stimuli. TLR3 is preferentially expressed on CD8+ DCs, TLR5 on CD8− DCs, and TLR7 and TLR9 primarily on plasmacytoid DCs (Edwards et al. 2003). This specialization suggests that different DC subsets respond to different infections, depending on their cell surface receptor profile. On the other hand, both CD8+ and CD8− DC subsets express CD40 in the steady state and mature in response to αCD40 injection. Antigen processing by different DC subsets also can differ. The CD8+ subset of DCs more effectively presents exogenous antigens onto MHC class I (Wilson et al. 2004), whereas plasmacytoid DCs are typically weak at processing nonreplicating antigens onto either MHC class I or II products. More recently, a type of DC termed the Tip-DC has been identified during infection of mice with Listeria monocytogenes. During this bacterial infection, Tip-DCs are evident in the white pulp of the spleen and produce high amounts of the protective molecules, TNF-α, and inducible nitric oxide synthase (iNOS; Serbina et al. 2003).
E.
Migration of DCs
The proper localization of DCs to secondary lymphoid tissues is a critical event for optimal immune responses (reviewed
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in Randolph et al. 2005). In the steady state, expression of lymphotoxin β receptor (LT-βR) by circulating DC precursors promotes DC homeostasis in the spleen and lymph nodes. Lymphotoxin 1 and 2, typically secreted by B cells, regulates cell division. Therefore, mice lacking LTβR have reduced numbers of DCs in lymphoid tissues (Kabashima et al. 2005). In the steady state, DCs also reside in peripheral tissues where they exert a sentinel function for incoming antigens (self, environmental, tumor, and microbial). Upon microbial contact or stimulation by inflammatory cytokines, these DCs traffic in increased numbers via the afferent lymphatics to the T cell areas of the lymph node to initiate immune responses. Increased expression of CCR7 is a characteristic feature of conventional DC maturation induced by many different stimuli, and the corresponding chemokines, CCL19 and CCL21, are expressed in vivo by lymphatic endothelium and stromal cells in the lymph nodes (Cyster 1999; Martin-Fontecha et al. 2003). However, additional lipid mediators also influence migration (Angeli et al. 2004; Kabashima et al. 2003; Robbiani et al. 2000). During maturation of plasmacytoid DCs in response to TLR7 or TLR9 agonists, the cells move outward from the central to peripheral (marginal zone) regions of the splenic white pulp nodule, and this maturation is dependent on type I interferons; in contrast, conventional DCs are only partially dependent on type I interferons for their maturation and do not require these cytokines for activation-induced migration from the marginal zone to the central T cell zones in the spleen (Asselin-Paturel et al. 2005). It is often not appreciated that, even in the absence of invading pathogens, some DCs are always migrating from tissues in lymphatics to lymph nodes. Studies of afferent lymph are currently difficult in mice, but in rats, for example, DCs along liver sinusoids move in hepatic lymphatics to celiac lymph nodes (Matsuno and Ezaki, 2000; Matsuno et al. 1996), and DCs from intestine migrate to mesenteric lymph nodes (Huang et al. 2000; Pugh et al. 1983). These cells are not found in the efferent lymph, indicating that most of the migrating DCs die after their arrival in lymphoid tissues. The DCs that migrate in the steady state might have several functions: to replenish immature populations, to transport self or environmental antigens, or to be on patrol to identify invaders. Thus, DC migration in vivo has many potential roles in DC homeostasis and function. DC migration is a regulated process, controlled, for example, at the level of chemokine production and chemokine receptor expression and function (reviewed in Randolph et al. 2005). Some immature DCs can express a repertoire of chemokine receptors (e.g., CCR1, CCR2, CCR5, CCR6, and CXCR4) that bind inflammatory chemokines (e.g., CCL5, CCL2, CCL3, CCL4, CCL20, and CXCL12). Subsequent DC activation and maturation are associated with the downregulation of chemokine receptors and the de novo expression of CCR7, the receptor for CCL19 and CCL21. The crucial role of CCR7 and its ligands is clearly observed in vivo in mice deficient for these proteins. In mice homozygous for an autosomal recessive
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mutation, paucity of lymph node T cells (plt), naive T cells fail to home to secondary lymphoid organs. The plt mutation is associated with a deficit in the expression of CCL21 within secondary lymphoid organs and a defect in CCL19 as well. As a consequence of the lack of CCL21 within secondary lymphoid organs, DCs from these mice fail to accumulate in the spleen and in the T cell areas of lymph nodes (Gunn et al. 1999). Similarly, CCR7−/− mice show defective architecture of secondary lymphoid organs, a defective homing of DCs and lymphocytes, and defective entry of DCs into lymphatic vessels at peripheral sites both in the steady state and inflammation conditions (Forster et al. 1999). CCL19 and CCL21 also increase the maturation and proinflammatory differentiation of DCs (Marsland et al. 2005). Therefore, chemokine–chemokine receptor interactions not only orchestrate the migration of DCs migration but also influence their immunogenic potential for T cells. There are many other examples in which specific chemokines control the traffic of select populations of DCs (Table 4-1). In skin exposed to ultraviolet light, LCs disappear and are replaced in 2 weeks. The recruitment of LC precursors from blood is dependent on their expression of CCR2 (Merad et al. 2004). During murine listeriosis, CCR2 is also required for Tip-DCs to migrate into the spleen (Serbina et al. 2003). Migration of plasmacytoid DCs into inflamed lymph nodes and their redistribution during inflammation depend on CXCR3 and CCR7. CCR6 is used by DCs to populate epithelial surfaces during inflammation (Dieu et al. 1998; Greaves et al. 1997; Iwasaki and Kelsall 2000). In the steady state, recent evidence shows that CXCL14 is important for LC progenitors to establish themselves in the skin (Schaerli et al. 2005). All of these findings indicate that the differential expression of chemokine receptors by DCs (and their subsets) at different stages of their life history determines their location in vivo.
VIII. A.
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STEINMAN
[Toll/IL-1 receptor domain-containing adaptor inducing IFN-β], TRAF6 [TNF receptor–associated factor], and IRFs) have benefited enormously from genetic alterations in mice (Alexopoulou et al. 2001; Hayashi et al. 2001; Hemmi et al. 2000, 2002; Hoebe et al. 2003; Honda et al. 2005; Kaisho and Akira 2001; Kawai et al. 1999; Kobayashi et al. 2003; Poltorak et al. 1998, Takaoka et al. 2005). Interestingly, simultaneous signaling through two TLRs, which use MyD88 and TRIF adapters, leads to greatly enhanced IL-12 production in a type I interferon– dependent manner (Gautier et al. 2005). In addition to TLR ligands, DCs can show evidence for maturation and IL-12 production in response to heat shock proteins, which may be released by the pathogen (Lazarevic et al. 2003), tumor cells (Somersan et al. 2001), or stressed cells (Basu et al. 2000). DCs and innate lymphocytes (e.g., NK, NKT, and γδ T cells) interact in ways that enhance the function of each cell type. DCs produce IL-12, IL-15, IL-2, and IFNα/β, which affect different facets of NK cell function, whereas the innate lymphocytes act back to mature the DCs (reviewed in Munz et al. 2005; Walzer et al. 2005). Injection of mature DCs leads to the recruitment of NK cells into the draining lymph nodes, whereas exposure of DCs to TLR stimuli induces IL-2 and IL-12 production, which in turn activates NK cells to produce IFN-γ. DCs also present different glycolipids on CD1d molecules to the invariant T cell receptor on NKT cells. These include endogenous lysosomal glycosphingolipids (e.g., iGb3; Zhou et al. 2004), microbial lipids (Kinjo et al. 2005; Mattner et al. 2005), or synthetic glycolipids (e.g., α-GalCer; Fujii et al. 2003; Hermans et al. 2003) to activate NKT cells in vivo. The interaction of innate lymphocytes and DCs is not yet known to involve TLR signaling pathways. Recall that many of the classic areas of DC function and maturation involve nonmicrobial settings, such as transplantation and contact sensitivity, so that there may well be signal transduction pathways for maturation that are independent of TLRs and other microbial sensors.
FUNCTIONS OF DCS
Innate Responses via Toll-Like Receptors
and Interactions with Innate Lymphocytes Innate responses are the first line of defense against microbial invasion and tumor growth. DCs, like other hematopoietic and nonhematopoietic cells, have important roles in innate immunity. On the one hand, some DCs directly mediate innate responses through TLRs that recognize microbial ligands expressed by viruses, bacteria, fungi, and protozoa. As a result, DCs can be the major source in vivo for the production of IL12 and interferon (IFN)-α in response to microbial stimulation (Dalod et al. 2002; Reis e Sousa et al. 1997). Although the function of TLRs in innate immunity was first evaluated in flies (Lemaitre et al. 1996) and human cells (Medzhitov et al. 1997), the dissection of TLR function and the identification of the molecules used during signal transduction (e.g., MyD88, TRIF
B.
Initiating Adaptive T Cell Immunity
As discussed in the section on features of DCs, immature DCs can endocytose a diverse array of antigens through multiple receptors on their cell surface. However, immature DCs typically express relatively low levels of surface MHC class I and II products and only low levels of costimulatory molecules (e.g., CD80 and CD86). MHC class II molecules are sequestered intracellularly in late endocytic lysosomal compartments (Inaba et al. 2000; Pierre et al. 1997; Turley et al. 2000). Upon receipt of a maturation stimulus, DCs undergo extensive differentiation, and they migrate in increased numbers to secondary lymphoid tissues. For example, maturation is required to reduce the acidity of lysosomes and permit proteolysis of antigens as well as the invariant chain chaperone for MHC class II products (Trombetta et al. 2003). In contrast, immature DCs may retain intact protein for relatively long periods and then
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form MHC class II–peptide complexes subsequently in maturing intracellular compartments (Delamarre et al. 2005). DC–T cell interactions in the lymph nodes have now been studied in the living state with two-photon microscopy (Bousso and Robey 2003; Hugues et al. 2004; Mempel et al. 2004; Miller et al. 2004; Shakhar et al. 2005). Antigen-specific T cells arrest on antigen-presenting DCs, and this stable interaction lasts for at least 18 hours. If DCs remain immature, this stable DC–T cell interaction leads to tolerance, whereas a maturation signal allows DCs to initiate immunity. Maturation includes a massive redistribution of MHC class II molcules from intracellular compartments to the plasma membrane, the upregulation of costimulatory molecules such as CD40, CD80, and CD86, and changes in the profiles of cytokine and chemokines such as TNF-α and IL-12. All of these changes probably contribute to the initiation of T cell immunity. The communication between DCs and T cells is a dialog in which the DCs also respond to T cells. For example, CD40 and TRANCE (TNF-related activation-induced cytokine)/RANK (receptor activator of nuclear factor-κB) receptor on DCs are ligated by the corresponding TNF family member expressed on activated and memory T cells, that is, CD40L and RANK-L (Anderson et al. 1997; Van Kooten and Banchereau 1997; Wong et al. 1997). This leads to increased DC survival and, in the case of CD40, upregulation of CD80 and CD86, secretion of IL-12, and release of chemokines such as IL-8 and macrophage inflammatory protein (MIP)-1α and -1β. The dialog between DCs and T cells thus allows for the expansion of T cell immunity.
C.
Controlling the Quality of the T Cell Response
The T cell repertoire generated in the thymus is formed by a spectrum of clones that together provide exquisite receptor diversity. After the generation of this repertoire, DCs are required for critical decisions such as clonal selection and expansion, tolerance versus immunity, T helper (Th) 1 versus Th2 cells, and even memory. In the presence of mature DCs producing IL-12 or interferons (as might occur when DCs are ligated by CD40L or infected with viruses), the CD4+ T cells differentiate along a Th1 pathway for IFN-γ production. The latter in turn activates the antimicrobial activities of macrophages and promotes killer T cell differentiation. In the presence of exogenous IL-4, however, DCs induce T cells to differentiate into Th2 cells, which secrete IL-4, IL-5, and IL-13. These cytokines help B cells to make antibodies of the immunoglobulin (Ig) G1 and IgE isotypes, activate eosinophils, and stimulate fibrosis. A new and striking pathway that was first discovered with human myeloid DCs involves the epithelium-derived cytokine, thymic stromal lymphopoietin (TSLP). This matures DCs to induce “inflammatory Th2 cells,” which produce TNF-α (rather than IL-10), in addition to IL-4, IL-5, and IL-13 (Soumelis et al. 2002). Recent studies have shown that TSLP receptor (TSLPR) knockout mice exhibit strong Th1
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responses, with high levels of IL-12, IFN-γ, and IgG2a, but low production of IL-4, -5, -10, and -13 and IgE (Al-Shami et al. 2005). DCs that are matured with either CD40L or TSLP are similar in appearance, being highly dendritic and rich in MHC class II and CD86 stimulatory molecules. However, they differ significantly in cytokine and chemokine production, and as mentioned, the functional consequences for T cells vary enormously (Soumelis et al. 2002).
D.
Initiating Different Types of Tolerance
The mechanisms used by DCs to initiate immunity against pathogens pose a major risk for the development of autoimmunity, allergy, and chronic inflammatory disease. Maturation takes place in response to microbial components that signal through Toll-like receptors, but an additional consequence of infection is the death of some infected cells as well as adjacent self-tissues. Therefore, when immature DCs are capturing pathogens, they are also capturing dying cells, which are processed efficiently (Iyoda et al. 2002; Liu et al. 2002). Likewise, at body surfaces, maturing DCs are also capturing environmental antigens to which the body must remain unresponsive. How then do maturing DCs focus the immune response on antigens derived from the pathogen and avoid inducing immunity to self and environmental antigens? It is becoming evident that DCs are responsible for different types of immune silencing or tolerance of reactivity to these self-antigens. First, DCs mediate central tolerance that takes place in the thymus. Self-antigens for negative selection in the thymus include universal self-antigens that are expressed by DCs and antigens that enter the thymus through the bloodstream for capture by DCs. Also in the thymus, medullary epithelial cells can express and tolerize to self-antigens, expressed under the control of the autoimmune regulator “AIRE” (Anderson et al. 2002). Nonetheless, self-reactive T cells capable of causing autoimmune disease can escape negative selection. Central tolerance also may not have a chance to operate if the expression of a self-antigen occurs after the lymphocyte repertoire has been produced, typically before birth in humans. Furthermore, the immune system must remain tolerant to harmless or “noninfectious” antigens in the environment to which we are exposed after the lymphocyte repertoire has already formed. Central tolerance, therefore, has its limitations and does not by itself fully prevent autoimmunity and chronic inflammatory diseases. DCs also mediate tolerance in the periphery. Recent studies show that DCs constantly carry innocuous antigens from the periphery, for example, from the skin, airways, stomach, intestine, and pancreas (reviewed in Steinman et al. 2003). An important finding is that bone marrow–derived cells in the pancreatic lymph nodes present peptides derived from insulinproducing β cells of the pancreatic islets. Presentation of tissue antigens to T cells in the draining lymph nodes leads to tolerance (Kurts et al. 1996, 1997). Accordingly DCs may be able
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to present many self-antigens, probably derived from the normal turnover of somatic cells, to T cells and thus induce tolerance to self-proteins that have no access to the thymus. In a recent study a subset of mouse DCs (CD8+ DEC-205+) was found to be specialized in capturing dying cells (Iyoda et al. 2002). In the steady state, the phagocytic DCs remained immature, as defined by a lack of increase in costimulatory molecules, even though the DCs efficiently presented antigens to CD8+ and CD4+ T cells. Although T cells underwent clonal expansion in vivo when antigen was presented by steady state DCs, tolerance eventually developed (Liu et al. 2002). Likewise, when DCs capture innocuous proteins from the airway, profound tolerance develops even though the T cells can initially proliferate extensively to the antigen-capturing DCs in the draining lymph nodes (Brimnes et al. 2003). Mechanisms for peripheral tolerance can be intrinsic (deletion and anergy) or extrinsic (through suppressor T cells) to the tolerized T cells. Interestingly, the former seems to require expression of B7 family members on the steady-state DCs, for example, PD-L1 and CD86, which then ligate PD-1 and CTLA-4 on the T cells to be tolerized. This requirement for “costimulation” is contrary to the classic view that tolerance is the result of presentation of MHC peptide (“signal one”) in the absence of costimulation (“signal two”). In mice, the targeting of antigen to DCs in vivo (using anti-DEC-205 antibody) leads to deletion of CD4+ and CD8+ T cells (Bonifaz et al. 2002; Hawiger et al. 2001). Previously, peripheral tolerance was difficult to induce, requiring large doses of antigen if successful, whereas the targeting of low doses of antigen to DCs can lead to tolerance. Importantly, DCs are able to expand CD4+ CD25+ regulatory T cells (Yamazaki et al. 2003), and these cells have been used to inhibit autoimmunity mediated by self-reactive T cells in a mouse diabetic model (Tarbell et al. 2004). Also, DCs are able to induce the Tr1 type of regulatory T cells (Levings et al. 2005; Menges et al. 2002), although more research is needed on this pathway in intact mice. In sum, DCs have the capacity to induce tolerance by several mechanisms, which enable DCs to subsequently generate resistance selectively to microbial antigens.
E.
Interactions with Antibody-Forming B Cells
DCs have important effects on B cell growth and immunoglobulin secretion. This involves both direct DC-B cell interactions as well as indirect activation through helper T cells primed by DCs (Boscardin et al. 2006). DCs are able to retain unprocessed antigens for presentation to naive B cells, which initiate an antigen-specific antibody response (Wykes et al. 1998). There is also evidence showing that monocyte-derived CD11clow, B220− DCs from the blood bind and transport bacterial antigen to the splenic marginal zone, where it becomes accessible to B cells for initiation of T cell–independent antibody production (Balazs et al. 2002).
CHARALAMBOUS,
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The DC-B cell interaction is important for B cell proliferation and survival. The classic pathway for a T cell–dependent response is that DCs activate and expand T helper cells, which then provide cytokine and cell-bound “help” to B cells to induce B cell growth and antibody production (Inaba and Steinman 1984, 1985). However, by secretion of soluble factors, including IL-12, DCs can directly stimulate the production of antibodies and the proliferation of B cells, which have also been stimulated by CD40L on activated T cells. Furthermore, during the DC-B cell interaction in the spleen bridging channel area and the lymph node medulla, DC-derived TNF-family ligands APRIL and/or BAFF enhance plasmablast survival and differentiation to plasma cells for a T cell–independent antibody response (Balazs et al. 2002; MacLennan and Vinuesa 2002). DCs influence immunoglobulin class-switching of B cells activated by T cells. Type I interferon stimulation of DCs in vivo contributes to the production of all IgG subclasses and longlived antibody responses (Le Bon et al. 2001). Intestinal DCs carrying commensal bacteria selectively induce production of protective IgA (Macpherson and Uhr 2004). In cultures of human cells, myeloid DCs are able to stimulate class switching to IgA (Fayette et al. 1997), whereas plasmacytoid DCs enhance antibody responses and induce nonselective Ig class switching (Jego et al. 2003). Although the traditional understanding is that CD40L on activated T cells binds CD40 on B cells and induces class switching, a recent study concludes that DCs can directly induce CD40-independent, Ig class switching (Litinskiy et al. 2002). The scenario is that DC upregulate BlyS/BAFF and APRIL upon exposure to IFN-α, IFN-γ, or CD40L. BlyS/BAFF and APRIL on DCs interact with their respective receptors on B cells. In the milieu of appropriate cytokines (such as IL-10, transforming growth factor-β, and IL-4), BlyS/BAFF and APRIL induce class-switch DNA recombination from Cµ to Cγ and/or Cα genes or Cε in B cells. These B cells differentiate along the plasma cell pathway for antibody secretion upon B cell antigen receptor engagement and exposure to IL-15. BlyS/BAFF and APRIL expression levels in DCs is controlled by SOCS1 (suppressor of cytokine-signaling 1). DCs from SOCS1-deficient mice express these molecules at high levels, and this expression is associated with autoantibody production (Hanada et al. 2003). In sum, different DC subsets in different anatomic locations can contribute to the development, activation, and survival of antibody-producing B cells.
IX.
SUMMARY OF APPROACHES TO ANALYZING DC FUNCTION IN MICE A.
DCs Studied Ex Vivo
Mouse spleen remains the major source for studying the properties of DCs ex vivo. In addition, mouse spleen DCs that
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have captured antigens can be transferred to naïve mice and used to study DC function in controlling immunity (Fujii et al. 2004; Inaba et al. 1990; Liu et al. 2005). Using positive selection with anti-CD11c mAbs, one obtains 1–2 million DCs from a spleen. These DCs are composed of at least three major subsets: CD8+ and CD8− myeloid DCs and plasmacytoid DCs. Most are in an immature state but begin to differentiate as soon as they are released from the spleen. One way to document the immature state of DCs taken from mice is to lightly fix the cells immediately after their isolation with formaldehyde. When DCs are isolated from mice in the steady state, the fixed cells are virtually inactive as mixed leukocyte reaction stimulators, but if the mice are given a maturation stimulus beforehand, the fixed DCs are potent stimulators (Fujii et al. 2003). Another way to follow DC maturation is to examine the intracellular distribution of MHC class II products, which are primarily localized within late endosomal compartments in immature cells and on the cell surface in mature ones (Pierre et al. 1997; Wilson et al. 2004).
B. DCs Generated Ex Vivo from Proliferating Progenitors As mentioned at the start of this chapter, research on DCs has been greatly stimulated by the ability to grow large numbers of these cells from bone marrow progenitors. This source of DCs has been especially valuable for cell biological studies of antigen presentation, especially for studying the properties of immature and mature stages of DC development. A valuable source of cells has been the D1 cell line, which also can be studied in immature and mature forms (Winzler et al. 1997). Ex vivo generated DCs are contributing to the science of immunotherapy, because the DCs can be loaded ex vivo with defined tumor or microbial antigens and then reinfused to initiate immunity (Inaba et al. 1993). It is important to add a maturation stimulus to achieve immunity (Schuurhuis et al. 2000). These experiments nevertheless have a major unresolved problem, which is the very low efficiency with which injected DCs are able to gain access to the draining lymph nodes (Josien et al. 2000).
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Hawiger et al. 2001, 2004). Previously one had to administer very large doses of antigens to induce peripheral tolerance, and often the tolerance was incomplete. In the presence of a maturation stimulus, immunity and memory can develop after receptor-mediated delivery of antigens to DCs. The immune responses include combined CD4+ and CD8+ immunity, protection against infection and tumors, and strong helper T cell–dependent antibody responses (Bonifaz et al. 2004; Boscardin et al. 2006; Trumpfheller et al. 2006). It will be important to assess different targets on DCs and other antigenpresenting cells, DC subsets, and maturation stimuli. Work in this field is just beginning, and we suspect that research in mice will set the stage for translation of the approach into nonhuman primates and humans.
D.
Creation of Improved Mice Carrying Human Immune Systems
Progress is being made in the creation of humanized mice because of the recognition that human DCs will need to be engrafted to gain more authentic control on the function of other human lymphoid components. The use of populations enriched in human hematopoietic stem cells represents one component of achieving engraftment with human DCs (Palucka et al. 2003). Another important step is to use mice that are not only deficient in mouse lymphocytes (RAG knockouts and scid mice) but also deficient in critical components of the innate immune system, such as the type I interferon receptor (Boyman et al. 2004; Nestle et al. 2005) and the common γ chain signaling molecule for many hematopoietins (Traggiai et al. 2004; Weijer et al. 2002). These developments are permitting the formation of organized human lymphoid organs in mice, which should allow mice to be used as a more direct preclinical model for studies of the human immune system.
X.
APPENDIX: PROTOCOLS FOR THE ISOLATION OF DCS
C.
Direct Targeting of Antigens to DCs within Intact
A.
Isolating Spleen Dendritic Cells
Lymphoid Tissues Materials: A new and promising approach to studying DC function in situ is to identify ligands that target selectively to these cells and to do this in either the steady state or after the coadministration of maturation stimuli. This identification has been achieved using mAbs to DCs, for example, with an antibody to the uptake receptor DEC-205/CD205. In the steady state, tolerance can be induced after DC targeting, but interestingly only small doses of antigen are needed (Bonifaz et al. 2002;
1. 2. 3. 4.
Hanks’ balanced salt solution (Gibco 14175-095) Collagenase D, 400 units/ml (Roche 1088882) EDTA solution, 0.5 M (Gibco 15575-038) FACS buffer, 2% fetal bovine serum (FBS) in 500 ml of phosphate buffered saline (PBS) 5. Automated magnetic cell sorting (MACS) buffer, 8.3 ml of 30% bovine serum albumin (BSA) + 2 ml of 0.5 M EDTA
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6. R5 medium, 5% FBS in 500 ml of RPMI medium 1640 + L-Glutamine + antibiotics + HEPES buffer 7. Anti-mouse CD11c MicroBeads (Miltenyi Biotec 130-042-201) 8. LS MACS separation columns (Miltenyi Biotec 130-042-401) Procedure: 1. Prepare Petri dishes with 4.5 ml of Hanks’ balanced salt solution/spleen. 2. Sacrifice mice by approved euthanasia techniques. 3. Remove spleen. 4. Add 500 µl of 400 units/ml collagenase D per dish. 5. Using a 3-ml syringe and a 25 5/8-gauge needle, take up 3 ml of the Hanks’/collagenase mixture and inject this into the spleen to balloon the organ. 6. Then tease the spleen into very small pieces. 7. Incubate at 37°C for 25 minutes. 8. Add 100 µl of 0.5 M EDTA and mix. 9. Incubate at 37°C for 5 minutes (spleen should be spindly in texture). 10. Place a cell strainer on a 15-ml Falcon tube and using a Pasteur pipette, pipette spleen up and down until dissolved. 11. Pass through strainer and wash dish with 5–10 ml of FACS buffer. 12. Spin down at 1200 rpm for 10 minutes. For low-density cell enrichment: a. Resuspend the cells in 3 ml of BSA and carefully lay 1 ml of cold PBS on top. b. Spin at 2200 rpm for 30 minutes at 4°C. c. Remove the cells at the interface between the BSA and PBS (low-density cells) collecting into 15-ml tubes. d. Wash twice with cold PBS and count the cells. Without low-density cell enrichment: a. Lyse red blood cells by resuspending in 1 ml of ACK lysing buffer/spleen (Biosource International p304-100) and incubating at room temperature for 4 minutes. b. Wash twice with FACS buffer and count the cells. 13. Resuspend the cells in 460 µl of FACS buffer and add 40 µl of anti-mouse CD11c MicroBeads/spleen. 14. Incubate at 4°C for 20 minutes. 15. After incubation, fill tube with FACS buffer and spin down for 10 minutes at 1200 rpm. 16. Place an LS column (two spleens/column) in the magnet. 17. Place a filter on top of the column, and calibrate the column with 3 ml of MACS buffer. 18. Resuspend the cells with 3 ml of MACS buffer, and pass through the column. 19. Wait until flow has stopped. 20. Wash tubes with 3 ml of MACS and pass through. 21. Repeat two more times. 22. Remove the column from the magnet and elute the positive cells with 5 ml of MACS buffer.
CHARALAMBOUS,
B.
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M.
STEINMAN
Isolating Bone Marrow Dendritic Cells
Materials: 1. 2. 3. 4. 5. 6.
RPMI medium 1640 (Gibco 11875-093) 1 M HEPES solution (Gibco 15630-080) Gentamicin (BioWhittaker 17-5192) Cell strainers (Falcon 352350) ACK lysing buffer (Biofluids P304-100) Rabbit complement (Pel-Freez 31038-100)
Procedure: 1. Sacrifice mice by prescribed and approved euthanasia techniques. 2. Remove femurs and tibias and place into a small Petri dish with RPMI medium 1640. 3. Clean bones with a gauze to remove adjacent muscles. 4. Incubate bones in 70% ethanol in a Petri dish (2 minutes). 5. Wash the bones with RPMI twice. 6. Cut off the tips of the bones. 7. Use a 3-ml syringe and 25-gauge needle to flush the cells out of the bones. 8. Cut up the bones into small pieces. 9. Disrupt the clumps with a pipette. 10. Filter everything through a cell strainer in a 50-ml tube. 11. Lyse the red blood cells (in 100 µl of ACK lysing buffer) for 1 minute at room temperature and count the cells. 12. Centrifuge (for 10 minutes at 1200 rpm). 13. Resuspend the cells at 10 million/ml in 5% FCS medium (RPMI) containing the following antibodies: a. Tib-120 (anti-I-A, MHC II) b. GK1.5 (anti-CD4) c. Tib-146 (anti-B220) d. Tib-211 (anti-CD8) e. Rabbit complement 14. Incubate at 37°C for 1 hour to lyse the B and T cells in bone marrow. Although this step adds time to the procedure, the resulting smaller numbers of bone marrow cells reduce the numbers of cultures and increase the occurrence of DCs, which saves time. 15. Wash the cells three times in RPMI and count. 16. Resuspend the cells at 1 million/ml in the following medium: a. RPMI medium 1640 b. 5% heat activated FCS c. 10 mM HEPES d. 50 mM 2-mercaptoethnol, gentamicin (The medium should have granulocyte-macrophage colonystimulating factor (GMCSF) (1:30 GMCSF containing supernatant from a stably transfected J558 cell line or recombinant GMCSF)
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17. Culture in a 24-well plate, 1 ml/well. 18. On day 2 and day 4 gently wash adherent cells by slowly going around the rim of the well with a Pasteur pipette. 19. Remove medium and replace with fresh GMCSF-containing medium. 20. On day 6, dislodge the clusters of immature DCs and transfer to a 100-mm culture dish at 1 million/ml, 7.5 ml/plate. 21. Incubate 24 h with 10 µg/ml of lipopolysaccharide. 22. Use the cells on day 7 or 8. Two hours before use, transfer the cells in a new dish to deplete adherent cells. The expected yield is between 2 and 5 million DCs/mouse.
ACKNOWLEDGMENT This work was supported by Grant AI13013 from the National Institutes of Health.
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Chapter 5 Mouse Models Revealed the Mechanisms for Somatic Hypermutation and Class Switch Recombination of Immunoglobulin Genes Maria D. Iglesias-Ussel, Ziqiang Li, and Matthew D. Scharff
I. The Generation of Antibody Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Somatic Hypermutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Class Switch Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Activation-Induced Cytidine Deaminase and Uracil N-Glycosylase . . . . . . III. Mismatch Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Error-Prone DNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. NHEJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. THE GENERATION OF ANTIBODY DIVERSITY The production of specific antibodies involves three diversification processes: V(D)J recombination, somatic hypermutation (SHM), and class switch recombination (CSR). At the beginning of B lymphocyte development in the bone marrow,
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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immunoglobulin (Ig) variable (V) regions are assembled by V(D)J and VJ rearrangements of heavy- and light-chain Ig genes, respectively, to produce a primary repertoire of IgM antibodies with typically low-affinity binding for the antigen. Once antigen appears and is taken up by the B cells through the surface IgM molecules, it is processed and presented by the major histocompatability complex (MHC) class II complex to helper T cells. This interaction results in the activation of the
Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
155
156
MARIA
D.
IGLESIAS-USSEL,
B cells and their rapid proliferation and migration into the dark zone of the germinal center in peripheral lymphoid organs, such as spleen, lymph node, tonsil, and Peyer’s patches, where they become centroblasts. The differentiation of the activated B cells into centroblasts is associated with the expression of activation-induced cytidine deaminase (AID), which alters the Ig genes by triggering SHM and CSR (Fig. 5-1). Once these processes are completed, the cells differentiate into centrocytes in the light zone of the germinal center. Centrocytes making antibodies that have acquired a higher affinity for antigen as a result of SHM successfully compete for antigen presented by follicular dendritic cells and are positively selected to differentiate into plasma or memory B cells, whereas those B cells that have not undergone affinity maturation or have become autoreactive either undergo apoptosis or become anergic (MacLennan 1994).
A.
Somatic Hypermutation
SHM occurs at a rate of ~1 × 10−3 mutations per base per generation (McKean et al. 1984; Rajewsky et al. 1987), which
Iµ Sµ V(D)J
ZIQIANG
Iγ1 Sγ1 Cµ3
Cδ
AND
MATTHEW
D.
SCHARFF
is nearly a million times higher than the rate of mutation of housekeeping genes. These mutations occur more often at G:C pairs within DGYW (D = A/G/T, Y = C/T, and W = A/T) and the complementary WRCH (R = A/G and H = A/C/T) hotspot motifs (Rogozin and Diaz 2004). Some hotspots are targeted more than others and transition mutations (purine to purine or pyrimidine to pyrimidine) are more frequent than transversions (purine to pyrimidine or pyrimidine to purine) (Golding et al. 1987). SHM begins 200 base pairs downstream from the V region transcriptional start site, occurs most frequently at the hypervariable regions (CDRs) that encode the antigen-binding site, and extends 1.5 kilobases downstream from the promoter. Transgenic mice with deletions in their Ig genes have been used to search for cis-acting sequences that target SHM to the V regions but not the C region of Ig genes. These in vivo studies showed that ectopically located Ig genes undergo SHM in an apparently normal fashion, suggesting that all of the information required for targeting SHM was present in the Ig gene and its immediate flanking sequences, including its promoter, intronic, and 3’ enhancers (Betz et al. 1994; Giusti and Manser 1993; O’Brien et al. 1987; Peters and Storb 1996; Sharpe et al.
Iγ Sγ3 Cµ
LI,
Iα Sα Cα
Cγ1
µGLT
γ1GLT
Activation-induced cytidine deaminase (AID) Somatic hypermutation (SHM)
Class switch recombination (CSR)
3
Cγ
Cµ
Sγ3 Cδ
V(D)J Sγ3 Cµ
Cδ
Sγ1 Cγ3
Sα Cγ1
Sα Cγ1
Cα Sγ3
Cα
Cδ
Cγ3
Cµ
***** V(D)J
Sµ
* * Sµ Sγ1
*mutations
** Sµ/γ1 Sµ/γ1 V(D)J
**
Sα Cγ1
Cα
Fig. 5-1 Schematic representation of the mouse heavy chain locus and the AID dependent changes that it undergoes during SHM and CSR. The right panel shows the model for class switch recombination. An example of switching of IgM to IgG1 is shown. After induction with specific cytokines, germline transcription takes place and the primary mRNA is spliced, so that the I and C exons are fused. AID initiates CSR (right panel) by introducing C to U mutations (*) into the donor (Sµ) and recipient (Sγ1) switch regions. As a result, double-stranded DNA breaks are generated, mediating the recombination of the involved switch regions, with the looping out of the intervening sequence as a circle. As a consequence, IgG1 is now expressed. The left panel represents the point mutations introduced by AID in the variable (V) region of the immunoglobulin, which results in a change in the affinity of the antibody. I = intronic region, which includes the promoter of germline transcription; S = switch region; Cµ = IgM constant region exon; Cδ = IgD constant region exon; Cγ = a IgG constant region exon; Cα = IgA constant region exon; GLT = germline transcript.
5. MOUSE
MODELS
REVEALED
THE
MECHANISMS
1991; Sohn et al. 1993; Storb et al. 1998; Tumas-Brundage et al. 1996, 1997). However, many different foreign sequences have been substituted for the Ig regulatory regions or for the V region itself without disrupting the targeting and characteristics of SHM, suggesting that no particular motifs are required for this process (reviewed in Jolly et al. 1996; Storb et al. 1998). Transcription is required for SHM (Fukita et al. 1998). This is supported by the finding that SHM also occurs in some non-Ig genes that are highly expressed in centroblast B cells such as Bcl-6 (Pasqualucci et al. 1998; Shen et al. 1998), fas/CD95 (reviewed in Muschen et al. 2002), mb1/Igα, and B29/Igβ (Gordon et al. 2003). In malignant cells, such as subtypes of diffuse large B cell lymphoma and AIDS-associated non-Hodgkin lymphoma, the proto-oncogenes PIM1, c-myc, RhoH/TTF, and Pax5 are also found to be mutated (Gaidano et al. 2003; Pasqualucci et al. 2001). In addition, AID itself has also been reported to hypermutate when overexpressed in cultured B and non-B cells (Martin and Scharff 2002). Nonetheless, in vivo these non-Ig genes mutate at rates that are at least 10 times lower than those in Ig genes whereas other genes that are highly expressed in centroblast B cells do not undergo mutation (Shen et al. 2000). Targeting of SHM to nonIg loci could be accomplished either by virtue of as yet unidentified cis-acting elements, by features such as a high density of hotspots, by the propensity to form susceptible DNA secondary structures, or as a result of an increase in chromatin accessibility (reviewed in Li et al. 2004).
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each of the S regions are differentially responsive to mitogens and cytokines and various combinations of these direct transcription and class switching to specific isotypes (reviewed in Stavnezer 2000). For instance, lipopolysaccharide (LPS) induces sterile transcription of and class switching to Cγ2b and Cγ3, whereas LPS plus interleukin (IL)-4 induces switching to Cγ1 and Cε (reviewed in Coffman et al. 1993). Enhancers that are located at the 3′ end of the heavy-chain locus appear to regulate the CSR, since the deletion of HS3b and HS4 is associated with the loss of switching to some isotypes (Pinaud et al. 2001). AID initiates CSR by introducing G:U mismatches into the donor Sµ and recipient S regions (see Fig. 5-1). Doublestranded DNA breaks (DSBs) most probably originate from closely spaced staggered single-stranded breaks resulting from the action of downstream repair processes (Arudchandran et al. 2004; Imai et al. 2003; Rush et al. 2004). The DSB mediates the recombination between the donor Sµ and recipient S regions. The role of the S regions has been supported by in vivo mouse studies in which either the donor Sµ or the recipient Sγ region have been removed or manipulated (reviewed in Chaudhuri and Alt 2004). As with SHM, it has not been possible to identify particular cis-acting motifs for DNA binding proteins that are required for CSR except possibly a p50 nuclear factor-κb binding site in the Sγ3 region (Kenter et al. 2004).
II.
ACTIVATION-INDUCED CYTIDINE DEAMINASE AND URACIL
B.
Class Switch Recombination
CSR exchanges the isotype of the antibody produced by a B cell from IgM or IgD to IgG, IgE, or IgA, adding diverse effector functions while preserving antigen specificity. The heavy-chain constant (CH) region in mouse contains eight different genes organized in the 5′ to 3′ order: Cµ, Cδ, Cγ3, Cγ1, Cγ2b, Cγ2a, Cε, and Ca (Fig. 5-1). CSR occurs by intrachromosomal recombination between large repetitive switch (S) regions located upstream of each CH region gene, except for δ, with the excision and loss of all intervening sequences (reviewed in Stavnezer 2000). When CSR occurs, the first CH (Cµ) expressed during development is replaced with one of the downstream CH exons (Cγ, Cε, or Cα). The CH region determines whether the antibody will fix complement, bind to Fc receptors on phagocytic cells, remain in the circulation, diffuse into tissues and body spaces, or polymerize to increase avidity. Like SHM, CSR requires transcription. These sterile or germline transcripts (GLTs) do not encode a protein and are spliced, so that the I exon and CH region are fused (Fig. 5-1). The nascent GLT corresponding to the S region remains stably associated with the DNA template strand and forms RNA-DNA hybrids, whereas the displaced G-rich nontemplate DNA strand exists in a single-stranded state. Such a structure is called an R loop (Shinkura et al. 2003; Yu et al. 2003). The promoters 5′ to
N-GLYCOSYLASE As described above, mice with engineered deletions of various parts of the Ig genes have been very useful in defining critical regulatory elements that are required for SHM and CSR and in revealing the need for transcription for both of these processes. In addition, the examination of some patients with immunodeficiencies has provided important insights (reviewed in Durandy et al. 2003, 2004). However, the major breakthroughs in our understanding of the enzymes responsible for SHM and CSR and of the relationship of these two processes have come primarily from studies with mice that are genetically deficient in the proteins involved (Table 5-1). This is best illustrated by the discovery of AID. Little was known about the biochemical basis of SHM and CSR until 1999 when “the master molecule” essential for the initiation of these two processes, AID, was discovered by Honjo and his colleagues (Muramatsu et al. 1999). They found a 10-fold increase in the expression of this previously unknown gene when a murine B lymphoma line, CH12F3-2, was induced to undergo CSR. They went on to show that AID is selectively expressed in germinal center B cells, where SHM and CSR occur. They then generated AID-deficient mice and found that these mice could not carry out either SHM or CSR (Muramatsu et al. 2000).
TABLE 5-1
PROTEINS DETERMINED TO BE INVOLVED IN SHM AND CSR FROM STUDIES WITH DEFICIENT MICE Effects in CSR in Its Absence Protein Knocked Out
Function
Effect in SHM in Its Absence
Isotype Switching
Targeting of Consensus Motif
Positioning of Junctions
Blunt Junctions
Microhomologies at Junctions
Insertions
References
ND
ND
ND
ND
In patients (Revy et al. 2000); in mice (Muramatsu et al. 2000; Petersen et al. 2001; Reina-SanMartin et al. 2003) Ehrenstein and Neuberger, 1999; Martin et al. 2003; Min et al. 2003; Phung et al. 1998; Rada et al. 1998; Schrader et al. 1999, 2002, 2003; Vora et al. 1999
AID
Cytidine deaminase in ssDNA.
Abolished
Abolished
ND
Msh2
MMR: recognition of point mutations, short mismatches (in complex with Msh6), large mismatches, insertions, deletions (with Msh3); recruitment of accessory proteins involved in processing of mismatch Recognizes mismatch but fails to repair it; signals apoptosis.
No effect in frequency of mutations to 5-fold reduction in mutation frequency; reduced mutations at A:T; increase in hotspot targeting
*Impairment: 80% reduction in switching to IgG3 and 35% reduction in switching to IgG1
Increase in ND consensus targeting at Sµ
Increase
Decreased length of microhomologies at Sµ-Sγ3 junctions.
Increase
Decrease in mutation frequency; reduced mutations at A:T; increase in Ts at G:C; increase in hotspot targeting No effect in mutation frequency; decrease in Ts at G:C in V
Impairment: 50% reduction in switching to IgG3 and 25% reduction in switching to IgG1
ND
ND
Decrease
Increase in long microhomologies
Increase in long insertions
Martin et al. 2003
No effect; decrease in Ts at G:C in Sµ regions
No effect in consensus targeting
No effect
No effect
No effect
Increase
Li et al. 2004; Wiesendanger et al. 2000
Decrease in mutation frequency (Wiesendanger); reduced mutations at A:T; increase in Ts at G:C; increase in hotspot targeting
Impairment; no effect in frequency of mutation at S regions to slight decrease; slight increase of G:C mutations at S regions; high increase of hotspot targeting at recombined Sµ-Sγ3 regions ND
Increase in Changed consensus targeting in Sγ3 but not in Sµ
No effect
No effect
No effect
Li et al. 2004; Martomo et al. 2004; Wiesendanger et al. 2000
ND
ND
ND
ND
ND
Wiesendanger et al. 2000
3.4-fold reduction in switching to IgG1 and 1.4-fold reduction in switching to IgG3
No effect in consensus targeting
Shifted toward ND 3′ part of Sµ and 5′ of Sγ1
Increase in long microhomologies
No effect
Cascalho et al. 1998; Ehrenstein et al. 2001; Kim et al. 1999; Kong and Maizels 1999; Schrader et al. 2002; Winter et al. 1998
~3-fold reduction in switching; increase of mutations at S regions
ND
ND
Increase in long microhomologies
No effect
Kim et al. 1999; Schrader et al. 2002, 2003
Msh2 G674A
Msh3
Msh6
MMR: recognition of large mismatches, insertions, deletions (in complex with Msh2) MMR: recognition of point mutations, short mismatches (in complex with Msh2)
Msh3/Msh6 double knockout
MMR
Pms2
MMR
Mlh1
MMR
Decrease in the frequency of mutation; reduced mutations at A:T; increase in hotspot targeting No effect/∼2-fold reduction in mutation frequency; high frequency of dinucleotide mutations; slight increase of mutations at G:C ∼2-fold reduction in mutation frequency (Kim); slight increase of mutations at G:C
ND
Msh2/Mlh1 double knockout
MMR
ND
UNG
Base excision repair: excises dU creating an abasic site MMR/base excision repair pathway
95% of Ts at G:C; unaffected pattern of mutations at A:T. Ablation of mutations at A:T pairs; 99% of Ts at G:C pairs Reduced mutations at A:T; increase in Ts at G:C; increase in hotspot targeting
Msh2/UNG double knockout Exo1
Exonuclease involved in MMR
DNA-PKcs
DSB repair by NHEJ
No effect
DNA-PKcs (mutant) Ku70
DSB repair by NHEJ
Small to no effect
DSB repair by NHEJ
No effect
Ku86
DSB repair by NHEJ
No effect
γH2AX
DSB repair
No effect
53BP1
DSB repair
No effect
ATM
DSB repair
No effect in SHM
Rad10 (mammalian homolog ERCC1)
Nucleotide excision repair ND endonuclease; cleavage of R loops in vitro
Xpg (homolog of XPG)
Nucleotide excision repair 3-fold reduction in endonuclease; cleavage of mutation frequency; R loops in vitro increase in mutations at A:T pairs
22–45% reduction in switching to all isotypes increase of mutations at S regions Severe impairment: reduced levels of IgG3; no effect in GLT Total ablation of CSR in vitro; reduction of 50% in vivo 70% reduction
ND
ND
ND
Increase in long microhomologies
Slight increase
Schrader et al. 2003
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
In mice (Begum et al. 2004; Rada et al. 2002); in patients (Imai et al. 2003) Rada et al. 2004
ND
ND
Increase
Increase in long insertions
Bardwell et al. 2004
Abrogated switching to all isotypes but not to IgG1 No effect
ND
No effect
ND
Decrease in long microhomologies and increase in short microhomologies ND
ND
Manis et al. 2002
ND
No effect
ND
ND
ND
Lack of CSR; no effect in GLT Severe impairment; absence of mutations at Sµ Impairment: 50–86% reduction in switching to IgG1; no effect in GLT; no effect in mutation at S regions Modest impairment of CSR: 10% reduction in switching to IgG and 20% reduction in switching to IgA; no effect in GLT Severely impaired CSR; no effect in GLT but reduced expression of postswitch transcripts; lower to normal frequency of hypermutation at S regions; no effect in intraswitch recombination 20–55% reduction in switching to all isotypes in vitro; increase in hotspot targeting at S regions No effect
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Bemark et al. 2000; Bosma et al. 2002 Gu et al. 1997; Manis et al. 1998 Casellas et al. 1998; Reina-SanMartin et al. 2003
ND
No effect
ND
No effect
ND
Petersen et al. 2001; Reina-SanMartin et al. 2003
ND
ND
ND
No effect
No effect
Manis et al. 2004
ND
ND
ND
Increased microhomology at Sµ-γ1 junctions/no effect
ND
Lumsden et al. 2004; Reina-SanMartin et al. 2004
ND
ND
ND
No effect
No effect
Schrader et al. 2004
ND
ND
ND
ND
ND
Shiomi et al. 2001; Tian et al. 2004
Continued
TABLE 5-1
PROTEINS DETERMINED TO BE INVOLVED IN SHM AND CSR FROM STUDIES WITH DEFICIENT MICE—cont’d Effects in CSR in Its Absence Protein Knocked Out NF-κB (p50)
Function
Effect in SHM in Its Absence
Isotype Switching
Transcription factor
ND
Decrease of CSR to
involved in B cell
IgG3 and no effect in
activation
CSR to IgG1
Polymerase ζ (antisense)
DNA replication; role in SHM
Polymerase η
DNA replication; role in SHM
Decreased frequency of mutation no effect in pattern of mutations Decreased mutations at A:T (mainly A to C or T to G changes)
ND
Increase of mutations at G:C in Sµ regions
Targeting of Consensus Motif Increase in
Positioning of Junctions
Blunt Junctions
Microhomologies at Junctions
Insertions
References
ND
ND
Decreased length of
ND
Kenter et al. 2004
consensus
microhomologies
targeting at Sγ3 ND
ND
ND
ND
ND
Diaz et al. 2001
ND
ND
ND
ND
ND
Delbos et al. 2005; Martomo et al. 2005
ND, not determined; S, switch region; Ts, transition mutations; GLT, germ line transcript; MMR, mismatch repair.
in Sµ-γ3 junctions
5. MOUSE
MODELS
REVEALED
THE
MECHANISMS
These mice were the equivalent of a subset of patients with hyper-IgM syndrome, who were found to have inactivating mutations in the AID gene (Revy et al. 2000). Thus, AID deficiency leads to the production of low-affinity, unmutated IgM antibodies and no IgG, IgE, or IgA. The IgM antibodies are able to protect mice from death after primary and secondary infections with influenza virus, but after the secondary infection AID−/− mice have a high morbidity, due to the absence of other Ig classes and/or high-affinity antibodies (Harada et al. 2003). Activated IgM B cells and IgM plasma cells accumulate in lymphoid tissues, especially in the intestine, but IgA is absent, resulting in proliferation of anaerobic bacteria and a change in the gut microflora (Fagarasan et al. 2002). When splenic B cells from these AID-deficient mice were stimulated in vitro with LPS and cytokines, they failed to undergo class switch recombination, although they expressed GLTs (Muramatsu et al. 2000). A primary role for AID was confirmed by showing that the ectopic expression of AID can induce CSR and/or SHM in B-cells at the wrong stage of differentiation (Martin et al. 2002), in non-B cells, such as fibroblasts (Okazaki et al. 2002; Yoshikawa et al. 2002) and Chinese hamster ovary cells (Martin and Scharff 2002), and even in Escherichia coli (Petersen-Mahrt et al. 2002; Ramiro et al. 2003; Sohail et al. 2003) and yeast (Poltoratsky et al. 2004). These findings suggest that AID might be the only B-cell specific factor required for these processes whereas the other factors involved in SHM and CSR are ubiquitously expressed. AID is a small protein of 198 amino acids containing a conserved cytidine deaminase motif. AID possesses a 34% amino acid identity to apolipoprotein B editing complex catalytic subunit 1 (APOBEC1) (Muramatsu et al. 1999). APOBEC1 deaminates the cytidine at position 6666 in the apolipoprotein B (ApoB) mRNA to uracil, creating a nonsense codon that results in a truncated ApoB100, ApoB48, which has an entirely different function. AID, based on its homology with APOBEC1 and by comparison of APOBEC1 with the three-dimensional structure of E. coli metabolic cytidine deaminase (Navaratnam et al. 1998), was predicted to have the same four functional domains: 1) an N-terminal α helix domain (residues 1–20), that contains a potential nuclear localization signal; 2) an active cytidine deaminase motif (residues 20–110), which contains the residues that coordinate Zn; 3) a linker motif (residues 110–148); and 4) a cytidine deaminase pseudoactive site (residues 148–198), which has homology with the active site of the enzyme but presumably has no enzymatic activity. The C terminus contains a nuclear export signal. Two models have been proposed to describe the role of cytidine deamination in SHM and CSR: the RNA editing model, that has been championed by Honjo, proposes that AID, like APOBEC1, deaminates C to U at a specific position of a yet to be identified messenger RNA, which encodes an unknown DNA endonuclease that would cleave the V or S regions.
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This hypothesis is supported by the finding that de novo protein synthesis is required for AID activity, suggesting that a messenger RNA edited by AID has to be translated (Doi et al. 2003) before the cleavage step (Begum et al. 2004). The hypothesis that AID was acting as an RNA editing enzyme became less tenable when it was shown that mice with a genetic deficiency in uracil N-glycosylase (UNG), a DNA uracil glycosylase, had a distorted spectrum of somatic mutation and a decrease in CSR (Rada et al. 2002). Just as the phenotype of the AID−/− mice revealed a critical role for cytidine deamination in initiating both SHM and CSR, the phenotype of the UNG−/− mice (see Table 5-1) tipped the balance between RNA and DNA as the potential substrates for AID. A role for UNG was also reported in chicken B cells in culture (Di Noia and Neuberger 2002), in bacteria (Petersen-Mahrt et al. 2002), and subsequently in humans (Imai et al. 2003). In fact, it seemed very unlikely that AID was editing a messenger RNA in non-B cells (Martin and Scharff 2002; Okazaki et al. 2002; Yoshikawa et al. 2002) and bacteria (Petersen-Mahrt et al. 2002; Ramiro et al. 2003; Sohail et al. 2003), in which AID itself is not normally expressed. The DNA deamination model for SHM was confirmed in vitro when semipurified AID deaminated dC in a SHM-like manner in single-stranded DNA (ssDNA), but not in double-stranded DNA, RNA, or DNA:RNA hybrids (Bransteitter et al. 2003; Pham et al. 2003). Other laboratoriess have also reported that ssDNA is the substrate for AID purified from B cells (Chaudhuri et al. 2003; Ramiro et al. 2003) and bacteria (Dickerson et al. 2003; Sohail et al. 2003). The biochemical studies with AID made from transfected insect cells and from B cells are especially interesting because they suggest that AID itself has the inherent ability to recognize the same hotspot motifs that are targeted in vivo (Chaudhuri et al. 2003; Pham et al. 2003). The finding that ssDNA was the substrate for AID provided an explanation for why transcription was required, since AID recognized the ssDNA in the nontranscribed strand in transcription bubbles and in the R loops in the S regions. Although biochemical studies suggest that AID acting alone can target hotspots on ssDNA, there is also evidence that this AID-initiated process is facilitated by replication protein A (RPA), a ssDNA binding protein (Chaudhuri et al. 2004) with roles in replication, repair, and recombination. RPA could facilitate the mutation of both V and S regions by binding to ssDNA and/or through its association with RNA polymerase II and UNG. Additional as yet undiscovered associated proteins may be required to target AID to the V or S regions. Alterations (deletion, insertion, or frameshift replacement) in the 8–17 amino acids in the C-terminal region of AID, found in patients with the hyper-IgM type 2 syndrome, cause complete loss of CSR activity whereas SHM activity is retained (Ta et al. 2003). Similarly, deletion of C-terminal amino acids 189–198 of AID also causes loss of CSR while unexpectedly displaying higher levels of SHM than the wild-type AID (Barreto et al. 2003). Mutants in the N-terminal region of AID have decreased
162
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IGLESIAS-USSEL,
LI,
AND
MATTHEW
D.
SCHARFF
2002). dU can be 1) replicated to produce a C to T (G to A on the other strand) transition mutation (Fig. 5-2a), 2) removed by UNG to create an abasic site that can be bypassed by errorprone DNA polymerase(s) to generate transition and transversion mutations in SHM (Figs. 5-2b and 5-3) recognized by the Msh2-Msh6 complex of the mismatch repair pathway so that the dU:dG mismatch is excised and resynthesized by errorprone polymerases that will create additional mutations including mutations at A:T base pairs (Fig. 5-2c). To initiate CSR, the dU created by AID in the S region is removed by UNG, creating an abasic site that is converted into a single-stranded nick by endonuclease. Close spaced nicks on both strands are converted into double-stranded breaks, which are then processed by NHEJ (reviewed in Chaudhuri and Alt 2004) (not shown).
ability to carry out SHM while retaining CSR activity (Shinkura et al. 2004). These results raise the possibility that AID activity in SHM and CSR depends on the recruitment of separate cofactors that have yet to be identified. Although both SHM and CSR take place in centroblast B cells, they can occur independently, since there are IgM antibodies with somatic mutations and IgG antibodies with unmutated V regions. Moreover, stimulation of spleen B cells in vitro induces CSR but not SHM. Although these two processes share factors such as AID, UNG, and mismatch repair (MMR) proteins and require transcription, they also differ in that CSR requires DSB, looping out and excision of S regions and the proteins involved in nonhomologous end joining (NHEJ) to complete the recombination process, whereas recombination and NHEJ are not required for SHM (Table 5-1). These similarities and differences, which were revealed primarily through the examination of genetically deficient mice (see Table 5-1), have led to a model (Rada et al. 1998) in which both SHM and CSR are initiated by AID-dependent deamination of dC either in the V region or the S region. For SHM, the dU created in the V region by AID may be subsequently processed in different ways (Petersen-Mahrt et al.
C
ZIQIANG
III.
MISMATCH REPAIR
Mismatch repair machinery recognizes and corrects the mismatched base pairs that arise during DNA replication or other
Co-factors
RPAAID G RNAP
RNA polymerase complex
AID U
AID-derived G-U mismatch
RPA AID G RNAP
Replication
Ts
T
UNG
G : abasic site
A C
Replication (error-prone DNA polymerases)
G
Phase 1a
C
or Ts
G
MLH1/PMS2/EXO1
G
Error-prone DNA polymerase ***** **
A:T mutations
T A
or
Phase 2
A
Tv or Tv
Msh2-Msh6
T G C
Phase 1b Fig. 5-2 Model of AID-induced mutations in SHM. An AID molecule along with some associated molecules is shown in a moving transcription bubble. AID deaminates deoxycytosines (C) to deoxyuracil (U) in ssDNA. The dU can be (a) replicated, creating C to T and G to A transition mutations (Ts); (b) removed by UNG to create an abasic site that can bypassed by error-prone DNA polymerases to create transition and transversion mutations (Tv); or (c) recognized by the mismatch repair complex Msh2-Msh6, excised, and resynthesized by error-prone DNA polymerases, creating additional mutations at A:T pairs. RNAP = RNA polymerase II; RPA = replication protein A.
5. MOUSE
MODELS
REVEALED
THE
MECHANISMS
FOR
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MSH6
163
MSH3
MSH2
Mismatch recognition MSH6 or MSH3 MSH2 RFC EXO1 MLH1
MSH6 or MSH3
PCNA
PMS2
RFC MSH2 PCNA
EXO1
Excision
MLH1 PMS2
DNA Polymerase
Resynthesis & ligation DNA Ligase
Fig. 5-3 The major known mismatch repair proteins are shown recognizing and repairing a mismatch. Small mismatches are recognized by Msh2 in complex with Msh6 and large mismatches by the Msh2-Msh3 complex. The heterodimer moves away from the mismatch and recruits additional mismatch repair proteins, such as Mlh1, Pms2, Exo1, PCNA and replication factor C (RFC). As a result, a DNA patch containing the mismatch is excised, and the resulting gap is filled in and repaired by DNA polymerase and DNA ligase.
DNA repair processes. Deficiency or malfunction in MMR results in genomic instability and, as a consequence, tumors in humans and in mice (reviewed in Wei et al. 2002). The basic process of MMR includes mismatch recognition, excision of the mismatch through digestion of the DNA containing the mismatch, and filling the gap by error-free DNA polymerases (reviewed in Wei et al. 2002) (Fig. 5-3). Mismatch recognition is usually carried out by mammalian MutS homolog (Msh) 2, which forms a heterodimer with either Msh6 or Msh3. The Msh2-Msh6 heterodimer mainly recognizes single base mismatches and short mismatches, whereas the Msh2-Msh3 heterodimer recognizes larger mismatches and insertion/deletion loop mismatches (reviewed in Buermeyer et al. 1999). Msh2 and Msh6 proteins have intrinsic ATP binding and hydrolysis activities that are not required for the recognition of the mismatch (Alani et al. 1997). However, the conversion of ATP to ADP is necessary for the Msh2-Msh6 dimers to move away from the mismatch (Blackwell et al. 1998) and allow the recruitment of downstream MMR repair proteins such as
exonuclease 1 (Exo1), postmeiotic segregation (Pms) 2, MutL homologue (Mlh) 1, proliferating-cell nuclear antigen (PCNA), and presumably an unknown endonuclease, which makes a nick at some distance from the mismatch. Then, the nicked DNA containing the mismatch can be digested by Exo1. Filling in the gap by DNA polymerases and the ligation by DNA ligases complete the MMR process (reviewed in Edelmann and Edelmann 2004; Kolodner 1996; Wei et al. 2002). Interestingly, when Pms2−/− mice were examined for SHM, the rate of SHM was found to be lower, as opposed to higher, as one would predict for MMR deficiency (Cascalho et al. 1998). These results indicate that MMR is necessary for efficient SHM. In addition, the pattern of SHM from Msh2−/−, Msh6−/−, or Exo1 mutant mice shows a loss of A:T mutations and, as a consequence, a very high percentage of mutations at G:C bases (reviewed in Li et al. 2004) (see Table 5-1). However, mice deficient in Pms2 or Mlh1, two important downstream MMR proteins (Fig. 5-3), do not have as significant a reduction in A:T mutations as Msh2−/−, Msh6−/−, or Exo1 mutant mice
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(see Table 5-1). Deficiency of either Pms2 or Mlh1 ablates mismatch repair activity. Thus, these two proteins could have different functions in SHM and MMR. Nonetheless, it appears that MMR proteins are needed to generate more mutations, including mutations at A:T bases, during SHM. A recent article (Rada et al. 2004) showing that in UNG/Msh2 double knockout mice there were no mutations at A:T bases and no transversion mutations, further supports the idea that MMR and UNG are competing pathways in processing the AID-generated G:U mismatches in SHM. MMR proteins are also implicated in efficient CSR. A deficiency in Msh2, Msh6, Mlh1, Pms2, or Exo1 results in reduced efficiency in CSR (see Table 5-1). In addition, in UNG/Msh2 double knockout mice there was virtually no switching (Rada et al. 2004). This suggests that MMR and UNG are alternative pathways for processing the AID-generated G:U mismatches to generate the DSBs required to initiate recombination and complete CSR. What is puzzling is that different MMR deficiencies exhibited different features in the recombined DNA segments. An increase in blunt junctions and insertions and a decreased length of microhomologies were observed in the switch junctions from Msh2−/− mice. Exo1 mutant mice also exhibited a similar, although less dramatic S junction pattern. Interestingly, Msh6−/− mice do not show these kinds of changes, even though both Msh2−/− and Msh6−/− mice exhibited a similar extent of reduction in CSR frequency. A deficiency in Msh6 increases the targeting of GAGCT/GGGGT consensus motifs in Sγ3 and in RGYW/ WRCY hotspot motifs and changes the usage of different subsegments of switch regions in CSR. In addition, Mlh1- or Pms2-deficient mice showed longer microhomologies in the switch junctions than Msh2−/− mice (see Table 5-1). Although Msh3-deficient mice have normal levels of CSR and SHM, they exhibited more insertions in the switch junctions, suggesting that Msh3 is not involved in processing of G:U mismatches but does participate in certain stages of CSR, possibly via mediation of the resolution of DNA breaks (Li et al. 2004). Collectively, MMR proteins may have other distinct functions in addition to processing G:U mismatches to generated DSBs to promote CSR (reviewed in Li et al. 2004). The role of MMR proteins in SHM and CSR appears to depend upon their ATPase activity, because mice harboring a point mutation in the ATPase domain of Msh2 (G674A) have a phenotype in CSR and SHM that is similar to that of Msh2−/− mice (Martin et al. 2003). Msh2 protein with this point mutation is able to recognize the mismatch but unable to initiate downstream MMR repair processes because of a lack of ATPase activity. Nonetheless, Msh2 protein lacking ATPase activity is able to trigger apoptosis, allowing the dissection of the role of Msh2 in SHM and in CSR without affecting other functions. Interestingly, these mice showed a mixed phenotype in switch junctions, that is, more insertions, which resembles findings in Msh2−/− mice, and longer microhomologies, like those found in Mlh1−/−, Pms2−/−, or Msh2−/−/Mlh1−/− mice.
ZIQIANG
IV.
LI,
AND
MATTHEW
D.
SCHARFF
ERROR-PRONE DNA POLYMERASES
Recently discovered error-prone DNA polymerases are not processive and are involved in bypassing various types of DNA lesions during DNA replication (reviewed in Tippin et al. 2004). Therefore, they could act on G:abasic sites, generating transition and transversion mutations during SHM (see Fig. 5-2b), which is essentially mediated by AID. Mice deficient in the catalytic subunit of polymerase ζ (Rev3) are embryonic lethal, making it difficult to evaluate the role of polymerase ζ in SHM in vivo. However, downregulation of Rev3 by antisense expression in transgenic mouse and in cell lines (Diaz et al. 2001; Zan et al. 2001) decreases the rate of SHM. In addition, complete deletion of polymerase ι in human Burkitt′s lymphoma BL-2 cells results in a significant reduction of SHM frequency (Faili et al. 2002), although 129/SvJ-derived strains of mice are naturally deficient in polymerase ι and show normal levels of SHM (McDonald et al. 2003). These two contradictory results could reflect the mechanistic difference in SHM between mouse and human or could be peculiar to the BL-2 cell line. Polymerase η, a very error-prone polymerase when one is replicating an undamaged DNA template, is clearly involved in SHM. When patients with xeroderma pigmentosum variant who have a polymerase η deficiency were examined for V region hypermutation, they exhibited a spectrum similar to that of Msh2- or Msh6-deficient mice, that is, a loss of mutations at A:T bases and, as a consequence, a high percentage of mutations at G:C bases (Zeng et al. 2001). The same result was observed in mice lacking polymerase η (Delbos et al. 2005; Martomo et al. 2005). Because of this finding, it was proposed that MMR and polymerase η are involved in the second phase of SHM to generate additional mutations via the error-prone replication of polymerase η when filling in the gap created by Exo1 during the resolution of the G:U mismatch repair (see Fig. 5-3). Indeed, in vitro studies showed that Msh2-Msh6 is able to bind to G:U mismatches (Gu et al. 2002; Wilson et al. 2005). The puzzling part is why polymerase η is used instead of a high-fidelity DNA polymerase during the final step of G:U mismatch repair in SHM.
V.
NHEJ
NHEJ is required for V(D)J recombination during B-cell development and DNA repair via its ability to repair DSBs. The key components include the DNA-dependent protein kinase, consisting of Ku70, Ku86, and the catalytic subunit (DNAPKcs), as well as p53 binding protein 1 (53BP1), Xrcc4, and ligase IV (reviewed in Chaudhuri and Alt 2004) (see Table 5-1). Because CSR requires the generation and repair of DSBs, NHEJ-deficient mice are defective in CSR but normal in SHM
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(see Table 5-1), supporting the idea that repairing DSBs is not required for SHM. In addition, proteins that are important for signaling the repair of DSBs are also implicated in CSR. Mice deficient in ataxia telangiectasia mutated (ATM) kinase (Lumsden et al. 2004; Reina-San-Martin et al. 2004) and H2AX (Reina-San-Martin et al. 2003) show defects in CSR. Indeed, phosphorylated H2A histone family member X (γ-H2AX), which facilitates DSB repair, forms foci that colocalized with IgH foci from in vitro stimulated primary B cells (Petersen et al. 2001). The presence of proficient CSR and SHM in homologous recombination-deficient mice, such as Rad54 (Essers et al. 1997) or Rad52 knockout mice (Rijkers et al. 1998), suggests that homologous recombination is not a major factor in CSR.
VI.
CONCLUSION
In the previous sections, we have reviewed much of the current state of our knowledge about mechanisms responsible in mice and humans for the production of high-affinity isotype switched antibodies that are distributed throughout the body to deal with pathogenic organisms and their products. In the course of this discussion and in Table 5-1, we have summarized the many genetically manipulated mice that form the basis for our understanding of the biochemical basis of SHM and CSR. Although genetic defects in humans and studies done with cultured cells and cell-free biochemical systems have been very important in advancing our knowledge about the enzymes involved in these processes, it should be evident that mouse models lacking each of these enzymes were absolutely crucial, not only because they revealed the various pathways and activities in which they were involved but also because they provided an understanding of the relative importance of each of these pathways in vivo.
ACKNOWLEDGMENTS Maria D. Iglesias-Ussel is supported by a Northeast Biodefense Center Postdoctoral Fellowship. Ziqiang Li is supported by a Cancer Research Institute Postdoctoral Fellowship and is a special fellow of the Leukemia and Lymphoma Society. Matthew D. Scharff is supported by the Harry Eagle Chair provided by the National Women’s Division of the Albert Einstein College of Medicine.
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Rush, J.S., Fugmann, S.D., Schatz, D.G. (2004). Staggered AID-dependent DNA double strand breaks are the predominant DNA lesions targeted to Sµ in Ig class switch recombination. Int Immunol 16, 549–557. Schrader, C.E., Edelmann, W., Kucherlapati, R., Stavnezer, J. (1999). Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes. J Exp Med 190, 323–330. Schrader, C.E., Vardo, J., Linehan, E., Twarog, M.Z., Niedernhofer, L.J., Hoeijmakers, J.H., et al. (2004). Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions. J Exp Med 200, 321–330. Schrader, C.E., Vardo, J., Stavnezer, J. (2002). Role for mismatch repair proteins Msh2, Mlh1, and Pms2 in immunoglobulin class switching shown by sequence analysis of recombination junctions. J Exp Med 195, 367–373. Schrader, C.E., Vardo, J., Stavnezer, J. (2003). Mlh1 can function in antibody class switch recombination independently of Msh2. J Exp Med 197, 1377–1383. Sharpe, M.J., Milstein, C., Jarvis, J.M., Neuberger, M.S. (1991). Somatic hypermutation of immunoglobulin κ may depend on sequences 3’ of Cκ and occurs on passenger transgenes. EMBO J 10, 2139–2145. Shen, H.M., Michael, N., Kim, N., Storb, U. (2000). The TATA binding protein, c-Myc and survivin genes are not somatically hypermutated, while Ig and BCL6 genes are hypermutated in human memory B cells. Int Immunol 12, 1085–1093. Shen, H.M., Peters, A., Baron, B., Zhu, X., Storb, U. (1998). Mutation of BCL6 gene in normal B cells by the process of somatic hypermutation of Ig genes. Science 280, 1750–1752. Shinkura, R., Ito, S., Begum, N.A., Nagaoka, H., Muramatsu, M., Kinoshita, K., et al. (2004). Separate domains of AID are required for somatic hypermutation and class-switch recombination. Nat Immunol 5, 707–712. Shinkura, R., Tian, M., Smith, M., Chua, K., Fujiwara, Y., Alt, F.W. (2003). The influence of transcriptional orientation on endogenous switch region function. Nat Immunol 4, 435–441. Shiomi, N., Hayashi, E., Sasanuma, S., Mita, K., Shiomi, T. (2001). Disruption of Xpg increases spontaneous mutation frequency, particularly A:T to C:G transversion. Mutat Res 487, 127–135. Sohail, A., Klapacz, J., Samaranayake, M., Ullah, A., Bhagwat, A.S. (2003). Human activation-induced cytidine deaminase causes transcriptiondependent, strand-biased C to U deaminations. Nucleic Acids Res 31, 2990–2994. Sohn, J., Gerstein, R.M., Hsieh, C.L., Lemer, M., Selsing, E. (1993). Somatic hypermutation of an immunoglobulin mu heavy chain transgene. J Exp Med 177, 493–504. Stavnezer, J. (2000). Molecular processes that regulate class switching. Curr Top Microbiol Immunol 245, 127–168. Storb, U., Peters, A., Klotz, E., Kim, N., Shen, H.M., Hackett, J., et al. (1998). Cis-acting sequences that affect somatic hypermutation of Ig genes. Immunol Rev 162, 153–160. Ta, V.T., Nagaoka, H., Catalan, N., Durandy, A., Fischer, A., Imai, K., et al. (2003). AID mutant analyses indicate requirement for class-switch-specific cofactors. Nat Immunol 4, 843–848. Tian, M., Shinkura, R., Shinkura, N., Alt, F.W. (2004). Growth retardation, early death, and DNA repair defects in mice deficient for the nucleotide excision repair enzyme XPF. Mol Cell Biol 24, 1200–1205. Tippin, B., Pham, P., Goodman, M.F. (2004). Error-prone replication for better or worse. Trends Microbiol 12, 288–295. Tumas-Brundage, K., Vora, K.A., Giusti, A.M., Manser, T. (1996). Characterization of the cis-acting elements required for somatic hypermutation of murine antibody V genes using conventional transgenic and transgene homologous recombination approaches. Semin. Immunol 8, 141–150. Tumas-Brundage, K.M., Vora, K.A., Manser, T. (1997). Evaluation of the role of the 3′α heavy chain enhancer [3′α E(hs1,2)] in Vh gene somatic hypermutation. Mol Immunol 34, 367–378. Vora, K.A., Tumas-Brundage, K.M., Lentz, V.M., Cranston, A., Fishel, R., Manser, T. (1999). Severe attenuation of the B cell immune response in Msh2-deficient mice. J Exp Med 189, 471–481.
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Wei, K., Kucherlapati, R., Edelmann, W. (2002). Mouse models for human DNA mismatch-repair gene defects. Trends Mol Med 8, 346–353. Wiesendanger, M., Kneitz, B., Edelmann, W., Scharff, M.D. (2000). Somatic mutation in MSH3, MSH6, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern. J Exp Med 191, 579–584. Wilson, T.M., Vaisman, A., Martomo, S.A., Sullivan, P., Lan, L., Hanaoka, F., et al. (2005). MSH2-MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes. J Exp Med 201, 637–645. Winter, D.B., Phung, Q.H., Umar, A., Baker, S.M., Tarone, R.E., Tanaka, K., et al. (1998). Altered spectra of hypermutation in antibodies from mice deficient for the mismatch repair protein PMS2. Proc Natl Acad Sci USA 95, 6953–6958.
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Yoshikawa, K., Okazaki, I.M., Eto, T., Kinoshita, K., Muramatsu, M., Nagaoka, H., et al. (2002). AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts. Science 296, 2033–2036. Yu, K., Chedin, F., Hsieh, C.L., Wilson, T.E., Lieber, M.R. (2003). R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol 4, 442–451. Zan, H., Komori, A., Li, Z., Cerrutti, M., Flajnik, M.F., Diaz, M., et al. (2001). The translesional polymerase ζ plays a major role in Ig and Bcl-6 somatic mutation. Immunity 14, 643–653. Zeng, X., Winter, D.B., Kasmer, C., Kraemer, K.H., Lehmann, A.R., Gearhart, P.J. (2001). DNA polymerase η is an A-T mutator in somatic hypermutation of immunoglobulin variable genes. Nat Immunol 2, 537–541.
Chapter 6 Mouse Natural Killer Cells: Function and Activation Francesco Colucci
I. Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Development and Surface Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Morphology and Surface Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Tissue Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cytokine and Chemokine Production . . . . . . . . . . . . . . . . . . . . . . . . . . C. Tumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Viral Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Inhibitory Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Activating Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. A Dynamic View of NK Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
HISTORICAL PERSPECTIVE
In the 1960s, Cudkowicz described a phenomenon that could not be explained by the current thinking in transplantation biology. Hybrid mice from a cross between two inbred strains accept grafts of solid tissues from either parents but, in certain combinations, they rejected parental bone marrow grafts. This exception to the laws of transplantation was called hybrid
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resistance (Cudkowicz and Stimpfling 1964). A decade later, Kiessling and Herberman independently discovered white blood cells in the mouse capable of killing tumors cells in vitro. Because the killing process was spontaneous and did not require any previous immunization, these lymphocytes were called natural killer (NK) cells (Herberman et al. 1975; Kiessling et al. 1975). Similar lymphoid cells were also described in humans (Ortaldo et al. 1977). The potential implications for tumor
Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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immunology were clear, but the recognition mechanisms of NK cells remained elusive. Kärre discovered that NK cells preferentially killed tumor cells with no or low expression of major histocompatability (MHC) class I antigens (Kärre et al. 1986). This observation led to a prediction of the existence of inhibitory MHC receptors that were later found on both murine and human NK cells (Colonna and Samaridis 1995; Karlhofer et al. 1992). Thus, hybrid resistance in mice could be explained on the basis of the missing self-MHC on the parental cells. The absence of one set of MHC class I antigens on the transplanted parental bone marrow cells triggered activation of the hybrid NK cells. Later it was discovered that, in addition to the activation that follows the absence of inhibitory signals, NK cells could also respond to the presence of activating signals expressed by malignant or infected cells. Experimental evidence in mice (Bukowski et al. 1985) and clinical observations in a child with multiple episodes of viral infections and defective NK cell functions (Biron et al. 1989) led to the appreciation of the role of NK cells in infectious biology. Resistance to mouse cytomegalovirus was genetically linked to a chromosomal region encompassing genes encoding for NK receptors (Scalzo et al. 1992), including a lectin-like activating receptor that, about a decade later, was found to directly bind a viral glycoprotein (Arase et al. 2002). Another activating receptor found in mouse and human recognizes MHC class I–like cellular molecules induced by stress, transformation, and viral infection (Raulet 2003). Work in the mouse, as well as in human cells, will help define the receptor-ligand systems and the intracellular signal transduction pathways that regulate NK cell functions and activation.
II.
DEVELOPMENT AND SURFACE MARKERS A.
Development
This chapter is focused on the function and activation of mouse NK cells, but a short section on their development and surface phenotype may be a useful complement. During embryonic development, NK cell precursors are found in the thymus, blood, liver, and spleen. NK cell precursors isolated from embryonic tissues give rise to NK, natural killer T (NKT), and T cells and are therefore referred to as common T/NK progenitors. NK-committed precursors (NKPs) can be found in the bone marrow of adult mice, and it is thought that they originate from common lymphoid precursors, under the action of earlyacting cytokine stem cell factor (SCF; also known as c-Kit ligand or KL), FMS-like tyrosine kinase 3 ligand (FLT3L; also known as fetal liver kinase 2 ligand, Flk2, or FL), and interleukin (IL)-7 (Colucci et al. 2003). NKP cells express the IL-2 receptor β chain (IL2Rβ; also known as CD122), which is also part of the IL-15 receptor.
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The expression of CD122 marks the commitment to the NK cell lineage, that is, NKPs lose the potential to differentiate in B or T lymphocytes or myeloid-erythroid lineages. At this stage of development, the precursors acquire the capacity to respond to a key cytokine in NK cell development, that is, IL-15. Bone marrow CD122+ NKPs do not express other NK cell markers such as NK1.1 (also known as NKR-P1C; expressed by NK cells in some mouse strains) or the integrin DX5 (α2 integrin, very late antigen-2; also known as CD49b, which is expressed on most mature NK cells in all mouse strains). Thus, NKP cells may be defined as CD122+ lymphoid cells that do not express any lineage-specific marker, including CD3, CD4, CD8, CD19, NK1.1, DX5, Mac-1 (also known as CD11b), Gr-1, and Ter119. NKP cells proceed along the pathway of NK cell development, and the next detectable event is the acquisition of the NK1.1 marker. At this stage, CD122+NK1.1+DX5−, immature NK cells are still incapable of cytotoxicity. This function is acquired coincidentally with the expression of DX5. Thus, the first lytic developmental intermediates are CD122+NK1.1+DX5+ cells. It is not clear how NK cell functions are regulated during ontogeny, but it is likely that the factors governing regulation of NK cell activity may be different in immature and mature NK cells. For example, the regulation of mature NK cells is mediated mostly by Ly49 receptors that bind MHC class I (see below), but cytotoxic, immature CD122+NK1.1+DX5+ cells do not express Ly49 receptors. Therefore, there may be alternative mechanisms of regulation of NK cell functions, some of which may be independent of MHC receptors. One possible mechanism of regulation during ontogeny is provided by the expression of CD94/NKG2 heterodimers that bind to nonclassic MHC class I. Early in ontogeny, virtually all NK cells express CD94/NKG2 receptors, whereas in adult mice most NK cells express Ly49 receptors, and CD94/NKG2 are found only in about half of NK cells. A similar sequence of events occurs also during human NK cell ontogeny. Ly49 receptors are the functional homologs of human killer cell immunoglobulin-like receptors (KIRs). Most of them are inhibitory, but some are activating. The factors dictating the expression of the Ly49 repertoire are not yet fully understood, but the induction of these receptors requires that developing NK cells interact with bone marrow stromal cells. The expression of Ly49 genes is regulated by a stochastic mechanism, is monoallelic, and is a late event during the developmental program of NK cells. Each NK cell, after expressing one Ly49, can go on expressing up to four distinct Ly49 receptors. However, each NK cell expresses at least one inhibitory receptor that binds to the MHC alleles present in the environment. Thus, host MHC shapes the Ly49 repertoire, both in term of selection and expression intensity. The final maturation step is marked by the acquisition of high levels of CD43 and CD11b, at which point NK cells are exported from the bone marrow out to peripheral lymphoid organs (Yokoyama et al. 2004). NK cells have a low rate of cell
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division in the steady state (<5%); however, when challenged with viral infections, tumors, or inflammatory cytokines, they undergo blastogenesis and enter the cell cycle. Experiments based on adoptive transfer of mature NK cells have suggested that the half-life of NK cells is around 10 days.
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(see below). Surface phenotype and functions of NK cells vary in the different NK cell populations, reflecting distinct differentiation pathways and/or the influence of the microenvironment. Most experimental procedures use splenic NK cells, and splenic NK cells are the gold standard against which surface markers and functions of other NK cell populations are compared.
Morphology and Surface Markers
Mature NK cells are mononuclear cells with little cytoplasm that are morphologically indistinguishable from other lymphocytes. But unlike B and T cells, NK cells are characterized by the presence of azurophilic granules, which become more obvious in activated NK cells. Because of the presence of these granules, NK cells are referred to as large granular lymphocytes. When NK cells are cultured in high doses of IL-2, they expand, become activated, and can kill more target cells, including some that are otherwise resistant to “resting” NK cells. Such in vitro activated cells are referred to as lymphokine activated killer (LAK) cells. If the culture contains T cells, CD8+ T cells will also respond to this treatment, expand, and acquire spontaneous killing activity, albeit lower than that of NK cells. Thus, LAK cells prepared from unpurified splenocytes contain a mixture of NK and CD8+ T cells. NKT cells are also capable of spontaneous cytotoxicity, and this activity is augmented by IL-2. When analyzed by flow cytometry, because they are slightly larger and more granular than B and T cells, NK cells fall in the upper right end of a standard lymphoid gate, which is set according to parameters of scattered light. Most mature NK cells express CD122, NK1.1 (in some strains of mice), DX5, Ly49s, CD11b and CD43, NKG2D, 2B4 (in some strains of mice), CD16, CD44, and CD2. Subsets of NK cells (10–50%) also express B220 (CD45R), Thy-1 (CD90), and c-kit (CD117).
C.
Tissue Distribution
NK cells represent a small fraction of lymphocytes found in primary and secondary lymphoid organs, as well as in the blood and in various tissues. They are quite rare in thymus and bone marrow, representing only about 0.1–1% of lymphocytes. Among secondary lymphoid organs, they are most abundant in the spleen, where they represent 2–5% of the lymphocyte population, reaching about 2–3 × 106 cells in total. NK cells are barely detectable in lymph nodes of naïve mice; however, they reach sizable populations in immunized mice. Peripheral blood lymphocytes comprise 5–15% NK cells. Similar percentages are found in the peritoneal cavity, as well as within resident lymphocytes of the liver and the lungs. The uterus of pregnant female mice contains the highest percentage of NK cells, where they can reach 90% of the resident lymphocytes at midgestation
III.
FUNCTION
A.
Cytotoxicity
Cellular cytotoxicity can be executed in response to various stimuli and, for the sake of clarity, it can be categorized as natural cytotoxicity (also known as spontaneous cytotoxicity or natural killing) and antibody-dependent cellular cytotoxicity (ADCC). Natural cytotoxicity defines the capacity of NK cells to spontaneously kill target cells without any prior immunization or stimulation of any sort. In contrast, ADCC is mediated by antigen-bound immunoglobulin (Ig) G antibodies, which are recognized by the FcγRIII expressed on NK cells. NK cells can kill targets using a variety of mechanisms that depend on cellto-cell contact or that rely on the secretion of soluble molecules. The cytotoxicity machinery of NK cells resembles that of cytotoxic T lymphocytes (CTLs); however, NK cells are poised to execute faster than T cells because they do not need a period of clonal expansion. Cell contact–dependent mechanisms include exocytosis of pore-forming perforin and apoptosis-inducing granzymes into the target cells. This is likely to be the predominant cytotoxicity pathway in murine NK cells. Perforin and granzymes are constitutively expressed in NK cells, as opposed to CTLs, in which perforin and granzymes are produced only upon activation. Perforin and granzymes are stored in cytotoxic granules that, upon contact with a susceptible target, move toward the site of contact with the target—the immune synapse—and are released in an orderly manner into the susceptible target. NK cells and CTLs protect themselves from the toxic granule content by means of cathepsin B that prevents perforin-mediated pore formation in the plasma cell membrane of NK cells and CTLs. An alternative pathway to target cell apoptosis is triggered by tumor necrosis factor (TNF) family members expressed on NK cells such as TNF-α, lymphotoxin, Fas ligand (FasL), and TNF-related apoptosis–inducing ligand (TRAIL). These molecules bind to death domain–containing receptors on target cells, leading to apoptosis in a perforin-independent way.
B.
Cytokine and Chemokine Production
NK cells are a rich source of cytokines and chemokines early during immune responses. The production of these factors can
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be induced by cell-to-cell contacts between NK cells and tumors or virus-infected cells. NK cells can also respond to soluble cytokines (interferon [IFN]-α/β, IFN-γ, IL-12, IL-15, IL-18, and IL-21) released in the local microenvironment by infected cells, T cells, macrophages, and dendritic cells. Once stimulated by these cytokines, NK cells show more potent spontaneous cytotoxicity, proliferate, and produce cytokines and chemokines, thereby amplifying the immune response. Mouse NK cells can produce and secrete IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor, and various chemokines such as macrophage-inflammatory protein (MIP)-1α (CCL3) and MIP-1β (CCL4). The capacity to rapidly produce IFN-γ is probably the most powerful feature of NK cells and is, second only to cytotoxicity, undoubtedly the best studied function. This cytokine activates macrophages, enhances MHC expression on antigen-processing cells, and, most probably through an effect on dendritic cells, guides the adaptive immune response toward a T helper (Th) 1 polarization. Besides its antimicrobial role, IFN-γ participates also in immune surveillance against cancer.
C.
Tumor Immunity
Ever since it was discovered that NK cells could kill various tumor cells in vitro, murine models have been advantageous in establishing the participation of NK cells in immunity against cancer in vivo. A variety of experimental approaches have shown that NK cells limit the emergence of induced tumors and control the growth and metastatic spread of transplanted tumor cells. In general, lack of MHC class I on tumor cells makes them a preferential target for NK cells. Numerous approaches relying on the use of mice in which NK cells were depleted by antibody treatment (classically anti-NK1.1 in mouse strains that express this marker or anti-asialo-GM1 and anti-CD122 in all mouse strains) have been used before transplantation of tumor cells. In these conditions, the tumor expanded and metastasized more in NK-cell–depleted mice. A similar approach has been used to study the role of NK cells in immune surveillance. NK-cell–depleted mice developed more carcinogen-induced tumors than NK-cell–competent mice. Despite the established role of NK cells in antitumor immunity, other cells (i.e., NKT cells and γδ T cells) can participate in this function. For example, NKT cell activation by the CD1d-reactive glycolipid α-galactosylceramide initiates a series of events that lead to downstream activation of cellular and cytokine networks, including NK cells and IFN-γ, which exert antitumor immunity (Smyth et al. 2005). Another general conclusion from the experiments described above is that the antitumor immunity of NK cells is neither absolute nor long lasting. On one hand, NK cell activity is not effective against a large number of transplanted tumor cells, whereas on the other, NK cell antitumor immunity does not induce immunological memory. Nevertheless, in some experimental conditions, the stimulation
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of NK cells with tumors appeared to induce activation of dendritic cells (DCs), which in turn initiated antigen-specific antitumor T cell responses that led to immunological memory. DCs and NK cells actively cross-talk during the initial phase of immune responses. These interactions appear to be bidirectional, so that NK cell activity is enhanced by DCs, and DCs are induced to mature and become activated under the influence of NK cells. Thus, the contribution of NK cells to antigenspecific adaptive immunity may be indirect and mediated by interactions with DCs.
D.
Viral Immunity
The first NK cell function to be discovered was the capacity to spontaneously kill tumor cells. However, it is likely that NK cells have evolved primarily under the selective pressure of infectious agents. Despite the fact that the antiviral function of NK cells overlaps with the antiviral function mediated by CTLs, it is complementary and essential in at least two ways. First, it provides an immediate response against viral infections, whereas CTLs require a lag phase of a few days before the antigen-specific clones have expanded to detectable numbers. Second, NK cells and CTLs have alternative modes of target recognition. CTLs are restricted by and depend on the presence of MHC class I, whereas NK cells preferentially attack cells that have lost MHC class I expression. Studies in the mouse system suggested that NK cells play key roles during innate immunity to herpesviruses, such as mouse cytomegalovirus (MCMV). Thus, mice in which NK cells had been depleted by antibody treatment, showed high titers of MCMV. On the other hand, adoptively transferred NK cells could provide protection against MCMV (Bukowski et al. 1985). In the late 1980s, Biron et al. (1989) described a child who developed recurrent herpesvirus infections and had deficient NK cell functions, but normal B and T cell functions. Human NK cells are reportedly reactive also to herpes simplex virus and Epstein-Barr virus (EBV), and NK cell deficiency correlates with susceptibility to these infections. Scalzo et al. (1992) genetically linked MCMV resistance to the distal region of mouse chromosome 6, which encompasses a complex of genes that encode NK cell receptors (NK gene complex [NKC]). Ly49 genes are included in this complex. The activating Ly49H receptor mediates the resistance to MCMV (Brown et al. 2001; Daniels et al. 2001; Lee et al. 2001), and it binds the viral m157 glycoprotein expressed on infected cells (Arase et al. 2002; Smith et al. 2002). Interactions between Ly49H and m157 on infected cells of resistant mouse strains activate NK cells that kill infected cells and produce IFN-γ and chemokines. The importance of NK cells in viral infections is highlighted by the number of genes that MCMV and other viruses dedicate to escape NK cell recognition. A general viral evasion strategy consists of downregulation of MHC class I on infected cells, thereby avoiding CTL recognition. This should expose infected
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cells to NK cell killing. However, MCMV has evolved several mechanisms to cope with this process. For example, in susceptible mouse strains that lack Ly49H expression, the viral m157 product binds the Ly49I inhibitory receptors, thus switching off NK cell activation. MCMV also has genes that can encode for other MCH class I–like products that may interact with other inhibitory NK receptors. Another receptor system targeted by MCMV is NKG2D and its ligands, retinoic acid–early inducible (RAE)-1, murine UL-16 binding protein (ULBP)–like transcript (MULT)-1, and H60. The expression of these ligands is either induced or upregulated during MCMV infection and the interaction of NKG2D with its ligands elicits cytotoxicity and IFN-γ production, therefore making NKG2D a crucial defense system against viral infection. However, MCMV dedicates at least three genes, m145 (downmodulation of MULT-1), m152 (downmodulation of RAE-1), and m155 (downmodulation of H60), to interfere with the expression of NKG2D ligands (Krmpotic et al. 2005). Although human cytomegalovirus (HCMV) and MCMV may share a common ancestor, they are different viruses, which display strict specificity for the host species. However, they seem to have been under similar selective pressure imposed by the host immune system, including NK cell functions. So, despite implementing different sets of genes, HCMV and MCMV use remarkably similar strategies aimed to escape NK cells. For example, the HCMV product UL16 downregulates human NKG2D ligands, such as ULBPs. Furthermore, the HCMV product UL40 selectively enhances nonclassic MHC class I HLA-E, which inhibits human NK cells. The capacity to quickly produce IFN-γ in large quantity makes NK cells important players in immune responses to bacterial and fungal infections also. Although it does not appear that NK cells directly recognize these microorganisms, they respond to inflammatory cytokines (IL-12, IL-15, IL-18, and IL-21) released in the site of infection by macrophages. IFN-γ secreted by NK cells activate macrophage effector functions, such as the production of antibacterial nitric oxide.
E.
Autoimmunity
Autoimmune diseases are generally caused by uncontrolled activation of B and T cells that leads to unwanted inflammatory responses, which culminate in tissue damage. Many reports have described altered NK cell repertoires associated with a number of human autoimmune diseases including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and type 1 diabetes (French and Yokoyama 2004). Experimental mouse models of autoimmune diseases support this notion. For example, NK-cell derived IFN-γ and TNF-α may exacerbate the inflammatory process of several autoimmune conditions. NK cells can also directly participate in the process of tissue damage. In vitro experiments have suggested
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that NK cells can kill neurons and insulin-producing pancreatic β cells. In vivo mouse models have suggested that the presence of NK cells in pancreatic infiltrates correlates with destructive autoimmunity and type 1 diabetes (Poirot et al. 2004). On the other hand, some studies suggested that NK cells can protect from autoimmune diseases. For example, depletion of NK cells renders mice susceptible to a murine model of multiple sclerosis induced by Theiler’s murine encephalitis virus. Similarly, depletion of NK cells caused a more aggressive pathological response in a mouse model of autoimmune colitis induced by adoptive transfer of T lymphocytes. These two experimental conditions suggest an immunoregulatory role for NK cells in autoimmune pathological changes. The mechanisms at play are not clear, but it is likely that the interactions between NK cells and DC may culminate in influencing pathogenic T cells. In addition, NK cells may also directly kill activated T cells and DCs.
F.
Reproduction
NK cells reside in the uterus and increase in numbers during pregnancy, eventually becoming the most abundant lymphocyte population at midgestation (Croy et al. 2003). The precise role of uterine NK (uNK) cells is not known, but IFN-γ produced by uNK cells is a key factor for the essential modifications that occur to the spiral arteries of the pregnant uterus. Consistent with this notion, mice lacking NK cells do not efficiently modify the architecture of spiral arteries. Thus, uNK cells may be important players in reproductive immunology. They may also modulate, in ways that are not yet understood, the fine balance that regulates maternofetal tolerance. Small, agranular NK cells are found in the uterus of sexually immature mice at 2 weeks of age. Contrary to NK cells purified from spleen and blood or those resident in other tissues (i.e., liver and lungs), both rodent and human uNK cells are noncytotoxic. In this respect, they resemble a subset of NK cells that are over-represented in human lymph nodes and are also found in small percentages in human blood but are not yet characterized in the mouse. This human NK cell subset seems to be specialized in cytokine production rather than cytotoxic activity. Incidentally, mouse lymph nodes are enriched in precursors for uNK cells. uNK cells remain small and agranular until implantation of the blastocyst and, by 5–7 days postcoitus, several changes occur. At this stage of pregnancy, uNK cells express perforin, produce IFN-γ, and proliferate. Precursors of uNK cells may arise in situ or may be joining the uterus from secondary lymphoid organs, most likely lymph nodes. The factors dictating uNK cell differentiation and activation are not fully characterized, although they may be similar to factors that regulate differentiation and activation of peripheral NK cells. For example, mice lacking IL-15 or any component of the IL-15 pathway fail to modify spiral arteries. Also, despite the crucial role of estrogen and progesterone, inflammatory
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cytokines IL-12 and IL-18 may regulate uNK cells like they regulate peripheral NK cells. uNK cells express both inhibitory and activating Ly49 receptors. In general, the effect of inhibitory receptors dominates over the activating receptors, preventing NK cell activation. Although this may be beneficial to prevent tissue damage caused by cytotoxicity, it can also be detrimental because it may prevent the production of IFN-γ. Inflammatory cytokines may be crucial, because they contribute essential positive inputs that help to overcome the downmodulatory effect of inhibitory receptors. In outbred populations, it is possible that the invading trophoblast cells also participate in activating uNK cells, since their MHC composition may be mismatched with the maternal uNK receptors and therefore escape inhibition (Hiby et al. 2004).
IV.
ACTIVATION
NK cells are poised to quickly produce inflammatory cytokines and to kill potential target cells. A fine balance of positive and negative intracellular signals tightly regulates such potent effector functions. The final outcome depends on the integration of these signals that are generated by inhibitory and activating receptors expressed on the cell surface.
A.
Inhibitory Receptors
Kärre and coworkers proposed that NK cells sense and attack cells that have lost or reduced the expression of self MHC class I molecules (Ljunggren and Kärre 1990). This theoretical framework led to the discovery of receptors on rodent and human NK cells that specifically bind to MHC class I and inhibit NK cell activation. Most NK cell receptors, inhibitory and activatory (sometimes globally indicated as NKRs), are found on two crucial gene clusters. One, called the NKC, is located on mouse chromosome 6; it encompasses crucial NK cell gene families, including Ly49 and NKG2/CD94. The other gene cluster, called leukocyte receptor complex (LRC), is located on mouse chromosome 7 and encompasses genes of the Ig superfamily that are expressed in multiple leukocyte lineages. The human LRC is located on chromosome 19 and it encompasses the KIR genes. The inhibitory receptors belong to three main families. KIRs are expressed by human NK cells whereas C-type lectin-like Ly49 receptors are expressed by mouse NK cells. The third family of C-type lectin-like receptors is composed of heterodimers CD94/NKG2, which are expressed by both human and mouse NK cells. In both species CD94/NKG2 receptors bind nonclassic MHC class I HLA-E in human and Qa1 in the mouse. These class I molecules present leader peptides from classic MHC class I molecules, and, therefore, NK cell recognition of HLA-E and Qa1 may represent a way of global MHC class I detection.
COLUCCI
Despite the differences in nature and structure, KIR, Ly49, and CD94/NKG2 receptors share similar signal transduction pathways. They have an intracellular tail containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Upon interaction of the inhibitory receptors with cognate ligands, ITIMs recruit intracellular phosphatases (PTPs) that have tandem Src-homology 2 (SH2) domains and that are known as SH2-containing phosphatases (SHP-1 and SHP-2). Activated PTPs dephosphorylate intracellular kinases, leading to inhibition of NK cell activation. The specific targets of these PTPs are still elusive; however, the best candidates are 1) the guanine exchange factor VAV, which activates the small GTPases of the RHO/RAC-family and 2) phospholipase C-γ (PLC-γ), which triggers the rise in intracellular calcium concentration and the activation of protein kinase C pathways.
B.
Activating Receptors
Molecules expressed on infected, malignant, and also normal cells can trigger NK cell activation by binding to one of the many NK cell–activating receptors (Colucci et al. 2002). Most activating receptors lack intrinsic signaling domains and enzymatic activity. The transmembrane portion of activating receptors associates with small adapters possessing intracellular signaling domains. This association is crucial to achieve the activation of signal transduction pathways. The adapters have a very short extracellular domain and do not participate in the binding to cognate ligands but are essential for the stabilization of the receptors in the plasma membrane and for recruitment and activation of downstream signaling components. NK cell receptors can associate with four known adapters. Three of these adapters, FcεRIγ, CD3ζ, and DNAX adapter protein (DAP)-12, contain ITAMs that activate signal transduction pathways, which are very similar to those activated by antigen receptors on B and T cells and by FcR on many leukocytes. Engagement of receptors associated to ITAM-containing adapters initiates a sequence of intracellular events, including activation of protein tyrosine kinases (PTKs) of the SRC family (such as LCK, FYN, YES, and LYN), followed by the recruitment and activation of SYK family PTKs (ZAP-70 and SYK). This sequence of events leads to the activation of downstream regulators such as adaptor molecules (LAT, SLP-76, and 3BP2), lipid enzymes including PLC-γ and phosphatidylinositol 3-kinase (PI3K), VAV family guanine nucleotide exchange factors, and RHO-RAC low-molecular-weight GTP-binding proteins and their effectors. By contrast, the fourth adaptor, DAP-10, does not contain ITAMs, but possesses instead a motif that initiates an alternative signal transduction pathway that resembles the pathway activated by costimulatory CD28 receptor on T cells. This pathway, through PI3K and possibly other signaling components, activates critical downstream regulators (VAV and PLC-γ), without the need for an SYK-dependent activation step.
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The best-characterized activating receptor on NK cells is the multisubunit immune recognition complex CD16, also called FcγRIIIA. CD16 is a low-affinity receptor for IgG that mediates ADCC. It couples with homodimers of FcεRIγ in mouse NK cells, whereas in human NK cells it couples with hetero- or homodimers of CD3ζ and FcεRIγ. Despite being one of the first identified activating receptors after CD16, NKR-P1C (also known as NK1.1; see above) is still an orphan receptor. This C-type lectin-like receptor couples with FcεRIγ, but its function is unknown. NKR-P1C belongs to a family that include four members. Two family members, the inhibitory NKR-P1D and the activating NKRP1F, both bind to C-type lectin-like glycoproteins encoded by genes within the mouse NKC. NKR-P1D is expressed on most NK cells in some strains of mice, and it recognizes a C-type lectin-like glycoprotein (Clr-b), which is expressed on hematopoietic cells. When a tumor cell line susceptible to NK cell lysis was induced to ectopically express Clr-b, susceptibility was reversed. This suggests that members of the NKR-P1 family may regulate NK cell functions by recognizing non-MHC ligands. A large group of activating receptors couple with DAP-12. These include, among others, 1) Ly49D, which binds to MHC class I, 2) Ly49H, whose only identified ligand is the MCMV product m157, and 3) NKG2C and NKG2E, which both bind to nonclassic MHC class I Qa1b. Ly49D, expressed on about half of the mouse NK cells of the C57BL/6 mouse strain, recognizes H-2Dd and plays a role in the rejection of allogeneic BALB/c bone marrow cells. The functional human homologs KIR2DS and KIR3DS may function in similar ways and have been implicated in the rejection of leukemic cells in an allogeneic bone marrow transplantation setting. Therefore, recognition of MHC class I molecules by KIRs and Ly49s appears to be a major mechanism in NK cell immunity to allogeneic hematopoietic cells. The presence in the same individual of activating and inhibitory receptors that bind to the same ligand poses a dilemma as to how NK cells can avoid autoimmunity when this ligand is expressed on normal cells (as it is the case for MHC class I). In general, each single NK cell expresses both the inhibitory and the activating receptor and the affinity of the inhibitory receptors is higher. This differential affinity is believed to prevent unwanted activation. A group of receptors identified in human NK cells and called natural cytotoxicity receptors (NCRs) includes NKp30, NKp44, and NKp46. NKp44 couples with DAP-12, whereas NKp30 and NKp46 interact with hetero- or homodimers of CD3ζ and FcεRIγ. Except for the evidence that NKp44 and NKp46 bind to the hemagglutinins of influenza and Sendai virus in vitro, no other ligands have been identified for these receptors. Among the NCRs, only a murine homolog of NKp46 has been described, called murine activating receptor-1 (MAR-1) or Ncr1. The activating receptor NKG2D is a C-type lectin-like receptor expressed on most mouse and human NK cells and on a subset of T cells. NKG2D is only distantly related to the NKG2 family.
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Contrary to the other members of the NKG2 family, NKG2D does not couple to CD94 but is expressed as a homodimer. In the mouse there are two variants of NKG2D, generated by alternative splicing. The long variant, NKG2D-L, is constitutively expressed. NKG2D-L couples with DAP-10, an adaptor molecule that contains a motif in its cytoplasmic tail that can recruit PI3K and growth factor receptor-bound protein-2 (GRB-2) upon phosphorylation. The short variant, NKG2D-S, is mostly expressed by activated NK cells and it can couple with both DAP-10 and DAP12. Human NK cells do not generate NKG2D-S. NKG2D can recognize multiple ligands that generally have a structural similarity to MHC class I and are induced upon stress, infection, and tumor transformation. Mouse NKG2D ligands include RAE-1, MULT-1, and H60. Human NKG2D ligands include MHC class I chain-related molecules (MIC-A and MIC-B), ULBPs, and RAE-1-like transcripts (RAET)-1E and 1G. Thus, NKG2D may play a pivotal role in immune surveillance of tumors and immune defense against viral infections. The 2B4 receptor (also known as CD244) is an Ig-like glycoprotein that belongs to the CD2 family and binds to CD48. 2B4 was initially described as an activating receptor on NK cells and on a subpopulation of T cells. However, 2B4 is wired to two opposing signal transduction pathways that may activate or inhibit NK cells. The intracellular tail of 2B4 contains a signaling motif called immunoreceptor tyrosine-based switch motif (ITSM). Upon binding to the relevant ligand (CD48), ITSM can recruit the signaling lymphocyte activation molecule-associated protein (SAP), which mediates activation of NK cells. However, the ITSM motif can also recruit SH2domain containing PTPs, thereby initiating inhibitory pathways. The outcome may depend on the local signaling context of the lipid rafts. Based on the switching property of the ITSM motif, the simultaneous engagement of other receptors and the activation of other signaling pathways may decide whether 2B4 will be activating or inhibitory. The importance of 2B4 in human is highlighted by a loss-of-function mutation of SAP that leads to a severe inherited immunodeficiency called X-linked lymphoproliferative disease (XLP). This disease is aggravated by EBV infection. NK cells may play a role in the early phase of EBV infection; thus, it is possible that altered signaling by ITSM-containing receptors, including 2B4, may contribute to the pathogenesis of XLP. Recently, 2B4-FYNSAP has been shown to be an ITAM-independent pathway that activates natural cytotoxicity (Bloch-Queyrat et al. 2005).
V.
A DYNAMIC VIEW OF NK CELL ACTIVATION
The encounter of an NK cell with a target cell is mediated in the first instance by integrins and adhesion molecules. Thus, a stable conjugate is formed that leads to the organization of an immune synapse. Further to their indispensable role in forming
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the cell conjugate, integrin and adhesion molecule interactions can also trigger signaling events. For example, interactions between the β2 integrin lymphocyte function–associated antigen-1 (LFA-1) on NK cells and the adhesion molecule intracellular adhesion molecule-1 (ICAM-1) on target cells can activate the guanine exchange factors of the VAV family, which in turn activate small RHO/RAC GTPases that rearrange the actin cytoskeleton of NK cells. This early signaling event may contribute to the recruitment of NK cell receptors into lipid rafts. When the signals from inhibitory receptors do not stop this process, the formation of the immune synapse goes on, and more receptor-ligand interactions can be established between the NK cell and the target. These early events are wired to multiple downstream signaling pathways, which revolve around tyrosine phosphorylation. Whether part of the pathways initiated by ITAM-bearing receptors (such as CD16, NKG2D-S, Ly49D, and Ly49H) or alternative pathways (such as NKG2DL-DAP10-PI3K or 2B4FYN-SAP), proximal tyrosine kinases phosphorylate VAV, PLC-γ, and extracellular regulated kinases. The activation of these key signaling components induces reorganization of the cytoskeleton, polarization of cytotoxic granules, and elevation of intracellular calcium concentration, which culminate in exocytosis of cytotoxic granules and cytokine/chemokine gene transcription. Despite sharing a number of key signaling components and activation pathways, cytokine production and cytotoxicity are independently regulated. Research based on the use of genetargeted mice lacking specific signal transduction proteins is contributing to define the minimal requirements for the two main NK cell effector functions (Colucci et al. 2002). Unlike the specific activation that follows antigen receptor recognition by B and T lymphocytes, the signal transduction pathways that activate NK cells are multiple in number and possibly redundant in nature. These features confer robustness and flexibility to cellular innate immunity, which can be activated by and respond to a vast array of challenges in a quick and effective way, while the organism builds the specific adaptive immune response.
VI.
SUMMARY
NK cells are armed with an array of surface receptors that enable them to detect and swiftly destroy malignant and infected cells in the body. Beside their ability to kill targets without prior immunization, NK cells can also deliver chemokines as well as antimicrobial and proinflammatory cytokines that activate macrophages and dendritic cells and guide lymphocytes to mount specific and long-lasting immunity. Cell-mediated cytotoxicity and production of cytokines are the two main effector functions of NK cells. A fine balance between opposing forces generated by activating and inhibitory
COLUCCI
receptors tightly regulates NK cell activation. These receptors dictate the roles that NK cells play in various phases of innate and adaptive immune responses, including tumor surveillance, control of metastatic spread, antiviral immunity and natural immunity to hematopoietic cells. NK cells play key roles in generating the crucial changes of vasculature that take place in the uterus during pregnancy. Moreover, NK cells have been implicated in modulating various autoimmune diseases. Therefore, NK cells carry out central functions in immunity in health and disease. The mouse represents an excellent model to study the function and activation of NK cells in the whole animal. REFERENCES Arase, H., Mocarski, E.S., Campbell, A.E., Hill, A.B., Lanier, L.L. (2002). Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 296, 1323–1326. Biron, C.A., Byron, K.S., Sullivan, J.L. (1989). Severe herpesvirus infections in an adolescent without natural killer cells. N Engl J Med 320, 1731–1735. Bloch-Queyrat, C., Fondaneche, M.C., Chen, R., Yin, L., Relouzat, F., Veillette, A., et al. (2005). Regulation of natural cytotoxicity by the adaptor SAP and the Src-related kinase Fyn. J Exp Med 202, 181–192. Brown, M.G., Dokun, A.O., Heusel, J.W., Smith, H.R., Beckman, D.L., Blattenberger, E.A., et al. (2001). Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science 292, 934–937. Bukowski, J.F., Warner, J.F., Dennert, G., Welsh, R.M., Kalwak, K., Gorczynska, E., et al. (1985). Adoptive transfer studies demonstrating the antiviral effect of natural killer cells in vivo. J Exp Med 161, 40–52. Colonna, M., Samaridis, J. (1995). Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 268, 405–408. Colucci, F., Caligiuri, M.A., Di Santo, J.P. (2003). What does it take to make a natural killer? Nat Rev Immunol 3, 413–425. Colucci, F., Di Santo, J.P., Leibson, P.J. (2002). Natural killer cell activation in mice and men: different triggers for similar weapons? Nat Immunol 3, 807–813. Croy, B.A., Esadeg, S., Chantakru, S., van den Heuvel, M., Paffaro, V.A., He, H., et al. (2003). Update on pathways regulating the activation of uterine natural killer cells, their interactions with decidual spiral arteries and homing of their precursors to the uterus. J Reprod Immunol 59, 175–191. Cudkowicz, G., Stimpfling, J.H. (1964). Deficient growth of C57BL marrow cells transplanted in F1 hybrid mice. Association with the histocompatibility-2 locus. Immunology 19, 291–306. Daniels, K.A., Devora, G., Lai, W.C., O’Donnell, C.L., Bennett, M., Welsh, R.M. (2001). Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H. J Exp Med 194, 29–44. French, A.R., Yokoyama, W.M. (2004). Natural killer cells and autoimmunity. Arthritis Res Ther 6, 8–14. Herberman, R.B., Nunn, M.E., Lavrin, D.H. (1975). Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic acid allogeneic tumors. I. Distribution of reactivity and specificity. Int J Cancer 16, 216–229. Hiby, S.E., Walker, J.J., O’Shaughnessy K.M., Redman, C.W., Carrington, M., Trowsdale, J., Moffett, A. (2004). Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J Exp Med 200, 957–965. Karlhofer, F.M., Ribaudo, R.K., Yokoyama, W.M. (1992). MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 6381, 66–70. Kärre, K., Ljunggren, H.G., Piontek, G., Kiessling, R. (1986). Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319, 675–678.
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Kiessling, R., Klein, E., Wigzell, H. (1975). “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells: specificity and distribution according to genotype. Eur J Immunol 5, 117–121. Krmpotic, A., Hasan, M., Loewendorf, A., Saulig, T., Halenius, A., Lenac, T., et al (2005). NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145. J Exp Med 201, 211–220. Lee, S.H., Girard, S., Macina, D., Busa, M., Zafer, A., Belouchi, A., et al. (2001). Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily. Nat Genet 28, 42–45. Ljunggren, H.G., Karre, K. (1990). In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol Today 11, 237–244. Ortaldo, J.R., Oldham, R.K., Cannon, G.C., Herberman, R.B. (1977). Specificity of natural cytotoxic reactivity of normal human lymphocytes against a myeloid leukemia cell line. J Natl Cancer Inst 59, 77–82.
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Poirot, L., Benoist, C., Mathis, D. (2004). Natural killer cells distinguish innocuous and destructive forms of pancreatic islet autoimmunity. Proc Natl Acad Sci USA 101, 8102–8107. Raulet, D.H. (2003). Roles of the NKG2D immunoreceptor and its ligands. Nat Rev Immunol 3, 781–790. Scalzo, A.A., Fitzgerald, N.A., Wallace, C.R., Gibbons, A.E., Smart, Y.C., Burton, R.C., et al. (1992). The effect of the Cmv-1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells. J Immunol 149, 581–589. Smith, H.R., Heusel, J.W., Mehta, I.K., Kim, S., Dorner, B.G., Naidenko, O.V., et al. (2002). Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci USA 99, 8826–8831. Smyth, M.J., Wallace, M.E., Nutt, S.L., Yagita, H., Godfrey, D.I., Hayakawa. Y. (2005). Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer. J Exp Med. 2005 201, 1973–1985. Yokoyama, W.M., Kim, S., French, A.R. (2004). The dynamic life of natural killer cells. Annu Rev Immunol 22, 405–429.
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Chapter 7 Cytokine-Activated JAK-STAT Signaling in the Mouse Immune System Bin Liu and Ke Shuai
I. II.
III.
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cytokine-Activated JAK-STAT Pathway . . . . . . . . . . . . . . . . . . . . . . . . B. Regulation of the JAK-STAT Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . 1. SOCSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. PIASs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. PTPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vivo Functions of JAK-STAT Signaling in the Mouse Immune System . A. JAKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Jak1-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Jak2-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Jak3-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Tyk2-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. STATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Stat1-Deficient and Stat1S727A Mutant Mice . . . . . . . . . . . . . . . . . 2. Stat2-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Stat3-Deficient and Stat3S727A Mutant Mice . . . . . . . . . . . . . . . . . 4. Stat4-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Stat5a- and Stat5b-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Stat6-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. SOCSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Socs1-Deficient and Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . 2. Socs3-Deficient and Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . 3. Cis Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Socs5 Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Socs2-Deficient and Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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D. PIASs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Pias1-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Piasy-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Piasx-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. PTPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
PREFACE
Genetic approaches using targeted gene disruption and transgenic mouse models have proven to be extremely powerful in the field of immune regulation. Genetic analysis can validate the results from biochemical studies and can address the physiological functions of proteins in the immune system at the whole animal level. Furthermore, genetic studies can reveal signaling specificities in vivo and they often lead to the discovery of unexpected gene functions. The cytokine-activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways have been extensively studied because of their important roles in immune regulation. The JAK-STAT pathway is regulated at multiple levels by various proteins, including suppressors of cytokine signaling (SOCSs), protein inhibitor of activated STATs (PIASs), and protein tyrosine phosphatases (PTPs). In this chapter, we review important findings using various genetic mouse models to study the key components as well as several regulators of the JAK-STAT pathways in the immune system.
II.
INTRODUCTION
Cytokines play essential roles in the regulation of immune responses. Cytokines exert their biological functions by binding to the cell surface receptors to activate various signaling pathways, resulting in the activation or repression of downstream genes.
A. Cytokine-Activated JAK-STAT Pathway The JAK-STAT pathway is one of the major signaling pathways activated by a variety of cytokines and growth factors (Igaz et al. 2001; Imada and Leonard 2000; Leonard and Spolski 2005; Levy and Darnell 2002) (Table 7-1). The binding of a cytokine to its receptor results in receptor dimerization and the subsequent activation of receptor-associated JAK kinases. Specific tyrosine residues on the receptor are then phosphorylated by activated JAKs and serve as docking sites for a family of latent cytoplasmic transcription factors termed STATs. STATs are phosphorylated by JAKs, then dimerize, and subsequently
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translocate to the nucleus, where they bind directly to DNA and mediate transcription (Fig. 7-1A). Four mammalian JAK proteins have been identified: JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase 2) (Aringer et al. 1999; Shuai and Liu 2003; Stark et al. 1998) (Fig. 7-1B). Each contains a conserved kinase domain (JH1) and a related, but catalytically inactivate, pseudo-kinase domain (JH2) at the carboxyl terminus (Fig. 7-1B). The pseudo-kinase domain may play a regulatory role in the activation of JAKs. The N-terminal domains JH3–JH7 are based on sequence similarities among JAK members. JH6 and JH7 have been implicated in the binding of JAKs to the receptors, whereas the functions of other regions, such as JH3 to JH5, remain to be elucidated. There are seven mammalian STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 (Darnell 1997;
TABLE 7-1
LIGAND-ACTIVATED JAK-STAT SIGNALING PATHWAYS Receptors
Ligands
JAKs
STATs
Heterodimeric
IFN-α/β IFN-γ IL-10 IL-2, IL-7, IL-9, IL-15 IL-21 IL-4 IL-13
JAK1, TYK2 JAK1, JAK2 JAK1, TYK2 JAK1, JAK3
STAT1, 2, 3, 5 STAT1, 3, 5 STAT1, 3 STAT5, 3, 1
JAK1, JAK3 JAK1, JAK3 JAK1, JAK2, TYK2 JAK2
STAT1, 3, 5 STAT6 STAT6
JAK1, JAK2, TYK2
STAT3, 1, 5
JAK2, TYK2 JAK2
STAT4 STAT5
JAK1, JAK2, TYK2 JAK1, JAK2, JAK3
STAT1, 3, 5
γ-C family
β-C family
IL-3, IL-5, GM-CSF gp-130 family IL-6, IL-11, OSM, CNTF, LIF, CT-1 IL-12 Homodimeric GH, PRL, EPO, TPO RTKs EGF, PDGF, CSF-1 G-protein-coupled Chemokines receptors SDF1-α, RANTES, MCP-1, MIP-1α Angiotensin-II
JAK2, TYK2
STAT5
STAT1, 3, 5
STAT1, 2
Reviewed in Igaz et al. 2001; Imada and Leonard, 2000; Leonard and Spolski, 2005; Levy and Darnell, 2002.
7. C Y T O K I N E - A C T I VAT E D
JAK-STAT
SIGNALING
JAK
B.
Transcription
JH7
JH6
JH5 JH4 JH3
Pseudo-kinase domain
Kinase domain
JH2
JH1
B
C
C
pY pY N-terminal domain STAT N
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Regulation of the JAK-STAT Pathway
The proper regulation of the JAK-STAT pathway is crucial for its biological functions. Dysregulated JAK-STAT signaling has been documented in various immune diseases. Recent studies have demonstrated that JAK-STAT signaling is regulated at multiple levels in the cytoplasm as well as the nucleus through distinct mechanisms. Key regulators include SOCSs, PIASs, and PTPs (Alexander 2002; Alexander and Hilton 2004; Shuai 2000; Shuai and Liu 2003) (Fig. 7-2A).
STAT
JAK N
MOUSE
SH2-pY
Y
A
THE
STAT tetramers and tyrosine dephosphorylation (Shuai et al. 1996; Xu et al. 1996).
Cytokine
JAK
IN
Coiled-coil DNA-binding Linker domain domain domain
SH2 Transactivation domain domain C pY pS
Fig. 7-1 The JAK-STAT pathway. A, A schematic representation of the JAK-STAT pathway. The activation of JAKs upon cytokine stimulation results in the phosphorylation of STATs, which then dimerize and translocate into the nucleus to regulate transcription. Y, tyrosine; SH2-pY, the interaction between SH2 domain and phosphotyrosine. B, The domain structure of JAK kinases. The domains JH1–7 are regions based on sequence similarity in four known JAKs. JH1 is the kinase domain, which contains two tyrosines that can be phosphorylated (pY) upon ligand stimulation. JH2 is the pseudo-kinase domain. C, The domain structure of STATs. STATs contain an SH2 domain, a DNAbinding domain, and a carboxyl-terminal transactivation domain (TAD). The activity of STATs can be regulated by protein modification, including tyrosine (pY) and serine phosphorylation (pS).
Levy and Darnell, 2002; Shuai 1999). The splice variants of STAT1 and STAT3 have also been identified. STAT proteins share several conserved regions, including a Src homology 2 (SH2) domain (Fu and Zhang 1993; Shuai et al. 1994), a DNAbinding domain (Horvath et al. 1995), and a transcription activation domain (TAD) (Muller et al. 1993; Shuai et al. 1993) (Fig. 7-1C). Each STAT contains a conserved tyrosine residue in the carboxyl-terminal region, which becomes phosphorylated upon cytokine stimulation (Schindler et al. 1992; Shuai et al. 1992). The interaction between intermolecular phosphotyrosine and the SH2 domain mediates STAT dimerization, which is required for subsequent nuclear translocation and DNA binding. Thus, tyrosine phosphorylation serves as a switching signal to activate STATs. STAT1, STAT3, STAT4, STAT5A, and STAT5B have also been shown to be modified by serine phosphorylation, which appears to be independent of the tyrosine phosphorylation and is thought to be required for the maximum transcriptional activity of STATs (Wen and Darnell 1997; Wen et al. 1995). The amino-terminal region of STATs is involved in regulating STAT activity, such as the formation of
1.
SOCSs
The SOCS family is composed of eight members: CIS (cytokine-inducible Src homology domain 2 protein) and SOCS1–SOCS7 (Greenhalgh and Hilton 2001; Hilton et al. 1998; Kile and Alexander 2001). All SOCS proteins contain an SH2 domain flanked by a variable amino-terminal domain and a conserved carboxyl-terminal SOCS box region (Kile et al. 2002) (Fig. 7-2B). SOCS proteins are generally expressed at low levels in unstimulated cells. Upon cytokine stimulation, SOCS proteins are rapidly induced and function as a classic negative feedback loop to inhibit cytokine signaling. Recent studies have demonstrated that SOCS proteins inhibit JAK-STAT signaling via distinct mechanisms. For example, SOCS1 directly inhibits the kinase activity of JAKs by binding to the tyrosine phosphorylated JAKs via its SH2 domain (Endo et al. 1997; Naka et al. 1997; Starr et al. 1997). In contrast, the inhibition of JAKs by SOCS3 requires binding of SOCS3 to the activated receptor (Nicholson et al. 1999; Sasaki et al. 2000). Instead of acting on JAKs, CIS appears to inhibit STAT activation by competing with STATs for binding to the receptor docking sites (Yoshimura 1998). Finally, SOCS proteins have been implicated in targeting signaling components for ubiquitin-proteasome-mediated degradation. It has been shown that the SOCS box can bind to elongins B and C, which are known components of the ubiquitin E3 ligase complex (Kamura et al. 1998; Zhang et al. 1999). 2.
PIASs
The PIAS proteins are specific inhibitors for activated STATs that possess SUMO E3 ligase activity (Shuai and Liu 2003). Four mammalian PIAS members have been identified: PIAS1, PIAS3, PIASx, and PIASy (Arora et al. 2003; Chung et al. 1997; Liu et al. 1998, 2001). Each PIAS, except for PIAS1, has two splice variants. Several conserved domains have been identified among PIAS members (Fig. 7-2C). All PIAS proteins contain a C3HC4 type RING-finger-like zinc-binding domain (RLD) required for their SUMO E3 ligase activity. In addition,
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SOCS
JAK
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JAK
Ub SH2-pY
Y
JAK PTP
STAT
Degradation
PTP PIAS
A
Transcription
N-terminal region
SH2 domain
SOCS box
SOCS N
C
B SAP/LXXLL PIAS N
PINIT
RLD
AD/SIM
S/T C
C Fig. 7-2 Negative regulation of the JAK-STAT pathway. A, The JAK-STAT pathway is regulated at multiple levels. JAKs can be negatively regulated by SOCS proteins, PTPs, and ubiquitin-mediated protein degradation. SOCS proteins, which are induced by cytokines, act as a negative feedback loop to switch off the activity of JAKs. Multiple PTPs participate in the regulation of JAKs. The regulation of JAK2 by ubiquitination has been suggested. STATs can be negatively regulated by PTPs in the cytoplasm and by PIAS proteins as well as PTPs in the nucleus. PIAS proteins interact with STATs in response to cytokine stimulation and inhibit the transcriptional activity of STATs through distinct mechanisms. B, Domain structure of SOCS proteins. The SOCS family has eight members: CIS and SOCS1–SOCS7. SOCS proteins contain an SH2 domain flanked by a variable amino-terminal domain and a carboxyl-terminal SOCS box. The SOCS box can bind to elongins B and C, which are known components of a ubiquitin E3 ligase complex. C, Domain structure of PIAS proteins. Four mammalian PIAS members have been identified: PIAS1, PIAS3, PIASx, and PIASy. SAP/LXXLL: SAF-A/B, Acinus, and PIAS domain. Within the SAP domain, there is a conserved LXXLL signature motif. The PINIT motif is present in all PIAS proteins except the PIASyE6 variant. RLD: RING finger-like zinc binding domain. AD/SIM: acidic domain. Within the AD, a SIM is found in all PIAS proteins except PIASy. S/T: serine-threonine-rich region. The S/T domain is absent in PIASy.
a putative domain named SAP (SAF-A/B, Acinus, and PIAS) is localized at the amino terminus of PIAS, which is implicated in binding to scaffold/matrix attachment regions of the chromatin (Aravind and Koonin 2000). The LXXLL signature motif is located within the SAP domain, which is known to mediate interactions between nuclear receptors and their coregulators. The PINIT motif located within a highly conserved region of PIAS proteins is present in all PIAS members except PIASyE6−, a splice variant of PIASy. The PINIT motif may be involved in the nuclear retention of PIAS. The carboxyl-terminal regions of
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PIAS proteins, which contain a highly acidic region (AD) and a serine/threonine rich (S/T) region, are the most diversified. A putative SUMO1 interaction motif (SIM) is present in the AD of all PIASs except PIASy, which also lacks the S/T region. The function of AD, S/T, or SIM of PIAS proteins has not been defined. PIAS proteins reside in the nucleus in most cases, and they do not interact with STATs in unstimulated cells. Upon cytokine stimulation, PIASs become associated with phosphorylated STATs. In vivo co-immunoprecipitation studies using specific antibodies against PIAS proteins have identified specific PIAS-STAT interactions (Arora et al. 2003; Chung et al. 1997; Liu et al. 1998, 2001). PIAS1, PIAS3, and PIASx interact with STAT1, STAT3, and STAT4, respectively, in response to cytokines. In addition, PIASy also binds to activated STAT1. The cytokine dependency of the PIAS-STAT interaction may be explained by the finding that PIAS1 binds to the dimeric, but not the monomeric form of STAT1 (Liao et al. 2000). Each member of the PIAS family has been shown to inhibit STAT-mediated gene activation, however, via distinct mechanisms. PIAS1 and PIAS3 can inhibit the DNA binding activity of STAT1 and STAT3, respectively. In contrast, PIASx and PIASy inhibit STAT4- and STAT1-dependent transcription without affecting their DNA binding activities. It is likely that PIASx and PIASy act as transcriptional co-repressors of STATs, possibly by recruiting HDACs. Lastly, whether the SUMO ligase activity of PIAS proteins is involved in the regulation of STAT signaling is controversial. 3.
PTPs
The JAK-STAT signaling pathway is regulated by PTPs in both cytoplasm and nucleus. Several PTPs have been suggested to regulate JAKs, including SHP-1, SHP-2, CD45, PTP1B, and TC-PTP (T-cell protein tyrosine phosphatase) (Neel and Tonks 1997; Shuai and Liu 2003). SHP-1 and SHP-2 are SH2 domain–containing PTPs (Neel 1993). SHP-1 is predominantly expressed in hematopoietic cells and has been shown to physically interact with the interleukin (IL)-3 receptor β chain, the c-Kit receptor, and the erythropoietin (EPO) receptor. SHP-1 has been implicated in the dephosphorylation of JAK1 and JAK2, whereas genetic studies suggest that SHP-2 is involved in the negative regulation of JAK1 (David et al. 1995; Klingmuller et al. 1995; You et al. 1999). CD45 is a receptor PTP highly expressed in hematopoietic cells and has a critical role in antigen receptor signaling in T and B cells (Penninger et al. 2001). CD45 can directly bind and dephosphorylate all JAKs and enhanced JAK phosphorylation is observed in Cd45null cells (Irie-Sasaki et al. 2001). PTP1B and TC-PTP, two highly related PTPases, have also been suggested to dephosphorylate JAKs. JAK2 and TYK2, but not JAK1, can serve as substrates of PTP1B, and elevated JAK2 phosphorylation has been observed in Ptp1b-null mouse embryonic fibroblasts (MEFs) (Myers et al. 2001). TC-PTP can dephosphorylate
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JAK1 and JAK3 and in Tc-ptp-null macrophages, an enhancement of interferon (IFN)-γ-induced tyrosine phosphorylation of JAK1, but not JAK2, is observed (Simoncic et al. 2002). Thus, multiple PTPs participate in the dephosphorylation of JAKs. STATs are also regulated by PTPs in both the cytoplasm and nucleus. For example, SHP-2 interacts with STAT5 and can directly dephosphorylate STAT5 in the cytoplasm, and dephosphorylation of STAT5 is inhibited in Shp-2-null cells (Chen et al. 2003; Chughtai et al. 2002). PTP1B has also been implicated in the dephosphorylation of STAT5 under overexpression conditions (Aoki and Matsuda 2000). However, whether STAT5 is a physiological substrate of PTP1B remains to be established. Through biochemical purification, TC45, the nuclear isoform of TC-PTP, has been identified as a nuclear PTP for STAT1 (ten Hoeve et al. 2002). TC45 can directly dephosphorylate STAT1, and in Tc-ptp-null cells, the nuclear dephosphorylation of STAT1 is defective. In addition to TC45, SHP-2 is also involved in the nuclear dephosphorylation of STAT1. However, the identities of the PTPs for other STATs remain unknown.
III.
IN VIVO FUNCTIONS OF JAK-STAT SIGNALING IN THE MOUSE IMMUNE SYSTEM
Genetic studies with targeted disruption of the key components of the JAK-STAT pathway have provided invaluable information on the in vivo functions of JAK-STAT signaling in the immune system. Studies with targeted deletions and transgenic mouse models of the JAK-STAT regulators, such as SOCS and PIAS proteins, further demonstrate the importance of the precise regulation of JAK-STAT signaling in immune functions. These mouse model systems provide insights regarding the in vivo specificity and the physiological relevance of each player of JAK-STAT signaling. In the following section we summarize the
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phenotypes of the mice carrying genetic alterations of the key components and regulators of the JAK-STAT pathway. A. 1.
JAKs
Jak1-Deficient Mice
Jak1-deficient mice died perinatally with profound defects in lymphoid development and neurogenesis (Rodig et al. 1998) (Table 7-2). They were small at birth and failed to nurse. Studies with Jak1-deficient cells showed that these cells fail to respond to three distinct families of cytokines, including IFNs, γ-C-dependent cytokines, such as IL-2, IL-4, IL-7, IL-9, and IL15, and gp130-dependent cytokines, including IL-6, IL-11, ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), oncostatin M (OSM), and leukemia inhibitory factor (LIF), indicating that JAK1 is essential for signaling by these cytokines. 2.
Jak2-Deficient Mice
Jak2 deficiency in mice resulted in embryonic lethality due to the absence of erythropoiesis (Neubauer et al. 1998; Parganas et al. 1998) (Table 7-2). Jak2-deficient cells failed to respond to EPO, thrombopoietin (TPO), IL-3, granulocytemacrophage colony-stimulating factor (GM-CSF), and IFN-γ, whereas the responses to granulocyte colony-stimulation factor (G-CSF), IL-6, and IFN-α/β are normal. These studies established the crucial role of JAK2 in erythropoiesis. 3.
Jak3-Deficient Mice
Although JAK1 and JAK2 are more ubiquitously expressed, JAK3 is primarily expressed in hematopoietic cell lineages. Jak3-null mice were viable and fertile but developed severe combined immune deficiency (SCID) that affects both B and T cell functions (Nosaka et al. 1995; Park et al. 1995; Thomis et al. 1995) (see Table 7-2). JAK3 is associated with the common cytokine receptor γ chain, which is shared by the receptors for
TABLE 7-2
IN VIVO FUNCTIONS OF JAKS JAK
Phenotypes of Null Mice
Cytokine Pathways Affected
References
Jak1
Perinatal lethality Small at birth, no nursing Impaired lymphopoiesis Defective neurogenesis
Rodig et al. 1998
Jak2
Embryonic lethality No erythropoiesis SCID
IFNs γ-C dependent: IL-2, IL-4, IL-7, IL-9, IL-15 gp-130 dependent: IL-6, IL-11, CNTF, CT-1, OSM, LIF EPO, TPO, IL-3, GM-CSF, IFN-γ
Jak3 Tyk2
Hypersensitivity to pathogens Resistance to LPS-induced shock
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γ-C dependent: IL-2, IL-4, IL-7, IL-9, IL-15 IFNs and IL-12
Neubauer et al. 1998; Parganas et al. 1998 Nosaka et al. 1995; Park et al. 1995; Thomis et al. 1995 Karaghiosoff et al. 2003; Shimoda et al. 2000
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IL-2, IL-4, IL-7, IL-9, and IL-15. The phenotypes of the γ chain–deficient mice are indistinguishable from those of Jak3null mice, indicating that JAK3 plays an essential role in γ chain–dependent lymphoid development (Cao et al. 1995; DiSanto et al. 1995). 4.
Tyk2-Deficient Mice
Tyk2-deficient mice were developmentally normal but exhibited hypersensitivity to viral and bacterial challenges, consistent with the essential role of TYK2 in IFN-α/β signaling (Karaghiosoff et al. 2000) (see Table 7-2). Tyk2-null cells were also defective in IL-12 signaling (Shimoda et al. 2000). Interestingly, Tyk2-null mice were resistant to lipopolysaccharide (LPS)-induced endotoxic shock, which was associated with impaired production of IFNs upon LPS challenge, but not proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-6 (Karaghiosoff et al. 2003). These data indicate that TYK2 and IFNs are essential effectors in LPS-induced toxicity.
B. 1.
STATs
Stat1-Deficient and Stat1S727A Mutant Mice
Biochemical studies have shown that STAT1 can be activated by a variety of cytokines and growth factors, including IFNs, IL-6, IL-10, growth hormone (GH) and epidermal growth factor (EGF) (see Table 7-1). Genetic studies with Stat1-deficient mice revealed that STAT1 plays a specific and essential role in IFN signaling, whereas other signaling pathways were intact in the absence of STAT1. Stat1-null mice were developmentally normal, but hypersensitive to viral and microbial infections, consistent with an essential role of STAT1 in IFN signaling (Durbin et al. 1996; Meraz et al. 1996) (Table 7-3). Stat1S727A mutant mice have recently been generated, in which the serine phosphorylation site of STAT1, Ser-727, was substituted with alanine (Varinou et al. 2003). The Stat1S727A mice showed hypersensitivity to pathogens and resistance to LPS-induced shock but not as severe as that for Stat1-null mice. Studies with Stat1S727A macrophages revealed reduced activation of certain IFNγ-dependent genes compared with that in wild-type cells, supporting the notion that serine phosphorylation is important for the full transcriptional activity of STAT1.
2.
Stat2-Deficient Mice
Stat2-deficient mice showed increased susceptibility to viral infections, but otherwise were viable and developmentally normal (Park et al. 2000) (Table 7-3). Stat2-null cells were specifically defective in IFN-α/β signaling, but maintain a normal response to IFNγ. These results indicate that STAT2 is required specifically for IFN-α/β signaling.
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Stat3-Deficient and Stat3S727A Mutant Mice
Stat3-null mice were embryonic lethal, suggesting that STAT3 is essential for mouse development (Takeda et al. 1997) (Table 7-3). Recent studies using the Cre-loxP system to generate tissue-specific Stat3-null mice have revealed specific roles of STAT3 in various tissues. Unlike other members of the STAT family, which appear to have relatively specific functions in certain cytokine pathways, STAT3 is involved in a wide variety of physiological processes and is important for various cytokine pathways (Table 7-3). For example, specific deletion of Stat3 in the liver revealed that STAT3 is essential for the acute-phase response, consistent with a role of STAT3 in IL-6 signaling (Alonzi, Maritano, et al. 2001). Mice with a Stat3 deletion in macrophages and neutrophils exhibited increased inflammatory responses and T helper (TH) 1 differentiation and developed chronic colitis (Kobayashi et al. 2003; Takeda et al. 1999). These phenotypes are similar to those observed in mice lacking IL-10, and Stat3-null macrophages were not responsive to IL-10, indicating an essential role of STAT3 in IL-10 signaling. Interestingly, STAT3 seems to mediate distinct functions in different tissues. For example, mice with STAT3 deficiency in T lymphocytes showed impaired IL-6-dependent cell survival (Takeda et al. 1998), whereas mice lacking STAT3 in bone marrow cells showed increased cell proliferation due to the impaired negative feedback control of G-CSF signaling (Lee et al. 2002). The in vivo functions of STAT3 have also been addressed in other tissues, including skin (Sano et al. 1999), thymic epithelium (Sano et al. 2001), mammary epithelium (Chapman et al. 1999), and neurons (Alonzi, Middleton, et al. 2001; Schweizer et al. 2002), which further revealed the versatile functions of STAT3 in cell proliferation, differentiation, migration, and apoptosis (Levy and Lee 2002). Serine727 phosphorylation of STAT3 has been shown to be important for the full transcriptional activity of STAT3 (Wen and Darnell 1997; Wen et al. 1995). Mice carrying a mutant Stat3 gene with the serine727 residue substituted to alanine (Stat3S727A) have been generated (Shen et al. 2004) (Table 7-3). Studies with the MEF cells from these mice (SA/SA) revealed reduced STAT3-dependent transcriptional activation in response to IL-6 and OSM. Surprisingly, the SA/SA mice were viable and grossly normal. Studies with SA/− mice showed perinatal lethality and growth retardation of these mice, suggesting a role of STAT3 serine phosphorylation in perinatal growth. In contrast, the surviving SA/− mice showed a normal STAT3-dependent acute-phase response in liver, indicating that serine phosphorylation of STAT3 is not required for this process. Two splice variants of STAT3, STAT3α and STAT3β, have been identified. Mice with specific deletion of STAT3β showed altered global gene expression profiles compared with those of wild-type mice (Yoo et al. 2002) (Table 7-3). These mice were hypersensitive to LPS-induced endotoxic shock, accompanied by hyperactivation of a subset of LPS-induced genes in the liver.
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TABLE 7-3
IN VIVO FUNCTIONS OF STATS STAT
Phenotypes of Mutant Mice
Cytokine Pathways Affected
Reference
Stat1 null
Hypersensitivity to pathogens Resistance to LPS-induced shock Hypersensitivity to pathogens Resistance to LPS-induced shock Hypersensitivity to pathogens Embryonic lethality
IFN-α/β, IFN-γ
Durbin et al. 1996; Meraz et al. 1996
IFN-γ
Varinou et al. 2003
IFN-α/β
Park et al. 2000 Takeda et al. 1997
EFG, HGF TGF-α, IL-6
Sano et al. 1999
Stat1S727A Stat2 null Stat3 null; Tissue-specific Stat3 null Skin Thymic epithelium T lymphocytes Macrophages/neutrophils
Bone marrow cells Mammary epithelium Liver Sensory neurons Motoneurons Antigen-presenting cells (APCs) Stat3S727A
Stat3β null
Stat4 null Stat5a null
Stat5b null
Stat5a/5b null
Stat6 null
Defective second hair cycle, wound healing, and cell migration Age-dependent hypoplasia Hypersensitivity to stress Defective IL-6-dependent survival and IL-2Rα expression Increased inflammatory responses and TH1 differentiation Chronic colitis Impaired negative feedback Enhanced granulopoiesis Suppression of apoptosis Delayed mammary involution Defective acute-phase response Insulin resistance Increased apoptosis Impaired survival after damage Aberrant antigen-specific T cell tolerance Reduced transcriptional activation in MEFs SA/SA mice are grossly normal SA/− mice show perinatal lethality, growth retardation, and decreased thymocytes Reduced DNA binding of STAT3 Decreased recovery from LPS shock Hyperactivation of a subset of LPS-induced genes in liver Altered global gene transcription Impaired TH1 differentiation Resistance to LPS-induced shock Lactation defects Defective mammary gland development Impaired GM-CSF-induced proliferation in BMMs Impaired IL-2-induced T cell proliferation and IL-2Rα expression Defective NK cell development Loss of sexually dimorphic growth Defective NK cell functions Impaired IL-2-induced T cell proliferation and IL-2Rα expression No NK cells Female infertility Impaired T cell proliferation Defective TH2 development
HGF, human growth factor; TGF, transforming growth factor.
Sano et al. 2001 IL-6, IL-2
Takeda et al. 1998
IL-10
Kobayashi et al. 2003; Takeda et al. 1999
G-CSF
Lee et al. 2002
GH, PRL
Chapman et al. 1999
IL-6
Alonzi, Maritano, et al. 2001
CNTF, LIF CT-1
Alonzi, Middleton, et al. 2001 Schweizer et al. 2002 Cheng et al. 2003
IL-6, OSM
Shen et al. 2004
IL-6, OSM, LIF
Yoo et al. 2002
IL-12
Kaplan, Sun, et al. 1996; Thierfelder et al. 1996 Feldman et al. 1997; Liu et al. 1997; Nakajima et al. 1997
PRL, IL-2, GM-CSF
GH, IL-2, IL-15
Imada et al. 1998; Udy et al. 1997
IL-2, IL-15, GH, PRL
Teglund et al. 1998
IL-4, IL-13
Kaplan, Schindler, et al. 1996; Shimoda et al. 1996; Takeda et al. 1996
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Thus, these studies indicate a nonredundant role of STAT3β in the control of systemic inflammation and the transcription of a subset of LPS-induced genes. 4.
Stat5a- and Stat5b-Deficient Mice
STAT5A and STAT5B are two closely related STATs sharing >90% sequence homology. Studies with Stat5a- and Stat5bdeficient mice revealed both redundant and nonredundant roles of STAT5A and STAT5B in cytokine signaling (Table 7-3). Stat5a-deficient mice appeared to be normal except for defects in mammary gland development and lactation, indicating an essential role of STAT5A in prolactin (PRL) signaling (Liu et al. 1997). Furthermore, bone marrow–derived macrophages (BMMs) from Stat5a-deficient mice showed impaired proliferation and gene transcription in response to GM-CSF (Feldman et al. 1997). In contrast, Stat5b-null mice showed defects in GH-mediated sexual dimorphic growth, indicating a specific role of STAT5B in GH signaling (Udy et al. 1997). Further studies with Stat5a- and Stat5b-null mice revealed that IL-2-mediated T cell proliferation and NK cell functions were defective in both mice, although the defects appeared to be more severe in Stat5b-null mice (Imada et al. 1998; Nakajima et al. 1997). Stat5a/5b double knockout mice showed even more severe phenotypes: These mice had no NK cells, and T cells from these mice were defective in anti-CD3mediated cell proliferation (Teglund et al. 1998). These studies demonstrate the specific as well as redundant functions of STAT5A and STAT5B in mediating cytokine pathways. 6.
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IL-4 and IL-13 but responded normally to other cytokines. These results confirmed the essential role of STAT6 in IL-4/IL-13 signaling.
Stat4-Deficient Mice
Biochemical studies suggest that STAT4 is activated mainly by IL-12. Consistent with this observation, Stat4-deficient mice showed impaired TH1 differentiation (Kaplan, Sun, et al. 1996; Thierfelder et al. 1996) (Table 7-3). In addition, decreased natural killer (NK) cell cytotoxicity and abrogated IFN-γ production upon IL-12 treatment had been observed in Stat4-null mice. These data indicate that STAT4 primarily mediates IL-12 signaling. 5.
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Stat6-Deficient Mice
STAT6 is mainly activated by IL-4 and IL-13. Stat6-null mice consistently exhibited defective TH2 responses and enhanced TH1 differentiation (Kaplan, Schindler, et al. 1996; Shimoda et al. 1996; Takeda et al. 1996) (Table 7-3). These mice showed abrogation of eosinophilia, airway inflammation, and airway hyperresponsiveness (Akimoto et al. 1998; Kuperman et al. 1998). In addition, Stat6-null mice were defective in IL-4/IL-13-mediated expulsion of the gastrointestinal parasite Nippostrongylus brasiliensis (Urban et al. 1998). Stat6-deficient lymphoid cells were not responsive to
C. 1.
SOCSs
Socs1-Deficient and Transgenic Mice
Genetic studies with Socs1−/− mice indicated that SOCS1 is essential for the regulation of IFN-γ signaling and T cell homeostasis (Alexander et al. 1999; Marine, Topham, et al. 1999) (Table 7-4). Socs1-null mice died within 3 weeks after birth with extensive fatty degeneration of the liver and hematopoietic infiltration of multiple organs, including liver, heart, and lung. Socs1−/− mice showed lymphopenia and increased apoptosis in lymphoid organs (Naka et al. 1998; Starr et al. 1998). In addition, aberrant T cell activation and altered CD4+ and CD8+ ratios of T lymphocytes were observed in Socs1−/− mice (Cornish et al. 2003; Marine, Topham, et al. 1999). Treatment of the newborn Socs1-null mice with neutralizing antibodies against IFN-γ rescued the lethal phenotype of these mice. Furthermore, Socs1−/−IFNγ−/− mice were healthy and showed no histological defects as observed in Socs1-null mice. These results demonstrate that dysregulated IFN-γ signaling is the main cause of the phenotypes of Socs1−/− mice. Interestingly, constitutive activation of T cells and altered CD4+ and CD8+ ratios were also observed in Socs1−/−IFNγ−/− mice, suggesting that these phenotypes are independent of IFN-γ (Metcalf, Di Rago, et al. 2000). In addition to IFN-γ signaling, Socs1−/− mice also showed defects in signaling by other cytokines, such as IL-4, IL-12, TNF-α, LPS, insulin, and prolactin (Eyles et al. 2002; Kawazoe et al. 2001; Kinjyo et al. 2002; Morita et al. 2000; Naka et al. 1998; Nakagawa et al. 2002). Although elevated serum levels of IFN-γ were observed in Socs1−/− mice, the main feature of Socs1−/− cells was that they were hypersensitive to IFN-γ stimulation. For example, BMMs from Socs1−/− mice showed comparable capability for clearing intracellular parasites Leishmania major after IFN-γ stimulation at a dose approximately 100-fold less than that required for wildtype cells (Alexander et al. 1999). Consistently, Socs1−/− mice showed resistance to viral infection. In agreement with these results, biochemical studies with Socs1−/− cells revealed prolonged STAT1 phosphorylation in these cells, whereas the magnitude of STAT1 phosphorylation was not increased (Brysha et al. 2001). These data indicate that SOCS1 specifically controls the duration of IFN-γ signaling but has no effect on the initial activation process of the signaling pathway. Genetic studies with Socs1-null mice revealed the important role of SOCS1 in T cell development independent of IFN-γ signaling. Studies with Socs1 transgenic mice with enforced expression of SOCS1 in T lymphocytes further illustrated the inhibitory role of SOCS1 in multiple T cell cytokines, including IFN-γ, IL-6, and the cytokines signaling through the
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TABLE 7-4
IN VIVO FUNCTIONS OF SOCS PROTEINS SOCS
Phenotypes of Null Mice
Transgenic Mice
Reference
Cis
No abnormalities
Li et al. 2000; Marine, McKay, et al. 1999; Matsumoto et al. 1999
Socs1
Neonatal lethality Liver degeneration Lymphopenia Hematopoietic infiltration Apoptosis in lymphoid organs Altered CD4+ and CD8+ ratio of T cells Aberrant T-cell activation Elevated serum levels of IFN-γ Hyperactivated IFN-γ signaling Hypersensitive to LPS-induced shock Defects in γ-C-dependent cytokines and IL-12, TNF-α, insulin, PRL pathways Gigantism Dysregulated GH signaling Embryonic lethality with placental insufficiency Defective IL-6, LIF pathways
Runted Lactation defects Reduced splenic γδT, NK, NKT Preferential TH2 differentiation Abnormal T cell receptor responses Suppressed cytokine signaling Defective T cell development Spontaneous T cell activation
Socs2 Socs3
Socs5 Socs6
Mild growth retardation
Socs7
Mild growth retardation 50% adult mice die of hydrocephalus
Gigantism Dysregulated GH signaling Embryonic lethality with anemia T cell-specific transgenic mice develop asthma and increased TH2 response
Repressed TH2 responses Inhibited IL-4 signaling Improved glucose metabolism
common γ chain receptor, such as IL-4 and IL-7 (Fujimoto et al. 2000; Trop et al. 2001). Consistently, these mice showed defective T cell development and aberrant T cell activation. These results, together with the evidence from Socs1−/− mice, strongly indicate that SOCS1 is an important negative regulator of multiple cytokine pathways in addition to IFN-γ signaling, both of which are essential for T cell homeostasis. 2.
Socs3-Deficient and Transgenic Mice
Socs3−/− mice were embryonic lethal, probably due to placental insufficiency and erythrocytosis (Marine, McKay, et al. 1999; Roberts et al. 2001; Takahashi et al. 2003) (Table 7-4). Studies with conditional Socs3 deletions in liver and macrophages have revealed a specific role of SOCS3 in the negative regulation of IL-6 signaling (Croker et al. 2003; Lang et al. 2003; Yasukawa et al. 2003). Both SOCS1 and SOCS3 are induced by IFN-γ and IL-6, and both can inhibit IFN-γ and IL-6 responses when overexpressed. However, genetic studies
Alexander et al. 1999; Brysha et al. 2001; Cornish et al. 2003; Eyles et al. 2002; Fujimoto et al. 2000; Kawazoe et al. 2001; Kinjyo et al. 2002; Marine, Topham, et al. 1999; Metcalf, DiRigo, et al. 2000; Morita et al. 2000; Naka et al. 1998; Nakagawa et al. 2002; Starr et al. 1998; Trop et al. 2001 Greenhalgh et al. 2002; Metcalf, Greenhalgh, et al. 2000 Croker et al. 2003; Lang et al. 2003; Marine, McKay, et al. 1999a; Roberts et al. 2001; Seki et al. 2003; Takahashi et al. 2003; Yasukawa et al. 2003 Seki et al. 2002 Krebs et al. 2002; Li et al. 2004 Krebs et al. 2004
have demonstrated the in vivo specificity of SOCS1 and SOCS3 in the negative regulation of cytokine signaling. In Socs3−/− macrophages, the activation of STAT1 and STAT3 by IL-6 was prolonged, whereas the activation of STAT1 by IFN-γ was normal. In contrast, in Socs1−/− macrophages, the opposite is true. In addition, although STAT3 is activated by both IL-6 and IL-10, the tyrosine phosphorylation of STAT3 induced by IL-6, but not IL-10, was prolonged in Socs3−/− macrophages. Thus, SOCS proteins display specificity toward cytokines but not JAKs or STATs. Interestingly, the prolonged STAT activation induced by cytokines in the absence of SOCS proteins resulted in altered cytokine responses. For example, microarray analysis showed that the gene activation profile induced by IL-6 in Socs3−/− macrophages mimics that induced by IFN-γ in wild-type cells, suggesting a role for SOCS3 in the control of the specificity of cytokine responses. A similar alteration of the IL-6 response has been observed in Stat3−/− cells, in which STAT1 activation was prolonged (Costa-Pereira et al. 2002). One explanation is that
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the expression of SOCS3 is STAT3 dependent. In the absence of STAT3, SOCS3 is not induced by IL-6; therefore, prolonged STAT1 activation leads to an IFN-γ-like inflammatory response. These studies indicate that SOCS1 and SOCS3 play important roles in defining the specificity of cytokine responses in vivo. Studies with Socs3 transgenic mice further revealed important functions of SOCS3 in cytokine signaling. Enforced expression of SOCS3 inhibited fetal erythropoiesis, resulting in embryonic lethality (Marine, McKay, et al. 1999). Recently, a T cell–specific Socs3 transgenic mouse model has been established (Seki et al. 2003). These mice showed enhanced TH2 responses and pathological features of asthma in an airway hypersensitivity model. Thus, these studies indicated that SOCS3 is important for the regulation of TH2-mediated allergic responses. 3.
Socs5 Transgenic Mice
Socs5 is expressed primarily in TH1 cells and is associated with the IL-4 receptor. Consistently, Socs5 transgenic mice showed defective TH2 differentiation and reduced TH2 production of cytokines, such as IL-4, IL-5, and IL-10 (Seki et al. 2002) (Table 7-4). These results suggest a role of SOCS5 in the regulation of IL-4 and STAT6 signaling. However, the function of SOCS5 in physiological settings needs to be addressed with Socs5-null mouse models. 5.
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TABLE 7-5
IN VIVO FUNCTIONS OF PIAS PROTEINS PIAS
Phenotypes of Null Mice
Reference
Pias1
Resistant to viral and bacterial infections Hypersensitivity to LPS Elevated serum levels of proinflammatory cytokines Growth retardation and perinatal lethality Enhanced STAT1 and NF-κB signaling No abnormality in 129 genetic background Mild defects in IFN and Wnt signaling in 129/C57 mice Viable and fertile Reduced testis weight and sperm count Increased apoptosis of testicular cells
Liu et al. 2004; 2005
Piasy
Piasx
Roth et al. 2004; Wong et al. 2004
Santti et al. 2005
Cis Transgenic Mice
Cis-deficient mice appeared to be normal (Marine, McKay, et al. 1999). Transgenic mice with widespread expression of Cis showed growth retardation and lactation defects, which is reminiscent of the phenotypes observed in Stat5a−/− and/or Stat5b−/− mice (Li et al. 2000; Matsumoto et al. 1999) (Table 74). In addition, T cells from Cis transgenic mice failed to proliferate in response to IL-2 and IL-2-induced STAT5 activation was absent in these cells. Furthermore, these mice showed reduced numbers of T and NK cells, as well as preferential TH2 differentiation. Similar phenotypes have been observed in Stat5−/− mice, suggesting that enforced expression of CIS may inhibit STAT5-mediated cytokine signaling. However, since Cis−/− mice appeared to be normal, the precise role of CIS in immune regulation in vivo remains unclear. 4.
LIU
Socs2-Deficient and Transgenic Mice
Socs2 deficiency in mice resulted in gigantism, implicating dysregulated GH signaling in these mice (Metcalf, Greenhalgh, et al. 2000) (Table 7-4). Socs2−/− cells showed prolonged STAT5 activation, and the gigantic phenotype of Socs2−/− mice was rescued when these mice were crossed onto the Stat5b−/− background. These data indicate that SOCS2 regulates GH signaling and STAT5 activation in vivo. Surprisingly, transgenic mice with widespread expression of Socs2 also showed the gigantism phenotype, indicating that SOCS2 can both positively
and negatively regulate GH signaling (Greenhalgh et al. 2002). One possible explanation is that when overexpressed, SOCS2 can prevent other negative regulators from binding to the GH receptor, thus resulting in increased growth. The function of SOCS2 in immune regulation has not been documented. D. 1.
PIASs
Pias1-Deficient Mice
Pias1-null mice were runted and showed perinatal lethality (Liu et al. 2004) (Table 7-5). Consistent with a role of PIAS1 as a negative regulator of activated STAT1, Pias1-null mice displayed resistance to viral infection and hypersensitivity to LPS-induced endotoxic shock. Detailed gene transcription profiling analyses revealed elevated expression of STAT1-dependent genes in Pias1-null cells. Interestingly, PIAS1 deficiency affected only a subset of IFN-inducible genes. Consistent with the in vitro studies suggesting that PIAS1 inhibits the DNA-binding activity of STAT1, chromatin immunoprecipitation assays revealed enhanced STAT1 binding to the endogenous promoters of PIAS1-regulated genes in Pias1-null cells. In addition to a negative regulatory role in IFN signaling, PIAS1 has been shown to inhibit nuclear factor (NF)-κB signaling (Liu et al. 2005). Elevated NF-κB-mediated transcription in response to TNF-α and LPS was observed in Pias1-null cells. Serum levels of proinflammatory cytokines, such as TNF-α and IL-1β, were consistently increased in Pias1- null mice. These studies established an essential role of PIAS1 in both STAT1 and NF-κB signaling pathways. 2.
Piasy-Deficient Mice
Piasy null mice have been reported to be either grossly normal or to show mild defects in IFN and Wnt signaling pathways (Roth et al. 2004; Wong et al. 2004) (Table 7-5).
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It has been shown that both PIAS1 and PIASy can interact with activated STAT1 in vivo and can regulate STAT1-dependent transcription (Liu et al. 1998, 2001). It remains to be determined whether the lack of profound phenotypes of Piasy null mice may be due to the redundant functions of other PIAS proteins, such as PIAS1. 3.
Piasx-Deficient Mice
Piasx knockout mice have been reported recently (Santti et al. 2005). Although Piasx null mice were viable and fertile, they displayed reduced testis weight and decreased sperm count. The increased number of apoptotic testicular cells was consistently observed in Piasx-null mice. These data indicate that PIASx is involved in spermatogenesis. E.
CONCLUSIONS
Genetic studies using various mouse models have contributed significantly to our understanding of the physiological TABLE 7-6
IN VIVO FUNCTIONS OF PTPS PTP
Phenotypes of Null Mice
Reference
Tcptp
Hematopoietic defects Impaired T and B cell functions Hypersensitivity to insulin Defects in leptin signaling
Simoncic et al. 2002; You-Ten et al. 1997 Cheng et al. 2002; Elchebly et al. 1999; Zabolotny et al. 2002
Defective thymic development Increased apoptosis and impaired TCR signaling Increased erythroid colony formation Enhanced antiviral activity Autoimmunity Hyperproliferation of myeloid cells Anemia Embryonic lethality Hematopoietic defects
Byth et al. 1996; IrieSasaki et al. 2001; Kishihara et al. 1993
Ptp1b
Cd45
Shp1
Shp2
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functions of the JAK-STAT signaling pathways in immune regulation. In addition, these genetic studies have revealed unexpected in vivo signaling specificity. Understanding the molecular basis of signaling specificity may be a benefit in the design of more efficient therapeutic strategies using cytokines. Clearly, genetic systems with the disruption of multiple components of the JAK-STAT pathways are needed to address the potential redundant functions of these molecules in the regulation of immune responses. Finally, genetic models that mimic immunological disorders should facilitate the development of therapeutic drugs. ACKNOWLEDGMENTS This work was supported by grants from National Institutes of Health (to K.S.). B.L. is a Leukemia and Lymphoma Special Fellow.
PTPs
Several phosphatases have been implicated in the regulation of the JAK-STAT pathway, including TC-PTP, PTP1b, SHP-1, SHP2, and CD45. Genetic studies with targeted deletions of these molecules revealed important functions of these proteins in the immune system (Table 7-6). However, these phosphatases have also been implicated in other pathways, such as the role of SHP1 and SHP-2 in B and T cell receptors and c-Kit receptor signaling. Therefore, the phenotypes of these mice may be the cumulative results of the defects in multiple pathways.
IV.
IN
Shultz et al. 1984; 1997
Qu et al. 2001; You et al. 1999
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7. C Y T O K I N E - A C T I VAT E D
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Chapter 8 Signal Transduction Events Regulating Integrin Function and T Cell Migration in the Mouse Lakshmi R. Nagarajan and Yoji Shimizu
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. T Cell Receptor Signaling and Integrin Regulation . . . . . . . . . . . . . . . . . . . A. Mouse Knockout Models with Defects in TCR-Mediated Integrin Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. ADAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Vav1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Rap1 and RapL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Itk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Phosphatidylinositol 3-Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. T Cell Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Chemokine Receptor Signaling and T Cell Migration . . . . . . . . . . . . . . . . . A. Use of Knockout Models to Identify Intracellular Signaling Proteins Critical for T Cell Migration . . . . . . . . . . . . . . . . . . 1. DOCK2 and Rac GTPases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. PI 3-K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. RapL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Itk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Dok-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Integrin Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I.
INTRODUCTION
Efficient immune surveillance of pathogens requires continuous trafficking of naïve T lymphocytes through secondary lymphoid tissues via the blood and lymphatic systems. Once in secondary lymphoid organs, naïve T cells survey the environment for dendritic cells displaying immunogenic peptides derived from foreign pathogens. These dendritic cells acquire antigen either while resident in lymphoid organs or before their migration from infected nonlymphoid tissue. Successful activation of a naïve T cell by antigen-laden dendritic cells involves engagement of the antigen-specific T cell receptor (TCR) complex, which generates an array of intracellular signals that leads to clonal expansion and the generation of effector functions such as cytotoxicity and cytokine production (Catron et al. 2004). Unlike naïve T cells, these effector T cells have the ability to exit secondary lymphoid tissue and migrate into nonlymphoid tissue. Some T cells ultimately differentiate into long-lived memory T cells that reside in both lymphoid and nonlymphoid tissue, ready to rapidly respond if the pathogen is encountered at a later time. The integrin family of adhesion receptors participates in T cell–mediated immune responses by mediating adhesive interactions to other cells and to extracellular matrix proteins that are critical for T cell migration and T cell recognition of antigen (Hynes 2002; Pribila et al. 2004). Integrins mediate efficient T cell adhesion to endothelial cells, which is essential for steadystate trafficking of naïve T cells into secondary lymphoid organs, as well as the migration of effector T cells into inflammatory sites. The lymphocyte function-associated antigen-1 (LFA-1) integrin, in particular, is critical for efficient T cell interaction with antigen-presenting cells and defines the outer ring of the immunological synapse (Huppa and Davis 2003). In addition, β1 integrins promote the interaction of T cells with extracellular matrix proteins found in the tissue extracellular environment. The functional activity of integrin receptors is regulated by intracellular signaling events generated by antigen receptors and chemokine receptors (Pribila et al. 2004), so that T lymphocytes cycle appropriately between adhesive and nonadhesive states suitable for different extracellular environments and conditions. Upon engagement with appropriate cell surface or extracellular matrix ligands, integrins also transduce signals critical for lymphocyte functional responses. Thus, intracellular signaling events play an essential role in integrin-dependent adhesive events critical for T lymphocyte function. Many of the early advances in our understanding of integrin function in the immune system came from in vitro approaches that allowed investigators to isolate and analyze specific integrin-ligand interactions. Such approaches, often utilizing human cells, were used to identify integrins and their ligands, as well as to define the contribution of integrins to cell-cell interactions in which multiple receptors play nonredundant roles. A particularly notable example is the use of parallel plate flow chamber systems to define the unique contribution of
integrins, selectins, and chemokines in the multistep cascade of adhesive events that occurs when lymphocytes interact with endothelium under conditions of vascular shear flow. In vitro approaches have also been critical in identifying intracellular signaling events that regulate integrin functional activity (Pribila and Shimizu 2003). In recent years, the physiological significance of integrins and integrin regulation has been tested with the use of various transgenic and knockout mouse models. In this chapter we highlight the mouse models that have provided key insights into integrin function and T cell migration (Table 8-1).
II.
T CELL RECEPTOR SIGNALING AND INTEGRIN REGULATION
Integrins expressed on resting T cells are typically unable to mediate strong adhesion to cells or extracellular matrix proteins. However, TCR activation results within minutes in a transient increase in integrin functional activity that does not require new protein synthesis. Thus, antigen-dependent stimulation of the TCR results in enhanced LFA-1-dependent interactions of T cells with antigen-presenting cells, allowing for more efficient transmission of critical signals between the T cell and the antigen-presenting cell. Integrins respond to activation signals initiated by the TCR (and other receptors, such as chemokine receptors) via two distinct mechanisms that probably work together to result in increased overall adhesive strength (Kinashi 2005). Structural studies have clearly demonstrated that integrins can alter their conformation and that these conformations have different ligand-binding affinities (Kinashi 2005). In addition to direct assessment of integrin affinity states using soluble integrin ligands, in many of these studies anti-integrin antibodies that recognize unique epitopes associated with specific integrin conformational states have been used. Almost all of these antibodies are specific for human integrins. The lack of availability of similar antibodies for mouse integrins has limited the analysis of the physiological relevance of changes in integrin conformation in response to T cell activation in the mouse. A second mechanism of integrin regulation involves activation-induced changes in the membrane distribution of integrins that result in clustering that can be detected by microscopic techniques. Integrin clustering involves transient changes in the attachment of integrins to the cytoskeleton (Kinashi 2005; Kucik et al. 1996). Engagement of ligand by integrins can promote integrin clustering (Kim et al. 2004), but clustering of some integrins can be observed before or in the absence of integrin ligand engagement (Medeiros et al. 2005). Integrin affinity modulation and clustering are both dependent on the association of integrin cytoplasmic tails with intracellular signaling proteins (Kinashi 2005). Although TCR signaling has been proposed to induce increased LFA-1 function primarily via integrin clustering (Stewart et al. 1996), a cooperative role
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8 . S I G N A L T R A N S D U C T I O N E V E N T S R E G U L AT I N G I N T E G R I N F U N C T I O N
TABLE 8-1
MOUSE TRANSGENIC AND KNOCKOUT MODELS OF INTRACELLULAR SIGNALING PROTEINS CRITICAL FOR INTEGRIN FUNCTION AND/OR T CELL MIGRATION Model
Type of Molecule
Adhesion and Migration Phenotype
ADAP−/−
Adapter protein
●
●
●
VASP−/−
Cytoskeletal protein
●
Vav1−/−
Rac GEF, adapter protein
●
●
●
●
Rac1 L61 mutant transgenic RhoA V14 mutant transgenic Rap1A V12 mutant transgenic Rap1A E63 mutant transgenic
GTPase
●
GTPase
●
GTPase
●
GTPase
●
●
●
RapL−/−
Rap1A effector
● ●
●
●
SPA-1−/−
Rap1A GAP
●
●
Rap1GAP1 transgenic
Rap1A GAP
●
●
●
C3G−/−
Rap1A GEF
● ●
Cbl-b−/−
E3 ubiquitin ligase
●
●
Impaired integrin-mediated adhesion of T cells after TCR stimulation Impaired integrin clustering on T cells after TCR stimulation Impaired T cell proliferation Enhanced thrombin-induced increases in integrin-mediated binding of fibrinogen by platelets Impaired integrin-mediated adhesion of T cells after TCR stimulation Impaired antigen-dependent conjugate formation Impaired integrin clustering on T cells after TCR stimulation Impaired actin polymerization after TCR stimulation Enhanced basal adhesion of thymocytes to fibronectin Enhanced basal adhesion of thymocytes to fibronectin Enhanced basal adhesion of thymocytes to β1 and β2 integrin ligands Enhanced basal adhesion of thymocytes to ICAM-1 Reduced T cell proliferation and IL-2 production Increased fraction of regulatory T cells Reduced lymph node cellularity Impaired homing of T and B cells to lymph nodes and spleen Impaired lymphocyte adhesion to integrin ligands following chemokine stimulation Impaired dendritic cell adhesion and migration Elevated and persistent Rap1A activation in T cells after anti-CD3 stimulation Development of a spectrum of myeloid disorders characteristic of human chronic myelogenous leukemia Impaired antigen-dependent conjugate formation Impaired activation of Rap1 after CTLA-4 ligation Enhanced T cell proliferation and IL-2 production Embryonic lethal Defective adhesion, cell spreading, and Rap1 activation of C3G−/− fibroblasts Increased adhesion to ICAM-1, LFA-1 clustering, and Rap1 activation after TCR stimulation Enhanced interaction between Crk-L and the Rap1 GEF C3G
Reference Griffiths et al. 2001 Peterson et al. 2001
Hauser et al. 1999 Ardouin et al. 2003 Krawczyk et al. 2002
Gomez et al. 2001 Corre et al. 2001 Sebzda et al. 2002 Li et al. 2005
Katagiri, Ohnishi, et al. 2004
Ishida, Kometani, et al. 2003; Ishida, Yang, et al. 2003
Dillon et al. 2005
Ohba et al. 2001
Zhang et al. 2003
Continued
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TABLE 8-1
MOUSE TRANSGENIC AND KNOCKOUT MODELS OF INTRACELLULAR SIGNALING PROTEINS CRITICAL FOR INTEGRIN FUNCTION AND/OR T CELL MIGRATION—cont’d Model
Type of Molecule
Adhesion and Migration Phenotype
Itk−/−, Itk−/− Rlk−/−
Tyrosine kinases
●
●
●
●
● ●
p110δD910A/D910A
Lipid kinase
●
●
●
mev/mev(inactive SHP-1)
Phosphatase
●
●
●
DOCK2−/−
Adapter protein
● ●
●
●
●
Rac2−/− p110γ−/−
GTPase Lipid kinase
● ●
●
●
● ●
SHIP−/−
Phosphatase
●
●
PTEN+/−
Phosphatase
●
PTEN−/−
Phosphatase
●
Dok-1−/− Hck−/−, Fgr−/−
Adapter protein Tyrosine kinases
●
Syk−/−
Tyrosine kinase
●
SLP-76−/−
Adapter protein
●
●
●
●
Impaired antigen-dependent conjugate formation Impaired adhesion of T cells to ICAM-2 after anti-CD3 stimulation Impaired actin polymerization after TCR stimulation Impaired membrane recruitment of Vav1 and impaired activation of WASp after TCR stimulation Impaired T cell chemotaxis in vitro Decreased T cell homing to lymph nodes Defective adhesion and migration of mast cells after stem cell factor stimulation Normal adhesion of T cells to fibronectin and ICAM-1 after TCR stimulation Reduced migration of B cells to CXCL13 and reduced B cell homing to Peyer’s patches and splenic white pulp cords Impaired integrin-dependent adhesion and spreading of macrophages Elevated levels of PI (3,4,5)P3 and membrane-associated PI 3-K activity Enhanced T cell chemotaxis to CXCL12 in vitro Defective T and B cell chemotaxis in vitro Reduced number of T and B cells in secondary lymphoid tissue Loss of marginal zone B cells and lymphoid follicle atrophy Loss of chemokine-induced activation of Rac and actin polymerization Impaired integrin-mediated adhesion of B cells, but not T cells, after chemokine stimulation Impaired T cell chemotaxis in vitro Impaired in vitro migration of neutrophils and macrophages in response to chemoattractants Impaired migration of neutrophils and macrophages into inflammatory sites in vivo Altered F-actin localization in migrating neutrophils Modest reduction in T cell chemotaxis in vitro Impaired dendritic cell migration in vitro and in vivo Enhanced migration of thymocytes, B cells, and hematopoietic precursors to CXCL12 in vitro Enhanced calcium mobilization and actin polymerization in response to CXCL12 Increased responsiveness of B cells to CXCL12-induced chemotaxis Impaired B cell migration to CXCL12 and CXCL13 Enhanced T cell chemotaxis to CXCL12 in vitro Impaired integrin-mediated degranulation, oxidative burst, and cell spreading of neutrophils Impaired integrin-mediated spreading of macrophages Impaired integrin-mediated degranulation, oxidative burst and cell spreading of neutrophils Profound block in T cell development Impaired integrin-mediated degranulation, oxidative burst, and spreading of neutrophils
Reference Finkelstein et al. 2005; Fischer et al. 2004; Labno et al. 2003; Takesono et al. 2004
Ali et al. 2004; Okkenhaug et al. 2002
Kim, Qu, et al. 1999; Roach et al. 1998
Fukui et al. 2001; Nombela-Arrieta et al. 2004
Croker et al. 2002 Del Prete et al. 2004; Hannigan et al. 2002; Hirsch et al. 2000; Li et al. 2000; Nombela-Arrieta et al. 2004; Reif et al. 2004; Sasaki et al. 2000
Kim, Hangoc, et al. 1999
Fox et al. 2002 Anzelon et al. 2003 Okabe et al. 2004 Lowell et al. 1996; Mocsai et al. 1999; Suen et al. 1999 Mócsai et al. 2002 Clements et al. 1998; Pivniouk et al. 1998; Newbrough et al. 2003
8 . S I G N A L T R A N S D U C T I O N E V E N T S R E G U L AT I N G I N T E G R I N F U N C T I O N
for both LFA-1 affinity modulation and clustering has recently been proposed (Kim et al. 2004). Chemokine stimulation results in detectable changes in both LFA-1 clustering and conformation (Constantin et al. 2000). The proximal signaling events triggered by TCR stimulation have been extensively characterized and involve 1) src family tyrosine kinase-mediated phosphorylation of tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) in CD3 subunit cytoplasmic domains, 2) ITAMdependent association of the tyrosine kinase ZAP-70 and subsequent src family tyrosine kinase–mediated phosphorylation of ZAP-70, resulting in enhanced ZAP-70 tyrosine kinase activity, 3) ZAP-70-mediated tyrosine phosphorylation of two key substrates, the adapter proteins LAT (linker for T cell activation) and SLP-76 (SH2 domain containing protein of 76 kDa), and 4) the formation of LAT-nucleated protein-protein interactions critical for initiation of downstream signaling responses, such as calcium mobilization, that result in changes in gene transcription in the nucleus. The essential role that these early signaling events play in TCR function suggests that these signaling proteins will also play a central role in TCR-mediated activation of integrin function. Although studies with human T cells have in fact demonstrated that ZAP-70 and LAT are critical for TCR signaling to integrins, the inability of T cells to develop in the absence of these molecules (and others, such as SLP-76) has made it impossible to study integrin regulation in these knockout models (Clements et al. 1998; Negishi et al. 1995; Pivnioud et al. 1998; Zhang et al. 1999). Although the block in T cell development is not as profound in Lck-deficient mice (Molina et al. 1992), an extensive analysis of integrin function in these mice has not been conducted. Integrin function has also not been extensively analyzed in mice lacking Fyn, the other major src family tyrosine kinase expressed in T cells, even though these mice do not have major defects in T cell development (Appleby et al. 1992). The ability of Lck and Fyn to play compensatory roles when the other kinase is limiting may complicate such analyses, as there is a complete block in thymic development in mice lacking both Lck and Fyn (Groves et al. 1996; Van Oers et al. 1996).
A.
Mouse Knockout Models with Defects in TCR-Mediated Integrin Activation
1.
ADAP
Adhesion and degranulation-promoting adapter protein (ADAP) is an intracellular adapter protein that becomes tyrosine phosphorylated and associates with the SLP-76 adapter protein after TCR stimulation. An analysis of mice lacking ADAP demonstrated that TCR stimulation of ADAP-deficient T cells results in impaired increases in β2 and β1 integrin–mediated adhesion to purified ligands, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and fibronectin (Peterson et al. 2001;
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Griffiths et al. 2001). This defect in TCR-mediated integrin activation is also associated with impaired integrin clustering after TCR stimulation (Peterson et al. 2001; Griffiths et al. 2001), suggesting a role for ADAP in regulating the interaction of integrins with the cytoskeleton. ADAP-deficient T cells also exhibit defects in TCR-mediated proliferation and cytokine production, even though key early signaling responses, including calcium mobilization and ERK activation, are not impaired. In addition to SLP-76, ADAP also interacts with other proteins, including Fyn, SKAP-55, and members of the Ena-VASP family of actin regulatory proteins. A SKAP-55 mouse knockout model has not as yet been reported, although in vitro studies using overexpression and RNA interference approaches suggested that loss of SKAP-55 may also impair TCR signaling to integrins (Jo et al. 2005; Wang et al. 2003). The role of Ena-VASP proteins in integrin regulation in T cells has also not been investigated using transgenic or knockout technology, although platelets from mice lacking expression of VASP exhibit enhanced, rather than reduced, integrin-dependent binding to fibrinogen after thrombin stimulation (Hauser et al. 1999). 2.
Vav1
Vav1 is a guanine nucleotide exchange factor (GEF) for the Rac1 and Rac2 small GTPases, and its multidomain structure allows it to function as an adapter molecule as well. Together with Wiskott-Aldrich syndrome protein (WASp), Vav1 plays an important role in inducing actin cytoskeleton reorganization, TCR clustering, and immunological synapse formation after TCR stimulation. T cells from Vav1 knockout mice demonstrate impaired integrin-dependent adhesion and integrin-dependent conjugate formation with antigen-presenting cells (Ardouin et al. 2003; Krawczyk et al. 2002). In contrast to ADAP-deficient T cells, Vav1-deficient T cells also exhibit defects in TCRmediated actin polymerization. The mechanism by which Vav1 controls integrin activation is unclear, but it appears to be independent of WASp, as WASp-deficient T cells do not exhibit defects in TCR-mediated increases in integrin-dependent adhesion or integrin clustering (Krawczyk et al. 2002). The enhanced basal integrin-dependent adhesion of thymocytes isolated from transgenic mice expressing a constitutively active form of Rac1 to fibronectin (Gomez et al. 2001) suggests that Vav1 may regulate integrin function via its role as a GEF for Rac1. Similar results were reported for thymocytes from transgenic mice expressing a constitutively active form of RhoA (Corre et al. 2001), consistent with the ability of Rho family GTPases to modify the actin cytoskeleton. 3.
Rap1 and RapL
Numerous studies using dominant-negative and constitutively active forms of the Rap1 GTPase have demonstrated that this member of the Ras family of small GTPases plays an important role in regulating both basal and activation-dependent integrin function in T lymphocytes (Bos et al. 2003). Development of
200 transgenic and knockout models in which Rap1 activity has been altered have confirmed these in vitro findings and further expanded our understanding of Rap1 and its role in regulating integrin function. Similar to transgenic mice expressing constitutively active Rac1 or RhoA, thymocytes from transgenic mice expressing constitutively active Rap1 (G12V) also have enhanced basal adhesion to β1 and β2 integrin ligands (Sebzda et al. 2002). Thymocytes from transgenic mice expressing a different active Rap1 construct (E63) also showed elevated LFA-1-dependent adhesion (Li et al. 2005). Despite elevated LFA-1 function, there are impaired T cell activation responses in Rap1 E63 transgenic mice that are associated with an increased number of regulatory T cells (Li et al. 2005). RapL is a recently identified effector of Rap1 that binds to active Rap1 as well as to the cytoplasmic domain of the β2 integrin (Katagiri et al. 2003). As described in further detail below, loss of RapL in mice results in impaired lymphocyte and dendritic cell migration (Katagiri, Ohnishi, et al. 2004). Although integrin function after TCR stimulation of RapL-deficient T cells was not analyzed in this study, in vitro studies predict impaired integrin function in the absence of RapL expression in mice. Other mouse models have focused on effects of loss of expression of proteins that modulate Rap1 activity. SPA-1 is a Rap1-specific GTPase activating protein (GAP), and naïve T cells from SPA-1-deficient mice exhibit elevated and persistent activation of Rap1 after stimulation of the TCR with anti-CD3 antibodies (Ishida, Yang, et al. 2003). Although T cell development is normal, SPA-1-deficient mice have an impaired recall response to antigen. In addition, a population of unresponsive CD44hi memory-like T cells develops in these mice as they age. These T cells have elevated levels of active Rap1, but impaired extracellular signal-regulated kinase (ERK) activation in response to anti-CD3 stimulation. This biochemical phenotype is consistent with the initial identification of Rap1 as an antagonist of Ras signaling and thus a potential factor critical in inducing T cell anergy. The relevance of these findings in SPA-1 knockout mice to potential perturbations in integrin function is currently unclear. However, it is interesting to note that SPA-1−/− mice also develop myeloid disorders characteristic of chronic myelogenous leukemia (Ishida, Kometani, et al. 2003), a disease that is associated with altered β1 integrin function and abnormal association of integrins with the cytoskeleton (Bhatia et al. 1999). Expression of the Rap1 GAP Rap1GAP1 in transgenic mice impairs Rap1 activation and integrin function in T cells, but these T cells exhibit enhanced proliferation and interleukin (IL)-2 production (Dillon et al. 2005). This study also demonstrated an ability of the inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) to activate Rap1, and CTLA-4 function was reduced in transgenic T cells expressing Rap1GAP1 (Dillon et al. 2005). Thus, this transgenic model is consistent with other mouse models, suggesting that Rap1 positively regulates integrin function but may negatively regulate other activation responses of T cells. One GEF that regulates Rap1 activity in T cells is C3G. Although C3G knockout mice die before embryonic day 7.5,
LAKSHMI R. NAGARAJAN AND YOJI SHIMIZU
fibroblast cell lines have been made from embryos rescued by expression of a human C3G transgene flanked by loxP recombination sites. When these cell lines were infected with Cre-expressing viruses, the resulting C3G-deficient cell lines exhibited defective adhesion, cell spreading, and Rap1 activation (Ohba et al. 2001). Mice with a conditional loss of C3G specifically in T cells have not as yet been developed. TCR stimulation of T cells from mice lacking the E3 ubiquitin ligase Cbl-b results in enhanced LFA-1 clustering and T cell adhesion to ICAM-1, as well as elevated levels of Rap1 activity (Zhang et al. 2003). In the absence of Cbl-b, the interaction between the adapter protein Crk-L and C3G is enhanced, suggesting that the association of Cbl-b and Crk-L serves to negatively regulate C3G-dependent Rap1 activation in T cells. 4.
Itk
TCR stimulation results in the association of the Tec family tyrosine kinase Itk to the LAT signaling complex, where it can modulate the activity of phospholipase C-γ1 (PLC-γ1). Consequently, T cells from Itk-deficient mice exhibit impaired PLC-γ1 tyrosine phosphorylation and calcium mobilization in response to TCR stimulation. Recent studies have also shown that Itk regulates actin cytoskeletal reorganization and integrindependent adhesion after TCR stimulation (Finkelstein and Schwartzberg 2004). T cells from Itk-deficient mice exhibit impaired integrin-dependent adhesion to purified integrin ligands after anti-CD3 stimulation (Finkelstein et al. 2005), as well as impaired antigen-dependent conjugate formation mediated by the LFA-1 integrin (Labno et al. 2003). T cells also express the Tec family tyrosine kinase Rlk and loss of Rlk together with Itk enhances the defect in TCR-mediated calcium mobilization observed in Itk−/− T cells. However, the defect in integrin function is comparable in Itk−/− T cells and Rlk−/−Itk−/− T cells (Finkelstein et al. 2005; Labno et al. 2003). Itk-deficient T cells also exhibit severe defects in actin polymerization upon interaction with antigen-pulsed antigen-presenting cells or anti-TCR antibody–coated latex beads. These defects in actin polymerization are associated with impaired recruitment of Vav1 to the contact site, as well as impaired activation of WASp (Labno et al. 2003). Thus, defects in TCR-mediated integrin activation in Itk-deficient T cells are consistent with a requirement for Itk in effective actin polymerization after T cell activation. The ability of Itk to regulate both PLC-γ1 activation and calcium mobilization may also be critical to the integrin defects observed in Itk knockout mice, as both PLC-γ1 and calcium have been implicated in TCR signaling to integrins using human T cell systems (Katagiri, Shimonaka, et al. 2004; Stewart et al. 1998). 5.
Phosphatidylinositol 3-Kinase
TCR-mediated integrin activation can be blocked by pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), a family of lipid kinases that participates in intracellular signaling by phosphorylating the D-3 position of the inositol
8 . S I G N A L T R A N S D U C T I O N E V E N T S R E G U L AT I N G I N T E G R I N F U N C T I O N
ring of phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate [PI (3,4,5)P3]. Analysis of PI 3-K and its role in integrin regulation in mice has been complicated by the heterodimeric structure of PI 3-K, the presence of multiple isoforms of both subunits, the embryonic lethality of some knockouts, and changes in expression of PI 3-K subunits when one subunit is deleted in the mouse germline (Okkenhaug and Vanhaesebroeck 2003). Defects in TCR signaling have been reported in mice in which the p110δ subunit of PI 3-K has been inactivated by a point mutation that renders it catalytically inactive (Okkenhaug et al. 2002). Although p110δ-mutant mast cells exhibit defective integrin-dependent adhesion to fibronectin after stem cell factor stimulation (Ali et al. 2004), there are no defects in the adhesion of p110δ-mutant T cells to fibronectin or ICAM-1 after TCR stimulation (Okkenhaug et al. 2002). In contrast, integrin-dependent adhesion and spreading of macrophages is elevated in moth-eaten mice, which lack src homology 2 domain phosphatase-1 (SHP-1) enzymatic activity (Roach et al. 1998). Loss of SHP-1 activity is also associated with elevated levels of PI (3,4,5)P3 and membrane-associated PI 3-K activity, suggesting a role for SHP-1 in negative regulation of PI 3-K-dependent integrin function in macrophages.
III.
T CELL MIGRATION
A series of sequential events that are required for a lymphocyte to interact with endothelial cells and successfully leave the bloodstream have been identified in in vitro systems. In this adhesion cascade, initial tethering or rolling of lymphocytes along the endothelial surface is typically mediated by selectins or, in some cases, by α4 integrins. Subsequent triggering of G protein–coupled receptors by chemokines or chemoattractants “presented” on the endothelial cell surface transiently activates integrins, resulting in stable, shear-resistant attachment of lymphocytes to the endothelial surface. Integrins also participate in the transmigration process, in which lymphocytes move between adjacent endothelial cells as they exit the bloodstream and enter the surrounding tissue. Although the specific selectins, chemokine receptors, and integrins that mediate T cell migration are dependent on the differentiation and activation state of the T cell, as well as the activation state of the endothelial cells, intravitral microscopic studies have confirmed that this adhesion paradigm is operative in vivo under a variety of steady state and inflammatory conditions. For naïve T cells, L-selectin, the CCR7 chemokine receptor, and LFA-1 play particularly critical roles in mediating the migration of naïve T cells out of the bloodstream into lymph nodes. Knockout mouse models have been particularly informative in elucidating the role of these molecules in steadystate trafficking of naïve T cells into lymph nodes. Mice lacking expression of L-selectin or CCR7 exhibit drastic reductions in lymph node cellularity due to impaired ability of naïve T cells lacking expression of these molecules to
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migrate into lymph nodes (Arbones et al. 1994; Forster et al. 1999). Similar defects in lymph node cellularity and naïve T cell trafficking into lymph nodes are also observed in mice lacking expression of the CCR7 chemokine receptor ligand, CCL21 (SLC) (Gunn et al. 1999). Analysis of mice lacking CCR7 or CCL21 also demonstrate a critical role for these molecules in trafficking of dendritic cells from nonlymphoid tissue into lymph nodes (Forster et al. 1999; Gunn et al. 1999). Mice lacking LFA-1 also have reduced lymph node cellularity and exhibit defects in naïve T cell trafficking into lymph nodes (Berlin-Rufenach et al. 1999). However, some residual trafficking of LFA-1-deficient T cells into lymph nodes is observed, consistent with some contribution of α4 integrins in naïve T cell migration into lymph nodes (Berlin-Rufenach et al. 1999). JAM-A (JAM-1), a molecule expressed in the tight junctions of endothelial cells as well as on dendritic cells, has been proposed to play a role in leukocyte transmigration, either via homophilic interactions or via interaction with the LFA-1 integrin (Ostermann et al. 2002). Recent studies have revealed that dendritic cells isolated from JAM-A knockout mice have increased migration to lymph nodes that is associated with enhanced contact hypersensitivity responses (Cera et al. 2004). Thus, these mice have revealed a novel negative regulatory role for JAM-A in dendritic cell migration. The migration of effector T cells into inflammatory sites can be mediated by a number of different chemokine receptors, chemokines, and chemoattractants and is dependent in large part on the nature of the inflammatory stimulus and where in the body it is occurring. Coupled with the large number of chemokine receptors and the ability of multiple chemokine receptors to recognize more than one chemokine, the issue of functional redundancy highlights the value of analyzing chemokine receptor knockout mouse models (Power 2003). For example, analysis of T cells lacking the CCR4 receptor, which is expressed by skin memory T cells, demonstrated that CCR4-independent migration is mediated by the CCR10 chemokine receptor interacting with the chemokine CCL27 (Reiss et al. 2001). Recent studies with knockout mice have also revealed that the interaction of the BLT1 chemoattractant receptor with the lipid mediator leukotriene B4 plays a critical role in mediating the migration of effector CD4 and CD8 T cells into inflammatory sites by regulating chemotaxis and shearresistant adhesion to endothelial cells under shear flow conditions (Goodarzi et al. 2003; Ott et al. 2003; Tager et al. 2003). Integrin knockout models have also provided new insights into effector T cell migration and function. The αEβ7 integrin is expressed on intraepithelial lymphocytes (IELs) and binds to E-cadherin expressed on epithelial cells. Mice lacking αE integrin expression show a dramatic reduction in the number of intestinal and vaginal IELs, as well as a reduced number of lamina propria T cells (Schön et al. 1999). Effector T cells also express the α1β1 integrin, which binds to the extracellular matrix protein collagen. Mice lacking α1 integrin expression exhibit attenuated immune responses in several different inflammatory disease models (De Fougerolles et al. 2000;
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LAKSHMI R. NAGARAJAN AND YOJI SHIMIZU
Krieglstein et al. 2002; Ray et al. 2004). Because α1β1 integrin has not been implicated in mediating T cell adhesion to endothelial cells, it seems likely that α1β1 integrin expression on effector T cells may be critical for T cell retention in nonlymphoid tissue, which is rich in collagen. Knockout models have also been informative in defining the molecular requirements for the movement of lymphocytes out of tissue. Sphingosine-1-phosphate is produced by sphingosine kinase–mediated phosphorylation of sphingosine, a lipid that is generated de novo or by breakdown of membrane sphingolipids such as sphingomyelin. The receptor for sphingosine-1-phosphate (S1P1) is a recently identified Gαi protein–coupled receptor that is expressed on naïve T and B cells, as well as mature thymocytes. Initial evidence that S1P1 is critical for lymphocyte egress from the thymus and secondary lymphoid organs was obtained using FTY720, a small molecular agonist that sequesters S1P1. Although S1P1 knockout mice have an embryonic lethal phenotype, analysis of S1P1-deficient fetal liver chimeras or mice carrying Lox-P flanked S1P1 alleles and thymically expressed cre-recombinase revealed that S1P1 expression in T cells is essential for egress from the thymus (Matloubian et al. 2004). These studies have also documented an essential role for S1P1 in egress of T and B lymphocytes from secondary lymphoid organs.
IV.
CHEMOKINE RECEPTOR SIGNALING AND T CELL MIGRATION
As discussed above, signaling initiated by G protein–coupled chemokine receptors results in increased integrin functional activity. In addition, leukocyte migration in response to chemokines and other chemoattractants involves the acquisition of a polarized morphology, characterized by Rac-dependent actin polymerization at the leading edge that drives extension of the pseudopod. The leading edge of migrating cells also contains an increased accumulation of chemokine receptors and lipid products generated by PI 3-K. A large number of signaling intermediates are known to be activated in response to chemokine stimulation, including mobilization of intracellular calcium and activation of PI 3-K, PLC, protein kinase C, and ERK, as well as activation of small GTPases such as Rac and Rap1. Mouse knockout models have been informative in linking perturbations in chemokine receptor signaling with changes in cellular responses critical for effective cell migration, such as integrin activation and actin polymerization.
A.
Use of Knockout Models to Identify Intracellular Signaling Proteins Critical for T Cell Migration
A number of knockout models have been identified in which defects in T cell migration have been reported. Typically, these
studies involve testing the migration of knockout T cells in response to various chemokines using in vitro assays with transwell chambers, as well as assessing the ability of knockout T cells to rapidly enter lymph nodes after adoptive transfer into syngeneic recipients. Although these studies have identified a number of signaling intermediates critical for effective T cell migration in response to chemokines, the mechanism by which these molecules regulate the migration process remains less well defined at present. 1.
DOCK2 and Rac GTPases
DOCK2 is a member of the CDM family of scaffolding proteins that regulates activation of the small GTPase Rac. Analysis of DOCK2−/− mice demonstrated that chemokineinduced activation of Rac and actin polymerization in T and B cells is dependent on DOCK2 expression (Fukui et al. 2001). Consequently, DOCK2−/− T and B cells exhibit defective migration in response to appropriate chemokines, and DOCK2- deficient mice have reduced numbers of T and B cells in secondary lymphoid tissue, a loss of marginal zone B cells and lymphoid follicle atrophy. Thus, the DOCK2 knockout model provided evidence that the formation of actin-rich lamellipodial extensions via Rac activation is critical for lymphocyte migration in response to chemokines. Consistent with these findings, Rac2-deficient T cells also exhibit in vitro defects in T cell migration and actin polymerization in response to chemoattractants (Croker et al. 2002). DOCK2 also regulates chemokine- mediated integrin activation, as the adhesion of DOCK2-deficient B cells to integrin ligands is impaired after CXCL13 stimulation (Nombela-Arrieta et al. 2004). Surprisingly, chemokine-induced integrin activation is normal in DOCK2-deficient T cells, suggesting lymphocytespecific differences in the regulation of chemokine receptor signaling to integrins. 2.
PI 3-K
Numerous studies with PI 3-K inhibitors have indicated that PI 3-K plays a central role in leukocyte migration. The analysis of mouse knockout models has revealed unexpected complexity in the role that different PI 3-K isoforms play in this process in different cell types. The p110γ isoform of PI 3-K is activated specifically by G protein–coupled receptors, and therefore it was not surprising to find that neutrophils and macrophages isolated from p110γ−/− mice exhibit defects in migration in response to various chemoattractants in vitro and impaired migration into inflammatory sites (Hirsch et al. 2000; Li et al. 2000; Sasaki et al. 2000). In addition, p110γ−/− neutrophils exhibit altered intracellular localization of F-actin at the leading edge in response to chemoattractant stimulation (Hannigan et al. 2002). Whereas T cell migration in response to chemokines in vitro and in vivo is also inhibited by PI 3-K inhibitors, p110γ−/− T cells exhibit only modest defects in migration both in vitro
8 . S I G N A L T R A N S D U C T I O N E V E N T S R E G U L AT I N G I N T E G R I N F U N C T I O N
and in vivo (Nombela-Arrieta et al. 2004; Reif et al. 2004). Chemotactic responses of p110γ-deficient B cells are also normal (Reif et al. 2004). In contrast, there is impaired in vitro chemotaxis and in vivo homing to Peyer’s patches and spleen of B cells isolated from mice expressing a catalytically inactive mutant form of the p110δ subunit (Reif et al. 2004; Okkenhaug et al. 2002). Finally, although the chemotactic response of naïve T cells to the CCR7 ligands CCL21 and CCL19 is only modestly reduced in the absence of p110γ expression, the migration of p110γ−/− dendritic cells in response to these same chemokines is significantly impaired (Del Prete et al. 2004). Although PI 3-K inhibitors have been used to demonstrate a role for PI 3-K in regulating chemokine-mediated integrin clustering events (Constantin et al. 2000), the status of integrin membrane localization on T cells isolated from these various PI 3-K knockout models has not as yet been analyzed. Phosphatases that antagonize PI 3-K function have also been shown to modulate T cell migration. SH2-containing inositol-5′-phosphatase (SHIP) hydrolyzes the 5′ phosphate of the PI 3-K lipid product PI (3,4,5)P3. Thymocytes, B cells, and hematopoietic progenitor cells isolated from SHIP−/− mice exhibit enhanced migration in response to CXCL12 (SDF-1) in vitro, as well as enhanced chemokine-mediated calcium mobilization and actin polymerization (Kim, Hangoc, et al. 1999). The phosphoinositide-3-phosphatase and tensin homolog (PTEN) also antagonizes PI 3-K function by dephosphorylating the 3′ position of PI (3,4,5)P3 and phosphatidylinositol 3,4-bisphosphate. Whereas one report documented increased migration of B cells isolated from PTEN heterozygous mice to CXCL12 in vitro (Fox et al. 2002), another study using B cells from mice with a conditional deletion of PTEN demonstrated decreased migration and enhanced Rac activation of PTEN−/− B cells in response to CXCL12 and CXCL13 (Anzelon et al. 2003). Finally, the migration of several hematopoietic cell types, including T cells, in response to CXCL12 is enhanced in viable moth-eaten mice, which lack SHP-1 enzymatic activity (Kim, Qu, et al. 1999). Actin polymerization and ERK activation induced by CXCL12 stimulation are also elevated in moth-eaten mice. The mechanism by which SHP-1 regulates chemokine receptor signaling events is not clear, although the finding of elevated levels of PI 3-K lipid products in macrophages from moth-eaten mice (Roach et al. 1998) is consistent with a role for PI 3-K in chemokine-dependent chemotaxis. Alternatively, SHP-1 may affect tyrosine phosphorylation events critical to regulating the actin cytoskeleton (see below). 3.
RapL
Similar to what has been observed with TCR stimulation, chemokine stimulation of T cells also activates the Rap1 GTPase. Mice lacking expression of the Rap1 effector RapL have dramatically reduced cell numbers in lymph nodes (Katagiri, Ohnishi, et al. 2004). In addition, T cells and B cells isolated from RapL−/− mice have impaired migration to lymph
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nodes and spleen when transferred into wild-type recipient mice. Consistent with in vitro studies, RapL−/− lymphocytes exhibit impaired adhesion to integrin ligands after chemokine stimulation. This suggests that the primary function of chemokinemediated activation of Rap1 is to regulate integrin functional activity during lymphocyte migration. Analysis of RapL knockout mice also revealed a function for RapL in dendritic cell adhesion and migration (Katagiri, Ohnishi, et al. 2004). 4.
Itk
In addition to Rap1, the tyrosine kinase Itk is also activated by both TCR signaling and chemokine receptor signaling (Fischer et al. 2004; Takesono et al. 2004). In vitro migration of Itk-deficient T cells in response to CXCL12 is reduced compared with that in wild-type T cells. In addition, there is an impaired ability of T cells isolated from Itk/Rlk-deficient mice to traffic into lymph nodes in vivo (Takesono et al. 2004). Thus, in contrast to integrin activation induced by the TCR, migratory responses of T cells appear to be dependent on both Itk and, to some extent, Rlk. Itk may participate in regulating both integrin activation and actin polymerization events, as Itk−/− T cells exhibit defects in T cell adhesion to fibronectin and actin polymerization after CXCL12 stimulation (Fischer et al. 2004). Impaired actin polymerization induced by chemokine stimulation of Itk−/− T cells is also consistent with a role for Itk in regulating chemokine-mediated activation of Rac (Takesono et al. 2004). 5.
Dok-1
Dok-1 (downstream of tyrosine kinase) is a 62-kDa adapter protein that associates with Ras GTPase-activating protein (RasGAP) that becomes tyrosine phosphorylated after stimulation with a number of cytokines, including platelet-derived growth factor and vascular endothelial growth factor. Stimulation of human T cells with CXCL12 also results in tyrosine phosphorylation of Dok-1 (Okabe et al. 2004). Consistent with proposed negative regulatory functions for Dok-1, T cells isolated from Dok-1−/− mice exhibit enhanced migration in response to CXCL12 in vitro (Okabe et al. 2004).
V.
INTEGRIN SIGNALING
Upon ligand engagement, integrins generate additional intracellular signals that can modulate cell function. Despite extensive in vitro studies characterizing integrin signaling in T cells and other cell types (Perez et al. 2003), mouse knockout models have not been extensively used to study integrin signaling in T cells. One recent study used adoptive transfer approaches with CD18−/− (β2 integrin) T cells expressing a transgenic TCR to demonstrate a role for LFA-1 in optimal
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priming of CD4 T cells in vivo (Kandula and Abraham 2004). Although these results are consistent with a role for LFA-1 in generating signals necessary for optimal T cell activation in vivo, the impaired response of CD18−/− T cells in vivo may also be due to a lack of effective LFA-1-dependent adhesion between T cells and antigen-presenting cells. Mouse knockout models have been most informative in delineating integrin signaling pathways in neutrophils and macrophages, where integrin function does not require “activation” induced via signaling from other receptors. Integrin-mediated degranulation, oxidative burst, and cell spreading are impaired in neutrophils lacking expression of the myeloid src family tyrosine kinases Hck and Fgr (Lowell et al. 1996; Mócsai et al. 1999), the Syk tyrosine kinase (Mócsai et al. 2002), or the SLP-76 adapter protein (Newbrough et al. 2003). Impaired integrin signaling is also observed in macrophages isolated from Hck/Fgr double knockout mice (Suen et al. 1999). These mouse models have been instrumental in defining key intermediates involved in integrin-mediated signaling in these cell types.
VI.
FUTURE DIRECTIONS
Transgenic and knockout mice have proven instrumental in advancing our understanding of integrin function and T cell migration. In addition to identifying novel functions for proteins in regulating T cell adhesion and migration, these mouse models have allowed investigators to assess the physiological significance of perturbing signaling pathways that regulate T cell adhesion and migration. These functional responses, such as the movement of lymphocytes into tissue sites, are not easily studied in vitro. Furthermore, the availability of TCR transgenic mice provides opportunities for assessing the impact of modulating these adhesive and migratory events on antigen-specific immune responses in vivo. However, there are some limitations to the use of mouse models that need to be considered. The critical role that many signaling proteins play in developmental processes makes it impossible to use conventional knockout models to study the role that these proteins play in mature lymphocytes. Alternative approaches, such as conditional or tissue-specific knockouts, are needed to study these molecules. In addition, redundant functions for signaling proteins may confound phenotypic changes in knockout mice, and in some cases it has been necessary to simultaneously delete genes encoding for two or more molecules that are capable of compensating for each other. Future advances in our understanding of T cell integrin function and migration will undoubtedly continue to utilize the mouse as a model system. The continued use of these proven approaches, together with new technologies such as RNA interference, will expand the utility of these models. A better understanding of these important cellular events in the context of physiological immune responses
will be critical to the development of novel therapeutic strategies targeting adhesion molecules and their modulators.
VII.
SUMMARY
Integrin adhesion receptors facilitate the movement of bloodborne cells into lymphoid and nonlymphoid tissue during both steady-state and inflammatory conditions. In addition, integrins promote the stable interaction of T lymphocytes with antigenpresenting cells that are necessary for optimal adaptive immune responses. Signal transduction initiated by antigen and chemokine receptors regulates the functional activity of integrin receptors to promote appropriate periods of strong adhesion during lymphocyte migration and antigen recognition. Recent advances in this area of immunological research have come from the identification of transgenic and knockout mouse models with defects in integrin function and/or T cell migration. These mouse models provide powerful new tools for elucidating the mechanisms by which integrin function and T cell migration are regulated. In addition, these models provide unique opportunities for examining the function of integrin-dependent responses in the context of physiological immune responses that can be initiated, manipulated, and monitored in mice.
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Chapter 9 Mouse Models of Negative Selection Troy A. Baldwin, Timothy K. Starr, and Kristin A. Hogquist
I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overview of Thymic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Positive and Negative Selection—Who and Where? . . . . . . . . . . . . . . . . . . III. Models for Central Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Anti-CD3/Peptide Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Endogenous Antigen Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. TCR Transgenics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Vβ Transgenics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Endogenous Superantigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Mediators of Negative Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The T Cell Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Costimulatory Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Signal Transducers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. ERK/JNK/p38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Grb2/RasGRP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. MINK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Nuclear Factor-κB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. AIRE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Nur77 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Molecules Involved in Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Death Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
207
208
TROY
I.
A.
BALDWIN,
TIMOTHY
INTRODUCTION
T lymphocytes play a critical role in adaptive immunity to pathogens. Their ability to function properly in this role, while not causing an immune reaction to host tissues, reflects cellular properties that are established as they develop in the thymus. For this reason, the thymus has long been studied by immunologists, and many mouse models that facilitate these analyses exist. In this chapter, we discuss the utility and limitations of transgenic and gene-deficient mouse strains to model thymic processes, specifically negative selection.
K.
STARR,
Overview of Thymic Development
Thymocytes are derived from bone marrow progenitors that migrate via the blood to the thymus. The thymus is composed of two distinct areas, the outer cortex and inner medulla. Development of thymocytes proceeds through a well-defined series of intermediates culminating with exit from the thymus and entry into the periphery (Starr et al. 2003). The most immature thymocytes enter the thymus at the cortical-medullary junction and do not express either coreceptor CD4 or CD8 and are thus called “double negative” (DN). After entering the thymus, DN thymocytes begin to traffic outward toward the capsule and continue to develop. Progression within the DN compartment is broken down further into four steps (DN1–DN4) identified by the dynamic expression of CD44 and CD25. At the DN3 stage, rearrangement of the T cell receptor (TCR) β locus occurs, and, if it is successful, the TCRβ chain pairs with a pre-TCRα chain, traffics to the cell surface, and initiates the process of β-selection. An assembly-dependent signaling complex induces a burst of proliferation, initiates rearrangement at the TCRα locus, and sends survival signals to DN3 thymocytes (Michie and Zuniga-Pflucker 2002). After completion of β-selection, expression of CD4 and CD8 is induced, resulting in a “double-positive” (DP) thymocyte. At this stage a mature αβ-TCR is expressed, and the DP thymocyte enters the cortex and begins a return journey toward the medulla. All subsequent selection events are mediated by the clonal αβ-TCR. The selection process is necessary for the further development of those DP thymocytes that express a “useful” TCR (positive selection) and the elimination of those cells that express an overtly self-reactive TCR (negative selection). Negative selection provides the basis for the phenomenon of central tolerance. Recently, the clinical importance of central tolerance has been directly demonstrated by the loss of a critical transcription factor, the autoimmune regulator (AIRE), which results in an inherited autoimmune syndrome in both mouse and man (Venanzi et al. 2004). One of the long-standing questions in developmental immunology is how engagement of the same TCR by different ligands can lead to dramatically different fates—life or death. The answer appears to be tied to the affinity of the TCR for
KRISTIN
A.
HOGQUIST
self-peptide major histocompatability complexes (MHCs) (Alam et al. 1996). The affinity paradigm proposes that if the TCR interacts with self-peptide MHCs with a high affinity, a program of death ensues. If, however, the TCR interacts with self-peptide MHCs with a low to moderate affinity, the cell will receive survival signals and continue development to either the CD4 single-positive (CD4SP) or CD8 single-positive (CD8SP) lineage. Although this appealingly simple model can explain much of the data, other factors have been demonstrated to play a significant role in these selection processes.
II. A.
AND
POSITIVE AND NEGATIVE
SELECTION—WHO AND WHERE? The cell type expressing the self-peptide MHC probably plays an important role in the decision of the DP thymocyte to live or die. As was stated previously, the thymus is divided into two principal regions, the cortex and the medulla. The cortex comprises mostly cortical thymic epithelial cells (cTECs), fibroblasts, and DP thymocytes, whereas the medulla comprises medullary thymic epithelial cells (mTECs) and medullary dendritic cells (mDCs). These different compartments and the cell types residing within them are necessary for all the selection events, yet they play different roles in the selection process. As the DP thymocyte travels through the cortex toward the medulla, it first encounters cTECs. By restricting expression of MHC class II or I to cTEC using a keratin 14 (K14) promoter in class II- or I-deficient mice, respectively, positive selection was restored (Capone et al. 2001; Laufer et al. 1996). Interestingly, negative selection was impaired, resulting in self-reactivity in the periphery, implying the importance of the medulla for central tolerance. Other evidence also suggests that deletion occurs in the medulla. For example, when deletion of superantigen reactive thymocytes in MHC-sufficient or -deficient animals was compared, apoptotic cells were detected in the medulla of MHC-sufficient animals (Surh and Sprent 1994). Additionally, when MHC expression was restricted to the DC compartment, and thus the medulla (Shortman et al. 1998), by the CD11c promoter, deletion was observed (Brocker et al. 1997; Cannarile et al. 2004). Furthermore, ligation of CD40 has been demonstrated to be essential for clonal deletion by endogenous superantigen (Foy et al. 1995). Stimulation of CD40 upregulated the expression of B7 exclusively in the medulla. Therefore, the medulla and more specifically the medullary DCs are thought to be prominent in mediating deletion. Finally, AIRE expression is restricted to medullary TECs (Zuklys et al. 2000), and either direct presentation by mTECs or cross-presentation by mDCs of high-affinity ligand is sufficient to induce deletion (Gallegos and Bevan 2004). Overall, one can generalize that positive selection occurs in the cortex and is mediated by cTECs, whereas negative selection is restricted to the medulla. Interestingly, CCR7 is thought to be important for the directed
9. MOUSE
MODELS
OF
NEGATIVE
migration of thymocytes to the medulla, but the Takahama group showed that neither CCR7 expression on the thymocyte nor CCL19 or 21 expression in the medulla was required for thymocytes to be deleted or to exit the thymus (Ueno et al. 2004). These studies, however, were not conducted in mixed bone marrow chimeras with a normal thymic architecture, and thus it remains possible that CCR7 does play a role in directing the progenitor to the medulla for deletion.
III.
209
SELECTION
MODELS FOR CENTRAL TOLERANCE
Over the past 15–20 years, there have been numerous models used to study negative selection in the mouse. Early work focused on examination of deletion in two principle models, the first model being superantigen-mediated deletion in wild-type mice and the second being deletion in TCR transgenic mice. Interestingly, these two models produced different conclusions regarding the developmental stage of deletion. These discrepancies have made it difficult to map the molecular factors/ pathways controlling deletion. In describing the different models, we will attempt to point out strengths and weaknesses of each model and how these might affect results obtained with the different models.
(non–antigen-specific) DP thymocytes were deleted (Martin and Bevan 1997). DP thymocytes are particularly sensitive to apoptosis before positive selection, so it is these cells that are lost. These findings highlight the major drawbacks to using injection of antiTCR or peptide as a model for studying negative selection. To overcome some of the limitations of the aforementioned models, researchers have used fetal thymic organ culture (FTOC) or reaggregate thymic organ culture as an alternative. These model systems use an intact fetal lobe or empty thymic lobe seeded with thymocytes. With these models many different TCR stimuli can be used; costimulation is provided by physiological thymic APCs, and mature T cells are not present to respond to the stimulus. In general, this is a better alternative than the in vitro systems, but is somewhat difficult to carry out (Anderson and Jenkinson, 1998).
B.
Endogenous self-antigen expression is utilized in many models. These are powerful in vivo model systems, and selfreactive cells are tracked either by use of a TCR transgene, peptide/MHC tetramers, or Vβ antibodies in the case of endogenous superantigens. 1.
A.
Anti-CD3/Peptide Models
There are a number of models of negative selection in which bulk thymocyte populations are stimulated in vitro or in vivo with antibodies to CD3 or in the case of TCR transgenics, MHC-binding peptides. The in vitro models have the advantage of being cost and time effective. They are also extremely easy to manipulate by changing the amount of TCR stimulation or the costimulation molecule engaged. However, in most cases the TCR stimulus is artificial, and if antigen-presenting cells (APCs) are used, often they are not thymically derived. The biggest drawback to these in vitro models is that the assay is not conducted in the context of an intact thymus, and the threedimensional architecture of the thymus appears to be important for appropriate selection (van Ewijk 1991). In conclusion, although the in vitro systems are easy to use, the physiological relevance of the models is highly questionable. The in vivo administration of anti-CD3 or peptide has the benefit that negative selection occurs in an intact thymus; however, this alone may not be sufficiently physiologic. For example, in these cases, deletion occurs early in development at the DP stage, which is now recognized to be a nonspecific consequence of peripheral T cell activation due to the production of glucocorticoids and cytokines (Brewer et al. 2002; Martin and Bevan 1997; Zhan et al. 2003). In fact, when mature antigenspecific T cells were adoptively transferred into a wild-type host and activated by peptide injection, the host-derived
Endogenous Antigen Models
TCR Transgenics
As an alternative to the artificial injection of anti CD3 or peptide, negative selection to endogenous self-antigens can be modeled in TCR transgenics (Table 9-1). In many of these models, deletion occurs either at the DP or cortical stage, whereas in other models, deletion occurs at the SP or medullary stage. Clearly, where the self-antigen is expressed can contribute to this difference; however, this location does not seem to explain all of the data. It has been proposed that TCR affinity dictates the stage of deletion (Sant’Angelo and Janeway 2002), because DP cells have a lower level of TCR and are presumed to be less sensitive. However, direct tests of this hypothesis revealed that DP cells are as sensitive as mature cells (Davey et al. 1998). An additional factor to consider regarding TCR transgenics is their elevated, premature TCR expression in DN cells. Recently, this early TCR expression was demonstrated to have an impact on negative selection in the HY TCR model such that when the TCR was expressed at the physiological DP stage, deletion occurred late in development (Baldwin et al. 2005). Therefore, although TCR transgenics provide a powerful means to model negative selection, deletion occurs at an early stage, which is nonphysiological, in many models and again can lead to confounding information about molecular mechanisms. 2.
Vβ Transgenics
One means to avoid the overwhelming monoclonality and problematic early α chain expression of TCR transgenics is to
TABLE 9-1
210
TCR TRANSGENIC MODELS Model of Negative Selection
Name
Reference
2C
McGargill et al. 2002; Sha et al. 1998 Auphan et al. 1994 Morahan et al. 1991
BM3.3 F3
MHC Class
TCR Transgenes
Positive Selection Background
I
Vα3/Vβ8
H-2b
I I
?/? Vα8/Vβ11
H-2k H-2bm1
Antigen
NP transgenic
I
H-2k, H-2kxd
N15
Ghendler et al. 1997
I
Vα8/Vβ5.2
H-2b (Kb)
NOD.AI4, AI4 OT-1
Choisy-Rossi et al. 2004
I
Vα8/Vβ2
I
Vα2/Vβ5
P14
Gallegos and Bevan 2004; McGargill et al. 2000 Pircher et al. 1989
H-2g7 (NOD) (Kd) H-2b (Kb)
I
Vα2/Vβ8.1
H-2b (Db)
P1ACTL
Sarma et al. 1999
I
Vα8/Vβ1
H-2d (Ld)
Lymphocytic choriomeningitis virus (LCMV) GP33 P1A 35–43
QM11
Suzuki et al. 1994
I
Vα10/Vβ4
H-2q
I-Ak alloreactive
RT1 ST40 ST42 T1
Yokosuka et al. 2002
Doucey et al. 2003
I I I I
Vα42/Vβ8.1 Va16/Vb1 Va16/Vb1 Vα?/Vβ?
H-2d (Dd) H-2b (Db) H-2b (Db) H-2b
TG-B 6C5
Geiger et al. 1992 Sullivan et al. 2002
I Ib
Vα5/Vβ8 Vα3.2/Vβ5.1
H-2k (Kk) H-2b or H-2d (Qa-1b) H-2b (H-2M3)
HIV-1 gp160 Adenovirus E1a 234–243 Adenovirus E1a 234–243 Plasmodium berghei circumsporozoite Ag SV40 large T antigen Human insulin B chain
Vα4.2/Vβ8.1
Ib
Vα10.2/Vβ5.2
1H3.1
II
Vα1/Vβ6
Viret and Janeway 2000
H-2b or H-2d (H2-M3) H-2b (I-Ab)
Listeria monocytogenes AttM f-MFFINILTL L. monocytogenes LemA protein Peptide from I-Ea chain
B6.H2g7 RIP-OVA, K14OVAp K14-OVA, Act-OVA Neonatal (chronic) LCMV infection P1A (no deletion)
I-E+/Mtv 6/9+ strains Mlsa
Eµmb-P1A Strains expressing I-Ak
RIP-SV40
I-Ab/Ea peptide
HOGQUIST
Ib
Vesicular stomatitis virus 52–59 Uncharacterized β cell antigen Chicken ovalbumin (OVA) 257–264
A.
C10.4 Berg et al. 1999 TCRtrans+ D7 Chiu et al. 1999
H-2Kb expressing strains
KRISTIN
Schonrich et al. 1991
Male mice
AND
Kb5.C20, Des
H-2b (Db)
K14-male antigen transgenic
STARR,
I
Male mice
I-E+/ Mtv8/9/11
K.
H-2b (Db)
Baldwin et al. 2005
Vα17/Vβ 8.2 Vα17/Vβ 8.2 ?/?
B6 H-2Kb expressing strains
TIMOTHY
I
Ld
A.
H-2b (Db)
Alloantigen
BALDWIN,
Influenza nucleoprotein 366–374 Mouse male antigen SMCY 738–764 Mouse male antigen SMCY 738–764 H-2Kb alloantigen
Vα4/Vβ11
Superantigen
TROY
“SIY” peptide K14 promoter Kb transgenics RIP-Kb
I
HY (B6.2.12) HYcd4
Transgenic Antigen
Ld alloantigen (synthetic peptide SIYRYYGL H-2Kb) H-2Kb/PBM1 alloantigen Kb alloantigen
Mamalaki et al. 1993; Wack et al. 1996 Kisielow et al. 1988
F5
Endogenous Antigen
3.L2
Haribhai et al. 2003; Kersh et al. 1998 Haribhai et al. 2003; Zhang et al. 2003 Schmidt et al. 1997
II
Vα18/Vβ8.3
H-2k (I-Ek)
II
Vα3/Vβ8.2
H-2k (I-Ak)
II
Vα4.1/Vβ11
H-2g7 (I-Ag7)
Bogen et al. 1993 Girgis et al. 1999 Lanoue et al. 1997; Jordan et al. 2001; Reed et al. 2003; Sarukhan et al. 1998 Zal et al. 1994
II II II
Vα1, Vβ8.2 Vα11, Vβ3 Vα4/Vβ8.2
H-2d (I-Ed) H-2a (I-Ek) H-2d (I-Ed)
II
Vα11.1/Vβ8.3
H-2a (I-Ek) (C5a negative)
Complement protein C5 107–121
ABLE ABM or 3BBM74
Reiner et al. 1998 Backstrom et al. 1998
II II
Vα8.2/Vβ4 Vα2/Vβ8
H-2d (I-Ad) H-2b
Leishmania major LACK I-Abm12
AND
Liu et al. 1997; Oehen et al. 1996; Vasquez et al. 1992
II
Vα11/Vβ3
I-Ek or I-Ab (I-Ek or I-Ab)
MCC 88–103/PCC 88–104
B5 BDC2.5
Granucci et al. 1996 Katz et al. 1993
II II
Vα4/Vβ14 Vα1/Vβ4
H-2d (I-Ad) H-2g7 NOD
IgG2ab 435–451 Uncharacterized islet cell antigen
D011.10
II
Vα?/Vβ8.2
H-2d (I-Ad)
Chicken ovalbumin 323–339
D10, D10.G4.1
Fremont et al. 1996; Ignatowicz, Kappler, Marrack, 1996; Ignatowicz, Kappler, Parker, et al. 1996; Kawahata et al. 2002; Liu et al. 1996; Vella et al. 1996; Sant’Angelo and Janeway 2002
II
Vα2/Vβ8.2
H-2k (I-Ak)
Chicken conalbumin 134–146
Dep
Klein et al. 1998
II
Vα11.2/Vβ5.1
H-2b (I-Ab)
Ea6 G2, 2.102 G286, B16.3 KB
Viret et al. 2000 Hsu et al. 1995
II II
H-2b (I-Ab) H-2k (I-Ek)
Tarbell et al. 2002
II
Vα23.1/Vβ6 endogeno us α/Vβ1 Vα4.5/Vβ1
He et al. 2002
II
Vα10/Vβ8
(I-As)
Human reactive C protein (hCRP) 89–101 I-Eα-chain 52–66 Mouse hemoglobin d allele 64–76 Mouse glutamic acid decarboxylase 65 286–300 Human collagen IV α2 chain 675–686
3A9 4.1, NY4.1 4B2A1 5C.C7 6.5, HA, TS1
A18, C5
Pigeon cytochrome c (PCC) 81–104 Human hemoglobin (Hbb) d allele 64–76 Hen egg lysozyme (HEL) 46–61 Uncharacterized β cell antigen MOPC315 IgG2 91–101 PCC/MCC 88–104 Influenza hemaglutinin (HA) 111–119
PCC transgenic 1) 2) 1) 2)
Mls-2a and Mls-3a
Hbbd Tg tet HEL/Hbd Tg HEL Tg tet HEL/Hbd Tg
NOD β cell antigen MOPC315 BCR Tg MX-MCC-Hel Tg Igk-HA, RIP-HA, SV40-HA, Pev-HA, β-myo HA CBA mouse strain (C5a+) B6.bm12
1) Kb-PCC transgenic 2) I-Ek MCC peptide transgenic
I-As
Ld-nOVA transgenic
I-Ab
SELECTION
H-2a (I-Ek)
NEGATIVE
Vα?/Vβ3
OF
II
MODELS
Berg et al. 1989
9. MOUSE
2B4
NOD, no overt deletion seen
H-2 b, q, d, f, r, and u hCRP Tg I-Eαd transgenic Hbbd+ strains NOD
211
Continued
212
TABLE 9-1
TCR TRANSGENIC MODELS—cont’d Model of Negative Selection
Bovine pancreas RNAse alloreactive to H-2g7 41–61
Huseby et al. 2001
II
Vα2/Vβ8
H-2u (I-Au)
Goverman et al. 1993 II Lafaille et al. 1994 II Gallegos and Bevan 2004 II
Vα2.3/Vβ8.2 Vα?/Vβ8 Vα2/Vβ5
H-2u (I-Au) H-2u (I-Au) H-2b (I-Ab)
Sep SM1 Smarta
Klein et al. 1998 McSorley et al. 2002 Oxenius et al. 1998
II II II
Vα4/Vβ8.3 Vα10/Vβ2 Vα2.3/Vβ8.3
H-2b (I-Ab) H-2b (I-Ab) H-2b (I-Ab)
Murine myelin basic protein (MBP) 121–150 Murine MBP 1–11 Murine MBP 1–11 Chicken ovalbumin protein 323–339 hCRP 80–94 Salmonella flagellin 427–441 LCMV GP 61–80
TCli
Wong et al. 2000
II
Vα18/Vβ6
H-2b (I-Ab)
TCRHNT TEa
Scott et al. 1994
II
Vα?/Vβ8
H-2d (I-Ad)
Grubinet al. 1997
II
Vα2/Vβ6
H-2b (I-Ab)
Human invariant chain CLIP fragment 81–104 Influenza virus hemagglutinin 126–138 Murine I-Ea-chain 52–68
DN (CD1) SP (CD1)
Cheng et al. 1996 Cheng et al. 1996
Vα4.4/Vβ9 Vα4.4/Vβ9
H-2d H-2d
Probable alloreactivity to CD1 Probable alloreactivity to CD1
MBP, 121-150 MBP, 172.1 MBP, 19G OT-II
Eα-glucose-6phosphate isomerase peptide transgenic Endogenous MBP
RIP-OVA hCRP Tg Neonatal LCMV infection
Alloantigen NOD
Strains expressing I-Ea
AND
H-2k (I-Ak)
Superantigen
STARR,
Vα4/Vβ6
Transgenic Antigen
K.
II
Endogenous Antigen
TIMOTHY
Kouskoff et al. 1996; Shih et al. 2004
KRN, R28
Antigen
BALDWIN,
Positive Selection Background
A.
TCR Transgenes
Reference
TROY
MHC Class
Name
KRISTIN A. HOGQUIST
9. MOUSE
MODELS
OF
NEGATIVE
use mice expressing only the β chain of a particular TCR. Such mice have a diverse repertoire, but in many cases, the antigenspecific precursors are detectable with tools such as peptide/MHC tetramers (Baldwin et al. 1999) or by knowing which Vα chains give reactivity (Holman et al. 2003; Mikszta et al. 1999). This is an excellent model for some questions. One drawback, however, is that the level of TCR expressed on DP cells usually precludes their analysis with tetramers, so studying the stage and biochemical mechanism of negative selection is difficult with this model. 3.
213
SELECTION
Endogenous Superantigens
Arguably the most physiological model of negative selection to date is endogenous superantigen deletion in wild-type mice, which was in fact the first way that negative selection was modeled experimentally (Kappler et al. 1987). TCRVβ+ DP cells were present, but mature SP thymocytes were absent, suggesting that deletion occurred after positive selection, as cells transitioned to the SP stage. Studies by Surh and Sprent (1994) demonstrated that in MHC+ mice, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)+ cells were present in the medulla, whereas in MHC− mice, those cells were absent, confirming a late deletion mechanism. This model has a number of benefits, including the use of nontransgenic mice and endogenous antigen presentation. This mechanism of deletion occurs most efficiently in I-E+ strains. However, the strains most commonly used for gene targeting are C57BL/6 and 129, which are I-E−; thus, extensive breeding is required to test the role of various genes using this model. Another drawback is that the interaction of MHC-superantigen and TCR is quite different from the interaction of TCR with peptide-MHC, possibly resulting in differential signal transduction.
In summary, we assert that the most physiological models of negative selection in mice are those that display late deletion mediated by an endogenous peptide antigen in a Vβ or TCR transgenic mouse.
IV.
MEDIATORS OF NEGATIVE SELECTION
Two nonmutually exclusive paradigms have emerged in the field of positive and negative selection, the “affinity” model and the “unique costimulus” model. The affinity model postulates that the affinity of the clonally expressed TCR for self-peptide–MHC complexes is responsible for inducing either positive or negative selection (Starr et al. 2003), whereas the unique costimulus model suggests that ligation of an additional costimulatory receptor is required for deletion but not for positive selection (Amsen and Kruisbeek 1998). Probably both are true. One can envision a situation in which a high-affinity ligand but not a low-affinity ligand can induce the expression of a costimulatory receptor on the DP thymocyte and that ligation of this costimulatory receptor in the medulla is necessary to complete negative selection. In either case, it remains unclear how TCR and/or coreceptor engagement leads to apoptosis. Additionally, a great deal of controversy surrounds some molecules that have been shown to be important for deletion in certain systems but not others, perhaps due to the plethora of models used and their inherent differences. Because positive selection is thought to precede negative selection, many of the proximal signaling molecules downstream of the TCR are required for both positive and negative selection. In this chapter we focus exclusively on the pathways and molecules uniquely involved in negative selection. Table 9-2 provides an overview of the molecules
TABLE 9-2
MOLECULES INVOLVED IN NEGATIVE SELECTION Family
Molecule Affected
Modulation
Model
Effect
Reference
TCR
TCRζ
ITAM deletion
TCRβ TCRα CD3γ
FG loop deletion α-CPM deletion CD3γ ∆-ITAM knockin CD40L−/−
HY Exogenous superantigen N15 peptide injection OT-I FTOC F5 CD3γ ∆-ITAM knockin in vitro HY Endogenous superantigen AND/H-2a
Impaired Impaired Impaired None None
Shores et al. 1997 Shores et al. 1997 Sasada et al. 2002 Werlen et al. 2000 Haks et al. 2002
None Impaired
Page et al. 1998 Foy et al. 1995
Impaired
Foy et al. 1995
Endogenous superantigen Exogenous superantigen P1ACTL Endogenous superantigen
Impaired
Buhlmann et al. 2003
None Impaired Impaired
Buhlmann et al. 2003 Gao et al. 2002 Gao et al. 2002
Costimulator
CD40L-CD40
B7-1/B7-2-CD28
B7-1/B7-2−/−
Perinatal Ab. blockade
Continued
214
TROY
A.
BALDWIN,
TIMOTHY
K.
STARR,
AND
KRISTIN
A.
HOGQUIST
TABLE 9-2
MOLECULES INVOLVED IN NEGATIVE SELECTION—cont’d Family
Molecule Affected
Modulation
Model
Effect
Reference
Death receptor
Fas
Fas−/− (lpr)
Superantigen
None Impaired Impaired Impaired (high dose)
Kotzin et al. 1988 Dautigny et al. 1999 Page et al. 1998 Kishimoto et al. 1998
Impaired (high dose)
Kishimoto et al. 1998
Impaired Impaired Impaired (low Ag dose)
Castro et al. 1996 Castro et al. 1996 Schmitz et al. 2003
FasL blockade
Signal transducers
TNFR1 TNFR2 TRAIL DR3
TNFR1−/− TNFR2−/− TRAIL−/− DR3−/−
FADD-dependent death receptors Erk
Dominant-negative FADD Tg Dominant negative MEK Tg Dominant negative JNK1 Tg JNK 2−/− SB203580 inhibitor Grb2+/-
JNK1 JNK2 p38 Grb2
Transcription factors
MINK
MINK RNA interference
NF-κB
Retroviral overexpression of IκBNS AIRE−/− Dominant negative Nur77 Tg Apaf-1−/−
AIRE Nur77 Apoptosis
Apaf-1 Caspases
Bcl-2
Caspase 3−/− Caspase 8−/− Caspase 9−/− zVAD
Baculovirus p35 Tg EµBcl-2 Tg lckpr Bcl-2 Tg
Bak/Bax
Bak/Bax−/−
Bim
Bim−/−
HY Exogenous superantigen (neonates) Peptide injection of D011.10 (neonates) Anti-CD3 in vivo Peptide injection of D011.10 VSV8 peptide treated N15 FTOC Endogenous superantigen Endogenous superantigen Endogenous superantigen Endogenous superantigen HY Superantigen HY HY
None None None None Impaired None None None
Pfeffer et al. 1993 Erickson et al. 1994 Cretney et al. 2003 Wang et al. 2001 Wang et al. 2001 Newtonet al. 1998 Newton et al. 1998 Alberola-Ila et al. 1995
Peptide-injected AND Tg
Impaired
Rincon et al. 1998
Anti-CD3 injection in vivo Inhibitor-treated HY FTOC HY Anti-CD3 injection in vivo Endogenous superantigen HY Peptide injection of OT-II Anti-CD3 treatment of retrovirally infected RTOC 3A9 insHEL Tg NP-injected F5 Exogenous superantigen HY HY FTOC Anti-CD3/CD28 in vitro Anti-CD3 in vitro Anti-CD3/CD28 in vitro OT-I in vitro peptide
Impaired Impaired Impaired Impaired Impaired Impaired Impaired Enhanced
Sabapathy et al. 2001 Sugawara et al. 1998 Gong et al. 2001 Gong et al. 2001 McCarty et al. 2005 McCarty et al. 2005 McCarty et al. 2005 Fiorini et al. 2002
Impaired Impaired None None Impaired None None Impaired None Impaired
HY Peptide injection of F5 Endogenous superantigen HY Endogenous superantigen HY Anti-CD3 in vitro Endogenous superantigen Exogenous superantigen FTOC Peptide injection of OT-II HY
None Impaired None None Impaired Impaired Impaired Impaired Impaired
Liston et al. 2003 Calnan et al. 1995 Calnan et al. 1995 Hara et al. 2002 Matsuki et al. 2002 Kuida et al. 1996 Salmena et al. 2003 Kuida et al. 1998 Doerfler et al. 2000 McGargill, Hogquist 1999 Doerfler et al. 2000 Izquierdo et al. 1999 Sentman et al. 1991 Tao et al. 1994 Strasser et al. 1991 Strasser et al. 1994 Rathmell et al. 2002 Rathmell et al. 2002 Bouillet et al. 2002
Impaired Impaired
Bouillet et al. 2002 Bouillet et al. 2002
9. MOUSE
MODELS
OF
NEGATIVE
demonstrated to play a role in negative selection and the models used to test their role.
A.
The T Cell Receptor
The central player in clonal deletion is the TCR complex. Ligation of the TCR by high-affinity antigen and subsequent signal transduction are required for negative selection. The TCR complex is composed of disulfide-linked TCRα and β chains and noncovalently associated CD3γ/ε, CD3δ/ε, and TCRζ/ζ dimers. Deletion of a segment of the TCRβ chain (the FG loop), which may be involved in the association of CD3ε with the TCR, blocked negative selection in VSV8 peptide–injected N15 TCR transgenic mice (Sasada et al. 2002). In support of a role for TCRβ in mediating apoptosis, deletion of the TCRβ transmembrane and cytoplasmic domains impaired the ability of peripheral T cells to undergo activationinduced cell death after antigen stimulation (Teixeiro et al. 2004). The effect of this deletion on negative selection in the thymus has yet to be investigated. In contrast, a mutation in the TCRα chain (α-CPM), which results in a failure to recruit CD3δ upon TCR ligation, had no effect on negative selection in FTOC (Werlen et al. 2000). Based on this evidence, one could speculate that TCRβ plays an important role in signal transduction, leading to negative selection. The CD3/TCRζ complex contains a total of 10 signal transducing immunoreceptor tyrosine-based activation motifs (ITAMs). These motifs have been mutated in an attempt to decipher whether individual CD3 chains provide unique signals for negative selection or whether deletion is dependent on the “amount” of signal (i.e., number of ITAMs). Work by Shores et al. (1997) suggested that a reduction in the number of TCRζ ITAMs (one instead of three) resulted in impaired negative selection. This was observed in both early (HY male) and late (superantigen) models of deletion. On the other hand, when CD3γ lacked an ITAM, positive selection was impaired, but using an in vitro model, negative selection was not (Haks et al. 2002). Additionally, the CD3ε ITAM was not required for positive selection, whereas negative selection was not directly tested (Sommers et al. 2000). Therefore, there does appear to be a requirement for a minimal number of ITAMs, but the molecular organization of the TCR/CD3 complex also appears to be critical for negative selection. These findings suggest that the “quality” of signal received by the thymocyte plays a key role in negative selection, supporting the affinity model of deletion.
B.
215
SELECTION
Costimulatory Molecules
The role of costimulatory molecules in negative selection has been controversial. Some of the most compelling evidence for the role of costimulatory molecules in negative selection
involves the requirement for CD40-CD40 ligand (CD40L) and B7/CD28 interactions. With use of antibody blockade or CD40L-deficient mice, deletion was attenuated when CD40 on thymic APCs was not ligated (Foy et al. 1995). Interestingly, CD40L acts in a non–cell-autonomous fashion such that when both CD40L-expressing and CD40L-deficient thymocytes are present in the thymus, both populations are equally susceptible to deletion (Williams et al. 2002). These data suggest that ligation of CD40 on thymic APCs results in the upregulation or induction of a costimulatory molecule on the APCs required for deletion and supports a “unique costimulus” model for deletion. However, the requirement for CD40-CD40L interactions in negative selection has been demonstrated in late models of deletion, whereas most early models of deletion show no such requirement (Page et al. 1998). One of the molecules shown to be regulated by the CD40-CD40L interaction is B7-2 (Foy et al. 1995). A number of early studies showed either a limited or no role for CD28/B7 interactions in negative selection; however, recent experiments using B7-1/B7-2 double knockouts have demonstrated impaired endogenous superantigen-mediated deletion (Buhlmann et al. 2003). Furthermore, perinatal blockade of B7-1 and B7-2 leads to a decrease in negative selection and an increase in pathogenic T cells in both transgenic and superantigen models (Gao et al. 2002). Other costimulatory molecules such as CD43 and CD5 have, in some circumstances, been shown to affect deletion. In most cases the effect is subtle. However, when ligated with antibodies in combination with anti-CD28, their effect is much more pronounced in in vitro assays (Li and Page 2001). Because no single costimulatory molecule appears essential for deletion in all models, there is some speculation that signal transduction through these receptors is redundant or that high-affinity receptors do not require costimulation for deletion. Further work needs to be done to address this issue. Based on the data regarding the role of various cell surface receptors, it is likely that affinity plays a key role in determining the fate of the DP progenitor, but it is also likely that a second costimulatory signal provided by a medullary APC is also required. We hypothesize that induction of the costimulatory receptor on the progenitor is a consequence of a high-affinity TCR stimulus whereas expression of the costimulatory ligand on the APC may be mediated by CD40-CD40L interactions. Therefore, we favor a combination of both the affinity and unique costimulus models in negative selection.
C.
Signal Transducers
For the progenitor to undergo death, signal transduction pathways emanating from the TCR and perhaps other cell surface receptors must be initiated. For the most part, the pathways involved in clonal deletion are incompletely understood. However, a few key signal transducing molecules and pathways important in negative selection have been identified.
216 1.
TROY
A.
BALDWIN,
TIMOTHY
Grb2/RasGRP
The canonical MAPK signaling pathway is initiated by the activation of the small GTPase, Ras, by a guanine nucleotide exchange factor (GEF). In a number of cell types, Ras activation is mediated through the adaptor Grb2 binding to the GEF, Sos. Haplo-insufficiency at the Grb2 locus does not impair positive selection (Gong et al. 2001), as Ras activation during positive selection has been shown to be dependent on the presence of another GEF, namely RasGRP (Dower et al. 2000). Interestingly, haplo-insufficiency at the Grb2 locus did impair negative selection in both the HY and anti-CD3 injection negative selection models (Gong et al. 2001). This haploinsufficiency at the Grb2 locus did not affect MAPK activation, but did impair JNK and p38 activation, indirectly supporting a role for these molecules in negative selection. Therefore, thymocytes may have evolved different mechanisms for differential activation of the MAPKs in positive versus negative selection. 3.
MINK
The MAP4K, MINK, was recently identified as a molecule induced in DP thymocytes, and “knockdown” of MINK expression by RNA interference did not affect positive selection but dramatically inhibited clonal deletion (McCarty et al. 2005). This impairment in negative selection was observed in superantigen, peptide injection, and the HY models of deletion. MINK was demonstrated to interact with Nck, which has been shown to associate with the TCR through CD3ε. MINK was also demonstrated to be required for phosphorylation of JNK and subsequent induction of Bim (another molecule necessary for deletion that will be discussed later). MINK knockdown had no effect on ERK activation (McCarty et al. 2005). Therefore, MINK may be a direct link from the TCR leading to deletion. It will be interesting to determine whether the affinity of the TCR differentially regulates MINK and what the other players in this pathway are.
STARR,
D.
ERK/JNK/p38
The mitogen-activated protein kinase (MAPK) pathways have received considerable attention in both positive and negative selection. The extracellular signal-regulated kinase (ERK) pathway, although required for positive selection appears to be dispensable for negative selection (Alberola-Ila et al. 1995). There is evidence for a role of the c-Jun NH2-terminal kinase (JNK) and p38 pathways in negative selection. DN JNK1expressing (Rincon et al. 1998) or JNK2-deficient thymocytes (Sabapathy et al. 2001) were partially impaired in apoptosis, and treatment of thymocytes with a p38 inhibitor (SB203580) also interfered with negative selection (Sugawara et al. 1998). However, these findings have yet to be confirmed in more physiologic models of negative selection. 2.
K.
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KRISTIN
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HOGQUIST
Transcription Factors
Differential changes in gene expression are thought to be important factors influencing the decision of the progenitor to live or die. Therefore, the identification of specific transcription factors that are necessary or important for negative selection has been intensely investigated. To this point, only a few such transcription factors have been identified. 1.
Nuclear Factor-κB
The nuclear factor (NF)-κB transcription factor family is involved in many immune cell processes including development and survival. A novel inhibitor of NF-κB, IκBNS, was identified as a transcript specifically induced in DP thymocytes after peptide injection of N15 TCR transgenic mice (Fiorini et al. 2002). Retroviral overexpression of IκBNS resulted in increased thymic negative selection in FTOC treated with anti-CD3. Therefore, by inhibiting NF-κB, DP thymocytes were more sensitive to this type of stimuli. Other evidence for the role of NF-κB in negative selection exists; however, NF-κB is such an important factor for numerous processes and cell types, it is difficult to separate its requirement in other processes with those in negative selection. 2.
AIRE
Mutations in the AIRE gene result in a severe, multiorgan autoimmune syndrome in both mouse and man (Anderson et al. 2002; Bjorses et al. 1998). This autoimmunity was shown to be a result of defective negative selection in the thymus (Liston et al. 2003). AIRE is critical for the antigen presentation of tissuespecific antigens (TSA) such as insulin, thyroglobulin, and others (Anderson et al. 2002). Interestingly, AIRE-dependent presentation of TSAs occurs within a subset of medullary epithelial cells, further supporting the role of the medulla as the primary site of negative selection (Derbinski et al. 2001). AIRE contains a DNA-binding domain and thus has been postulated to be a transcription factor, driving transcription of these TSAs in the thymus (Pitkanen et al. 2000). Additionally, AIRE possesses an E3 ubiquitin-ligase activity, implicating a role for AIRE in antigen presentation by directing proteins to the proteosome as opposed to simply providing TSA transcripts (Uchida et al. 2004). In fact, work by the Matsumoto group demonstrated transcription of fodrin in AIRE-deficient mice, yet antigen presentation of fodrin was still impaired in this situation, resulting in autoimmunity highlighting the importance of the E3 ubiquitin-ligase activity in deletion (Kuroda et al. 2005). It will be of great interest to determine other transcription factors responsible for TSA expression in the absence of AIRE and the role of the E3 ubiquitin-ligase activity of AIRE.
9. MOUSE
3.
MODELS
OF
NEGATIVE
Nur77
Another transcription factor identified through a genetic screen for transcripts induced during apoptosis in T cell hybridomas was Nur77 (Liu et al. 1994). Nur77 is a member of the orphan steroid receptor transcription factor family. Nur77 is highly upregulated after TCR stimulation in DP thymocytes (Liu et al. 1994), and expression of a dominant negative form of Nur77 prevented negative selection in NP peptide–injected F5 TCR transgenic mice but not superantigen-mediated deletion (Calnan et al. 1995). Interestingly, Nur77-deficient thymocytes still undergo deletion (Lee et al. 1995), but it is thought that the Nur77 family member NOR1 can compensate for the loss of Nur77 in this situation (Cheng et al. 1997). Interestingly, in analysis of genes induced by Nur77 overexpression, two novel genes were identified. One gene, NDG1 (Nur77 downstream gene 1), induced apoptosis when expressed in 293 cells, but was blocked by the caspase 1 and 8 inhibitor, CrmA (Cheng et al. 1997). It will be of interest to identify other genes regulated by Nur77 in hopes of identifying factors involved in negative selection.
E.
Molecules Involved in Apoptosis
Although there is a considerable amount of information on the pathways leading to apoptosis in many cell types and a lot of effort has gone into understanding how apoptosis is mediated by negative selection, a clear paradigm has not yet emerged. There are two general pathways leading to death, one involving death receptor signaling (type I), and one not (type II). There are commonalities between these pathways, such as caspase activation and disruption of the mitochondria, but there are differences as well, including the mechanism of caspase activation. 1.
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Death Receptors
Death receptor expression and ligation in peripheral T cells results in apoptosis through activation of caspases. Caspases can be recruited to death receptors through the association with death domain–containing adaptor proteins, such as Fas-associated death domain (FADD), and subsequently activated by proteolytic cleavage. For the most part, death receptor ligation does not appear to be required for negative selection. Deficiency of tumor necrosis factor receptor (TNFR) 1 (Pfeffer et al. 1993), TNFR2 (Erickson et al. 1994), death-inducing receptor-3 (Wang et al. 2001), and TNF-related apoptosis-inducing ligand (TRAIL) (Cretney et al. 2003) has little or no impact on negative selection. However, the role of Fas in negative selection is somewhat controversial. Early work in Fas-deficient mice demonstrated no impact on endogenous superantigen-mediated deletion (Kotzin et al. 1988), whereas in more recent experiments,
Fas does appear to play some role in negative selection (Castro et al. 1996; Kishimoto et al. 1998; Page et al. 1998; Schmitz et al. 2003). In some instances, ligation of Fas can augment deletion at high antigen concentrations (Kishimoto et al. 1998), whereas in others, Fas appears to have an affect at low antigen doses (Schmitz et al. 2003). Perhaps the most compelling evidence against the involvement of death receptors that utilize the adaptor protein FADD was obtained when expression of a dominant negative version of FADD had no effect on negative selection in numerous models (Newton et al. 1998). It will be of interest to determine whether other TNFR family members play a role in deletion. Recent gene array experiments from the Goodnow group indicated that in negative selection conditions, members of the TNFR superfamily including OX40 and 4-1BB were upregulated (Liston et al. 2004). It is possible that one of these receptors may be important for negative selection. 2.
Mitochondria
Mitochondria have been shown to play a critical role in apoptosis in all cell types. After initiation of the apoptotic program, there is a loss of outer mitochondrial membrane integrity and release of mitochondrial constituents into the cytosol, including cytochrome c. Cytochrome c then binds to caspase 9 and apoptotic protease activating factor-1 (apaf-1) to form the apoptosome and activates downstream caspases, ultimately leading to apoptosis. There is conflicting evidence for the role of apaf-1 in negative selection (Hara et al. 2002; Matsuki et al. 2002), and most individual caspases (3, 8, and 9) seem not to be required for negative selection (Kuida et al. 1996, 1998; Salmena et al. 2003). The use of broad-spectrum caspase inhibitors such as baculovirus p35 and zVADfmk in a number of different models (Doerfler et al. 2000; Izquierdo et al. 1999; Villunger et al. 2004) generally did not have an effect on negative selection. Therefore, the role for caspases in negative selection remains an open question. On the other hand, there is ample evidence for a role for bcl-2 family members in negative selection. The early approaches using transgenic overexpression of bcl-2 led to conflicting results. Two individual Bcl-2 transgenic mice were constructed and in one transgenic mouse, negative selection was unaffected in superantigen and HY models (Sentman et al. 1991; Tao et al. 1994), whereas using the same models of negative selection, the other Bcl-2 transgenic mouse showed impaired deletion (Strasser et al. 1991, 1994). The reason for this difference is currently unknown; however, one could postulate that differences in Bcl-2 expression levels due to copy number, integration site, or promoter strength may be the cause. In Bim-deficient animals, negative selection is highly impaired in a number of different models including exogenous superantigen-mediated deletion and in the HY system (Bouillet et al. 2002; Villunger et al. 2004). Bax and Bak are likewise required (Rathmell et al. 2002). Bim, Bax, and Bak
218
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TIMOTHY
are also required for death of DP thymocytes via pathways outside of the antigen receptor (such as glucocorticoids and irradiation) suggesting that they are generally important in regulating life and death in DP thymocytes (Bouillet et al. 1999; Rathmell et al. 2002). It is unclear at this point how the TCR regulates these bcl-2 family members, and further research is needed to address this crucial point.
V.
CONCLUSIONS
Clonal deletion is a fundamental process necessary to maintain tolerance to self-antigens. As demonstrated by the occurrence of multiorgan autoimmunity in the face of impaired negative selection to tissue-specific antigens in AIRE-deficient mice or humans, understanding the factors controlling deletion is of great importance. When one is using mice as a model system to study negative selection, it is critical to choose an appropriate model as data concerning the role of a molecule in negative selection from one model are not always the same as those in another model. Given that the medulla is replete with potent antigen-presenting cells, late deletion models appear to be the most physiological. Further work examining the molecular pathways leading to deletion, specifically those involved in delivering the apoptotic signal, is greatly needed at this juncture.
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Lee, S.L., Wesselschmidt, R.L., Linette, G.P., Kanagawa, O., Russell, J.H., Milbrandt, J. (1995). Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77). Science 269, 532–535. Li, R., Page, D.M. (2001). Requirement for a complex array of costimulators in the negative selection of autoreactive thymocytes in vivo. J Immunol 166, 6050–6056. Liston, A., Lesage, S., Gray, D.H., O’Reilly, L.A., Strasser, A., Fahrer, A.M., et al. (2004). Generalized resistance to thymic deletion in the NOD mouse; a polygenic trait characterized by defective induction of Bim. Immunity 21, 817–830. Liston, A., Lesage, S., Wilson, J., Peltonen, L., Goodnow, C.C. (2003). Aire regulates negative selection of organ-specific T cells. Nat Immunol 4, 350–354. Liu, C.P., Kappler, J.W., Marrack, P. (1996). Thymocytes can become mature T cells without passing through the CD4+ CD8+, double-positive stage. J Exp Med 184, 1619–1630. Liu, C.P., Parker, D., Kappler, J., Marrack, P. (1997). Selection of antigenspecific T cells by a single IEk peptide combination. J Exp Med 186, 1441–1450. Liu, Z.G., Smith, S.W., McLaughlin, K.A., Schwartz, L.M., Osborne, B.A. (1994). Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77. Nature 367, 281–284. Mamalaki, C., Elliott, J., Norton, T., Yannoutsos, N., Townsend, A.R., Chandler, P., et al. (1993). Positive and negative selection in transgenic mice expressing a T-cell receptor specific for influenza nucleoprotein and endogenous superantigen. Dev Immunol 3, 159–174. Martin, S., Bevan, M.J. (1997). Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection. Eur J Immunol 27, 2726–2736. Matsuki, Y., Zhang, H.G., Hsu, H.C., Yang, P.A., Zhou, T., Dodd, C.H., et al. (2002). Different role of Apaf-1 in positive selection, negative selection and death by neglect in foetal thymic organ culture. Scand J Immunol 56, 174–184. McCarty, N., Paust, S., Ikizawa, K., Dan, I., Li, X., Cantor, H. (2005). Signaling by the kinase MINK is essential in the negative selection of autoreactive thymocytes. Nat Immunol 6, 65–72. McGargill, M.A., Derbinski, J.M., Hogquist, K.A. (2000). Receptor editing in developing T cells. Nat Immunol 1, 336–341. McGargill, M.A., Hogquist, K.A. (1999). Antigen-induced coreceptor downregulation on thymocytes is not a result of apoptosis. J Immunol 162, 1237–1245. McGargill, M.A., Mayerova, D., Stefanski, H.E., Koehn, B., Parke, E.A., Jameson, S.C., et al. (2002). A spontaneous CD8 T cell-dependent autoimmune disease to an antigen expressed under the human keratin 14 promoter. J Immunol 169, 2141–2147. McSorley, S.J., Asch, S., Costalonga, M., Reinhardt, R.L., Jenkins, M.K. (2002). Tracking Salmonella-specific CD4 T cells in vivo reveals a local mucosal response to a disseminated infection. Immunity 16, 365–377. Michie, A.M., Zuniga-Pflucker, J.C. (2002). Regulation of thymocyte differentiation: pre-TCR signals and β-selection. Semin Immunol 14, 311–323. Mikszta, J.A., McHeyzer-Williams, L.J., McHeyzer-Williams, M.G. (1999). Antigen-driven selection of TCR in vivo: related TCR α-chains pair with diverse TCR β-chains. J Immunol 163, 5978–5988. Morahan, G., Hoffmann, M.W., Miller, J.F. (1991). A nondeletional mechanism of peripheral tolerance in T-cell receptor transgenic mice. Proc Natl Acad Sci USA 88, 11421–11425. Newton, K., Harris, A.W., Bath, M.L., Smith, K.G., Strasser, A. (1998). A dominant interfering mutant of FADD/MORT1 enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes. EMBO J 17, 706–718. Oehen, S., Feng, L., Xia, Y., Surh, C.D., Hedrick, S.M. (1996). Antigen compartmentation and T helper cell tolerance induction. J Exp Med 183, 2617–2626. Oxenius, A., Bachmann, M.F., Zinkernagel, R.M., Hengartner, H. (1998). Virus-specific MHC-class II-restricted TCR-transgenic mice: effects on humoral and cellular immune responses after viral infection. Eur J Immunol 28, 390–400.
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MODELS
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SELECTION
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221 Vella, A.T., Scherer, M.T., Schultz, L., Kappler, J.W., Marrack, P. (1996). B cells are not essential for peripheral T-cell tolerance. Proc Natl Acad Sci USA 93, 951–955. Venanzi, E.S., Benoist, C., Mathis, D. (2004). Good riddance: thymocyte clonal deletion prevents autoimmunity. Curr Opin Immunol 16, 197–202. Villunger, A., Marsden, V.S., Zhan, Y., Erlacher, M., Lew, A.M., Bouillet, P., et al. (2004). Negative selection of semimature CD4+8−HSA+ thymocytes requires the BH3-only protein Bim but is independent of death receptor signaling. Proc Natl Acad Sci USA 101, 7052–7057. Viret, C., He, X., Janeway, C.A., Jr. (2000). On the self-referential nature of naive MHC class II-restricted T cells. J Immunol 165, 6183–6192. Viret, C., Janeway, C.A., Jr. (2000). Functional and phenotypic evidence for presentation of Eα52–68 structurally related self-peptide(s) in I-Eα-deficient mice. J Immunol 164, 4627–4634. Wack, A., Ladyman, H.M., Williams, O., Roderick, K., Ritter, M.A., Kioussis, D. (1996). Direct visualization of thymocyte apoptosis in neglect, acute and steady-state negative selection. Int Immunol 8, 1537–1548. Wang, E.C., Thern, A., Denzel, A., Kitson, J., Farrow, S.N., Owen, M.J. (2001). DR3 regulates negative selection during thymocyte development. Mol Cell Biol 21, 3451–3461. Werlen, G., Hausmann, B., Palmer, E. (2000). A motif in the αβ T-cell receptor controls positive selection by modulating ERK activity. Nature 406, 422–426. Williams, J.A., Sharrow, S.O., Adams, A.J., Hodes, R.J. (2002). CD40 ligand functions non-cell autonomously to promote deletion of self-reactive thymocytes. J Immunol 168, 2759–2765. Wong, P., Goldrath, A.W., Rudensky, A.Y. (2000). Competition for specific intrathymic ligands limits positive selection in a TCR transgenic model of CD4+ T cell development. J Immunol 164, 6252–6259. Yokosuka, T., Takase, K., Suzuki, M., Nakagawa, Y., Taki, S., Takahashi, H., et al. (2002). Predominant role of T cell receptor (TCR)-α chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system. J Exp Med 195, 991–1001. Zal, T., Volkmann, A., Stockinger, B. (1994). Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen. J Exp Med 180, 2089–2099. Zhan, Y., Purton, J.F., Godfrey, D.I., Cole, T.J., Heath, W.R., Lew, A.M. (2003). Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation. Proc Natl Acad Sci USA 100, 1197–1202. Zhang, M., Vacchio, M.S., Vistica, B.P., Lesage, S., Egwuagu, C.E., Yu, C.R., et al. (2003). T cell tolerance to a neo-self antigen expressed by thymic epithelial cells: the soluble form is more effective than the membrane-bound form. J Immunol 170, 3954–3962. Zuklys, S., Balciunaite, G., Agarwal, A., Fasler-Kan, E., Palmer, E., Hollander, G.A. (2000). Normal thymic architecture and negative selection are associated with Aire expression, the gene defective in the autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). J Immunol 165, 1976–1983.
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Chapter 10 Peripheral Tolerance of T Cells in the Mouse Vigo Heissmeyer, Bogdan Tanasa, and Anjana Rao
I. II. III. IV. V. VI.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Mouse as a Model System for the Study of Autoimmune Disease . . . Scope of This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Context of Antigen Recognition Determines T Cell Activation . . . . . Processes Involved in T Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . T Cell Receptor Signal Transduction Leading to Productive T Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Control of T Cell–Mediated Autoimmunity through Central Tolerance . . . VIII. TCR Stimulation in the Absence of Costimulation: Induction of Peripheral T Cell Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Contributions from T Cell Homeostasis and T Cell Apoptosis . . . . . . . . . . X. Negative Regulation of T Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . A. Humoral Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Negative Costimulatory Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. E3 Ubiquitin Ligases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Adaptor Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Phosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
One of the most crucial evolutionary advances made by higher vertebrates has been to create an adaptive immune system that protects against foreign substances and invading organisms. Unfortunately, the newly acquired effectiveness in
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blocking potential pathogens has also brought about a propensity to autoimmune self-destruction. The likelihood of developing autoimmunity has arisen as a consequence of the new mode of “adaptive” antigen recognition, in which lymphocytes in the immune system can recognize millions of possible chemical target structures in specific molecular, cellular, and humoral contexts.
Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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Inappropriate recognition of host antigens by T cells in the periphery is a relatively common feature of peripheral T cells, which are positively selected in the thymus for having productively rearranged their T cell antigen receptor (TCR) genes so as to yield receptors with some degree of cross-reactivity to the particular antigens and major histocompatibility complex (MHC) molecules that are present in the host. Concomitant negative selection in the thymus filters out T cells that have strong autoreactivity, but negative selection by itself is not completely effective for eliminating self-reactive T cell clones. As a result, a plethora of cellular programs and molecular mechanisms have coevolved with the adaptive immune system to deal with self-reactive lymphocytes and silence their autoreactivity. In healthy individuals, these mechanisms operate synergistically and redundantly to ensure that self-reactivity is minimized and that immune and inflammatory responses are ended as promptly as is consistent with effective clearance of pathogens.
II.
THE MOUSE AS A MODEL SYSTEM FOR THE STUDY OF AUTOIMMUNE DISEASE
Study of the underlying mechanisms of peripheral tolerance has been an area of intensive research, since knowledge of how to modulate immune function is invaluable for the treatment of autoimmune disease and the avoidance of transplant rejection. The use of the laboratory mouse has been critical in this endeavor. Initially, immunologists used mice to recapitulate human autoimmune diseases and to establish a causal relationship between the antigen, the reactive immune cell type, and the development of the autoimmune phenotype. Later, T cells capable of transferring or attenuating autoimmune disease were cloned, either from mouse strains that are genetically prone to develop autoimmune disease or from mice that were immunized deliberately with antigens capable of inducing an autoimmune attack on a specific tissue (e.g., myelin basic protein, pancreatic β-cell antigens, and collagen or other joint antigens in mouse models of autoimmune demyelinating diseases, autoimmune diabetes, and autoimmune arthritis, respectively). The molecular cloning of rearranged T cell receptor genes from these T cell clones allowed the subsequent generation of TCR-transgenic mice, which have been invaluable as a means of manipulating at will the development of autoimmune disease, especially in adoptive transfer models. These mice are also a source of homogeneous T cell populations, which can be stimulated with a defined antigen in vitro to assess their biochemical responses to antigen and other signaling inputs. Finally, current advances in gene targeting in mice, combined with emerging results from genome-wide screens in which mice treated with mutagenic substances are evaluated for autoimmune phenotypes, have provided investigators with a growing list of molecules whose mutation or loss of function alters the susceptibility of a mouse strain to develop autoimmune disease.
III.
SCOPE OF THIS CHAPTER
In this chapter we attempt to provide a comprehensive list of gene products that have been implicated as negative regulators of autoimmunity in T cells, as judged by the fact that mice deficient in or mutant for the gene in question present either with frank autoimmunity or show increased susceptibility to developing autoimmunity in experimental models of autoimmune disease (Table 10-1). Given space constraints, we regret that we cannot include a discussion of the many known autoimmune loci, which are genomic regions linked to specific autoimmune diseases that in general contain large numbers of candidate genes. The focus of this chapter will be on the biological mechanisms intrinsic to T cells that are involved in preventing autoimmunity and not on the pathological differences in the autoimmune phenotypes that arise after autoreactive T cells start to attack host tissue. We have excluded genes whose genomic deletion is associated with autoimmune disease in mice, but whose products are not expressed in T cells; or genes whose deletion gives rise to an autoimmune phenotype that is not T cell autonomous. In the following sections, we will summarize established and potential molecular mechanisms that contribute to peripheral T cell tolerance, briefly discussing possible mechanisms through which selected gene products that are thought to be involved in T cell tolerance or otherwise may act to prevent autoimmunity.
IV.
THE CONTEXT OF ANTIGEN RECOGNITION DETERMINES T CELL ACTIVATION
T lymphocytes bind with their antigen receptors only to antigens that are presented on a matching MHC molecule. The specific recognition of antigen that leads to T cell activation occurs during an extended period of time, during which the T cell forms close membrane contacts with specialized cell types known as antigen-presenting cells. These close contacts occur in the context of a macromolecular structure known as the immunological synapse, which is suited for intense information exchange between the T cell and the antigen-presenting cell (Krogsgaard et al. 2003). Antigen is presented particularly effectively by a well-characterized subtype of antigen-presenting cells known as dendritic cells, which mature in a specific humoral milieu established by cells of the innate immune system that produce proinflammatory cytokines in response to infection by pathogens (Germain 2004). T cell activation therefore results from an encounter with an antigen-loaded antigen-presenting cell and is not simply a function of interaction strength and local concentration of the TCR and complexes of antigen-MHC. Rather, the T cell sums inputs from multiple interactions that occur within the immunological synapse, between surface receptors (TCR, integrins, and costimulatory receptors) on the T cell and their ligands on the
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TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES Molecule Name [aliases] [gene name description]
Molecular Function
Mouse Knockout Phenotype
AIRE [autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy)]
Probable transcriptional regulator protein that binds to DNA as dimer and tetramer, highly expressed in thymus stromal cells, contains SAND and PHD domains. It is localized to speckled domains in the nucleus and shows colocalization with the cytoskeletal filaments. The PHD domain of AIRE mediates E3 ubiquitin ligase activity.
AKT1 [PKB] [thymoma viral proto-oncogene 1]
A serine/threonine-protein kinase, widely expressed, contains a PH domain. A mediator of survival factors and a regulator of cell cycle, AKT1 activation occurs through PI3K.
BCL2L11 [Bim] [Bcl-2-like 11 (apoptosis facilitator)]
A member of the BCL-2 family, acts as an apoptosis activator. Contains a BH3 domain and forms heterodimers with other members of the BCL-2 family, including BCL2 and BCL-XL. Expressed in lymphoid lineage; it is sequestered to the microtubular dynein motor complex.
BHLHB2 [Stra13] [basic helix-loop-helix (bHLH) domain containing, class B2]
Contains a bHLH domain and may function as a transcription factor. Able to heterodimerize with E47/TCFE2A, associates with UBC9 that targets the molecules for proteolysis by the ubiquitindependent proteasome pathway.
1. The Aire−/− thymic medullary epithelial cells showed a specific reduction in ectopic transcription of genes encoding peripheral antigens. Aire−/− mice exhibited multiorgan autoimmunity and an increased level of autoantibodies, with a similar pattern of organ distribution (Anderson et al. 2002). 2. Aire-deficient mice developed Sjögren’s syndrome– like pathological changes in the exocrine organs, and these were associated with autoimmunity against a ubiquitous protein, α-fodrin, whose gene expression was retained in the Aire-deficient thymus (Kuroda et al. 2005). 3. Mutations in human AIRE are responsible for autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) (The Finnish-German APECED Consortium, 1997). 4. Deficiency of Aire expression is observed in severe immunodeficiencies with autoimmune manifestations. In Omenn syndrome patients, hypomorphic mutations in RAG1 and RAG2 genes impair the process of VDJ recombination, leading to the generation of T cells with a highly restricted receptor repertoire that infiltrate the skin, the gut, the liver, and the spleen (Cavadini et al. 2005). 1. In transgenic mice expressing myristoylated Akt (mAkt), a constitutively active form of AKT, T cells were less dependent on CD28 costimulation, grow rapidly, and secrete IL-2 and IFN-γ in the absence of CD28 ligation. mAkt-transgenic T lymphocytes resist death-by-neglect and accumulate memory T and B cells. Many aged mAkt-transgenic mice developed autoimmunity with immunoglobulin deposits on kidney glomeruli and displayed increased incidence of lymphoma (Rathmell et al. 2003). 1. In Bim−/− mice, the numbers of both the CD4-8-pro-T cells and the mature T cells CD4+ or CD8+ are elevated. The absence of Bim augmented survival of the resting T and B cells and protected the cells against apoptosis induced by cytokine withdrawal. With age, Bim deficiency led to progressive lymphadenopathy, autoimmune glomerulonephritis, and vasculitis. The number of plasma cells was dramatically increased and immunoglobulin (Ig) G autoantibodies reached very high concentrations (Bouillet et al. 1999). 2. TCR ligation upregulated Bim expression and promoted interaction of BIM with BCL-XL, inhibiting its survival function. Thymocytes lacking Bim are refractory to apoptosis induced by TCR-CD3 stimulation. In transgenic mice expressing autoreactive TCRs that cause widespread clonal deletion, Bim deficiency severely impaired thymocyte killing (Bouillet et al. 2002). 1. T cell development is normal in Stra13−/− mice. Stra13 deficiency affect clonal expansion, differentiation, and AICD. Ineffective deletion of activated T and B cells leads to lymphoid hyperplasia and Stra13−/− mice develop autoimmune disease characterized by circulating autoantibodies, infiltration in multiple organs, and Continued
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TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES—cont’d Molecule Name [aliases] [gene name description]
CBLB [Casitas B-lineage lymphoma b]
Molecular Function
A signal transduction protein, contains an EFhand-like calcium-binding domain and a a RING-type zinc finger that mediates the binding to an E2 ubiquitin–conjugating enzyme.
Mouse Knockout Phenotype
1.
2.
CD274 [B7-H1; PD-L1] [CD274 antigen]
PDCD1 [PD-1] [programmed cell death 1]
CD276 [B7H3] [CD276 antigen]
CTLA-4 [CD152] [cytotoxic T lymphocyte–associated protein 4]
PD-L1, a type I membrane protein, essential for T lymphocyte proliferation and production of IL-10 and IFN-γ, in an IL-2-dependent and a PD-CD1-independent manner. Contains Ig-like C2-type and V-type domains
1.
PD-CD1, a type I membrane protein, a possible inducer of apoptosis. Belongs to the immunoglobulin superfamily, contains an Ig-like V-type domain. Interaction between PD-CD1 and PD-L1 inhibits T cell proliferation and cytokine production. A type I membrane protein, serves as a negative regulator of T cell activation and function.
3.
A type I membrane protein, contains 1 Ig-like V-type domain and transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal.
2.
1.
1.
2.
E2F2 [E2F transcription factor 2]
A member of the E2F family of transcription factors; acts cooperatively with DP family members and regulates the genes involved in cell cycle regulation or in DNA replication. E2F2 binds specifically to RB1 protein, in a cell cycle–dependent manner.
1.
deposition of immune complex in glomeruli. The phenotype of Stra13−/− mice is similar of that of IL2-and IL-2 receptor-deficient mice, which show normal T cell development but impaired expansion and apoptosis after lymphocyte activation (Sun et al. 2001). Resting Cblb−/− lymphocytes display increased proliferation and IL-2 production upon TCR stimulation, with minimal requirement for CD28 costimulation. Cblb−/− mice develop spontaneous autoimmunity characterized by infiltration of activated T and B lymphocytes into multiple organs, organ damage, and autoantibody production (Bachmaier et al. 2000). Thymic positive and negative selections of T cells in Cblb−/− mice are normal. Mice deficient in Cblb are highly susceptible to experimental autoimmune encephalomyelitis (Chiang et al. 2000). T cell responses, including cytokine production, were enhanced in Pdl1−/− mice. Pdl1−/− mice develop an autoimmune-like phenotype, which is delayed in onset compared with Ctla4−/− mice (Latchman et al. 2004). Pdcd1−/− mice die prematurely on a BALB/c background, but not on a C57BL/6 background. Pdcd1-deficient BALB/c mice develop dilated cardiomyopathy that leads to heart failure and exhibit high-titer autoantibodies (Nishimura et al. 2001). C57BL/6-Pdcd1−/− mice spontaneously developed lupus-like glomerulonephritis and destructive arthritis as they age. The onset and severity of both glomerulonephritis and arthritis in B6-Pdcd1−/− mice is accelerated by the additional lymphoproliferation (lpr) mutation (Nishimura et al. 1999). B7H3 negatively regulates T helper (Th) 1 responses. B7h3-deficient mice developed experimental autoimmune encephalomyelitis and severe airway inflammation and accumulated increased concentrations of autoantibodies. T helper cells differentiate toward a Th1 rather than a Th2 phenotype (Suh et al. 2003). Ctla4-deficient T cells proliferated strongly and spontaneously after TCR stimulation, and they were sensitive to FAS-mediated apoptosis. Lymph nodes and spleens of Ctla4−/− mice, but also liver, heart, lung, and pancreas tissue, were infiltrated with activated T cells. The concentration of serum immunoglobulins was elevated (Waterhouse et al. 1995). Ctla4-deficient mice develop lymphoproliferative disease with multiorgan infiltration and tissue destruction, with particularly severe myocarditis and pancreatitis (Tivol et al. 1995). In E2f2-deficient mice, thymic negative selection is intact, and T cells show an enhanced proliferation but normal sensitivity to FAS-triggered apoptosis and/or IL-2 withdrawal. Autoreactive effector/memory T lymphocytes accumulate and appear to be responsible for causing late-onset autoimmune features, represented by widespread inflammatory infiltrates, antinuclear antibodies, and glomerular deposition of immune complexes (Murga et al. 2001).
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TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES—cont’d Molecule Name [aliases] [gene name description]
Molecular Function
FAS [CD95; TNFR6] [Fas (TNF receptor superfamily member)]
FAS, type I membrane protein, contains a death domain involved in the binding of Fas associated death domain (FADD). FADD recruits caspase-8 to the activated receptor, and the resulting death-inducing signaling complex (DISC) performs caspase-8 proteolytic activation, which initiates the subsequent cascade of caspases mediating apoptosis.
FASL [CD95L; TNFSF6; CD178] [Fas ligand (TNF superfamily, member 6)]
FASL, type II membrane protein, belongs to the TNF family. Homotrimer, binds to FAS.
FOXJ1 [FKHL13] [forkhead box J1]
Contains one forkhead DNA-binding domain.
FOXO3A [FKHRL1] [forkhead box O3a]
Contains one forkhead DNA-binding domain. May trigger apoptosis by inducing the expression of genes that are critical for cell death.
FOXP3 [JM2; scurfin] [forkhead box P3]
Contains 1 fork-head DNA-binding domain.
GADD45A [Ddit1] [growth arrest and DNA-damage-inducible 45 α]
Belongs to the GADD45 family binds to proliferating cell nuclear antigen (PCNA), interacts with cell division protein kinase (CDK) complexes; stimulates DNA excision repair and inhibits entry of cells into S phase.
Mouse Knockout Phenotype 2. T cells lacking E2f1 and E2f2 proliferate more extensively in response to antigenic stimulation. E2f1/E2f2-null mutant mice are predisposed to the development of tumors and exhibit signs of autoimmunity (Zhu et al. 2001). 1. Mice carrying the lpr mutation have defects in the Fas gene. The lpr mice develop lymphadenopathy and suffer from a systemic lupus erythematosus–like autoimmune disease (Watanabe-Fukunaga et al. 1992). 2. Fas-null mice show a massive proliferation of lymphocytes and a substantial liver hyperplasia (Adachi et al. 1995). 3. Autoimmune lymphoproliferative syndrome (ALPS) type IA, characterized by nonmalignant lymphadenopathy, autoimmunity, and expanded populations of CD4−CD8− lymphocytes, is caused by mutation in the human Fas gene (Fisher et al. 1995; Rieux-Laucat et al. 1995). 4. An autosomal recessive mutation generalized lymphoproliferative disease (gld) determines the development of massive lymphoid hyperplasia with antinuclear antibodies and hypergammaglobulinemia, a pattern that resembles the phenotype induced by the lpr mutation (Roths et al. 1984). 5. Fasl−/− mice exhibited splenomegaly and lymphadenopathy, multiple organ infiltration, and autoimmune disease (Karray et al. 2004). 6. ALPS type IB is caused by mutation in the human Fasl gene (Wu et al. 1996). 1. Foxj1−/− mice die in utero because of hydrocephalus and/or heterotaxy. Foxj1−/− chimeras displayed evidence of systemic autoimmune inflammation, including T cell infiltrates of the salivary glands, liver, lung, and kidney. Naïve Foxj1−/− T cells produced elevated quantities of IL-2 and IFN-γ after TCR stimulation and Foxj1−/− Th cells demonstrated increased NF-kB1 activation (Lin, Spoor, et al. 2004). 1. Foxo3a-deficient mice were generated using the gene trap targeting strategy. Foxo3a deficiency leads to spontaneous lymphoproliferation and the inflammation of the salivary gland, lung, and kidney. No apoptotic defects were observed. Helper T cells were hyperactivated hyperproliferated, and produced large amounts of cytokines. NF-kB1 activity in Foxo3a-deficient mice was elevated (Lin, Hron, et al. 2004). 1. Scurfy is an X-linked recessive mouse mutant characterized by CD4+ T cell hyperproliferation, extensive multiorgan infiltration, and elevation of numerous cytokines. The gene defective in sf mice is Foxp3 (Brunkow et al. 2001). 2. FOXP3 is specifically expressed in CD4+CD25+ regulatory T cells, and it is required for their development. Foxp3-null mice develop a lethal autoimmune syndrome (Fontenot et al. 2003). 1. Gadd45a−/− T cells hyperproliferate after TCR stimulation. Gadd45a−/− mice develop an autoimmune disease that is similar to human systemic lupus erythematosus, with high titers of autoantibodies, hematological disorders, and autoimmune glomerulonephritis. The development of autoimmunity was accelerated in mice deficient in both Gadd45a and Cdkn1a (p21) (Salvador et al. 2002). Continued
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TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES—cont’d Molecule Name [aliases] [gene name description]
Molecular Function
Mouse Knockout Phenotype
GPR132 [G2a] [G protein–coupled receptor 132]
Integral membrane protein, belongs to the G protein-coupled receptor 1 family, receptor for lysophosphatidylcholine. Induced by DNA-damaging agents and may serve as a mechanism for T and B cells to slow their proliferation and repair damaged DNA to ensure proper replication.
ICOS [inducible T cell costimulator]
Type I membrane protein, contains 1 immunoglobulin-like V-type domain, expressed on activated T cells and resting memory T cells. Homodimer, enhances proliferation, secretion of lymphokines, and effective help for antibody secretion by B cells. Does not increase the production of IL-2, but induces IL-10 expression. Prevents the apoptosis of preactivated T cells and plays a critical role in CD40-mediated class switching of Ig isotypes. Belongs to the α/β interferon family. Monomer, has antiviral, antibacterial, and anticancer activities.
1. Gpr132-deficient T cells are hyperresponsive to TCR stimulation and exhibit enhanced proliferation. Old Gpr132−/− mice develop lymphoid hyperplasia that is associated with abnormal expansion of both T and B lymphocytes, multiple organ infiltration, glomerular deposition of immune complexes, antinuclear autoantibodies, and a progressive wasting syndrome (Le et al. 2001). 1. T cell activation and proliferation are defective in Icos-deficient mice. Icos−/− T cells fail to produce IL-2, IL-4, or IL-13. Icos−/− mice showed enhanced susceptibility to experimental autoimmune encephalomyelitis (Dong et al. 2001).
IFNB1 [interferon-β1, fibroblast]
IL-2 [interleukin 2]
Belongs to the IL-2 family and produced by T cells in response to antigenic or mitogenic stimulation. The expression of this gene in mature thymocytes is monoallelic. Required for T cell proliferation.
IL-2RA [CD25] [interleukin 2 receptor, α chain]
The IL-2RA and IL-2RB chains, together with the common γ chain IL-2RG, constitute the high-affinity IL-2 receptor. Homodimeric α chains IL-2RA result in low-affinity receptor, whereas homodimeric IL-2RB chains produce a medium-affinity receptor. Type I membrane protein, contains two Sushi domains.
1. Ifnb1−/− mice are susceptible to experimental autoimmune encephalitis and develop severe and chronic neurological symptoms with extensive central nervous system inflammation and demyelination. T cells proliferate normally, but have increased effector functions, as measured by IFN-γ and IL-4 production (Teige et al. 2003). 2. The IFN-RI/CD118 deficiency leads to lymphoproliferative syndrome and autoantibody production. The IFN-RII/CD119 deficiency protected MRL/lpr mice from the development of autoimmuneassociated lymphadenopathy, autoantibodies, and autoimmune renal disease. Mice deficient for both IFN-RI and IFN-RII developed an autoimmune phenotype intermediate between that of wild-type and IFN-RII-deficient animals (Hron and Peng 2004). 1. T cell development in Il2−/− mice is normal. The number of activated T and B cells is elevated, as well as the serum immunoglobulin levels. The null mutants develop adult onset autoimmune disease, with death due to hemolytic anemia. Survivors develop an inflammatory bowel disease with anti-colon antibodies (Sadlack et al. 1993). 2. Il2−/− mice on a BALB/c background develop a lymphoproliferative syndrome with autoantibodies and of various specificities hemolytic anemia and die within 1 month of age (Sadlack et al. 1995). 3. Il2−/− mice on a C3H background, after immunization with myosin, develop severe myocarditis with high titers of autoantibodies (Grassl et al. 1997). 1. The development of T and B cells in Il2ra−/− is normal. Activation-induced cell death is impaired, T and B cells expand, and adult mice develop enlargement of secondary lymphoid organs. Older Il2ra-deficient mice develop autoimmune disorders, including hemolytic anemia and inflammatory bowel disease (Willerford et al. 1995).
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TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES—cont’d Molecule Name [aliases] [gene name description]
Molecular Function
Mouse Knockout Phenotype
IL-2RB [CD122] [interleukin 2 receptor, β chain]
The intermediate- and high-affinity forms of the receptor are involved in transduction of mitogenic signals from IL-2 and in receptor-mediated endocytosis. Type I membrane protein, contains one fibronectin type III domain.
IL-10 [CSIF] [interleukin 10]
Homodimer, belongs to the IL10 family. Inhibits the synthesis of a number of cytokines, including IFN-γ, IL-2, IL-3, and TNF produced by activated macrophages and by helper T cells.
ITCH [itchy]
A E3 ubiquitin-protein ligase, contains one HECT-type E3 ubiquitin-protein ligase domain.
LAT [linker for activation of T cells]
Type III membrane protein, involved in TCR signal transduction pathway. Tyrosine phosphorylation by ZAP-70 leads to the recruitment of multiple signaling molecules.
MGAT5 [GlcNAc-TV] [mannoside acetylglucosaminyl transferase 5]
Type II membrane protein, on Golgi apparatus; glycosyltransferase is involved in the synthesis of protein-bound and lipid-bound oligosaccharides.
1. In Il2rb−/− mice, T cells are spontaneously activated, and B cells differentiate into plasma cells and produce high serum concentrations of IgG1 and IgE. Autoantibodies cause hemolytic anemia, and animals died after about 12 weeks (Suzuki et al. 1995). 2. Transgenic thymic expression of the Il2rb in Il2rbdeficient mice prevents autoimmunity and restores the normal production of B lymphocytes (Malek et al. 2000). 3. Autoimmunity and imbalanced T cell homeostasis were prevented by the adoptive transfer of normal CD4+CD25+ T cells to Il2rb−/− mice (Malek et al. 2002). 1. In Il10-deficient mice, lymphocyte development and antibody responses are normal, but growth is retarded and the mice are anemic and suffer from chronic enterocolitis. Mutants kept under specific pathogen-free conditions develop only a local inflammation limited to the proximal colon (Kuhn et al. 1993). 1. An inversion in Itch is responsible for the immunological phenotype of the non–agouti-lethal 18H mice. On the JU/Ct background, the mice develop an inflammatory intestinal disease. On the C57BL/6J background, they develop a lethal disease characterized by chronic inflammation of the lung, stomach, and skin, resulting in scarring due to constant itching and the hyperplasia of lymphoid cells (Perry et al. 1998). 2. Itch−/− T cells show an activated phenotype and enhanced proliferation, with hyperproduction of IL4 and IL5 after stimulation and increased serum concentrations of IgG1 and IgE. Itch deficiency leads to immune and inflammatory disorders and constant itching of the skin (Fang et al. 2002). 1. In Lat−/− deficient mice, B cell populations are normal, but T cell development is blocked at the CD4−CD8− stage (Zhang et al. 1999). 2. The phenotype of distal four tyrosines knockin mice was identical to the phenotype of Lat−/− mice: Thymocyte development was arrested at the CD4−CD8− stage, and no mature T cells were detected (Sommers et al. 2001). 3. Mice homozygous for a single tyrosine mutation in Lat developed a polyclonal lymphoproliferative syndrome and signs of autoimmune disease: elevated serum concentrations of IgG1, IgE, and IgM and nuclear autoantibodies. IL2 production and T cell apoptosis were affected (Sommers et al. 2002). 4. Mice homozygous for a single tyrosine mutation in Lat showed hampered T cell development but accumulated polyclonal T cells having a Th2 phenotype. Th2 differentiation leads to eosinophilia and maturation of plasma cells secreting IgE and IgG1 (Aguado et al. 2002). 1. Mgat5−/− mice showed an autoimmune-mediated glomerulonephritis, increased susceptibility to experimental autoimmune encephalomyelitis, and enhanced delayed-type hypersensitivity. Mgat5 deficiency lowers the T cell activation threshold by promoting TCR clustering (Demetriou et al. 2001). Continued
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TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES—cont’d Molecule Name [aliases] [gene name description]
Molecular Function
NFAT1 [Nfatc2; Nfatp] [nuclear factor of activated T cells, cytoplasmic, calcineurindependent 2]
Cytoplasmic for the phosphorylated form and nuclear after activation that is controlled by calcineurin-mediated dephosphorylation. Rapid nuclear exit of NFAT is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals.
PIK3CD [p110δ] [phosphatidylinositol 3-kinase catalytic δ polypeptide]
Contains a PI3/PI4 kinase domain, belongs to the PI3/PI4 kinase family; expressed predominantly in leukocytes, involved in signaling pathways regulating cell growth.
PIK3R1 [p50α; p55α; p85α] [phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1]
PI3K phosphorylates the inositol ring of phosphatidylinositol at the 3′ position. The enzyme comprises a 110-kDa catalytic subunit and a regulatory subunit of either 85, 55, or 50 kDa. Acts as an adapter, mediating the association of the p110 catalytic unit to the plasma membrane. Belongs to the PI3K p85 subunit family, one SH3 domain and one SH2 domain. The protein encoded this gene is a PIP3 and contains one tensin domain and one tyrosine protein phosphatase domain. A tumor suppressor that is mutated in a large number of cancers. Defects in PTEN are a cause of Cowden disease, Lhermitte-Duclos disease, Bannayan -Zonana syndrome, and Proteus syndrome.
PTEN [TEP1; MMAC1] [phosphatase and tensin homolog]
Mouse Knockout Phenotype 2. Mgat5−/− mice produce more IFN-γ and less IL-4 compared with wild-type cells (Morgan et al. 2004). 1. Nfat1−/− mice showed increased primary responses to Leishmania major. Accumulation of eosinophils and increased levels of IgE were observed in an in vivo model of allergic inflammation in Nfat1−/− mice (Xanthoudakis et al. 1996). 2. Nfat1−/− mice have splenomegaly with hyperproliferation of both B and T cells. Early IL-4 expression in Nfat1−/− mice is impaired, but production of IL-4 at later time points as well as Th2 differentiation is increased. IL-2 and IFN-γ are minimally affected. The mice display defects in Cd40l and Fasl transcription (Hodge et al. 1996). 3. Nfat1 deficiency leads to the accumulation of activated T cells, enhanced T cell responses after secondary stimulation, and defects in the termination of immune responses. Nfat1−/− T cells do not show defects in FAS-mediated apoptosis (Schuh et al. 1998). 4. Nfat1/Nfat2−/− T cells were deficient in cytokine production and cytolytic activity. Doubly deficient B cells were hyperactivated, serum IgG1 and IgE were elevated, and multiple organs were infiltrated (Peng et al. 2001). 5. The Nfat1−/− Nfat4−/− T cells showed increased proliferation and differentiate into Th2 cells. Nfat1/Nfat4 deficiency lowers the activation threshold and makes CD28 ligation dispensable (Rengarajan et al. 2002). 1. Pik3cd was inactivated by point mutation instead of deletion to prevent changes in the expression levels of the other PI3K catalytic and regulatory subunits. Thymocyte development was normal and Pik3cdD910A/D910A homozygous mice did not exhibit anatomical or behavioral abnormalities. B and T cell responses were impaired and the Pik3cdD910A/D910A mice developed an inflammatory bowel disease (Okkenhaug et al. 2002). 1. The transgenic mice expressing an active form of PI3K (p65, a truncation mutant of p85) showed prolonged CD4+ T cell survival and developed an infiltrating lymphoproliferative disorder and kidney autoimmune disease, with increased numbers of memory T lymphocytes and anti-double-stranded (ds) DNA antibodies. The signs were similar to those developed by heterozygous Pten+/− mice. p65 Tg p53−/− mice develop T cell lymphomas (Borlado et al. 2000). 1. Pten+/− mutants develop an autoimmune disorder with glomerulopathy, hypergammaglobulinemia, and autoantibodies reacting against nuclear antigens. FAS-mediated apoptosis was impaired in Pten+/− mice, and T cells showed increased proliferation upon activation. The features were similar to those observed in Fas-deficient mutants (Di Cristofano et al. 1999). 2. In Ptenflox/− mice, the negative selection is diminished, T cells are spontaneously activated, hyperproliferate, secrete increased levels of cytokines, and resist apoptosis. The mice display hypergammaglobulinemia
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10. PERIPHERAL TOLERANCE OF T CELLS IN THE MOUSE
TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES—cont’d Molecule Name [aliases] [gene name description]
Molecular Function
PTPN22 [PEP; Ptpn8] [protein tyrosine phosphatase, nonreceptor type 22 (lymphoid)]
PEP is a cytoplasmic phosphatase expressed exclusively in hematopoietic cells and associates with C-terminal c-Src kinase (CSK) to inhibit TCR signaling through dephosphorylation of LCK.
PTPN6 [SHP1; moth-eaten] [protein tyrosine phosphatase, nonreceptor type 6]
Belongs to the protein tyrosine phosphatase family, a regulator of tyrosine protein kinase CSK in T cells.
PTPRC [CD45 ] [protein tyrosine phosphatase, receptor type C]
Type I membrane protein, belongs to protein tyrosine phosphatase family. Expression is restricted to the hematopoietic cells, required for T cell activation through TCR.
RasGRP1 [RAS guanyl-releasing protein 1]
A member of a family of genes characterized by the presence of a Ras superfamily guanine nucleotide exchange factor (GEF) domain. Contains an EF-hand motif.
RC3H1 [Roquin] [RING CCCH (C3H) domains 1]
Contains RING-type zinc finger and C3H1-type zinc finger domains. Acts as an ubiquitin ligase that regulates ubiquitin-dependent control of AU-rich mRNA. Represses ICOS function and plays a central role in the prevention of production of autoantibodies by repressing follicular helper T cells and germinal centers.
Mouse Knockout Phenotype and elevated levels of autoantibodies (Suzuki et al. 2001). 3. Mice heterozygous for both Pten and Ship developed lymphoproliferation, hypergammaglobulinemia with high autoantibody titers, and renal disease. The pathological changes were more severe than those in Pten+/− mice (Moody et al. 2003). 1. Ptpn22 deficiency had minimal effects on naïve T cell activation, but the effector and memory T cells demonstrated increased expansion and function. Germinal centers develop spontaneously in the spleen and Peyer’s patches, and the serum levels of IgG1, IgG2, and IgE were elevated (Hasegawa et al. 2004). 2. 1858T, a functional polymorphism in Ptpn22, which encodes the amino-acid substitution R620W, affects the interaction with CSK and confers susceptibilities to rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, and Graves disease (Siminovitch, 2004). 1. Mice with the recessive moth-eaten (me) or the viable moth-eaten (mev) mutations develop a severe autoimmune and immunodeficiency syndrome (Tsui et al. 1993). 2. Sequence analyses revealed that both the me and mev mutations are point mutations that result in aberrant splicing of the Shp1 transcript (Shultz et al. 1993). 1. In Cd45−/− mice, thymocyte development is inhibited at two distinct stages: CD4−CD8− to CD4+ CD8+ transition and double-positive into single-positive transition. Apoptosis is impaired in response to TCR signals, but it can be induced normally in Cd45-null thymocytes by non-TCR-mediated signals (Byth et al. 1996). 2. A single point mutation E613R inactivates the inhibitory wedge of CD45 and causes polyclonal lymphocyte activation, leading to lymphoproliferation and severe lupus nephritis with anti-dsDNA antibodies. Both homozygous and heterozygous mice develop the autoimmune pathological changes (Majeti et al. 2000). 1. The thymi of Rasgrp-null mutant mice are deficient in mature, single-positive thymocytes (Dower et al. 2000). 2. A mutation in Rasgrp1 that prevented translation seems to be responsible for an autoimmune lymphoproliferative syndrome similar to systemic lupus erythematosus. Rasgrp1lag T cells spontaneously adopted a memory phenotype, were skewed toward IL-4 production and became resistant to activation-induced cell death. Autoantibodies were detected in older mice (Layer et al. 2003). 1. The sanroque mutation was generated by ethylnitrosourea mutagenesis, disrupts Roquin, a repressor of ICOS, and leads to excessive production of IL-21 by T cells. The san/san mice develop signs of systemic lupus erythematosus: glomerulonephritis with deposition of immune complexes, autoantibodies against dsDNA, hepatitis, anemia, and autoimmune thrombocytopenia. It is believed that the failure to repress autoimmunity Continued
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TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES—cont’d Molecule Name [aliases] [gene name description]
SH2D2A [Lad; TSAd] [SH2 domain protein 2A]
Molecular Function
Contains one SH2 domain, a T cell–specific adapter protein involved in the control of T cell activation, upregulated substantially after T cell activation.
Mouse Knockout Phenotype
1.
2.
STAT4 [signal transducer and activator of transcription 4]
Belong to the transcription factor STAT family. STAT4 and STAT6 contain one SH2 domain and carry out a dual function: signal transduction and activation of transcription.
1.
STAT6 [signal transducer and activator of transcription 6]
STAT6 is involved in IL-4 signaling.
Sts-1 [Cbl-interacting protein Sts-1] and Sts-2 [Ubash3a] [ubiquitin associated and SH3 domain containing type A]
Sts-1 contains one SH3 domain, an ubiquitin-associated (UBA) domain, and a domain with similarities to the catalytic motif of phosphoglycerate mutase. Sts-2/Ubash3a contains one SH3 domain and one UBA domain.
1.
TGFB1 [transforming growth factor β1]
Belongs to the TGFB family. Controls proliferation and differentiation. Defects in TGFB1 are the cause of Camurati-Engelmann disease. TGFB1, TGFB2, and TGFB3 function through the same receptor signaling systems.
1.
2.
TRAF6 [TNF receptor–associated factor 6]
Contains RING-type zinc finger and TRAF-type zinc finger domains. Adapter protein and signal transducer, homotrimer.
2.
1.
2.
ZAP-70 [Srk] [ζ chain (TCR)–associated protein kinase]
Belongs to the Tyr protein kinase family, SYK/ZAP-70 subfamily. Contains two SH2 domains.
1.
in the sanroque strain does not correspond to defects in any of the known tolerance mechanisms (Vinuesa et al. 2005). Sh2d2a-deficienct mice develop glomerulonephritis, autoantibodies against single-stranded DNA and dsDNA, and hypergammaglobulinemia and accumulate large numbers of activated T and B cells in spleen. The autoimmune phenotype in Sh2d2a−/− mice is associated with defective T cell death (Drappa et al. 2003). In Sh2d2a−/− mice, T cell proliferation and production of IL2 and IFNG, but not IL4, were defective (Rajagopal et al. 1999). Stat4-deficient NZM mice develop nephritis in the absence of high levels of autoantibodies and in the presence of reduced levels of IFN-γ. In contrast, Stat6-deficient NZM mice exhibit a significant reduction in the incidence of the kidney disease despite the presence of high levels of autoantibodies (Jacob et al. 2003). Stat4 deficiency leads to an increase in type 2 cytokine responses and to a decrease in type 1 cytokine production. Stat6 deficiency or anti-IL-4 antibody treatment decreases type 2 cytokine responses and ameliorates the kidney disease (Singh et al. 2003). Sts2−/− mice did not succumb to any unusual pathological condition. Sts1/2−/− T cells are hyperresponsive to TCR stimulation, with a marked increase in cytokine production. Double knockout mice are susceptible to experimental autoimmune encephalomyelitis, but triple knockout mice, lacking both Sts proteins and Cblb, developed the disease comparable in incidence and severity with Sts1/2−/− mice (Carpino et al. 2004). Tgfb1−/− mice succumb to a wasting syndrome accompanied by multifocal inflammation and tissue necrosis that leads to organ failure and death (Shull et al. 1992). Tgfb1−/− mice develop a rapid wasting syndrome and die by 4 weeks of age. Pathological examination of Tgfb1−/− mice revealed massive infiltration of lymphocytes and macrophages in many organs and primarily in heart and lungs (Kulkarni et al. 1993). Traf6−/− mice die prematurely. In Traf6−/− chimeras, T cell development is normal, and T cells are polarized toward a Th2 phenotype. The mice exhibit cachexia, scaling, and itching and develop a progressive lethal inflammatory disease associated with massive organ infiltration. Complementation of Rag2-deficient mice with Traf6-deficient lymphocytes leads to the development of a similar inflammatory disease (Chiffoleau et al. 2003). Autoimmunity was induced by grafting Traf6−/− fetal thymic stroma into athymic nude mice. The number of regulatory T cells was dramatically reduced in the Traf6−/− thymus (Akiyama et al. 2005). Zap70−/− mice had neither CD4 nor CD8 single-positive T cells, but expression of wild-type human ZAP-70 reconstituted both CD4 and CD8 single-positive populations (Negishi et al. 1995).
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10. PERIPHERAL TOLERANCE OF T CELLS IN THE MOUSE
TABLE 10-1
GENES WHOSE KNOCKOUTS IN MICE DISPLAY HYPERPROLIFERATIVE SYNDROMES, MANIFESTATIONS OF AUTOIMMUNITY, AND/OR AUTOIMMUNE DISEASES—cont’d Molecule Name [aliases] [gene name description]
Molecular Function
Mouse Knockout Phenotype 2. Spontaneous point mutation in the SH2 domain of ZAP-70 causes chronic autoimmune arthritis in mice that resembles human rheumatoid arthritis. Alterations of the ZAP-70 activity changes the thresholds of thymic selection and leads to the positive selection of otherwise negatively selected autoimmune T cells (Sakaguchi et al. 2003).
The molecules were selected based on the phenotype ontology descriptions (Smith et al. 2005) in the Mouse Genome Database (Eppig et al. 2005), which are associated with the defined criteria for autoimmunity (Rose and Bona 1993; Witebsky et al. 1957). The molecular functions are given based on biochemical characterizations and functional studies, and the information is available in the UCSC Genome Browser Database (Karolchik et al. 2003), NCBI Gene (Maglott et al. 2005), and Swiss-Prot/Uniprot (Bairoch et al. 2005). References are given for the descriptions of the autoimmune phenotype of the knockout or knockin mouse.
antigen-presenting cell, and then makes an integrated response. The response may be further influenced by simultaneous contact with regulatory T cells or through auto- or paracrine stimulation with pro- or anti-inflammatory cytokines. Finally, transduction of the signal that emanates from a single TCR-antigen–MHC complex is subject to regulation through negative factors that attenuate the signal and positive regulators that amplify it. The amplitude of these modulations and the kinetics with which they come into play with respect to antigen encounter are influenced by the prior activation history of the T cells, either determining the response threshold before the antigen encounter or altering the overall duration of the antigen response by influencing the kinetics of negative feedback mechanisms that curtail the activating response. Thus, the overall output response of a T cell to an activating stimulus reflects the history of the cell’s previous encounters with antigen, cytokines, surface receptors on antigen-presenting cells, and other environmental inputs which, together with intrinsic genetic influences, establish the gene expression profile within the T cell at the instant of primary or secondary antigenic stimulation. Over time, changes in gene expression measure the persistence of antigen and thereby allow opposing responses in situations of acute versus chronic stimulation.
V.
PROCESSES INVOLVED IN T CELL ACTIVATION
The use of very focused biological, biophysical, or biochemical methods to describe complex processes such as T cell activation results in an inevitable reductionism. However, the enormous medical importance of T cell activation has inspired many different ways of examining and evaluating this process in all its complexity, and these efforts will eventually provide
us with a molecular picture that approaches reality. Before we try to connect the T cell receptor with intracellular signaling cascades that culminate in the nucleus, with the objective of identifying the critical players, it seems appropriate to emphasize the complex and dynamic nature of T cell activation. It is possible that the molecular mechanisms that prevent inappropriate activation do not simply block signal transduction but dynamically downmodulate responses. On the microscopic level, antigen recognition by T cells leads to cellular changes such as the acquisition of cellular polarity. The stimulated T cells also show clustering of T cell receptors (capping) in the membrane of the contact zone. Inside the cell, a reorientation of the microtubule organizing center occurs, which now allows for directed endo- and exocytosis from and into the membrane patch that forms the contact to the antigenpresenting cell (Kupfer and Kupfer 2003). The cytoskeleton becomes rearranged, providing an adjusted structure in which cytoskeleton-associated signal transduction and transport can influence signaling and cellular processes. In parallel, the immunological synapse that forms at the contact surface of the T cell with the antigen-presenting cell adopts a typical structure, with a central area containing T cell receptor molecules bound to the MHC-antigen complex, and a peripheral ring in which the integrin lymphocyte function–associated antigen-1 (LFA-1) is bound to intercellular adhesion molecules. This structure is itself a result of signal transduction and is usually stable over prolonged periods of time, a property correlated with productive T cell activation (Bromley et al. 2001). Biochemistry provides another view: rapidly after TCR crosslinking, a burst of tyrosine phosphorylation can be detected. Phosphorylation not only activates downstream enzymes such as phospholipase C-γ (PLC-γ) and Vav, but also allows phosphorylation-dependent interactions so that multiprotein complexes form and provide physical interactions for efficient enzyme-substrate
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interaction (Singer and Koretzky 2002). Fusion of lipid rafts occurs after triggering of the T cell receptor, and the subsequent changes in local membrane composition bring in a large number and variety of signaling molecules, which either bind to specific lipids/phospholipids or interact with molecules that constitutively or transiently associate with the rafts. The catalytic activity of enzymes such as phospholipase C locally produces second messenger molecules that amplify the signal, because their rapid diffusion generates a concentration gradient and activate signal transduction molecules along the way. Inactivation of genes encoding signaling proteins in the mouse has validated many biochemical findings, previously generated in cell lines by studying changes in protein-protein interactions, protein modifications, or protein localization during T cell activation. However, it has often been difficult to assign a given protein a specific position in the signal transduction cascade by gene ablation alone, either because the entire pathway is inactivated or because of compensation by redundant members of a multigene family. Nevertheless, targeted gene disruption and genome-wide mutagenesis strategies have led to many unexpected findings and have in many instances brought about a readjusted view. The large number of molecules, many assembled in multiprotein complexes, and the different cell membrane and cytoskeletal processes that participate in T cell activation, allow differential modulation of the preexisting, T cell–intrinsic gene expression pattern, skewing transduction of the signal so that several very different outcomes can be generated. In particular, T cell stimulation can lead under different conditions to effector cytokine production, cell growth and proliferation, cell differentiation, activation-induced cell death (AICD), or a state of functional unresponsiveness known as anergy.
VI.
T CELL RECEPTOR SIGNAL TRANSDUCTION
LEADING TO PRODUCTIVE T CELL ACTIVATION In a very simplified view of T cell activation, the signal starts with the recognition of antigen-MHC complexes by the T cell receptor, which then allows Src-family kinases to phosphorylate the LAT adaptor protein. Phosphorylated LAT is bound by ZAP-70, and this triggers activation of PLC-γ1, a critical enzyme that catalyzes hydrolysis of phosphatidylinositol 4,5bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate (IP3), two very important second messengers. IP3 binds to the IP3 receptor and triggers Ca2+ release from intracellular endoplasmic reticulum (ER) stores. By an unknown mechanism, the now-emptied ER stores couple to the opening of Ca2+ channels of unknown identity in the plasma membrane, inducing a strong rise in intracellular Ca2+. The elevation of intracellular Ca2+ activates the phosphatase calcineurin, which dephosphorylates the transcription factor NFAT in the cytoplasm and allows it to enter the nucleus and activate transcription. The other second messenger, diacylglycerol, is a
lipid that attracts signaling proteins containing C1 domains to the plasma membrane. Among these are proteins such as protein kinase (PK) Cθ and RasGRP, key signaling molecules that trigger the activation of Ras/mitogen-activated protein kinase (MAPK)/c-Jun NH2-terminal kinase (JNK) and inhibitor of nuclear factor (NF)-κB (IκB)-kinases. These signaling cascades culminate in nuclear activation of AP-1 (activator protein-1) and NF-κB transcription factors (Wange 2000). Together, transcriptional activation of NFAT, AP-1, and NF-κB is required to productively induce T cell proliferation and induce expression of effector cytokines such as IL-2, IFN-γ, and tumor necrosis factor (TNF)-α. Although TCR stimulation alone is able to activate all three transcription factors NFAT, AP-1, and NF-κB, the latter two transcription factors, AP-1 and NF-κB, are more potently activated through coengagement of CD28/B7 (Acuto and Michel 2003). The cis-regulatory regions of cytokine genes (i.e., IL-2 and IFN-γ) often contain composite sites that are bound by an NFAT-AP1 complex with much higher affinity than by NFAT or AP-1 individually; this ensures strong gene expression only if CD28 is triggered in combination with the TCR. TCR stimulation also leads to increased phosphoinositide 3-kinase (PI3K) activity, which is potentiated if CD28 is engaged as well. Ligation of CD28 leads to tyrosine phosphorylation in its cytoplasmic tail, which can then be bound by the p85 SH2 (Src homology) domain of PI3K. PI3K activity phosphorylates phosphatidylinositol 4-5-bisphosphate at the 3′ position and produces another important second messenger, phosphatidyl inositol 3,4,5-trisphosphate (PIP3). PIP3 allows membrane localization and activation of PH domain-containing proteins such as Vav, a GTP/GDP exchange factor for the small GTPases, Rac, and Cdc42, which couples TCR/CD28 stimulation to cytoskeletal rearrangement. Another very well-studied outcome of PI3K signaling is activation of the PH domain–containing kinase, Akt/PKB. Akt has been shown to critically regulate T cell survival through activation of the transcription factor NF-κB, leading to the induction of Bcl-XL (Kane and Weiss 2003).
VII.
CONTROL OF T CELL–MEDIATED
AUTOIMMUNITY THROUGH CENTRAL TOLERANCE T lymphocytes that mature in the thymus are subject to positive and negative selection. These processes have been described in detail in chapter 9 and will therefore not be discussed here. In brief, thymocytes are positively selected for their ability to cross-react to self-antigen, as a measure of having a productively rearranged T cell receptor. However if the thymocyte displays a strong reaction against self-antigens, death is induced, and the T cell with the self-reactive TCR is clonally deleted (negative selection).
10. PERIPHERAL TOLERANCE OF T CELLS IN THE MOUSE
Mutations in two categories of genes that influence negative selection can lead equivalently to an autoimmune phenotype. Gene inactivation of the putative transcriptional regulator AIRE results in flagrant autoimmune disease in mice. The aire-deficient mouse shows a phenotype very similar to the disease found in human patients with APECED, who also have mutations in the AIRE gene. AIRE appears to de-repress transcription of tissuespecific self-antigens in the medulla of the thymus, enabling the thymocytes to experience the full repertoire of antigens in the periphery and providing the molecular basis for negative selection (Anderson et al. 2002, 2005; Liston, Gray, et al. 2004). Surprisingly, however, point mutations in signaling proteins that participate in TCR signal transduction (i.e., LAT, RasGRP, and ZAP-70), which create hypomorphic alleles, also evoke an autoimmune phenotype in the mouse. As demonstrated for the knockin Y136F mutation in LAT, a decreased ability of LAT to couple to PLC-γ1 activation leads to a partial block in T cell development, but the T cells that enter the periphery display autoreactivity and cause autoimmune disease (Aguado et al. 2002; Sommers et al. 2002). The underlying mechanism of such autoimmunity can be seen as a shift in the threshold for positive and negative selection, as observed for point mutations in both LAT and ZAP-70 (Sakaguchi et al. 2003; Sommers et al. 2005). A possible interpretation for the observed autoimmune phenotype of these mutant mice could be that only those T cells that have high affinity for self are positively selected, since these compensate for compromised signaling. Among the positively selected T cells, incomplete negative selection due to overall reduced responsiveness or due to the absence of specific selfepitopes in the thymus will allow escape of self-reactive T cells to the periphery and create autoimmune potential. The mechanisms of cell death during negative selection are not entirely clear. One step, however, appears to be the induction of proapoptotic proteins, among them the BH3 domain-containing protein Bim. Gene deficiency for Bim leads to decreased sensitivity of double-positive thymocytes as well as mature T cells to apoptotic stimuli such as ionomycin and cytokine deprivation. Older Bim-deficient mice display accumulation of single-positive T cells and the development of autoimmune disease (Bouillet et al. 1999). The inefficient deletion of self-reactive T cells that was observed in the autoimmune diabetes-prone NOD mouse strain (Kishimoto and Sprent 2001) is associated with reduced upregulation of Bim in CD4-positive T cells that recognize selfantigen in the thymus (Liston, Lesage, et al. 2004).
VIII.
TCR STIMULATION IN THE ABSENCE OF
COSTIMULATION: INDUCTION OF PERIPHERAL T CELL TOLERANCE Although peptide-MHC recognition by the T cell receptor is the central component in T cell activation, its impact on T cell
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cytokine production and proliferation is greatly enhanced through costimulation. Conversely, strong cross-linking of the TCR in vitro without CD28 stimulation induces a state of unresponsiveness termed anergy. In this cell intrinsic unresponsiveness the T cell stays alive but fails to respond with IL-2 production and proliferation to optimal stimulation through its TCR and costimulatory receptors. It seems that a partial stimulus induces the anergic state since altered peptide ligands can also induce unresponsiveness in the presence of costimulation (Schwartz 2003). The in vivo correlate of an anergizing condition can be produced when TCR-transgenic T cells are adoptively transferred into a host that presents the recognized antigen (Schwartz 2003), in this case initial T cell activation that soon converts into an unresponsive state of the T cells occurs. Partial stimulation in vivo could also be provided by quiescent DCs that have not received stimulation through innate immune pathways (i.e., Toll-like receptors), and have not upregulated expression of B7.1 and B7.2 molecules that are the ligands for the costimulatory receptor CD28 (Medzhitov 2001). On the transcriptional level, stimulation of the TCR in the absence of costimulation is associated with efficient calcium mobilization, which induces nuclear translocation of NFAT. In the absence of costimulation, however, the TCR signal does not sufficiently activate AP-1 and NF-κB. In fact, NFAT transcription in the absence of AP-1 induces anergy-associated genes, consistent with induction of anergy through the Ca2+ ionophore ionomycin and the observed inability of T cells to produce IL-2 in response to TCR stimulation when they have been transduced to ectopically express constitutively active NFAT (Macian et al. 2002). Targeted deletion of NFAT family members has shown that NFAT expression in lymphocytes is required for activation and full effector function (Hodge et al. 1996; Ranger, Hodge, et al. 1998). It has also been observed that NFAT1 or NFAT1 and 4 deficiency triggers hyperactivation of T cells (Ranger, Oukka, et al. 1998; Rengarajan et al. 2002; Xanthoudakis et al. 1996) and in combined knockouts (NFAT 1 and 2 or NFAT1 and 4) the development of autoimmune disease (Peng et al. 2001; Ranger, Oukka, et al. 1998). Peripheral T cell tolerance is not only mediated through cell-intrinsic unresponsiveness, but also contains a dominant suppressive component mediated by regulatory T cells (Treg). These cells can be considered as a separate CD4+CD25+ T helper lineage, characterized by high FoxP3 expression and suppressor function (Hori et al. 2003). FoxP3 deficiency in the naturally occurring scurfy mouse mutant prevents the development of regulatory T cells, leading to an autoimmune phenotype in mice that resembles human immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, also caused by mutations in FoxP3 (Fontenot et al. 2003). It is clear that development of Treg cells requires T cell receptor–dependent recognition of antigen. Moreover Tregs that naturally develop in the thymus can also be induced in the periphery (Apostolou and von Boehmer 2004). It is tempting to suggest that T cell tolerance utilizes acute mechanisms such as
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clonal deletion in the thymus and T cell intrinsic unresponsiveness to cope with self-antigen recognition but that there is also a connection of T cell activation to the developmental process that generates regulatory T cells. Indeed, antigen recognition is known to be needed for Treg development, both in the thymus and also in an adaptive process in the periphery (Apostolou and von Boehmer 2004; Jordan et al. 2001).
IX.
CONTRIBUTIONS FROM T CELL
HOMEOSTASIS AND T CELL APOPTOSIS The importance of T cell homeostasis to prevent autoimmune disease is best illustrated by the behavior of T cells during experimental or disease- or stress-induced lymphopenia. Under lymphopenic conditions, T cells engage in compensatory proliferation in an attempt to fill up the empty lymphoid compartment and this proliferation then allows self-reactive T cells to expand in the periphery and cause autoimmune disease, with IL-21 as one factor that may trigger the homeostatic expansion (King et al. 2004). Indeed, many experimental autoimmune disease models make use of the lymphopenia-induced proliferation and activation of self-reactive T cells that occurs when small numbers of T cells are injected into immune-compromised (e.g., RAG-deficient) hosts. Under these conditions, the expanding T cells very effectively cause autoimmune diseases, for example, inflammatory bowel disease. Normal immune responses also bring about enormous expansion of effector T cells, but after elimination of the pathogen it is essential that the population of T lymphocytes is induced to contract massively. This contraction is achieved through AICD, in which T cells are killed in a Fas ligand (FasL)-dependent fashion. FasL expression in T cells may be regulated through activation-associated increases in levels of the transcription factor Stra13. T cells from Stra13 knockout mice showed reduced cytokine and FasL induction and reduced AICD, and older mice developed autoimmunity with accumulation of T and B cells in the periphery as well as multiorgan infiltration (Sun et al. 2001). FasL/CD95 ligation effectively induces death in T cells that contain a low ratio of antiapoptotic (Bcl-2 and Bcl-XL) to proapoptotic (Bim, Bid, and Bax) factors (for review, see Krueger et al. 2003). The antiapoptotic factors Bcl-2 and Bcl-XL are induced either as a result of costimulationinduced NF-κB activation or, in the case of memory T cells, in response to stimulation with cytokines that promote cell survival. NF-κB activation in T cells appears to be regulated through the kinase activity of Akt/PKB, which in turn depends on membrane levels of PIP3 (Kane and Weiss 2003); in fact, hypermorphic Akt alleles caused NF-κB activation, Bcl-XL expression, and increased T lymphocyte survival (Jones et al. 2000) as well as the development of autoimmune disease with immunoglobulin deposits on kidney glomeruli (Rathmell et al. 2003).
Notably, various genetic manipulations of the Akt pathway, which either introduce an activator or inactivate an inhibitor of upstream or downstream Akt signaling, lead to induction of autoimmune disease. Thus, transgenic expression of a constitutively active PI3K p85 deletion mutant (p65), as well as targeted disruption of genes encoding the PIP3 phosphatases PTEN and SHIP (SH2-containing inositol-5′-phosphatase), results in upregulation of PIP3 levels and consequent Akt activation, and both manipulations are associated with increased survival of lymphocytes, appearance of T lymphomas, and the development of autoimmune disease (Borlado et al. 2000; Di Cristofano et al. 1999; Moody et al. 2003; Suzuki et al. 2001). Similarly, downstream of Akt, targeted deletions of Foxj and Foxo3 have implicated these transcription factors as negative regulators of NF-κB, with one mode of inhibition being a role in the basal expression of IκBβ or IκBβ and IκBε (Lin, Hron, et al. 2004; Lin, Spoor, et al. 2004). Balanced T cell homeostasis is especially important in the CD4+CD25+ subset of regulatory T cells to prevent autoimmune disease. These cells critically depend on the CD28-B7 interaction (Lohr et al. 2003; Salomon et al. 2000) as well as on IL-2 that is produced by effector T cells, since neutralization or IL-2 deficiency leads to autoimmune disease owing to reduced numbers of regulatory T cells (Setoguchi et al. 2005).
X.
NEGATIVE REGULATION OF T CELL ACTIVATION
Our overall understanding of negative signaling in T cells is much more limited than our understanding of positive signaling. Negative regulators have been identified mostly through analysis of knockout mice that displayed exaggerated responses to T cell stimulation, with respect to proliferation or cytokine production. Negative regulation has been described at many levels of T cell biology. These include humoral factors, costimulatory molecules, adaptor molecules, E3 ubiquitin ligases, phosphatases, and transcription factors.
A.
Humoral Factors
Humoral factors, especially the anti-inflammatory cytokines IL-10 and transforming growth factor-β (TGFβ), are well known to influence the responsiveness of T cells. Both cytokines can inhibit T cell responses, both directly and through their effects on the functions of antigen-presenting cells. IL-10 and TGFβ are particularly effective in preventing the development of colitis and CD8 T cell–mediated inflammation, respectively. Additionally, IL-10 is implicated in the function and TGFβ in the induction of regulatory T cells (von Boehmer 2005).
10. PERIPHERAL TOLERANCE OF T CELLS IN THE MOUSE
B.
Negative Costimulatory Molecules
Negative costimulatory molecules of the CD28 family include CTLA-4 and PD-1. CTLA-4 deficiency causes severe lymphoproliferative and autoimmune disease (Tivol et al. 1995). CTLA-4 is expressed on activated T cells and shows trafficking to the immunological synapse upon TCR stimulation, in a manner that depends on the strength of the activating stimulus (Egen and Allison 2002). In vivo findings also demonstrate that CTLA-4 levels on T cells tune the threshold of activation (Eggena et al. 2004): ovalbumin (OVA)-specific T cells in a CTLA-4-deficient background, when injected into lymphopenic hosts, caused autoimmune destruction of the target organ (β islets expressing OVA from a rat insulin promoter), whereas self-reactive T cells from wild-type mice in the same experimental setting needed to be primed with antigen and adjuvants. CTLA-4 has been proposed to mediate inhibition through competition with CD28 for B7 binding (Collins et al. 2002; Linsley et al. 1994), thereby reducing costimulation and elevating the signal threshold of activated T cells. In addition, CTLA-4 might also itself transduce signals, either to the inside of the same cell by activating Rap1 and thereby inducing LFA-1 clustering and adhesion (Schneider et al. 2005) or to other cells via B7 ligation. In fact, it was recently shown that T cells from B7-deficient mice were not sensitive to suppression by regulatory T cells (Paust et al. 2004). Suppression could be restored by transduction of constructs expressing full-length B7 molecules but not by B7 molecules that lack the cytoplasmic tail, even though both types of B7 molecules were equally costimulatory if transduced into antigen-presenting cells (Paust et al. 2004). The PD-1/PD-L1 system is another very important costimulatory pathway that prevents autoimmunity. PD-1 negatively regulates T cell activation and counteracts CD28-dependent IL-2 production; however, PD-1 effects on proliferation can be reversed through increased CD28 ligation or exogenous IL-2 (Carter et al. 2002). PD-1 ligation inhibited expression of the Bcl-XL gene, possibly by recruiting SHP proteins (Src homology 2 domain phosphatase) (Chemnitz et al. 2004). PD1-deficient mice develop different autoimmune diseases depending on the strain background, exhibiting arthritis and a lupus-like phenotype on the C57BL/6 background but dilated cardiomyopathy on the BALB/c background, the latter associated with and likely induced by antibodies against cardiac troponin I (Nishimura et al. 1999, 2001; Okazaki et al. 2003). PD-1 or PD-L1 blockade precipitated autoimmune diabetes in NOD mice, consistent with a general role of PD-1 in autoimmune diseases (Ansari et al. 2003).
C.
E3 Ubiquitin Ligases
Polyubiquitination has been recognized for a long time as a way for cells to flag cytoplasmic and nuclear proteins for degradation mediated by the proteasome. However, during the
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last few years there has been an enormous increase in our understanding of how ubiquitin regulates cellular processes. Specifically, we now know that the cellular pathway influenced by formation of branched ubiquitin chains depends on the specific lysine residue in ubiquitin that is used for this purpose: branched-chain formation using lysine 48 of ubiquitin targets proteins for proteasome-mediated degradation, whereas branched-chain formation using lysine 63 activates the IκB kinase (IKK) signaling cascade. Moreover, it is now established that mono- and di-ubiquitination regulate the sorting of membrane proteins to the endosomal-lysosomal compartment, stemming from findings that mono- and di-ubiquitin can serve as protein-protein interaction motifs for ubiquitin-binding domains. In other recently investigated functions, mono- and di-ubiquitination have been shown to serve as activating histone modifications and also appear to play a role in DNA-repair. It is therefore not surprising that a rather large number of E3 ubiquitin ligases are involved in the regulation of T cell responses. T cell unresponsiveness has been shown to be a result of unproductive activation of T cells through their T cell receptor in the absence of costimulation (Schwartz 2003). Such signaling triggers the induction of many anergy-associated genes, which are upregulated primarily through activation of the Ca2+/calcineurin/NFAT signaling pathway (Macian et al. 2002), and impose a cell-intrinsic unresponsiveness due to defects in proximal TCR signal transduction (Schwartz 2003). As a result of reduced signal transduction, the formation of the immunological synapse becomes destabilized (Heissmeyer et al. 2004) in a characteristic pattern of disintegration of the LFA-1 ring, that can also be observed if T cells are stimulated with altered peptide ligands (Sumen et al. 2004). Among the anergy-associated genes are those encoding the E3 ligases Cbl-b, Itch, and Grail (Anandasabapathy et al. 2003; Heissmeyer et al. 2004). These E3 ligases are not only upregulated at the transcriptional level but Itch and the structurally related E3 ligase Nedd4 also display membrane relocalization to lipid rafts after anergy induction. Furthermore, Itch is able to ubiquitinate PLC-γ1 after coexpression, PKCθ is mono-ubiquitinated after anergy induction, and both molecules appear at reduced levels in anergic T cells during restimulation. These findings suggest that during restimulation of an anergized T cell, E3 ligases target signaling proteins for degradation via the endosomal/lysosomal degradation pathway (Heissmeyer et al. 2004). Resistance of Itch and Cbl-b-deficient T cells to anergy induction was demonstrated by evaluating their proliferation; furthermore, reduced effectiveness of anergy induction was observed in Cbl-b-deficient T cells as measured by synapse stability and Ca2+ mobilization (Heissmeyer et al. 2004; Jeon et al. 2004). Targeted disruption of either cbl-b or itch genes is associated with an autoimmune phenotype in mice (Bachmaier et al. 2000; Chiang et al. 2000; Fang et al. 2002; Perry et al. 1998). Recently, another E3 ubiquitin ligase, Roquin, was identified in a genome-wide mouse mutagenesis screen. Roquin activity is required to inhibit the development of an autoimmune
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phenotype akin to systemic lupus erythematosus in mice (Vinuesa et al. 2005). Roquin function appears critical in a specific subset of T cells called follicular helper T cells that provide T cell help to B cells (Vinuesa et al. 2005). Decreased Roquin activity, traced to a point mutation in the roquin gene, resulted in increased levels of the costimulatory ICOS molecule in follicular helper T cells. It is possible that this increase in the levels of ICOS and other factors whose expression might be restrained by Roquin, enables autoreactive follicular helper T cells, which recognize self antigens in the absence of costimulation to differentiate and provide help to B cells, thereby triggering autoantibody production.
D.
Adaptor Molecules
Combined inactivation of the related adapter molecules T cell receptor signaling (Sts)-1 and Sts-2 in mice results in an autoimmune phenotype, implying a negative role for these molecules in TCR signal transduction. The same proteins have a role in signal transduction through the epidermal growth factor (EGF) receptor, by binding the adaptor-type E3 ubiquitin ligase Cbl and regulating the ubiquitination-dependent endosomal/lysosomal degradation of the EGF receptor. Sts-1 and Sts-2 have ubiquitinassociated domains as well as an SH3 domain, and as demonstrated using T cells from double Sts-1, Sts-2-deficient mice, they destabilize ubiquitinated forms of ZAP-70 in T cells and inhibit ZAP-70 activity through a mechanism that is not yet understood (Carpino et al. 2004). Cbl and Cbl-b, the presumed binding partners of Sts-1 and Sts-2 in T cells, cooperate in activation-induced downregulation of the TCR (Naramura et al. 2002), suggesting that one way to prevent autoimmunity is to terminate TCR signal transduction by targeting the TCR itself for degradation. In this context, it was recently shown that OVAreactive T cells, when adoptively transferred into a host that systemically presented this soluble antigen, did not cause disease in a host that contains B cells since the T cells became anergized in vivo; this effect on T cell function correlated with dramatic downregulation of the TCR in a B cell– and antigen-dependent fashion (Knoechel et al. 2005). Further analysis is required to understand the importance of TCR downregulation in T cell anergy and tolerance and to identify the molecules involved. One other family of adaptor proteins has shown a pronounced link to autoimmunity. The protein T cell specific adaptor (TSAd) contains an SH2 domain, four conserved tyrosines that can be phosphorylated by protein tyrosine kinases, and two proline stretches, providing multiple interaction surfaces to interact with phosphorylated SH2 motifs or SH2 and SH3 domains in other proteins (Marti et al. 2005). Gene ablation of TSAd leads to spontaneous development of a lupus-like autoimmune disease apparent in older mice, as well as much higher susceptibility to immunization-induced lupus in young Tsad-deficient mice (Drappa et al. 2003). The molecular pathway in which TSAd deficiency is required to prevent autoimmunity is not yet
clear, but it has been proposed that positive regulation of TCRtriggered IL-2 expression and apoptosis induction through the mitochondrial pathway might be most important (Drappa et al. 2003; Marti et al. 2005).
E.
Phosphatases
The genetic linkage of the ptpn22 gene to human autoimmune disease provides compelling evidence for the involvement of the tyrosine phosphatase (PTP) N22 in diabetes, rheumatoid arthritis, systemic lupus erythematosus, and Graves thyroiditis (for review, see Siminovitch 2004). In mice, PTPN22−/− T cells display increased positive selection and no defects in negative selection or AICD but rather an overall increased signaling by T cells with higher levels of Ca2+-flux, prolonged Lck activation, and increased proliferation (Hasegawa et al. 2004). In fact, mice deficient for this phosphatase show increased numbers of effector T cells in the periphery, elevated IgG1, IgG2, and IgE levels and spontaneous germinal center formation in the spleen and Peyer’s patches that depended on T cell help. Surprisingly, and different from the situation in humans, neither young nor old PTPN22−/− mice develop overt autoimmune symptoms (Hasegawa et al. 2004). Not only phosphorylated proteins but also phospholipids are targets of phosphatases that inhibit the development of autoimmunity. The control of cellular PIP3 levels by the phosphatases PTEN and SHIP, two phosphatases that dephosphorylate the 3′ and the 5′ position of PIP3, respectively, has been shown to prevent autoimmunity (Moody et al. 2003), and both PTEN and SHIP are important regulators of T cell apoptosis and homeostasis (see earlier).
XI.
CONCLUDING REMARKS
Gene targeting in mice, inactivating single genomic loci at a time, has elucidated a predominant role for T cells in various autoimmune diseases. Mice carrying such mutations develop fatal systemic or organ-specific damage, the symptoms of which correlate well with many detrimental human autoimmune diseases. Genetic linkage analysis of human patients points to a complex multigenic basis for many autoimmune syndromes, and the associated genes or genetic regions show considerable overlap with genes linked to autoimmunity in mice. Although our knowledge of general “autoimmune factors” is limited at present, some of the genes identified could have diagnostic and therapeutic potential. Experimental access to cells and tissues in mice has allowed investigators to study perturbations in processes such as thymic selection, T cell homeostasis, differentiation, or activation that have been shown to result in autoimmunity, and critical molecules and pathways of signal transduction have been identified.
10. PERIPHERAL TOLERANCE OF T CELLS IN THE MOUSE
The constantly growing list of genes that participate in T cell signaling and autoimmune disease indicates that our understanding of these processes is at an early stage and still incomplete. Although there is evidence from the clinic that the onset of autoimmune disease shows a crucial environmental influence, the critical factors have not been identified experimentally. Also, how infection shapes the immune system and thereby determines the type or severity of autoimmune disease an individual will develop, is not understood. Progress in all of these areas is needed to discover treatments for autoimmunity that target critical points in the disease and alleviate a patient’s burden with minimal side effects.
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Vinuesa, C.G., Cook, M.C., Angelucci, C., Athanasopoulos, V., Rui, L., Hill, K.M., et al. (2005). A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 435, 452–458. von Boehmer, H. (2005). Mechanisms of suppression by suppressor T cells. Nat Immunol 6, 338–344. Wange, R.L. (2000). LAT, the linker for activation of T cells: a bridge between T cell-specific and general signaling pathways. Sci STKE 63, RE1. Watanabe-Fukunaga, R., Brannan, C.I., Copeland, N.G., Jenkins, N.A., Nagata, S. (1992). Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356, 314–317. Waterhouse, P., Penninger, J.M., Timms, E., Wakeham, A., Shahinian, A., Lee, K.P., et al. (1995). Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270, 985–988. Willerford, D.M., Chen, J., Ferry, J.A., Davidson, L., Ma, A., Alt, F.W. (1995). Interleukin-2 receptor α chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3, 521–530.
Witebsky, E., Rose, N.R., Terplan, K., Paine, J.R., Egan, R.W. (1957). Chronic thyroiditis and autoimmunization. JAMA 164, 1439–1447. Wu, J., Wilson, J., He, J., Xiang, L., Schur, P.H., Mountz, J.D. (1996). Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. J Clin Invest 98, 1107–1113. Xanthoudakis, S., Viola, J.P., Shaw, K.T., Luo, C., Wallace, J.D., Bozza, P.T., et al. (1996). An enhanced immune response in mice lacking the transcription factor NFAT1. Science 272, 892–895. Zhang, W., Sommers, C.L., Burshtyn, D.N., Stebbins, C.C., DeJarnette, J.B., Trible, R.P., et al. (1999). Essential role of LAT in T cell development. Immunity 10, 323–332. Zhu, J.W., Field, S.J., Gore, L., Thompson, M., Yang, H., Fujiwara, Y., et al. (2001). E2F1 and E2F2 determine thresholds for antigen-induced T-cell proliferation and suppress tumorigenesis. Mol Cell Biol 21, 8547–8564.
Chapter 11 The Genetics of Mouse Models of Systemic Lupus Srividya Subramanian and Edward K. Wakeland
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Models of Murine Lupus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Spontaneous Lupus Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. MRL.lpr . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. BXSB.yaa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. [NZW × NZB]F1: Insights into MHC and Non-MHC Susceptibility Loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. NZM2410 and the Congenic Derivatives . . . . . . . . . . . . . . . . . . . . . . . E. Congenic Dissection of NZM2410 . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Epistatic Interactions in the Development of Pathogenic Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Epistatic Interactions in the Suppression of Pathogenic Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Insights into Lupus, Pathways, and Epistasis from Genetically Manipulated Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Antigen and Immune Complex Clearance . . . . . . . . . . . . . . . . . . . . . . . B. Regulation of Proliferation and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . C. Lymphocyte Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
The prototype of systemic autoimmune disorders, systemic lupus erythematosus (SLE), is characterized by a loss in immunological tolerance to a variety of ubiquitous self-antigens, such as chromatin, double-stranded (ds) DNA, ribonucleoproteins, and complement. A variety of immunological aberrations THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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ultimately culminate in the different pathogenic processes of SLE, which can include glomerulonephritis (GN), nondeforming arthritis, pericarditis, vasculitis, pleuritis, and serosititis (Wakeland et al. 2001). These immune irregularities have been postulated to be a consequence of changes in the activation thresholds and functions of immune cells, variations in the cytokine milieu produced, alterations in the clearance of apoptotic Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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and necrotic debris by phagocytotic cells, and modulations in antigen presentation capacities (reviewed in Banchereau et al. 2004; Carroll 1998; Hoffman 2004; Lipsky 2001). Whereas the factors involved in the initiation of SLE are poorly understood, numerous researchers have clearly demonstrated that genetic predisposition is the major element dictating lupus susceptibility. SLE has all the features of a complex, polygenic trait, including a requirement for multiple genes, incomplete penetrance, and genetic heterogeneity (Vyse and Kotzin 1998; Wandstrat and Wakeland 2001). An understanding of the genetic basis of lupus susceptibility is further complicated by emerging evidence indicating that the predisposing causal variants are common alleles that per se are nondeleterious, as most clearly exemplified by the human leukocyte antigen (HLA) polymorphisms associated with autoimmunity (Rioux and Abbas 2005). This makes conclusive identification of the causal polymorphisms difficult, as they are relatively frequent within the population, and their phenotypic consequences are subtle. If the predisposing allelic variations are commonly found in the general population, how then do they elicit systemic autoimmunity? It is postulated that different combinations of polymorphic genes, due to their specific genetic interactions, termed epistasis, can lead to imbalances in immune regulation/function that potentiate the development and progression of autoimmunity (Subramanian and Wakeland 2005).
II.
MODELS OF MURINE LUPUS
The overall complexity of the genetics of human SLE susceptibility has led many investigators to turn to murine lupus models. These studies are greatly facilitated by the availability of both spontaneous and engineered mouse strains that develop lupus. These murine models share many of the immunological and pathological abnormalities observed in human patients with lupus, including a loss in tolerance to nuclear antigens and consequent high titers of serum autoantibodies (autoAbs), as well as the development of immune complex–mediated GN. Spontaneous models are particularly useful as they represent genomes, which through different breeding strategies, contain enough susceptibility genes so that their “autoimmune potential” is maximized, and the animals develop overt disease. Linkage analysis has been a powerful tool in the identification of predisposing loci in these spontaneous lupus models, as summarized in Table 11-1. However, the 95% confidence intervals obtained for these loci often contain hundreds of potential candidate genes, making functional identification of the causal gene challenging and providing little insight into the contributions of individual loci to the disease process. Consequently, congenic dissection of these strains has proven to be invaluable. This involves introgressing the susceptibility locus of interest onto a non-autoimmune prone background, such as C57Bl/6J (B6), and assessing the clinical and immunological phenotypes
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mediated by the locus in isolation. Importantly, these congenic lines can then be used to generate recombinants for fine mapping of the susceptibility locus. A major caveat to this approach is that the phenotype mediated by the locus in isolation must be reasonably robust. An alternative strategy is to move corresponding resistance loci onto the autoimmune background and assess for disease amelioration. Lupus-like phenotypes have also been observed in mice in which genetic ablation and transgenic overexpression of genes has been performed. In addition, by introgressing these genetic modifications onto autoimmune-prone spontaneous strains, insight has been gained into the requirements for certain molecules in disease initiation and pathogenesis. Both the spontaneous and engineered models provide tools with which to better identify genes contributing to underlying genetic susceptibility and shed light onto how abnormalities in various pathways may contribute to the overall disease process (Jorgensen et al. 2004; Vyse and Kotzin 1998; Wakeland et al. 2001).
III.
SPONTANEOUS LUPUS MODELS
A.
MRL.lpr
The lpr mutation was originally discovered during the derivation of the MRL/MpJ strain, and on this background resulted in hypergammaglobulinemia, anti-dsDNA autoAbs, profound lymphadenopathy and splenomegaly, and immune complex (IC)-mediated GN. MRL.lpr mice were also characterized by the emergence of an unusual CD4−CD8−CD3+B220+ T cell population. It was later discovered that lpr encodes for the apoptosis-signaling receptor Fas antigen and that the mutation results in premature termination of Fas transcription (Watanabe-Fukunaga et al.1992). The mechanism by which lack of Fas-mediated signaling results in autoimmunity is postulated to be due to the emergence of self-reactive T and B cells that would normally undergo apoptosis during thymic, bone marrow (BM), and peripheral selection events (Rioux and Abbas 2005; Vyse and Kotzin 1998). However, the effect of the lpr mutation is highly genome dependent, as evidenced by the varying degrees of autoimmunity seen when it is introgressed onto non-autoimmune strains, such as B6, C3H, and AKR. This illustrates the necessity for epistatic interactions between lpr and the rest of the genome for full disease expression (Kelley and Roths 1985). Furthermore, Fas-intact MRL mice develop a late-onset autoimmunity, culminating in 50% mortality by 18 months, suggesting that lpr is an accelerating factor, as opposed to a causal mutation (Watson et al. 1992). Consistent with this result, CD4+ T cells from Fas-intact MRL mice have an intrinsic hyperresponsive phenotype after engagement with high- and low-affinity peptide-MHC complexes and a lower threshold for CD3-mediated activation. Thus, the
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TABLE 11-1
MAJOR SPONTANEOUS LUPUS MODELS AND LOCI LINKED TO AUTOIMMUNE PHENOTYPES Autoimmune Mouse Strain
Linked Loci
Chromosome (peak QTL cM)
Associated Phenotype(s) GN GN SM, LA, dsDNA SM, LA, dsDNA SM, LA, dsDNA SM, LA, GN dsDNA dsDNA SM, ANA, GN dsDNA SM, ANA, GN GN ANA, GN
MRL.lpr
Lrdm1 Lrdm2 Lmb1 Lmb2 Lmb3 Lmb4
BXSB.yaa
Bxs1 Bxs2 Bxs3 Bxs4 Nba1 Nba2
7 (6.0) 12 (35.0) 4 (57.5) 5 (41.0) 7 (28.0) 10 (41.0) 2 11 1 (32.8) 1 (61.3) 1 (100.0) 1 (7.7) 4 (75.0) 1 (95.0)
Nba3
7 (42.6)
ANA, GN
Lbw1 Lbw2 Lbw5 Lbw6 Lbw7 Sle1a Sle1b
17 (19.1) 4 (42.6) 7 (23.0) 18 (47.0) 1 (90.0) 1 (88.0) 1 (89.5)
ANA, GN SM, GN GN GN SM, ANA ANA SM, ANA
Sel1c Sleld Sle2a Sle2b Sle3
1 (106.3) 1 4 (33.0) 4 (42.6) 7 (16.0)
ANA GN SM, dsDNA, GN SM, dsDNA, GN SM, ANA, GN
Sle5
7 (0.0)
dsDNA, ANA
Sles1
17 (19.0)
Sles2 Sles3
4 (58.0) 3 (35.0)
SM, ANA, dsDNA, GN (suppressed) ANA (suppressed) ANA (suppressed)
[NZB × NZW]F1
NZM2410
Postulated Gene(s)
Ifi202
SLAM/Cd2 family Cr2
References Vidal et al. 1998 Vidal et al. 1998 Watson et al. 1992 Watson et al. 1992 Watson et al. 1992 Watson et al. 1992 Gu et al. 1998 Gu et al. 1998 Haywood et al. 2004 Haywood et al. 2004 Haywood et al. 2004 Haywood et al 2004 Drake et al. 1994 Drake et al. 1994; Rozzo et al. 2001 Drake et al. 1994; Vyse, Drake, et al. 1996 Kono et al. 1994 Kono et al. 1994 Kono et al. 1994 Kono et al. 1994 Kono et al. 1994 Morel et al. 2001 Morel et al. 2001; Wandstrat et al. 2004 Morel et al. 2001 Morel et al. 2001 Xu et al. 2005 Xu et al. 2005 Mohan, Morel, et al. 1999; Morel et al. 1994; Morel, Mohan, et al. 1999 Mohan, Morel, et al. 1999; Morel, Mohan, et al. 1999 Morel, Tian, et al. 1999; Subramanian et al. 2005 Morel, Tian, et al. 1999 Morel, Tian, et al. 1999
Listed are the various loci identified in the different spontaneous lupus models discussed in the text, their chromosomal positions in centimorgans (cM) (based on the Mouse Genome Informatics database, if available), the various phenotypes associated with the loci, and postulated causal genes. ANA, antinuclear autoantibodies; dsDNA, anti-dsDNA IgG; GN, glomerulonephritis; LA, lymphadenopathy; SM, splenomegaly.
phenotypic variations of the Fas mutation may be a consequence of strain-specific differences in the intrinsic responsiveness of the T cell compartment (Vratsanos et al. 2001; Zielinski et al. 2005). Further insights into mechanisms leading to autoimmunity in the MRL.lpr strain come from studies investigating how genetic ablation of various molecules potentiates or ameliorates disease. The absence of molecules such as interferon (IFN)-RI, the C3 inhibitory factor decay-accelerating factor (DAF), interleukin (IL)-10, and the costimulatory molecule CD80, have been to shown to exacerbate various aspects of disease (Hron and Peng 2004; Liang et al. 1999; Miwa et al. 2002; Yin et al. 2002).
In contrast, deficiency in a variety of molecules involved in immune functions, such as interferon receptor II (IFN-RII), colony-stimulation factor (CSF), leukocyte function-associated antigen-1 (LFA-1), Nuclear-factor E2–related factor (NrF2), terminal deoxynucleotidyl transferase (Tdt), IL-2, IL-12p40, Factor B, CD28, CD86, and IFN-γ receptor (IFNγR), were observed to confer a protective effect on specific disease phenotypes (Hron and Peng 2004; Kevil et al. 2004; Kikawada et al. 2003; Lenda et al. 2004; Liang et al. 1999; Morito et al. 2004; Robey et al. 2004; Schwarting et al. 1998; Tada et al. 1999; Watanabe et al. 2000; Xiao et al. 2003).
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It must be kept in mind that in many of these studies, the deficiencies were studied on mixed genetic backgrounds, which can significantly influence the magnitude of disease phenotypes. This situation was seen in the case of interleukin-18 receptor (IL-18R), where, in contrast to a previous report on a less homogenous genetic background, deficiency of the IL18R, and consequently IL-18-mediated signaling, was found to have no effect on disease development (Lin and Peng 2005). Deficiency of the common Fcγ receptor (FcγR) chain was also shown to have no impact on disease development (Matsumoto et al. 2003). Linkage analyses have been performed on the MRL.lpr strain to identify loci contributing to the strain-specific effects of the lpr mutation described above. By using [MRL.lpr × Cast/Ei]F1 × MRL.lpr backcross animals, MRL-derived loci on chromosomes 7 and 12 (Lrdm1–2) were identified as contributing to renal disease (Watson et al. 1992). Another cross utilizing [MRL.lpr × B6.lpr]F2 intercross progeny identified four loci with significant linkage to lymphadenopathy and/or splenomegaly on chromosomes 4, 5, 7, and 10, termed Lmb1–4, respectively. Lmb1–3 were also associated with antidsDNA autoAbs, whereas Lmb4 was associated with GN (Vidal et al. 1998). An analysis of [MRL.lpr × BALB/c]F2 mice revealed linkage for anti-dsDNA autoAbs with chromosomes 2 and 11 (Gu et al. 1998). Other linkage studies have been performed to identify loci involved in the development of arthritis and collagen disease in the MRL.lpr strain, which has also been used as a model of polyarteritis, rheumatoid arthritis, Sjögren’s syndrome, and autoimmune lymphoproliferative syndrome (Kamogawa et al. 1999, 2002; Nakatsuru et al. 1999; Nishihara et al. 1999). The specific identities of any of these genes have not yet been determined.
B.
BXSB.yaa
The BXSB.yaa strain is a recombinant inbred strain derived from B6 and SB/Le. Because of the presence of the as-yet unidentified Y chromosome autoimmune accelerator (yaa) locus, male mice from this strain spontaneously develop humoral autoimmunity to a plethora of self-antigens and lupus nephritis, ultimately culminating in 100% mortality by 6 months of age. Analyses of different consomic strains, carrying the BXSB-derived Y chromosome have revealed that the ability of yaa to mediate severe autoimmunity is, like that of lpr, highly dependent on interactions between it and other susceptibility loci from the autosomal genome (Hudgins et al. 1985). Moreover, the addition of yaa to a variety of F1 crosses, in which the progeny are normally non-autoimmune, such as [New Zealand white (NZW) × B6]F1 and [New Zealand black (NZB) × B6]F1 crosses, can lead to the development of significant systemic autoimmunity (Hudgins et al. 1985; Kikuchi et al. 2005; Santiago et al. 1998). Importantly, yaa on the B6
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background does not lead to fulminant autoimmunity, and it has been postulated that the capacity of yaa to promote autoimmunity is dependent on the ability of the autosomal background to promote the spontaneous development of autoantibodies (Merino et al. 1994). The course of disease in BXSB.yaa males is highly similar to that seen in other murine lupus models. In addition to clinical features, such as GN and the emergence of immunoglobulin (Ig) G autoAbs specific for different self-antigens, the animals develop with age a profoundly activated immune system and hypercellularity of the spleen and lymph nodes (LNs). This is characterized by dramatic increases in the percentage of CD4+ T cells having effector memory (CD62L−CD45RBloCD44hi) and activated (CD25+) phenotypes. The CD4+ cells have been shown to be refractive to both anti-CD3 and phorbol myristic acid/ionomycin-induced proliferation in vitro, yet produced increased amounts of IFN-γ and IL-4 and decreased IL-2 in response to both of these stimuli (Chu et al. 1994). Notably, decreased IL-2 production is also a feature of human lupus T cells (Chowdhury et al. 2005; Crispin and Alcocer-Varela 1998; Juang et al. 2005). These changes in the functional responses of CD4 T cells are also characteristic of replicative senescence. Treatment with cytotoxic T lymphocyte-associated Ag (CTLA-4) Ig was shown to prevent different CD4 cell surface and functional phenotypes and reduced both GN and anti-chromatin autoAbs, presumably via negative regulation of T cells. Cessation of treatment, however, restored the CD4 effector memory phenotype (Chu et al. 1996). BXSB.yaa mice lacking the cyclin-dependent kinase inhibitor p21 showed increased survival and a reduction in GN scores, anti-chromatin IgG, and hypergammaglobulinemia. CD4 cells were reduced in overall number and had a less activated phenotype. Importantly, these CD4s made less IFN-γ, and more of the CD4+CD44hi cells were undergoing apoptosis, had incorporated bromodeoxyuridine, and were in the G1 versus G0 phase of the cell cycle (Lawson et al. 2004). Hence, it may be that preventing the accrual of these CD4 cells can ameliorate some of the more pathogenic phenotypes and ameliorate morbidity. There are numerous B cell–associated differences in BXSB.yaa mice as well, some of which appear to be specifically associated with the yaa locus and are independent of the development of systemic autoimmunity. Resting splenic B cells from both BXSB.yaa and B6.yaa mice were shown to proliferate more in response to a variety of different stimuli. These data suggested that yaa acts downstream of initial signaling events and is not Ca2+ or PKC dependent. Because this difference in B cell responsiveness was seen in both BXSB.yaa and B6.yaa, it indicated that such a change is not sufficient for the development of autoimmunity. In contrast, similar experiments performed with purified T cells from young B6.yaa mice showed no differences in proliferative responses to various stimuli. This increased B cell responsiveness was not amplified with age, and by 5 months, similar to the CD4 T cells, BXSB.yaa B cells showed reduced proliferative responses,
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which were also mitigated in BXSB.yaa|p21-mice (DesJardin et al. 1996, Lawson et al. 2004). The ultimate goal of identifying the causal gene within a susceptibility locus is unusually confounded in the case of yaa. In most murine positional cloning efforts naturally occurring meiotic recombination events and subsequent phenotypic characterizations are used to narrow the locus to a susceptibility interval amenable for causal gene identification. This is not possible for Y chromosome–located genes, and investigators have used other approaches to help elucidate the nature of the yaa gene. Mixed BM chimeric experiments have demonstrated that only yaa- bearing B cells produce autoantibodies and that the presence of yaa- bearing T cells is not sufficient to make nonyaa- bearing B cells make autoantibodies (Merino et al. 1991). Further studies using both mixed BM chimeric experiments and BXSB.yaa/TCRα−/− mice implicated CD4 T cells as being necessary for severe disease but demonstrated that these cells do not need to express yaa to mediate severe disease (Fossati et al. 1995; Lawson et al. 2001). These data indicate that although CD4 T cells are necessary, only the B cells actually need to functionally express yaa for disease development to occur in this lupus model. Despite the difficulties associated with identifying yaa, a variety of linkage studies have been conducted to determine the positions of autosomal genes that interact with yaa to mediate lupus. Analyses of [NZW × B6.yaa]F1 × B6 males, to identify dominant NZW contributions, revealed a major dominant locus contributing to severe GN and anti-dsDNA IgG susceptibility on chromosome 7. Interestingly, whereas there was no significant linkage of either of these phenotypes to chromosome 17 by itself, there appeared to be interactions between the chromosome 7 locus and chromosome 17, such that heterozygotes at chromosome 7 and B6 homozygotes at chromosome 17 had the highest titers. Somewhat surprisingly, no significant linkage for these phenotypes was obtained with chromosome 1 (Santiago et al. 1998). In a similar study of [NZB × B6.yaa]F1 × B6 backcross males NZB-contributed GN susceptibility was mapped to chromosomes 1 and 13 but not to chromosomes 7 and 17. Interestingly, anti-DNA and anti-chromatin IgG mapped to an NZB contribution on chromosome 1 but to B6 homozygosity at chromosome 17 (Kikuchi et al. 2005). Congenic analyses of various autosomal yaa-interacting loci, identified via such linkage studies, have revealed some interesting findings. The chromosome 1 Bxs1–4 loci, originally identified in [BXSB.yaa × B10]F1 reciprocal backcrosses, were moved in various combinations onto the B10.yaa background. Bxs1 appeared to have an impact on kidney pathological changes in the absence of significant antinuclear autoantibody (ANA) production, Bxs2 specifically contributed to antidsDNA IgG, whereas the most centromeric locus, Bxs4, appeared to interact with Bxs1 to mediate moderate GN. It is evident, however, that the major BXSB-derived locus on
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chromosome 1 is Bxs3, which can interact with yaa on the B10 background to recapitulate most, if not all, of the phenotypes observed in BXSB.yaa (Haywood et al. 2004). Interestingly, Bxs3 corresponds to the same genomic interval as the NZBand NZM2410-derived loci Nba2 and Sle1, respectively, both of which can also interact with yaa on the non-autoimmune B6 background to generate systemic autoimmunity (Croker et al. 2003; Kikuchi et al. 2005; Morel et al. 2000). A limitation in the interpretation of the above-described studies is that none of the Bxs chromosome 1 loci have been studied in the absence of yaa on the B10 background. Given the importance of epistatic interactions in the development of various lupus phenotypes, it would be informative to assess the component phenotypes of individual loci. Similar studies with the NZB-derived chromosome 1 and 7 loci, Nba2 and Nba5, respectively, demonstrated that both these loci can interact with yaa on the B6 background as well, in both cases resulting in increased 15-month mortality and severe GN. Only B6.Nba2|yaa mice showed increased anti-DNA and antichromatin IgG relative to both female controls and B6.yaa (Kikuchi et al. 2005). C.
[NZW × NZB]F1: Insights into MHC and Non-MHC Susceptibility Loci
One of the most well-characterized models of murine lupus is the F1 hybrid of the NZB and NZW inbred mouse strains (BWF1). These mice develop progressive, severe GN, and high levels of antinuclear antigen–specific IgG autoAbs, similar to both BXSB.yaa and MRL.lpr, but unlike those models, no single gene mutation is necessary for full disease expression. Interestingly, neither parental strain develops severe lupus phenotypes, although each is associated with a late-onset mild autoimmunity, illustrating the importance of genetic interactions in the F1 genome for full expression of lupus phenotypes (Morel and Wakeland 1998). As observed in the other spontaneous lupus models, a variety of immunological abnormalities are observed in BWF1 mice. B cells from young BWF1 mice demonstrate elevated expression of costimulatory molecules ex vivo and show increased IgM secretion, proliferation, and expression of costimulatory molecules after both cytokine and CD40 cross-linking stimulation in vitro (Wither, Roy, et al. 2000). Furthermore, using different IgM Tg systems, it was found that whereas BWF1 mice had intact central B cell tolerance, the frequency of anti-dsDNA IgG–producing follicular B cells was increased, even in the setting of a very restricted B cell repertoire, suggesting significant alterations in the peripheral selection mechanisms in these mice (Wellmann, Letz, et al. 2001; Wellman, Werner, et al. 2001). Ectopic expression of CXCL13, a B lymphocyte chemoattractant, has been observed in myeloid dendritic cells in the thymus, kidney, and lungs of BWF1 mice, as well as impaired and aberrant B1 trafficking
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(Ishikawa et al. 2001; Ito et al. 2004; Sato et al. 2004). Treatment of BWF1 mice with CTLA-4 Ig was shown to decrease the expansion of autoreactive B cells, inhibit the Ig class switch, and decrease numbers of activated CD4+ T cells, although this effect was not permanent (Mihara et al. 2000). Numerous linkage studies with a variety of different crosses have been undertaken to identify susceptibility loci in the BWF1 murine lupus model. In most of these studies the concentration has been on assessing NZB contributions to disease. As recently reviewed, loci associated with suggestive or significant linkage have been implicated on >14 of the autosomal chromosomes (Jorgensen et al. 2004). Consistent linkages have been observed with distal chromosome 1, mid-distal chromosome 4, chromosome 7, and the MHC region of chromosome 17, as described below. Backcross analyses of [BWF1 × NZW]F1 progeny revealed a strong linkage on chromosome 4, termed Nba1 (New Zealand Black autoimmunity) (Drake et al. 1994; Vyse, Drake, et al. 1996). This interval was also implicated in an independent backcross study of similar design and in an analysis of [SWR × NZB]F1 × NZB backcross progeny (Hirose et al. 1994; Xie et al. 2001). Linkage studies of BWF2 intercross progeny also identified a chromosome 4 locus, designated Lbw2 (Lupus-NZB × NZW), as being involved in mortality, GN, and splenomegaly but not anti-chromatin autoAbs. Genome-wide linkage studies with two different non-autoimmune, H2z congenic strains, revealed that whereas chromosome 4 was linked to nephritis in [BALB.H2z × NZB]F1 × NZB backcross progeny, it was not when B6.H2z was used instead of BALB.H2z (Rozzo et al. 1996). This latter study illustrates the impact genetic interactions with loci from the non-autoimmune strain can have in such crosses. Linkage to anti-chromatin autoAbs and nephritis was also seen on chromosome 4 in a study of [B6 × NZB]F2 mice (Tucker et al. 2000). Like chromosome 4, chromosome 1 has been implicated in a number of different experimental crosses. An NZB-derived locus, designated Nba2, has been mapped in [NZB × SM/J]F1 × NZW, [B6.H2z × NZB]F1 × NZB, and [BALB.H2z × NZB]F1 × NZB backcross and [B6 × NZB]F2 progeny as contributing to nephritis, hypergammaglobulinemia, autoAbs, and polyclonal B cell activation phenotypes (Drake, Rozzo, Vyse, et al. 1995; Rozzo et al. 1996; Vyse et al. 1997; Wither, Patterson, et al. 2000). Lbw7, an NZB-derived locus on chromosome 1 contributing to anti-chromatin autoAbs, was mapped in BWF2 intercross progeny in the same region as Nba2 (Kono et al. 1994). Evidence for NZW contributions to IgG autoAb production on chromosome 1 has also been revealed using [BALB/c × NZW]F1 × NZW backcross progeny (Vyse, Morel, et al. 1996). Mice made congenic for the NZB-derived Nba2 interval on the B6 background, (B6.Nba2), broke tolerance to chromatin, and had increased percentages of CD69+ B cells and increased in vitro IgM secretion (Atencio et al. 2004; Rozzo et al. 2001). The causal gene has been postulated to be Ifi202, an IFNinducible gene showing differential expression, identified via
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microarray analyses. Sequencing of the promoter region of Ifi202 revealed eight different promoter polymorphisms between B6 and NZB, and the one at position 95 correlates with known expression differences between mouse strains. However, the congenic interval introgressed in the study was >30 cM in length and contained >100 genes, making it impossible to definitively conclude that Ifi202 is the causal gene within Nba2 (Rozzo et al. 2001). The third non-MHC region consistently implicated in multiple studies is chromosome 7. In BWF2 intercross progeny, the chromosome 7 locus Lbw5 was linked more strongly with heterozygosity than with NZB or NZW homozygosity, and similar results were observed in BWF1 × NZB backcross progeny as well (Kono et al. 1994; Vyse et al. 1999). A dominant NZB chromosome 7 contribution, Nba3, was identified as contributing to nephritis and autoAbs in [NZB × SM/J] F1 × NZW and [BWF1 × NZW] backcross progeny, respectively (Drake, Rozzo, Vyse, et al. 1995; Vyse, Drake, et al. 1996). Linkage of the murine MHC on chromosome 17 with lupus susceptibility, similar to the observation in human studies, has proved to be very complicated and background specific. Studies of the BWF1 model, which has a heterozygous MHC genotype, have implicated separate strong contributions from both H2d (NZB) and H2z (NZW). Analysis of BWF2 intercross progeny identified Lbw1 on chromosome 17, where H2dz heterozygosity conferred increased risk for mortality, GN, and anti-chromatin antibodies (Abs) (Kono et al. 1994). Linkage of various lupus phenotypes to MHC heterozygosity has also been observed in BWF1 × NZW, BWF1 × NZB, and [BALB/c × NZW]F1 × NZW backcross progeny (Hirose et al. 1994; Vyse, Drake, et al. 1996; Vyse, Morel, et al. 1996; Vyse et al. 1999). In contrast to chromosome 4, [B6.H2z × NZB]F1 × NZB backcross progeny showed linkage to the MHC, but when BALB.H2z was used, this association was not observed, again illustrating the importance of non-MHC background loci in these two crosses (Rozzo et al. 1996). In addition, B6 MHC contributions were observed in linkage studies of [B6 × NZB]F2 progeny, in which maximum susceptibility was observed for H2b/b (Tucker et al. 2000). No MHC effect was observed in [NZB × SM/J] F1 × NZW backcross progeny, indicating that H2zd and H2zv confer equal risk (Drake, Rozzo, Hirschfeld, et al. 1995). To evaluate the risk conferred by the class II IEz in the development of lupus, linkage analysis comparing [B6.H2z × NZB]F1 × NZB and [B6.IEz × NZB]F1 × NZB backcross progeny was undertaken. In the latter cross, B6 mice express transgenic Class II IEz, but have no other H2z contributions. Interestingly, no linkage was observed with transgenic class II IEz expression and lupus nephritis development, although the previously observed MHC association was again seen in the control [B6.H2z × NZB]F1 × NZB backcross progeny (Vyse et al. 1998). A similar lack of association was observed when C57BL/10 (B10) mice expressed a transgene for class II IAz (Rozzo et al. 1999). This suggests that class II IAz and IEz are
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not the culprit genes within the MHC and that mixed haplotype effects cannot explain the increased susceptibility observed in BWF1 mice, as has been long postulated (Drake et al. 1994; Morel et al. 1994; Rozzo et al. 1996; Vyse, Drake, et al. 1996; Vyse, Morel, et al. 1996; Zhang et al. 2004).
D.
NZM2410 and the Congenic Derivatives
The major difficulty associated with studying the BWF1 model of lupus, the spontaneous model most reminiscent of human lupus in terms of both disease phenotypes and underlying genetic complexity, is the fact that the hybrid genome is required for disease expression. This prevents the full utilization of the genetic uniformity found in inbred strains to study this complex trait. Fortuitously, a series of 27 inbred strains, termed the New Zealand Mixed (NZM) strains, were derived from [NZB × NZW]F1 × NZW backcross progeny and show varying degrees of susceptibility to SLE. The NZM2410 (NZM) strain is one of the most severely affected BWF1 derivatives and spontaneously develops autoAbs by 4–6 months and lupus nephritis by 6 months. Linkage analyses of [NZM × B6]F1 × NZM (BC1) progeny were performed to map recessive NZM loci linked to GN and anti-dsDNA IgG production. Three NZM loci were found to be linked to GN susceptibility: Sle1, Sle2, and Sle3 on chromosomes 1, 4, and 7 respectively. Heterozygosity at a locus on chromosome 17, initially termed Sle4, was found to be associated with GN. This study also demonstrated that the incidence of disease correlated with the number of susceptibility loci that had segregated in the BC1 progeny according to a threshold liability model, the first such demonstration in an animal model of autoimmunity (Morel et al. 1994). Subsequently, a [NZM × B6]F1 intercross was performed to identify dominant NZM genes contributing to GN and humoral autoimmunity, the effects of which would have been masked in the original BC1 study. The age of GN onset in these F2 animals was more delayed than that in the BC1 progeny, consistent with the recessive inheritance of loci contributing to this phenotype. This study again identified linkage for Sle1 and Sle3, as well as a new locus on chromosome 7 termed Sle5, but not for Sle2 and Sle4 (Morel, Mohan, et al. 1999). To study the component phenotype of these loci, each was introgressed onto the B6 background and studied as a B6.Sle strain.
E.
Congenic Dissection of NZM2410
B6.Sle1 mice were found to develop a progressive loss in tolerance, such that by 9–12 months, they made high titers of anti-chromatin IgG, as well as some degree of anti-dsDNA IgG autoAbs, but did not develop GN (Mohan et al. 1998; Morel et al. 1997; Subramanian et al. 2005). In addition, the mice
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developed mild splenomegaly and an age-associated increase in activated splenic B and T cells. Despite this intrinsic activation phenotype, B and T cells were shown to have normal in vitro proliferative responses to different stimuli and in vivo IgM and IgG responses to T-dependent antigens. Furthermore, lymphocytes from B6.Sle1 mice had comparable rates of spontaneous and receptor-engagement induced apoptosis (Mohan et al. 1998; Morel et al. 1997). Mixed BM chimera experiments revealed that Sle1 is expressed in a BM-derived population and that the ability of a B cell to produce anti-chromatin autoantibodies and express an increased activation status is contingent on the B cell expressing Sle1 (Sobel et al. 1999). Subsequent experiments, using B6.Sle1|TCRα−/− and B6.Sle1|µMT mice, revealed that conventional T cells were not required for the increased levels of total, anti-chromatin, and anti-ssDNA IgM or for the manifestation of cell surface B cell activation phenotypes but were necessary for the generation of high-tittered IgG autoantibodies. In the absence of B cells, there were still increased percentages of activated CD4 T cells, suggesting that Sle1 is expressed functionally at the level of both the B and T cell (Sobel, Satoh, et al. 2002). Fine mapping of the Sle1 locus revealed that it is actually a cluster of functionally related subloci termed Sle1a–1d. All of these loci, with the exception of Sle1d, are associated with varying degrees of humoral autoimmunity, with B6.Sle1b mice recapitulating most of the phenotypes observed for the entire locus, making it the most potent of the subloci (Morel et al. 2001). It has been proposed that the causal gene for the Sle1c locus is the complement receptor Cr2/Cr1, as the allele from Sle1c has a novel glycosylation site that results in lowered functional responses to low and intermediate stimuli (Boackle et al. 2001). Fine mapping and subsequent sequencing of the Sle1b interval linked extensive functional polymorphisms in the SLAM/CD2 family of genes with the autoimmune phenotypes of B6.Sle1b (Wandstrat et al. 2004). Each family member is expressed in a specific set of immune cell lineages, and their expression can be altered by different stimuli, including antigen and Toll-like receptor triggering, as well as cytokines. Studies have demonstrated that this family can affect numerous immune functions, including macrophage, natural killer cell and T cell activation, cell-cell interactions, cytokine secretion, and cytotoxicity (Mooney et al. 2004; Wang et al. 2004). Altogether, these data strongly indicate that the SLAM/CD2 family has the ability to modulate immune responses in a highly flexible fashion and hence may function as “fine-tuners” of the immune response (reviewed in Veillette and Latour 2003). It was also shown that the Sle1b SLAM/CD2 family haplotype is the more prevalent version in both non-autoimmune and autoimmune-prone laboratory mouse strains and that only in the context of the B6 genome does this haplotype elicit autoimmunity, indicative of epistatic interactions (Wandstrat et al. 2004).
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Characterization of the NZW-derived Sle3/5 locus initially revealed primarily T cell phenotypes including elevated CD4/CD8 ratios and increased CD4 T cell activation phenotypes and proliferative responses but reduced activation-induced cell death (AICD). With age, splenic B cells showed increased expression of activation markers and produced anti-chromatin autoAbs. Unlike B6.Sle1, there was no evidence for splenomegaly in aged mice, but the spleens showed an agedependent change in splenic architecture. Despite the low-grade IgG humoral autoimmune response, these mice had a GN penetrance of ~20% (Mohan, Yu, et al. 1999). Mixed BM chimera experiments revealed that, like Sle1, Sle3/5 needed to be expressed in a BM-derived, radiationsensitive population. Interestingly however, non-Sle3/5 bearing T and B cells can express the phenotypes associated with this locus, such as elevated CD4/CD8 ratios and the production of anti-chromatin Abs, provided they develop in the presence of Sle3/5-bearing BM. This finding suggested that Sle3/5 is expressed in a non-B cell APC population or that Sle3/5 T cells have both autocrine and paracrine effects (Sobel, Morel, et al. 2002), which was subsequently shown to be a consequence of hyperstimulatory dendritic cells and macrophages that had more activated phenotypes and were less apoptotic, more pro-inflammatory, and better costimulators (Zhu et al. 2005). In contrast to Sle1 and Sle3/5, the phenotypes associated with the chromosome 4 locus, Sle2, were very B cell specific. There was no evidence of splenomegaly, lymphadenopathy, or changes in T cell percentages, activation status, or functional response. The B cells, however, were larger, had elevated activation marker expression, secreted increased levels of IgM, and were more responsive to different stimuli in vitro. Both T-independent and T-dependent IgM in vivo responses were heightened, whereas there was no change in the secondary IgG response, consistent with Sle2 not affecting the T cell compartment (Mohan et al. 1997). Starting at an early age, there was an increase in the peritoneal cavity B1a population in B6.Sle2 mice, with a concomitant decrease in the conventional B2 population, which was also seen in the spleen at an older age. This B1a expansion was independent of the housing conditions of the mice and was recently shown to be CD5 independent (Mohan et al. 1997; Xu et al. 2004). Mixed fetal liver chimeras, the early source of the B1a population, further revealed that Sle2 must be expressed in the B cell for this B1a expansion to occur (Xu et al. 2004). Congenic dissection hence allowed the complex, polygenic lupus phenotypes characteristic of the NZM2410 strain to be broken down and “assigned” to a specific chromosome interval. Characterization of the various B6 congenic lines revealed a variety of phenotypes, many of which are indicative of an overall heightened responsiveness to stimuli. This finding suggests that a more self-reactive immune system may be a consequence of the fact that the different immune cell populations bearing these susceptibility alleles are more prone to respond.
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The above studies also clearly demonstrate that in isolation on the B6 background, none of these susceptibility loci are sufficient to mediate systemic autoimmunity. On the basis of the original modeling analysis, which demonstrated that disease liability increased as a function of the number of susceptibility alleles present, it was predicted that combining these loci would reconstitute the severe lupus phenotypes associated with NZM2410 (Morel et al. 1994). To test this hypothesis, a series of bi- and tricongenic lines were created to assess the role of their different genetic interactions in the development of disease, as detailed below. F.
Epistatic Interactions in the Development of Pathogenic Autoimmunity
The combination of Sle1 and Sle3/5 on the B6 background resulted in significant humoral and pathogenic autoimmunity, much greater than what would be predicted by the phenotypes of these loci in isolation. By 9–12 months of age, these mice had significant mortality, 75% penetrance of GN, severe splenomegaly, and increased reactivity to a spectrum of chromatin components, as well as increased numbers of glomerular basement membrane–binding Abs. There were also increased percentages of both B and T cells expressing activation and effector-phenotype markers in the spleen (Mohan, Morel, et al. 1999). In contrast, B6.Sle1|Sle2 mice had a slight increase in mortality (18%) with a low penetrance of proliferative GN, but like B6.Sle1|Sle3/5 mice had spleen weights comparable to those of NZM2410. In addition, the level of humoral autoimmunity was not very high in this bicongenic line and was similar to that seen in B6.Sle1 (Morel et al. 2000). These data indicate that the degree of epistatic interaction between Sle1 and Sle3/5 is greater than that seen between Sle1 and Sle2. It is tempting to speculate that this finding may be due to the fact that both loci in the former combination have effects on both T and B cells, whereas this is not true for Sle2 in the latter combination. Interestingly, the combination of Sle2 and Sle3 on the B6 background does not result in phenotypes significantly different from the parental congenics, suggesting that these loci do not interact in a multiplicative fashion. The kidneys from this particular bicongenic combination, however, display numerous hyaline deposits and mesangial lesions. These types of kidney pathological changes are not observed in the parental congenic strains and are almost indistinguishable from those seen in NZW (Morel et al. 2000). Although Sle1 and Sle3/5 are sufficient to mediate a high degree of fatal lupus nephritis on the B6 background, the combined effects of these loci does not fully recapitulate the severity and kinetics of disease observed in NZM2410. The combination of Sle1, Sle2, and Sle3/5 on the B6 background, however, results in fully penetrant lupus nephritis. The degree of proliferative lesions, splenomegaly, and 5-month anti-dsDNA IgG autoantibodies was in fact higher than that seen in NZM2410
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(Morel et al. 2000). In addition, B6.Sle1|Sle2|Sle3/5, like NZM2410, accumulated long-lived splenic plasma cells that do not migrate normally in response to CXCL12, with a corresponding absence of BM plasma cells (Erickson et al. 2003). These data demonstrate that this combination of NZM2410 loci, originally identified via linkage analyses, fulfills the genetic equivalent of Koch’s postulates, as they are sufficient to reconstitute the development of fatal lupus nephritis on the non-autoimmune B6 background. Sle1, Sle2, and Sle3/5 were also individually combined on the B6 background with the BXSB-derived Y chromosome locus, yaa, to test the degree of epistatic interactions between this amplifying locus and the NZM2410-derived lupus susceptibility loci. Interestingly, only Sle1 interacts epistatically with yaa, culminating in fatal lupus nephritis and immunological characteristics similar to those with BXSB.yaa, whereas yaa combined with either Sle2 or Sle3/5 does not result in phenotypes that are significantly different from those observed by these loci in isolation (Croker et al. 2003; Morel et al. 2000). What stands out from these different studies is the necessity for the break in tolerance to chromatin, mediated by Sle1, for the development of pathogenic and systemic autoimmunity. Only the bicongenic combinations in which Sle1 is present (Sle1|Sle2, Sle1|Sle3/5 and Sle1|yaa) resulted in significant potentiation of autoimmunity, indicating that the breach in immune tolerance to chromatin mediated by Sle1 is key to the initiation of fatal autoimmunity in these models. G.
Epistatic Interactions in the Suppression of Pathogenic Autoimmunity
The importance of epistatic interactions in the development of autoimmunity is clearly exemplified by the amplified phenotypes elicited when different susceptibility loci are combined. The demonstration that Sle1, Sle2, and Sle3|5 are necessary and sufficient on the B6 background to reconstitute the fatal autoimmunity seen in NZM2410, while validating the results of the original linkage analysis, also begets an important question. Because Sle1, Sle3/5, and part of Sle2 are derived from the non-autoimmune NZW parent of NZM2410, why does the NZW strain not develop penetrant and fatal lupus nephritis? A genome wide analysis of [B6.Sle1 × NZW]F1 × NZW backcross progeny was conducted to identify putative, recessive epistatic suppressive modifiers. These analyses revealed four recessive Sles (SLE suppressor) loci: Sles1, Sles2, Sles3, and Sles4 on chromosomes 17, 4, 3, and 9, respectively, where B6/NZW heterozygosity was associated with increased susceptibility. Multivariate analysis of disease penetrance as a function of suppressive alleles supported the hypothesis that the cumulative effects of these four loci accounted for the lack of autoimmunity in NZW (Morel, Tian, et al. 1999).
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The described linkage analyses identified the chromosome 17 locus, Sles1, as the strongest of the four suppressive modifier loci. Introgression of Sles1 onto B6.Sle1, B6.Sle2, and B6.Sle3/5 was performed, via marker-assisted selection, to test its ability to modulate the various phenotypes associated with each of the single congenics. Interestingly, Sles1 was able to completely suppress the anti-chromatin IgG autoAbs and increased B cell CD86 expression of Sle1, yet had no effect on the increased peritoneal cavity B1a/B2 ratio and anti-dsDNA IgG phenotypes of Sle2 and Sle3/5, respectively (Morel, Tian, et al. 1999). This result is consistent with the nature of the original linkage analysis, which was designed to identify suppressive modifiers in the context of Sle1 homozygosity. Impressively, when B6.Sle1|Sles1 is crossed to NZW, instead of the severe GN, high autoAb titers, and splenomegaly seen in [B6.Sle1 × NZW]F1 progeny, the mice have no autoimmune phenotypes (Morel, Tian, et al. 1999). These data indicate that homozygosity at Sles1 is sufficient to suppress the autoimmunity elicited by homozygosity at Sle1 in this particular lupus model and that Sles1 interacts specifically with Sle1. It is important to note, however, that in the BC1 progeny, a percentage of the mice that are NZW homozygous at Sles1 still develop fatal GN, again illustrating the importance of additional epistatic interactions in the modulation of these phenotypes. Furthermore, the lack of anti-dsDNA IgG suppression in the B6.Sle3/5|Sles1 bicongenic would indicate that Sles1 is not a global suppressor of humoral autoimmunity. Recently, fine mapping of Sles1 with truncated congenic intervals localized it to a ~956 kilobase segment of mouse chromosome 17, and classic genetic complementation tests suggested that the non-autoimmune 129/SvJ strain possesses a Sles1 allele complementary to NZW, as evidenced by the complete lack of autoimmunity in [129 × B6.Sle1|Sles1]F1 crosses (Subramanian et al. 2005).
IV.
INSIGHTS INTO LUPUS, PATHWAYS, AND EPISTASIS FROM GENETICALLY MANIPULATED MODELS
The ability to manipulate the mouse genome provides powerful tools with which to understand the functions of genes classically either via transgenic overexpression or genetic ablation. Such systems have provided tremendous insight into the requirements for the expression of different molecules in the development and functional responses of the various lineages and subsets of the immune system. The utility of such models in the analysis of SLE susceptibility would be expected to be more limited, as the causal mutations for such a complex genetic trait are predicted to be minor polymorphisms resulting in slightly altered function, rather than complete ablation or aberrantly high overexpression (Subramanian and Wakeland 2005).
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However, such knockouts and transgenics can provide insight into the requirement for certain molecules, such as cytokines, in the development of various phenotypes when introgressed onto spontaneously autoimmune strains, as has been reviewed in the previous sections. Interestingly, and somewhat surprisingly, a number of strains with targeted deletions of different molecules that are involved in a variety of cellular functions upon aging develop lupus-like phenotypes, such as GN and autoantibody production (Morel 2004). Although many reflect a legitimate alteration of the pathways leading to autoimmunity, recent work has demonstrated that in many cases these late-onset autoimmune phenotypes may be a consequence of epistatic interactions between the 129 genome, derived from the ES cell in which the targeted deletion was made, and that of B6, the strain that many of knockouts are bred onto. Further proof of this theory comes from the fact that the SLE phenotypes oftentimes disappear when moved onto pure backgrounds. In the next section, the various genes implicated by either knockout or transgenic technologies as influencing the development of systemic autoimmunity are organized according to pathways relevant to their function. Any evidence for the background genome having an impacting on the autoimmune effects is discussed as well. In addition, Table 11-2 provides information regarding their murine and human chromosomal locations, the strain of origin of the ES cell used (for knockouts), the background genome used, and any modulations in phenotypes observed when the genes are studied on different backgrounds.
A.
Antigen and Immune Complex Clearance
The components of the complement pathway play important roles in both the clearance of ICs and the determination of activation thresholds in lymphocytes. Hence, it has been postulated that complement deficiencies lead to an autoreactive B cell repertoire, perhaps due to altered peripheral tolerance (Carroll 1998). Although deficiencies in the early components of complement, C1q and C4, act in an almost monogenic fashion to mediate SLE in humans, the data from murine studies is less definitive and much more dependent on background genome effects (Manderson et al. 2004). The effects of c1q deficiency have been investigated on the B6/129 F2 and the pure 129, B6, and MRL (Fas intact) backgrounds, and these studies provide clear examples of background genome effects. On both the pure B6 and 129 backgrounds, c1q deficiency does not lead to either autoAb production or GN (Botto et al. 1998; Mitchell et al. 2002). However, c1q− B6/129 F2 mice developed both autoAbs (54%) and severe GN (25%), significantly higher than that seen in B6, 129, and wild-type (WT) B6/129 F2 controls. However, of the WT B6/129 F2 controls, 33% developed IgG autoAbs and 4% developed GN, both of which were not observed in the
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parental B6 and 129 strains, clearly demonstrating the epistatic interactions occurring between these two genomes (Botto et al. 1998). On the autoimmune-prone MRL background, c1q deficiency led to increased mortality, GN, autoAb production, larger spleen weights, monocytosis, and increased B and T cell activation phenotypes, with a concomitant increase in plasma cells (Mitchell et al. 2002; Trendelenburg et al. 2004). These data illustrate that the lack of c1q serves to amplify an existing predisposition to autoimmunity. In the case of C4 deficiency, although the severity of the phenotypes varies, on all backgrounds some autoimmune phenotypes are present relative to appropriate controls. Compared with WT B6/129 F2 controls, C4−/− mice had ICmediated GN, elevated autoAb titers, splenomegaly, increased numbers of activated B and T cells, and increased CD11b+ percentages (Chen et al. 2000). When the effects of C4 deficiency on the B6, B6/129 F2, and [B6X129] F1 × BALB/c backgrounds were examined separately, in all cases, relative to WT controls, the C4-deficient mice had increased anti-dsDNA autoAbs (Paul et al. 2002). Serum amyloid P component (SAP) has been shown to bind, in a Ca2+-dependent manner, chromatin on the surface blebs of apoptotic cells and in nuclear debris (Casciola-Rosen et al. 1996; Cocca et al. 2002; Korb and Ahearn 1997; Nauta et al. 2002; Navratil et al. 2001). These functional data suggest that SAP could play an important role in efficient removal of key SLE self-antigens. The results of the targeted disruption of the Apcs gene, which encodes for SAP, are very similar to that seen for C1q: highly background-specific effects on lupus phenotypes. The first study examining Apcs−/− mice on the B6/129 F2 background, showed an increase in anti-chromatin and antidsDNA autoAbs and GN relative to WT F2 controls (Bickerstaff et al. 1999). A follow-up study examining the lack of SAP on the pure B6 and 129 backgrounds demonstrated that although on the B6 background Apcs−/− mice had increased autoAbs and increased incidence of GN (75%), there was no “lupus” phenotype on the 129 background. Furthermore, transgenic expression of human SAP did not prevent the development of the SLE phenotypes on the B6 background, suggesting that the mutation per se may not be causal (Gillmore et al. 2004). A second study examining the lack of SAP on B6/129 F2 progeny demonstrated that in WT F2 mice, if the chromosome 1 interval was fixed as 129 and compared with Apcs−/− mice of the same background, there were no differences in the antichromatin autoAbs between the two groups (Bygrave et al. 2004). These data clearly indicate that the chromosome 1 segment derived from 129 has the potential to mediate loss in tolerance to chromatin on the appropriate background, independent of the effects of targeted deletion of SAP. This finding was further supported by the break in tolerance to chromatin seen in two independently derived 129 chromosome 1 congenic lines on the B6 background (Bygrave et al. 2004; Wandstrat et al. 2004). As previously mentioned, this 129 interval includes
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TABLE 11-2
SUBSET OF GENES IMPLICATED IN LUPUS SUSCEPTIBILITY THROUGH KNOCKOUT AND TRANSGENIC STRATEGIES Gene*
Protein
Mouse Chromosome†
Human Chromosome (Cytoband)
ES Cell‡
Ptprc Tnfsf6 Frcgr2b
CD45 FasL FcRII
1 (138.0) 1 (161.8) 1 (171.0)
1q31.3 1q24.3 1q23.3
129 — 129
Apcs
SAP
1 (173.0)
1q23.2
129
Rasgrp1 Bcl2l11 Mertk Sh2d2a Lyn E2f2 C1qa
RASgrp1 Bim C-mer TSAd E2F2 C1q
2(117.1) 2 (127.9) 2 (128.4) 3 (88.3) 4 (3.6) 4 (134.6) 4 (135.4)
15q14 2q13 2q13 1q23.1 8q12.1 1p36.12 1p36.12
129 129 129 129 129 129
Gadd45a Bhlhb2 Tnfrsf13b Prkcd
GADD45a Stra13 TACI PKCδ
6 (67.1) 6 (109.1) 11 (60.7) 14 (26.6)
1p31.2 3p26.1 17p11.2 3p21.1
129 129 129 129
Background Genome§
Autoimmune Phenotypes¶
129/B6 C3H/HeJ 129/B6 BALB/c (N12) B6 (N12) 129/B6 129 (N6) B6 (N6) 129/B6 129/B6 B6 (N10) B6 (N10) 129/B6
GN, autoAbs AutoAbs None None GN, autoAbs GN, autoAbs None GN, autoAbs GN, autoAbs GN, autoAbs AutoAbs GN, autoAbs GN, autoAbs GN, autoAbs GN, autoAbs None None GN, autoAbs GN, autoAbs GN, autoAbs GN, autoAbs GN, autoAbs
129/B6 B6 (N12) 129 (N12) MRL/+ (N12) 129/B6 129/B6 129/B6 129/B6
Listed by murine chromosomal locations are the various genes discussed in the text that have been implicated in lupus susceptibility through transgenic or knockout methods. *Genetically ablated or overexpressed genes. †Chromosomal positions of genes that were knocked out and enhancer/promoter-type used in transgenics. ‡Strain of ES cell line. —, spontaneous mutation; not applicable. §Background genome. (strain A/strain B): [strain A × strain B]F2; N(x): number of backcross generations if known. ¶Autoimmune phenotypes: none, GN, or autoAbs.
the same SLAM/CD2 family haplotype as that seen in Sle1b, demonstrating the functional equivalency of this region from both strains in mediating a humoral autoimmune phenotype. Intriguingly, the second F2 study demonstrated that in both WT and knockout F2 mice, a B6 interval on chromosome 17, encompassing the Sles1 region, had suggestive association with increased GN susceptibility (Bygrave et al. 2004). The facts that 129 and B6 share the same MHC haplotype (in particular, class II alleles) and that 129 mice are non-autoimmune despite an autoimmune-promoting SLAM/CD2 family haplotype provides further evidence that, like NZW, a suppressive modifier allele may be harbored within the 129 MHC region. Two other molecules for which roles in “antigen clearance” have been postulated to explain the lupus phenotypes seen in their absence are the nuclease DNase I and the membrane tyrosine kinase c-mer. DNase I is a nuclease expressed at sites of high cell turnover such as the gastrointestinal tract, skin, and hematopoietic system, and DnaseI-deficient B6/129 F2 mice developed higher levels of autoAbs than those observed in WT controls, whereas the incidence of GN was not significantly increased (Napirei et al. 2000). The effects of this mutation on
a pure background have not been reported. The membrane tyrosine kinase c-mer regulates macrophage cytokine profiles and indirectly binds phosphatidyl serine. On the B6 background, c-mer−/−mice had decreased in vitro and in vivo phagocytosis of apoptotic cells, increased titers of autoAbs of various specificities, and increased tumor necrosis factor (TNF)-α production (Cohen et al. 2002). This increase in proinflammatory cytokine production coupled with impaired apoptotic clearance may be responsible for the increased humoral autoimmune phenotype.
B.
Regulation of Proliferation and Apoptosis
Gadd45α (growth arrest and DNA damage-inducible) has roles in various cellular processes including cell growth and apoptosis, is regulated by the tumor suppressor gene p53, and is expressed in many tissues, including resting T cells. It also interacts with the cyclin-dependent kinase, p21, which is involved in the inhibition of cell cycle progression (Salvador et al. 2002, 2005). Targeted deletion of Gadd45α and p21, both on the B6/129 F2 background, revealed similar phenotypes
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including increased mortality, GN, and autoAb production, and these effects were exacerbated in the absence of both genes. Both knockouts had increased T cell numbers, but their responses to various T cell stimuli differed. Whereas Gadd45α−/− mice had increased proliferative responses to T cell receptor (TCR) stimulation in terms of both kinetics and magnitude, p21-mice had increased responses only to IL-2, and both phenotypes were seen in the double knockout. These data indicate that p21 and Gadd45α act to negatively regulate cytokineinduced and TCR-mediated T cell proliferation, respectively. Notably, they both normally act via inhibition of cell cycle progression, but not by increasing apoptosis. Neither targeted deletion affects the B cell compartment (Balomenos et al. 2000; Salvador et al. 2002). The protein tyrosine phosphatase Pten has been shown to play a role in proliferation, differentiation, and apoptosis. In the absence of Pten activity, there is increased activation of the survival-promoting factor protein kinase B (PKB/Akt) due to increased phosphatidylinositol 3-kinase (PI3K) activity. Although Pten-is an embryonic lethal mutation, it has been demonstrated that Pten haploinsufficiency on the B6/129 F2 background led to a severe lymphoproliferative disorder with increased mortality, IC-mediated GN, and increased autoAbs. These mice also had an expansion of splenic CD4 T cells and increased peripheral B and T cell activation and Fas expression, with impaired AICD and responsiveness to Fas stimulation (Di Cristofano et al. 1999). Furthermore, Pten deletion, specifically in either B or T cells, resulted in autoAb production, hypergammaglobulinemia, hyperproliferation, and resistance to apoptosis. Interestingly, lymphocyte-specific Pten deletion was not reported to result in increased GN (Suzuki et al. 2001, 2003). Consistent with the idea that increased cell survival mediated by the PI3K/Akt axis can increase susceptibility to autoimmunity was the demonstration that T cell—specific expression of a constitutively active form of PI3K led to IC-mediated GN, autoAbs, and an expansion of the CD4 compartment. These T cells developed an effector memory phenotype and in vitro display increased survival and decreased cell death (Borlado et al. 2000). More recently, in vivo inhibition of PI3Kγ was shown to increase survival in the MRL.lpr mouse strain (Barber et al. 2005). Protein kinase Cd (PKCd) has been shown to be involved in apoptosis and the inhibition of cell differentiation and growth, and PKCd deficiency on the B6/129 F2 background led to GN and autoAb production. Pkcd−/−mice had an expansion in their peripheral B cell population, and B cells in vitro displayed increased proliferative potential and IL-6 production (Miyamoto et al. 2002). Similarly, genetic ablation of a member of the E2F family, E2F2, involved in regulation of the cell cycle, differentiation, and apoptosis, resulted in GN, splenomegaly, and serum autoAbs on the B6/129 F2 background. T cells from E2f−/− mice showed increased proliferation in vivo and in vitro, with no change in responses to apoptotic stimuli. A matter of some concern regarding the studies on
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the E2f−/− mice is the fact that the authors reported WT F2 spleen weights in excess of 650 mg by 8–12 weeks of age, making interpretation of “splenomegaly” a difficult issue (Murga et al. 2001). Members of the Bcl2 family of molecules have also been implicated in dysregulation of apoptotic processes in lupus. Transgenic overexpression of the cell survival–promoting Bcl2 in B cells led to IC-mediated GN and serum autoAbs, with B cell accumulation in the BM and periphery. Interestingly, these B cells had a quiescent phenotype indicative of increased survival but not proliferation (Strasser et al. 1991). Similarly, the absence of Bim, a proapoptotic member of the Bcl2 family, on the B6/129 F2 background, resulted in GN, splenomegaly, and autoAbs, with elevated numbers of both B and T cells (Bouillet et al. 1999).
C.
Lymphocyte Signaling
Early indications that aberrations in proximal signaling molecules can influence the development of autoimmunity came from studies of mice deficient in the Src family protein tyrosine kinase (PTK) Lyn. Despite a significant reduction in peripheral B cells and impairment in B cell receptor (BCR)–mediated signaling, Lyn−/− mice developed GN, splenomegaly, IgG autoAbs, and increased total IgM and numbers of plasma cells (Hibbs et al. 1995; Nishizumi et al. 1995). Additional deletion of another Src family PTK, Fyn, resulted in decreased survival, presumably due to the increased kidney disease seen in the double knockout (Yu et al. 2001). For the receptor protein tyrosine phosphatase CD45, targeted knockin of a mutation that prevents negative regulation of CD45 signaling, led to IC-mediated GN, splenomegaly, and serum autoAbs in an allele dose-dependent fashion, with concomitant activation of both B and T cells (Majeti et al. 2000). These data suggest that the lack of the inhibitory functions normally mediated by these molecules can result in the development of systemic autoimmunity on the appropriate genetic background. The impact of deficiency in the inhibitory Fcγ receptor, FcγRIIb, on the development of autoimmunity, has a highly background-dependent effect. On the mixed B6/129 F2 and on the pure BALB/c backgrounds, FcγrIIb−/− mice did not develop autoimmunity. However, on a pure B6 background FcγRIIb deficiency led to mortality, GN, splenomegaly, and autoAb production, as well as an activated lymphocyte population. In fact, with the exception of mortality and GN, B6.FcgrIIb−/− mice had phenotypes almost identical to that seen for B6.Sle1b (Bolland and Ravetch 2000). The FcγR gene cluster is just proximal to the SLAM/CD2 family on chromosome 1, and 129 shares the same haplotype as that seen in Sle1b (Wandstrat et al. 2004). It is hence tempting to speculate that the increased mortality and GN seen in B6.FcgrIIb−/− mice is a consequence of the lack of the inhibitory FcγRIIb in the context of the
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predisposition to humoral autoimmunity conferred by the SLAM/CD2 haplotype. The importance of expression levels of FcγRIIb in maintaining peripheral tolerance, however, was revealed in a recent report. In these studies, it was shown that a modest increase in the levels of FcγRIIb on B cells, using retroviral transduction, ameliorated the pathogenic and humoral phenotypes seen in NZM2410, BXSB.yaa, and B6.FcgrIIb−/− mice (McGaha et al. 2005). Deficiencies in molecules that function downstream of initial signal transduction events have also been implicated in predisposition to lupus-like autoimmunity. Mice that lack the T cell adaptor molecule (TSAd), which is expressed in thymocytes and activated T cells, and mice deficient in the transcriptional repressor Stra13, normally induced upon naïve T cell activation, both developed with age IC-mediated GN, splenomegaly, and autoAb production. In addition, both knockouts showed increased T and B cell activation phenotypes and impaired AICD, IL-2, and IFN-γ induction in activated T cells (Drappa et al. 2003; Sun et al. 2001). T cells from young Stra13−/− mice were also shown to have impaired proliferative and cytokine responses in vitro and in vivo (Sun et al. 2001). Mice with a spontaneous deficiency in the Ras guanidine nucleotide exchange factor, Rasgrp1, which normally serves to activate Ras after TCR stimulation, also developed the characteristic lupus phenotypes of GN, splenomegaly, and autoAbs. Interestingly, Rasgrp−/−mice were shown to have impaired thymic development that prevented selection of low-affinity TCRs, yet showed an accumulation of splenic effector memory CD4 T cells with reduced numbers of CD8 T cells. This finding is in accordance with data showing that low-affinity TCRs are highly dependent on Rasgrp1 signals for positive selection and suggests that in its absence only high-affinity TCRs undergo positive selection (Layer et al. 2003). The phenotypes of Rasgrp−/− mice are, in fact, very similar to those seen for knockin mice with a point mutation in LAT (Y136F) that disrupts PLCγ activation upon TCR signaling (Aguado et al. 2002; Sommers et al. 2002). Recently, members of the TNF superfamily of ligands and receptors have also emerged as modulators of systemic autoimmunity. Transgenic overexpression of BLyS, a potent B cell–activating cytokine, both in a ubiquitous and in a B cell–specific manner, resulted in the characteristic murine lupus phenotypes of GN, splenomegaly, and autoAb production. Although there were no gross changes in B cell development, there was increased in vivo B cell numbers and in vitro B cell viability in BLyS transgenic mice (Gross et al. 2000; Khare et al. 2000). Consistent with the idea that overexpression of BLyS and consequent B cell activation can result in murine lupus phenotypes, deficiency in the inhibitory receptor for BLyS-mediated signaling, TACI, expressed on B cells and CD4 T cells, resulted in murine lupus. In addition to decreased survival, GN, splenomegaly, and autoAbs, TACI−/− mice had increased B cell percentages and in vitro proliferative responses and Ig secretion
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(Seshasayee et al. 2003). These studies again emphasize the importance of appropriate inhibition of signaling in the prevention of autoimmunity. Studies have also provided evidence for the idea that subtle modulations in the expression of key signaling molecules can facilitate the development of systemic autoimmunity. CD19 is a BCR coreceptor that undergoes rapid tyrosine phosphorylation upon BCR engagement, allowing it to interact with a variety of downstream signaling molecules. A transgenic line that overexpresses CD19 by just 20% was shown to develop humoral autoimmunity in the absence of GN, with titers of IgG autoAbs comparable to that seen in a line overexpressing two times the normal levels of CD19. Significantly, this small increase in CD19 expression did not result in detectable alterations in phosphorylation patterns of downstream signaling molecules or any changes in B cell numbers or percentages (Sato et al. 2000).
V.
CONCLUSIONS
The various murine lupus models have provided great insight into the many immune irregularities underlying lupus, and, cumulatively, the data suggest a multistep genetic pathway for the development of systemic autoimmunity, as illustrated in Fig. 11-1. This model postulates that a key step for the initiation and development of pathogenic autoimmunity is mediated by loci such as the Sle1a–c subloci, all of which cause a loss in tolerance to chromatin and are associated with minimal pathogenicity. It is believed that these loci modulate immune cell interactions, antigen clearance, and response to antigen. These effects can be considered to be similar to the phenotype of seropositivity seen in first-degree relatives of SLE probands. Genes such as Sle2, Sle3/5, lpr, and yaa amplify and interact with the pathways modulated by the “first-step” genes, via unknown mechanisms, resulting in the development of pathogenic autoimmunity. It may be postulated that the genes encoded by these loci could contribute to overall immune responsiveness and these particular allelic variants result in slightly dysregulated immune function on a non-autoimmune-prone background. The final class of susceptibility genes, such as Sle1d, Sle6, IFNα, and FcγrIIb, are believed to potentiate end-organ damage, via a variety of mechanisms such as a modification of effector functions, an increase in inflammatory processes, or an alteration of end-organ susceptibility. This last class of genes may be responsible for the wide diversity in clinical end-organ pathogenesis seen in human lupus patients. This lupus model provides a framework with which to interpret the genetic interactions that progressively increase disease pathogenesis in mouse model systems. The remaining questions focus on the identification of the specific disease alleles and the characterization of the molecular mechanisms responsible for driving disease. Although identification of the disease-causing genes still
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Susceptible Individuals Pathway 1 Chronic lymphocytic activation
Sle1b, Sle1c, Roquim
Sles1, Sles2, Sles3
Anti-nuclear Autoantibodies 2-4% of population ANA positive Familial aggregation
Pathway 2 Disruption of immune regulation
Sle2, Sle3, Fas Yaa, Lyn - SHP- 1
Sles1
Pathogenic Autoimmunity Familial aggregation
Pathway 3 End-organ targeting
FcRIIb, Sle1d, IFNα nephritis
neurologic disorders
arthritis
vasculitis
Fig. 11-1 Model pathway demonstrating the interactions of potentiating and suppressive genes leading to systemic autoimmunity. We postulate that genes involved in mediating susceptibility to SLE have an impact on one or more of the three pathways shown in the figure. The epistatic interactions between these loci, which can be potentiating or suppressive, culminate in the development of systemic autoimmunity. Adapted from Wakeland et al. 2001.
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Chapter 12 Inhibitory Receptors and Autoimmunity in the Mouse Menna R. Clatworthy and Kenneth G.C. Smith
I. II.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitory Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. IgG Fc Receptors—The Archetypal Activatory and Inhibitory Receptor Pair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Inhibitory Receptors and Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . A. FcγRIIb and Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Genetic Linkage Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. FcγRIIb Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. FcγRIIb-Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. FcγRIIb and B Cell Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Inhibitory Receptors and B Cell Activation Thresholds . . . . . . . . . . . . 1. CD22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. PD-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Inhibitory NK Receptors for MHC Class I . . . . . . . . . . . . . . . . . . . . . . 1. KIRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Ly49 Natural Killer Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. T Cell Inhibitory Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. CTLA-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. BTLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Inhibitory Receptor Pathways and Autoimmunity . . . . . . . . . . . . . . . . . . . . A. Lyn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. SHP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. SHIP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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MENNA
I.
INTRODUCTION
The role of the immune system is to recognize and eliminate invading microorganisms. It has evolved a number of complex mechanisms to achieve this goal, which center on an ability to distinguish between self and non-self and to limit autoreactivity. Critical to its function is the capacity to rapidly mount a potentially lethal response to pathogens but yet to limit and terminate the relevant pathways when they are no longer required. Thus, the immune system has evolved activatory mechanisms that facilitate a rapid response that are counterbalanced by inhibitory pathways. Loss of inhibitory signaling pathways is associated with both autoreactivity and excessive inflammatory responses, emphasizing their critical role.
II.
INHIBITORY RECEPTORS
Paired activatory and inhibitory receptors have been identified on most cells of the immune system (Table 12-1). In B cells inhibitory receptors, including FcγRIIb, CD22, and CD72, downregulate B cell receptor (BCR)–triggered activation (Pritchard and Smith 2003). In addition, inhibitory motif–containing Fc receptor homologs (FcRHs) have been identified in B cells, although their role in inhibitory signaling remains to be characterized (Davis et al. 2001; Ehrhardt et al. 2003). Natural killer (NK) cells have major histocompatability (MHC)–recognizing receptors, such as killer cell immunoglobulin (Ig)-like receptors (KIRs) and lectin-like Ly49 receptors, which mediate the capacity of NK cells to preferentially kill targets lacking MHC class I (Chiesa et al. 2005). In T cells
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SMITH
three inhibitory receptors that inhibit immune activation after binding to B7 family members, namely cytotoxic T lymphocyte–associated antigen-4 (CTLA-4), programmed death-1 (PD-1), and B and T lymphocyte attenuator (BTLA) have been identified (Chen 2004). The best-characterized activatoryinhibitory receptor pair is that of the activatory and inhibitory Fc receptors (FcRs) (Ravetch and Bolland 2001). The generation of the inhibitory Fc receptor (FcγRIIb)–deficient mouse nearly a decade ago has allowed the detailed characterization of inhibitory signaling pathways and the definition of several general paradigms for the class of inhibitory receptors as a whole; therefore, much of this chapter will focus on FcγRIIb. Two broad classes of inhibitory receptor exist—most are of the immunoglobulin superfamily whereas the remainder are lectin-like molecules. They share a number of structural and functional similarities and can be identified by a consensus amino acid sequence, the immunoreceptor tyrosine-based inhibitory motif (ITIM). Each inhibitory receptor contains one or more ITIMs within its cytoplasmic domain. The ITIM consists of a prototype six amino acid sequence, (Ile/Val/Leu/Ser)-X-Tyr-X-X-(Leu/Val), where X denotes any amino acid (Muta et al. 1994). Inhibitory receptors function to attenuate signals generated by activatory receptors, often those containing immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains, and mediate this function upon clustering with their activatory counterpart on the cell surface (Daeron, Malbec, et al. 1995). Ligation of the inhibitory receptor with an ITAM-containing activatory receptor results in phosphorylation of the tyrosine residue within the ITIM, often by a Src family kinase (Malbec et al. 1998). This allows binding and activation of phosphatases containing an Src homology 2 (SH2) domain. Two classes of SH2-containing inhibitory phosphatases have been identified; the tyrosine
TABLE 12–1
INHIBITORY RECEPTORS Inhibitory Receptor
Activatory Counterpart
Expression Pattern
Phenotype in Knockout Mouse
FcγRIIb CD22
FcγRI, III, IV FcεR, BCR BCR
B cells, macrophages, dendritic cells, mast cells, activated neutrophils B cells
PD-1
TCR, BCR
T cells, B cells, myeloid cells
CD72
BCR
B cells
PIR-B CD5 CTLA-4
BCR BCR CD28
B cells B1 cells T cells
BTLA KIRs Ly49 CD94/NK
TCR
Th1 cells, B cells NK cells NK cells NK cells/ T cells
Spontaneous SLE-like disease on C57BL/6 background Hyperactive B cells, development of autoantibodies, but no overt autoimmune disease Development of SLE-like disease and autoimmune dilatory cardiomyopathy Hyperactive B cells, no autoantibodies or autoimmune disease Hyperactive B cells, enhanced graft versus host disease Hyperactive B cells and autoantibodies Rapid infiltration of organs with T cells, autoimmunity No obvious spontaneous disease — — —
Ly49/DAP10
12. INHIBITORY
RECEPTORS
AND
AUTOIMMUNITY
phosphatases SHP-1 and SHP-2, and the phosphoinositol phosphatases SHIP1 and SHIP2 (Tamir et al. 2000). These classes have separate downstream signaling pathways through which they modulate cellular inhibition. In general, each class interacts with the ITIMs of different inhibitory receptors, but each inhibitory receptor appears to act predominantly through one class of phosphatase. A.
IgG Fc Receptors—The Archetypal Activatory and Inhibitory Receptor Pair
FcγRs are widely distributed cell surface molecules that recognize the Fc portion of IgG (Daeron 1997). There are three activatory FcγRs in mice, FcγRI (CD64), FcγRIII (CD16), and the recently described FcγRIV (CD16-2) (Nimmerjahn et al. 2005), which are multichain receptors made up of a ligandbinding FcR α subunit associated with one or two signal transduction subunits that contain ITAMs (Fig. 12-1). In humans, but not in mice, there is another FcγR activatory receptor, FcγRIIa, which is a single-chain receptor with an ITAM in its cytoplasmic domain. The inhibitory receptor FcγRIIb is a single-chain low-affinity IgG receptor and is found on B cells, monocytes, macrophages, activated neutrophils, and mast cells. Activatory
Inhibitory
Activatory
FcγRIIb
FcγRIII
FcγRI
s-s
+
s-s
+
+
−
+
+ +
γ chain
α chains
ε−γ chain+
Key Ig-like domain
+ ITIM
− ITIM
Fig. 12-1 Murine activatory and inhibitory FcγRs. FcγRs are members of the Ig superfamily, with variable numbers of disulfide-bonded immunoglobulin domains extracellularly. FcγRI and FcγRIII are activatory and are associated ITAM-containing accessory molecules such as γ chains. FcγRIIb is an inhibitory receptor containing a cytoplasmic ITIM.
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It is a 40-kDa protein that consists of two extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic domain that contains one ITIM (Latour et al. 1996) (Fig. 12-1). It binds to multivalent soluble antigen as immune complexes or to cell membranes. FcγRIIb is encoded by a single gene (Fcgr2b) on chromosome 1 in both humans (1q23–24) and mice (1 92.3) (Hogarth et al. 1991). Control of FcγRIIb transcription lies in two methylated regions in the promoter and in the intron between the two exons coding for the leader peptide (intron 3) (Bonnerot et al. 1992). Sequence analysis of the 5′-untranslated region (UTR) of the mouse FcγRIIb gene indicated the presence of regulatory elements including three binding sites for the transcription factor Sp1, an activator protein-4 (AP-4) binding site, and a tandem glucocorticoid response element upstream of the transcription initiation site. In addition, sites of methylation that regulate gene expression were also located at the 5′ end of the mouse FcγRIIb gene (Bonnerot et al. 1988; Hogarth et al. 1991). Several isoforms of the receptor are found on different cell types; in B cells FcγRIIb1 contains an intracytoplasmic motif that prevents its internalization whereas in myeloid cells FcγRIIb2 can mediate phagocytosis (Latour et al. 1996) FcγRIIb, when signaling through its cytoplasmic ITIM, functions to inhibit activatory signals generated through adjacent ITAM-containing activatory receptors (Muta et al. 1994). It has been shown in vitro to be capable of inhibiting ITAM signals from activatory Fc receptors, the BCR and the T cell receptor (TCR) (Daeron, Latour, et al. 1995; Muta et al. 1994). Coligation of FcγRIIb to the activatory receptor leads to tyrosine phosphorylation of the ITIM by the tyrosine kinase Lyn (Malbec et al. 1998). Once phosphorylated, FcγRIIb recruits the cytoplasmic phosphatase SHIP through its SH2 domain and activates it by phosphorylation(Ono et al. 1996). SHIP then inhibits ITAM activation signaling by dephosphorylation of phosphatidylinositol triphosphate (PIP3), which is the product of receptor activation (Ono et al. 1996). This leads to disassociation of Btk and phospholipase Cγ (PLCγ) from the activatory complex at the cell membrane, producing blockade of Ca2+ flux through the capacitancecoupled channel and inhibition of calcium dependent signaling (Bolland et al. 1998). Calcium-dependent processes such as degranulation, phagocytosis, antibody-dependent cellmediated cytotoxicity, and cytokine release are all blocked (Fig. 12-2). SHIP has a role in prevention of proliferation in B cells, but the precise mechanism remains uncertain. By dephosphorylation of PIP3, SHIP can prevent recruitment of the survival factor Akt (Liu et al. 1999). It also functions to recruit p62dok to the membrane where it is activated by Lyn to downregulate mitogen-activated protein (MAP) kinase activity (Yamanashi et al. 2000). These two FcγRIIb-mediated inhibitory effects on Ca2+-dependent signaling and prevention of proliferation appear to be independent, since FcγRIIb in dok-deficient B cells is unable to arrest BCR-mediated
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Co-crosslinking by IgG immune complexes
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Fig. 12-2 Inhibitory FcγRIIb signaling. Cross-linking of FcγRIIb to the activatory FcγR (or the BCR) by immune complexes containing IgG leads to tyrosine phosphorylation of the ITIM by the kinase Lyn. This allows recruitment of the phosphatase SHIP, which mediates inhibition of activation by 1) blockade of Ca2+ flux and calcium-dependent signaling by dephosphorylation of PIP3 by SHIP and subsequent disassociation of Btk and PLCγ and 2) recruitment of p62dok to the membrane where it is activated by Lyn to downregulate MAP kinase (MAPK) activity. Ag, antigen; PIP2, phosphatidylinositol bisphosphate; DAG, diacylglycerol; IP3, inositol 3-phosphate.
proliferation while retaining its ability to inhibit a Ca2+ response. An additional regulatory function of FcγRIIb in the B cell is the induction of apoptosis on co-aggregation of the receptor without BCR signaling. In this circumstance an apoptotic signal is generated through Btk and Jnk, which is abrogated by the recruitment of SHIP that occurs on cross-linking with the BCR (Pearse et al. 1999).
III.
INHIBITORY RECEPTORS AND
A.
FcγRIIb and Autoimmunity
Three lines of evidence implicate defective FcγRIIb function in the pathogenesis of autoimmunity: • Genetic linkage studies in polygenic mouse models of autoimmune diseases, such as systemic lupus erythematosus (SLE). • A naturally occurring murine FcγRIIb polymorphism is associated with autoimmunity. • FcγRIIb-deficient mice have an increased susceptibility to both inducible and spontaneous autoimmune diseases.
AUTOIMMUNITY As mentioned previously, one of the central features of the immune system is that potentially lethal responses are directed against pathogens whereas the host itself is spared. Inhibitory receptors play a critical role in maintaining self-tolerance, and an absence or dysfunction of these receptors can lead to a variety of autoimmune diseases.
1.
Genetic Linkage Studies
Genetic studies of polygenic murine autoimmune diseases implicate FcγRIIb in pathogenesis. Seven independent linkage studies in murine models of autoimmune disease have identified a susceptibility locus that contains Fcgrb2 (Wakeland et al. 2001). It should be emphasized that the region at the distal end
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of chromosome 1 containing Fcgr2 also contains a large number of other candidate genes, for example, Cr2 (Boackle et al. 2001). The situation is made more complex because it is now clear that contributions to disease pathogenesis are made by at least three independent subdivisions of this locus that have been identified by congenic studies (Morel et al. 2001). 2.
FcγRIIb Polymorphisms
The earliest murine FcγRIIb polymorphism described was that of the Ly-17 alloantigenic system (Hibbs et al. 1985). The mouse Ly-17 locus has two alleles, Ly-17a and Ly-17b, encoding the Ly17.1 and Ly17.2 antigens, respectively. Ly-17 polymorphisms have been defined in a number of strains of inbred mice. Although all autoimmune strains examined in this study were of the Ly-17.1 allotype (BXSB, MRL/lpr, NZB, and NZW), so were a number of non-autoimmune-prone strains (C3H/HeJ, SM/J, SJL/J, and WySnJ); therefore, it is unlikely that this polymorphism contributes to a predisposition to autoimmunity (Slingsby et al. 1997). More recently, four further polymorphic sites were identified in the gene encoding FcγRIIb: two in the third intron and two in the promoter (Jiang et al. 2000; Pritchard et al. 2000) (Fig. 12-3). The 13-base pair deletion within the promoter, 110 base pairs upstream from the start site, is found in most autoimmune-prone inbred strains including NZB, BXSB, MRL, and NOD and is associated with reduced surface expression of the receptor on macrophages (Pritchard et al. 2000) and activated B cells (Jiang et al. 2000). The polymorphism and associated reduction in FcγRIIb expression in inbred strains correlated with increased antibody titers after immunization (Jiang et al. 2000) and with macrophage hyperactivity as evidenced by enhanced calcium fluxes and phagocytosis (Pritchard et al. 2000). Furthermore, a luciferase reporter assay indicated that the deletion was associated with reduced transcriptional activity, probably due to reduced binding of the transcription factor AP-4 as shown by Southwestern analysis (Xiu et al. 2002). Interestingly, C57BL/6 mice congenic for a region containing the promoter deletion also show downregulation of FcγRIIb in germinal center B cells and higher levels of antibody after immunization. These studies are highly suggestive that this promoter polymorphism contributes to immune hyperactivity and a predisposition to autoimmunity. 3.
FcγRIIb-Deficient Mice
Some of the most compelling evidence for the involvement of FcγRIIb in autoimmunity has been produced by studying the FcγRIIb knockout mouse. FcγRIIb-deficient mice on a 129/SvC57BL/6 background have augmented humoral responses after immunization with both T-dependent and -independent antigens but do not develop autoantibodies spontaneously (Takai et al. 1996). FcγRIIb deficiency renders normally resistant strains of mice susceptible to a number of antibody- or immune complex–dependent induced models of autoimmunity, including
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collagen-induced arthritis (CIA), Goodpasture’s syndrome, and immune complex–mediated alveolitis. The CIA model is antibody-dependent arthritis and occurs after immunization with bovine type II collagen. H2q haplotype mice (e.g., DBA/1) are susceptible to this disease but H2b (e.g., C57BL/6 and 129) are resistant. Deficiency in FcγRIIb renders H2b mice as susceptible to disease as H2q mice (Yuasa et al. 1999). Enhanced arthritis in FcγRIIb-deficient BALB/c mice after the administration of anticollagen antibodies and cytokines has also been reported (Kagari et al. 2003). In CIA, signaling through FcγRIIb appears to inhibit the degradation of aggregan and collagen by matrix metalloproteases, causing inhibition of severe cartilage destruction (Blom et al. 2003). In the model of Goodpasture’s syndrome,FcγRIIbdeficient mice develop pulmonary hemorrhage and crescentic glomerulonephritis in response to immunization with bovine type IV collagen, whereas no control animals developed disease (Nakamura et al. 2000). Similarly, in cryoglobulin-associated membranoproliferative glomerulonephritis induced by overexpression of thymic stromal lymphopoietin, deletion of FcγRIIb caused a massive influx of macrophages and increased cellular proliferation, aggravating disease progression (Muhlfeld et al. 2003). Mice deficient in FcγRIIb also develop alveolar inflammation in response to subthreshold concentration of immune complexes (Clynes et al. 1999). This inflammation is characterized by the influx of neutrophils into the lungs and by heightened tumor necrosis factor-α secretion by macrophages. FcγRIIb knockout mice also have an enhanced susceptibility to a murine model of multiple sclerosis, myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. By contrast, γ chain–deficient mice were protected from disease (Abdul-Majid et al. 2002). In addition, FcγRIIb−/− mice derived on a 129/Sv/C57BL/6 background backcrossed for 10 generations onto a C57BL/6 background develop hypergammaglobulinemia, autoantibodies (to antigens such as chromatin and doublestranded [dsDNA]), and an immune complex-mediated autoimmune disease resembling SLE (Bolland and Ravetch 2000). This is characterized by an immune complex–mediated glomerulonephritis, renal failure, and a reduced life expectancy. Transfer studies show that the disease is fully transferable and dependent on B cells. FcγRIIb−/− myeloid cells are not necessary for disease development in this system (Bolland and Ravetch 2000), but a role for them in determining severity has not been excluded. The differences seen between the knockouts on different backgrounds are postulated to be produced by differences in strain-specific epistatic modifiers of autoimmunity. Finally, even mice heterozygous for deletions in FcγRIIb exhibit only a modest reduction in protein expression but have a predisposition to autoimmunity (Bolland et al. 2002). 4.
FcγRIIb and B Cell Tolerance
The work outlined above in the FcγRIIb knockout mouse has left little doubt that abnormalities of this inhibitory receptor can contribute to the development of autoimmunity, but the precise
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Ly-17 polymorphisms 116 161 166 AA: Ly-17.1 Pro Gln Thr Ly-17.2 Leu Leu Pro
258 Ile Ser
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FcγRIIb regulatory region polymorphisms
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Coding region
Untranslated region
Unmethylated region
Fig. 12-3 Murine FcγRIIb polymorphisms. The Ly-17 antigenic system consists of three single-nucleotide polymorphisms (SNPs) within the area of the gene encoding the second extracellular domain and a further SNP in exon 8, encoding the intracytoplasmic domain. The second group of polymorphisms are deletions within regulatory regions in the promotor region and in intron 3. Autoimmune-prone inbred strains of mice (such as NZB, BXSB, MRL, and NOD) have deletions in polymorphic regions 1, 2, and 3. The 13-base pair deletion in region 1 is associated with reduced gene transcription, probably due to disruption of the binding site for the transcription factor AP-4.
mechanism remains a subject of intense investigation. However, it seems that FcγRIIb plays a role in the development or maintenance of self-tolerance. It is now widely accepted that despite a number of developmental checkpoints (central tolerance), self-reactive cells do escape into the periphery (Bouneaud et al. 2000). In addition, mechanisms that enhance antibody diversity, such as somatic hypermutation, can generate potentially autoreactive antigen receptors in the adult (Marion et al. 2003). Thus, checkpoints operate in the periphery and are critical for maintaining tolerance to self-antigens that only appear after maturity. Inhibitory signaling is a critical feature of peripheral tolerance, providing a means for establishing thresholds of activation and for deletion of autoreactive cells. FcγRIIb is the only Fc receptor expressed by B cells, and its expression is required for the maintenance of tolerance (Bolland and Ravetch 2000). As mentioned previously, C57BL/6 FcγRIIb-deficient mice develop lupus-like autoimmunity, including a fulminant glomerulonephritis (Bolland and Ravetch 2000). Studies of bone marrow transfer into recombinase-activating gene (RAG)-deficient mice suggest that FcγRIIb deficiency in the B cell compartment is most likely responsible for the loss of tolerance seen in these mice. Furthermore, several autoimmune-prone inbred strains of
mice such as NZB, NOD, and BXSB have also been shown to have reduced FcγRIIb expression in germinal centers (Jiang et al. 2000). These results suggest that the absolute level of FcγRIIb expressed on B cells may regulate the ability of these cells to maintain tolerance and that relatively small changes may allow the persistence and expansion of autoimmune cells. Further support for this concept has been provided by studies in which B cell FcγRIIb expression was normalized by retroviral transduction of bone marrow cells from NZB, BXSB, and FcγRIIb-deficient mice (McGaha et al. 2005). Mice were irradiated, and bone marrow was reconstituted with autologous marrow transduced with FcγRIIb-expressing retrovirus. These mice exhibit reduced levels of antinuclear antibodies and antibodies to DNA and chromatin, reduced renal immune complex deposition, and improved survival compared with mice with control transfected bone marrow. The only caveat to these observations is that although FcγRIIb upregulation after retroviral transduction was seen in 40% of B cells, 20% of immature thymocytes and 10% of macrophages also showed increased expression, so the role of the inhibitory receptor in restoring tolerance may be related to expression on these other cell types. The precise mechanism by which FcγRIIb contributes to the maintenance of peripheral tolerance is still unclear, but there is
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evidence to suggest that it may alter receptor editing and control the expansion of autoreactive plasma cells (Fukuyama et al. 2005). It has also been suggested that FcγRIIb serves to differentiate between high-affinity cognate antigen binding and low-affinity (and hence potentially cross-reactive) specificities in the germinal center. Antigen in the germinal center is retained in the form of immune complexes bound to follicular dendritic cells, and hence B cell stimulation can occur either through FcγRIIb stimulation alone, resulting in apoptosis, or through FcγRIIb and the BCR, producing survival (Pearse et al. 1999). In addition to its controlling role in peripheral tolerance, FcγRIIb may also play a role in central B cell tolerance (Brauweiler and Cambier 2004). FcγRIIb is expressed on pre-B cells, and aggregation (in the absence of B cell aggregation) can induce cell death and inhibit migration (Brauweiler and Cambier 2004). However, if the pre-B cell receptor is cross-linked, then FcγRIIb can inhibit apoptosis (Kato et al. 2002). B.
Inhibitory Receptors and B Cell Activation Thresholds
Upon antigen binding to the BCR, B cell activation thresholds are determined by the net effects of positive and negative regulatory molecules. A number of inhibitory receptors have been identified on B cells, including FcγRIIb, CD22, PD-1, CD72, CD5, CD66a, leukocyte-associated Ig-like receptor-1 (LAIR-1), paired Ig-like receptor-B (PIR-B), and Ig-like transcript (ILT-2). Some of these will be discussed in detail but overall, mice deficient in these molecules have hyperactive B cells and are prone to autoimmunity. 1.
CD22
CD22 is a B cell–specific surface glycoprotein of the Ig superfamily in the sialoadhesin subclass (O’Keefe et al. 1996). It is made up of seven extracellular Ig-like domains, a transmembrane region, and an intracytoplasmic tail that contains six highly conserved tyrosine residues, three of which are within ITIMs (Smith and Fearon 1999). The ligand for CD22 is Siaα2,6Galβ1-4GlcNAc, a glycosylated sialic acid residue expressed at high levels on lymphocytes and inflamed endothelial cells (Hanasaki et al. 1994). CD22 is constitutively associated with the BCR and is phosphorylated on stimulation through it by the tyrosine kinase Lyn. Lyn also controls the basal levels of CD22 phosphorylation and SHP-1 association (Smith et al. 1998). Phosphorylation of the ITIMs within the cytoplasmic tail of CD22 allows association with and phosphorylation of SHP-1. Ligation of CD22 to the BCR and subsequent SHP-1 activation inhibits B cell activation by inhibiting the MAP kinases extracellular signal-regulated kinase-2 (ERK2), c-Jun NH2-terminal kinase (JNK), and p38
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and dephosphorylating molecules involved in the early events of BCR-mediated activation (Smith and Fearon 1999). Coligation of CD22 to the BCR reduces B cell activation while sequestering it away from the BCR, as would occur if CD22 bound to its ligand on adjacent cells, results in B cell hyperactivity. Thus, the interaction of CD22 with its ligand may promote B cell activation in appropriate lymphoid environments. Alternatively, increased levels of ligand on inflamed endothelium would recruit CD22 and make B cell activation by inflamed self less likely (Neuberger et al. 1999). CD22-deficient mice have an expanded B1 cell population and increased serum IgM levels. They also have B cells that are hyper-responsive to stimulation via their BCR (O’Keefe et al. 1996). With age they develop high-affinity, isotype-switched autoantibodies to dsDNA, myeloperoxidase, and cardiolipin, although not overt autoimmune disease (O’Keefe et al. 1999). Heterozygous CD22 knockout mice have a reduced but significant autoimmune phenotype and mice heterozygous for CD22, Lyn, and SHP-1 show reduced B cell tolerance in the HEL-antiHEL transgenic system (Cornall et al. 1998). These data imply that even a partial defect in CD22 function may contribute to the development of autoimmune disease. Mice deficient in other molecules involved in the inhibitory pathway, such as Lyn and SHP-1, also display B cell hyperresponsiveness and autoimmune disease (Hibbs et al. 1995). The CD22 gene has been shown to lie within a susceptibility locus for the development of lupus in the NZBW/F1 and related NZM2410 models of lupus (the relevant locus in the NZM2410 being of NZW origin (Kono et al. 1994; Morel et al. 1994, 1997). This region has also been linked to the development of glucose intolerance in the NOD mouse (Ghosh et al. 1993) and to experimental allergic encephalomyelitis (EAE) (Butterfield et al. 1999). A number of autoimmuneprone strains of mice, including the NZW mouse, express the CD22a or CD22c alleles, which are associated with abnormal processing of CD22 mRNA leading to heterogeneous 5′-UTRs and truncated exon-4 coding sequence (Mary et al. 2000). This defect is associated with reduced surface expression of CD22 on resting B cells and reduced ability of lipopolysaccharideactivated B cells to upregulate CD22. Heterozygous expression of CD22a with the Y chromosome–linked autoimmune accelerator Yaa promoted autoantibody production, supporting a link between this CD22 allele and autoimmune disease. 2.
PD-1
PD-1 is a 55-kDa transmembrane protein of the Ig superfamily and is expressed on resting B cells, T cells, and macrophages (Agata et al. 1996). It is composed of a single extracellular Iglike domain and a transmembrane domain and has two tyrosine residues in its cytoplasmic tail, one of which forms part of an ITIM (Nishimura and Honjo 2001). Two PD-1 ligands have been identified; These are membrane
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proteins of the Ig superfamily expressed constitutively on dendritic cells and on heart, lung, thymus, and kidney (Freeman et al. 2000; Latchman et al. 2001). In vitro studies on a B cell lymphoma line using a chimeric molecule with an FcγRIIb extracellular domain and a PD-1 cytoplasmic domain have shown that ligation of the PD-1 cytoplasmic domain to the BCR can inhibit signaling through it (Okazaki et al. 2001). Mice lacking PD-1 have defects in T cell selection and in the maintenance of peripheral T cell tolerance. Splenic B cells from these mice have enhanced responses to anti-IgM stimulation in vitro and an enhanced IgG3 response to T-independent antigen. On a C57BL/6 background, PD-1-deficient mice develop autoantibodies, an immune complex–mediated glomerulonephritis (similar to that seen in human SLE), and a deforming arthritis resembling rheumatoid arthritis (Nishimura et al. 1999). When the C57BL/6 PD-1 knockouts were crossed onto the lpr/lpr mouse, they developed high titers of anti-dsDNA autoantibodies and accelerated glomerulonephritis and arthritis. BALB/c PD-1−/− mice develop dilated cardiomyopathy, with IgG deposition on the myocardium associated with the development of isotype switched autoantibodies to a cardiac myocyte–specific protein (Nishimura et al. 2001). PD-1−/− mice were protected from disease and the development of autoantibodies on the RAG2−/− background, and disease could successfully be transferred to these mice with spleen or bone marrow cells from diseased mice. Therefore, lymphoid cells appear to be crucial in the development of autoimmune disease in PD-1-deficient mice. Although defective PD-1 on myeloid cells may not be critical to transfer of disease, this result does not rule out an additional role for myeloid cells in the disease process. More recent studies have also implicated PD-1 in the development of glucose intolerance in the NOD mouse (Ansari et al. 2003) and in the progression of EAE in a murine model (Salama et al. 2003). A polymorphism of PD-1 has now been identified in humans. An intronic SNP in PD-1 (also called PDCD1) is associated with the development of SLE in Europeans (found in 12% of affected individuals versus 5% of control subjects). The SNP alters a binding site for the runt-related transcription factor 1 (RUNX1) located in an intronic enhancer leading to aberrant regulation of PD-1 expression, and perhaps a release of autoreactive cells from inhibitory restraint (Prokunina et al. 2002).
C.
Inhibitory NK Receptors for MHC Class I
One critical characteristic of the immune system is its ability to distinguish self from non-self. This is illustrated by the capacity of NK cells to preferentially target cells lacking MHC class I. NK cell–mediated surveillance for class I expression is mediated by three classes of NK inhibitory receptors that
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functionally dominate triggering signals induced by activatory receptors: the KIRs, the Ly49 receptors, and the heterodimeric CD94-NKG2A-B receptor, which recognizes nonclassic MHC class I molecules (Chiesa et al. 2005). All three classes of NK inhibitory receptors have one or more ITIMs in their cytoplasmic domain. 1.
KIRs
In humans, NK cell receptors are encoded by KIR genes. They were initially though to be absent in mice, but a number of murine KIR genes have now been identified (Hoelsbrekken et al. 2003). After MHC recognition, downstream events include recruitment of SHP-1 and the subsequent inhibition of Vav1. Vav1 plays a central role in the regulation of actin-dependent changes during cytotoxic lymphocyte activation (Stebbins et al. 2003). 2.
Ly49 Natural Killer Receptors
Mouse NK receptors are encoded by the C-type lectin Ly49 multigene family that maps onto chromosome 6, in a region termed the NK gene complex (NKC). The genes encoded within the NKC display allelic polymorphism, which in conjunction with alternative mRNA splicing results in expanding of the Ly49 repertoire. It is possible that at least 23 Ly49 members exist, of which 13 are inhibitory (Ortaldo and Young 2005). Ly49 receptors are expressed at the cell surface as transmembrane disulfide-bonded homodimeric type II transmembrane proteins, each composed of a C-type lectin domain connected to the cell membrane by an α-helical stalk. Inhibitory Ly49 NK cell receptors contain an ITIM in their cytoplasmic domain, which recruits SHP-1 on ITIM phosphorylation (Nakamura et al. 1997). Activatory Ly49 receptors are associated noncovalently with the ITAM-containing adaptor protein DAP-12. The inhibitory receptors dominate, and studies indicate that NK cells require two activatory signals (e.g., activatory Ly49 receptor engagement and treatment with interleukin-12) to overcome the overriding inhibitory receptor–mediated blockade (Ortaldo and Young 2003). These receptors are likely to be important in the immune response to viruses and in tumor immunity, but as yet there are no data on their role in autoimmunity.
D.
T Cell Inhibitory Receptors
On T cells, three receptors have been noted to inhibit T cell activation after binding to B7 family members: CTLA-4, PD-1 (discussed above), and BTLA. 1.
CTLA-4
CTLA-4 is the most extensively characterized of the T cell inhibitory receptors. Whereas CD28 recognition of B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells (APCs)
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provides effective costimulation for T cells recognizing antigen via their TCRs, recognition of B7 by CTLA-4 markedly inhibits T cell activation (Taylor et al. 2004). CTLA-4-deficient mice show polyclonal T cell proliferation and a lymphoproliferative disorder that culminates in early mortality (Tivol et al. 1995; Waterhouse et al. 1995). The mechanism of CTLA-4-mediated inhibition is unclear, but this receptor does not contain a classical ITIM in its cytoplasmic domain. It may act by competing for B7 binding, thus preventing the association of activation-mediating molecules such as CD28. More recently some data suggest a more complex mechanism in which CTLA-4 modulates tryptophan catabolism in APCs (Fallarino et al. 2003; Grohmann et al. 2002). The ligation of APC B7 by CTLA-4 triggers interferon (IFN)-γ production, which in turn promotes the synthesis of an enzyme that catabolizes tryptophan (indoleamine 2, 3-dioxygenase [IDO]). Tryptophan is an essential constituent of all proteins and is also required for the function of two biochemical pathways, the generation of 5-hydroxytryptophan, and the IDO-catalyzed formation of a series of biologically active metabolites referred to as the kynurenines. By mechanisms that are as yet unclear, activation of IDO and/or production of kynurenines can regulate T cell proliferation and survival and promote an immunomodulatory rather than a stimulatory phenotype (Grohmann, Fallarino, Puccetti, et al. 2003). Dendritic cells from autoimmune-prone NOD mice exhibit a defect in IFN-γ-induced IDO activity and thus fail to develop a tolerizing phenotype (Grohmann, Fallarino, Bianchi, et al. 2003). In humans and in the NOD mouse, a CTLA-4 polymorphism that gives rise to alternatively spliced, soluble forms of CTLA-4 and has been mapped to susceptibility regions for a variety of autoimmune diseases including Graves’ disease, autoimmune hypothyroidism, and type 1 diabetes in humans has been identified (Ueda et al. 2003). 2.
BTLA
BTLA is an Ig domain–containing transmembrane receptor that has two ITIMs in its cytoplasmic tail, both of which can recruit SHP-1 and SHP-2 (Gavrieli et al. 2003; Watanabe et al. 2003). BTLA is expressed only on activated B cells and on developing T helper (Th) 1 and Th2 cells. It is subsequently lost from fully developed Th2 cells but is retained by Th1 cells. Binding of its putative ligand B7-H4 inhibits TCR-induced cytokine production and cell cycle progression. Furthermore, administration of the fusion protein for the putative ligand (B7H4-Ig) inhibits antigen-specific T cell responses in vivo (Sica et al. 2003). In the BTLA knockout mouse T cells show increased proliferation in response to antigen. B cells are also hyperactive and BTLA-deficient mice have increased levels of antigen-specific IgG titers after T-dependent immunization. These mice are also susceptible to peptide antigen-induced EAE (Watanabe et al. 2003).
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Inhibitory receptors are subserved by remarkably similar signaling pathways. Mice with deficiencies or abnormalities in these downstream signaling molecules also have a predisposition to autoimmunity, providing further evidence of the importance of these pathways in the pathogenesis of autoimmune disease.
A.
Lyn
Lyn is a Src family kinase that phosphorylates ITIMs in both the SHIP- and SHP-1–mediated inhibitory receptor pathways. Lyn is expressed in many hemopoietic cells, but much of the work to date has focused on its function in B cells (Burkhardt et al. 1991; Takata et al. 1994). A significant proportion of Lyn molecules are constitutively associated with the BCR, and it becomes rapidly activated on BCR cross-linking. Lyn is involved in both the activation and inhibition of the B cell. Cross-linking of the BCR in Lyndeficient mice leads to delayed and reduced phosphorylation of Syk and several other substrates within the activatory pathway. However, there is sufficient phosphorylation by other Src kinase family members to generate a B cell response (Takata et al. 1994). The nonredundant role of Lyn appears to be inhibitory, however, because the B cells of Lyndeficient mice are hyper-responsive to BCR cross-linking (Smith et al. 1998). B cells in lyn−/− mice show exaggerated proliferative responses after BCR cross-linking (Wang et al. 1996) and have increased numbers of peripheral mature B cells and elevated serum IgM and IgA levels. B cells from Lyn-deficient anti-HEL transgenic mice show a delay in the initial antigen–induced Ca2+ flux, but overall Ca2+ flux was increased (Cornall et al. 1998). This suggests that Lyn may be involved in the initiation of intracellular Ca2+ release but overall has an inhibitory effect upon it. The Lyn knockout mice develop isotype switched autoantibodies, lymphadenopathy, splenomegaly, and immune complex–mediated glomerulonephritis similar to that seen in SLE (Hibbs et al. 1995). Lyn-deficient mice develop worse disease than do mice deficient in single inhibitory receptors, presumably because Lyn deficiency interrupts the function of multiple inhibitory receptors.
B.
SHP-1
SHP-1 is the protein tyrosine phosphatase most widely utilized in the inhibitory receptor signaling pathways. It contains two amino-terminal SH2 domains, a phosphatase domain, and
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two conserved carboxyl-terminal tyrosine residues (Tamir et al. 2000). SHP-2 has an additional carboxyl-terminal domain that may allow interaction with SH3-containing proteins (Hof et al. 1998). Although both molecules are activated after binding through their amino-terminal SH2 domains to phosphorylated ITIMs, they appear to bind with different affinities (Barford and Neel 1998; Vely et al. 1997). These differences in structure and binding affinities appear to confer significant differences in the signaling functions of the two molecules. SHP-1 is a broadly inhibitory molecule and plays the predominant role of the two in regulation through ITIMs, although increasingly evidence suggests that SHP-2 may have an additional activatory role (Huyer and Alexander 1999). Clearly these molecules have an important role in regulation of a normal immune system that is due, at least in part, to their recruitment by inhibitory receptors. Consistent with its role in mediating inhibitory receptor function, SHP-1 deficiency results in the development of spontaneous autoimmune disease. However, the situation is complicated because this is not the only group of receptors it subserves. SHP-1 also associates with BCRs, FcRs, growth factor, complement, and cytokine receptors (Bolland and Ravetch 1999). Despite these complicating factors, much of the knockout phenotype is consistent with SHP-1 having a predominant role in the inhibitory receptor pathways. The “moth-eaten” (me) and “moth-eaten viable” (mev) mice are naturally occurring SHP-1 mutants (Greiner et al. 1986). The me mutation completely stops production of SHP-1, whereas the mev mutation is a single base pair deletion that disrupts an mRNA splice site, leading to production of aberrant SHP-1 protein with 10–20% of normal activity (Tsui et al. 1993). The me and mev mice have a broadly similar phenotype, although it is milder in the mev. These mice have reduced numbers of B cells but a higher proportion of B1 cells. The mice have B cells that are hyper-responsive to BCR stimulation, have raised levels of serum immunoglobulin, and develop autoantibodies (Sidman et al. 1986). Both strains develop severe autoimmune disease with immune complex deposition in skin, lung, and kidney, patchy alopecia, splenomegaly, and inflamed paws. The lifespan of a homozygous me mouse is 3 weeks whereas that of a homozygous mev mouse is 9 weeks (Shultz et al. 1984). The double mutant mev and RAG-1−/− mice develop the full phenotype but do not develop autoantibodies. Thus, SHP-1 deficiency produces such severe immune dysregulation that B cells do not appear to be necessary for the development of disease, although their contribution is demonstrated by the fact its phenotype is altered in their absence (Yu et al. 1996). The severity of the disease that is seen in SHP-1–deficient mice is clearly worse than that seen in mice with deficiencies of individual inhibitory receptors. This is most likely due to the effects of disruption of multiple inhibitory receptor pathways, although the fact that SHP-1 has functions in addition to mediating inhibitory receptor suppression should be kept in mind. In a number of genetic studies of mouse models of disease, susceptibility loci that contain SHP-1 have been identified,
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although a role for them in disease pathogenesis has not been proven (Mary et al. 2000; McIndoe et al. 1999).
C.
SHIP
SHIP is an SH2-containing inositol phosphatase related to SHIP-2, and they share a conserved N-terminal SH2 catalytic domain. In the B cell, SHIP acts predominantly on the FcγRIIb signaling pathway. In humans, it occurs in a number of isoforms, the most common of which is 145 kDa in size. The molecule is highly conserved between humans and mice (96% homology) and is widely expressed in myeloid and lymphoid lineages (Huber et al. 1999). SHIP acts to dephosphorylate PIP3 and inositol-1,3,4,5-tetra phosphate, and because PIP3 is produced by the action of phosphoinositide 3-kinase (PI3K) on phosphatidylinositol bisphosphate, in so doing it serves to counteract PI3K activity (Damen et al. 1996). Through this mechanism activation of SHIP leads to reduced BCR-mediated phosphoinositide hydrolysis and Ca2+ mobilization (Brauweiler et al. 2000). The pattern of B cell abnormalities seen in SHIP-deficient mice is consistent with this inhibitory role in B cell signaling. Splenic B cells have an activated phenotype with lower surface levels of IgM and higher levels of IgD and are hyper-responsive to BCR-mediated stimulation measured by the activation markers CD69 and CD86 (Helgason et al. 1998). SHIP-deficient B cells also demonstrate prolonged Ca2+ influx and enhanced proliferation in vitro in response to BCR stimulation, which was associated with increased phosphorylation of MAP kinase and Akt and also with increased cell cycling and survival (Helgason et al. 2000; Liu et al. 1998). SHIP-deficient mice also have elevated serum immunoglobulin levels with enhanced IgG responses to thymus-independent (TI) antigen (Helgason et al. 2000). However, the mice do not develop autoantibodies or B cell–mediated autoimmune disease. They die prematurely (50% mortality by 10–12 weeks) with consolidation of the lungs brought about by myeloid cell infiltration (Helgason et al. 1998). Thus, mice deficient in these three signaling molecules develop autoimmune disease, emphasizing the important role inhibitory pathways play in the maintenance of tolerance.
V.
CONCLUSION
The innate and adaptive immune systems utilize a range of inhibitory receptors, which control the strength and duration of an immune response. They facilitate the development of a measured response and play an important role in preventing autoreactivity. An increasing number of immune inhibitory receptors have been identified in mice and humans. The generation of mice deficient in both inhibitory receptors and molecules downstream in inhibitory pathways has provided
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compelling evidence that inhibitory mechanism are critical for the prevention of autoimmunity. A better understanding of these pathways will also allow the development of therapeutic agents that might modify inhibition and prevent or reduce autoimmune diseases.
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Taylor, P.A., Lees, C.J., Fournier, S., Allison, J.P., Sharpe, A.H., Blazar, B.R. (2004). B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions. J Immunol 172, 34–39. Tivol, E.A., Borriello, F., Schweitzer, A.N., Lynch, W.P., Bluestone, J.A., Sharpe, A.H. (1995). Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3, 541–547. Tsui, H.W., Siminovitch, K.A., de Souza, L., Tsui, F.W. (1993). Motheaten and viable motheaten mice have mutations in the haematopoietic cell phosphatase gene. Nat Genet 4, 124–129. Ueda, H., Howson, J.M., Esposito, L., Heward, J., Snook, H., Chamberlain, G., et al. (2003). Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423, 506–511. Vely, F., Olivero, S., Olcese, L., Moretta, A., Damen, J.E., Liu, L., et al. (1997). Differential association of phosphatases with hematopoietic co-receptors bearing immunoreceptor tyrosine-based inhibition motifs. Eur J Immunol 27, 1994–2000. Wakeland, E.K., Liu, K., Graham, R.R., Behrens, T.W. (2001). Delineating the genetic basis of systemic lupus erythematosus. Immunity 15, 397–408. Wang, J., Koizumi, T., Watanabe, T. (1996). Altered antigen receptor signaling and impaired Fas-mediated apoptosis of B cells in Lyn-deficient mice. J Exp Med 184, 831–838. Watanabe, N., Gavrieli, M., Sedy, J.R., Yang, J., Fallarino, F., Loftin, S.K., et al. (2003). BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1. Nat Immunol 4, 670–679. Waterhouse, P., Penninger, J.M., Timms, E., Wakeham, A., Shahinian, A., Lee, K.P., Thompson, C.B., et al. (1995). Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270, 985–988. Xiu, Y., Nakamura, K., Abe, M., Li, N., Wen, X.S., Jiang, Y., et al (2002). Transcriptional regulation of Fcgr2b gene by polymorphic promoter region and its contribution to humoral immune responses. J Immunol 169, 4340–4346. Yamanashi, Y., Tamura, T., Kanamori, T., Yamane, H., Nariuchi, H., Yamamoto, T., et al. (2000). Role of the rasGAP-associated docking protein p62(dok) in negative regulation of B cell receptor-mediated signaling. Genes Dev 14, 11–16. Yu, C.C., Tsui, H.W., Ngan, B.Y., Shulman, M.J., Wu, G.E., Tsui, F.W. (1996). B and T cells are not required for the viable motheaten phenotype. J Exp Med 183, 371–380. Yuasa, T., Kubo, S., Yoshino, T., Ujike, A., Matsumura, K., Ono, M., et al. (1999). Deletion of Fcγ receptor IIB renders H-2b mice susceptible to collagen-induced arthritis. J Exp Med 189, 187–194.
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Chapter 13 Mouse Models of Immunodeficiency B. Anne Croy, James P. Di Santo, Marcus Manz, and Richard B. Bankert
I. II.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Commonly Used Natural (Spontaneous) Mutant Models and Their Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Athymic “Nude” Mouse (Genotype Foxn1nu; Forkhead Box N1; Chromosome 11, cM 45.0) . . . . . . . . . . . . . . . . . . . . . . . . . . B. SCID Mice (Genotype Prkdcscid; Protein Kinase, DNA Activated, Catalytic Polypeptide; Chromosome 16 cM 9.2) . . . . . . . . . . . . . . . . . C. Nonobese Diabetic Strains (Genotype Idd#; Insulin-Dependent Diabetes Susceptibility Genes; Multiple Chromosomes) . . . . . . . . . . . D. NOD-scid (NOD-Prkdc) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Commonly Used, Genetically Engineered Immune-Deficient Strains and Their Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Rag1 or Rag2 Gene Deleted (T and B Cell–Deficient; Chromosome 2, cM 56.0) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Common Cytokine Chain γ Null (T and NK Cell–Deficient; Il2rg; Interleukin 2 Receptor γ; X Chromosome) and Related Cytokine Signaling-Deficient Strains . . . . . . . . . . . . . . . . . . . . . . . . . . C. Alymphoid Mice (T, B, and NK Cell–Deficient) . . . . . . . . . . . . . . . . . D. Lymphotoxin and Lymphotoxin Receptor Null Mutants (Lta, Chromosome 17 cM 19.059; Ltb, Chromosome 17 cM 19.061; Ltbr, Chromosome 6 cM 60.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Complement Cascade Disrupted Strains . . . . . . . . . . . . . . . . . . . . . . . . F. Gene Overexpression Resulting in Impaired Immune Responsiveness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Point Mutagenesis to Create Immunologically Deficient and Immune-Modified Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Specialized Allogeneic and Xenogeneic Transplantation Applications Using Immune-Deficient Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Analysis of Mouse Lymphohematopoietic Cell Lineage Differentiation B. Analyses of Differentiation of Human Lymphohematopoietic Cell Lineages . . . . . . . . . . . . . . . . . . . . . . . . . . C. Analyses of Therapeutic Strategies for Cancer Patients . . . . . . . . . . . . D. Analyses of the Maternal-Fetal Interface . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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INTRODUCTION
There are many types of genetically immune-deficient mice. Immune-deficient mice include spontaneously mutant (Joliat and Shultz 2001) and genetically engineered deleted or transgenic and mutagen exposed lines (Nelms and Goodnow 2001; Schorle et al. 1991; Ziljstra et al. 1989). The numbers of immunedeficient lines and strains available for research continue to increase through intercrossing of established lines and by new genetic engineering and mutagenesis (Maurer et al. 2002; Nelms and Goodnow 2001). Murine immune deficits, like those in humans, range from very mild to complete absence of multiple immune cell lineages (Fischer et al. 1997). Genetic loss of one or more lymphocyte lineage or its significant impairment creates an absolute requirement for specialized husbandry to provide a barrier to microbiological agents that would compromise the health of these animals. Most modern animal facilities have barrier areas. It is ideal that mice with even mild immune deficiencies be housed under barrier conditions and regularly monitored for disease through post-mortem histopathological analysis. Post-mortem examinations of immune-deficient mice will have unusual findings (Ward et al. 2000). These will be specific for genotype, and it is helpful for the investigator to advise the pathologist of anticipated findings that are “normal” for the strain being examined. Findings may include missing organs [i.e., thymus (Pantelouris 1968) or lymph nodes (Rennert et al. 1996; Kim et al. 2000)], unusually sized organs (the spleen is small in some strains and dramatically enlarged in others (Nilsson and Bertoncello 1994) and modified microscopic architecture (e.g., absence of nasal-associated lymphoid tissue (Ying et al. 2005), absence of Peyer’s patches (Rennert et al. 1996), or great reductions in bone marrow cavity space (Tagaya et al. 2000), or more subtle distortion of subregions within a lymphoid structure (Junt et al. 2002). Serological surveillance requires use of immune-competent sentinels because seroconversion due to pathogen exposure does not occur in most severely immune-compromised mice.
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Immune deficiencies encompass not only the lymphocyte lineages but all other immune cell types such as granulocytes, macrophages, and dendritic cells (Guleria and Pollard 2001). Expression of immune deficiency may result from alterations in properties intrinsic to immune cells themselves, such as absence of a cytokine receptor (di Santo 2000) or downstream receptor signaling molecule (Kaplan et al. 1998) or may result from changes extrinsic to the immune cell. The latter would include a reduction or absence in cytokine support (Guleria and Pollard 2001; Kennedy et al. 2000; Leonard 2001; Ranson et al. 2003) or altered hormone-mediated effects (Thurmond et al. 2000). Often, when a series of distinct deletions is made in a common pathway, the resulting animals are similar. For example, deletion of the common cytokine receptor chain γ (γc), blocks signaling by interleukin (IL)-15 and several additional cytokines. The resulting mice are similar to those in which IL-15 is deleted as well as those in which Jak3 (Janus-family tyrosine kinase-3) is deleted. Jak3 is a downstream transcription factor from the IL-15 receptor (Fischer et al. 1997). The vast range of immune deficits and the large number and continuing development of new mutant lines make it impractical to discuss murine immune-deficient models briefly but comprehensively. For example, in 1996, mice deleted for the proinflammatory cytokine tumor necrosis factor-α (TNF-α) were reported (Pasparakis et al. 1996). Today (mid-2006), strain panels are being reported in which TNF-α deletion occurs only in T cells, only in B cells, and only in macrophages (Grivennikov et al. 2005). Use of the panel permits identification of critical, nonredundant functions of TNF-α in promotion of pathogenesis and health. Readers will find the most up-to-date information in electronic databases. Of particular interest is the Wellcome Trust (UK) Library of Immune Variants. Table 13-1 provides current Web addresses for governmentally supported consortia providing mouse genomic and phenomic information. It is essential to assess immune parameters in phenotyping of all new strains. Deletions of a number of molecules not primarily associated with the immune system have resulted in major immune deficiencies. Examples are the severe natural killer (NK) cell deficit in mice deleted for the vitamin D
TABLE 13-1
DATABASES PROVIDING ACCESS TO ESTABLISHED AND EMERGING IMMUNE-DEFICIENT MICE Name of Database
Address
Wellcome Trust (UK) Library of Immune Variants Comparative Resources Medicine Directory Canadian Mutant Mouse Resource European Mutant Mouse Archive MRC Mammalian Genetics Unit, Harwell Riken BioResource Center Australian Phenomics Facility Mouse Genome Informatics International Mouse Strain Resource
http://www.apf.edu.au/resources/wt/data.shtml http://www.ncrr.nih.gov/ncrrprog/cmpdir/RODENT.asp http://www.cmmr.ca/index.html http://www.emma.rn.cnr.it http://www.mgu.har.mrc.ac.uk http://www.brc.riken.jp/lab/animal/en http://www.apf.edu.au/index.shtml http://www.informatics.jax.org http://www.jax.org/resources/mouse_resources.html
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upregulated protein 1 (Lee et al. 2005) and the resistance to induction of autoimmune disease in mice deleted for an amine oxidase known as vascular adhesion protein-1 (Stolen et al. 2005). In this chapter we will focus on commonly used, severely immune-deficient strains that have been cornerstones for studying the immune system via genetic functional deletion. It should be noted that a single gene may be mutated in multiple ways at a single point or, because of its length, at different points (Gauen et al. 1994). Alterations at key residues within the one gene may affect the immune system differentially, causing immune hypersensitivity, autoimmunity, or immune deficiency. Investigations of these types of variations and defining the phenotypes of newly induced mutations in genes that affect the immune system represent a major effort in current studies of the mouse immune system (Nelms and Goodnow 2001). The common immune-deficient strains are most often used to define the steps in lymphohematopoietic cell differentiation and its regulation (Traggiai et al. 2004; Vosshenrich et al. 2005) and to define susceptibility, development, progression, pathogenesis, and therapy of infectious diseases (Mercer et al. 2001) and tumors (Dore et al. 1987; Hess et al. 2003). The success of many of these studies has depended upon adoptive cell transfer and the ability of immune-deficient mice to support not only syngeneic cells but also allogeneic and xenogenic cells and tissues. The “humanized” immune-deficient mouse has been and continues to be of unique value in providing new models to approach experimental therapeutic manipulations. Examples of these applications conclude our chapter.
II.
COMMONLY USED NATURAL (SPONTANEOUS) MUTANT MODELS AND THEIR HYBRIDS
A.
The Athymic “Nude” Mouse (Genotype Foxn1nu; Forkhead Box N1; Chromosome 11, cM 45.0)
In 1968, a spontaneous mouse mutation presenting as hairless and designated nu/nu was reported to be athymic (Pantelouris 1968). Nude mice do not develop thymus-derived T cells, but they retain functional extrathymic T cell differentiation pathways and low levels of T cells. Distribution of this mutant and its commercial development triggered research with immune-deficient mice and fueled the development of new types of caging and husbandry procedures now taken as routine for successful work with immune-deficient rodents (Committee on Immunologically Compromised Rodents 1989). Nude mice accept a wide range of xenografts (Manning et al. 1973), but they are best known as hosts for murine or xenogeneic tumor transplants. Because some types of transplanted tumors metastasize in patterns similar to those seen in in situ disease, tumor-transplanted nude mice have been extensively used to
277 investigate antioncogenic therapies. At the beginning of April 2005, PubMed held ~30,000 publications on nude mice with >21,000 related to tumor biology. The nude mutation, now formally designated Foxn1nu/Foxn1nu, blocks normal development of epithelium (Brissette et al. 1996; Lee et al. 1999; Nehls et al. 1994) by alteration of a wingedhelix/forkhead transcription factor. This gives the characteristic hairless state with twisted toenails. In normal mice at fetal gestation day 10–11, the aortic arches bud off the epithelial anlage of the thymus. This does not happen in the Foxn1nu/Foxn1nu nude mouse. Development of most T cells requires transit through the thymus, where maturation and clonal deletion occur. Thus, T cell deficiency is the major immune defect in nude mice. Because T cells provide cytokine help in B cell differentiation and activation, reduced B cell function is seen in these mice. Nude mice have highly activated NK cells that can be undesirable for some research applications (Clark et al. 1981). The residual mucosally associated T cells, activated innate immunity, and B cell responsiveness make nude mice less susceptible to microbial challenge than immune-deficient mice lacking more than a one lineage (Bonnez et al. 1993; Croy and Percy 1993). Nude mice have elevated rates of some types of spontaneous tumors compared with their heterozygote littermates or wild-type mice, but these do not usually restrict experiments (Dore et al. 1987). By backcrossing, a number of other spontaneous mutants have been combined with nude. One of these, “beige/nude/Xid,” is a mouse homozygously deleted for three spontaneous immune deficiency genes, each impairing a separate lymphocyte lineage. Beige/nude/Xid was the first pan-lymphocyte-impaired strain and it served as a forerunner to alymphoid strains now available. Beige (Lystbg) reduces NK cell lytic but not cytokine functions, nude (Foxn1nu) removes thymus-derived T cells, and Xid, X-linked Bruton agammaglobulinemia tyrosine kinase (Btkxid), greatly reduces the number of B cells (Brandt et al. 1981; Cancro et al. 2001). Beige/nude/Xid was the first strain routinely successful for study of engrafted normal human lymphohematopoietic stem cells (Kamel-Reid and Dick 1988). The most recently defined T cell subset is a minor population referred to as “regulatory” T cells (Treg). These cells are mostly CD25+ (IL-2 receptor α chain) CD4+CD8−, appear lineage committed by the forkhead transcription factor FOXP3 and function in promotion of self-tolerance, i.e., suppression (Fontenot and Rudensky 2005). Treg arise in the thymus and require signals from T cell receptors during their differentiation. This was established by studies in which Treg failed to develop when major histocompatability complex (MHC) class I-restricted T cell receptor transgenic mice were crossed to Rag null mice who are unable to construct T cell receptors (see Section III.A later). Autoimmune diseases arise in the absence of functional Treg. Mice deleted in IL-2, its receptor chains, FOXP3, the glucocorticoid-inducible tumor necrosis factor receptor (GITR), CD80, CD86 and the spontaneous mutant scurfy have been valuable in establishing Treg as a separate functional lineage (Fontenot et al. 2005; Stephens et al. 2004; von Boehmer 2005).
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SCID Mice (Genotype Prkdcscid; Protein Kinase,
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Nonobese Diabetic Strains (Genotype Idd#;
DNA Activated, Catalytic Polypeptide;
Insulin-Dependent Diabetes Susceptibility
Chromosome 16 cM 9.2)
Genes; Multiple Chromosomes)
Reduced plasma immunoglobulin is indicative of compromise to the B cell lineage. The SCID mutation (scid/scid, severe combined immune deficiency older nomenclature; formally Prkdc gene and Prkdcscid genotype) was found as spontaneous agammaglobulinemia in an immunoglobulin genotyping experiment conducted in barrier-raised C.B-17 mice and was first reported in 1983 (Bosma et al. 1983). At the time of writing of the chapter, PubMed included >8000 citations for the “SCID mouse.” If the search term is “SCID or RAG and mouse” citations number just over 12,000. C.B-17-scid mice are mutated in the DNA-dependent protein kinase necessary for joining nonhomologous ends of double-stranded DNA. These ends are created by recombinase activating gene (RAG) products during somatic rearrangement of immunoglobulin molecules (B cell receptors) and T cell receptors. Thus, SCID and the RAG1 null and RAG2 null mice subsequently created (Mombaerts et al. 1992; Shinkai et al. 1992) lack both T and B cell lineages. These three T and B cell-deficient strains can be used interchangeably for many applications. Because C.B-17-scid mice have reduced ability to repair DNA, they have been found to be very sensitive to irradiation compared with other strains, including RAG1 null and RAG2 null (Essers et al. 2000). They also frequently develop very large primary thymic lymphomas that can almost fill the thoracic cavity (Martina et al. 2003). This results in wasting and can lead to death, although humane euthanasia is strongly recommended once wasting is observed. Thymomas are not usually seen in animals younger than 16 weeks of age unless they have been irradiated or subjected to other DNA-damaging events. Another feature of the C.B-17-scid mouse is the development of “leakiness” with age (Bosma and Carroll 1991). This refers to detection of low concentrations of antibodies in plasma or serum as the mice age. The amounts of antibody never reach the concentrations seen in immune-competent animals, and the antibodies are of very limited specificity (i.e., oligoclonal) (Gibson et al. 1989). For some applications, endogenous antibodies may complicate experimental interpretation. It should be noted that maternal immunoglobulins will pass to the pups during lactation (Broen and Cafruny 1993) if the dam is leaky or immune competent (i.e., heterozygote mother or wild-type mother receiving C.B-17-scid conceptuses by embryo transfer). Thus, serological testing for immunoglobulin should not be undertaken during the first 2 weeks after weaning of C.B-17-scid pups because falsepositive results may be found. If the lactating mother is immunoglobulin negative, testing can be undertaken at weaning. Genotype, but not leakiness, can be determined by DNA analysis.
Many autoimmune mouse strains have been bred from the original lines referred to as nonobese diabetic (NOD). A series of polygeneic interactions accounts for the phenotype. Many of these genes have been mapped and are called insulin-dependent diabetes susceptibility genes (Leiter 1997). The NOD model of type 1 juvenile diabetes has a generalized dysregulation of the immune system that contributes to inflammation and destruction of pancreatic islets. The immunological problems in NOD mice include deficits in antigen presentation, T cell regulation, NK cell function, cytokine production, and the absence of the complement protein C5 (Serreze and Leiter 1988). The NOD mouse has been backcrossed to create further immune deficiencies and cytokine deficiencies.
D.
NOD-scid (NOD-Prkdc)
The multiple defects in innate immunity known in NOD mice and the T and B cell defects in adaptive immunity of C.B-17-scid mice led to crossing of the strains and development of the NOD-scid strain reported in 1995 (Shultz et al. 1995). The NOD-scid mouse does not display diabetes but retains the cytokine aberrations of the NOD strain as well as defects in NK cell lysis, macrophage lipopolysaccharide response, and complement C5 (Greiner et al. 1998; Shultz et al. 1995). NOD-scid mice show hypocellularity of their bone marrow and a mild macrocytic anemia with normal hematocrits (i.e., reduced erythrocyte numbers with an increased mean cell volume). Only 10% of NOD-scid mice show immunoglobulin “leakiness,” but there is an elevated rate of thymic lymphoma. The NOD-scid mouse has been widely used as a host for human hematopoietic cells since 1996, when it was reported to accept 5- to 10-fold more human hematopoietic cells than the other immune-deficient mice available at that time (Greiner et al. 1998).
III.
COMMONLY USED, GENETICALLY
ENGINEERED IMMUNE-DEFICIENT STRAINS AND THEIR HYBRIDS A. Rag1 or Rag2 Gene Deleted (T and B Cell–Deficient; Chromosome 2, cM 56.0) Gene ablation technology has produced many immunedeficient mutations. Mice deleted in either Rag1 or Rag2 lack
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adaptive immunity and present a phenotype resembling C. B-17-scid. Rag1 and Rag2 are located next to each other in the mouse genome and in the genomes of all jawed vertebrates that have been investigated (Litman et al. 1999). The RAG1 and RAG2 proteins are only coexpressed in B and T lymphocytes. In these cells, RAG1 and RAG2 form a complex that participates at the earliest steps of somatic gene rearrangement for B cell receptors (immunoglobulin molecules) and T cell receptors by recognizing specific signal sequences and cleaving the DNA. In later stages of rearrangement, the RAG1/RAG2 complex interacts with additional proteins to conduct repair of the nicked DNA and provide a functionally rearranged receptor gene (Fugmann et al. 2000). Thus, loss of function of either Rag1 or Rag2 is sufficient to produce a mouse lacking both T and B cells (Mombaerts et al. 1992; Shinkai et al. 1992). Lymphoid organs in Rag1- or Rag2-deleted mice are small, and these strains do not show the leakiness of oligoclonal lymphocyte rearrangement seen in C.B-17-scid mice.
B.
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Common Cytokine Chain γ Null
(T and NK Cell–Deficient; Il2rg; Interleukin 2
C.
Alymphoid Mice (T, B, and NK Cell–Deficient)
γc null mice provided a starting point for generating viable, nonleaky, alymphoid (i.e., B, T and NK cell–deficient) mouse strains. The complete NK cell deficiency in γc null mice was crossed to the completely B and T cell–deficient RAG null strains to create double mutants. RAG/γc double knockout mice are viable, breed normally, and have a normal lifespan when maintained in protected, specific pathogen–free conditions. In comparison with the RAG null or γc null single mutants, RAG/γc doubly null mice are more susceptible to infections (bacterial, fungal, or viral) and are less efficient in rejecting tumors. The more severe immune deficiency of RAG/γc doubly null mice also extends to their inability to reject allogeneic or xenogeneic tissues. RAG/γc doubly null mice fail to reject H-2 mismatched tissues and accept rat and human xenografts of normal tissue. This last feature of RAG/γc doubly null mice has provided the greatest opportunities to date for creation of human/mouse tissue chimeras that are useful for the study of human physiology as well as pathological conditions (see Section V). NOD/RAG/γc triple null mice capitalize on the same feature (Hiramatsu et al. 2003; Ishikawa et al. 2005; Ito et al. 2002).
Receptor γ; X Chromosome) and Related Cytokine Signaling-Deficient Strains D. Cytokines are soluble proteins that act in autocrine or paracrine fashions to play important roles in hematopoiesis in general and lymphopoiesis in particular. A group of cytokines that includes IL-2, -4, -7, -9, -15, and -21, use a shared signaling component—γc (Vosshenrich and di Santo 2001). These cytokines are essential for normal lymphocyte development and function. Humans deficient in γc manifest X-linked severe combined immunodeficiency disease (SCIDX1), a lethal condition (if untreated) that is characterized by cellular and humoral immunodeficiencies due to the absence of T cells and NK cells. Mice deficient in γc are likewise immune deficient, having reduced (but not absent) T cell development and a complete block in NK cell generation. γc null mice, unlike SCIDX1 patients, have reduced numbers of B cells. The immunophenotype of γc deficiency is copied in mice and humans that are deficient in JAK3 tyrosine kinase, which physically and functionally associates with γc-dependent signaling pathways (Fischer et al. 1997). The immune defects in γc null mice result from the additive effects of inhibiting individual γc-dependent cytokine pathways. Thus, IL-7 deficiency reduces B and T cell development in mice and IL-15 deficiency blocks NK development. γc null mice therefore manifest the sum total of abnormalities found in IL-2, -4, -7, -9, -15, and -21 deficient mice, which further underlines the specificity of individual γc-dependent pathways and functions in vivo (di Santo 2000).
Lymphotoxin and Lymphotoxin Receptor
Null Mutants (Lta, Chromosome 17 cM 19.059; Ltb, Chromosome 17 cM 19.061; Ltbr, Chromosome 6 cM 60.4) A series of mice deleted in lymphotoxin (LT) family members has been important in defining development and organization of murine secondary lymphoid tissue, i.e., lymph nodes, spleen, and intestinal Peyer’s patches (Cupedo and Mebius 2005; Rennert et al. 1996). LTα and LTβ are members of the TNF superfamily. LTα is lymphocyte restricted and secreted as a homotrimer and binds to either of the TNF receptors, TNF-R55 or TNF-R75. LTβ is a surface molecule that only forms heterotrimers which include LTα. These heterotrimers bind to a unique LTβ receptor (LTβR), not to the TNFR (Rennert et al. 1996). Deletion of Lta-generated mice lacking lymph nodes and Peyer’s patches and having disorganized splenic architecture (Detogni et al. 1994; Fu et al. 1997), whereas deletion of TNF (formally Tnfsf1a) generated mice lacking only Peyer’s patches (Matsumoto, Fu, et al. 1997). Deletion of Ltb provided mice with no peripheral lymph nodes or Peyer’s patches but retention of mesenteric and cervical lymph node development. Deletion of Ltbr gave mice without any lymph nodes (Fu and Chaplin 1999). Functional immune deficits accompanied these changes in secondary lymphoid tissues.
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From these initial observations, understanding of murine lymph node development has rapidly advanced (Cupedo and Mebius 2005). Three components that are coordinately involved in establishing the environment for attracting and retaining circulating hematopoietic cells are characterized. These are “lymphoid tissue inducer” cells, stromal organizer cells, and induction of expression of adhesion and vascular addressin molecules. It appears that the various lymph nodes in the body depend upon slightly different signaling events. This fact represents the beginning in understanding the mechanisms for tissue and organ-specific immune monitoring and organ-specific immune responsiveness.
E.
Complement Cascade Disrupted Strains
Complement proteins were among the first described proteins of the immune system. They are found in plasma, extravascular fluids, and cell membranes. The complement system comprises >20 proteins that become activated to provide humoral immunity. Antigen-antibody complexes mediate classical complement activation; the alternative pathway is triggered in antibody-independent interactions, for example, by endotoxin. Complement activation is a proteolyic process that produces cleaved proteins that serve as ligands for receptors carried by polymorphonuclear leukocytes, eosinophils, macrophages, mast cells, and other cell types. This makes the complement system particularly important in host resistance to microbial infection. Genetic complement deficiencies are well characterized in both humans (Colten 1992) and in mice. Mouse genome informatics currently lists ~40 genes associated with complement, including its receptors and inhibitors. A number of immune-deficient strains are available that lack specific components of the complement system (Coxon et al. 1996; Fischer et al. 1996; Matsumoto, Fukuda, et al. 1997; Xu et al. 2000). Mice deleted in the negative complement regulatory protein Crry are of particular interest because of embryo lethality. Investigations of this genotype found that the onset of fetal loss occurred after gestation day 9.5 and was associated with complement activation. In normal mice, CRRY is expressed by gestation day 7.5 on trophoblast cells committed to placental differentiation. In Crry null mice, complement deposits onto the surface of the placenta, leading to extreme implantation site inflammation and fetal death. Only the alternate and not the classic complement activation pathway contributes to this breeding disruption that was subsequently associated with a block to placental angiogenesis (Mao et al. 2003; Xu et al. 2000).
F.
Gene Overexpression Resulting in Impaired Immune Responsiveness
Gene deletion and loss of function mutations are not the only strategies for producing immune-deficient mice. Some immune
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defects are created in mice with generalized, organ-specific, or lymphocyte subset-specific forced overexpression of molecules (Araujo et al. 2003; Fehniger et al. 2001; Kondo et al. 1997; Temann et al. 2002). It is becoming routine to contrast deletion and overexpression mutations for complete examination of immune effects of key gene products. Overexpression of genes associated with apoptosis induction or cell survival, such as Fas, Bcl2, Bcl-xL (formally Bcl2l1), and Xiap (formally Birc4) disturbs immune homeostasis and leads to specific immune impairments that accompany overactivity in other components of the immune system (Chuang et al. 2002; Conte et al. 2001; Van Parijs et al. 1998). Overexpression of cytokine genes, such as Il15, can result in chronic inflammation that leads to overproduction of some lymphocyte subsets with relative deficits in others. Il15 overexpressing mice rapidly develop a fatal leukemia (Fehniger et al. 2001).
IV.
POINT MUTAGENESIS TO
CREATE IMMUNOLOGICALLY DEFICIENT AND IMMUNE-MODIFIED MICE Because of the importance of the mouse as a genetic model for most mammalian physiological and pathological processes and the availability of the full genome, an international effort has been organized to identify the functions of all mouse genes using a process of chemical mutagenesis. One of the thrusts in this initiative is to discover new, immunologically variant strains (Nelms and Goodnow 2001). The mutagen N-ethylN-nitrosourea (ENU) is used in this phenotype-driven rather than genotype-driven search for new strains. Usually male mice are mutagenized and then are screened and bred and have their sperm cryopreserved. Generation 2 mice are intercrossed, and generation 3 animals are investigated using 10 generation 3 animals per mutagenized founder. The immune phenotype screen includes humoral immunity and autoimmunity. Sera are collected at 8 weeks of age, followed by immunization with an immunoglobulin-coupled hapten to induce IgG1 and a killed bacterium to induce IgG2a. Fifteen days later, the primary antibody response is measured and is indicative of polarized Th2 versus Th1 responses. A second round of immunization is undertaken 4 weeks after the primary immunization using the immunoglobulin-coupled hapten and a T cell–independent antigen (NP-Ficoll). Sera are collected for evaluation after 7 more days. The antibody titers provide assessment of affinity maturation and humoral memory. Sera are also screened for antinuclear antibodies. According to the Web site for the Wellcome Trust (UK) Library of Immune Variants (http://www.apf.edu.au/resources/wt/data.shtml) “extended immune phenotypes that include hematological cytopenia,” a reflection of immune deficiency, have been obtained. Currently listed as ENU5WT45 is a mouse without B cells.
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ENU4AT92 and ENU4AT13 are lymphopenic, whereas a number of strains show low IgG or low responses to NP-Ficoll. ENU4AT37 has a killing defect. Mice generated in the ENU mutagenesis and phenome screening programs are available to investigators internationally. This rapidly changing resource site should be consulted by investigators requiring up-to-date information for protocol design.
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SPECIALIZED ALLOGENEIC AND
XENOGENEIC TRANSPLANTATION APPLICATIONS USING IMMUNE-DEFICIENT HOSTS A. Analysis of Mouse Lymphohematopoietic Cell Lineage Differentiation Like all hematopoietic lineages, lymphocytes derive from hematopoietic stem cells (often called HSCs) that are produced during embryonic, fetal, and adult life. One extremely useful approach to study hematopoietic development has been the use of murine hematopoietic stem cell chimeras, whereby HSCs are transferred into appropriately conditioned recipients. The transferred hematopoietic stem cells are subsequently capable of long-term reconstitution of all differentiated blood cell types (white blood cells, red blood cells, platelets, etc.) in the recipient animal. Several aspects of the host-conditioning regimen influence the success of hematopoietic stem cell engraftment. These include the availability of environmental niches (or stem cell “space”) as well as immunological tolerance of the host since hematopoietic stem cells can be rejected by host T or NK cells. Alymphoid RAG/γc null mice have proven to be the recipients of choice for hematopoietic stem cell transfer because these hosts have normal radiation sensitivity (Mazurier et al. 1999) unlike C.B-17-scid mice that are highly radiation sensitive (Fulop and Phillips 1990) and due to their complete deficiency in alloreactive lymphocytes, especially NK cells (Colucci et al. 1999). Direct comparisons of RAG null versus RAG/γc null show that the latter are superior hosts for allogeneic hematopoietic stem cells and especially those from embryos (Colucci et al. 2000; Cumano et al. 2001). Because recipient RAG/γc null mice are completely alymphoid, all lymphocytes present after hematopoietic stem cell transfer are, by definition, donor-derived. The recent derivation of RAG/γc null mice bearing different H-2 alleles (H-2b, H-2d, or H-2k; Traggiai et al. 2004) and hematopoietic marker allotypes (CD45.1 or CD45.2) provides an additional means to unambiguously distinguish the origins of nonlymphoid, but hematopoietic-derived, cells after transfer of HSC into RAG/γc null recipients.
B.
Analyses of Differentiation of Human Lymphohematopoietic Cell Lineages
For ethical and practical reasons, most research on human cells and tissues has been restricted to in vitro assays that have brief time courses and lack the major components and complexity of a living organism. With the increasing availability of immunodeficient mice that have reduced capacities for rejection of xenogenic grafts, it has become possible to develop in vivo models to study aspects of normal human lymphohematopoiesis. In these models, human hematopoietic cells and tissues and/or hematopoietic stem and progenitor cells are transplanted into immunodeficient recipients. Human cells and subcellular components can then be detected in the xenogenic hosts, simply by using human-specific markers for fluorescenceactivated cell sorting, enzyme-linked immunosorbent assay, polymerase chain reaction, immunostaining, or in situ hybridization. Within the last two decades, tremendous progress has been made in shaping xenogenic human to mouse transplantation models to more and more faithfully resemble the human lymphohematopoietic system. Table 13-2 summarizes selected publications on human to mouse xenotransplantation models. In pioneering work during the late 1980s, three groups initiated the broad use of mice as recipients for normal human hematopoietic cells: Mosier et al. (1988) transferred human peripheral blood leukocytes into severe combined immunodeficient mice (hu-PBL-SCID mice), McCune et al. (1988; McCune 1996) transplanted human fetal liver hematopoietic cells, fetal bone, fetal thymus, and fetal lymph nodes into C.B-17-scid mice (SCID-hu mice), and Kamel-Reid and Dick (1988) engrafted beige/nude/X-linked immunodeficient mice with human bone marrow cells. Both hu-PBL-SCID mice that maintain B and T cells over a limited time and SCID-hu mice that support de novo differentiation of B and T cells are used to study some aspects of adaptive immune responses, for example, in viral infections such as human immunodeficiency virus (HIV) (Mosier 2000). However, these models are associated with substantial limitations. Hu-PBL-SCID mice produce human recall, but rarely primary immune responses (Delham et al. 1998; Lapenta et al. 2003; Mosier 2000; Mosier et al. 1988; Sandhu et al. 1994), and activation of transferred human xenoreactive T cells can cause graft-versus-host disease (Pflumio et al. 1993; Tary-Lehmann et al. 1994). SCID-hu mice require multiple fetal tissues and are labor and cost intensive, and primary human immune responses to vaccinations are only detectable upon transplantation of additional nonhematopoietic human tissues such as skin (Carballido et al. 2000). To study human hematopoietic stem cells and early hematopoiesis, models were improved (for review see Greiner et al. 1998). Initial studies with both beige/nude/X-linked immunodeficient as well as C.B-17scid mice showed rather low levels of hematopoietic engraftment. This, however, changed dramatically once nonobese diabetic-scid (NOD-scid) mice were generated. NODscid mice display additional defects in the innate immune system
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TABLE 13-2
SELECTED PUBLICATIONS ON HUMAN TO MOUSE XENOTRANSPLANTATION MODELS Cell/Tissue Source for Transplantation
Route of Transplantation
Type of Human Cell Engraftment
i.p. Surgery
B, Ig, T, myelomonocytic B, Ig, T, myelomonocytic
Adult Adult
PBMNC FL, FTh, FLN, FBone, Fskin BM CB, BM, PB
i.v. i.v.
Myelomonocytic B, myelomonocytic
NOD-scid/β2m−/−
Adult
CB, BM, PB
i.v.
B, myelomonocytic
NOD-scid anti-IL-2Rβ NOD-scid β2m−/− NOD-scid
Adult Newborn Adult
CB CB CB
i.v. i.v., i.p., i.h. i.v.
NOD-scid/γc (NOG)
Adult
CB
i.v.
Rag2−/−gγc−/− (huAIS-RG) NOD-scid/γc−/− (complete) NOD-scid/γc−/− (complete) Rag2−/−gγc−/− (huAIS-RG)
Newborn
CB
i.h.
Adult
PB +/-IL-7
i.v.
Newborn
CB CD34+
i.v.
Newborn
FL
i.p.
B, T, myelomonocytic B, T, myelomonocytic B, myelomonocytic, DC, IPC B, Ig, T, NK, myelomonocytic B, Ig, T, myelomonocyte, DC, IPC B, Ig, T, NK, myelomonocytic, IPD B, Ig, T, myelomic, DC, IPC, mouse FDC B, T, myelomonocytic, onocytic, erythrocytes, megakaryocytes
Recipient Mouse
Recipient Age
SCID (hu-PBL-SCID) SCID (SCID-hu)
Adult Adult
Bnx NOD-scid
Reference Moiser et al. 1988 Carballido et al. 2000; McCune 1996; McCune et al. 1988 Kamel-Reid and Dick 1988 Greiner et al. 1998; Lowry et al. 1996; Pflumio et al. 1996 Glimm et al. 2001; Kollet et al. 2000 Kerre et al. 2002 Ishikawa et al. 2002 Cravens et al. 2005; Palucka et al. 2003 Hiramatsu et al. 2003; Ito et al. 2002; Yahata et al. 2002 Gimeno et al. 2004; Schotte et al. 2003 Shultz et al. 2005 Traggiai et al. 2004 Ishikawa et al. 2005
PBMNC, peripheral blood mononucleated cells; i.p., intraperitoneal; i.v., intravenous; i.h., intrahepatic; B, B cells; Ig, human immunoglobulins; T, T cells; myelomonocytic, myelomonocytic cells; FL, fetal liver; FTh, fetal thymus; FLN, fetal lymph node; FSkin, fetal skin; Bnx, beige/nude/X-linked immunodeficiency; BM, bone marrow; CB, cord blood; PB, peripheral blood; IL-2Rβ, interleukin-2 receptor β; DC, dendritic cells, IPC, natural interferon type I–producing cells, NK, NK cells; FDC, follicular dendritic cells.
(complement, macrophage, and NK cell deficiency as described earlier in this chapter) that promote about 1 log-fold better human stem and progenitor cell engraftment levels than C.B-17scid mice (i.e., human cells in some animals were found to account for up to 80% of all nucleated cells in hematopoietic tissues). Since then, NOD-scid mice have become the standard human to mouse xenotransplantation model for researchers studying early human hematopoiesis (Greiner et al. 1998; Hogan et al. 1997; Lowry et al. 1996; Pflumio et al. 1996; Wang et al. 1997). Upon human stem and progenitor transfer, NOD-scid mice maintain donor stem cells for a limited time (up to about 1/2 year) and develop B cells as well as some myelomonocytic cells. In addition, development of dendritic cells and natural type I interferon-producing cells was recently reported (Cravens et al., 2005; Palucka et al. 2003). However, no significant human T cell development occurs in engrafted NOD-scid mice, and B cells do not mature to immunoglobulin-secreting plasma cells, probably because of the lack of T cell help. Thus, human lymphohematopoietic reconstitution remained incomplete and stem and progenitor cell transplanted NOD-scid mice were not suitable models to study all aspects of human lymphohematopoietic reconstitution and function.
Most recently, relevant T cell development and consecutive B cell maturation to immunoglobulin-secreting cells was observed, when residual mouse NK cells were eliminated, and incoming human stem and progenitor cells (isolated from fetal liver, cord blood, bone marrow, and peripheral blood as cytokine mobilized stem and progenitor cells) were given an environment highly supportive for immune system development (Glimm et al. 2001; Kollet et al. 2000). NK cell elimination was achieved by either antibody-induced depletion (anti-IL-2 receptor β chain; Kerre et al. 2002) or by genetic deletion of the signaling part or of the complete common cytokine receptor chain γ, a receptor component essential for NK cell development. The most supportive environmental conditions were achieved by transplanting cells into newborn Rag2 null/γc null or NOD-scid/γc null mice of specific H-2 backgrounds, ready to expand their immune system, or by adding human IL-7 to adult transplanted NOD-scid/γc null animals (Gimeno et al. 2004; Hiramatsu et al. 2003; Ishikawa et al. 2002, 2005; Ito et al. 2002; Schotte et al. 2003; Shultz et al. 2005; Traggiai et al. 2004; Yahata et al. 2002) (Table 13-2). In mice in which human B cells, T cells, NK cells, myelomonocytic cells, dendritic cells, natural interferon-producing cells, and even some human erythrocytes and megakaryocytes develop, human
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cells form primary and secondary lymphoid organ structures and even induce some mouse lymphoid tissue development, as shown by the formation of mouse follicular dendritic cells, probably induced by lymphotoxin α expression on human B cells. This implies substantial conservation and cross-reactivity of some important elements of the lymphohematopoietic system across species barriers. The availability of these “complete” human immune system reconstituted animals will certainly improve research on human immune system–specific viruses such as HIV and Epstein-Barr virus (EBV), may be useful for the generation of human monoclonal antibodies, and will probably allow “preclinical” testing of vaccinations or substances that specifically target the human lymphohematopoietic system. However, although NOD-scid mice and now NOG (NOD-scid/γc null) and huAIS-RG mice (human adaptive immune system RAG/γc null) provide powerful models to study aspects of human lymphohematopoietic cell development and function, they are still probably far from perfect. In terms of human T cell selection in the thymus and MHC restriction in a mouse, it might be useful to transgenically express human MHC on nonhematopoietic cells, such as thymic stroma, in future models. In terms of “parking” human hematopoietic stem cells for maintenance, it may become useful to provide human cytokines and chemokines or additional human nonhematopoietic cell support. In this way, further improvements will be made to produce models that even more faithfully resemble the development and function of the human lymphohematopoietic-system.
C.
Analyses of Therapeutic Strategies for Cancer Patients
The ability to engraft human tumors, human immunocompetent cells, and nondisrupted tumor biopsy tissues has been successfully exploited to evaluate a wide variety of therapeutic approaches to cancer (Bankert et al. 2001). The simplest and most widely utilized version of these mouse models uses single cell suspensions of human tumor cell lines implanted orthotopically or ectopically into nude (Foxn1nu/Foxn1nu or Prkdcscid) or other immunodeficient mice. Both conventional as well as novel chemotherapeutic agents have been evaluated (Guilbaud et al. 1997; Hua and Pero 1997; Jounaidi and Waxman 2000; Kamishohara et al. 1996; Kuo et al. 1993; Nielson et al. 1999; Sharma et al. 1997; Silver and Piver 1999; Tanaka et al. 1994; Tang et al. 1998; Teicher et al. 1997). The ectopic route, in which tumors are implanted subcutaneously, is the most common application because it is possible to assess drug effects upon the tumor by periodically measuring changes in xenograft volume (usually by use of calipers) after administration of test agents systemically or by direct tumor inoculation. In addition to chemotherapeutic drugs that have direct effects upon tumors, agents that act indirectly upon tumors have been
283 successfully evaluated in SCID mouse-human tumor models. For example, monoclonal antibodies specific for endothelial cells have been used to inhibit angiogenesis and shown to inhibit the progression of human breast tumor xenografts (Brooks et al. 1995). The orthotopic route, in which tumors are implanted into the organ in which the tumor originated, are more physiologically relevant for studying therapeutic effects upon tumor progression and metastases. This approach is less frequently utilized because of the difficulty in monitoring tumor growth over time. To overcome this limitation, tumor cells have been transfected with marker genes (i.e., prostate-specific antigen), and their progression has been monitored by quantifying the level of the tumor marker protein in sera from the tumor-bearing mice (Conway et al. 2000). With this approach the effects of antibody-directed cytotoxic drug-loaded immunoliposomes upon the growth and metastasis of human lung tumor xenografts established orthotopically in the lungs of C.B-17-scid mice were successfully monitored. Several other orthotopic human tumor xenograft models have been developed and used successfully for evaluation of different therapeutic strategies (Boehle et al. 2000; Mohammad et al. 1998). Co-engraftment of human tumors and human immunocompetent cells into immunodeficient mice has been used to evaluate immune-based cancer therapies. These more complex models pose several pitfalls and challenges including the complications of graft-versus-host disease, host-versus-graft disease, and the spontaneous development of EBV-positive human B cell lymphomas that can rapidly kill the host mouse. These limitations and their possible solutions are discussed further in Bankert et al. (2001). One of the earliest successful uses of the co-engraftment model demonstrated that human cytotoxic T cells were able to suppress the growth and spontaneous metastases of human melanomas and that exogenous IL-2 enhanced this cytotoxic T lymphocyte (CTL)–mediated antitumor immunity (Sabzevari and Reisfeld 1993). Additional researchers have used co-engraftment of tumors and effector cells to confirm the efficacy of both allogeneic (Conlon et al. 1996; de Kroon et al. 1997; Riedle et al. 1998) and autologous (Stenholm et al. 1998) tumor-specific CTL or lymphokine activated killer cells (Takahashi et al. 1993) to suppress the growth of human tumors. Novel strategies designed to enhance the delivery of effector cells to tumors, that is, bispecific antibodies (Bohlen et al. 1997; Cochlovius et al. 1999; Manzke et al. 1997; Weiner et al. 1993), enhance the function and duration of effector cells with targeted cytokine delivery (Becker et al. 1996; Dolman et al. 1998; Gillies et al. 1998), or modulate coregulatory molecules (Chen et al. 1995; Foy et al. 1998; Hirano et al. 1999; Kim et al. 1998; Lazarus et al. 1999; Murphy et al. 1999; Parney et al. 1997; Sabel et al. 2000) have been successfully evaluated in C.B-17-scid mice co-engrafted with human tumors and human immunocompetent effector lymphocytes. A relatively simple and effective approach for demonstrating the presence of tumor-specific T cells in the peripheral blood
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leukocytes (PBLs) of tumor-bearing patients is to mix varying numbers of the patient’s PBLs with the patient’s own dissociated tumor cells and then inject the mixed cell suspensions subcutaneously into C.B-17-scid mice. Effector T cells inhibit the development and growth of the tumor xenograft in a titratable, dose-dependent manner (Egilmez et al. 2002). This model is called the SCID/Winn assay because of its analogy to the original Winn model designed to evaluate murine effector cells (Winn 1960). The dissociated tumor cell models discussed above are limited by the absence of a human tumor microenvironment. The microenvironment is a major contributor to tumor progression or arrest, and it is therefore likely to significantly affect responsiveness to therapy. By surgically implanting intact (i.e., not disrupted) small pieces of human tumor biopsy samples into C.B-17-scid mice, xenografts can be established in which the tissue architecture, including tumor-associated leukocytes, endothelial cells, pericytes, tumor cells, and other stromal cells, is functionally preserved (Sugiyama et al. 2001; Williams et al. 1996). This xenograft model established the fact that delivery of exogenous IL-12 into the tumor microenvironment mobilizes human tumor-associated leukocytes to kill tumors in situ by indirect mechanisms that are dependent upon human interferon-γ (Broderick et al. 2005; Hess et al. 2003). Future human-SCID mouse chimeric models for evaluating cancer immunotherapies will probably focus upon engraftment of nondisrupted patient tumor tissue to establish xenografts that retain an intact microenvironment. Investigators should standardize their routes, doses, and schedules of the inoculation of human tissues and the administration of therapeutic agents (e.g., drugs, cytokines, effector cells, and antibodies). This standardization would make it possible to compare results from one laboratory to another more reliably. Ultimately it will be necessary to determine how closely the responses to therapy in xenograft models reflect the responses that are observed in human patients.
D.
Analyses of the Maternal-Fetal Interface
The observation that pregnancies occur normally in most lines of immune-deficient mice has been important in focusing investigations to strains in which pregnancy fails, such as the Crry gene deleted embryo (see Section III.G above) (Xu et al. 2000), to elucidate critical components in the maternal-fetal relationship. A transient influx of NK cells or NK-like cells occurs early in gestation in all species studied to date (Stewart 1998). These cells achieve extremely high numbers in mice, rats, and humans. Histological investigation of various lymphocyte-depleted strains was instrumental in defining the lineage of these pregnancy-associated lymphocytes whereas adoptive cell transfers to pregnant alymphoid hosts has advanced understanding of their biology and in their promotion of placenta-associated angiogenesis (Croy et al. 2003). In the absence of uterine
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NK cells, the midgestational structural changes that enlarge the capacity of placental arteries do not occur (Croy et al. 2003). The use of immune-deficient animals in cross-breeding and embryo transfer studies was critical in establishing roles for indoleamine-2,3-dioxygenase in T cell tolerance of the fetus (Mellor et al. 2002; Munn et al. 1998) and in regulatory T cell function at the fetal-maternal interface (Aluvihare et al. 2004; Zenclussen et al. 2005). The mouse is among the species that, like humans, have invasive hemochorial placentae. This, plus the limited access to the human implantation site and the descriptive/correlative nature of the data human uterine samples provide, makes the genetically modified mouse one of the best tools for developing insights into the constantly changing relationships and structures in implantation sites. The ability of alymphoid mice to provide in vivo support for differentiation of early human trophoblast explants (Poehlmann et al. 2004) and for their uterine arteries to undergo the quantifiable, pregnancy-associated vascular changes when exposed to human proteins (Croy et al. 2003), suggest that humanized mouse models addressing the physiology and pathology of human pregnancy can be developed. The mice we have discussed and the transplantation studies we have briefly reviewed are simply examples that illustrate the wide range of immune-deficient lines available and their value in contributing new information on specific research questions. There are numerous additional research areas in which studies using immune-deficient mice have provided seminal understanding. One of these is the arena of infectious diseases (Mercer et al. 2001). By cross-breeding, transfer of embryos or lymphocytes, and/or tissue grafting, powerful novel in vivo models can be created that will continue to advance understanding of processes that promote health and disease.
VI.
SUMMARY
Genetically defined immune-deficient mice are important research tools. Their study has provided deep insights into the differentiation, functions, and regulatory relationships of the immune system. Types of immune-deficient mice range from those with complete absences of multiple immune cell lineages to others with mild alterations. Success in use of immunedeficient mice requires specialized attention to husbandry that will maintain their environment free from microbial contaminants. Under these conditions, most immune-deficient stains are fertile and long lived. Immune-deficient strain development is ongoing with the use of gene-targeting manipulations in embryos, cross-breeding, and mutagenesis. Immune-deficient mice have found special applications as recipients of transplanted murine or xenogenic cells and tissues. They support more complex interactions than most in vitro models and provide an in vivo environment for studies of disease pathogenesis and treatment. in this chapter we have highlighted key features of the major,
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historically significant immune deficient strains that have been widely used and provided comparative information to help readers optimize their selection of an immune-deficient strain.
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Chapter 14 Mouse Models to Study the Pathogenesis of Allergic Asthma Chad E. Green, Nicholas J. Kenyon, Scott I. Simon, and Fu-Tong Liu
I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mouse Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IgE and Mast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trafficking of Eosinophils Involving Cytokines, Chemokines, Selectins, and Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Role of Cytokines and Chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Selectins and Their Sialylated Ligands . . . . . . . . . . . . . . . . . . . . . . . . . C. Leukocyte Integrins and Their Ligands . . . . . . . . . . . . . . . . . . . . . . . . . VI. Contribution of Eosinophils to AHR and Airway Remodeling . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
INTRODUCTION
Allergic asthma is a chronic inflammatory disease of the airways characterized by airway inflammation, reversible bronchoconstriction, and airway hyperresponsiveness (AHR). Recent advancements have led to the identification of a number of cellular and molecular components that contribute to airway inflammation. T cells play a central role in this process and cytokines produced by T helper (Th) 2 cells contribute to the initiation of the inflammatory response. As with other adaptive immune responses, dendritic cells are responsible for antigen presentation to T cells and are now recognized as sentinel cells THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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in the immune and inflammatory response in the lung. Patients with allergic asthma often have elevated IgE levels against environmental allergens and thus IgE levels specific to the allergens are believed to contribute to the asthmatic phenotype. Mast cells are important effector cells, and the peptide and lipid mediators as well as cytokines that they secrete contribute to the inflammatory response. Cytokines and chemokines combined with mast cell–released mediators lead to the upregulation of adhesion molecules on the vascular endothelium and the recruitment to the airways of inflammatory cells, in particular, eosinophils, the most notable inflammatory component of allergic asthma. Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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The bronchial epithelium itself is an active participant. Not only is it a target of the mediators and cytokines mentioned above, but also it produces substances that directly or indirectly contribute to or amplify the asthmatic response. Some of the effector cells and molecules described above contribute to AHR. They are probably also associated with the asthmatic phenotype of a progressive loss of lung function over time, a process attributed to dysregulated remodeling of the airways, which is characterized by thickening of the airway wall and increased collagen. The development of allergic asthma and the progression of the disease therefore involve complex interactive processes that require both molecular and mechanical coordination between regulatory and effector cells. Studies of human materials have gradually unraveled many of the pathological processes described above. However, this approach lacks the detailed examination of the time course of morphological changes occurring in airways and the potential for genetic manipulation of the cell phenotype. In this respect, animal models provide an opportunity for a more thorough evaluation of the mechanisms underlying the development of asthma. The most common animal model is the mouse; this model has many attributes including wellestablished technologies for generating transgenic mice, well-defined physiology, short breeding periods, and ease of maintenance. Indeed this model has afforded valuable insights into the hierarchy of events culminating in asthma progression. The purpose of this chapter is to provide an overview of the mouse model as well as the molecular and cellular mechanisms linking T-cell and mast cell activation with the recruitment of eosinophils to the airways and development of AHR.
II.
MOUSE MODELS
In general, mice are immunized systemically with an antigen and subsequently challenged with the same antigen through the airways. Wide variations exist in the protocols used to sensitize and challenge the mice with regard to dosage of the antigen, the number of times the antigen is administered, and timing (as described in articles cited in the following sections). In most studies, mice are immunized intraperitoneally with an antigen adsorbed on alum as adjuvant. In the majority of cases the antigen used is ovalbumin (OVA). One immunization is usually sufficient for sensitizing the mice (Mayr et al. 2002), although two immunizations 2 weeks apart are commonly used (Zhang et al. 1997). In selected cases, immunization is performed with a soluble antigen without adjuvant; however multiple immunizations are required to sensitize the mice sufficiently (Hessel et al. 1995; Mayr et al. 2002; Williams and Galli 2000). The airway antigen challenge is typically performed 14 days after the immunization, either by exposing the mice to the aerosolized antigen once a day for several consecutive days or giving the antigen intranasally a number of times (Mayr et al. 2002; Zhang et al. 1997). The aerosol is generated in a Plexiglas
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chamber connected to a nebulizer. More recently, a chronic asthma model with structural changes characteristic of airway remodeling has been developed. In this model, sensitized mice are exposed to aerosolized antigen daily or several times a week for weeks (Kenyon et al. 2003; Kumar and Foster 2002; Tanaka et al. 2001). The OVA mouse model is invariably associated with airway eosinophilia developed after the airway antigen challenge. Bronchoalveolar lavage fluid from the mice contains a large number of eosinophils as well as elevated levels of different Th2 cytokines. The model also exhibits AHR and reversible airflow obstruction, two cardinal features of human allergic asthma. Mice demonstrate increased AHR in response to methacholine compared with mice exposed to filtered air (Hamelmann et al. 1997; Kenyon et al. 2003; Temelkovski et al. 1998). Furthermore, when these methacholine-exposed mice are subsequently allowed to breathe filtered air or are treated with a bronchodilator, airflow obstruction is ameliorated and airway resistance decreases to baseline levels. Measurement of AHR in mice exposed to OVA is done by several techniques and this topic has been reviewed thoroughly (Bates and Irvin 2003). The least invasive technique and one of the most popular involves monitoring anesthetized, unrestrained mice in a whole body plethysmograph. Plethysmograph pressure changes are measured and analyzed with changes in the respiratory pattern. OVA-exposed mice treated with nebulized methacholine develop rapid, shallow breathing; changes in the resulting pressure waveform may reflect changes in the caliber of the airways. The calculated output, enhanced pause (Penh), is a dimensionless value and the interpretation of Penh remains a point of contention (Lundblad et al. 2002). Several factors are difficult to control when measuring Penh, including changes in gas temperature and humidity with respiration. Still, Penh measurements have been correlated with changes in lung mechanics (e.g., increased lung resistance) by more formal methods in several studies (Hemelmann et al. 1997b). We believe that unrestrained plethysmography is useful for screening larger groups of mice, particularly when testing potential therapeutic inhibitors (Kenyon et al. 2003). This technique can limit the usage of animals in such trials. Any promising results in these screening trials should be repeated with measurements of lung mechanics by more formal methods (e.g., Kenyon and Last 2005; Zhang et al. 1997; Zuberi et al. 2004). The most well-studied and accepted measurements of lung mechanics in mice are respiratory system impedance, resistance, and compliance, which are measured before and after exposure to methacholine. For these measurements, mice are deeply anesthetized, often paralyzed, and mechanically ventilated through a tracheotomy cannula. A single pressure transducer is needed to determine impedance, and pressure and flow transducers are needed to calculate respiratory system resistance and compliance. Impedance is determined by measuring the change in pressure at the tracheotomy tube, while the mouse is ventilated at a constant flow rate on a volume-cycled mode. The more
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conventional technique for determining compliance and resistance requires independent measurements of pressure and flow. Generally, the pressure transducer is in-line with the ventilator, whereas the flow transducer is connected to the sealed mouse plethysmograph. Dynamic compliance, for example, is calculated by dividing the change in volume, derived from the flow-time signal, over the change in pressure. Measurement of lung, rather than respiratory system, resistance requires eliminating the contribution of the chest wall by opening the mouse chest cavity. However, in the deeply anesthetized and paralyzed mouse, the chest wall and diaphragmatic contributions to total respiratory system resistance are probably minimal. Changes in dynamic compliance and resistance in response to methacholine probably reflect changes in the airways. Methacholine is the most commonly used bronchoconstrictive agent in experiments designed to quantify AHR. It can be nebulized through the ventilator for anesthetized mice and nebulized into a mixing chamber for freely roaming mice. Although nebulized administration is most popular, intravenous methacholine is preferred by some investigators and the doses range from <10 µg/kg to >3000 µg/kg in various studies. Significant strain variation exists in the doses of methacholine required to promote AHR, and this has been well described (Reinhard et al. 2002). In general, no comparisons can be made between the severity of AHR in mice and human asthma based on the doses of methacholine given. On the basis of differences between the mouse and human lung, there is frequent discussion that the OVA mouse model is not a model of asthma. This is true; the OVA mouse model is a model of allergic airway inflammation and AHR. Mice do not experience the wheezing episodes that characterize persistent asthma. Several important anatomical differences between human and mice lungs must be considered when one makes
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comparisons between the OVA mouse model and human asthma. Such differences include a relatively large airways diameter in mice, an increased airway/lung parenchyma ratio in mice, and a single lobe in the left lung (Bates and Irvin et al. 2003). The sum of these anatomical differences appear to result in less resistance to airflow in the airways of mice as a percentage of total lung resistance compared with the airways of humans. Whether this can explain why mice do not wheeze is not known. Other important differences include the presence of an inflammatory alveolitis and the absence of airway thickening and fibrosis in mice exposed to OVA for 1 week. In the chronic asthma model, in which mice are exposed to aerosolized OVA for 4–8 weeks, the animals do develop airway remodeling, and this may reflect human asthma better (Kenyon et al. 2003; Temelkovski et al. 1998). In addition, mice are obligate nose breathers, and measurements of respiratory function in unrestrained mice must factor in the contribution of the upper airways. Thus, as with any case of using an animal model to understand human disease, interpretation of the mechanisms underlying asthma are preliminary until confirmed in humans.
III.
CYTOKINES
A body of evidence implicates a role for cytokines in allergic airway inflammation and AHR. A mainstay of the data comes from transgenic and gene knockout mice, whereas others are from mice given recombinant proteins or neutralizing antibodies. Unless otherwise stated, the cited studies involved mice sensitized with OVA and challenged by the same antigen through the airways. This section focuses on interleukin (IL)-4, IL-5, IL-12, and IL-13 and the relevant studies on other cytokines are listed in Table 14-1.
TABLE 14-1
PHENOTYPE OF TRANSGENIC MICE OVEREXPRESSING OR DEFICIENT IN A CYTOKINE Cytokines IL-4 IL-4 IL-5 IL-5 IL-9 IL-10 IL-10 IL-10 IL-11 IL-12 IL-13 IL-15 IL-18
Transgenic (tg) or Knockout (ko)
Airway Eosinophilia
ko ko ko ko tg ko ko ko tg ko ko tg ko
↓ ↓ ↓ ↓ ↑ ↓ ↑ ↔ ↓ ↓ ↓ ↓ ↑
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AHR ↓ ↓ ↑ ↑ ↓ ↔
References Brusselle et al. 1994; Hogan et al. 1997 Hamelmann et al. 2000 Mould et al. 1997; Wang et al. 1998 Foster et al., 1996; Hamelmann et al. 2000 McLane et al. 1998 Yang et al. 2000 Tournoy et al. 2000 Mäkelä et al. 2000 Wang et al. 2000 Wang et al. 2001 Wang et al. 2000; Webb et al. 2000 Whittaker et al. 2002 Campbell et al. 2000
↑, ↓, and ↔ indicate that the response is higher than, lower than, and comparable with that of wild-type mice, respectively.
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IL-4 and IL-13 are major Th2 cytokines known to play an important role in the allergic response, including the production of immunoglobulin (Ig) E. IL-5 is another Th2 cytokine that regulates eosinophil development. A number of groups have shown that IL-4−/− mice developed substantially fewer eosinophils in the airways compared with wild-type mice (Brusselle et al. 1994; Hogan et al. 1997). These mice also mounted reduced AHR when challenged with methacholine (Hamelmann et al. 2000). The role of IL-4 has also been shown by experiments in which mice were given in vitro generated antigen-specific Th2 cells and then exposed to nebulized antigen. It was found that Th2 cells from IL-4−/− mice did not accumulate in the lung, suggesting that endogenous IL-4 in Th2 cells contributes to cell recruitment to the lung (Cohn et al. 1997). These authors subsequently found that although mice receiving Th2 cells from wild-type mice developed airway eosinophilia and AHR, those receiving cells from IL-4−/− mice exhibited markedly reduced airway eosinophilia. However, there was no difference in AHR (Cohn et al. 1997). Thus, it was concluded that IL-4 production by Th2 cells is critical for the accumulation of eosinophils into the airways but not essential for the induction of AHR. Another report emphasized the finding that IL-4−/− mice still developed airway eosinophilia, albeit at reduced levels, and AHR comparable to those in wild-type mice (Hogan et al. 1998). Only when these mice were treated with anti-IL-5 mAb before the airway antigen challenge was airway eosinophilia and AHR abolished (Hogan et al. 1997). However, in another study, IL-4−/− mice pretreated with anti-IL-5 monoclonal antibody (mAb) still developed AHR, although airway inflammation was substantially reduced (Hogan et al. 1998). Finally, by using a chronic asthma model and IL-4−/− mice, it was concluded that IL-4 is responsible for allergen-induced airway remodeling (Komai et al. 2003). A number of reports showed that airway eosinophilia was diminished in IL-5−/− mice (Foster et al. 1996; Hamelmann et al. 2000; Mould et al. 1997; Wang et al. 1998). It was also demonstrated that delivery of vectors expressing IL-5 restored the response (Foster et al. 1996; Wang et al. 1998). These findings, together with reports demonstrating that airway eosinophilia is diminished by treatment of the mice by anti-IL-5 monoclonal antibody, convincingly established the essential role of IL-5 in development of airway eosinophilia. Some of these studies also demonstrated the role of IL-5 in the development of AHR. However, such a role as well as the issue of whether eosinophils contribute to AHR remains unresolved (see section VI for more discussion). Finally, in a study using IL-5 receptor–deficient mice, IL-5 transgenic mice, and antiIL-5 antibody, convincing evidence was provided that IL-5 is an important contributor of airway remodeling (Tanaka et al. 2004). Another group reached the same conclusion by using IL-5−/− mice (Cho et al. 2004). Inhibition of IL-13 by neutralizing antibody results in reduction of airway eosinophilia and AHR (Grünig et al. 1998;
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Wills-Karp et al. 1998). Subsequently, it was shown that compared with wild-type animals, IL-13−/− mice exhibited diminished accumulation of eosinophils in the airways; however, AHR was still present (Kumar et al. 2002; Webb et al. 2000). An important role of IL-13 was further supported by studying transfer of OVA-specific CD4+ T cells that were wild type or deficient in IL-13 to nonsensitized mice that were then challenged with nebulized OVA. This model clearly demonstrates that T cell– derived IL-13 plays a key role in regulating airway eosinophilia and AHR (Mattes et al. 2001). Additional studies showed that the contribution of IL-13 to the airway response does not overlap with that of IL-4. Thus, the reduced airway inflammation in IL-13−/− mice could be further reduced by treatment with antibody to IL-4 (Webb et al. 2000). Moreover, mice in which both IL-4 and IL-13 were depleted displayed a marked reduction in airway eosinophilia and AHR to an extent greater than in those deficient in either one of the cytokines (Webb et al. 2000). Finally, results from studies of IL-13−/− mice demonstrated that IL-13 plays an important role in airway remodeling in a chronic asthma model (Kumar et al. 2002). IL-12 is a primary Th1 cytokine and contributes significantly to eosinophil recruitment, as IL-12−/− mice exhibited substantially reduced airway eosinophilia (Wang et al. 2001). A number of researchers demonstrated that mouse recombinant IL-12 administered to mice systemically at the time of immunization or during airway antigen challenge abolished the airway eosinophilia and AHR (Gavett et al. 1995; Kips et al. 1996; Riezman et al. 1997). Delivery of IL-12 locally into the airway is more effective than systemic IL-12 in inhibiting AHR (Sur et al. 2000). The mechanism of IL-12 action may be secondary to its role in increasing interferon (IFN)-γ and decreasing IL-4 and IL-5 expression (Gavett et al. 1995). It was also reported that the effects of IL-12 were partially inhibited by anti-IFN-γ mAb and that administration of IL-12 during daily aerosol exposure significantly inhibited eosinophilia in both IFN-γ−/− mice and wild-type controls (Gavett et al. 1995; Brusselle et al. 1997). With an adenovirus-mediated gene transfer approach, the conclusion in another study was that IL-12-mediated inhibition of airway eosinophilia was mainly IFN-γ independent (Stampfli et al. 1999). Finally, using IL-10-deficient mice, it was shown that inhibition of the airway response by IL-12 is independent of IL-10 (Tournoy et al. 2001).
IV.
IgE AND MAST CELLS
IgE-activated mast cells are a primary source of inflammatory cytokines, chemokines, and lipid mediators during the progression of asthma. It is known that asthma can result from sensitization to various allergens and IgE specific to these allergens are present in individuals with asthma. The importance of IgE and IgE-dependent mast cell activation in human asthma is underlined by the close association between asthma and serum
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IgE levels, as well as skin-test reactivity to allergens (Milgrom et al. 1999). The most convincing evidence for the role of IgE is provided by clinical trials on anti-IgE therapy. This therapy is based on a monoclonal antibody that binds IgE and inhibits its binding to IgE receptors. The therapy causes a dramatic reduction in the IgE level (Casale et al. 1997; Corne et al. 1997; MacGlashan et al. 1997) and the efficacy of this therapy has been established in multiple clinical trials (reviewed in Bousquet et al. 2005; Brownell and Casale 2004; Bush 2002; Holgate et al. 2004). In mice, it has been reported that germinal centers containing IgE- and IgG-producing plasma cell are formed in the parenchyma of inflamed lungs after airway antigen challenge (Chvatchko et al. 1996). Zuberi et al. (2000) showed that IgE secreted locally in a mouse model of asthma can capture the antigen presented to the airways, and the immune complexes are able to augment the allergic airway response in an Fc ε receptor I (FcεRI)-dependent manner. Thus, IgE present in airway secretions may facilitate antigen-mediated allergic airway inflammation. However, allergic airway inflammation and AHR can be elicited in mice in the absence of IgE (Hamelmann et al. 1999; Mehlhop et al. 1997) or all classes of immunoglobulins (Corry et al. 1998). In addition, both IL-4−/− and CD40−/−mice mount airway responses to antigen challenge even though antigenspecific IgE is not detectable in the sera (Brusselle et al. 1994; Hogan et al. 1997). Furthermore, administration of IL-12 to actively immunized mice during the daily airway antigen exposure abolished airway eosinophilia and AHR but did not influence the production of antigen-specific IgE (Bursselle et al. 1997). The results suggest that allergic airway disease can occur via pathways that operate independently of allergen-specific IgE. In view of the demonstrated clinical efficacy of anti-IgE therapy on human asthma, it is of interest to know the effect of neutralizing anti-IgE antibody in mouse models of asthma. In this regard, there is a report that anti-IgE antibody is able to inhibit eosinophilic infiltration induced by airway antigen challenge (Coyle et al. 1996). However, results of another study show that the effect of anti-IgE treatment is dependent on the method used to sensitize the mice. In mice sensitized to dust mite allergen in conjunction with human respiratory syncytial virus infection, anti-IgE treatment was effective in reducing the airway inflammatory response. However, the same treatment had no effect in mice sensitized to the same antigen in the absence of concomitant viral infection (Tumas et al. 2001). These studies illustrate well the fact that for mouse models of asthma to be useful for preclinical testing of drugs, protocols tailored for the purpose may need to be developed. The role of IgE is also revealed by studying mice deficient in its receptor, FcεRI. In general, FcεRI-deficient (FcεRI−/−) mice exhibited reduced airway responses only under experimental conditions in which immunization or antigen challenge is suboptimal. We found that FcεRI−/− mice developed reduced airway eosinophilic inflammation compared to wild-type mice, when
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they were sensitized with OVA in the absence of adjuvant and then challenged with OVA through the airways. OVA-sensitized FcεRI−/− mice also showed reduced airway inflammation when they were challenged with OVA intranasally, but not when they were challenged with nebulized OVA (Mayr et al. 2002). The role of mast cells in allergic airway inflammation and bronchial reactivity has been investigated using W/Wv mast cell–deficient mice, which demonstrate attenuated eosinophil airway inflammation (Kung et al. 1995; Ogawa et al. 1999) and a lack of IgE-induced pulmonary responsiveness to cholinergic stimulation (Martin et al. 1993). However, in a number of studies mast cell participation in allergic airway inflammation and AHR was not revealed (Brusselle et al. 1994; Nagai et al. 1996; Takeda et al. 1997). More recently, Kobayashi et al. (2000) used W/Wv mice sensitized and challenged by suboptimal amounts of OVA and found reduced AHR compared with that in normal congenic mice. However, the mice developed AHR that was comparable to that in normal mice when they were more highly sensitized by using either a higher dose of the antigen or an increased number of immunizations. Likewise, Williams and Galli (2000) noted that W/Wv mice developed significantly reduced responses when the mice were immunized with OVA in the absence of adjuvant and then challenged with OVA intranasally. In contrast, W/Wv mice that were sensitized with OVA in alum and then challenged with aerosolized OVA exhibited airway responses similar to those in unsensitized mice. Thus, it appears the role of mast cells in development of AHR is dependent on the protocols used to induce airway responses. Hence, AHR can be induced by different mechanisms, and only certain protocols enlist mechanisms that involve IgE-mediated mast cell activation.
V.
TRAFFICKING OF EOSINOPHILS INVOLVING CYTOKINES, CHEMOKINES, SELECTINS, AND INTEGRINS A.
Role of Cytokines and Chemokines
Inflammatory responses are typically initiated when cytokines and chemokines trigger selectin-mediated adhesion of leukocytes to the vascular endothelium. The principal function of the cytokines and chemokines are to 1) induce expression of adhesion molecules on the luminal surface of vascular endothelium, 2) signal increased release of eosinophils from the bone marrow, and 3) chemoattract circulating eosinophils to the inflamed airways. Allergen-challenged T cells produce Th2 cytokines that combine to elicit numerous responses from adjacent epithelial cells including release of chemokines that function as attractants for eosinophils. The most abundant chemoattractants are IL-8 (or the murine homolog KC), RANTES (regulated on activation, normal T-cell expressed and
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secreted), MCP-1 (monocyte chemoattractant protein-1), MIP1α (macrophage inflammatory protein-1α), and eotaxin (Erger et al. 1995; Li et al. 1999; Kameyoshi et al. 1992; Ponath et al. 1996). Some of these chemokines also mediate eosinophil adhesion to inflamed airway endothelium (Gonzalo et al. 1996; Holgate et al. 1997; Lamkhioued et al. 1997; McNulty et al. 1999). In most cases, these chemokines signal cell arrest of eosinophils on endothelium through activation of the β1- and β2-integrins (Burke-Gaffney et al. 1996; Walsh et al. 1991). Data from both asthmatic patient and mouse models suggest that expression of CXC and CC chemokines may serve to coordinate the eosinophil recruitment. In particular, eotaxin-1 and eotaxin-2 are CC chemokines that specifically activate and chemoattract eosinophils through binding to CCR3 (Ponath et al. 1996; Ravensberg et al. 2005). Eotaxin-1 may be expressed very early in asthma, providing a primary activator of arrest and transmigration of eosinophils. In this regard, IL-13-induced eosinophil recruitment to the lung is inhibited in eotaxin−/− mice (Pope et al. 2001). Moreover, antigen-induced eosinophil recruitment to the airways is decreased in eotaxin-1−/− mice (Rothenberg et al. 1997) and inhibited by antibody to eotaxin (Kim et al. 2001). However, another study showed that eotaxin-1−/− mice developed airway eosinophilia and AHR after airway antigen challenge, indicating that this chemokine is not essential for the development of antigen-induced airway responses in mice (Tomkinson et al. 2001). A subsequent study also using eotaxin-1−/− mice showed that this chemokine is required for the acute but not chronic allergic airway response in mice sensitized to and then challenged by the fungus Aspergillus fumigatus (Schuh et al. 2002). The roles of a number of chemokine receptors in the allergic airway response have been studied by using mice deficient in respective receptors, as listed in Table 14-2.
TABLE 14-2
PHENOTYPE OF MICE DEFICIENT IN A CHEMOKINE AND CHEMOKINE RECEPTOR Chemokine/Chemokine Receptor Eotaxin-1 Eotaxin-1 Eotaxin-1 Eotaxin-2 Eotaxin-1/2 CCR2 CCR2 CCR3 CCR4 CCR6 CCR8
Airway Eosinophilia ↓ ↔ ↓ ↓ ↓↓ ↑ ↔ ↓↓ ↓ ↓ ↔
AHR ↔
↑ ↔ ↓ ↓
References Rothenberg et al. 1997 Tomkinson et al. 2001 Pope et al. 2005 Pope et al. 2005 Pope et al. 2005 Kim et al. 2001 MacLean et al. 2000 Pope et al. 2005 Schuh et al. 2002 Lukacs et al. 2001 Chung et al. 2003
↑, ↓, ↓↓, and ↔ indicate the response is higher than, lower than, substantially lower than, and comparable with that of wild-type mice, respectively. Eotaxin-1/2 represents mice deficient in both eotaxin-1 and -2.
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With expression of adhesion molecules and release of chemoattractants, the bronchial postcapillary endothelium is primed for leukocyte infiltration. The process of eosinophil recruitment to inflamed vasculature is characterized as a multistep continuum of receptor-ligand binding events (Springer 1994; Wardlaw 1999). The initial phase of capture and rolling is dependent upon low-affinity interactions between opposing selectin molecules on leukocytes and inflamed endothelium. Subsequent firm adhesion and migration requires higher affinity binding between leukocyte integrins and endothelial intercellular adhesion molecules. Although structurally diverse, these adhesion receptors function sequentially and synergistically in mediating leukocyte arrest under the constant shear force of blood.
B.
Selectins and Their Sialylated Ligands
The pattern of upregulated expression of chemokines, selectins, and their fucosylated and sialyl Lewisx ligands begins to explain the sequence by which eosinophils are recruited during asthma. Eosinophils express P-selectin glycoprotein ligand 1 (PSGL-1), the most widely expressed selectin ligand that is recognized by endothelial P-selectin and E-selectin, as well as L-selectin on leukocytes. PSGL-1 is essential for optimal eosinophil tethering and subsequent diapedesis into the airway of asthmatic mice (Borchers et al. 2001). The role of P-selectin in eosinophil recruitment to the airways is demonstrated by the lack of eosinophil tethering and accumulation in P-selectin−/− mice after airway antigen challenge (Broide et al. 1998). The essential role of both P-selectin and E-selectin in allergen-induced airway eosinophilia and AHR was similarly established using mice lacking either or both of these selectins (Lukacs et al. 2002). L-selectin is, on the other hand, not required for recruitment into the airways, as no decrease in eosinophil infiltration into the lung was evident in OVA challenged Lselectin−/− mice relative to wild-type mice (Fiscus et al. 2001).
C.
Leukocyte Integrins and Their Ligands
Within minutes of tethering and rolling via selectins, leukocytes arrest on inflamed endothelium through engagement of integrins. Of the integrins, eosinophils utilize the β2-integrins (CD18), specifically LFA-1 (lymphocyte function–associated Ag, αLβ2, CD11a/CD18) and Mac-1 (αMβ2, CD11b/CD18), in mediating adhesion. Eosinophils also use VLA-4 (very late antigen-4, α4β1, CD49d/CD29), which participates in cell rolling and arrest through binding primarily to vascular cell adhesion molecule-1 (VCAM-1). VLA-4 also binds with high affinity to the CS-1 region of fibronectin that is positioned outside of the more common RGD binding motif (Elices et al. 1990). The primary ligand for the β2-integrins on inflamed endothelium is intercellular adhesion molecule-1 (ICAM-1)
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(Diamond et al. 1990; Dustin and Springer, 1988). Collectively, sequential and cooperative engagement of endothelial cell adhesion molecules by the β1- and β2-integrins contributes to slow rolling and ultimately firm arrest of eosinophils on inflamed endothelium (Dunne et al. 2002). Mouse models have helped establish these adhesive interactions. Thus, intraperitoneal injection of anti-ICAM-1 reduced infiltration of eosinophils into the lung of OVA challenged mice by >70% (Chin et al. 1997). Systemic administration of anti-VCAM-1 significantly reduced eosinophil infiltration to the lung in OVA challenged mice (Borchers et al. 2001; Chin et al. 1997). Similarly, small molecule antagonists of VCAM-1 have been shown to inhibit airway eosinophilia in this model (Kudlacz et al. 2002). Therefore, evidence suggests that there is cooperation between the β1- and β2-integrins in recruitment of eosinophils to the lung.
VI.
CONTRIBUTION OF EOSINOPHILS TO AHR AND AIRWAY REMODELING
In mouse models of asthma, both development of airway eosinophilia and AHR are observed. Although the relationship between the two has been extensively investigated, the contribution of eosinophils to AHR remains to be completed resolved. Transgenic mice constitutively expressing IL-5 in the lung epithelium showed a large number of eosinophils in the peribronchial areas. In addition, these animals displayed AHR to methacholine even in the absence of airway antigen challenge (Lee et al. 1997). The results seem to support the contribution of eosinophils to AHR. However, in another study IL-5 transgenic mice did not show AHR, despite the presence of eosinophils in the lungs (Hisada et al. 1999). Results obtained from studying IL-5-deficient mice or mice treated with anti-IL-5 mAbs are also controversial, and some of the studies have already been described in the section on cytokines. For example, studies by Nagai et al. (1996) and Corry et al. (1996) showed that the anti-IL-5 antibody treatment inhibited airway eosinophilia but not AHR. Kobayashi et al. (2000) also described markedly inhibited airway eosinophilia without a concomitant reduction in AHR in mice treated with anti-IL-5 antibody. However, it has been shown that anti-IL-5 mAb prevented the development of AHR (Hamelmann et al. 1997a), and there are studies showing that IL-5−/− mice develop neither eosinophilia nor AHR (Foster et al. 1996), supporting an association between these two processes. Furthermore, it has been observed that repeated intranasal administration of IL-5 to mice resulted in eosinophil infiltration into the airways followed by development of AHR (Van Oosterhout et al. 1995), which also supports the relationship between these two responses. A number of studies suggested that the contribution of eosinophils to the development of AHR in mice might be
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masked by coexisting pathways that operate independently of eosinophils. First, whereas in IL-4−/− mice, AHR persisted after treatment with anti-IL-5 mAb that caused a diminished eosinophil response, AHR was abolished when the mice were treated also by anti-CD4+ mAb (Hogan et al. 1998). Second, whereas anti-IL-5 treatment did not reduce AHR significantly in wild-type mice, the same treatment inhibited AHR in IL-13−/− mice (Webb et al. 2000). Third, anti-IL-5 treatment caused a substantial reduction in AHR in FcεRI−/− mice but not in wildtype mice (Mayr et al. 2002). Thus, pathways dependent on CD4+ T cells, IL-13, and IgE-mediated mast cell activation, respectively, may mask the contribution of eosinophils to the development of AHR. In the absence of one of these pathways, a positive correlation between eosinophilia and AHR can be more easily demonstrated. In the third case, the results also suggest that there is a synergy between mast cells and eosinophils in the induction of AHR. It is possible that mast cells and eosinophils secrete different mediators that may activate bronchial smooth muscle through distinct pathways and these pathways cooperate synergistically in inducing heightened bronchial responsiveness. As mentioned in the section on cytokines, various studies have demonstrated the role of IL-5 in development of airway remodeling, which indirectly support the role of eosinophils in this process. Importantly, a direct role for eosinophils as effector cells in airway remodeling has been established by experiments in which the eosinophil lineage was ablated (Humbles et al. 2004).
VII.
CONCLUSIONS
The use of transgenic mice together with the use of specific neutralizing antibody or small molecule antagonists has enabled the discovery of cytokines, chemokines, adhesion molecules, and cell types responsible for development and progression of airway inflammation and AHR. In this role, mouse asthma models have provided tremendous insight into the mechanisms regulating human disease. Ongoing research indicates that asthma is a complex disease involving T cells, mast cells, macrophages, epithelial cells, and endothelial cells. These cells produce and respond to cytokines, chemokines, and small molecule mediators that ultimately result in AHR and recruitment of eosinophils to the bronchial airways through specific interactions between adhesion molecules. However, for a number of different molecules, variable and sometimes contradictory results were obtained from different research groups and thus the roles of these molecules in the airway response in mice remain to be elucidated. Studies also show that the role of a given molecule and cell sometimes depends on the protocol used to induce the airway response. In addition, the conclusions achieved with mouse models remain to be tested in humans before they can be accepted as applicable to human asthma.
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Nevertheless, mouse models will continue to be useful for elucidation of the pathogenesis of human asthma. They will also be valuable for discovering or validating molecules and cells as targets for treatment for asthma as well as for testing agonists/antagonists directed at these molecules and cells.
VIII.
SUMMARY
Allergic asthma is a disease of the airways characterized by reversible bronchial obstruction and airway hyperresponsiveness associated with allergic inflammation. Discovery of the individual molecular and cellular components of asthma has depended heavily on animal models that mimic human disease. In this regard, these models have enabled description of a complex sequence of events involving T cells, B cells, dendritic cells, mast cells, macrophages, eosinophils, epithelial cells, and endothelial cells. Differential responses of these various cell types to triggers of asthma results in a hierarchy of cytokine, chemokine, and mediator release, leading to homing and activation of immune and inflammatory cells in the pulmonary vasculature and culminating in allergic inflammation. Some of these cells and molecules are also responsible for development of airway hyperresponsiveness. Mouse models have been particularly valuable, especially in conjunction with the use of genetically engineered animals with heightened expression or deficiency of certain gene products. In this chapter, we discussed briefly various methodological aspects of mouse models and then reviewed some of the critical components in progression of asthma for which the models have provided insights, including 1) cytokine release, 2) mast cell activation and degranulation, and 3) homing of eosinophils.
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Kenyon, N.J., Last, J.A. (2005). Reversible and irreversible airway inflammation and fibrosis in mice exposed to inhaled ovalbumin. Inflamm Res 54, 57–65. Kenyon, N.J., Ward, R.W., Last, J.A. (2003). Airway fibrosis in a mouse model of airway inflammation. Toxicol Appl Pharmacol 186, 90–100. Kenyon, N.J., Ward, R.W., McGrew, G., Last, J.A. (2003). TGF-β1 causes airway fibrosis and increased collagen I and III mRNA in mice. Thorax 58, 772–777. Kim, J., Merry, A.C., Nemzek, J.A., Bolgos, G.L., Siddiqui, J., Remick, D.G. (2001). Eotaxin represents the principal eosinophil chemoattractant in a novel murine asthma model induced by house dust containing cockroach allergens. J Immunol 167, 2808–2815. Kim, Y., Sung, S.-s. Kuziel, W.A., Feldman, S., Fu, S.M., Rose, C.E., Jr. (2001). Enhanced airway Th2 response after allergen challenge in mice deficient in CC chemokine receptor-2 (CCR2). J Immunol 166, 5183–5192. Kips, J.C., Brusselle, G.J., Joos, G.F., Peleman, R.A., Tavernier, J.H., Devos, R.R., Pauwels, R.A. (1996). Interleukin-12 inhibits antigen-induced airway hyperresponsiveness in mice. Am J Respir Crit Care Med 153, 535–539. Kobayashi, T., Miura, T., Haba, T., Sato, M., Serizawa, I., Nagai, H., Ishizaka, K. (2000). An essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model. J Immunol 164, 3855–3861. Komai, M., Tanaka, H., Masuda, T., Nagao, K., Ishizaki, M., Sawada, M., Nagai, H. (2003). Role of Th2 responses in the development of allergeninduced airway remodelling in a murine model of allergic asthma. Br J Pharmacol 138, 912–920. Kudlacz, E., Whitney, C., Andresen, C., Duplantier, A., Beckius, G., Chupak, L., et al. (2002). Pulmonary eosinophilia in a murine model of allergic inflammation is attenuated by small molecule α4β1 antagonists. J Pharmacol Exp Ther 301, 747–752. Kumar, R.K., Foster, P.S. (2002). Modeling allergic asthma in mice: pitfalls and opportunities. Am J Respir Cell Mol Biol 27, 267–272. Kumar, R.K., Herbert, C., Yang, M., Koskinen, A.M., McKenzie, A.N., Foster, P.S. (2002). Role of interleukin-13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma. Clin Exp Allergy 32, 1104–1111. Kung, T.T., Stelts, D.M., Zurcher, J.A., Adams, G.K., Egan, R.W., Kreutner, W., et al. (1995). Involvement of IL-5 in a murine model of allergic pulmonary inflammation: prophylactic and therapeutic effect of an anti-IL-5 antibody. Am J Respir Cell Mol Biol 13, 360–365. Lamkhioued, B., Renzi, P.M., Abi-Younes, S., Garcia-Zepada, E.A., Allakhverdi, Z., Ghaffar, O., et al. (1997). Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation. J Immunol 159, 4593–4601. Lee, J.J., McGarry, M.P., Farmer, S.C., Denzler, K.L., Larson, K.A., Carrigan, P.E., et al. (1997). Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 185, 2143–2156. Li, L., Xia, Y., Nguyen, A., Lai, Y.H., Feng, L., Mosmann, T.R., et al. (1999). Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells. J Immunol 162, 2477–2487. Lukacs, N.W., John, A., Berlin, A., Bullard, D.C., Knibbs, R., Stoolman, L.M. (2002). E- and P-selectins are essential for the development of cockroach allergen-induced airway responses. J Immunol 169, 2120–2125. Lukacs, N.W., Prosser, D.M., Wiekowski, M., Lira, S.A., Cook, D.N. (2001). Requirement for the chemokine receptor CCR6 in allergic pulmonary inflammation. J Exp Med 194, 551–555. Lundblad, L.K., Irvin, C.G., Adler, A., Bates, J.H. (2002). A reevaluation of the validity of unrestrained plethysmography in mice. J Appl Physiol 93, 1198–1207. MacGlashan, D.W., Jr., Bochner, B.S., Adelman, D.C., Jardieu, P.M., Togias, A., McKenzie-White, J., et al. (1997). Down-regulation of FcεRI expression on human basophils during in vivo treatment of atopic patients with anti-IgE antibody. J Immunol 158, 1438–1445. MacLean, J.A., De Sanctis, G.T., Ackerman, K.G., Drazen, J.M., Sauty, A., DeHaan, E., et al. (2000). CC chemokine receptor-2 is not essential for the
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SCOTT
I.
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AND
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LIU
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Chapter 15 The Mouse Trap: How Well Do Mice Model Human Immunology? Christopher C.W. Hughes and Javier Mestas
I. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Immune System–Related Genetic Disorders . . . . . . . . . . . . . . . . . . . . . . . . III. Response to Viral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Response to Mycobacterium tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . V. Response to Helminth Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis . . . . VII. Delayed-Type Hypersensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
OVERVIEW
Huge advances have been made in the understanding of human immune responses through the study of mice. However, therein lies a trap: mice have proved to be such good models that it is now tempting to assume that what is true in mice—in vivo veritas—is necessarily true in human. As we describe below, the differences between human and mouse immunology are significant and numerous. Although recent sequencing efforts reveal only 300 or so genes that are unique to mouse or human (Waterston et al. 2002), it should not be forgotten that the two species diverged around 75 million years ago, differ hugely in both size and lifespan, and have evolved in quite different ecological niches in which widely different pathogenic challenges need to be met. THE MOUSE IN BIOMEDICAL RESEARCH, 2ND EDITION
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Notably, humans are about 3000 times larger than mice and have a correspondingly larger number of cells, the result of perhaps 105 more mitoses (Rangarajan and Weinberg 2003). Added to this, humans live 30–50 times longer, resulting in a greatly increased potential for generating cells bearing mutations predisposing to cancer. As such, it might be predicted that changes in immune surveillance may have evolved to combat this risk. Most, if not all, of the differences we have noted between mouse and human immunology (Table 15-1) have probably become fixed during the 75 million years since our divergence because they provide some selective advantage. In all likelihood these adaptations are in response to new pathological challenges from microorganisms, which have very short generation times and often have high mutation rates (McDade and Worthman 1999). Such rapid changes in microorganisms have Copyright © 2007, 1980, Elsevier Inc. All rights reserved.
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TABLE 15-1
SUMMARY OF SOME KNOWN IMMUNOLOGICAL DIFFERENCES BETWEEN MOUSE AND HUMAN
Hematopoiesis in spleen Presence of BALT Neutrophils in peripheral blood Lymphocytes in peripheral blood Hematopoietic stem cells CD34− MSC expression of CXCR5 CD34− MSC expression of CCR2 TLR2 expression on PBLs TLR3 TLR9
TLR10 Expression of IRAK splice variants Response to endotoxin Sialic acid Neu5GC expression CD33 Leukocyte defensins Paneth cell defensins Paneth cell defensins iNOS in macrophages Murine macrophage NO CD4 on macrophage Predominant T cells in skin and mucosa γ/δ T cells respond to phospho-antigens CD1 genes NK inhibitory receptors for MHC I NKG2D ligands
Mouse
Human
Active into adulthood Significant 10–25%
Ends before birth Largely absent in healthy tissue 50–70%
Pabst and Gehrke 1990 Doeing et al. 2003
75–90%
30–50%
Doeing et al. 2003
c-kithigh, flt-3− Absent
c-kitlow, flt-3+ Present
Sitnicka et al. 2003 von Luttichau et al. 2005
Present
Absent
von Luttichau et al. 2005
Low (induced on many cells including T cells) Expressed on DCs, macrophages; induced by LPS Expressed on all myeloid cells, plasmacytoid DCs and B cells
Constitutive (but not on T cells)
Binds lipopeptides
Rehli 2002
Expressed by DC; no LPS induction Expressed only on B cells, plasmacytoid DCs and neutrophils Widely expressed IRAK1b, IRAK1c
Binds dsRNA
Heinz, Haehnel et al. 2003; Rehli 2002 Lund et al. 2003; Trevani et al. 2003
Pseudogene All IRAK2 variants, IRAK1s, IRAK1b Requires high dose; no physiological response Widespread Expressed on granulocytes
Low dose induces fever, tachycardia Absent Expressed on monocytes
Toll receptor regulation
Rao et al. 2005 Copeland et al. 2005
Binds pathogens Binds sialic acids
Yes
Kaufmann 1996
CD1d 8–18 Ly49 (except Ly49D and H), 2 KIR H-60, Rae1β
CD1a,b,c,d 9–14 KIR, 1 LY49
Dutronc and Porcelli 2002 Lanier 1998; Parham 2005
MIC A, MIC B, ULBP Expressed by all
Low Absent
High Present
FcγRIIA, C Serum IgA
Absent Mostly polymeric
Present Mostly monomeric
Ig classes
IgA, IgD, IgE, IgG1, IgG2a,* IgG2b, IgG3, IgM Shorter, less diverse IgMhi B cells in periphery
IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM Longer, more diverse No peripheral B cells
Normal pre-B and immature B Leaky block at pro-B to pre-B transition
Blocks pro-B to pre-B transition Blocks pro-B to pre-B transition
Neutrophils
Varki 2001 Brinkman-Van der Linden et al. 2003 Risso 2000 Cunliffe et al. 2001; Ghosh et al. 2002 Ouellette and Selsted 1996 Schneemann et al. 1993 Weinberg 1998 Crocker et al. 1987 Elbe et al. 1996
Present Stored as pro-form; processed by trypsin Two Absent Induced by IFN-α/β Present α/β TCRs
Only on activated and memory
Btk deficiency λ5 deficiency
Binds CpG
References
Absent Processed by MMP7; stored preprocessed At least 20 Present Induced by IFN-γ and LPS Absent γ/δ TCRs (dendritic epidermal T cells) No
NKG2D expression on αβ CD8+ T cells fMLP receptor affinity FcαRI
Ig CDR-H3 region BLNK deficiency
Notes
NK activating receptors Costimulatory naïve human T
Raulet 2003 Maasho et al. 2005 Gao and Murphy 1993 Monteiro and Van De Winkel 2003 Daeron 1997 Monteiro and Van De Winkel 2003 Martin and Lew 1998 Zemlin et al. 2003 Minegishi et al. 1999; Pappu et al. 1999 Conley et al. 2000 Conley et al. 2000
15. THE
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TRAP:
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MODEL
HUMAN
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IMMUNOLOGY?
TABLE 15-1
SUMMARY OF SOME KNOWN IMMUNOLOGICAL DIFFERENCES BETWEEN MOUSE AND HUMAN—cont’d
Osteopontin expression in lymph node CD38 expression on B cells B cell CD5 and CD23 expression IL-13 effect on B cells Thy1 expression
Mouse
Human
Absent on follicular DCs
Widespread including follicular DCs High on GC B cells and plasma cells Coexpression
Low on GC B cells, off in plasma cells Mutually exclusive None Thymocytes, peripheral T cells
Notes
Li et al. 2005 Gordon et al. 2001 Gordon et al. 2001
Induces switch to IgE Absent from all T cells, expressed on neurons Loss of T and NK cells, but B cell numbers normal Phenocopies γc deficiency Only blocks T cell development
Snapper and Finkelman 1999 Tokugawa et al. 1997
Effect of γc deficiency
Loss of T, NK, and B cells
Effect of Jak3 deficiency Effect of IL-7R deficiency
Phenocopies γc deficiency Blocks T and B cell development
ZAP70 deficiency
No CD4+ or CD8+ T cells
Caspase 8 deficiency
Embryonic lethal
No CD8+ T cells but many nonfunctional CD4+ Viable, immunodeficiency
Caspase 10 IFN-α promotes Th1 differentiation Induction of IL-12R β2
Absent No
Present Yes
IFN-γ
IFN-α/β but not IFN-γ
Th expression of IL-10 IL-4 and IFN-γ expression by cultured Th cells CD26 expression on T cells
Th2 Either/or
Th1 and Th2 Sometimes both
CD4 CD8 DN thymocytes
CD4+ and CD8+ thymocytes, memory T Widespread, including T cells
Complement R
On 80% of CD4+, 50% of CD8+ Cyclin D2, no cyclin D3
D3→ rapid response?
CD46 expression CD28 expression on T cells Cyclin D in resting CD8+ memory T cells ICOS deficiency
Only on spermatozoa and testes germ cells On 100% of CD4+ and CD8+ Cyclin D3 Normal B cell numbers and function, normal IgM levels
B cells immature and severely reduced in number, low IgM
B7-H3 effects on T cells
Inhibits activation
Promotes activation
ICAM3
Absent
Present
P-selectin promoter GlyCAM MHC II expression on T cells
Activated by TNF and LPS Present Absent
Unresponsive to inflammation Absent Present
MUC1 on T cells
Absent
Present
Granulysin CXCR1
Absent Absent
Present Present
IL-8, NAP-2, ITAC, MCP-4, HCC-1, HCC-2, MPIF-1, PARC, eotaxin-2/3 MRP-1/2, lungkine, MCP-5
Absent
Present
Present
Absent
Protective in EAE
Exacerbates MS
IFN-γ effects in demyelinating disease
References
Related to syk level?
Mutant stat2 in mice
Fischer et al. 1997; Leonard 1999 Buckley 1999 Peschon et al. 1994; Roifman et al. 2000 Chu et al. 1999; Elder et al. 2001 Chun et al. 2002; Tibbetts et al. 2003 Tibbetts et al. 2003 Farrar et al. 2000 Rogge et al. 1997; Szabo et al. 1997 Del Prete et al. 1993
Ohnuma et al. 2005 Kemper et al. 2005 Lenschow et al. 1996
Possibly age-related Grimbacher et al. 2003; McAdam et al. 2001; Tafuri et al. 2001 Chapoval et al. 2001; Suh et al. 2003 DC-SIGN ligand Geijtenbeek et al. 2000; van Kooyk and Geijtenbeek 2002 Pan et al. 1998 Crommie and Rosen 1995 Barnaba et al. 1994; Lombardi et al. 1996; Denton et al. 1999; Taams et al. 1999 Regulates Correa et al. 2003 migration? In CTL Flynn and Chan 2001; Olson and Ley 2002; Zlotnik and Yoshie 2000 Chemokines Olson and Ley 2002; Zlotnik and Yoshie 2000 Chemokines
Olson and Ley 2002; Zlotnik and Yoshie 2000 Heremans et al., 1996; Lublin et al. 1993; Panitch et al. 1987 Continued
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TABLE 15-1
SUMMARY OF SOME KNOWN IMMUNOLOGICAL DIFFERENCES BETWEEN MOUSE AND HUMAN—cont’d Mouse
Human
Notes
Protective in EAE
Exacerbates MS
Exacerbates EAE
Protects in MS
Neutrophil-rich
Lymphocyte-rich
Constitutive MHC II on ECs ECs present antigen to CD4+ T cells
Absent No
Present Yes
CD58 (LFA-3) T cell dependence on CD2-ligand interactions CD2-ligand interaction CD40 on ECs
Absent Low
Present High
Selmaj et al. 1991; van Oosten et al. 1996 Loss of Treg in mu. Kohm et al. 2003; Bielekova et al. 2004 Crowle 1975; Dumonde et al 1982 Choo et al. 1997 Memory T only Mestas and Hughes 2001; Murphy et al. 1999; Pober et al. 2001 CD2 ligand van der Merwe 1999 van der Merwe 1999
Lower affinity, with CD48 Absent
Higher affinity, with CD58 Present
Vascularized grafts tolerogenic? Success with microchimerism inducing graft tolerance CD24 or CD25 mAbs induce tolerance Passenger leukocytes
Yes
No
High
Low (experiments in nonhuman primates) No
TNF effects in demyelinating disease IL-2 mAb effects in demyelinating disease DTH lesions
High-dose IL-2 for anticancer treatment
Yes Account for graft immunogenicity Clears B16 melanoma
Do not account for graft immunogenicity Little to no benefit
References
van der Merwe 1999 Ensminger et al. 2003; Karmann et al. 1995 Sykes 2001 Monaco 2003 Sachs 2003 Wood 2003 Parkinson et al. 1990; Sparano et al. 1993
BALT, bronchus-associated lymphoid tissue; MSC, mesenchymal stem cell; TLR, toll-like receptor; DC, dendritic cell; LPS, lipopolysaccharide; ds, doublestranded; PBL, peripheral blood lymphocytes; IRAK, IL-1 receptor kinase; MMP, matrix metalloproteinase; iNOS, inducible nitric oxide synthase; NO, nitric oxide; fMLP, formyl-methionyl-leucyl-phenylalanine; GC, germinal center; DN, double negative; ICAM, intercellular adhesion molecule; NCP, neutrophilactivating peptide; ITAC, interferon-inducible T cell α chemoattractant; MCP, monocyte chemoattractant protein; HCC, hemofiltrate CC chemokine; MPIF, myeloid progenitor inhibitory factor; PARC, pulmonary and activation-regulated chemokine; MRP, multidrug resistance protein; LFA, leukocyte (lymphocyte) function-associated antigen. *Absent in C57BL/6, /10, SJL, and NOD mice, which have IgG2c.
driven rapid evolution of mammalian major histocompatability (MHC) molecules, and consequently, natural killer (NK) cell inhibitory receptors (Martin et al. 2002; Pabst and Gehrke 1990; Parham 2005). By assuming that what is true in mice is also true in human we run the risk of overlooking aspects of human immunology that do not occur or cannot be modeled in the mouse. Included in this subset are differences that may preclude a successful preclinical trial in mice becoming a successful clinical trial in human. The literature is littered with examples of therapies that work well in mice but fail to provide similar efficacy in humans (Monaco 2003; Oehler and Bicknell 2000; Panitch, Hirsch et al. 1987; Shepherd and Sridhar 2003; Sykes 2001; van Oosten et al. 1996; Wood 2003). When the ultimate goal of research in mice is to improve human health, then efforts to identify ways we respond differently to immunological challenges will surely help avoid undue early focus on therapies that are destined to be unsuccessful in the clinic.
So, why do we focus so much immunological research on the mouse? The simple answer is that, in comparison, doing these experiments in people is very hard. Humans are outbred and do not live in controlled environments; obtaining institutional review board approvals is time-consuming and costly; and the ethics of placebo-controlled trials are still being debated. Some of these issues were recently reviewed (Steinman and Mellman 2004). Several groups, including our own, have used SCID mice engrafted with various components of the human immune system, including purified T cells, and these models have proven to be a useful intermediate between mouse studies and clinical trials (Bankert et al. 2001; Davis and Stanley 2003; Murray et al. 1994; Sultan et al. 1997). However, there are dangers in these models, too, as some cell-cell interactions do not cross species barriers. For example, human T cells do not efficiently cross mouse endothelium (Murray et al. 1994). We recently published a detailed discussion of the immunological differences between the species (Mestas and Hughes 2004),
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and these are summarized with updates in Table 15-1. In this chapter, we will focus on specific disease processes that are known to differ between mice and humans.
II.
IMMUNE SYSTEM–RELATED GENETIC DISORDERS
Important differences have been noted between patients with mutations in BLNK (SLP-65) and mice having targeted mutations in this gene. BLNK is an adapter protein that is rapidly phosphorylated by Syk after cross-linking of the B cell antigen receptor. It then serves as a scaffold for downstream signaling components such as Grb2, Vav, Nck, and phospholipase C-γ. A naturally occurring mutation in the human BLNK protein has been identified that results in a splicing defect preventing protein expression. In this patient there was a block in the pro-B to pre-B transition, resulting in a complete absence of B cells in the periphery. In contrast, although B cell development in mice lacking BLNK was also blocked at the pro-B to pre-B transition, there were low numbers of IgM+ B cells in the periphery; however, no mature IgMloIgDhi B cells were observed (Pappu et al. 1999). These findings suggest a more severe block in human B cell development than in mouse in response to defects in BLNK (Minegishi et al. 1999). Btk is a tyrosine kinase associated with B cell receptor (BCR) signaling, and both human X-linked agammaglobulinemia (XLA) and mouse X-linked immunodeficiency are associated with defects in this gene (Satterthwaite and Witte 2000). Mutation of Btk in human XLA results in a severe block in B cell development at the pre-B cell stage. In contrast, deletion of the gene in mice results in normal numbers of pre-B and immature cells and ~50% of the normal number of mature B cells. In addition, these mice have near normal concentrations of IgG1, IgG2a, and IgG2b, although levels of IgG3 and IgM are low (Conley et al. 2000). Importantly, when mice have been generated with mutations identical to those seen in human disease, these differences persisted (Buckley 1999; Thomas et al. 1993). Similarly discrepant phenotypes are seen in mice and humans lacking a functional λ5 gene. λ5 is the light chain component of the pre-BCR and in λ5-deficient patients there is again an almost complete block in B cell differentiation at the pro-B to pre-B cell stage (Conley et al. 2000). In stark contrast, mice that lack λ5 also have a block at this stage but it is “leaky,” such that mature mice have 10–20% of the normal number of B cells and antibody responses occur normally. Overall, the data suggest that B cell development is more tightly regulated in human than in mouse. Human and mouse X-linked severe combined immunodeficiency disease (SCID) is the result of mutations in the gene for the cytokine receptor common γ chain, γc. Several cytokine receptors, including those for interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15, share this signaling component. Perhaps not surprisingly,
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deletion or mutation of this gene, which is on the X chromosome, results in severe immunological defects. Interestingly, these differ between human and mouse X-linked SCID (Fischer et al. 1997; Leonard 1999). Numerous mutations have been identified in the human γc gene that inhibit function, and in most of these cases the result is a dramatic decrease in the number of T cells and NK cells. B cell development, however, is normal, although function is impaired, probably because of the lack of T cell help. In marked contrast, B cell numbers are greatly diminished in γc-null mice. Given that IL-7 receptor deficiency in mouse blocks both T and B cell development (Peschon et al. 1994) but only blocks T cell development in humans (Roifman et al. 2000), it is likely that B cell development in humans is independent of IL-7. The major signal transducer for γc is JAK3 (Janus tyrosine kinase-3) and mutation of this gene phenocopies the γc mutation in both mouse and human, that is, a lack of T and NK cells in human with the addition of a severe B cell defect in mice (Buckley 1999). Interesting differences have also been noted in ZAP70deficient mice and humans. ZAP70 is essential for T cell receptor (TCR) signaling in both development and activation and compromised signaling results in SCID. In humans the defect results in normal numbers of CD4+ T cells and the absence of CD8+ T cells. The CD4+ T cells are, however, nonfunctional. In contrast, an identical mutation introduced into the mouse ZAP70 results in a block in differentiation of both T cell subsets at the double-positive stage (Elder et al. 2001). It has been suggested that the “leakiness” of the human mutant is due to incomplete downregulation of the protein tyrosine kinase Syk in human thymocytes, compared with mouse thymocytes (Chu et al. 1999). To become fully activated T cells require both a primary, antigen-dependent signal and a second, antigen-independent or costimulatory signal. Costimulation is provided by antigenpresenting cells (APCs) expressing ligands for costimulatory receptors on the T cells. Inducible T cell costimulator (ICOS) is an important costimulatory molecule and a human ICOS deficiency was recently reported (Grimbacher et al. 2003). Whereas in mouse the loss of ICOS does not affect either the number of mature B cells, their maturation status, or their secretion of IgM (McAdam et al. 2001; Tafuri et al. 2001), the loss of ICOS in human resulted in a severe reduction in B cell number, maturation status, and secretion of IgM (Grimbacher et al. 2003).
III.
RESPONSE TO VIRAL INFECTIONS
Human cytomegalovirus (HCMV) infection has been widely modeled in mice using mouse cytomegalovirus (MCMV), and despite earlier reports to the contrary MCMV can indeed establish latent infections in mice, similar to those from HCMV in human (Reddehase et al. 2002). Using different strains of mice a susceptibility locus (cmv1) was identified and later shown to encode the Ly49 family of proteins (Webb et al. 2002). There are some 8–18
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members of the family, and most are expressed on NK and NK T cells, in which the majority act as NK inhibitory receptors for MHC I molecules (Parham 2005). Apart from a related gene with a different function, the Ly49 family is absent in human, which uses the KIR family as NK inhibitory receptors (Lanier 1998; Parham 2005). KIR proteins are highly diverged from the Ly49 family and have immunoglobulin rather than C-type lectin domains in their extracellular domain; however, similarly to Ly49, they also recognize MHC class I. Ly49H is an activating receptor implicated in the pathogenesis of MCMV infection and binds an MHC-like protein encoded by MCMV (Arase et al. 2002). The NKG2D receptor appears to play an analogous role in human (Raulet 2003) and is important for control of viral replication. The ligands for mouse and human NKG2D differ: in human, NKG2D binds the polymorphic MHC class I–like molecules MHC class I chain related A antigen (MIC A) and MHC class I chain related B antigen (MIC B) and the UL16 binding protein (ULBP) family, whereas in mouse NKG2D binds to H-60 and Rae1β. NKG2D is more widely expressed in human than in mouse and in human acts also as a T cell costimulator (Maasho et al. 2005). The significance of these differences to cytomegalovirus infection and to NK biology in general have not been determined. There has been considerable interest in generating mouse models of human immunodeficiency virus (HIV) infection. Currently the best model utilizes SCID mice transplanted with human peripheral blood mononuclear cells or fetal liver and fetal thymus. These models have proven to be a necessary complication as mouse macrophages, in contrast to human macrophages, do not express CD4 (Crocker et al. 1987). As a consequence, the infection of macrophages by HIV in the early phase of disease is not easily modeled in mice. Recently, transgenic mice have been generated that express human CD4 and CCR5. The T cells of these mice are infectible with R5-tropic HIV-1 strains, although the virus does not replicate (Unutmaz et al. 1998).
IV.
RESPONSE TO MYCOBACTERIUM TUBERCULOSIS
The CD1 family of molecules along with γ/δ T cells have been implicated in the pathogenesis of tuberculosis (TB) (Flynn and Chan 2001; Kaufmann 2001); however, there are significant differences between mouse and human responses. Mouse epidermis contains a large fraction of cells bearing a Vγ5-Vδ1 TCR, and these are known as dendritic epidermal T cells (DETCs). DETCs represent the predominant T cell in mouse skin, whereas cells bearing α/β receptors predominate in human skin and are found mostly in the dermis. Indeed, a cell with DETC characteristics has not been identified in human (Elbe et al. 1996). The differing expression of CD1 genes between mice and humans may turn out to affect activation of both α/β and γ/δ T cells in TB, as both subsets can recognize a variety of antigens presented by CD1 molecules.
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However, only human γ/δ T cells are thought to recognize antigen presented by CD1 molecules, in particular CD1b in human (Dutronc and Porcelli 2002). Interestingly, of the five CD1 molecules found in human (designated CD1a, b, c, d, and e), only CD1d is expressed in mice (Dutronc and Porcelli 2002). Some other differences in mouse models of M. tuberculosis infection include the limited role of reactive nitrogen intermediates in human and the absence in mice of granulysin, a potent killer of mycobacteria that along with perforin is present in human cytotoxic lymphocyte granules (Flynn and Chan 2001).
V.
RESPONSE TO HELMINTH INFECTIONS
The existence of polarized T cell populations was first demonstrated by Mossman et al. (1986) and since then has become a guiding principle for T cell activation. Although polarization is relatively easy to observe in mice, the paradigm has never been as clear-cut in the human system. For example, in mice IL-10 is considered to be a Th2 cytokine, whereas in human both Th1 and Th2 cells can make IL-10 (Del Prete et al. 1993). In mice T cells make either IL-4 or interferon (IFN)-γ when polarized, whereas it is relatively easy to induce human T cells to make both cytokines in culture. Epidemiological data suggest that a Th2 response involving eosinophils and IgE may be key to combating schistosomiasis infection in humans (Hagan 1993), whereas in mice effector cell activation by IFN-γ, a Th1 response, is essential for clearance of the parasite (Pearce and Sher 1991).
VI.
MULTIPLE SCLEROSIS AND EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS
Demyelinating diseases provide a fine example of both differences and similarities between mouse and human immunology. Multiple sclerosis (MS) is a multifactorial disease that appears to have a large autoimmune component (Pedotti et al. 2003). Experimental autoimmune (allergic) encephalomyelitis (EAE) is a widely used model for MS that mimics the demyelination seen in central and peripheral nerves in MS. Several studies have indicated that IFN-γ is protective in EAE as neutralizing antibodies exacerbate disease, potentially by blocking induction/activation of suppressor activity (Lublin et al. 1993; Heremans et al. 1996). It was surprising, therefore that clinical trials were not successful; indeed they were stopped because treatment with IFN-γ was found to exacerbate disease (Panitch et al. 1987). A similar, but opposite conundrum has arisen with anti-IL-2 receptor (CD25) therapy. A humanized anti-CD25 monoclonal antibody (mAb) was recently shown to reduce by >75% the appearance of new MS lesions and to promote a significant improvement in clinical outcome (Bielekova et al. 2004). In sharp contrast, anti-CD25 treatment of mice with EAE led to
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exacerbation of disease (Kohm et al. 2003), probably because of a decline in CD4+CD25+ T regulatory (Treg) cells (Furtado et al. 2002). Interestingly, only a subset of human Treg cells have the CD4+CD25+ phenotype seen in mice (Mills 2004). A third example of the failure of mouse models to accurately predict the efficacy of MS treatments in humans comes from the tumor necrosis factor (TNF) field. Anti-TNF antibodies have been shown to be highly effective in abrogating autoimmune demyelination in EAE mice (Selmaj et al. 1991); however when transferred to the clinic, neutralizing mAbs to TNF led to enhanced lesions detectable by magnetic resonance imaging and an increase in cerebrospinal fluid leukocyte count and immunoglobulin (Ig) G index (van Oosten et al. 1996). On the plus side, however, studies in mice suggested that blocking of the VLA-4 (very late antigen-4, α4β1 integrin)-VCAM-1 (vascular cell adhesion molecule-1) interaction might help in MS (Yednock et al. 1992), and this blockade has indeed been carried through successfully into human trials (Miller et al. 2003).
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once they reach a draining lymph node and upregulate costimulators such as CD80 and CD86. A teleological argument can be made for the need to present antigen locally in human but not necessarily in mouse. It has been estimated that once a cell enters the lymphatics in human, it takes approximately 24 hours to return to the circulation if it is not retained in a node (Freitas 1999). On the basis of the higher cardiac output of mice as a proportion of their total blood volume compared with humans (5–10 ml/min, 2 ml total volume in mouse; 5 L/min, 5 L total volume in human), it is reasonable to suppose that the return of lymph is at least as fast in mouse as it is in human. Then it becomes a matter of scale. We calculate that an antigen traveling from toe to an inguinal lymph node in the groin should take ~12 hours in human and 20 minutes in mice. As the human DTH response begins ~4 hours after secondary antigen challenge, we propose that triggering of recall responses may occur by different mechanisms in mouse and human, involving draining of antigen to lymph nodes in mice, compared with local antigen presentation, possibly by ECs, in human.
DELAYED-TYPE HYPERSENSITIVITY VIII.
An interesting difference exists in the appearance of delayedtype hypersensitivity (DTH) reactions in mice and human. In human, ~4 hours after antigen challenge neutrophils can be seen forming a “cuff” around the venules. This is followed by a dramatic influx of mononuclear cells, such that by 24–48 hours the lesion is mostly mononuclear with a mix of T cells and macrophages (Dumonde et al. 1982). Paradoxically, in mice in which peripheral blood has a relative paucity of neutrophils compared with human blood (Table 15-1), the DTH response tends to be more neutrophil rich (Crowle 1975). In some cases, however, the response may be neutrophil rich at 24 hours but then evolves to a mononuclear-rich infiltrate by 48 hours. In addition, elicitation of murine DTH requires much higher concentrations of antigen than in human, and lesions cannot be assessed by the degree of redness and induration; rather, DTH in mouse skin is usually measured by swelling of the footpad or pinna of the ear. There is now considerable evidence that human endothelial cells (ECs) can present antigen to resting memory CD4+ and CD8+ T cells (Mestas and Hughes 2001; Murphy et al. 1999; Pober et al. 2001), whereas in mouse CD8+ T cells can be activated by ECs (Kreisel et al. 2002), but CD4+ T cells cannot (Kreisel et al. 2002). As CD4+ T cell–mediated activation of macrophages is thought to drive human DTH responses the suggestion has arisen that EC may be one of the local APCs that trigger the recall, CD4+ T cell–dependent, phase of DTH in human. It is not clear in mice whether the recall phase involves local antigen presentation or whether antigen is carried to the lymph node by Langerhans cells that then present the antigen to T cells in the node. There is good evidence that Langerhans cells express low levels of costimulators and are poor APCs (Geissmann et al. 2002), only becoming fully functional APCs
TRANSPLANTATION
The antigen presenting ability of human ECs may have significant consequences for transplantation. For example, in many rodent models vascularized grafts are tolerizing, whereas such grafts are rapidly rejected in human (Sykes 2001). Numerous studies have shown that purging mouse tissues of CD45+ cells before transplantation dramatically extends the life of the graft, sometimes even inducing tolerance. In sharp contrast, purging human tissues of CD45+ cells provided no benefit as the grafts were still rapidly rejected (Wood 2003). In addition, the establishment of microchimerism in mouse has been quite successful in inducing tolerance, whereas this has not been the case in human (Monaco 2003). The implication of these findings is that there are major differences between mouse and human in their responses to grafted tissue, and that there are cells in human grafts that are CD45– and are able to trigger host T cell activation. Taken together, these studies provide compelling evidence that human ECs can act as APCs to CD4+ T cells whereas mouse ECs cannot.
IX.
SUMMARY
These studies highlight how caution is required when results from mouse studies are extrapolated to the clinic. In many cases not only do successful mouse therapies fail to work in the clinic, they actually have opposite effects in patients, leading to exacerbation of disease. Although the mouse will continue to be an important preclinical model system, it is a dangerous trap to fall into if one believes that what is true in mouse must be true in human.
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ACKNOWLEDGMENTS Because of space limitations, we have in many cases cited reviews rather than the primary literature, and we offer our apologies to those authors we have been unable to acknowledge directly. We thank the following people for helpful suggestions: Jeffrey Bender, Mark Boothby, Alfred Bothwell, Michael Cahalan, David Camerini, Paolo Casali, George Chandy, Cheong Chang, Nick Crispe, Olja Finn, Bruce Freedman, David Fruman, Geoff Kansas, Mitch Kronenberg, Tom Lane, Klaus Ley, Ruslan Medzhitov, Andre Ouellette, Peter Parham, Howard Petrie, Jordan Pober, Bruce Rosengard, Markus Schneemann, Michael Selsted, Carl Ware, Marian Waterman, Arthur Weiss, and Miriam Wittmann. We acknowledge the Journal of Immunology for allowing us to use parts of our previously published review in this chapter. This work was supported by a grant from the National Institutes of Health (RO1 AI40710).
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Index A Activation-induced cytidine deaminase, 95, 156–157, 161–162 Adaptive immune system activation of, 87 cells of, 38–48 description of, 3, 109–110 T cells in, 144–145, 208 Adaptor molecules, 238 Adaptor protein-1, 3 Adhesion and degranulation-promoting adapter protein, 199 Aire, 68 Airway eosinophilia, 294 Airway hyperresponsiveness cytokines in, 293–294 description of, 291–292 eosinophils’ role in, 297 methacholine uses, 293 Airway remodeling, 297 Allergic asthma bronchial epithelium in, 292 cytokines in, 293–294 description of, 291 development of, 292 lung measurements, 292–293 mast cells in, 295 mouse models of, 292–293 summary of, 298 Alymphoid mice, 279 Anaphylatoxins description of, 7 receptors for, 11, 13 Antibody anti-CD11c, 139 classes of, 22–23 complementarity-determining regions, 21 diversity of, 155–157 Fab fragment, 21 H chains, 21 structure of, 21–22 subclasses of, 22–23 Antibody-dependent cellular cytotoxicity, 5, 171 Antibody-forming B cells, 146 Anti-CD3/peptide central tolerance models, 209 Anti-CD11c antibodies, 139 Antigen processing, 18–20 Antigen-antibody complexes, 280 Antigen-binding complex, 24–25
Antigen-presenting cells, 35, 268–269, 309 Antigen-specific cells, 59 Antiintegrin antibodies, 196 AP-1, 234 APECED, 235 ApoB48, 161 Apoptosis death receptors’ role in, 217 mitochondria’s role in, 217–218 regulation of, 253–254 T cell, 236 Athymic nude mice, 277 Autoimmune diseases mouse as model system for studying, 221 natural killer cells and, 173 systemic lupus erythematosus. See Systemic lupus erythematosus Autoimmune regulator, 68, 208, 216 Autoimmunity FCγRIIB and, 264–267 pathogenic, 250–251 T cell–mediated, 234–235
B B cell(s) activation of, 25, 267–268 affinity maturation of, 88 antibody-forming, 146 B-1 cells, 46, 81–82, 86 description of, 7, 43–44 development of, 44–48, 59–60, 307 effector, 91 extrafollicular differentiation of, 92–93 extrinsic signals, 82–83 follicular, 82, 85–86 germinal center differentiation of, 94 selection of, 95–96 homeostasis of, 82–85 intrinsic defects, 61 intrinsic signals, 83–85 maturation of, 45, 282 memory, 91 migration of description of, 47, 76–77 to lymphoid follicles, 78–82 negative selection of, 84–85 ontogeny of, 60–61 plasma cell differentiation from, 91 positive selection of, 84–85
B cell(s) (continued) precursors of, 61 resting splenic, 246 signaling of, 81 signals of, 87 survival of, 82 T cells and, 62, 91–96 tolerance of, 48, 61, 265–266 transitional, 85 turnover of, 82 B cell receptor BAFF and, 85 coreceptors, 24 description of, 23, 43 diversity of, 45 membrane Ig, 83 NF-κB activation via, 84 signaling of, 27–28 B cell stimulating factor-1, 30 B lineage cells, 60 B lymphocyte attenuator, 262 B-1 cells, 46, 81–82, 86 B-2 cells, 45–46 B7-1, 26–28, 268 B7-2, 26–28, 268 B220, 142–143 BAFF B cell receptor and, 85 description of, 82 marginal zone B cells affected by, 86 BAFF-R, 82–83, 85 Bapx1, 76 Basophils, 35 Bat1, 127 Bcl-6, 93, 94 Blimp-1, 93 BLNK, 307 Bone marrow anatomy of, 59–60 cellular organization of, 60–62 dendritic cells from, 140, 148–149 hematopoietic stem cells in, 33 hypertrophy of, 60 natural killer cell development, 37 structure of, 60 thymocytes from, 208 Brd2, 123 Bronchial-associated lymphoid tissue, 69 Btk, 307 BTLA, 269 BXSB.yaa mice, 246–247
313
314
INDEX
C C1, 8 c1q deficiency, 252 C3aR, 11 C3G, 200 C3H/HeJ mice, 110 C4 deficiency, 252 C4A, 124 C4B, 124 C4, 124 C5aR, 11 C5L2, 13 C5 convertase, 8 C57BL/10ScCr mice, 110 Cancer, 283–284 CARMA1, 84 Caspase recruitment domains, 114 C.B-17-scid mice, 278 CCL21, 201 CD1, 20–21, 141 CD3, 24 CD4, 26 CD4+ T cells, 39–40 CD8, 26 CD8+ T cells description of, 39–40 effector, 90 CD11b, 170 CD11b, 33 CD16, 175 CD19, 24, 84 CD21, 94 CD22, 83, 267 CD25, 42, 308–309 CD27, 93 CD35, 94 CD40, 28–29 CD40 ligand, 28–29, 94, 142 CD43, 170 CD45, 84, 254 Cd45, 189 CD49b, 170 CD62L, 90 CD70, 93 CD72, 83 CD74, 19 CD80, 245 CD86, 94, 142 CD91, 141 CD94/NKG2, 174 CD94/NKG2 receptors, 15 CD103, 43 CD122, 170 CD154, 29 CD206, 13 CD244, 175 Central supramolecular activation cluster, 89 Central tolerance models of, 209 T cell–mediated autoimmunity control through, 234–235 Centroblast B cells, 162 Centroblasts, 93
Centrocytes description of, 95 somatic hypermutation of, 94–96 terminal differentiation of, 96 cFLIPL, 95 CH12F3-2, 157 Chemokine(s) dendritic cell migration regulated by, 143–144 description of, 29, 31, 34, 47, 64 in inflammatory responses, 295 in lymphoid organogenesis, 75 natural killer cell production of, 171–172 from radiation-resistant stroma, 93 transcription of, 75 Chemokine receptors description of, 29, 64 signaling of, 202–204 CIITA, 93 Ciliary neurotrophic factor receptor, 41 Cis transgenic mice, 188 c-Kit, 35 Class II-associated invariant chain peptide, 19 Class switch recombination description of, 157 mismatch repair proteins involved in, 164 Clonal deletion, 215, 218 Clonal expansion, 68 Cluster of differentiation, 32 Collectins, 14 Complement protein 3, 8 Complement proteins, 280 Complement receptors characteristics of, 10 CR1, 10–11 CR2, 10–11 CR3, 11 CR4, 11 Complement system activation of, 8 functions of, 8 lectin pathway of, 8 overview of, 8–10 pentraxin pathway of, 8 Complementarity-determining regions, 21 Cortical epithelial cells, 63, 65 Cortical thymic epithelial cells, 208 Costimulatory molecules, 215 CR1, 10–11 CR2, 10–11, 24 Cr2, 11 CR3, 11 CR4, 11 Crry, 280 Cryptopatches, 69 CTLA-4, 237, 268–269 C-type lectin, 36 C-type lectin receptors, 13–14 C-type lectin-like glycoprotein, 175 CXCL13, 75, 78 CXCR5, 78 Cytochrome c, 217
Cytokine(s) in airway hyperresponsiveness, 293–294 description of, 29, 279 in inflammatory responses, 295 natural killer cell production of, 171–172 receptors for, 30–31 suppressors of cytokine signaling, 180–181 Cytokine-activated JAK-STAT pathway, 180–181 Cytokine-like factor-1, 41 Cytomegalovirus human, 307–308 mouse human cytomegalovirus modeled using, 307–308 natural killer cells, 172–173 Cytoplasmic receptors, 114 Cytosolic pathway, 18 Cytotoxic T cell-associated antigen 4, 26, 200 Cytotoxic T lymphocytes, 171, 283
D 33D1, 139 Death receptors, 214, 217 DEC-205, 139 Decay-accelerating factor, 245 Dectin-1, 14 ß-defensins, 32 Delayed-type hypersensitivity, 309 Delta-like 1, 63 Dendritic cells antigen handling by, 141–142 bone marrow, 140, 148–149 CD1 expression, 141 CD86 levels, 142 chemokine effects on, 143–144 definition of, 35 dendrites of, 141 dermal, 140 description of, 3 differentiation of, 36, 142 discovery of, 136 ex vivo studies of, 146–147 follicular, 71, 78, 140 functions of, 144–146 generation of, 139 granulocyte-macrophage colony-stimulating factor system for creation of, 139 in immature state, 142, 144–145 interleukin-12 production and, 144 interstitial, 36 intestinal, 146 isolation protocols for, 147–149 Langerhans cells and, 140 life span of, 141 lymphoid tissue distribution of, 139–140 major histocompatibility complex expression by, 138 in marginal zone, 78 maturation of, 142 migration of, 87, 143–144
315
INDEX
Dendritic cells (continued) monoclonal antibodies, 138 morphological properties of, 138, 141 mouse strains used to study, 136–138 myeloid, 112 natural killer cells and, 172 in nonlymphoid tissue, 140–141 phenotypic changes, 87 physical properties of, 138 plasmacytoid description of, 37, 76–77, 142–143 maturation of, 143 toll-like receptor signaling in, 114 spleen, 146–147 subsets of, 142–143 surface composition of, 138 T cell interactions with, 88–89, 145 in T cell regions, 139–140 in thymus, 139 tolerance mediated by, 145–146 turnover of, 141 Dendritic epidermal T cells, 308 Deoxyuridine, 95 Diacylglycerol, 234 DNA polymerases, 164 DNAX adapter protein-12, 174 DOCK2, 202 Dok-1, 203 Double negative thymocytes, 208 Double-stranded RNA, 112 Drosophila, 64–65
E E3 ubiquitin ligases, 237–238 Effector B cells, 91 Endo 180 receptor, 13 Endocytic pathway, 18 Endocytosis, 19 Endogenous antigen models, 209–213 Enhanced green fluorescent protein, 140 Eosinophil(s) in airway hyperresponsiveness, 297 description of, 33–34 inflammatory role of, 34 Eosinophilia, 294 Eotaxin-1, 296 Eotaxin-2, 296 Epidermal growth factor receptor, 238 Epidermal T cells, 308 Epithelial cells, 6 Epstein-Barr virus, 283 ERK/JNK/p38, 216 Erythropoietin receptor, 182 Experimental autoimmune encephalomyelitis, 308–309 Eya, 65
F Fab fragment, 21 Fas-associated death domain, 217 Fc receptors FCαRI, 8 FCγRI, 7 FCεRI, 7–8, 295
Fc receptors (continued) FCγRIIB autoimmunity and, 264–266 B cell tolerance, 265–266 description of, 6–7, 142, 254 genetic linkage studies, 264–265 mice deficient in, 265 polymorphisms, 265 signaling of, 263 FCγRIII, 7 FCγRIV, 7 homologs, 262 IgG, 263–264 Langerhans cell expression of, 140 neonatal, 6 overview of, 5 poly-Ig receptor, 5–6 transmembrane ligand-binding protein, 5 Fcgr2, 264–265 Fetal-maternal interface, 284 Fibroblast growth factor-7, 66 Fibroblast growth factor-10, 66 FLICE, 95 Follicle-associated epithelium, 69 Follicular dendritic cells B cell recruitment affected by, 93 description of, 71, 78, 140 Forkhead box N1, 277 Foxl1, 74 Foxn1nu, 277 Foxp3, 42 Foxp3+CD4+CD25+ Treg cells, 43 fsh, 123 FTY720, 202
G Gadd45α, 253–254 GATA-1, 34 Gene overexpression, 280 Gene targeting, 238 Genomic segmental polymorphism, 120 Germinal centers architecture of, 93 B cells differentiation of, 94 selection of, 95–96 IgE, 295 involution of, 96 kinetics of, 93 stromal support for, 93–94 Glucocorticoid-inducible tumor necrosis factor receptor, 277 Glycogen synthase kinase 3ß, 67 Glycolipids, 141 Glycosylphosphatidylinositol, 3, 110 Graft-versus-host disease, 283 Grb2/RasGRP, 216 GTPase activating protein, 200 Guanine nucleotide exchange factor, 199, 216
H H2-DM, 20 H2-DO, 20
H2-M4, 129 H2-Q region, 126–127 H2-T region, of Mhc, 127–128 Hassall’s corpuscles, 63 Helminth infections, 308 Helper T cells cytokines secreted by, 91–92 description of, 40–41, 89–90 Hematopoiesis, 60 Hematopoietic stem cells description of, 33, 60 T cell precursor derivation from, 63 Hereditary hemochromatosis, 121 Hoxa3, 64–65 Human ß-defensin-1, 32 Human killer cell immunoglobulin-like receptors, 170
I Immature dendritic cells, 144–145 Immune response, 87–88 Immune system adaptive cells of, 38–48 description of, 3 innate cells of, 32–38 description of, 3 ligands of, 3–15 receptors of, 3–15 Immunoglobulins IgA, 23 IgD, 23 IgE, 23, 294–295 IgG, 22–23, 263–264 IgG2b, 8, 22 IgM, 8, 23 membrane-anchored, 23 structure of, 43–44 Immunoreceptor tyrosine-based activation motifs, 83, 199, 215 Immunoreceptor tyrosine-based inhibitory motif, 6, 262 Immunoreceptor tyrosine-based switch motif, 28 Inducible costimulator, 28 Inducible costimulator ligand, 28 Inflammation eosinophils in, 34 modulation of, 7 Inflammatory responses, 295 Inhibitory receptors B cell activation thresholds and, 267–268 classes of, 262–263 description of, 262 natural killer cell receptors, 268 pathways of, 269–270 T cell, 268–269 Innate immune system activation of, 87 cells of, 32–38, 109 description of, 3
316
INDEX
Innate immune system (continued) ligands of, 3–15 receptors of, 3–15 Integrins clustering of, 196 description of, 196 knockout models, 201 leukocyte, 296–297 signaling, 203–204 T cell receptor-mediated activation defects, 199–201 Intercellular adhesion molecule-1, 81 Interferon-τ, 30–31, 172 Interferon regulatory factor-2, 143 Interferon regulatory factor-5, 114 Interleukin-1, 30 Interleukin-1 receptor-associated kinase, 113 Interleukin-2, 30, 170 Interleukin-2 receptor, 42 Interleukin-3, 30 Interleukin-4, 30, 43, 294 Interleukin-5, 30, 34, 297 Interleukin-6, 30, 93 Interleukin-7 deficiency of, 279 description of, 30, 74 Interleukin-8, 295 Interleukin-10, 30, 236 Interleukin-12, 30, 41, 144, 284, 294 Interleukin-13, 30, 294 Interleukin-15 deficiency of, 279 description of, 30, 173 Interleukin-17, 42 Interleukin-23, 41 Interstitial dendritic cells, 36 Intestinal crypts, 69 Isolated lymphoid follicles, 69 Itk, 200, 203
J J chains, 6 Jak1-deficient mice, 183 Jak2-deficient mice, 183 Jak3, 74 JAK3 tyrosine kinase, 279 Jak3-deficient mice, 183–184 JAK-STAT pathway cytokine-activated, 180–181 regulation of, 181–183 signaling of, 183–189 JAM-A, 201 Januse kinases, 180, 183–184
K Keratinocytes, 32 Killer cell immunoglobulin-like receptors, 262 Knockout mice integrin, 201 T cell migration, 197–198, 202 with T cell receptor-mediated integrin activation defects, 199–201
L Langerhans cells, 139–140, 309 Langerin, 14 Leucine-rich repeat motifs, 3, 110 Leukocyte(s) derivation of, 32–33 description of, 32–33 integrins, 296–297 peripheral blood, 283–284 Leukocyte receptor complex, 174 Lipopolysaccharides description of, 5 strains sensitive to, 110 L-selectin, 77 LTßR, 73 Ly-17, 265 Ly49 receptors, 14–15, 170, 175, 268 Lymph nodes B cell entry and exit from, 76–77 development of, 69–70 peripheral, 69 Lymphocyte(s) B. See B cell(s) lymphoid organ entry of, 77 natural killer cells, 37–38 recruitment of, 75 signaling of, 254–255 T. See T cell(s) Lymphocyte function-associated antigen-1, 176, 196, 233, 296 Lymphohematopoietic cell lineages, 281–283 Lymphoid enhancing factor, 67 Lymphoid follicles B cell migration to, 78–82 description of, 78 Lymphoid organs bone marrow. See Bone marrow location of, 59 molecular regulation of, 70–75 overview of, 59 primary, 59–68 secondary anatomy of, 68 B cell zones in, 78 cryptopatches, 69 dendritic cells, 143 formation of, 79–80 immune response function of, 68 location of, 69 lymphatic drainage to, 76 lymphocyte entry into, 77 microdomains in, 78–82 natural killer cells in, 171 overview of, 68–70 Peyer’s patches, 69 spleen. See Spleen thymus. See Thymus Lymphoid tissue-inducing cells description of, 73 development of, 73–74 LTα1ß2 induction on, 74 recruitment of, 74 Lymphotoxin, 279–280
Lymphotoxin receptor null mutants, 279–280 Lyn, 269
M M1, 128 M5, 129 M6, 129 M10, 128 Macrophage(s) characteristics of, 33 FCγRI expression on, 7 from TLR2-/- mice, 110 in granulomatous processes, 33 Macrophage inflammatory protein, 172 Macrophage inflammatory protein-1, 35 Macrophage inflammatory protein-1α, 296 Macrophage inflammatory protein-2, 14 Macrophage mannose receptor, 13 Macropinocytosis, 142 MAdCAM-1, 77 Major histocompatibility complex antigen processing, 18–20 CD1 genes, 20–21 dendritic cell expression of, 138 description of, 15 molecules class I, 15, 17–18, 308 class II, 17–18, 123 description of, 120 overview of, 119–120 receptors, 170 Marginal zone absence of, in neonatal period, 81 definition of, 78 macrophages, 14, 81 Mast cells in allergic asthma, 295 description of, 35 IgE-activated, 294–295 Maternal fetal interface, 284 mDC-SIGN, 14 Medullary epithelial cells, 63, 65, 67 Membrane attack complex, 8 Membrane-anchored immunoglobulin, 23, 83 Memory B cells, 91 Memory T cells, 90 Mesenchyme, 65 Methacholine, 293 Mhc class I region, 125–126, 130 class II molecules, 123, 130 class III region, 124–125, 130 comparative map of, 121–130 DM, 123 DO, 123 DP, 123 DQ, 123 DR, 123 H2-M region, 128 H2-M4, 129 H2-Q region, 126–127 H2-T region, 127–128
317
INDEX
Mhc (continued) M region, 129–130 M5, 129 M6, 129 non–class II molecules, 123 overview of, 119–120 polymorphism of, 120 regions of, 120 sequencing of, 120–121 ß2-Microglobulin, 15, 17 MINK, 216 Mismatch repair, 162–164 Mitochondria, 217–218 Mitogen-activated protein kinase kinase 6, 113 Mitogen-activated protein kinase pathways, 216 Mixed leukocyte reaction, 138 Monocyte(s), 33 Monocyte chemoattractant protein-1, 296 Mouse cytomegalovirus human cytomegalovirus modeled using, 307–308 natural killer cells, 172–173 Mouse genome project, 120 Mouse mammary tumor virus, 123 MRL.lpr mice, 244–246 mSIGNR2, 13–14 Mucosa-associated lymphoid tissue, 69 Mucosal epithelia, 32 Multiple sclerosis, 308–309 Murine activating receptor-1, 175 Mycobacterium tuberculosis, 308 MyD88, 113–114 Myeloid dendritic cells, 112 Myeloid differentiation factor 88, 3
N Nasal-associated lymphoid tissue, 69 Natural cytotoxicity receptors, 175 Natural killer cell(s) activating receptors, 174–175 activation of, 174–176 autoimmune diseases and, 173 cytokine and chemokine production by, 171–172 cytotoxicity functions of, 171 dendritic cells and, 172 description of, 37–38 development of, 170–171 discovery of, 169–170 function of, 171–174 inhibitory receptors, 174 interleukin-2 and, 171 major histocompatibility-recognizing receptors, 262 morphology of, 171 reproductive functions of, 173–174 summary of, 176 surface markers of, 171 tissue distribution of, 171 tumor immunity applications of, 172 uterine, 173, 284 viral immunity applications of, 172–173
Natural killer cell receptors description of, 14–15, 125 inhibitory, 268 Ly49, 268 Natural killer cell-committed precursors, 170 Natural killer T cells, 43 Negative costimulatory molecules, 237 Negative selection anti-CD3/peptide central tolerance models, 209 cell death mechanisms during, 235 costimulatory molecules in, 215 endogenous antigen models, 209–213 mediators of, 213–218 overview of, 208–209 signal transducers in, 215–216 T cell receptor in, 215 transcription factors in, 216–217 Neonatal Fc receptors, 6 Neutrophil(s) definition of, 33 functions of, 33 NHEJ, 164–165 NKG2D, 15, 173, 175 NKR-P1C, 175 Nod2, 114 NOD-scid mice, 278 Nuclear factor-κB B cell receptor activation of, 84 description of, 72, 216 T cell activation of, 236 Nude mice, athymic, 277 Nur77, 217 NZM2410 strain, 249–250 [NZW × NZB]F1 mice, 247–249
O Organogenesis LTßR ligation during, 73 signaling mutations effect on, 75 Ovalbumin, 237, 292
P p21, 254 Paired Ig-like receptor-B, 267 Pathogen-specific molecular patterns, 110 Pathogenic autoimmunity, 250–251 Pax1, 65 Pax9, 65 PD-1, 237, 267–268 PD-L1, 237 PD-L2, 28 Peptidoglycan, 110 Peripheral blood leukocytes, 283–284 Peripheral tolerance, 146, 266 Peyer’s patches B cell entry and exit from, 76–77 description of, 69 Phagocytes, 10 Phagocytosis, 19 Pheromones, 129 Phosphatases, 174, 238 Phosphatidylinositol 3-kinase, 200–203
Phosphoinositide 3-kinases, 234 Phospholipase C-τ, 233 Plasma cells, 91 Plasmablasts, 92–93 Plasmacytoid dendritic cells description of, 37, 76–77, 142–143 maturation of, 143 toll-like receptor signaling in, 114 Plethysmography, 292 Point mutagenesis, 280–281 Poly-Ig receptor, 5–6 Polyubiquitination, 237 Positive selection, 208–209 Pou5fl, 127 Prkdcscid, 278 Programmed death-1, 28 Protein inhibitor of activated STATs description of, 180–182 in vivo functions of, 188 Pias1-deficient mice, 188 Piasx-deficient mice, 189 Piasy-deficient mice, 188–189 Protein kinase Cd, 254 Protein tyrosine kinase, 254 Protein tyrosine phosphatases description of, 180, 189 in vivo functions of, 189 JAK-STAT pathway regulation by, 182–183, 189 Proteoglycans, 35 P-selectin glycoprotein ligand 1, 296 Ptp1b, 189 PU.1, 34
Q Qa2, 126
R Rac GTPases, 202 RAG-1, 48 Rag1, 278–279 rag-1/2, 61 RAG-2, 48 Rag2, 278–279 RAG/τc, 279 Rap1, 199–200 RapL, 199–200, 203 Rasgrp1, 255 Receptor activator of NF-κB ligand, 71 Receptor editing, 48 Receptor recombinase, 38 Recombination signal sequences, 38, 44 Regulatory T cells, 42 Replication protein A, 161 Reproduction, natural killer cells’ role in, 173–174 Respiratory system, 292 Retinoic acid inducible gene-I, 114 Retinoic acid–early inducible-1, 173 Ring3, 123 Roquin, 237–238 RT1-N, 128 Runt-related transcription factor 1, 268
318
INDEX
S S1P1, 202 Salmonella typhimurium, 112 Selectins, 296 Self-tolerance, 42–43 Serum amyloid P, 252 Severe combined immune deficiency description of, 183 mouse models, 278, 307 SH2-containing protein tyrosine phosphatase, 83 SHIP, 263–264, 270 Short consensus repeat units, 10 SHP-1, 263, 269–270 Shp1, 189 SHP-2, 28, 263 Shp2, 189 Six1, 65 SKAP-55, 199 Skin barrier function of, 32 cornification of, 32 functions of, 32 Sle1, 249–250 Sle2, 251 Sle3/5, 250–251 Slp, 124 SLP-76, 199 Socs5 transgenic mice, 188 Somatic hypermutation cytidine deamination in, 161 description of, 94–96, 156–157 mismatch repair proteins in, 164 proteins involved in, 158–160 SP-A, 14 SPA-1, 200 SP-D, 14 Specific intracellular adhesion molecule-3 grabbing non-integrin family, 13 Spi-B, 95 Spleen anatomy of, 77 dendritic cells, 147–148 description of, 69 development of, 75–76 red pulp of, 77 white pulp of, 77 STATs description of, 180–181 in vivo functions of, 185 Stat1-deficient mice, 184 Stat1S727A-mutant mice, 184 Stat2-deficient mice, 184 Stat3-deficient mice, 184, 186 Stat3S727A-mutant mice, 184, 186 Stat4-deficient mice, 186 Stat5a- and Stat5b-deficient mice, 186 Stat6-deficient mice, 186 Stem cell factor, 170 Stem cells, hematopoietic, 33 Sts-1, 238 Sts-2, 238 Superantigens, 213
Suppressors of cytokine signaling proteins Cis transgenic mice, 188 description of, 180–181 in vivo functions of, 187 Socs1-deficient mice, 186–187 Socs2-deficient mice, 188 Socs3-deficient mice, 187–188 Socs5 transgenic mice, 188 Surfactant proteins, 14 Syk kinases, 23 Syndecan-1, 93 Systemic lupus erythematosus antigen clearance, 252–253 description of, 251–254 immune complex clearance, 252–253 murine models of BXSB.yaa, 246–247 description of, 244 MRL.lpr, 244–246 NZM2410, 249–250 [NZW × NZB]F1, 247–249 summary of, 255–256 overview of, 243–244
T T cell(s) γδ, 39 activation of adaptor molecules, 238 antigen recognition and, 221, 233 description of, 88–90 E3 ubiquitin ligases, 237–238 humoral factors in, 236 negative costimulatory molecules, 237 negative regulation of, 236–239 phosphatases, 238 processes involved in, 233–234 adaptive immunity, 144–145, 208 anergic, 237 antigen receptor binding of, 224 antigen-specific, 68, 145 apoptosis of, 236 autoimmunity mediated by, 234–235 B cells and, 62, 91–96 CD4+, 39–40 CD8+, 39–40 dendritic cells and, 88–89, 145 dendritic epidermal, 308 development of, 38–39, 62, 282, 307 differentiation of, 88–90 effector, 201, 236 epidermal, 308 homeostasis of, 236 integrin expression on, 196 knockout models, 197–198, 202, 204 memory, 90 migration of description of, 47, 201–202 knockout models, 197–198, 202, 204 transgenic models, 197–198, 204 natural killer, 43 nuclear factor-κB activation in, 236 phenotypic differentiation of, 40–41
T cell(s) (continued) precursors of, 63 priming of, 89 regulatory, 42, 235, 277 response of, 145 self-reactive, 42 self-tolerance, 42–43 Th cells cytokines secreted by, 91–92 description of, 40–41, 89–90, 145 thymic, 39, 62 tolerance of, 235–236 transgenic models, 197–198, 204 T cell adaptor molecule, 255 T cell coreceptors, 25–26 T cell inhibitory receptors, 268–269 T cell receptor αß, 38 γδ rearrangement, 39 antigen-binding complex, 24–25 in clonal deletion, 215 description of, 24–25, 196 negative selection mediated by, 215 signal transduction, 234 signal-transducing complex, 25 signaling, 196–201 stimulation of, 235–236 transgenics, 209–212 V-D-J rearrangement, 38, 61 T cell specific adaptor, 238 T cell zone, 82 T1 cells, 85 T2 cells, 85 Tap 1/2, 123 TAPA, 24 Tapasin, 18, 125 Tbsp, 123 Tcptp, 189 Terminal deoxynucleotidyl transferase dUTP nick-end labeling cells, 213 TFH cells, 90 Th cells, 40–41, 89–90, 145, 291 Th17 cells, 41–42 Thymic epithelial cells cortical, 208 description of, 62, 67 origin of, 65 Thymic stromal lymphopoietin, 145 Thymocytes bone marrow, 208 description of, 64 double negative, 208 T cell-epithelium interactions during selection of, 67–68 from transgenic mice, 200 Thymus anatomy of, 62 cortical epithelial cells of, 63, 65 corticomedullary junction of, 63 dendritic cells in, 139 development of, 64–67, 208 epithelium of, 63 medullary epithelial cells of, 63, 65 mesenchyme of, 65
319
INDEX
Thymus (continued) structure of, 62–63 T cell development in, 62–64 Thymus-independent antigen, 270 Tissue-specific antigens, 216 TLR4, 110 Tnsf1a, 279 Tolerance B cell, 48, 61 dendritic cell mediation of, 145–146 peripheral, 146, 266 T cell, 42–43, 235–236 Toll/interleukin-1 receptor homology domain, 3, 110 Toll-like receptors characteristics of, 3 definition of, 110 description of, 110 innate responses via, 144 signaling pathway for, 113–114 TLR1, 3, 110–111 TLR2, 3, 110–111 TLR3, 5, 112 TLR4, 5, 110 TLR5, 5, 112 TLR6, 5, 110–111 TLR7, 5, 112, 114 TLR8, 5, 112 TLR9, 5, 112, 114 TLR10, 5
Toll-like receptors (continued) TLR11, 5, 112–113 types of, 3–5 Toxoplasma gondii, 112 Transcription factors, 216–217 Transferrin receptor, 8 Transforming growth factor-ß, 31, 236 Transgenic mice Cis, 188 cytokine expression, 293 Socs5, 188 suppressors of cytokine signaling, 186–187 T cell migration, 197–198 thymocytes from, 200 Transgenics T cell receptor, 209–212 Vß, 209, 213 Transplantation, 309 Transporter associated with antigen processing proteins, 18–19 Treg cells, 42, 235 TRIF-dependent pathway, 114 Tumor necrosis factor-α, 31 Tumor necrosis factor-ß, 31 Tumor necrosis factor immediate family, 75 Tumor necrosis factor receptors, 70–71, 217 Tumor necrosis factor-receptor superfamily description of, 70–71 signaling events, 71–72
Tumor necrosis factor-related apoptosis–inducing ligand, 171, 217 Tyk2-deficient mice, 184
U UL-16 binding protein–like transcript-1, 173 Uracil N-glycosylase, 161 Uterine natural killer cells, 173, 284
V Vß transgenics, 209, 213 Vascular cell adhesion molecule-1, 81, 296 Vav1, 199 V-D-J rearrangement, 38, 61, 155–156
W Wnt glycoproteins, 67
X Xiap, 280 X-linked immunodeficiency, 307
Z ZAP-70, 199, 234, 307
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Cumulative Index A A strain development, 2:636–637 nonneoplastic conditions, 2:637 pathogen susceptibility, 2:638 phenotype, 2:637 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:637–638 AA4.1 antigen, 3:160 AAV. See Adeno-associated virus ABC transporters. See ATP-binding cassette transporters Abdomen anatomy of, 3:2 necropsy evaluations, 3:482 Abducens nerve, 3:7 Abductor digiti longus, 3:5 Abnormal behavior, 3:517–518 Absolute decreased red cell mass, 3:147 Absolute differential white blood cell count, 3:141 Absolute reticulocyte width, 3:141 Accelerated erythropoiesis, 3:161, 3:163 Accessorius nerve, 3:7 Accessory glands, 3:96 Acetylcholinesterase description of, 3:53 megakaryocyte precursors and, 3:154 Acetyl-coenzyme A carboxylase-beta, 1:313 Acidification, of drinking water, 3:418 Acinar cells, 3:64 Acini, 3:51 Actinobacillus muris. See Pasteurellaceae Activated partial thromboplastin time, 3:141, 3:157 Activation-induced cytidine deaminase, 4:95, 4:156–157, 4:161–162 Acyl CoA:cholesterol acyltransferase, 3:187 Adaptive immune system activation of, 4:87 cells of, 4:38–48 description of, 4:3, 4:109–110 T cells in, 4:144–145, 4:208 Adaptor molecules, 4:238
Adaptor protein-1, 4:3 Adductor, 3:6–7 Adeno-associated virus, gene transfer vectors, 1:272 Adenosine monophosphate-activated protein kinase, 3:183 Adenosine triphosphate-binding cassette transporter A1, 3:186, 3:549 Adenovirus description of, 3:391 gene transfer vectors, 1:271–272 genome and structure, 1:271–272 vaccine vector development, 1:275–276 Adhesion, of embryo, 3:102 Adhesion and degranulation-promoting adapter protein, 4:199 Adip2, 3:624 Adipocytes, 3:184 Adipogenesis inhibitory factor, 3:192 Adipokines description of, 3:184 measurement of, 3:185 Adiponectin functions of, 3:618 insulin and, 3:184 Adiposity, 3:128 Adiposity index, 3:627 Adrenal demedullation, 3:466 Adrenalectomy, 3:466 Adrenalitis, murine cytomegalovirus myocarditis model, 2:24 Adrenocorticotrophic hormone, 3:201 Advanced intercross lines breeding, 1:71 gene mapping, 1:128 nomenclature, 1:85, 1:87 Advanced recombinant inbred lines, gene mapping, 1:128 Affinity chromatography, 3:740 A/Fg mice, 3:180 Aflatoxins, 3:346–348 Afp, 3:208 Age-related pathology. See also specific organisms amyloidosis, 2:632–633 hematopoietic neoplasms, 2:633 Aging accelerated, 3:651–653 antagonistic pleiotropy theory of, 3:650 biomarkers of chronological age and, 3:654
Aging (continued) description of, 3:668 end stage, 3:653, 3:655–658 life span, 3:652–653 nonlethal, 3:654–655 pathology data and, 3:657–658 purpose of, 3:658 rate, 3:653–655 selection criteria, 3:654 state, 3:653–655 summary of, 3:658 uses of, 3:653 classification of, in mice, 3:644 definition of, 3:640 disease models of, 3:652–653 endocrine theory of, 3:664–665 evolutionary theory of, 3:650 free radical theory of, 3:661 hematopoietic stem cells, 3:666 metabolic theory of, 3:665 senescence vs., 3:640, 3:642–643 stem cell theory of, 3:665–666 Aging clocks detection of, 3:662–663 evidence of, 3:661 number of, 3:661–662 operational definition of, 3:662 research, 3:664 in segregating populations, 3:663 Aging mice erythrocytes in, 3:161 hematology of, 3:161–162 leukocytes in, 3:161–162 lymphocytes in, 3:162 vitamin requirements in, 3:368 Aging research advances in, 3:639 availability of aged mice, 3:647–648 mouse models for description of, 3:639 F1 hybrids, 3:646–648 4-way cross stocks, 3:647, 3:649 heterogeneous lines, 3:648 inbred strains, 3:645–646, 3:648 outbred stock, 3:648 overview of, 3:639 terminology associated with, 3:639–644 AgNOR staining, ribosomal DNA, 1:150 Agouti mouse, 3:620 Agouti-related peptide, 3:184, 3:620 Agriculture, house mice impact, 1:29–30
321
322 Agtpbplpcd, 3:678 A/He mice, 3:260 Ahr, 3:579 AI. See Artificial insemination Aibl, 3:624 AILs. See Advanced intercross lines Air class 100, 3:299 quality of, in ventilated cages, 3:295 Air exchange rate, 3:412 Air monitoring, 3:413 Aire, 4:68 Airway eosinophilia, 4:294 Airway hyperresponsiveness cytokines in, 4:293–294 description of, 3:59–60, 4:291–292 eosinophils’ role in, 4:297 methacholine uses, 4:293 Airway pressure, 3:56 Airway pressure time index, 3:56 Airway reactivity, 3:63 Airway remodeling, 4:297 Airway resistance, 3:63 Airway responsiveness, 3:59 Airway segments, 3:55 AKR mice cholesterol studies in, 3:65 development, 2:638 life span of, 3:651 phenotype, 2:638 production index for, 3:260 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:639 nonneoplastic conditions, 2:638–639 pathogen susceptibility, 2:639 related strains, 2:639 AKR/J mice diet for, 3:107 protein requirements, 3:332 Akt, prostate cancer signaling, 2:599–600 Alanine aminotransferase description of, 3:198 reference range for, 3:181 Alb1, 3:208 Albendazole, Giardia muris management, 2:521 Albumin description of, 3:206 reference range for, 3:181 Alcohol, 3:426 Aldolase, 3:200 Aldrin, 3:346 Alkaline phosphatase description of, 3:198 intestinal, 3:198 reference range for, 3:181 tissue nonspecific, 3:198 Allantoin, 3:73
CUMULATIVE
Alleles diversity of, in wild-derived mice, 3:650–651 inheritance modes, 1:57–59 naming, 1:91 overview, 1:56–57 in random bred colonies, 3:267 Allergic asthma bronchial epithelium in, 4:292 cytokines in, 4:293–294 description of, 4:291 development of, 4:292 lung measurements, 4:292–293 mast cells in, 4:295 mouse models of, 4:292–293 summary of, 4:298 Allometric scaling, 3:67 Alopecia, C3H mice, 2:644 Alpha-1-fetoprotein, 3:208 Altered Schaedler’s flora, 3:228–229 Alternative splicing. See Splice variants Alveolar duct, 3:51 Alveolar fluid transport, 3:61 Alveolar pressure, 3:52 Alymphoid mice, 4:279 Alzheimer’s disease, 3:683–685 Ambient temperature metabolism affected by, 3:68 water consumption affected by, 3:76 Amino acids arginine, 3:333 autoclaving effects on, 3:362 description of, 3:331–332 requirements for, 3:332–333 in urine, 3:72 4-Aminoantipyrine, 3:187 4-Amino-5-imidazolecarboxamide, 3:73 Ammonia, 3:293 Amnion, 1:190 Amoxicillin Clostridium perfringens infection management, 2:357 Helicobacter infection management, 2:428–429 Ampicillin Clostridium perfringens management, 2:357 Corynebacterium bovis management, 2:401 Ampulla coli, 3:15 Ampullary glands, 3:96 Amy-1, 3:200 Amy-2, 3:200 Amylase, 3:66, 3:200 Amylin, 3:183 Amyloid precursor protein, 3:683–684 Amyloidosis Balb/cJ mice, 2:641 C57BL mice, 2:649 definition, 2:672–673 features, 2:692–694 SJL/J mice, 2:654 A strain mice, 2:637 Swiss mice, 2:658–659 Amyotrophic lateral sclerosis, 3:678–679
INDEX
Anabolic phase, of estrous cycle, 3:100 Anagen phase, of hair growth, 3:706–707 Analgesics, 3:480–481 Analytes age effects on, 3:180 cyclic biorhythm effects on, 3:180 dietary effects on, 3:180 ELISA kits, 3:177–178 historical methods of measuring, 3:175 immunologic methods to detect, 3:175–176 in insulin resistance, 3:185 lipid metabolism, 3:188 Anaphylatoxins description of, 4:7 receptors for, 4:11, 4:13 Anatomy body regions, 3:2–3 cupula diaphragmatis, 3:14 digestive tract, 3:15 female genital organs, 3:20–21 heart, 3:8–11 kidneys, 3:17 liver, 3:16 lungs, 3:10–11 lymph nodes, 3:21–22 male genital organs, 3:18–19 mammary gland, 3:3 skeleton, 3:4 stomach, 3:16 thoracic cavity, 3:12 Androgen(s) definition of, 3:130 erythropoiesis stimulated by, 3:161 in estrogen production, 3:130 functions of, 3:202 testosterone. See Testosterone Androgen receptor, 3:130 Anemia of chronic disease, 3:148 Anesthesia cardiovascular effects of, 3:30–31 care after, 3:479–480 cryoanesthesia, 3:464–465 for echocardiography, 3:48 for ear tagging, 3:441 hypothermic, 3:464–465 for imaging, 3:492 inhalants, 3:463–464 injectable anesthetics, 3:461–463 ketamine combinations for, 3:462 local anesthetics, 3:461 methods of, 3:179 neuroleptanesthetics, 3:462 for orbital sinus bleeding, 3:455 pentobarbital, 3:462–463 postanesthetic care, 3:479–480 preanesthetic medications, 3:460–461 side effects of, 3:31 tribromoethanol, 3:461–462 Anesthesia machine, 3:464 Angelman syndrome, 3:568 Angiogenesis in cancer, 3:601 corneal growth factor-stimulated, 3:611 ocular, 3:599–601
CUMULATIVE
323
INDEX
Angiogenic inhibitors, 3:611 Angioplasty, 3:554 Angiopoietin-1, 3:599 Angiopoietin-2, 3:599 Angiotensin I, 3:70 Angiotensin II, 3:70–71 Angiotensin type 1 receptors, 3:38 Angiotensinogen, 3:70–71 Animal bite, management, 2:735 Animal caretakers, 3:387 Animal husbandry, 2:626, 3:660–661 Anisocytosis prevalence of, 3:143 red cell distribution width, 3:143 Anonymous DNA segments, nomenclature, 1:89–90 Ansa subclavia dextra et sinistra, 3:10 Anserine, 3:180 Antagonistic pleiotropy, 3:650 Anterior segment dysgenesis definition of, 3:597 genes involved in, 3:603 histologic findings, 3:604 Anterior vena cava, 3:27 Anthelmintics, in drinking water, 3:309 Antibacterial agents, 3:228 Antibiotic therapy. See specific antibiotics Antibiotic-infused drinking water, 3:309 Antibody anti-CD11c, 4:139 classes of, 4:22–23 complementarity-determining regions, 4:21 diversity of, 4:155–157 Fab fragment, 4:21 H chains, 4:21 mouse mouse hepatitis virus diagnostics, 2:161 mousepox virus prevention, 2:89 Sendai virus prevention, 2:298 structure of, 4:21–22 subclasses of, 4:22–23 Antibody-dependent cellular cytotoxicity, 4:5, 4:171 Antibody-forming B cells, 4:146 Antibody-production tests, 3:746–747 Anti-CD11c antibodies, 4:139 Anti-CD3/peptide central tolerance models, 4:209 Anticonvulsants, 3:566 Antidiuretic hormone, 3:70, 3:77 Antiepileptic drugs, 3:566, 3:571, 3:582, 3:586 Antigen processing, 4:18–20 Antigen sensitization, 3:62 Antigen-antibody complexes, 4:280 Antigen-binding complex, 4:24–25 Antigenic assays, 3:190–191 Antigen-presenting cells, 4:35, 4:268–269, 4:309 Antigen-specific cells, 4:59 Antiintegrin antibodies, 4:196 Antioxidants, 3:550–551 Antisepsis, 3:424
Antithrombin II, 3:553 Anucleate erythroid cells, 3:159 Anus, 3:3, 3:20 Anxiety, 3:517 Aorta abdominalis et vena cava caudalis, 3:14, 3:20 Aorta descendens, 3:18 Aorta-gonad-mesonephros, 3:160 Aortic aneurysm, 3:553 Aortic valve, 3:27 Aorticus lymph node, 3:22 AP-1, 4:234 Apafl, 3:675 APECED, 4:235 Apex ceci, 3:15 Apicomplexa, general features, 2:528–529 ApoB48, 4:161 Apocrine glands, 3:713 Apolipoproteins ApoA-I, 3:186 ApoA-II, 3:186 ApoA-IV, 3:186 ApoB-48, 3:186 ApoB100, 3:185–186 ApoC-I, 3:186 ApoC-II, 3:186 ApoC-III, 3:186 ApoC-IV, 3:186 ApoE, 1:309, 3:186, 3:538, 3:556, 3:683–684 definition of, 3:185 measurement of, 3:188 types of, 3:186 Apoptosis death receptors’ role in, 4:217 defects in, 3:674–675 dermal cells, 3:709 description of, 3:142 mitochondria’s role in, 4:217–218 mouse polyoma virus anti-apoptotic responses, 2:111–112 regulation of, 4:253–254 in retinal degeneration, 3:607 T cell, 4:236 Apposition, 3:102 Aquaporins, 3:61 Ar, 3:130 Arachidonic acid metabolites, 3:196–198 Arcus aortae, 3:9–11 Arcus costalis, 3:2 Arginine, 3:333, 3:370 Arginine vasopressin. See Vasopressin Aromatase, 3:130 Arsenic, 3:336, 3:345 Arteria axillaris dextra, 3:10–11 Arteria axillaris sinistra, 3:11 Arteria carotis communis dextra, 3:9–10 Arteria carotis communis sinistra, 3:9–11 Arteria cervicalis superficialis dextra, 3:9, 3:11 Arteria cervicalis superficialis dextra et sinistra, 3:10 Arteria cervicalis superficialis sinistra, 3:9 Arteria ductus deferentis sinistra, 3:19
Arteria et vena circumflexa ilium dextra, 3:17 Arteria et vena circumflexa ilium sinistra, 3:17 Arteria et vena ovarica dextra, 3:17 Arteria et vena ovarica sinistra, 3:17 Arteria et vena renalis dextra, 3:17 Arteria et vena renalis sinistra, 3:17 Arteria intercostalis suprema, 3:9 Arteria ovarica sinistra, 3:21 Arteria pulmonalis dextra et sinistra, 3:10–11 Arteria subclavia dextra, 3:9–10 Arteria subclavia sinistra, 3:9–10 Arteria testicularis sinistra, 3:19 Arteria thoracica interna dextra, 3:9, 3:11 Arteria thoracica interna sinistra, 3:9, 3:11 Arteria vertebralis dextra, 3:9–10 Arteria vertebralis sinistra, 3:9–11 Arteriae mesentericae caudales, 3:17 Arterial blood collection, 3:457–458 Arterial catheterization, 3:475 Arteritis features, 2:699 strain 129 mice, 2:635 Arthritis, 3:191 Artificial insemination, 1:212, 3:227 Arytenoids, 3:50 AS spermatogonia, 3:96 Ascorbic acid, 3:370 Aseptic hysterotomy, for germfree mice derivation, 3:226 Aseptic technique, 3:465–466 Ashing process, 3:336 Asp1, 3:579–580 Aspartate, 3:584 Aspartate aminotransferase description of, 3:198–199 reference range for, 3:181 Aspergillus spp., 3:348, 3:423 Aspiculuris spp. A. tetraptera diagnosis, 2:556 differentiation from Syphacia obvelata, 2:553 life cycle, 2:556 morphology, 2:555 research-confounding effects, 2:556 treatment, 2:556–559 description of, 3:398 Assays antigenic, 3:190–191 bioassays, 3:751 functional, 3:190 immunoassays. See Immunoassays Limulus amebocyte lysate, 3:742 mouse antibody production, 3:400 Assisted reproduction. See also Reproduction artificial insemination, 1:212, 3:227 in vitro fertilization principles, 1:212–213 rederivation, 1:220–221 intracytoplasmic sperm injection principles, 1:213–214
324 Assisted reproduction (continued) somatic nuclear transfer, 1:221–222 steps, 1:214 transgenic mouse preparation, 1:216 parthenogenesis, 1:214 Associated animal, 3:218 Association for Gnotobiotics, 3:232 Association of Official Analytical Chemists, 3:324 Asthma characteristics of, 3:61–62 murine models of, 3:61–63 Astrocytes, 3:599 Ataxia Friedreich’s, 3:682 spinocerebellar, 3:680–681 Ataxia-telangiectasia, 3:679–680 Ataxin-7, 3:681 Atherosclerosis accelerated, 3:539 analytes associated with, 3:188 aortic, 3:541 characteristics of, 3:186 cholesterol levels and, 3:537 clinical features of, 3:537 description of, 3:544 disorders associated with, 3:186 lesion quantification, 3:541–542 magnetic resonance imaging evaluations, 3:556 modifiers of antioxidants, 3:550–551 cell adhesion molecules, 3:545–546 chemokines, 3:545 cytokines, 3:545 estrogen replacement therapy, 3:551–552 foam cells, 3:546 genetic, 3:544–550 growth factors, 3:545 infectious agents, 3:552 inflammatory mediators, 3:545 matrix remodeling, 3:546 nongenetic, 3:550–552 mouse models of accelerated, 3:539 aortic aneurysm, 3:553 apoE-/-, 3:544 cardiovascular disease induction in, 3:553–554 characteristics of, 3:539 description of, 3:537–538 genetic modification, 3:538–540 heart failure, 3:553 LDLR-/-, 3:544 linked genes, 3:549–550 myocardial infarction, 3:553–554 restenosis, 3:554 plaque bone marrow transfer studies of, 3:549 characteristics of, 3:540–543 development of, 3:548 matrix metalloproteinase effects on, 3:548
CUMULATIVE
Atherosclerosis, murine cytomegalovirus myocarditis model, 2:24 Athymic nude mice, 4:277 Atipamezole, 3:461 ATP-binding cassette transporters, 1:310 Atresia, of follicles, 3:99 Atrial natriuretic factor promoter, 3:38 Atrioventricular valves, 3:27 Atropine, 3:461 Attenuation, 3:495–496 Auchense hairs, 3:696 Audiogenic seizures definition of, 3:578 description of, 3:292, 3:415 in epilepsy prone mice, 3:580–581 experimental epilepsy model use of, 3:578–581 Frings mice, 3:571 genetic studies of, 3:580 interstrain variability in, 3:578–579 long-term potentiation and, 3:579 monogenic, 3:571–573 neurochemical findings, 3:579–580 neuropathologic findings, 3:579 serotonin levels and, 3:580 sudden unexpected death in epilepsy and, 3:579 susceptibility to, 3:578–579 Auricula dexter, 3:9, 3:11 Auricula sinister, 3:9, 3:11 Autoantibodies, 3:191 Autoclaves data loggers used with, 3:421 decontamination uses of, 3:303 description of, 3:225 ethylene oxide used in, 3:422 gnotobiotic uses of, 3:225 maintenance of, 3:421 noise caused by, 3:415 placement of, 3:415 precautions when working with, 3:421 pressurization of, 3:421 Autoclaving of automatic watering systems, 3:420 of diet description of, 3:225, 3:304–305, 3:362 irradiation of diet vs., 3:366 moist heat, 3:362 nutrients affected by, 3:362–3:363 physical changes caused by, 3:363 toxicological risks with, 3:363 Autocrine hormones, 3:124 Autoimmune diseases description of, 3:191 mouse as model system for studying, 4:221 natural killer cells and, 4:173 systemic lupus erythematosus. See Systemic lupus erythematosus Autoimmune regulator, 4:68, 4:208, 4:216 Autoimmunity FCγRIIB and, 4:264–267 pathogenic, 4:250–251 T cell–mediated, 4:234–235
INDEX
Automated hematologic analyzers, 3:140, 3:142 Automatic watering systems chlorine added to, 3:308–309 cleaning of, 3:420 description of, 3:241, 3:285–286, 3:419–420 flush systems, 3:419–420 pressure in, 3:420 recirculating systems, 3:419–420 steam autoclaving of, 3:420 Avertin, 3:30–31 Axenic animal, 3:218 Axillaris accessorius lymph nodes, 3:21–22 Axillaris proprius lymph nodes, 3:21–22 Azaperone, 3:461 Azotemia, 3:207
B B7-1, 4:26–28, 4:268 B7-2, 4:26–28, 4:268 B220, 4:142–143 B cell(s) activation of, 4:25, 4:267–268 affinity maturation of, 4:88 antibody-forming, 4:146 B-1 cells, 4:46, 4:81–82, 4:86 description of, 3:162, 4:7, 4:43–44 development of, 4:44–48, 4:59–60, 4:307 effector, 4:91 extrafollicular differentiation of, 4:92–93 extrinsic signals, 4:82–83 follicular, 4:82, 4:85–86 germinal center differentiation of, 4:94 selection of, 4:95–96 germinal center differentiation, 4:94 homeostasis of, 4:82–85 intrinsic defects, 4:61 intrinsic signals, 4:83–85 lactate dehydrogenase-elevating virus response, 2:224 lymphocytic choriomeningitis virus persistence role, 2:200–201 maturation of, 4:45, 4:282 memory, 4:91 migration of description of, 4:47, 4:76–77 to lymphoid follicles, 4:78–82 mouse adenovirus type 1 response, 2:60 mouse hepatitis virus response, 2:155, 2:157 mousepox response, 2:82 murine cytomegalovirus immune response, 2:28–29 myocarditis role, 2:23 negative selection of, 4:84–85 ontogeny of, 4:60–61 plasma cell differentiation from, 4:91 positive selection of, 4:84–85 precursors of, 4:61 resting splenic, 4:246 Sendai virus response, 2:296 signaling of, 4:81
CUMULATIVE
325
INDEX
B cell(s) (continued) signals of, 4:87 survival of, 4:82 T cells and, 4:62, 4:91–96 tolerance of, 4:48, 4:61, 4:265–266 transitional, 4:85 turnover of, 4:82 B cell receptor BAFF and, 4:85 coreceptors, 4:24 description of, 4:23, 4:43 diversity of, 4:45 membrane Ig, 4:83 NF-kB activation via, 4:84 signaling of, 4:27–28 B cell stimulating factor-1, 4:30 B-1 cells, 4:46, 4:81–82, 4:86 B-2 cells, 4:45–46 B lineage cells, 4:60 B lymphocyte attenuator, 4:262 B6 mice, 3:53 B6;129 mouse phenotype, 2:655 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:655 nonneoplastic conditions, 2:655 BAC. See Bacterial artificial chromosome Bacillus thetaiotaomicron, 3:228 Bacitracin Clostridium difficile infection management, 2:360 Clostridium perfringens infection management, 2:357 Back, 3:2 Backward migration, 3:257 Bacteria. See also specific bacteria antibacterial agents for, 3:228 feces examination to identify, 3:230 in gastrointestinal tract, 3:227 irradiation resistance by, 3:364 microbiological quality control testing for, 3:742–743 thermal death of, 3:310 urease positive, 3:230 Bacterial artificial chromosome clones, 3:623–624 genome sequencing, 1:100–101, 1:107 BAFF B cell receptor and, 4:85 description of, 4:82 marginal zone B cells affected by, 4:86 BAFF-R, 4:82–83, 4:85 Bait stations, 3:429 BALB/c mice blood volume in, 3:138 erythrocyte life span in, 3:142 isoflurane anesthesia in, 3:463 phenotype, 2:639–640 production index for, 3:260 spontaneous diseases
BALB/c mice (continued) comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:641–642 nonneoplastic conditions, 2:640–641 pathogen susceptibility, 2:642–643 related strains, 2:643 urea nitrogen levels in, 3:207 BALB/cBY mice description of, 3:181, 3:666 phenotype, 2:639–640 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:641–642 nonneoplastic conditions, 2:640–641 pathogen susceptibility, 2:642–643 related strains, 2:643 Band neutrophils, 3:153 Bapx1, 4:76 Barbering, 3:388 Basal cells, 3:50, 3:712 Basal metabolic rate description of, 3:67 nonshivering thermogenesis and, 3:68 Basement membrane, 3:72 Basophils, 3:150, 4:35 Bat1, 4:127 Batch washers, 3:302, 3:310, 3:417 Bax, 3:675 B-cell attracting chemokine-1, 3:195 B6C3F1 mouse phenotype, 2:655–656 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:656–657 nonneoplastic conditions, 2:656 pathogen susceptibility, 2:657 Bcl-6, 4:93–94 Bcl-2, 3:675 Bcl-xL, 3:142 BDNF. See Brain-derived neurotrophic factor Bdnf, 3:708 Bedding. See also Nests ammonia production, 3:293 autoclaving of, 3:421 breeding colony, 3:240–241 cellulose products as, 3:306 certified, 3:306 contact, 3:240 contaminated, 3:280 corncob as, 3:306 disposal of, 3:280 hardwood, 3:306
Bedding (continued) materials used for, 3:306 microbiological status of, 3:423 selection of, 3:305–306 soiled, 3:277, 3:285, 3:401 storage of, 3:423 Bedding dispensers, 3:285 Bedding sentinel animals, 3:395 Bedding transfer, 3:394 Behavior abnormal, 3:517–518 continuous monitoring of, 3:516 description of, 3:238 domains of, 3:518–520 evoked, constrained, 3:516–517 factors that affect, 3:514–516 high throughput test batteries, 3:515 homeostatic functions of, 3:514–516 information sources, 3:515–516 measurement of, 3:516–518, 3:520–524 naturalistic observation of, 3:516 phenotypes, 1:247–249, 3:514–516, 3:519 quantitative traits, 3:517–518 subdomains of, 3:518–520 temperature effects on, 3:107 Behavior of the Laboratory Rat, The, 3:531 Behavioral test acclimatization to testing room, 3:529 accuracy of, 3:520–521 apparatus used in, 3:525–526, 3:529–531 automated scoring of, 3:526 behavior observations during, 3:529 “blind” testing, 3:528–529 cage enrichment effects, 3:525 in colony room, 3:527–529 conducting of, 3:526–530 development of, 3:518 disease control, 3:525 equipment used in, 3:525–526, 3:529–530 handling during, 3:529 information sources, 3:530–531 lab environment, 3:527 light-dark cycle effects, 3:524–525 local conditions that affect, 3:527 maturity effects, 3:524 Morris water maze, 3:518, 3:520 multiple, 3:520 order of, 3:527 planning of, 3:524–526 precision of, 3:521 protocol for, 3:527 recommendations for, 3:531 refinement of, 3:518 reliability of, 3:521–522 replicability of, 3:522 sample protocol, 3:528 scoring of, 3:526 transport of mice for, 3:529 validity of, 3:522–524 video tracking of, 3:526 Bel2, 3:674 Benzoylphenyl urea, 3:430 Bernard-Soulier syndrome, 3:155 Beta-carotene, 3:550 Beta-catenin, 3:699
326 Bfq1, 3:624 Bgeq2, 3:624 Biceps brachii, 3:5–6 Biceps femoris, 3:4, 3:6 Bile collection of, 3:454 daily production of, 3:454 description of, 3:65 Bile acid, 3:66, 3:206 Bile acid CoA ligase, 3:206 Bile acid-CoA:amino acid N-acyltransferase, 3:206 Bile duct cannulation of, 3:454 catheterization of, 3:478–479 Bilirubin clearance of, 3:205–206 conjugated, 3:205 description of, 3:65 reference range for, 3:181 unconjugated, 3:205 Bioassays, 3:751 Bioexclusion facilities, for breeding colony cage level, 3:246–247 cleaning of, 3:244 combined techniques, 3:247 conventional, 3:243 disinfection of, 3:244 group level, 3:245 room level, 3:243–245 water supply, 3:244–245 Biofilms, 3:285 Biologic assays, 3:178 Biological control, house mice, 1:46 Biological hazards, 3:280 Biological indicator, 3:421 Biological safety cabinets Class I, 3:300–301 Class II, 3:301, 3:313 Class III, 3:302 illustration of, 3:281–282 Biologicals accidental human exposure to, 3:733 bioassays of, 3:751 contamination of, 3:401–402, 3:733–734 Creutzfeldt-Jakob disease contamination, 3:734 definition of, 3:732 derivation of, 3:732–734 efficacy of, 3:751 history of, 3:732 impurities in, 3:750 modern production of, 3:732 mouse antibody production assay, 3:400 mycoplasma contamination, 3:734 polymerase chain reaction testing, 3:400 prions, 3:734 purity and potency of, 3:750–751 quality control of cell-substrate characterization, 3:749–750 microbiological. See Microbiological quality control nonmicrobiological, 3:749–751 out-of-specification results, 3:751–752
CUMULATIVE
Biologicals (continued) product purity and potency, 3:750–751 raw materials, 3:738 regulation of, 3:732–733 testing of, 3:400–401 transmissible agents in, 3:733 viral agents in, 3:400–401 Bioluminescence imaging, 3:506–507 Biomarkers, of aging chronological age and, 3:654 end stage, 3:653, 3:655–658 identification of, 3:174 life span, 3:652–653 nonlethal, 3:654–655 pathology data and, 3:657–658 purpose of, 3:658 rate, 3:653–655 selection criteria, 3:654 state, 3:653–655 summary of, 3:658, 3:668 uses of, 3:653 Biomedical research mammals used in, 3:173 mouse in, 3:173 Biopharmaceuticals, 3:732, 3:750 Biopsy, testicular, 3:470 Biotin, 3:341–342, 3:369 Bisphenol A, 3:294, 3:307, 3:417 Bite. See Animal bite Biuret method, 3:208 Bladder. See Urinary bladder Blastocele, 3:102 Blastocyst microinjection chimeric mouse generation, 1:218 principles, 1:216–218 Blattella germanica, 3:429 Blatticides, 3:430 Bleeding orbital sinus, 3:454–455 submandibular, 3:457–458 Bleeding time, 3:155, 3:157 Blimp-1, 4:93 “Blind” testing, 3:528–529 BLNK, 4:307 Blood handling of, 3:138–139 refrigerated, 3:139 volume of, 3:137–138 Blood collection arterial, 3:457–458 cardiac puncture, 3:455–456 caudal vena cava, 3:458 after decapitation, 3:458 description of, 3:137–139 dorsal pedal vein, 3:456 heart, 3:455–456 hemolysis concerns during, 3:182 jugular vein, 3:179, 3:456–457 lateral tail vein, 3:456–457 after limb amputation, 3:458 orbital sinus, 3:454–455 restraints for, 3:454 saphenous vein, 3:456 sites for, 3:179
INDEX
Blood collection (continued) submandibular vascular bundle, 3:457–458 techniques for, 3:454 Blood concentration, 3:201 Blood islands, 3:158–159 Blood pressure fluctuations in, 3:31–32 monitoring methods electrocardiography. See Electrocardiography indwelling fluid-filled catheters, 3:33–34 invasive, 3:33–36 noninvasive, 3:32–33 radiotelemetry, 3:34–35 tail-cuff measurements, 3:32–33, 3:555 tethering techniques, 3:33 transducer-tipped catheters, 3:35–36 Blood smears evaluation of, 3:140, 3:145 platelet appearance on, 3:155 preparation of, 3:139 reticulocyte count on, 3:145 Blood urea nitrogen, 3:207 B10.LP mice, 3:142 B-mode echocardiography, 3:45 Body fat, 3:630–631 Body plethysmography, 3:54 Body regions, 3:2–3 Body size, 3:524 Body temperature heart rate affected by, 3:37 maintenance of, during electrocardiography, 3:37 thermoneutrality zone, 3:67–68 Body weight aging and, 3:655 diet restriction effects on, 3:366–367 water intake based on, 3:75 Bomb calorimetry, 3:324 Bone characteristics of, 3:124 hormones that affect, 3:127–131 phytoestrogen effects on, 3:350 regulation of, 3:124 x-ray imaging of, 3:494–495 Bone cells, 3:125, 3:136 Bone marrow age-related changes in, 3:161 anatomy of, 4:59–60 cellular organization of, 4:60–62 cellularity of, 3:145 collection of, 3:139, 3:458 dendritic cells from, 4:140, 4:148–149 evaluation of, 3:140, 3:145 hematopoietic stem cells in, 4:33 hypertrophy of, 4:60 megakaryocytes in, 3:154 natural killer cell development, 4:37 smear of, 3:139–3:140 structure of, 4:60 thymocytes from, 4:208 transplantation of, lethal irradiation for, 3:453–454
CUMULATIVE
327
INDEX
Bone marrow transfer experiments, 3:549 Bone morphogenetic proteins BMP-4, 3:93 signaling in hair follicle morphogenesis, 3:701–702 in nail development, 3:714 in sebaceous gland development, 3:711 in volar pad development, 3:716–717 Bone remodeling cells involved in, 3:125 mechanism of, 3:126–127 Bone resorption, 3:130–131 Boric acid, 3:430 Boron, 3:336 Borrelia, 2:729 Bottles. See Watering systems, bottles Bovine spongiform encephalopathy, 3:749 Boyle’s law, 3:54 Brain, 3:7 Brain-derived growth factor, 3:677 Brain-derived neurotrophic factor, 1:283 Brd2, 4:123 Breathing patterns, 3:52–53 Breeders replacement of, 3:238 retirement of, 3:261 Breeding. See also Reproduction ages to begin, 3:239 breeder selection, 1:54 chemical mutagenesis screening dominant phenotypes, 1:235 modifying genes dominant modifiers, 1:238 recessive modifiers, 1:238–239, 1:241 recessive phenotypes, 1:235–236 region-specific recessive mutations, 1:236–237 chromosomal aberration considerations, 1:72–73 commercial, 3:237 complexity of, 3:237–238 conditional mutagenesis strain maintenance, 1:71–72 economic considerations, 3:237–238 environmental factors that affect, 1:55–56, 3:108 genetic fundamentals alleles, 1:56–57 inheritance modes, 1:57–59 lethality and sterility, 1:59–60 genetic monitoring. See Genetic monitoring house mice characteristics intensity, 1:37 season length, 1:35–36 mating systems for, 3:252 mosaic populations, 1:75 noncommercial, 3:237 olfactory stimuli effects on, 3:239 pheromone responses, 1:54 protein requirements for, 3:332 random breeding, 1:75 record keeping, 1:56 strategies, 1:61, 1:74
Breeding (continued) summary of, 3:267–268 troubleshooting, 1:56 Breeding colony. See also Breeding stocks animal identification, 3:265–266 bedding for, 3:240–241 commercial, 3:237 culling of animals, 3:265 diet for, 3:239–240 economic considerations, 3:237–238 evaluation of, 3:110 exclusionary status, 3:248 F1 hybrids, 3:258 facilities for. See Breeding facilities feed, 3:239–240 founder animals, 3:251–252 genetically modified mice, 3:258–259 health monitoring of, 3:248–249 health status of, 3:105 hybrid strains, 3:266–267 inbred, 3:253–254, 3:266–267 inclusionary status, 3:248 litter consolidation, 3:261 maintenance of, 3:104–105 management of, 3:262 microbiological status of, 3:247–248 mismatchings, 3:266–267 noncommercial, 3:237 nutritional requirements, 3:240 outbred, 3:254–258 overview of, 3:236–237 planning for, 3:250–251 production colony sectioning, 3:259 production index for, 3:259 productivity information, 3:251 pup inventories, 3:265 quality assurance program testing of bacterial agents, 3:397–398 enzyme-linked immunosorbent assay, 3:396 frequency of, 3:395 Helicobacter spp., 3:397 hemagglutination inhibition assays, 3:396 immunofluorescence antibody testing, 3:396 Mycoplasma arthritidis, 3:397 Mycoplasma pulmonis, 3:397 number of animals to be tested, 3:395 parasites, 3:397–398 Pasteurella pneumotropica, 3:397 serologic tests, 3:395–396 sick or deceased animals, 3:394 variations in results, 3:394–395 viral genetic material, 3:396–397 random bred, 3:267 records. See Breeding records retirement, 3:261 sampling program for, 3:248–249 sentinel animals co-housed with, 3:394 specific pathogen free status of, 3:247–248 structure of, 3:252–259 timed matings, 3:261–262 transgenic mice, 3:258–259
Breeding colony (continued) troubleshooting the performance of, 3:109–110 water for, 3:241 Breeding facilities bioexclusion cage level, 3:246–247 cleaning of, 3:244 combined techniques, 3:247 conventional, 3:243 disinfection of, 3:244 group level, 3:245 room level, 3:243–245 water supply, 3:244–245 caging, 3:242–243 costs of, 3:238 isolators. See Isolators microisolation caging, 3:247 overview of, 3:241–242 Breeding records cage-level, 3:262–263 description of, 3:104 pedigree card, 3:262–263 pedigree ledger, 3:263, 3:265 pedigree tree, 3:265 production report, 3:263–264 Breeding stocks. See also Breeding colony advanced intercross lines, 1:71 background effects, 1:64–65 coisogenic strains, 1:64 congenic strains, 1:66–69 conplastic strains, 1:70–71 consomic strains, 1:69–70 hybrids, 1:65–66 inbred strains, 1:62–63 outbred stocks, 1:73, 1:75 recombinant congenic strains, 1:71 recombinant inbred strains, 1:71 segregating backgrounds, 1:66 segregating inbred strains, 1:63–64 Breeding unit configuration of, 3:109 description of, 3:104 Bromodeoxyuridine, sister chromatid exchange detection, 1:149 Bronchial circulation, 3:51–52 Bronchial-associated lymphoid tissue, 4:69 Bronchioles, 3:50 Bronchoalveolar lavage, 3:483 Bronchoconstrictor agents, 3:63 Bronchopulmonary inflammation, 3:48 Bronchopulmonary lavage, 3:459 Bronchus principalis dexter et rami arteriae et venae pulmonalis, 3:13 Bronchus principalis sinister, 3:13 Bruce effect, pheromone response, 1:54 Bsbob, 3:624 Btk, 4:307 BTLA, 4:269 Buccinatorius, 3:5 Buffy coat, 3:140 Bulboglandularis, 3:19 Bulbourethral glands, 3:96 Bulbus olfactorius, 3:7 Burst forming unit-erythroid, 3:142
328
CUMULATIVE
Butorphanol, 3:462 Bwq5, 3:624 BXSB.yaa mice, 4:246–247
C C1, 4:8 C3, 3:190 C3 mice, 3:53 C3aR, 4:11 C4 deficiency, 4:252 C4A, 4:124 C4B, 4:124 C5, 3:190 C5aR, 4:11 C5L2, 4:13 11C, 3:503 C58, 2:651 C4, 4:124 C5 convertase, 4:8 CACNA1A, 3:572–573 Cacnal a, 3:676 CACNB4, 3:572–573 Cacng2, 3:574 Cadmium, 3:345 Caesarean section, 3:251, 3:404 CAF1 mice, 3:157 Cage ammonia monitoring in, 3:294 behavior monitoring in, 3:516 cleaning of, 3:415–416 decontamination of, 3:280 enrichment of, 3:239, 3:525 environmental enrichment, 3:314 filter top, 3:242, 3:246, 3:293 flooding of, 3:307 health monitoring program based on, 3:248 history of, 3:294 infectious disease monitoring in, 3:394 isolators. See Isolators lighting in, 3:413–414 mass air displacement units in, 3:299–302 metal, 3:417 open top, 3:242 plastic, 3:417 radionuclide decontamination, 3:281 records on, 3:262–263 room temperature in, 3:239 size of, 3:242–243 static microisolator, 3:294–295 sterilization of, 3:280–281 ventilated air quality concerns, 3:295 blowers, 3:298 considerations in using, 3:298 description of, 3:284 excessive ventilation, 3:297–298 individually, 3:295–299 intracage supply/intracage exhaust, 3:295–296 intracage supply/perimeter capture, 3:295–296 water bottles in, 3:286–3:290 Cage filters, 3:417–418
Cage sanitation area chemical storage in, 3:277 clean activity, 3:277 description of, 3:276–277 location of, 3:273–274 mechanical equipment in, 3:277 soiled activity, 3:277, 3:285 Cage wash area clean cage wash, 3:311 description of, 3:277–278 soiled cage wash, 3:311–312 Cage washers alkali residues, 3:417 batcher, 3:302, 3:310, 3:417 cumulative heat factor, 3:416 efficacy evaluations, 3:417 filter screens, 3:417 improper loading of, 3:416 objective of, 3:415 operation of, 3:416 performance recommendations for, 3:416 pressure gauges, 3:416 quality assurance program for, 3:416 spray nozzles, 3:417 temperature gauges, 3:416 tunnel washer, 3:302–303, 3:310, 3:415–416 types of, 3:415 Calcα, 3:129 Calcaneus et ossa tarsi, 3:4 Calcitonin, 3:129 Calcitonin gene-related peptide, 3:129 Calcium daily requirements for, 3:336 dystrophic calcinosis, 3:338 reference range for, 3:181 serum levels of, 3:207–208 Calcium channels, voltage-gated, 3:572–573 Calcium:phosphorus ratio, 3:338 Calodium hepaticum, 1:46 Caloric restriction, for epileptic seizures, 3:583 Calories calculation of daily requirements, 3:445 definition of, 3:324 Cancer, 4:283–284 Cannibalism, 3:109 Cannulation bile duct, 3:454 vascular, 3:452 Caput epididymidis, 3:19 Caput laterale musculi gastrocnemii, 3:4, 3:6 Caput mediale musculi gastrocnemii, 3:7 CAR. See Cocksackie-adenovirus receptor CAR bacillus. See Cilia-associated respiratory bacillus Cara1, 3:624 Carbohydrates digestion of, 3:66 metabolism of, 3:185 Carbon dioxide asphyxiation using, 3:481 description of, 3:293 euthanasia uses of, 3:481 narcosis caused by, 3:464
INDEX
Carbon monoxide delivery of, 3:61 diffusing capacity for, 3:55 Carboxypeptidase E, 3:621 Carcinogenic compounds, 3:367 Cardiac output, 3:42–43 Cardiac puncture, 3:455–456 Cardiomyocytes, 3:29 Cardiovascular disease antioxidants and, 3:550 in atherosclerotic mice, 3:553–554 estrogen replacement therapy and, 3:551–552 in humans, 3:536 mortality caused by, 3:536 phenotyping, 1:245–246 prevalence of, 3:536 risk factors for, 3:536 Cardiovascular phenotyping, 3:555–556 Cardiovascular system ex-vivo techniques, 3:29–30 functions of, 3:41 heart. See Heart in-vivo techniques anesthesia effects, 3:30–31 blood pressure. See Blood pressure cardiac output, 3:42–43 electrocardiography. See Electrocardiography exercise tolerance assessment, 3:40–42 heart rate, 3:39–40 noninvasive imaging, 3:43–48 isolated heart preparation, 3:29–30 magnetic resonance imaging evaluations, 3:501–502 overview of, 3:25–26 Cardioviruses. See Encephalomyocarditis virus; Theiler’s murine encephalomyelitis virus Caretakers, 3:387 CARMA1, 4:84 Carnosine, 3:180 Carotid artery catheterization, 3:476 Carp1, 3:624 Carpal pads, 3:715 Casein, 3:354 Caspase recruitment domains, 4:114 Caspase-3 apoptosis inhibitor, 3:610 CAST/Ei mice, 3:651 Catabolic phase, of estrous cycle, 3:100 Catagen phase, of hair growth, 3:706–708 Caterpillar pads, 3:715 Cathepsins description of, 3:126 K, 1:310 Catheter(s) advantages and disadvantages of, 3:33–34 indwelling fluid-filled, 3:33–34 intragastric, 3:446 over-the-needle, 3:474 transducer-tipped, 3:35–36 vascular, 3:474 Catheterization arteries, 3:475 bile duct, 3:478–479
CUMULATIVE
329
INDEX
Catheterization (continued) carotid artery, 3:476 catheters used in, 3:474 colonic, 3:478 duodenal, 3:478 femoral artery, 3:476–477 femoral vein, 3:476–477 gastric, 3:477–478 ileal, 3:478 indications for, 3:474 intrathecal, 3:479 jejunal, 3:478 jugular vein, 3:475 nonsurgical approach, 3:474 nonvascular, 3:477–479 portal vein, 3:476 renal artery, 3:477 tail vein, 3:475 urinary bladder, 3:479 Catnb, 2:599, 2:602–603 Cauda epididymidis, 3:19 Caudal abdomen, 3:2 Caudal vena cava blood collection, 3:458 CBA, 2:646–647 CBA/J mice blood volume in, 3:138 water intake in, 3:76 CBA/JPh mice, 3:142 CBA/N mice, 3:189 C-banding, 1:149–150, 1:156 C57BL mice development, 2:647 phenotype, 2:647 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:649–650 nonneoplastic conditions, 2:647–649 pathogen susceptibility, 2:650 related strains, 2:650–651 C57BL/6 mice age-related diseases in, 3:646 atherosclerosis models, 3:539 breeding colony, 3:262 corticosterone levels in, 3:201 hyaloid development in, 3:600 immunoglobulins in, 3:189 isoflurane anesthesia in, 3:463 leptin levels in, 3:185 pentobarbital effects, 3:463 production index for, 3:260 protein requirements, 3:332 reference ranges for, 3:181 retinal vascular development in, 3:600 serum protein profiles of, 3:174 testosterone in, 3:202 trabecular meshwork in, 3:602 urea nitrogen levels in, 3:207 C57BL/10 mice, 3:138 C57BL/129 mice, 3:185 C57BL/6J mice asthma models, 3:62
C57BL/6J mice (continued) breathing patterns in, 3:52 cholesterol studies in, 3:65 diet for, 3:107 kidneys in, 3:71 C57BL/10ScCr mice, 4:110 C.B-17-scid mice, 4:278 CCL21, 4:201 CCR5, 1:308 CD1, 4:20–21, 4:141 CD3, 4:24 CD4, 4:26 CD8, 4:26 CD11b, 4:170 CD16, 4:175 CD19, 4:24, 4:84 CD21, 4:94 CD22, 4:83, 4:267 CD25, 4:42, 4:308–309 CD27, 4:93 CD30L, 3:194 CD35, 4:94 CD40, 3:193, 4:28–29 CD40 ligand, 3:194, 4:28–29, 4:94, 4:142 CD43, 4:170 CD45, 4:84, 4:254 CD49b, 4:170 CD62L, 4:90 CD70, 4:93 CD72, 4:83 CD74, 4:19 CD80, 4:245 CD86, 4:94, 4:142 CD91, 4:141 CD94/NKG2, 4:174 CD103, 4:43 CD122, 4:170 CD154, 4:29 CD206, 4:13 CD244, 4:175 Cd45, 4:189 CD-1 mice cholesterol studies in, 3:65 isoflurane anesthesia in, 3:463 reference ranges for, 3:181 CD4+ T cells, 4:39–40 CD8+ T cells description of, 4:39–40 effector, 4:90 CD11b, 4:33 Cdh1, 3:699 CD94/NKG2 receptors, 4:15 Cecal volume, in germfree mice, 3:219, 3:227, 3:482 Ced4, 3:675 Cell adhesion molecules, 3:545–546 Cell bank, 3:738–739 Cell cycle, 3:675 Cell transplantation therapy, embryonic stem cell studies, 1:286 Cell-substrate characterization, 3:749–750 Cellular retinaldehyde binding protein, 3:607 Cellular senescence, 3:640 Cellulose, 3:306
Cenpb, 3:622 Central nervous system, 3:674 Central supramolecular activation cluster, 4:89 Central tolerance models of, 4:209 T cell–mediated autoimmunity control through, 4:234–235 Centroacinar cells, 3:64 Centroblast B cells, 4:162 Centroblasts, 4:93 Centrocytes description of, 4:95 somatic hypermutation of, 4:94–96 terminal differentiation of, 4:96 Centromere, fluorescent in situ hybridization, 1:153 Cerebellum, 3:7 Cerebrospinal fluid collection, 3:458 Certified diet, 3:353 Ceruloplasmin, 3:206, 3:208 Ceruminous glands, 3:711 Cervical collars, 3:448 Cervical dislocation, 3:481 Cervical vagotomy, 3:473 Cervicales profundi lymph nodes, 3:21 Cervicales superficiales lymph nodes, 3:21 Cervicalis profundus caudalis lymph node, 3:22 Cervix, 3:98–99 Cervix uteri, 3:20 CF1 mice, 3:106 cFLIPL, 4:95 CFTR. See Cystic fibrosis transmembrane conductance regulator C3G, 4:200 C3H mice description of, 3:260 development, 2:643 phenotype, 2:643 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:645–646 nonneoplastic conditions, 2:643–645 pathogen susceptibility, 2:646 related strains, 2:646–647 Chagas disease, 3:45 Channelopathies, 3:676–677 Charge coupled device, 3:506 Chemical mutagenesis breeding strategies dominant phenotypes, 1:235 modifying genes dominant modifiers, 1:238 recessive modifiers, 1:238–239, 1:241 recessive phenotypes, 1:235–236 region-specific recessive mutations, 1:236–237 chlorambucil, 1:230–231 classification, 1:229 cyclophosphamide, 1:232
330 Chemical mutagenesis (continued) diethyl sulfate, 1:232 embryonic stem cells applications, 1:253 mutagens ethylmethanesulfonate, 1:255 ethylnitrosourea, 1:255 ICR191, 1:255–256 trimethylpsoralen, 1:256 phenotyping, 1:256 principles, 1:253–255 ethylmethanesulfonate, 1:231 ethylnitrosourea advantages, 1:233 clinical relevance, 1:233 description of, 1:230 effectiveness measures, 1:235 gene mapping, 1:241 historical perspective, 1:232–233 principles, 1:109 resources, 1:110 genotyping denaturing high-performance liquid chromatography, 1:256–257 single-strand conformational polymorphism, 1:256 temperature gradient capillary electrophoresis, 1:257 methyl methanesulfonate, 1:231 phenotyping. See Phenotyping procarbazine, 1:229–230 screening programs, 1:233–234 strain selection, 1:233–235 trimethylenemelamine, 1:230 Chemical mutagenicity, 3:175 Chemically defined diet, 3:352–353 Chemiluminescent immunoassays, 3:176 Chemogenomics, 1:294–295 Chemokine(s) atherosclerosis affected by, 3:545 CCR1, 3:197 CCR2, 3:196–197 CCR3, 3:196–197 CCR4, 3:196–197 CCR5, 3:196–197 CCR6, 3:197 CCR7, 3:196–197 CCR8, 3:196–197 CCR9, 3:197 CCR10, 3:197 dendritic cell migration regulated by, 4:143–144 description of, 3:195–196, 4:29, 4:31, 4:34, 4:47, 4:64 function of, 3:197 homeostatic, 3:195 inflammatory, 3:195 in inflammatory responses, 4:295 in lymphoid organogenesis, 4:75 natural killer cell production of, 4:171–172 from radiation-resistant stroma, 4:93 transcription of, 4:75 Chemokine receptors CCR2, 3:544
CUMULATIVE
Chemokine receptors (continued) description of, 4:29, 4:64 signaling of, 4:202–204 Chemosterilants, 3:431 Chenodeoxycholic acid, 3:206 Chest, 3:2 CH12F3-2, 4:157 C3H/Fg mice, 3:180 C3H/HeJ mice, 3:52, 4:110 Chief cells, 3:64 Chilomastix bettencourti, 2:525 Chimeric mouse blastocyst microinjection, 1:216–218 morula aggregation, 1:218 Chitin, 3:430 Chlamydia muridarum culture, 2:333 developmental cycle, 2:331–332 genital infection course, 2:339–340 immune response, 2:341–342 pathologic response, 2:340–341 genome, 2:331 history of study, 2:326–328 metabolism, 2:332–333 morphology, 2:330–331 pathogenesis, 2:334–335 respiratory infection course, 2:335–336 immune response, 2:337–339 pathologic response, 2:336–337 strains, 2:333–334 structure, 2:331 taxonomy, 2:333 Chlamydia spp. C. pneumoniae description of, 3:552 history of study, 2:342–343 mouse infection studies, 2:330, 2:343–344 taxonomy, 2:333 C. psittaci, 2:330, 2:344 C. trachomatis history of study, 2:327–328 mouse infection studies, 2:329–330, 2:339–342 taxonomy, 2:333 Chloral hydrate, 3:31 α-Chloralose, 3:31 α-Chlordane, 3:346 τ-Chlordane, 3:346 Chloride daily requirements for, 3:336 measurement of, 3:207 reference range for, 3:181 Chlorinated drinking water, 3:308–309, 3:372, 3:418–419 Chlorine dioxide description of, 3:309, 3:426 isolator sterilization using, 3:224 microbicidal efficacy of, 3:309 in research facilities, 3:309–310 shelf life of, 3:310 Chlorpyriphos, 3:346 Cholecalciferol, 3:339, 3:342
INDEX
Cholecystokinin, 3:64 Cholesterol. See also Lipoprotein(s) atherosclerosis risks, 3:537. See also Atherosclerosis description of, 3:65 efflux of, 3:187 excess, 3:187 measurement of, 3:187–188 mouse levels of, 3:537 serum, 3:187–188 total plasma, 3:188 Cholesterol ester hydrolase, 3:187 Cholesterol ester transfer protein, 3:186, 3:538 Cholesterol esters, 3:537 Cholic acid, 3:206 Choline, 3:340, 3:342 Chorion, 1:190 Chromatin, 3:149 Chromatography, 3:740 Chromium, 3:336 Chromosomal aberrations breeding considerations, 1:72–73 nomenclature, 1:94–97, 1:154–155 Chromosomal sex, 3:92 Chromosome(s). See Meiotic chromosomes; Mitotic chromosomes Chronobiology, 3:108–109 Chylomicrons, 3:66, 3:185, 3:537 CIITA, 4:93 Cilia, 3:696–697 Cilia-associated respiratory bacillus clinical features, 2:455–456 control of, 2:459 culture, 2:455 description of, 3:391 diagnosis culture, 2:458 electron microscopy, 2:459 enzyme-linked immunosorbent assay, 2:458 histopathology, 2:458–459 immunohistochemistry, 2:459 polymerase chain reaction, 2:459 geographic distribution, 2:457 history of study, 2:454–455 host range, 2:457–458 pathogenesis, 2:456–457 pathology, 2:456 prevalence of, 2:458 prevention of, 2:459 properties, 2:455 strains, 2:455 transmission, 2:458 treatment, 2:459 Ciliary neurotrophic factor, 3:599 Ciliary neurotrophic factor receptor, 4:41 Ciliated cells, 3:50 Ciliated fimbria, 3:98 Circular paired mating system, 3:255 Circulating immune complexes, 3:191 Cis transgenic mice, 4:188 Citrobacter rodentium, 3:391 classification, 2:373 clinical features, 2:375–376
CUMULATIVE
331
INDEX
Citrobacter rodentium (continued) diagnosis, 2:376 epizootiology, 2:376 history of study, 2:373–374 pathogenesis, 2:374–375 properties, 2:374 treatment and control, 2:377 c-Kit, 4:35 c-kit, 3:142 C57L, 2:651 Clara cells, 3:51 Class 100 air, 3:299 Class II-associated invariant chain peptide, 4:19 Class switch recombination description of, 4:157 mismatch repair proteins involved in, 4:164 Clavicula, 3:4–6 Clean cage wash, 3:311 Cleidobrachialis, 3:5–6 Cleidocephalicus, 3:4–6 Cleidooccipitalis, 3:5 Clindamycin Clostridium difficile infection management, 2:359 Clostridium perfringens infection management, 2:357 Clinical chemistry changes in, 3:175 ELISA kits, 3:177–178 information sources, 3:179 instrumentation used in, 3:174 multiplex technology, 3:176–177 quality assurance, 3:182 sampling, 3:179–180 services available, 3:176 statistics, 3:182 techniques used in, 3:174 Clinical descriptive terms, 3:137 Clitoral glands, 3:711 Clitoris, 3:21 Clitoris et orificium urethrae externum, 3:3 Clitoris et seccio transversalis clitoridis, 3:20 Clock, mutation phenotyping, 1:247 Clonal deletion, 4:215, 4:218 Clonal expansion, 4:68 Cloning. See Somatic nuclear transfer Closed formula diet, 3:355 Clostridium spp. C. difficile clinical features, 2:358–359 control and prevention, 2:360 culture, 2:358 diagnosis, 2:359 epizootology, 2:359 history of study, 2:357–358 pathogenesis, 2:359 properties, 2:358 strains and antigenic relationships, 2:358 treatment, 2:359–360 C. perfringens clinical features, 2:356 control and prevention, 2:357
Clostridium spp. (continued) culture, 2:356 description of, 3:228 diagnosis, 2:357 epizootology, 2:357 history of study, 2:355 pathogenesis, 2:356–357 properties, 2:356 strains and antigenic relationships, 2:356 treatment, 2:357 C. piliforme clinical features, 2:351–353 control and prevention, 2:355 culture, 2:351 description of, 3:392 diagnosis, 2:353–355 epizootology, 2:353 history of study, 2:350 pathogenesis, 2:353 properties, 2:350 strains and antigenic relationships, 2:350 treatment, 2:355 Cluster of differentiation, 4:32 c-mpl, 3:142, 3:154 c-Myc, 3:712–713 Coagulase test, 3:742 Coagulation time prolongation, 3:158 Coat color, genetics and nomenclature, 2:628–630 Cobalamin, 3:341 Cobalt, 3:336 Cobblestone pads, 3:715 Cocaine amphetamine regulated transcript, 3:130 Coccygeus, 3:18 Coccygeus dorsalis, 3:7 Coccygeus ventralis, 3:7 Cockroaches, 3:429–430 Cocksackie-adenovirus receptor, 2:54 Coelomys, 1:14 Coenzyme Q10, 3:550 Coisogenic strains breeding, 1:64 nomenclature, 1:87 Cold adaptation, 1:31, 3:68–69 Collars cervical, 3:448 polyethylene restraint, 3:444 Collectins, 4:14 Collection procedures blood. See Blood collection bone marrow, 3:139 Colliculi caudales, 3:7 Colliculi rostrales, 3:7 Collimation, 3:502 Colobomas, 3:598 Colon ascendens, 3:15 Colon descendens, 3:15, 3:17 Colon transversum, 3:15 Colonic catheterization, 3:478 Colonization resistance, 3:228 Colony. See also Breeding stocks animal identification, 3:265–266
Colony (continued) bedding for, 3:240–241 commercial, 3:237 culling of animals, 3:265 diet for, 3:239–240 economic considerations, 3:237–238 evaluation of, 3:110 exclusionary status, 3:248 F1 hybrids, 3:258 facilities for. See Breeding facilities feed, 3:239–240 foundation. See Foundation colony founder animals, 3:251–252 genetically modified mice, 3:258–259 health monitoring of, 3:248–249 health status of, 3:105 hybrid strains, 3:266–267 inbred, 3:253–254, 3:266–267 inclusionary status, 3:248 litter consolidation, 3:261 maintenance of, 3:104–105 management of, 3:262 microbiological status of, 3:247–248 mismatchings, 3:266–267 noncommercial, 3:237 nutritional requirements, 3:240 outbred, 3:254–258 overview of, 3:236–237 planning for, 3:250–251 production colony sectioning, 3:259 production index for, 3:259 productivity information, 3:251 pup inventories, 3:265 quality assurance program testing of bacterial agents, 3:397–398 enzyme-linked immunosorbent assay, 3:396 frequency of, 3:395 Helicobacter spp., 3:397 hemagglutination inhibition assays, 3:396 immunofluorescence antibody testing, 3:396 Mycoplasma arthritidis, 3:397 Mycoplasma pulmonis, 3:397 number of animals to be tested, 3:395 parasites, 3:397–398 Pasteurella pneumotropica, 3:397 serologic tests, 3:395–396 sick or deceased animals, 3:394 variations in results, 3:394–395 viral genetic material, 3:396–397 random bred, 3:267 records. See Breeding records retirement, 3:261 sampling program for, 3:248–249 sentinel animals co-housed with, 3:394 specific pathogen free status of, 3:247–248 structure of, 3:252–259 timed matings, 3:261–262 transgenic mice, 3:258–259 troubleshooting the performance of, 3:109–110 water for, 3:241
332 Colony forming unit-erythroid cells, 3:142 Colony log, 3:263–3:264 Colony stimulating factor-1, 3:131 Color flow Doppler echocardiography, 3:46 Column chromatography, 3:740 Commensal impact, 1:28–29 Commercial breeding colonies, 3:237 Committee for Proprietary Medicinal Products, 1:298 Comparative genome analysis ancestral chromosome structure, 1:106–107 conserved noncoding sequences, 1:105–106 conserved synteny, 1:106 gene orthology, 1:105 genome size, 1:105 polymorphisms and haplotypes, 1:106 prospects, 1:111, 1:315 purposes, 1:104 repetitive sequences, 1:104 Comparative tumor biology. See Tumor pathology, in genetically engineered mice Competence, 3:698 Complement protein 3, 4:8 Complement proteins, 4:280 Complement receptor(s) characteristics of, 4:10 CR1, 4:10–11 CR2, 4:10–11 CR3, 4:11 CR4, 4:11 Complement receptor 1 related gene/protein Y, 3:190 Complement system activation of, 4:8 description of, 3:190–191 functions of, 4:8 lectin pathway of, 4:8 overview of, 4:8–10 pentraxin pathway of, 4:8 Complementarity-determining regions, 4:21 Complete blood count, 3:141 Complex Trait Consortium, 3:632 Compound A, 3:464 Computed tomography contrast-enhanced, 3:496 facilities for, 3:282–283 high-resolution x-ray, 3:43–44 single-photon emission, 3:43, 3:504, 3:509 x-ray, 3:43–44, 3:493–494 Conditional mutagenesis, 1:264–265 Conduction system, 3:27 Confidence limit, 1:143 Congenic strains analysis of, 3:628–629 breeding, 1:66–69 definition, 2:628 development of, 1:67–69, 3:173 gene mapping, 1:128–129 marker-assisted development, 1:69 nomenclature, 1:87
CUMULATIVE
Congenic strains (continued) production of, 3:629 recombinant, 1:71, 1:84, 3:629–630 Conjugated bilirubin, 3:205 Conjunctivitis, 2:484–485 Conplastic strains breeding, 1:70–71 nomenclature, 1:87, 1:89 Conservation, 1:30–31 Consomic strains breeding, 1:69–70 gene mapping, 1:129 nomenclature, 1:87 Constant volume-reheat, 3:288 Constrictor vulvae, 3:20 Construct validity, 3:653 Contact bedding, 3:240 Contaminants adventitious agents description of, 3:735–736 prevention of, 3:737–739 cell banks used to prevent, 3:738–739 chlorinated hydrocarbons, 3:346 contaminant removal or inactivation, 3:739–740 description of, 3:345 drinking water, 3:241 endogenous retroviruses, 3:736–737 heavy metals, 3:345–347 laboratory practices to prevent, 3:739 manufacturing practices to prevent, 3:739 maximum amounts of, 3:345–346 murine leukemia virus, 3:737 mycotoxins, 3:347–348 nitrosamines, 3:350–351 operator-induced, 3:739 organophosphates, 3:346 pesticides, 3:345, 3:347 phytoestrogens, 3:348–350 polycarbon biphenyls, 3:347 saponins, 3:351 sources of, 3:736 viruses, 3:739–740 Contrast, 3:491 Contrast agents computed tomography use of, 3:496 magnetic resonance imaging use of, 3:502 ultrasound use of, 3:497–498 uses of, 3:492 Conventional animal, 3:218 Copper, 3:336 Copulation plug, 3:101–102, 3:110, 3:261–262 Cor in pericardii, 3:12 Corncob bedding, 3:306 Corneal dystrophy, Balb/c mice, 2:640 Corneal growth factor-stimulated angiogenesis, 3:611 Corniculate cartilage, 3:50 Cornu uteri dextrum, 3:20–21 Cornu uteri sinistrum, 3:20–21 Coronary arteries anatomy of, 3:27–28 magnetic resonance imaging of, 3:45
INDEX
Coronary veins, 3:28 Coronavirus, 3:396 Corpora lutea, 3:101, 3:103 Corpus cavernosum clitoridis, 3:20 Corpus cavernosum penis, 3:96 Corpus ceci, 3:15 Corpus pancreatis, 3:14 Corpus ventriculi, 3:16 Cortex telencephali, 3:7 Cortical epithelial cells, 4:63, 4:65 Cortical thymic epithelial cells, 4:208 Corticosterone adrenocorticotrophic hormone affected by, 3:201 circulation of, 3:200–201 definition of, 3:200 reference ranges for, 3:181 Cortisol-binding globulin, 3:200 Corynebacterium spp. C. bovis clinical features, 2:400 culture, 2:399 description of, 3:220 diagnosis, 2:401 epizootiology, 2:400–401 properties, 2:399 strains, 2:399 treatment and control, 2:401–402 C. kutscheri clinical features, 2:402–403 culture, 2:402 diagnosis, 2:403–404 epizootiology, 2:403 properties, 2:402 strains, 2:402 treatment and control, 2:404 Costa ultima, 3:9 Costimulatory molecules, 4:215 Coumestrol, 3:349 Cowper’s glands, 3:96 COX. See Cyclooxygenase COX-2 inhibitors, 3:480 Cpe, 3:621 CPMP. See Committee for Proprietary Medicinal Products c1q deficiency, 4:252 CR1, 4:10–11 CR2, 4:10–11, 4:24 CR3, 4:11 CR4, 4:11 Cr2, 4:11 Cranial abdomen, 3:2 CRB1, 3:607 Crb1rd8 mice, 3:607 C-reactive protein, 3:190 Creatine kinase description of, 3:199–200 mitochondrial, 3:199 Creatinine, 3:181, 3:207 Creatinine/creatine ratio, 3:73 Creutzfeldt-Jakob disease, 3:734 Crhr2, 3:520 Cricoid cartilage, 3:50 Critical infection risks, 3:424
CUMULATIVE
333
INDEX
Cross-linkage mapping backcross, 1:120–124 backcross-intercross, 1:123 guidelines, 1:120 intercross, 1:122–124 ordering loci in multipoint crosses, 1:124 penetrance, expressivity, or modifier loci, 1:124–125 strain selection databases, 1:116 description of, 1:120 disequilibrium mapping, 1:127 marker types, 1:117–120 mutant genes, 1:116–117, 1:241 quantitative trait loci, 1:126–127 Cross-validation, 3:653 Crry, 4:280 Crumbs homolog 1 gene, 3:607 Cryoanesthesia, 3:464–465 Cryopreservation description of, 3:114 embryos, 1:218–219, 1:235 germplasm, 1:219 ovarian tissue, 1:219–220, 1:235 sperm, 1:235 “Cryptic” immunodeficiency, 3:389, 3:396 Cryptopatches, 4:69 Cryptosporidium muris cell biology, 2:539 clinical features, 2:539 diagnosis, 2:539 history of study, 2:538 life cycle, 2:538–539 prevention, 2:539–540 research implications, 2:540 treatment and control, 2:539–540 Crypts, 3:65–66, 3:98 Crypts of Lieberkuhn, 3:66 CSTB, 3:570 CTLA-4, 4:237, 4:268–269 C-type lectin, 4:36 C-type lectin receptors, 4:13–14 C-type lectin-like glycoprotein, 4:175 C-type particles, 3:737 64Cu, 3:503 Cuboidal epithelial cells, 3:97 Cumulative exposure hypothesis, 3:664 Cumulative heat factor, 3:416 Cumulus cells, 3:102 Cuneiform cartilage, 3:50 Cupula diaphragmatis, 3:9, 3:14 Current Protocols in Neuroscience, 3:531 Curvatura major, 3:16 Curvatura major ventriculi, 3:17 Curvatura minor, 3:16 Cutaneus trunci, 3:5–6 Cut-like 1 signaling, 3:702 CXCL3, 3:196 CXCL4, 3:196 CXCL6, 3:196 CXCL9, 3:196 CXCL10, 3:196 CXCL11, 3:196 CXCL12, 3:196 CXCL13, 4:75, 4:78
CXCR2, 3:195 CXCR3, 3:195, 3:197 CX3CR1, 3:197 CXCR4, 3:195, 3:197 CXCR5, 3:195, 3:197, 4:78 CXCR6, 3:195, 3:197 Cyanocobalamin, 3:341 Cyclic adenosine monophosphate, 3:125 Cyclic biorhythms, 3:180 Cyclin-dependent kinase 5, 3:576, 3:683 Cyclohexylamine, 3:362 Cyclooxygenase description of, 3:196 inhibitors of, 3:480 knockout mouse phenotypes, 1:313 Cyclophosphamide, 1:232 Cyp19a1, 3:130 Cyp1b1−/−, 3:604 CYPs. See Cytochrome P1:450 Cystatin B, 3:569–571 Cystic fibrosis transmembrane conductance regulator, 1:309 Cystocentesis, 3:460 Cytochrome c, 4:217 Cytochrome P450 CYP1A2 humanized mouse, 1:312 knockout mouse phenotype, 1:311–312 CYP2D6 knockout mouse phenotype, 1:312 CYP2E1 knockout mouse phenotype, 1:312 CYP3A4 humanized mouse, 1:312 pharmacokinetic studies in vitro versus in vivo, 1:313–314 Cytochrome-oxidase activity, 3:68 Cytogenetic analysis. See Meiotic chromosomes; Mitotic chromosomes Cytokine(s) in airway hyperresponsiveness, 4:293–294 atherosclerosis affected by, 3:545 description of, 4:29, 4:279 in inflammatory responses, 4:295 leukocytes affected by, 3:153 natural killer cell production of, 4:171–172 receptors for, 4:30–31 suppressors of cytokine signaling, 4:180–181 Cytokine-activated JAK-STAT pathway, 4:180–181 Cytokine-like factor-1, 4:41 Cytomegalovirus atherosclerosis and, 3:552 description of, 3:105, 3:391 human, 4:307–308 mouse. See Mouse cytomegalovirus Cytoplasmic receptors, 4:114 Cytosolic pathway, 4:18 Cytotoxic T cell-associated antigen 4, 4:26, 4:200 Cytotoxic T lymphocytes, 4:171, 4:283 Cytotoxic T-lymphocyte associated antigen-8, 3:193
D 33D1, 4:139 Daidzein, 3:348–349 Dam, foster, 3:112 DAPI banding, 1:149 Dark:light cycle behaviors affected by, 3:524–525 description of, 3:291, 3:414 mating during, 3:101 Data loggers, 3:421 DBA mice development, 2:651 phenotype, 2:651 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:651–652 nonneoplastic conditions, 2:651–652 pathogen susceptibility, 2:651–653 DBA/2 mice cholesterol studies in, 3:65 pentobarbital effects, 3:463 production index for, 3:260 DBA/2J mice adult onset glaucoma in, 3:610 audiogenic seizure susceptibility in, 3:579 diet for, 3:107 pigmentary glaucoma in, 3:605–606 protein requirements, 3:332 testosterone in, 3:202 water intake in, 3:76 DC. See Dendritic cell DDT, 3:346 Death receptors, 4:214, 4:217 DEC-205, 4:139 Decapitation blood collection after, 3:458 description of, 3:481–482 Decay-accelerating factor, 4:245 Decidual response, 3:102 Decontamination. See also Autoclaving; Disinfectants; Sterilization definition of, 3:424 monitoring of procedures for, 3:427 of cage, 3:280 of diet, 3:361–366 Dectin-1, 4:14 ß-defensins, 4:32 Defined flora, 3:227–229, 3:247 Defined flora animal, 3:218, 3:247 Definitive erythropoiesis, 3:135, 3:158 Definitive hematopoiesis, 3:160 Dehydration postoperative, 3:479 red cell mass affected by, 3:146 signs of, 3:146 Dehydroepiandrosterone, 3:371, 3:647, 3:667 Dehydroretinol, 3:339 Delayed-type hypersensitivity, 4:309 Deletion mapping, 1:130
334 Delta-like 1, 4:63 Deltoideus, 3:5 Demodex musculi clinical features, 2:568 diagnosis, 2:568 host range, 2:568 life cycle, 2:568 morphology, 2:568 pathobiology, 2:568 prevention and control, 2:568 treatment, 2:568 Denaturing high-performance liquid chromatography, 1:256–257 Dendritic cells antigen handling by, 4:141–142 bone marrow, 4:140, 4:148–149 CD1 expression, 4:141 CD86 levels, 4:142 chemokine effects on, 4:143–144 definition of, 4:35 dendrites of, 4:141 dermal, 4:140 description of, 4:3 differentiation of, 4:36, 4:142 discovery of, 4:136 ex vivo studies of, 4:146–147 follicular, 4:71, 4:78, 4:140 functions of, 4:144–146 generation of, 4:139 granulocyte-macrophage colony-stimulating factor system for creation of, 4:139 immature, 4:142, 4:144–145 interleukin-12 production and, 4:144 interstitial, 4:36 intestinal, 4:146 isolation protocols for, 4:147–149 Langerhans cells and, 4:140 life span of, 4:141 lymphocytic choriomeningitis virus persistence role, 2:199 lymphoid tissue distribution of, 4:139–140 major histocompatibility complex expression by, 4:138 in marginal zone, 4:78 maturation of, 4:142 migration of, 4:87, 4:143–144 monoclonal antibodies, 4:138 morphological properties of, 4:138, 4:141 mouse strains used to study, 4:136–138 murine cytomegalovirus immune response, 2:27 myeloid, 4:112 natural killer cells and, 4:172 in nonlymphoid tissue, 4:140–141 phenotypic changes, 4:87 physical properties of, 4:138 plasmacytoid description of, 4:37, 4:76–77, 4:142–143 maturation of, 4:143 toll-like receptor signaling in, 4:114 spleen, 4:146–147 subsets of, 4:142–143 surface composition of, 4:138
CUMULATIVE
Dendritic cells (continued) T cells and, 4:88–89, 4:139–140, 4:145 in thymus, 4:139 tolerance mediated by, 4:145–146 turnover of, 4:141 Dendritic epidermal T cells, 4:308 Dentatorubral and pallidoluysian atrophy, 3:681–682 Deoxycytidine, 3:73 Deoxynivalenol, 3:346, 3:348 Deoxyuridine, 4:95 Depolarization, 3:37 Depressor labii inferioris, 3:5 Dermal administration of drugs, 3:447–449 Dermal papilla, 3:694–695 Dermal sheath, 3:694 Dermatitis, 3:371 Dermatophytosis fungal diagnosis, 2:511 epidemiology, 2:510–511 history of study, 2:510 pathology, 2:511 taxonomy, 2:510 treatment and control, 2:511 ringworm in humans clinical signs, 2:731 reservoir and incidence, 2:730 transmission, 2:730 DES. See Diethyl sulfate Developmental degenerations amyotrophic lateral sclerosis, 3:678–679 ataxia-telangiectasia, 3:679–680 cell cycle failure, 3:675 channelopathies, 3:676–677 dentatorubral and pallidoluysian atrophy, 3:681–682 DNA double strand break repair, 3:675 Friedreich’s ataxia, 3:682 homeostasis failures, 3:675–677 Huntington’s disease, 3:680 inherited human, mouse models of, 3:678–685 motor neuron diseases, 3:678–679 nervous mouse, 3:678 Niemann-Pick type C disease, 3:677–678 patterning failure, 3:674 programmed cell death defects, 3:674–675 progressive motor neuronopathy, 3:677 Purkinje cell degeneration, 3:678 retina, 3:679 spinal and bulbar muscular atrophy, 3:681 spinal muscular atrophy, 3:678 spinocerebellar ataxia, 3:680–681 summary of, 3:685 triplet repeat disease models, 3:680–682 wobbler mouse, 3:677 Developmental glaucoma, 3:603 Dexamethasone, 3:131 Diabetes mellitus definition of, 3:183 future research, 3:631–632 maternal effects, 3:631 mouse models of chemically mutagenized, 3:625–626
INDEX
Diabetes mellitus (continued) congenic strain analysis, 3:628–629 diet-induced, 3:630–631 haplotype analysis, 3:627–628 knockout, 3:621–623 multigenic, 3:626–630 natural alleles, 3:626–630 recombinant congenic strain, 3:629–630 recombinant inbred strain, 3:629–630 spontaneous single gene mutations, 3:620–621 transgenic, 3:623–624 N-ethyl-N-nitrosourea studies, 3:625 non-insulin dependent, 3:618, 3:631 obesity and, 3:184–185 phenotype, 3:630 prevalence of, 3:618 diabetes mutation, 3:620 Diacylglycerol, 4:234 Diaphragm, 3:52 Diazepam, 3:461 Diazinon, 3:346 Dibromoacetaldehyde, 3:461 Dichlorvos-containing pellets, 3:399 Dieldrin, 3:346 Diestrus, 3:100–3:101 Diet. See also Feed; Nutrients; Water amino acid requirements, 3:332–333 analytes affected by, 3:180 autoclaving of description of, 3:225, 3:304–305, 3:362, 3:421–422 irradiation of diet vs., 3:366 moist heat, 3:362 nutrients affected by, 3:362–363 physical changes caused by, 3:363 toxicological risks with, 3:363 breeding colony, 3:239–240 calcium:phosphorus ratio, 3:338–339 certified, 3:353 chemically defined, 3:352–353 closed formula, 3:355 contaminants in chlorinated hydrocarbons, 3:346 description of, 3:345 heavy metals, 3:345–347 maximum amounts of, 3:345–346 mycotoxins, 3:347–348 nitrosamines, 3:350–351 organophosphates, 3:346 pesticides, 3:345, 3:347 phytoestrogens, 3:348–350 polycarbon biphenyls, 3:347 saponins, 3:351 decontamination of, 3:361–366 energy requirements correlated with, 3:325 ethylene oxide sterilization of, 3:362 expanded, 3:240 extruded description of, 3:353–354 hardness of, 3:356 manufacturing process of, 3:357 softening of, 3:357 fat content of, 3:335
CUMULATIVE
335
INDEX
Diet (continued) fenbendazole added to, 3:305, 3:353 fiber content of, 3:335 fixed-formula, 3:355 forms of, 3:353–355 gamma-irradiated, 3:305 gelled, 3:240 ground, 3:354 hardness of, 3:355–357 high-quality, 3:373 house mice, 1:28 humidity effects on, 3:361 importance of, 3:323 infectious diseases affected by, 3:369–371 irradiation of autoclaving of diet vs., 3:366 description of, 3:361, 3:363–364 dosages for, 3:364 factors that affect, 3:364 microorganisms affected by, 3:364–365 nutrient effects, 3:365–366 packaging, 3:364 process involved in, 3:364 reviews of, 3:364 laboratory testing of, 3:351 least-cost formula, 3:355 liquid, 3:354–355 meals, 3:353 medicated, 3:353 microbiological spoilage of, 3:360 mineral requirements, 3:336–337 moisture in, 3:360 natural-ingredient blending of, 3:358 description of, 3:239, 3:304–305, 3:351 extrusion of, 3:358–359 manufacturing of, 3:358–359 packaging of, 3:359 pelleting of, 3:358 phytoestrogens in, 3:349 raw materials used in, 3:358 storage of, 3:360, 3:423 nutrient composition of, 3:325 open formula, 3:355 packaging of, 3:359 pasteurization of, 3:361 pelleted autoclaving of, 3:356–357, 3:422 description of, 3:240, 3:353 hardness of, 3:355 illustration of, 3:445 manufacturing process of, 3:357 natural-ingredient diets, 3:358 quality of, 3:356 steam sterilization of, 3:422 summary of, 3:357 pest control issues, 3:361 protein. See Protein, dietary purified for carcinogenicity studies, 3:352 composition of, 3:351–352 description of, 3:337, 3:351–352 manufacturing of, 3:359 vitamin levels in, 3:342 semisolid, 3:445
Diet (continued) shelf life of, 3:305 sterilization of, 3:304–305, 3:361 storage of, 3:360–361 summary of, 3:372–373 temperature effects on, 3:360 ultrafiltered, 3:354 variable-formula, 3:355 Diet restriction body weight affected by, 3:366–367 carcinogenic compound sensitivity affected by, 3:367 dehydroepiandrosterone effects, 3:667 form of, 3:366 immunity effects of, 3:371 protein, 3:368 spontaneous tumors and, 3:366 stem cell aging and, 3:666 survival affected by, 3:366–367 tumor incidence affected by, 3:366–367 undernutrition vs., 3:367–368 vitamin requirements and, 3:368–369 Diethyl sulfate, 1:232 Diethylhydroxylamine, 3:667 Differential white blood cell count, 3:151 Diffusing capacity for carbon monoxide, 3:55 Diffusion capacity of lungs, 3:55, 3:61 Digastricus, 3:5, 3:8 Digestive system description of, 3:63 esophagus, 3:63–64 exocrine pancreas, 3:64 gallbladder, 3:64–65 intestine, 3:65–66 liver, 3:64–65 oral cavity, 3:63 stomach, 3:64 Digestive tract, 3:15 Digital pads, 3:715 Dihydrotestosterone 5a-, 3:93, 3:130 description of, 3:202 1,25-Dihydroxy vitamin D3, 3:129–130 2,3-Diphosphoglycerate, 3:161 Disease definition of, 3:642 senescence vs., 3:642–644, 3:668 Diseases, house mice, 1:43–45 Disinfectants. See also Decontamination; Sterilization alcohol, 3:426 chemical bases for, 3:425 chlorine dioxide, 3:426 cleaning before application of, 3:424 description of, 3:423–424 dilution of, 3:425 formaldehyde gas, 3:426 high-level, 3:424 infection risk and, 3:424 intermediate, 3:424 iodophors, 3:425 low level, 3:424 monitoring of, 3:425 peroxymonosulfate, 3:426
Disinfectants (continued) quats, 3:425 rotation of use, 3:425 selection of, 3:424–425 sodium hypochlorite, 3:426 training in use of, 3:425 vaporized hydrogen peroxide, 3:426 Disinfection definition of, 3:237, 3:424 of room level bioexclusion facilities, 3:244 ultraviolet, 3:307–308 Disulfaton, 3:346 Diuretics, 3:74 Diverticulum glandulae bulbourethralis, 3:18–19 Dixenic animal, 3:218 DLCO. See Diffusing capacity for carbon monoxide DNA double strand break repair, 3:675 DNA microarray drug target elucidation, 1:293–294 gene expression analysis, 1:108 limitations, 1:294 DNA polymerases, 4:164 DNA vaccine, gene transfer, 1:270 DNA-binding fluorochrome test, 3:743 DNA-PKc, 3:675 DNAX adapter protein-12, 4:174 DOCK2, 4:202 Dok-1, 4:203 Domains, mouse proteins, 1:103 Domestication, 3:75 Dominant allele, 1:57 Dopaminergic neurons, 3:574 Doppler echocardiography, 3:45–46 Doppler effect, 3:497 Dorsal pedal venipuncture, 3:456–457 Double negative thymocytes, 4:208 Double stranded ribonucleic acid, 3:195 Double-stranded RNA, 4:112 Drinking water acidification of, 3:308, 3:372, 3:418 additives in, 3:308 anthelmintics added to, 3:309 antibiotics added to, 3:309 automatic systems for, 3:241, 3:285–286 bisphenol A contamination, 3:307 chlorine added to, 3:308–309, 3:372, 3:418–419 contaminants in, 3:241 delivery of, 3:241, 3:286–3:290, 3:307. See also Watering systems drug administration in, 3:444–445 hydrochloric acid added to, 3:308, 3:418 irradiation of, 3:372 mechanical filtration of, 3:372 microorganisms in, 3:371 monitoring of, 3:420 municipal sources of, 3:241, 3:307 ozone treatment of, 3:372 processing of, 3:307–308 quality of, 3:371–372, 3:418 restriction of, 3:371 reverse osmosis of, 3:307, 3:372 steam sterilization of, 3:308
336 sterilization of, 3:225, 3:308 ultraviolet disinfection of, 3:307–308 variations in, 3:306–307 Drosophila, 1:4–5, 4:64–65 Drug administration considerations before, 3:444 in drinking water, 3:444–445 enteral methods of, 3:444–447 external jugular vein, 3:451–452 food supply for, 3:445 footpad injections, 3:453 intrabronchial, 3:449 intracranial, 3:452 intradermal, 3:449 intragastric, 3:445–447 intramuscular injection, 3:445, 3:450 intranasal, 3:449 intranodal, 3:452–453 intraosseous, 3:452 intraperitoneal, 3:445, 3:450–451 intrasplenic, 3:453 intrathecal injection, 3:447 intrathoracic, 3:451 intratracheal, 3:449 intravascular, 3:451–452 intravenous bolus, 3:445 intravenous slow infusion, 3:445 lateral tail vein, 3:451 neonatal, 3:453 oral, 3:445 parenteral methods of, 3:447–452 rectal, 3:446–447 retro-orbital, 3:452 subcutaneous description of, 3:445 injection, 3:449–450 osmotic pumps, 3:450 pellets, 3:450 topical, 3:447–449 vascular cannulation, 3:452 in water, 3:444–445 Drug development costs of, 3:174 regulation of, 3:174–175 Drug discovery classical approach, 1:293 drug targets classification, 1:307 elucidation chemogenomics, 1:294–295 DNA microarray, 1:293–294 phenotype-driven selection, 1:296 validation, 1:295–296 historical perspective, 1:296–297 mouse contributions, 1:296–297 pharmacogenetic traits in mice, 1:304 pharmacogenomic study prospects in mice, 1:314–315 preclinical testing mouse versus rat, 1:297–298 pharmacodynamic studies, 1:299–300 pharmacokinetic studies, 1:300 safety studies carcinogenicity, 1:302–303 genotoxicity, 1:302
CUMULATIVE
Drug discovery (continued) immunotoxicity, 1:304 local tolerance, 1:301–302 overview, 1:300–301 photo-safety, 1:303 reproduction toxicity, 1:302 toxicokinetics, 1:301 quantitative trait loci mapping of drug response genes, 1:305–306 regulatory agencies, 1:298–299 transgenic, knockout, and knockin mouse examples, 1:306–313 Dry heat sterilization, 3:362 Ductile cells, 3:64 Ductus deferens, 3:96 Ductus deferens dexter, 3:19 Ductus deferens sinister, 3:18 Ductus parotideus, 3:5 Duffy, 3:197 Duodenum anatomy of, 3:14, 3:16 catheterization of, 3:478 Duodenum ascendens, 3:15 Duplication-deficiency mapping, 1:130–131 Dynamic lung compliance, 3:56, 3:58, 3:60 Dyslipidemia, 3:539 Dystrophic calcinosis, 3:338 Dystrophin, 1:313
E E3 ubiquitin ligases, 4:237–238 Ear neural degeneration in, 3:679 punching of, 3:265, 3:441–3:442 tagging of, 3:441–3:442 Early transposon-related elements features, 2:276 insertional mutagenesis, 2:272 Eccrine glands, 3:713 Echocardiography anesthesia for, 3:48 B-mode, 3:45 disadvantages of, 3:48 Doppler, 3:45–46 heart evaluations using, 3:46–48 limitations of, 3:48 magnetic resonance imaging vs., 3:44 M-mode, 3:45–46 transthoracic, 3:555–556 two-dimensional, 3:45 ultrasound principles, 3:45 Ecogenetics, 1:292 ECT. See Electroconvulsive threshold testing Ectodysplasin-A, 3:702, 3:711, 3:720 Ectromelia virus, 3:391 EDIM. See Rotavirus EEG. See Electroencephalography Effector B cells, 4:91 Egg cylinder, 1:188, 1:190 Eicosanoids, 3:51 Eimeria cell biology, 2:531 clinical features, 2:531 diagnosis, 2:531
INDEX
Eimeria (continued) life cycle, 2:529, 2:531 prevention, 2:531 research implications, 2:531–532 taxonomy, 2:529 treatment and control, 2:531 Ejaculation, 3:101 EL1, 3:585 EL2, 3:585 EL3, 3:585 EL4, 3:585 EL mouse description of, 3:581 emotional stress in, 3:582 GFAP-positive cells in, 3:584 hippocampus of, 3:584–585 seizures in antiepileptic drugs for, 3:582 aspartate and, 3:584 caloric restriction testing, 3:583 induction of, 3:581–582 inheritance patterns, 3:585 ketogenic diet testing, 3:582–583 pathogenesis of, 3:583–585 PTZ-induced, 3:582 sexual dysfunction in, 3:583 Electrocardiography description of, 3:36–37 in mice, 3:37–38, 3:555 phenotyping uses of, 3:38–39, 3:555 recording platform for, 3:38 Electrochemiluminescent immunoassays, 3:176 Electroconvulsive shock, 3:576–577 Electroconvulsive threshold testing, 1:250 Electroencephalography, 1:250 Electrolytes calcium, 3:207–208 chloride, 3:207 measurement of, 3:207–208 phosphorus, 3:207 potassium, 3:207 sodium, 3:207 Electromyography, 1:250 Electron microscopy cilia-associated respiratory bacillus diagnostics, 2:459 Mycoplasma pulmonis diagnostics, 2:451 ELISA. See Enzyme-linked immunosorbent assay Elizabethan collars, 3:448 Embryo cryopreservation of, 3:114, 3:226 germfree mice, 3:226–227 implantation of, 3:102–103 in utero development of, 3:103 preimplantation development of, 3:102 Embryo transfer breeding colony production, 3:251 description of, 3:113–114, 3:226 Embryoid bodies, 3:159 Embryology comparative human advantages of working with mouse embryos, 1:183–185
CUMULATIVE
337
INDEX
Embryology (continued) early postimplantation period, 1:188, 1:190–192 gland development in later pregnancy, 1:192–197 inner cell mass fate, 1:186, 1:188 pregnancy stage duration, 1:185–186 preimplantation period, 1:186, 1:188 computer-aided methodologies gene expression databases, 1:199–200 overview, 1:197–198 text-based anatomical databases, 1:198–199 three-dimensional reconstructions of sectioned embryos overview, 1:200 Theiler stage 14 embryos, 1:201–203 Theiler stage 20 embryos, 1:203–204 cryopreservation of embryos, 1:218–219, 1:235 developmental stages, 1:168–169 embryo isolation early postimplantation, 1:171, 1:173, 1:175–177 preimplantation, 1:167, 1:169–170 gestational age determination, 1:167 histological analysis, 1:178–179, 1:181, 1:183 magnetic resonance imaging studies, 1:204–205 optical projection tomography studies, 1:205–206 rederivation, 1:220–221 Embryonal carcinoma cell growth factor requirements, 1:283 sources and culture, 1:282 Embryonic germ cell growth factor requirements, 1:283 sources and culture, 1:282 Embryonic stem cells chemical mutagenesis applications, 1:253 genotyping denaturing high-performance liquid chromatography, 1:256–257 single-strand conformational polymorphism, 1:256 temperature gradient capillary electrophoresis, 1:257 libraries of clones, 1:285 mutagens ethylmethanesulfonate, 1:255 ethylnitrosourea, 1:255 ICR191, 1:255–256 trimethylpsoralen, 1:256 phenotyping, 1:256 principles, 1:253–255 description of, 3:159 differentiation induction, 1:284 gene expression, 1:282–283 gene function studies, 1:285–286 gene-specific mutagenesis conditional mutagenesis, 1:264–265 gene targeting, 1:262–263 gene trapping, 1:263–264
Embryonic stem cells (continued) inducible mutagenesis, 1:265 RNA interference, 1:265–266 targeted trapping, 1:264 transgenic mouse, 1:262 growth factor requirements, 1:283 insertional mutagenesis, 1:285 karyotype, 1:283–284 sources and culture, 1:282 transplantation studies, 1:286 EMCV. See Encephalomyocarditis virus EMG. See Electromyography EMS. See Ethylmethanesulfonate Encephalitozoon cuniculi cell biology, 2:541 clinical features, 2:541–542 description of, 3:392 diagnosis, 2:542 life cycle, 2:541 prevention, 2:542–543 research implications, 2:543 taxonomy, 2:540–541 treatment and control, 2:542–543 Encephalomyocarditis virus antigenic properties, 2:315 biophysical properties, 2:312 clinical features, 2:315–316 control and prevention, 2:320 diagnosis, 2:319–320 epizootiology, 2:318–319 genome, 2:312–313 history of study, 2:311–312 propagation, 2:315 receptors, 2:314–315 structure, 2:313–314 Endo 180 receptor, 4:13 Endocrine hormones, 3:124 Endocrine system, 3:93–94 Endocrine theory of aging, 3:664–665 Endocrinology, 3:124 Endocytic pathway, 4:18 Endocytosis, 4:19 Endogenous antigen models, 4:209–213 α-Endosulfan, 3:346 ß-Endosulfan, 3:346 Endothelial nitric oxide synthase, 3:551 Endotoxin, 3:742 Endrin, 3:346 Energy gross, 3:324 measurement of, 3:324 metabolizable, 3:324, 3:330 peak intake of, 3:330 Energy density, 3:324 Energy requirements body composition effects on, 3:325 environmental temperature effects on, 3:325 estimates of, 3:324–330 factors that affect, 3:324–327 during gestation, 3:327, 3:329 for growth, 3:328–329 during lactation, 3:327, 3:329 maintenance-related, 3:325–328 nutrient composition of diet and, 3:325
Energy requirements (continued) of pups, 3:330 sex effects on, 3:325 strain effects on, 3:325 Engrailed-1, 3:717 Engrailed-2, 3:675 Enhanced green fluorescent protein, 4:140 Enrofloxacin Citrobacter rodentium management, 2:377 Corynebacterium bovis management, 2:401 Entamoeba muris cell biology, 2:527 clinical features, 2:527–528 diagnosis, 2:528 life cycle, 2:527 morphology, 2:527 prevention, 2:528 research implications, 2:528 taxonomy, 2:527 treatment and control, 2:528 Enteral administration, of drugs drinking water, 3:444–445 food supply, 3:445 intragastric, 3:445–447 intrathecal injection, 3:447 oral, 3:445 rectal, 3:446–447 water, 3:444–445 Enteric nervous pathway, 3:65 Enterobacteriaceae. See also specific organisms culture media, 2:373 genera, 2:366–367 growth characteristics, 2:367 virulence factors and pathogenicity islands, 2:367–369 Enterococci, 3:227 Enterohepatic circulation, 3:206 Environment puberty affected by, 3:94 reproduction affected by enrichment, 3:108 feed, 3:107–108 housing, 3:109 human interaction, 3:109 humidity, 3:107 light, 3:106, 3:238, 3:291 sound, 3:106 temperature, 3:106–107, 3:239 time of year, 3:108–109 vibration, 3:106 of research facilities air quality in, 3:295 ammonia, 3:293 carbon dioxide concentrations, 3:293 enrichment of, 3:314 lighting, 3:291–292 macroenvironment, 3:288–293 microenvironment, 3:292–294 monitoring of, 3:292–293 noise, 3:292 overview of, 3:287–288 relative humidity, 3:291 temperature, 3:290–292
338 Environmental Protection Agency, 3:371 Enzyme(s) alanine aminotransferase, 3:181, 3:198 aldolase, 3:200 alkaline phosphatase, 3:198 amylase, 3:200 aspartate aminotransferase, 3:181, 3:198–199 creatine kinase, 3:199–200 glutamate dehydrogenase, 3:200 lactate dehydrogenase, 3:181, 3:199 ornithine transcarbamoylase, 3:199 sorbitol dehydrogenase, 3:200 Enzyme-linked immunosorbent assay cardiovirus applications, 2:319 cilia-associated respiratory bacillus applications, 2:458 Clostridium piliforme applications, 2:354 Corynebacterium kutscheri applications, 2:404 description of, 3:396 Helicobacter applications, 2:427–428 kits for complement quantification using, 3:191 description of, 3:177–178, 3:188 lactate dehydrogenase-elevating virus applications, 2:227–228 lymphocytic choriomeningitis virus applications, 2:203–204, 2:722 mammalian reovirus applications, 2:257 minute virus of mice applications, 2:100 mouse adenovirus applications, 2:61 mouse cytomegalovirus applications, 2:32 mouse parvovirus applications, 2:100 mouse thymic virus applications, 2:34 mousepox virus applications, 2:87, 2:160–161 Mycoplasma pulmonis applications, 2:450 Pasteurellaceae applications, 2:495–496 pneumonia virus of mice applications, 2:304 rotavirus applications, 2:245 Sendai virus applications, 2:297–298 Eosinopenia, 3:414 Eosinophil(s) in airway hyperresponsiveness, 4:297 characteristics of, 3:150 description of, 4:33–34 inflammatory role of, 4:34 morphology of, 3:150 transcription factors that affect, 3:149 Eosinophilia, 4:294 Eotaxin, 3:196 Eotaxin-1, 4:296 Eotaxin-2, 4:296 Eperythrozoon coccoides, 3:145 Epfpq2, 3:624 Epfq1, 3:624 Epfq2, 3:624
CUMULATIVE
Epicardium, 3:27 Epidermal growth factor receptor, 3:597, 4:238 Epidermal growth factor signaling, 3:702 Epidermal T cells, 4:308 Epididymis anatomy of, 3:95–96 description of, 3:18 sections of, 3:96 sperm maturation in, 3:95 Epigenetics genome analysis, 1:108 overview of mouse studies, 1:10 Epiglottis, 3:50 Epilepsy anticonvulsants for, 3:566, 3:571 antiepileptic drugs, 3:566, 3:571 incidence of, 3:566 ketogenic diet for, 3:582–583 Mendelian forms of, 3:567 mouse models of cystatin B, 3:569–571 description of, 3:567 EL mouse. See EL mouse Frings mice, 3:571 GABAA ß3 subunit, 3:568–569 gene screening, 3:586 jerky mice, 3:571 Lafora disease, 3:570–571 progressive myoclonus epilepsies, 3:569–571 summary of, 3:585–586 SWXL-4 mice, 3:585 ubiquitin protein ligase E6-AP, 3:568–569 voltage-gated calcium channels, 3:572–573 voltage-gated K+ channel KCNA 1, 3:568 voltage-gated Na+ channel SCN2A, 3:567–568 multifactorial, 3:576–586 orphan mouse mutants description of, 3:573 Nhe1, 3:575–576 p35, 3:576 serotonin receptor, 3:575 stargazer, 3:573–574 weaver, 3:574–575 progressive myoclonus, 3:569–571 sexual dysfunction and, 3:583 slow wave, 3:575 sudden unexpected death in, 3:579 symptomatic, 3:576–578 Epilepsy prone mice, 3:580–581 Epilepsy-associated repeat, 3:571 Epileptic seizures characteristics of, 3:566 classification of, 3:566 EL mouse model. See EL mouse electroconvulsive shock-induced, 3:576–577 generalized, 3:566 kainic acid-induced, 3:577–578
INDEX
Epileptic seizures (continued) partial, 3:566 PTZ-induced, 3:577, 3:582 Epinephrine insulin release suppressed by, 3:183 reference ranges for, 3:181 Episodic ataxia type 1, 3:568 Epithelial cells, 3:51, 4:6 EPM2A, 3:570 Epstein-Barr virus, 4:283 Equipment alkali residues, 3:417 autoclave. See Autoclave; Autoclaving cage. See Cage laminar flow, 3:421 management of, 3:410–411 watering systems. See Watering systems Erbb2, transgenic mice, 2:598–602 Ergocalciferol, 3:339 ERK/JNK/p38, 4:216 ERV. See Expiratory reserve volume Erythrocyte(s) in aging mice, 3:161 blood smear evaluations, 3:145 decreased production of, 3:148 development of, 3:142 2,3-diphosphoglycerate concentrations at birth, 3:161 functions of, 3:142 hemagglutination of, 3:746 hematocrit, 3:144 histology of, 3:143 increased production of, 3:142–143, 3:148 life span of, 3:142, 3:147–148 morphology of, 3:143, 3:161 parameters for, 3:143–146 parasites that affect, 3:145 red blood cell count changes in, 3:146–148 description of, 3:141, 3:143–144 in young mice, 3:161 red blood cell indices, 3:144 red cell distribution width description of, 3:141, 3:143 hemorrhage effects on, 3:147 red cell mass absolute, 3:147 absolute decreased, 3:147 decreases in, 3:147–148 increases in, 3:146–147 relative decreased, 3:147 senescence of, 3:640 volume of description of, 3:143 splenectomy effects on, 3:162–163 Erythrocyte mass absolute, 3:147 absolute decreased, 3:147 decreases in, 3:147–148 increases in, 3:146–147 relative decreased, 3:147 Erythroid cells anucleate, 3:159 enucleation of, 3:159
CUMULATIVE
339
INDEX
Erythroid cells (continued) erythropoietin’s role, 3:142 from hemangioblasts, 3:158–159 in primitive hematopoiesis, 3:158–159 Erythropoiesis accelerated, 3:161, 3:163 androgen stimulation of, 3:161 primitive erythropoietin’s function during, 3:159 macrophages in, 3:159 megakaryocytes in, 3:159 signaling molecules in, 3:159–160 in spleen, 3:163 stress, 3:142 Erythropoietin in definitive erythropoiesis, 3:160 in erythrocyte development, 3:142 functions of, 3:142 in primitive erythropoiesis, 3:159 Erythropoietin receptors, 3:142, 3:159, 4:182 ES cell. See Embryonic stem cell Escherichia coli clinical features, 2:377 description of, 3:346 diagnosis, 2:377 properties, 2:377 Esophagus, 3:10–11, 3:16, 3:63–64 Esr1, 3:130 Essential fatty acids, 3:334 Estradiol, 3:202 Estrogen atheroprotective effects of, 3:551 definition of, 3:130 preovulatory increase in, 3:103 Estrogen receptors, 1:307–308, 3:130, 3:551 Estrogen replacement therapy, 3:551–552 Estrus cycle length of, 3:239, 3:262 onset of, 3:94 ovulation during, 3:99 phases of, 3:100 postpartum, 3:239 stages of, 3:100–101 ESTs. See Expressed sequence tags Ethion, 3:346 Ethosuximide, 3:573, 3:577 Ethylene diaamine tetraacetate, 3:138–139 Ethylene oxide sterilization, 3:362, 3:422 Ethylmethanesulfonate chemical mutagenesis, 1:231 embryonic stem cell mutagenesis, 1:255 Ethylnitrosourea advantages, 1:233 clinical relevance, 1:233 description of, 1:230 effectiveness measures, 1:235 gene mapping, 1:241 historical perspective, 1:232–233 principles, 1:109 resources, 1:110 Eumorphia, 3:531 Eustachian tube, 3:49
Euthanasia carbon dioxide asphyxiation, 3:481 cervical dislocation, 3:481 decapitation, 3:481–482 of fetuses, 3:482 after gavage, 3:446 of neonates, 3:482 Office of Laboratory Animal Welfare guidelines, 3:480–481 Excitement, leukocytes affected by, 3:152–153 Exclusion level, 3:311 Excretory ducts, 3:95–96 Exercise heart rate during, 3:40 stress test cardiovascular system responses to, 3:41 description of, 3:40–42 heart rate responses to, 3:40 swimming, 3:41 treadmill, 3:41–42 Ex-germfree animal, 3:218 Exocrine pancreas, 3:64 Exogen phase, of hair growth, 3:706, 3:708 Expansion colony definition of, 3:236 purpose of, 3:253–254 Expansion colony segment, 3:236 Experimental autoimmune encephalomyelitis, 4:308–309 Experimental design, 3:644–645 Expiration, 3:52 Expiratory reserve volume, 3:53–54 Expressed sequence tags, nomenclature, 1:90 Expressivity linkage mapping, 1:124–125 phenotype, 1:72 Extensor carpi radialis, 3:6 Extensor carpi radialis longus, 3:5 Extensor carpi ulnaris, 3:5 Extensor digitorum communis, 3:5 Extensor digitorum lateralis, 3:5–6 Extensor digitorum longus, 3:6 External jugular vein injection, 3:451–452 Extravascular hemolysis, 3:148 Extruded diet description of, 3:353–354 hardness of, 3:356 manufacturing process of, 3:357 softening of, 3:357 Eya, 4:65 Eye(s) anatomy of, 3:601–602 angiogenesis of, 3:599–601 aqueous outflow system in, 3:602 development of, 3:596 drainage structures for, 3:601–602 examination techniques for, 3:596 illness signs, 3:388 mutant phenotyping, 1:244–245 retina, 3:596
Eye research advances in, 3:596 glaucoma. See Glaucoma phenotypic variations, 3:597–598 relevance of mice in, 3:596–597 retinal degeneration. See Retinal degeneration
F 18F,
3:503, 3:505 F1 hybrids advantages of, 3:646–647 aging research using, 3:646–648 definition of, 2:628, 3:646 description of, 3:258 disadvantages of, 3:647 life span of, 3:646 Fab fragment, 4:21 Face masks, 3:313 Facialis nerve, 3:7 Facies diaphragmatica, 3:16 Facies parietalis, 3:16 Facies visceralis, 3:16 Familial Alzheimer’s disease, 3:683 Familial hypertrophic cardiomyopathy, 3:39 Farnoquinone, 3:340 Fas ligand, 3:194 Fas-associated death domain, 4:217 Fascia cruris, 3:6 Fascia lata, 3:6 Fascia thoracolumbalis, 3:4 Fat(s) body, 3:630–631 dietary description of, 3:334–335 free radical oxidation of, 3:365 infectious diseases and, 3:370 in purified diets, 3:359 Fat insulin receptor knock out mice, 3:665 Fat pad, 3:97 Fat-soluble vitamins, 3:339–340, 3:345, 3:365 Fatty acids, 3:334–335 Fc receptors FCαRI, 4:8 FCγRI, 4:7 FCεRI, 4:7–8, 4:295 FCγRIIB autoimmunity and, 4:264–266 B cell tolerance, 4:265–266 description of, 4:6–7, 4:142, 4:254 genetic linkage studies, 4:264–265 mice deficient in, 4:265 polymorphisms, 4:265 signaling of, 4:263 FCγRIII, 4:7 FCγRIV, 4:7 homologs, 4:262 IgG, 4:263–264 Langerhans cell expression of, 4:140 neonatal, 4:6 overview of, 4:5
340 Fc receptors (continued) poly-Ig receptor, 4:5–6 transmembrane ligand-binding protein, 4:5 Fcgr2, 4:264–265 FDA. See Food and Drug Administration Feces collection of, 3:458 microscopic examination of, 3:230 Federal Food, Drug, and Cosmetic Act, 3:428 Federal Insecticide, Fungicide, and Rodenticide Act, 3:428 Feed. See also Diet autoclaving description of, 3:304–305 hardness increased by, 3:356–357 packaging of, 3:359 autoclaving of description of, 3:225, 3:304–305, 3:362, 3:421–422 irradiation of diet vs., 3:366 moist heat, 3:362 nutrients affected by, 3:362–363 physical changes caused by, 3:363 toxicological risks with, 3:363 contamination of, 3:422 dichlorvos-containing pellets, 3:399 drug administration in, 3:445 estimating requirements for, 3:330–331 fenbendazole, 3:305, 3:353, 3:399, 3:403 gamma-irradiated, 3:305 insect contamination of, 3:422 packaging of, 3:359 pelleted autoclaving of, 3:356–357, 3:422 description of, 3:240, 3:353 hardness of, 3:355 illustration of, 3:445 manufacturing process of, 3:357 natural-ingredient diets, 3:358 quality of, 3:356 steam sterilization of, 3:422 summary of, 3:357 pigmented, 3:305 purified, 3:304 quality of, 3:422 reproduction affected by, 3:107–108 in research facilities, 3:304–305 shipping of, 3:422 specialty, 3:305 sterilization of description of, 3:225 ethylene oxide, 3:422 using autoclave. See Feed, autoclaving of storage of, 3:360–361, 3:422 Female(s) artificial insemination of, 3:227 breeding age for, 3:239 donor, for germfree mice production, 3:226–227 energy requirements for, 3:325 retirement of, 3:261 stress responses, 3:109 Female genital organs, 3:20–21
CUMULATIVE
Female germ cells, 3:93 Female reproductive tract anatomy of, 3:97–99 cervix, 3:98–99 description of, 3:97 evaluation of, 3:110 ovary, 3:97 oviduct, 3:97–98 uterus, 3:98 vagina, 3:99 Femoral artery catheterization, 3:476–477 Femoral vein catheterization, 3:476–477 Femur, 3:4 Fenbendazole, for pinworm management description of, 2:558–559 in feed, 3:305, 3:353, 3:399, 3:403 in water, 3:419 Fenitrothion, 3:346 Fentanyl citrate-fluanisone, 3:461–462 Fentanyl-fluanisone-midazolam, 3:31 Ferric chloride, 3:337 Ferric sulphate, 3:337 Fertility assessment of, 3:110 of males, 3:104, 3:111–112 Fertilization description of, 3:102 temperature effects on, 3:107 Fetal bovine serum, 3:735 Fetal hemoglobin, 3:160 Fetal-maternal interface, 4:284 Fetus euthanasia of, 3:482 resuscitation of, 3:467–468 Fever, 3:69 FGF. See Fibroblast growth factor Fiber, 3:335 Fibrinogen, 3:206, 3:208 Fibrinogen concentration, 3:141, 3:157–158 Fibroblast growth factors -2, 3:599, 3:720 -5, 3:708 -7, 3:703, 4:66 -10, 4:66 pluripotent cell culture, 1:283 Fibula, 3:4 Filter top cage, 3:242, 3:246, 3:293 Filth flies, 3:430–431 Fipronil, 2:568 Fire response, 1:42 Firefly luciferase, 3:506 First messengers, 3:124 FISH. See Fluorescent in situ hybridization Fissura interlobaris, 3:13 Fixatives, 3:483 Fixed-formula diet, 3:355 Flea human interaction, 2:735 rodent species, 2:735 Flexible film isolators, 3:219–220, 3:245 Flexor carpi radialis, 3:6 Flexor carpi ulnaris, 3:6 Flexor digiti longus, 3:6–7 Flexor digitorum longus et tibia, 3:7 Flexor digitorum profundus, 3:6
INDEX
FLICE, 4:95 Flies, 3:429–431 Flk-1, 3:158 Flow cytometer, 3:178 Fluid-filled catheters, indwelling, 3:33–34 Fluorescence imaging, 3:506–508 Fluorescence resonance energy transfer, 3:507 Fluorescent in situ hybridization chromosome banding, 1:153 chromosome structures, 1:153 cytogenetics, 1:150–152 gene mapping, 1:129–130 high resolution technique, 1:152 meiotic chromosomes, 1:158–159 painting of chromosomes, 1:153 primed in situ technology, 1:152 Fluoride, 3:336, 3:345 Fluoroimmunoassay, 3:176 Flying insects, 3:429 Foam cells, 3:51, 3:546 Folacin, 3:341 Folate, 3:342 Folic acid, 3:341 Follicle hair. See Hair follicles ovarian development of, 3:99–100 follicle stimulating hormone effects on, 3:201–202 function of, 3:99–100 Graafian, 3:99 number of, 3:99 primordial stage of, 3:99 secondary, 3:99 Follicle stimulating hormone description of, 3:94, 3:201–202 plasma levels of, 3:202 reference ranges for, 3:181 secretory pattern for, 3:202 spermatogenesis initiation by, 3:97 Follicle-associated epithelium, 4:69 Follicular dendritic cells B cell recruitment affected by, 4:93 description of, 4:71, 4:78, 4:140 Food. See Feed; Nutrients Food and Drug Administration, drug development role, 1:298 Food deprivation, 3:76. See also Diet restriction Food intake insulin effects on, 3:618 neuropeptides that affect, 3:619 phenotyping, 1:251 Footpad injections, 3:453 Forced oscillation technique, 3:59 Forceps restraint, 3:439, 3:443 Forelimb muscles of, 3:5–6 regions of, 3:2 Forkhead box N1 description of, 4:277 signaling, 3:702, 3:714 Formaldehyde gas, 3:224, 3:426 Forward migration, 3:256–257
CUMULATIVE
341
INDEX
Fostering of pups, 3:112 Fosterlings, 3:112 Foundation colony definition of, 3:236 development of, 3:252 founder animals, 3:251–252 isolator housing of, 3:245 of inbred stock, 3:253 of outbred stock, 3:255–256, 3:267 pedigree card for, 3:263 Foundry robots, 3:285–286 4-way cross stocks, 3:647, 3:649 Foxc1, 3:603–604 Foxc1w/Foxc1w, 3:704 Foxl1, 4:74 Foxn1nu, 4:277 Foxn1nu/Foxn1nu, 3:704 Foxn1w/Foxn1w, 3:710 Foxp3, 4:42 Foxp3+CD4+CD25+ Treg cells, 4:43 Friedreich’s ataxia, 3:682 Friend of GATA-1, 3:155 Frings mice, 3:571 Frizzled 6, 3:701 Fructose, 3:66 fsh, 4:123 FTY720, 4:202 Fumonisin B1, 3:348 Functional assays, 3:190 Functional magnetic resonance imaging, 3:502 Functional residual capacity, 3:44, 3:54, 3:57 Fungal infection. See also Dermatophytosis; Pneumocystis spp. animal models, 2:513–514 systemic and opportunistic infection diagnosis, 2:513 epidemiology, 2:511–512 history of study, 2:511 pathology, 2:512–513 taxonomy, 2:511 treatment and control, 2:513 Fungi, 3:742–743 Fur dying, 3:440 Fur mites. See also Demodex musculi; Myobia musculi; Myocoptes musculinus; Ornithonyssus bacoti; Psorergates simplex; Radfordia; Trichoecius romboutsi description of, 3:105 diagnostic testing for, 3:391, 3:394, 3:398 nits, 3:398 Fusarium roseum, 3:349 Fusion, 1:222 FVB mice, 3:260 FVB/N mice description of, 3:185 development, 2:653 phenotype, 2:653 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690
FVB/N mice (continued) nonneoplastic conditions, 2:672–681 neoplasia, 2:653 nonneoplastic conditions, 2:653 pathogen susceptibility, 2:654
G 67Ga,
3:503 GABAA ß3 subunit, 3:568–569 Gabrb3, 1:308 GABRB3, 3:569 Gadd45α, 4:253–254 Gadolinium-enhanced magnetic resonance imaging, 3:502 Gallbladder, 3:64–65 Gametogenesis, 1:226–227 Gamma camera, 3:502 Gamma-irradiated diets, 3:305 Ganglia cardiaca, 3:10 Ganglion cervicothoracicum sinistrum, 3:11 Ganglion stellatum dextrum et sinistrum, 3:10 Ganglion venae cavae cranialis sinistrae, 3:10 garbrb3, 3:569 Gas dilution tests, 3:54–55 Gas exchange, 3:50 Gaster, 3:14–15 Gastric cancer, 2:425–426 Gastric catheterization, 3:477–478 Gastritis, 2:424–425 Gastrointestinal stem cells, 3:66 Gastrointestinal tract bacteria in, 3:227, 3:229 drug administration into, 3:445–447 illness signs, 3:388 immune functions of, 3:66 necropsy evaluations, 3:482–483 Gastrulation, 1:171 GATA-1 description of, 4:34 megakaryocytes deficient in, 3:154–155 in splenic hematopoiesis, 3:163 GATA-3, 1:286 GATA-4, 3:159 Gaussian distribution, 3:182 Gavage, 3:445–447 G-banding, 1:148–149, 1:156 GBASE. See Genetic Database of the Mouse Gelled diet, 3:240 Gelled water, 3:419 Gene(s) annotation approaches, 1:101–102 databases, 1:102 families, 1:103 human gene comparison with mouse set, 1:102–103 mapping of. See Deletion mapping; Duplication-deficiency mapping; Fluorescent in situ hybridization; Linkage mapping; Mitotic mapping; Quantitative trait loci; Radiation hybrid mapping; Somatic cell hybrids
Gene(s) (continued) naming, 1:89, 1:290 orthology, 1:105 symbols, 1:89 Gene mutations, 3:25 Gene overexpression, 4:280 Gene targeting, 1:262–263, 3:537, 3:567, 4:238 Gene transfer chromosomal versus extrachromosomal insertion, 1:268–269 disease model development, 1:274–275 ex vivo, 1:268 expression regulation studies, 1:275 gene therapy overview, 1:268–269 preclinical studies, 1:275 in vivo, 1:268 vaccine development, 1:275–276 vectors conditional replication vectors, 1:269 development, 1:275 nonviral vectors, 1:268–270 replication deficient vectors, 1:269 viral vectors, 1:268, 1:270–274 Gene trapping applications, 1:264 description of, 3:621 loci nomenclature, 1:92 principles, 1:263–264 screening, 1:264 targeted trapping, 1:264 GeneNetwork/WebQTL, 3:530 General anesthesia, 3:179 Generalized seizures, 3:566, 3:574 Genetic Database of the Mouse, historical perspective, 1:7 Genetic linkage mapping, 1:5 Genetic mapping, 3:648–650 Genetic monitoring biochemical assays, 1:138–139 DNA tests, 1:140–143 genetic quality control program components, 1:137 monitoring program development, 1:137–138 mouse room procedures, 1:137 immunoassays, 1:139–140 origins of genetic differences, 1:136 overview, 1:135–136 Genetic structure aging research results affected by, 3:645 F1 hybrids, 3:646–647 4-way cross stocks, 3:647, 3:649 guidelines for selection of, 3:648–650 heterogeneous lines, 3:648 inbred strains, 3:645–646, 3:648 outbred stock, 3:648 Genetically engineered mice. See also specific strain description of, 3:648 tumor pathology studies in comparative human pathology accuracy, 2:582, 2:584, 2:612 digital imaging, 2:614
342 Genetically engineered mice (continued) morphometrics, 2:615 prospects, 2:616–617 reporting of results, 2:613–614 spontaneous tumor surveillance, 2:615–616 validation, 2:612–613 experimental design, 2:587, 2:592, 2:594 gene targets, 2:591 historical perspective, 2:584 Internet resources, 2:591–592 nomenclature conventions, 2:594–595 oncogenic event considerations molecular alterations and microscopic structure, 2:601–603 spontaneous and carcinogen-induced tumors, 2:595–596 uniqueness of genetically engineered mouse tumors, 2:596–601 progression metastasis versus microinvasion, 2:611–612 sequential microscopic changes, 2:605–606, 2:608–611 promoters for tissue-specific expression, 2:592, 2:601–602 signature phenotypes, 2:586 tissue context effects microscopic structure, 2:604–605 strain effects, 2:604–605 tumor biology, 2:603–604 weak oncogenes, 2:603 Genistein, 3:349 Genital hair follicles, 3:697 Genital organs. See also Reproductive tract female, 3:20–21 male, 3:18–19 Genital ridge description of, 3:93 primordial germ cells, 3:93 Genital system development and differentiation of, 3:92–93 gonads, 3:92–93 Genome sequencing approach, 1:100–101 historical perspective, 1:8 organization of gene expression, 1:108 reagents, 1:107 Genome size, 1:105 Genomic segmental polymorphism, 4:120 Geobacillus stearothermophilus, 3:225, 3:421 Geographic distribution of house mice, 1:16–18, 1:26–28 Germ cells, 3:92–93 Germfree mice. See also Breeding colony; Gnotobiotics altered Schaedler’s flora introduced to, 3:228–229 aseptic hysterotomy derivation of, 3:226 bacterial “cocktails” given to, 3:227–228 cecal volume in, 3:219, 3:227, 3:482 colonization resistance, 3:228
CUMULATIVE
Germfree mice (continued) defined flora associated with, 3:227–229, 3:247 definition of, 3:218 derivation of, 3:226–227 donor females, 3:226–227 feces examination, 3:230 genetically altered, 3:220 historical highlights of, 3:219 immune system in, 3:219 long-term maintenance of, 3:219 mating of, 3:226 opportunistic organisms and, 3:228 pups, 3:251–252 rearing of, 3:219 Germinal centers architecture of, 4:93 B cells differentiation of, 4:94 selection of, 4:95–96 IgE, 4:295 involution of, 4:96 kinetics of, 4:93 stromal support for, 4:93–94 Gerontological research advances in, 3:639 mechanism studies, 3:661–666 mouse models for, 3:639 overview of, 3:639 terminology associated with, 3:639–644 Gestation. See also Pregnancy description of, 3:103 energy requirements during, 3:327, 3:329 Gestational age, 1:167 Ghrelin description of, 3:128 food intake affected by, 3:184–185 Ghrl, 3:128 Giardia muris cell biology, 2:520–521 clinical features, 2:521 diagnosis, 2:521 life cycle, 2:520 morphology, 2:520 prevention, 2:521 research implications, 2:521, 2:523 taxonomy, 2:520 treatment and control, 2:521 Giemsa staining, chromosomes, 1:148 GIRK1, 3:574 GIRK2, 3:574 Girk2, 3:676 Girk2Lc, 3:677 Girk2wv, 3:676 Glandula adrenalis dextra, 3:17 Glandula adrenalis sinister, 3:17 Glandula ampullaris, 3:19 Glandula bulbourethralis, 3:18–19 Glandula clitoridis, 3:20–21 Glandula coagulationis, 3:19 Glandula lacrimalis extraorbitalis, 3:4 Glandula preputialis, 3:18–19 Glandula sublingualis, 3:8 Glandula vesiculosa, 3:18–19
INDEX
Glandular lacrimalis extraorbitalis et eius ductus, 3:5 Glandular mandibularis, 3:5, 3:8 Glandular parotidea, 3:5, 3:8 Glaucoma anatomy of, 3:601–602 characteristics of, 3:601 developmental, 3:603 genetic factors, 3:603–604 histologic findings, 3:604 in humans, 3:601 intraocular pressure, 3:601 myocillin and, 3:604–605 physiology of, 3:601–602 pigmentary forms of, 3:605–606 primary open angle, 3:604–605 stochastic factors, 3:603–604 Glomerular filtration rate, 3:73 Glomerulonephritis, 3:334 Glossopharyngeus nerve, 3:7 Gloves isolator, 3:221 latex, 3:313 nitrile, 3:314 personal protective equipment uses of, 3:313–314 Glucagon receptor, 1:309 Glucocorticoid-inducible tumor necrosis factor receptor, 4:277 Glucocorticoids adipose differentiation and, 3:618 description of, 3:130–131 erythroblast expansion affected by, 3:142 Gluconeogenesis, 3:182 Glucose determinations of, 3:183 function of, 3:182 insulin effects on, 3:182–183, 3:618 nonfasting level of, 3:655 plasma levels of, 3:183 reference range for, 3:181 Glucose tolerance tests, 3:183–184 Glue boards, 3:429–430 GLUT-1, 3:183 GLUT-7, 3:183 Glutamate, 3:584 Glutamate dehydrogenase, 3:200 Glutamic oxaloacetate transaminase, 3:198 Glutamic pyruvic transaminase, 3:198 Glutathione S-transferase, 1:312 Gluteus medius, 3:6 Gluteus superficialis, 3:4, 3:6 Glycerol-phosphate, 3:187 Glycogen synthase kinase 3ß, 4:67 Glycogenolysis, 3:182 Glycolipids, 4:141 Glycosylphosphatidylinositol, 4:3, 4:110 Gnotobiote, 3:218 Gnotobiotic animal, 3:218 Gnotobiotics. See also Germfree mice application of, 3:220 Association for Gnotobiotics, 3:232 autoclaves, 3:225 definition of, 3:218 historical highlights of, 3:218–220
CUMULATIVE
343
INDEX
Gnotobiotics (continued) isolators. See Isolators programs in, 3:219 Goblet cells, 3:66 Gonad development, 3:92–93 Gonadal sex, 3:92 Gonadotrophin-releasing hormone description of, 3:94 luteinizing hormone secretion affected by, 3:201 Good manufacturing practices, 3:735, 3:751 Gpnmb, 3:605 G-protein-coupled receptor kinases, 1:310 Graafian follicle description of, 3:99 granulosa cells of, 3:202 Gracilis, 3:7 Graft-versus-host disease, 4:283 Granulocyte macrophage colony stimulating factor, 3:148 Granulocyte-colony stimulating factor description of, 3:148 splenic erythropoiesis affected by, 3:163 Granulopoiesis, 3:163 Granulosa cells, 3:99 Grb2/RasGRP, 4:216 Green fluorescent protein, 3:506, 3:624 Grey lung agent, 2:454 Grid2, 3:676 Griseofulvin, 2:511 GRKs. See G-protein-coupled receptor kinases GRO, 3:192 Gross energy, 3:324 Ground diet, 3:354 Growth energy requirements for, 3:328–329 protein requirements for, 3:332 Growth factors atherosclerosis affected by, 3:545 leukocytes affected by, 3:153 neuronal, 3:677 receptors for, 3:677 Growth hormone description of, 3:127 reference ranges for, 3:181 transgenic mouse phenotype, 1:309 Growth hormone secretogogue receptor, 3:128 Growth rate, of house mice, 1:31–32 GST. See Glutathione S-transferase GTPase activating protein, 4:200 Guanine nucleotide exchange factor, 4:199, 4:216 Guard hairs, 3:695 Guidelines for Environmental Infection Control in Health Care Facilities, 3:423, 3:427 Gulo, 3:550 Gut description of, 3:65–66 embryogenesis of, 1:177 Gut-associated lymphoid tissue, 3:369
H H antigen, 2:367–369 Habitat selection, house mice, 1:37–38 Haemophilus influenzaemurium. See Pasteurellaceae Hair regrowth of, as aging biomarker, 3:655 shedding of, 3:708 Hair bonnets, 3:313 Hair cone, 3:698 Hair follicles anagen phase of, 3:706–708 catagen phase of, 3:706–708 cilia, 3:696–697 cuticle layers of, 3:695 cycling wave of, 3:710 development of bone morphogenetic proteins, 3:701–702 differentiation phase, 3:701 epithelial invagination, 3:699, 3:701 epithelial-mesenchymal interactions, 3:699 follicular formation, 3:701 inductive phase, 3:698–699 morphogenetic phase, 3:699–701 patterning, 3:701 exogen phase of, 3:706, 3:708 genital, 3:697 growth patterns of, 3:705–707 molecular signaling of, 3:701–705, 3:708–709 morphology of description of, 3:694–697 stages, 3:697–698 mystacial, 3:694, 3:705 in nude mice, 3:702–703 patterning of, 3:701 pelage, 3:694–696 perianal, 3:697, 3:711 pluripotent neural crest stem cells, 3:710 research of, 3:693 stem cells, 3:693, 3:709–710 steroid hormone effects on, 3:709 tail, 3:697 telogen phase of, 3:706, 3:708–709 tissue types, 3:694 vibrissae, 3:694, 3:696, 3:705 Hair germ, 3:698 Hair peg, 3:698 Hairless mice. See also Nude mice bedding considerations for, 3:241, 3:306 production index for, 3:260 Halogens, 3:309 Handling, 3:439–440, 3:529 Hantavirus, 3:391, 3:747 Haploid spermatids, 3:96–97 Haplotype analysis, 3:627–628 Hardwood bedding, 3:306 Harem breeding system description of, 3:252, 3:254, 3:258 males in, 3:261 Hassall’s corpuscles, 4:63
Hazardous agents biological, 3:280 classification of, 3:280 containment of, 3:279–281 radionuclides, 3:280–281 volatile agents, 3:280 waste as, 3:280 HCB, 3:346 α-HCH, 3:346 ß-HCH, 3:346 H2-DM, 4:20 H2-DO, 4:20 Head, 3:5 Hearing, 3:106 Heart anatomy of, 3:8–11, 3:27–29 atria of, 3:27 blood collection from, 3:455–456 conduction system of, 3:27 coronary arteries of, 3:27–28 coronary veins of, 3:28 description of, 3:26–27 echocardiography assessments, 3:46–48 electrical activity in, 3:37 embryogenesis, 1:176–177 fibrous connective tissue of, 3:27 layers of, 3:27 magnetic resonance imaging of, 3:45 mineralization of, 3:338 myocytes of, 3:29 necropsy evaluations, 3:483 size of, 3:26 three-dimensional reconstructions of sectioned embryos, 1:203–204 valves of, 3:27 ventricles of, 3:27 Heart failure, 3:553 Heart rate activity-based variations in, 3:40 body temperature effects on, 3:37 description of, 3:39–40 during exercise, 3:40 exercise stress responses, 3:40 radiotelemetry measurement of, 3:35 resting, 3:39–40 Heat adaptation, 3:69 Heating, ventilation, and air-conditioning system description of, 3:272–273, 3:288–290 maintenance of, 3:413 Heavy metals, 3:345–347 Hedgehog signaling, 3:711–712 Heinz bodies, 3:144 Helicobacter spp. classification, 2:409–410 colony management in prevention, 2:429–430 description of, 3:392, 3:397, 3:552 diagnosis culture, 2:427 enzyme-linked immunosorbent assay, 2:427–428 histology, 2:428 polymerase chain reaction, 2:427 epizootiology, 2:426–427
344 Helicobacter spp (continued) genomic diversity analysis, 2:428 H. bilis, 2:417–418 H. felis, 2:424 H. ganmani, 2:419 H. hepaticus history of study, 2:426 oxidative stress and cytotoxicity biomarkers, 2:416–417 pathogenesis, 2:410, 2:412 susceptibility of host, 2:412–415 tumorigenesis mechanisms, 2:415–416 H. mastomyrinus, 2:420–421 H. muricola, 2:420 H. muridarum, 2:419–420 H. pylori, 2:408, 2:424–425 H. rappini, 2:421 H. rodentium, 2:419 H. trogontum, 2:424 H. typhlonius, 2:419 history of study, 2:409–410 host range and tissue tropism, 2:409 human disease, 2:408 human infection, 2:729 mouse models gastric cancer, 2:425–426 gastritis, 2:424–425 hepatitis and hepatic cancer, 2:421–422 inflammatory bowel disease, 2:422–424 overview, 2:410 phylogenetic relationships, 2:408 properties of mouse isolates, 2:410–411 research-confounding effects, 2:426 sentinel mice in monitoring, 2:430 treatment, 2:428 Heligmosomoides polygyrus, 1:45, 2:559 Helminths. See also specific organisms cestodes, 2:559–561 description of, 4:308 human infection, 2:732–733 nematodes, 2:559 oxyurids, 2:552–556 Helper T cells cytokines secreted by, 4:91–92 description of, 3:189, 4:40–41, 4:89–90 Hemacytometer, 3:140 Hemagglutination description of, 3:746 inhibition assays, 3:396 Hemangioblasts description of, 3:158 erythroid cells derived from, 3:158–159 Hematocrit aging biomarker use of, 3:655 in aging mice, 3:161 definition of, 3:144 parameters for, 3:141 phlebotomy effects on, 3:138 Hematologic analyzers, 3:140, 3:142, 3:146 Hematologic tests artifacts associated with, 3:138 automated analyzers, 3:140, 3:142 clinical descriptive terms, 3:137 factors that affect, 3:136 guidelines for, 3:136
CUMULATIVE
Hematologic tests (continued) manual methods, 3:139–140 microhematocrit tube, 3:140 parameters used to assess, 3:141 reference intervals for, 3:136 results of, 3:136–137 statistical evaluation, 3:137 Hematology of aging mice, 3:161–162 information resources on, 3:135 overview of, 3:135 preanalytical variables, 3:135–136 textbook resources on, 3:135 of young mice, 3:161 Hematopoiesis definitive characteristics of, 3:160 description of, 3:135, 3:158 description of, 4:60 primitive description of, 3:135, 3:158 erythroid cells in, 3:158–159 onset of, 3:158 spleen’s role in, 3:162–163 stages of, 3:135 Hematopoietic stem cells description of, 3:641, 3:666, 4:33, 4:60 T cell precursor derivation from, 4:63 Hemicelluloses, 3:335 Hemispherium cerebri, 3:7 Hemizygous matings, 3:260 Hemoglobin concentration of, 3:141, 3:144 fetal, 3:160 α-globins, 3:160 mean cell, 3:141, 3:144 switching of, 3:160–161 in young mice, 3:161 Hemoglobin genes, 3:160–161 Hemolysis during blood collection, 3:182 ex vivo, 3:148 extravascular, 3:148 in vivo, 3:148 intravascular, 3:148 overt, 3:139 phenylhydrazine-induced, 3:163 red cell mass decreases caused by, 3:147–148 Hemolytic uremic syndrome, 2:366 Hemorrhage, 3:147. See also Bleeding Hemostasis description of, 3:157 parameters for, 3:141 platelet’s role in, 3:155 tests for evaluating, 3:157–158 Hepar-lobus dexter lateralis, 3:14 Hepatectomy, 3:466 Hepatitis Helicobacter hepaticus model, 2:422 lymphocytic choriomeningitis virus model, 2:194–196 murine cytomegalovirus myocarditis model, 2:21–22 Hepatitis virus. See Mouse hepatitis virus
INDEX
Hepatocytes bile acids secreted by, 3:206 description of, 3:65 Heptachlor, 3:346 Hereditary hemochromatosis, 4:121 Hermaphroditism, 2:634 Herpesviruses. See Mouse cytomegalovirus; Mouse thymic virus Heterogeneous lines, 3:648 Hexamethylene tetramine, 3:224 High performance liquid chromatography, 1:292 High-density lipoproteins description of, 3:65, 3:186 measurement of, 3:188 High-efficiency particulate air filters advantages of, 3:412 description of, 3:245, 3:300 evaluation of, 3:421 integrity of, 3:421 High-level disinfectants, 3:424 High-resolution x-ray computed tomography, 3:43–44 Hindlimb muscles of, 3:6–7 regions of, 3:2 Histamine, 3:369–370 Histidine, 3:333 H2-M4, 4:129 Holding rooms for behavior test, 3:529 description of, 3:274–276 lighting in, 3:292 temperature in, 3:291 Homeostasis failures, 3:675–677 Homeostatic chemokines, 3:195 Homologous recombination, 1:262–263 Homovanillic acid, 3:73 Homozygosity, 3:649 Horizontal flow mass air displacement units, 3:300, 3:302 Hormone(s) androgens, 3:130 binding of, 3:124 calcitonin, 3:129 1,25-dihydroxy vitamin D3, 3:129–130 estradiol, 3:202 estrogen atheroprotective effects of, 3:551 definition of, 3:130 preovulatory increase in, 3:103 follicle stimulating description of, 3:94, 3:201–202 plasma levels of, 3:202 reference ranges for, 3:181 secretory pattern for, 3:202 spermatogenesis initiation by, 3:97 ghrelin, 3:128 glucocorticoids, 3:130–131 growth hormone, 3:127 insulin-like growth factor-I, 3:127 insulin-like growth factor-II, 3:127–128 leptin, 3:130 luteinizing blood concentration affected by, 3:201
CUMULATIVE
345
INDEX
Hormone(s) (continued) α-chain of, 3:201 ß-chain of, 3:201 functions of, 3:201 reference ranges for, 3:181 secretory pattern for, 3:202 testosterone synthesis promoted by, 3:202 mechanism of action, 3:124–125 oxytocin, 3:203–204 parathyroid, 3:205 parathyroid hormone, 3:128–129 parathyroid hormone-related peptide, 3:128–129 progesterone, 3:203 prolactin, 3:203 renal regulation of, 3:70 testosterone. See Testosterone thyroid stimulating hormone, 3:128 thyroxine, 3:204–205 triiodothyronine, 3:204–205 types of, 3:124 vasopressin, 3:204 Hormone replacement therapy, 3:551–552 Hormone response elements, 3:125 Host cell proteins, 3:751 House flies, 3:429 Housing. See Cage Howell-Jolly bodies, 3:143 Hox homeobox signaling, 3:703, 3:717–718 Hoxa3, 4:64–65 Hoxc13, 3:703, 3:714 Hoxc13neo/Hoxc13neo, 3:704 Hoxd13, 3:622 HPLC. See High performance liquid chromatography H2-Q region, 4:126–127 Hr, 3:703 HS-40, 3:160 H2-T region, of Mhc, 4:127–128 Human ß-defensin-1, 4:32 Human interactions, 3:109 Human killer cell immunoglobulin-like receptors, 4:170 Humerus, 3:4 Humidity diet affected by, 3:361 relative, 3:291 reproduction affected by, 3:107 research facility environment, 3:291 Humoral immunity. See B cell Huntingtin, 3:680 Huntington’s disease, 3:680 HUS. See Hemolytic uremic syndrome HVAC system. See Heating, ventilation, and air-conditioning system Hyaline cartilage, 3:50 Hyaloid artery, 3:599 Hyaloid vascular system, 3:599–600 Hybrid strains authenticity of, 3:266–267 breeding, 1:65–66 definition, 1:82–83 F1, 3:258
Hybrid strains (continued) genetic quality control for, 3:266–267 nomenclature, 1:83 Hydrobromic acid, 3:461 Hydrocephalus, C57BL mice, 2:647 Hydrochloric acid corrosion caused by, 3:418 in drinking water, 3:308, 3:418 Hydrodynamic injection, gene delivery, 1:270 Hydrogen peroxide peracetic acid and, for isolator sterilization, 3:224 vaporized, 3:310 Hydrographs, 3:413 Hydroperoxyeicosatetraenoic acid, 3:186 6-Hydroxydopamine, 3:682 5-Hydroxyperoxyeicosatetraenoic acid, 3:197 Hymenolepis diminuta features and management, 2:560–561 human infection, 2:732 Hypercalcemia, 3:208 Hyperchromasia, 3:146 Hypergammaglobulinemia, 3:208 Hyperglycemia, 3:183 Hyperlipoproteinemia, 3:538 Hypermorphosis, 3:640–642 Hyperthermia, 3:69 Hypertrophic cardiomyopathy, 3:39 Hypoalbuminemia, 3:206 Hypochromasia, 3:146 Hypocretin-1, 3:184 Hypocretin-2, 3:184 Hypoglossus nerve, 3:7 Hypoglycemia, 3:183 Hypophysis, 3:7 Hypothalamus, 3:7, 3:94 Hypothermia anesthetic use of, 3:464–465 description of, 3:68 postoperative, 3:479 Hysterectomy derivation of germline mice, 3:112–113, 3:226 technique for, 3:467–468 Hysteresis, 3:55–56 Hysterotomy description of, 3:467–468 germfree mice derivation, 3:226
I 123I,
3:503 124I, 3:503 125I, 3:503 131I, 3:503 IAP. See Intracisternal A particles IBD. See Inflammatory bowel disease IC. See Inspiratory capacity ICH. See International Conference on Harmonization ICM. See Inner cell mass ICR191, embryonic stem cell mutagenesis, 1:255–256
ICSI. See Intracytoplasmic sperm injection Identification methods description of, 3:265–266, 3:440, 3:661 permanent, 3:441–443 semipermanent, 3:441 subcutaneous radio frequency transponders, 3:266, 3:441–443 temporary, 3:440–441 Iditol dehydrogenase, 3:200 IFA. See Immunofluorescence assay Igf1, 3:127 Igf2, 3:127–128 Ileum anatomy of, 3:15 catheterization of, 3:478 Iliaci externi lymph nodes, 3:22 Iliacus internus lymph node, 3:22 Iliofemoralis lymph node, 3:22 Illness, 3:387–388 Imaging advantages of, 3:491–492 anesthesia for, 3:492 bioluminescence, 3:506–507 computed tomography. See Computed tomography contrast agents used in, 3:492 cross-contamination issues, 3:493 data handling, 3:493 disadvantages of, 3:492 fluorescence, 3:506–508 future of, 3:509 general features of, 3:490–491 high-resolution x-ray computed tomography, 3:43–44 image characteristics, 3:490–491 infection control considerations, 3:493 logistical considerations, 3:492–493 longitudinal, 3:509 magnetic resonance imaging. See Magnetic resonance imaging magnetic resonance spectroscopy. See Magnetic resonance spectroscopy microCT scanners, 3:43–44, 3:494, 3:497 modalities for, 3:490–491 monitoring during, 3:492–493 multimodality, 3:509 noninvasive methods of, 3:43–48 nuclear. See Nuclear imaging optical, 3:506–508 overview of, 3:489–490 positioning for, 3:493 summary of, 3:509 ultrasound. See Ultrasound x-ray, 3:493–494 x-ray computed tomography, 3:493–494 Imaging facilities, 3:282–283 Immature dendritic cells, 4:144–145 Immune complexes, circulating, 3:191 Immune response, 4:87–88 Immune system adaptive cells of, 4:38–48 description of, 4:3 gastrointestinal tract’s role in, 3:66
346 Immune system (continued) innate cells of, 4:32–38 description of, 4:3 ligands of, 4:3–15 receptors of, 4:3–15 mutant phenotyping, 1:247 nutrition effects on, 3:369 Immunity, 3:370–371 Immunoassays chemiluminescent, 3:176 electrochemiluminescent, 3:176 fluoroimmunoassay, 3:176 immunoglobulin quantification using, 3:189–190 multiplex, 3:176, 3:178–179 viral contamination detected using, 3:746–747 Immunodeficiency, 3:389 Immunofluorescence antibody testing, 3:396 Immunofluorescence assay Clostridium piliforme diagnostics, 2:354 lymphocytic choriomeningitis virus diagnostics, 2:203–204, 2:722 mouse thymic virus diagnostics, 2:34 mousepox virus diagnostics, 2:160–161 Immunoglobulins classes of, 3:189 composition of, 3:189 in germfree mice, 3:219 IgA, 4:23 IgD, 4:23 IgE, 4:23, 4:294–295 IgG, 3:189, 4:22–23, 4:263–264 IgG2b, 4:8, 4:22 IgM, 4:8, 4:23 immunoassay quantification of, 3:189–190 membrane-anchored, 4:23 prenatal transfer of, 3:189 structure of, 4:43–44 Immunologic senescence, 3:642 Immunoreceptor tyrosine-based activation motifs, 4:83, 4:199, 4:215 Immunoreceptor tyrosine-based inhibitory motif, 4:6, 4:262 Immunoreceptor tyrosine-based switch motif, 4:28 Impressio duodenalis, 3:16 Impressio esophagica, 3:16 Impressio jejunalis, 3:16 Impressio ventricularis, 3:16 Imprinting chromosomal anomalies, 1:95 mutations, 1:80 111In, 3:503 In vitro fertilization principles, 1:212–213 rederivation, 1:220–221 Inbred strains. See also specific strains advantages of, 3:645–646 aging research using, 3:645–646, 3:648 atherosclerosis models, 3:536 attributes, 2:661 authenticity of, 3:266–267 breeding, 1:62–63, 3:253–254
CUMULATIVE
Inbred strains (continued) definition, 1:82, 2:628 description of, 3:105 development of, 3:173, 3:645 disadvantages of, 3:646 genetic quality control for, 3:266–267 nomenclature, 1:83 recombinant, 1:71, 1:84, 3:173, 3:629–630 record keeping information, 3:262 Incipient inbred, 2:628 Incisura cardiaca, 3:13 IND. See Investigational New Drug application Indexing washers, 3:302–303, 3:310 Indian hedgehog, 3:159 Indirect calorimetry, 3:325 Individually ventilated cage, 3:295–299, 3:412 Indocyanine green, 3:206 Inducible costimulator, 4:28 Inducible costimulator ligand, 4:28 Inducible mutagenesis, 1:265 Indwelling fluid-filled catheters, 3:33–34 Infanticide, 3:109 Infectious diseases atherosclerosis and, 3:552 from commercial vendors, 3:401–402 dietary influences on, 3:369–371 eradication of, 3:403–404 leukocyte response to, in aging mice, 3:162 monitoring for, 3:391–392 quality assurance programs to prevent, 3:389 from research colonies, 3:402–403 sources of, 3:401–403 types of, 3:391–392 from vermin, 3:402 Infectious ectromelia. See Mousepox Infectivity assays, 3:749 Inflammation eosinophils in, 4:34 interleukin-1 modulation of, 3:191 leukocytes affected by, 3:153 modulation of, 4:7 Inflammatory bowel disease, 2:422–424 Inflammatory chemokines, 3:195 Inflammatory diseases, 3:149 Inflammatory mediators, 3:545 Inflammatory responses, 3:62, 4:295 Infraspinatus, 3:5 Inguinalis superficialis lymph node, 3:22 Inhalant anesthesia, 3:463–464 Inhibitory receptors B cell activation thresholds and, 4:267–268 classes of, 4:262–263 description of, 4:262 natural killer cell receptors, 4:268 pathways of, 4:269–270 T cell, 4:268–269 Injectable anesthetics, 3:461–463 Injections contrast agents, 3:492 external jugular vein, 3:451–452
INDEX
Injections (continued) footpad, 3:453 intra-arterial, 3:451 intracranial, 3:452 intramuscular, 3:445, 3:450 intraosseous, 3:452 intraperitoneal, 3:450–451 intrathecal, 3:447 intrathoracic, 3:451 lateral tail vein, 3:451 retro-orbital, 3:452 subcutaneous, 3:449–450 Innate immune system activation of, 4:87 cells of, 4:32–38, 4:109 description of, 4:3 ligands of, 4:3–15 receptors of, 4:3–15 Inner cell mass, 1:186, 1:188, 3:102 Insect growth regulators, 3:430 Insecticides, 3:430 Insensible water loss, 3:76 Insertio musculi sartorii, 3:7 Insertio pleurae, 3:12 Inspiration, 3:52 Inspiratory capacity, 3:53, 3:57 Inspiratory reserve volume, 3:53–54 Insulin adiponectin and, 3:184 epinephrine effects on, 3:183 food intake regulated by, 3:618 glucose release rate affected by, 3:182–183 hormones that affect, 3:183 hypoglycemic response to, 3:183 nonfasting level of, 3:655 visfatin and, 3:184 Insulin receptor substrate-2, 3:184 Insulin resistance analytes involved with, 3:185 inducement of, 3:184 Insulin-like growth factor-I, 3:127 Insulin-like growth factor-I receptor, 3:127 Insulin-like growth factor-II, 3:127–128 Insulin-like growth factor-II receptor, 3:127–128 Integrated pest management program, 3:428–429 Integrins ß-, 3:160 clustering of, 4:196 description of, 4:196 knockout models, 4:201 leukocyte, 4:296–297 signaling, 4:203–204 T cell receptor-mediated activation defects, 4:199–201 Intercellular adhesion molecule-1, 4:81 Intercostales dorsal, 3:9 Interdigital pads, 3:715 Interferon(s) -α, 3:195 -τ, 3:187, 3:195, 4:30–31, 4:172 description of, 3:194–195 lactate dehydrogenase-elevating virus response, 2:222–223
CUMULATIVE
347
INDEX
Interferon(s) (continued) lymphocytic choriomeningitis virus persistence role, 2:201–202 mammalian reovirus response, 2:257 mouse adenovirus type 1 response, 2:59 mouse hepatitis virus response, 2:155 murine cytomegalovirus response, 2:27–28 Sendai virus response, 2:292–295 Interferon regulatory factor-2, 4:143 Interferon regulatory factor 5, 4:114 Interfollicular skin, 3:712–713 Interleukin-1 characteristics of, 3:191–192 description of, 4:30 inflammation modulation by, 3:191 murine cytomegalovirus response, 2:28 Interleukin-1 receptor, 3:191–192 Interleukin-1 receptor-associated kinase, 4:113 Interleukin-2, 3:192, 4:30, 4:170 Interleukin-2 receptor, 4:42 Interleukin-3, 3:150, 3:192, 4:30 Interleukin-4, 3:150, 3:192, 4:30, 4:43, 4:294 Interleukin-5, 3:150, 3:192, 4:30, 4:34, 4:297 Interleukin-6 characteristics of, 3:192 description of, 4:30, 4:93 insulin resistance induced by, 3:184 murine cytomegalovirus response, 2:28 Interleukin-7 deficiency of, 4:279 description of, 3:192, 4:30, 4:74 Interleukin-8, 3:192, 4:295 Interleukin-10, 3:192, 4:30, 4:236 Interleukin-11, 3:192–193 Interleukin-12, 3:150, 3:193, 4:30, 4:41, 4:144, 4:284, 4:294 Interleukin-13, 3:193, 4:30, 4:294 Interleukin-15 deficiency of, 4:279 description of, 4:30, 4:173 Interleukin-17, 3:193, 4:42 Interleukin-18, 3:193 Interleukin-20, 3:193 Interleukin-21, 3:193 Interleukin-23, 4:41 Intermediate-density lipoprotein, 3:188 International Conference on Harmonization, drug development role, 1:298–299 International System of Units, 3:139, 3:324 Interstitial cells of Cajal, 3:65 Interstitial dendritic cells, 4:36 Interstitial pneumonitis, murine cytomegalovirus myocarditis model, 2:20–21 Intertransversarii, 3:7 Intestinal alkaline phosphatase, 3:198 Intestinal crypts, 4:69 Intestinal loops, 3:468 Intestine anatomy of, 3:65–66 motility in, 3:65
Intestine (continued) necropsy evaluations, 3:483 small, 3:65–66, 3:483 Intestine roll, 3:483 Intra-arterial injection, 3:451 Intrabronchial administration, 3:449 Intracisternal A particles classification, 2:276 description of, 3:737 insertional mutagenesis, 2:272 Intracranial administration, 3:452 Intracrine hormones, 3:124 Intracytoplasmic sperm injection principles, 1:213–214 somatic nuclear transfer, 1:221–222 steps, 1:214 transgenic mouse preparation, 1:216 Intradermal administration, 3:449 Intragastric administration, 3:445–447 Intragastric catheters, 3:446 Intramuscular injection, 3:445, 3:450 Intranasal administration, 3:449 Intranodal administration, 3:452–453 Intraocular pressure, 3:601 Intraosseous injection, 3:452 Intraperitoneal administration, 3:445, 3:450–451 Intrasplenic administration, 3:453 Intrathecal catheterization, 3:479 Intrathecal injection, 3:447 Intratracheal administration, 3:449 Intravascular administration, 3:451–452 Intravascular hemolysis, 3:148 Intraventricular pressure, 3:30 Introitus vaginae, 3:3 Inulin, 3:73 Invertebrate pests, 3:429 Investigational New Drug application, drug development, 1:299 In-vivo imaging. See Imaging Iodine daily requirements for, 3:336 lability of, 3:337 Iodophors, 3:425 Ionizing radiation, 3:453–454 Iron daily requirements for, 3:336 reference range for, 3:181 Irradiation ionizing radiation, 3:453–454 of diet autoclaving of diet vs., 3:366 description of, 3:361, 3:363–364 dosages for, 3:364 factors that affect, 3:364 microorganisms affected by, 3:364–365 nutrient effects, 3:365–366 packaging, 3:364 process involved in, 3:364 reviews of, 3:364 of drinking water, 3:372 of parasites, 3:364 IRV. See Inspiratory reserve volume Ischiocavernosus, 3:19 Isoelectric focusing, 1:228
Isoflavones, 3:348 Isoflurane, 3:31, 3:463, 3:492 Isolated heart preparation, 3:29–30 Isolated lymphoid follicles, 4:69 Isolators air supply in, 3:223, 3:245 ancillary components, 3:221 animal manipulation inside of, 3:221 bacterial contamination of, 3:230–231 components of, 3:220–221 contamination of, 3:230–232 definition of, 3:220 ergonomics of, 3:225–226 flexible film, 3:219–220, 3:245 germicidal dip tank with, 3:223 gloves attached to, 3:221 HEPA filtration, 3:245 history of, 3:218–219 inspection of, 3:230 leakage testing of, 3:223 monitoring of, 3:231–232 positive pressure in, 3:223 PVC flexible film, 3:220–221 quarantining of animals in, 3:278 research facility use of, 3:299 semirigid, 3:220–221, 3:245, 3:299–300 sleeve length of, 3:225 stainless steel, 3:219, 3:221 sterilization of chemical, 3:223–225 chlorine dioxide for, 3:224 documentation of, 3:224 formaldehyde gas for, 3:224 hydrogen peroxide and peracetic acid for, 3:224 irradiation, 3:225 peracetic acid for, 3:223–224 steam, 3:225, 3:279 supply cylinders used with, 3:222–223 transfer port used with, 3:221–222 viral contamination of, 3:231 Isoleucine, 3:333 Itk, 4:200, 4:203 Itraconazole, 2:511 Ivermectin description of, 3:403 Heligmosomoides polygyrus management, 2:559 Myobia musculi management, 2:571–572 Myocoptes musculinus management, 2:574 pinworm management, 2:557–558 Polyplax serrata management, 2:568 IVF. See in vitro fertilization
J J chains, 4:6 Jacket, 3:443, 3:448 Jackson Laboratory, 1:4, 1:7–8 Jak3, 4:74 JAK3 tyrosine kinase, 4:279 Jak1-deficient mice, 4:183 Jak2-deficient mice, 4:183 Jak3-deficient mice, 4:183–184
348
CUMULATIVE
JAK-STAT pathway cytokine-activated, 4:180–181 regulation of, 4:181–183 signaling of, 4:183–189 JAM-A, 4:201 jams1, 3:571, 3:580 Januse kinases, 4:180, 4:183–184 Jejunum anatomy of, 3:15 catheterization of, 3:478 jerky, 3:571 Jerky mice, 3:571 Joule, 3:324 Jugular vein blood collection from, 3:179, 3:456–3:457 catheterization of, 3:475–476 injection into, 3:451–452 Juvenile monoclonic epilepsy, 3:570 Juxtacrine hormones, 3:124 Juxtaglomerular apparatus, 3:70
K Kainic acid-induced epileptic seizures, 3:577–578 Kallikrein-kinin system, 3:71 Karyotyping, 3:750 KCNA1, 3:568 Kcnj10, 3:577 K14–Cre mice, 3:699 K14–Cre(neo) mice, 3:699 K14–deltaN87 mice, 3:699 Keratin, 3:712 Keratinocytes, 4:32 Ketamine, 3:462 Ketamine-xylazine-acepromazine, 3:31 Ketogenic diet, 3:582–583 Ketone bodies, 3:583 Kidney(s) anatomy of, 3:17 autoimmune diseases that affect, 3:74 blood supply to, 3:74 clearance by, 3:73–74 diuretics response by, 3:74 embryologic development of, 3:70 function of, 3:69–70 function tests, 3:206–207 genetic abnormalities of, 3:74 genetic factors that affect, 3:72 glomerular volume of, 3:71 hormone regulation by, 3:70 juxtaglomerular apparatus of, 3:70 mineralization of, 3:338 morphophysiology of, 3:70–71 nephrectomy, 3:468 nephrons of, 3:70–71 polycystic, 3:74 prostaglandins, 3:71–72 protein-induced damage to, 3:334 sexual dimorphism of, 3:72 transplantation of, 3:468 tubular system of, 3:70 vascular system of, 3:70–71, 3:74 weight of, 3:71 zones of, 3:71
Killer cell immunoglobulin-like receptors, 4:262 KK mice, 3:627 Klebsiella sp. clinical features, 2:380 diagnosis, 2:380 epizootiology, 2:380 history of study, 2:379–380 K. pneumoniae, 3:392 properties, 2:380 Klossiella muris cell biology, 2:533 clinical features, 2:533 diagnosis, 2:533–534 history of study, 2:533 life cycle, 2:533 prevention, 2:534 research implications, 2:534 treatment and control, 2:534 Knock-in mouse drug target study examples, 1:306–313 mutations, 1:92 Knockout mice description of, 3:173, 3:557 drug candidate screening using, 3:175 drug target study examples, 1:306–313 historical perspective, 1:8 integrin, 4:201 limitations of, 3:621–622 obesity studies using, 3:621–623 resources, 3:621 T cell migration, 4:197–198, 4:202 with T cell receptor-mediated integrin activation defects, 4:199–201 K14-NOG mice, 3:700, 3:714 KRT-14-cre, 3:699 KRT-14–Dkk1 mice, 3:699 Kv1.1. channel, 3:568 K-virus, 3:391
L LAA. See Laboratory animal-associated allergy Label-retaining cells, 3:709 Laboratory animal-associated allergy allergens, 2:736 clinical signs, 2:738–739 diagnosis, 2:737–738 incidence, 2:735–737 pathogenesis, 2:737 treatment and prevention, 2:738–739 Laboratory Code, 1:84 Lacrimal fluid collection, 3:459 Lactate dehydrogenase description of, 3:199 reference range for, 3:181 Lactate dehydrogenase-elevating virus classification, 2:216 control of, 2:228 description of, 3:180, 3:391, 3:400–401, 3:746–747 diagnosis enzyme-linked immunosorbent assay, 2:227–228
INDEX
Lactate dehydrogenase-elevating virus (continued) lactate dehydrogenase activity, 2:227 polymerase chain reaction, 2:227 history of study, 2:216 host range, 2:226 immune response autoimmunity, 2:225 B cells, 2:224 cytokines, 2:222–223 immunosuppression, 2:224–225 macrophages, 2:223 natural killer cells, 2:223 T cells, 2:223–224 lactate dehydrogenase clearance impairment, 2:221–222 morphology, 2:216 pathology, 2:222 persistence, 2:222 physicochemical properties of, 2:216–217 polioencephalomyelitis, 2:225–226 prevention of, 2:228 receptor, 2:221 replication cell culture studies, 2:220–221 kinetics, 2:220 RNA synthesis, 2:218 sites, 2:219–220 stability, 2:218–219 strains, 2:219 structure of, 2:216–217 transmission, 2:226–227 tumor studies, 2:226 Lactation energy requirements during, 3:327, 3:329 environmental temperature effects on, 3:330 milk composition, 3:329 Lactobacillus spp. description of, 3:227 L. murinus, 3:229 Lafora bodies, 3:570 Lafora disease, 3:570–571 Laforin, 3:570–571 Lamina propria, 3:49 Laminar flow equipment, 3:421 Langendorff-work-performing heart preparation, 3:29–30 Langerhans cells, 4:139–140, 4:309 Langerin, 4:14 Laparotomy pack, 3:465 Large intestine, 3:65–66 Laryngeus recurrens sinister, 3:10 Laryngeus recurrens sinister et dexter et trachea, 3:11 Laryngopharynx, 3:49 Larynx, 3:49–50 Late-onset neurodegenerative diseases Alzheimer’s disease, 3:683–685 description of, 3:682 Parkinson’s disease, 3:682–683 tauopathies, 3:683 Lateral tail vein blood collection from, 3:456–457 injection into, 3:451
CUMULATIVE
349
INDEX
Latex gloves, 3:313 Latissimus dorsi, 3:4, 3:6 LCMV. See Lymphocytic choriomeningitis virus L-Deprenyl, 3:666–667 LDV. See Lactate dehydrogenase-elevating virus Lead, 3:345 leaner, 3:676, 3:685 Least-cost formula diet, 3:355 Leber congenital amaurosis, 3:607, 3:609 Lecithin:cholesterol acyltransferase, 3:186 Lee-Boot effect, pheromone response, 1:54 Lef1 null mice, 3:699, 3:723 Left anterior descending coronary artery, 3:28 Left atrium, 3:27 Left circumflex artery, 3:28 Left ventricle anatomy of, 3:27 M-mode echocardiography uses, 3:46 pressure measurements, 3:36 Left ventricular mass, 3:44–45 Lentivirus, 1:273–274 Lep, 3:130 Lepob, 3:630 Lepr, 3:130 Leprdb, 3:130, 3:630 Leptin description of, 3:94, 3:130 interleukin-1ß modulation by, 3:184 knockout mouse phenotype, 1:308–309 obesity and, 3:184, 3:618 production of, 3:184 proopiomelanocortin stimulated by, 3:184 resistance to, 3:185 Leptin receptor, 3:618, 3:620 Leptospirosis clinical signs, 2:726 control, 2:726–727 diagnosis, 2:726 host range, 2:725 humans, 2:726 reservoir and incidence, 2:726 serovars, 2:725–726 transmission, 2:726 Lethal mutations, 1:59–60 Leucine, 3:333 Leucine-rich glioma-inactivated 1, 3:571 Leucine-rich repeat motifs, 4:3, 4:110 Leukemia inhibitory factor, 1:283 Leukocyte(s) in aging mice, 3:161–162 alterations in number of, 3:152–154 automated hematologic analyzer identification of, 3:140 basophils, 3:150, 4:35 derivation of, 4:32–33 description of, 3:148, 4:32–33 differential count of, 3:151 enumeration of, 3:151–152 eosinophils. See Eosinophil(s) excitement effects on, 3:152–153 inflammation effects on, 3:153 integrins, 4:296–297
Leukocyte(s) (continued) lymphocyte. See Lymphocyte(s) monocytes, 3:150-151, 4:33 morphology of, 3:151–152 neoplasia effects on, 3:153 neutrophils. See Neutrophil(s) peripheral blood, 4:283–284 protease release by, 3:195–196 stress effects on, 3:152–153 Leukocyte adhesion defects, 3:154 Leukocyte receptor complex, 4:174 Leukotriene A4, 3:197 Levator labii superioris proprius, 3:5 Levator nasolabialis, 3:5 Lewy body, 3:682 Leydig cells, 3:95 Lgr7, 1:307 Lice. See Polyplax serrata Lien, 3:14–3:17 LIF. See Leukemia inhibitory factor Life span as biomarker of aging, 3:652–653 data analysis, 3:656 intervention to increase, 3:666–668 long-lived models, 3:651 maximum, 3:657 of erythrocytes, 3:142, 3:147–148 of F1 hybrids, 3:646 senescence and, 3:655–656 short-lived models, 3:651–652 Ligamentum arteriosum, 3:9 Ligamentum coronarium sinistrum, 3:16 Ligamentum falciforme et ligamentum teres hepatis, 3:16 Ligamentum hepatorenale, 3:16 Ligamentum pulmonale, 3:13 Ligamentum triangulare sinistrum, 3:16 Ligamentum vesicae laterale, 3:18, 3:20 Ligamentum vesicae laterale dextrum, 3:21 Ligamentum vesicae laterale sinistrum, 3:21 Ligamentum vesicae medianum, 3:17-18, 3:20–21 Light cycle breeding affected by, 3:238 14L:10D, 3:106 reproduction affected by, 3:106, 3:238, 3:291 Light traps, 3:429–430 Light:dark cycle behaviors affected by, 3:524–525 description of, 3:291, 3:414 Light-emitting diodes, 3:414 Lighting color temperature of, 3:414 description of, 3:413–414 intensity of, 3:413 research facility environment, 3:291–292 Lignans, 3:335, 3:348–349 Limb amputation, 3:458 Limulus amebocyte lysate assay, 3:742 Lindane, 3:346 Line, 3:236 Line outbreeding system, 3:256 LINEs. See Long interspersed nuclear elements
Lingua, 3:15 Linkage mapping, cross backcross, 1:120–124 backcross-intercross, 1:123 guidelines, 1:120 intercross, 1:122–124 ordering loci in multipoint crosses, 1:124 penetrance, expressivity, or modifier loci, 1:124–125 strain selection databases, 1:116 description of, 1:120 disequilibrium mapping, 1:127 marker types, 1:117–120 mutant genes, 1:116–117, 1:241 quantitative trait loci, 1:126–127 Linoleic acid, 3:334 Linolenate, 3:334 Linolenic acid, 3:334 Lipase, 3:66 Lipids functions of, 3:185–186 metabolism of, enzymes involved in, 3:188–189 types of, 3:185–186 Lipohyaline, 3:74 Lipooxygenases, 3:186, 3:197 Lipopolysaccharides description of, 3:69, 4:5 strains sensitive to, 4:110 virulence factors, 2:367–369 Lipoprotein(s). See also Cholesterol description of, 3:65 high-density description of, 3:65, 3:186 measurement of, 3:188 low-density description of, 3:186 measurement of, 3:188 oxidized, 3:186–187, 3:550 trapping of, 3:186 metabolism of, 3:538 transport of, 3:537 very low-density description of, 3:185–186 measurement of, 3:188 Lipoprotein (a), 3:538 Lipoprotein lipase, 3:185 Liquid diet, 3:354–355 Listeria monocytogenes, 3:228 Lithium, 3:336 Litter consolidation of, in breeding colony, 3:261 handling of, 3:440 milk energy content based on size of, 3:329 size of, in house mice, 1:36–37 Little mouse, 3:620–621 Liver anatomy of, 3:16, 3:64–65 cholesterol production, 3:65 comparative embryology, 1:196 hepatectomy, 3:466 hepatocytes, 3:65
350 Liver (continued) mouse urinary protein production in, 3:71 necrosis of, 3:205 regeneration of, 3:64–65 urea production by, 3:206 Liver function tests, 3:205–206 LKB1, 3:183 LNNA. See Local lymph node assay LOBUND Institute, 3:219–230 Lobus accessorius pulmonis dextri, 3:12–13 Lobus caudalis pulmonis dextri, 3:12–13 Lobus caudatus, 3:14 Lobus caudatus hepatis, 3:16 Lobus cranialis pulmonis dextri, 3:12–13 Lobus dexter hepatis lateralis, 3:15, 3:17 Lobus dexter hepatis medialis, 3:15, 3:17 Lobus dexter medialis, 3:14 Lobus dexter medialis hepatis, 3:16–17 Lobus dexter medialis lateralis, 3:16 Lobus medialis pulmonis dextri, 3:12–13 Lobus pancreatis dexter, 3:14 Lobus pancreatis sinister, 3:14 Lobus sinister hepatis lateralis, 3:15 Lobus sinister hepatis medialis, 3:15 Lobus sinister lateralis hepatis, 3:16 Lobus sinister medialis, 3:14 Lobus sinister medialis hepatis, 3:16 Local anesthetics, 3:461 Local lymph node assay, 1:304 Locus variants, nomenclature, 1:91–92 Long interspersed nuclear elements, 1:104 Long terminal repeats, 1:104 Long-lived models, 3:651 Long-term potentiation, 3:579 Loop of Henle, 3:71 Lou Gehrig’s disease. See Amyotrophic lateral sclerosis Low-density lipoprotein description of, 3:65, 3:186 measurement of, 3:188 oxidized, 3:186–187, 3:550 trapping of, 3:186 Low-density lipoprotein receptor, 3:185, 3:187, 3:557 Low-density lipoprotein receptor-related protein-1, 3:187, 3:548 Lower respiratory tract, 3:50–51 Low-level disinfectants, 3:424 Lowry method, 3:208 LPS. See Lipopolysaccharide L-selectin, 4:77 LTßR, 4:73 LTRs. See Long terminal repeats Lumbales aortici lymph nodes, 3:22 Lumbosacrocaudalis dorsalis lateralis, 3:7 Lumbosacrocaudalis ventralis lateralis, 3:7 Lung(s) acini, 3:51 alveolar duct, 3:51 anatomy of, 3:10–12, 3:50–51 blood supply to, 3:51–52 comparative embryology, 1:193–194 diffusion capacity of, 3:55, 3:61 embryology of, 1:193–194
CUMULATIVE
Lung(s) (continued) function assessments airway pressure, 3:56 description of, 3:53 diffusion capacity of lungs, 3:61 ex-vivo techniques, 3:55–56, 3:60–61 forced oscillation technique, 3:59 invasive methods, 3:56–58, 3:60–61 in-vivo techniques, 3:56–61 noninvasive methods, 3:58–61 pulmonary function tests. See Pulmonary function tests function phenotyping, 1:245 gas exchange in, 3:50 high-resolution x-ray computed tomography of, 3:44 lateral view of, 3:12 lobes of, 3:50–3:51 medial view of, 3:13 murine vs. human, 3:51 parenchyma of, 3:51 Lung compliance dynamic, 3:56, 3:58 pressure-volume curves for estimating of, 3:55 Lung volumes, 3:53–55 Luteal development, 3:103 Luteinizing hormone blood concentration affected by, 3:201 chain of, 3:201 ß-chain of, 3:201 circadian effects on, 3:99 description of, 3:94 functions of, 3:201 ovulation and, 3:99 reference ranges for, 3:181 secretory pattern for, 3:202 spermatogenesis initiation by, 3:97 testosterone synthesis promoted by, 3:202 Luteotropin, 3:103 Ly-17, 4:265 Ly49 receptors, 4:14–15, 4:170, 4:175, 4:268 Lymph, 3:460 Lymph nodes B cell entry and exit from, 4:76–77 description of, 3:21–22 development of, 4:69–70 peripheral, 4:69 Lymphocyte(s) in aging mice, 3:162 B. See B cell(s) description of, 3:148 excitement effects on, 3:153 lymphoid organ entry of, 4:77 morphology of, 3:150–3:151 natural killer cells, 4:37–38 recruitment of, 4:75 signaling of, 4:254–255 T. See T cell(s) Lymphocyte function-associated antigen-1, 4:176, 4:196, 4:233, 4:296 Lymphocytic choriomeningitis virus autoimmune disease susceptibility, 2:203 behavioral effects, 2:203
INDEX
Lymphocytic choriomeningitis virus (continued) classification, 2:180–181 contamination of biological material, 2:202 control and prevention, 2:204 description of, 3:226, 3:391, 3:733-744, 3:747 diagnosis molecular detection, 2:204 serology, 2:203–204 genetic susceptibility, 2:197–198 history of study, 2:180 host range, 2:186 in humans clinical signs, 2:722 diagnosis, 2:722 incidence of, 2:721 overview, 2:186–187, 2:720–721 reservoir, 2:721 susceptibility, 2:722 transmission mode, 2:721–722 treatment, 2:722 immunosuppression, 2:202–203 in laboratory mice autoimmune disease in transgenic mice, 2:197 hematopoietic disorders, 2:196–197 hepatitis, 2:194–196 immunopathogenesis, 2:187–188 prenatal and neonatal infection endocrine disorders, 2:191 immune complex disease, 2:189–191 wasting disease, 2:194 major histocompatibility complex, 2:197–198 natural history, 2:184–185 persistence roles B cells, 2:200–201 dendritic cells, 2:199 interferons, 2:201–202 natural killer cells, 2:201 overview, 2:198–199 T cells CD4+, 2:200 CD8+, 2:199–200 propagation cells, 2:183 mouse bioassays, 2:184 replication, 2:181–182 safety, 2:204–205 strains antigenic and genetic relationships, 2:182 biologic differences, 2:183 transmission horizontal, 2:185 vertical, 2:185–186 virion structure, 2:181 Lymphohematopoietic cell lineages, 4:281–283 Lymphoid enhancing factor, 4:67 Lymphoid follicles B cell migration to, 4:78–82 description of, 4:78
CUMULATIVE
351
INDEX
Lymphoid organs bone marrow. See Bone marrow location of, 4:59 molecular regulation of, 4:70–75 overview of, 4:59 primary, 4:59–68 secondary anatomy of, 4:68 B cell zones in, 4:78 cryptopatches, 4:69 dendritic cells, 4:143 formation of, 4:79–80 immune response function of, 4:68 location of, 4:69 lymphatic drainage to, 4:76 lymphocyte entry into, 4:77 microdomains in, 4:78–82 natural killer cells in, 4:171 overview of, 4:68–70 Peyer’s patches, 4:69 spleen. See Spleen thymus. See Thymus Lymphoid tissue-inducing cells description of, 4:73 development of, 4:73–74 LTα1ß2 induction on, 4:74 recruitment of, 4:74 Lymphonodi axillary proprius, 3:6 Lymphonodi cervicales superficiales, 3:6 Lymphonodi colici, 3:15 Lymphonodi ileocolicus, 3:15 Lymphonodi mandibulares, 3:8 Lymphonodus axillaris accessorius, 3:5 Lymphonodus jejunalis, 3:15 Lymphonodus pancreaticoduodenalis, 3:15 Lymphonodus retropharyngeus lateralis, 3:8 Lymphonodus subiliacus, 3:3 Lymphopoietin-1, 3:192 Lymphotoxin, 3:194 description of, 4:279–280 Lymphotoxin receptor null mutants, 4:279–280 Lyn, 4:269 Lysine, 3:333 Lysophosphatidic receptor, 1:307
M M1, 4:128 M5, 4:129 M6, 4:129 M10, 4:128 Machado-Joseph disease, 3:681 Macrocytosis, 3:146 Macrophage(s) apoptosis of, 3:187 characteristics of, 4:33 FCγRI expression on, 4:7 from TLR2-/- mice, 4:110 in granulomatous processes, 4:33 lactate dehydrogenase-elevating virus response, 2:223 murine cytomegalovirus immune response, 2:27
Macrophage(s) (continued) pneumonia. See Pneumonia in primitive hematopoiesis, 3:159 Macrophage colony-stimulating factor, 3:126 Macrophage inflammatory protein, 4:172 Macrophage inflammatory protein-1, 4:35, 4:296 Macrophage inflammatory protein-2, 4:14 Macrophage mannose receptor, 4:13 Macropinocytosis, 4:142 MAdCAM-1, 4:77 Magnesium daily requirements for, 3:336 nephrocalcinosis and, 3:338 reference range for, 3:181 Magnetic resonance imaging atherosclerosis progression evaluations, 3:556 cardiovascular applications of, 3:501–502 contrast-enhanced, 3:502 description of, 3:44–45 echocardiography vs., 3:44 embryo studies, 1:204–205 facilities for, 3:282–283 functional, 3:502 gadolinium-enhanced, 3:502 heart evaluations using, 3:45 left ventricular mass evaluations, 3:44–45 magnetic field gradients, 3:499 neuroimaging applications of, 3:500–501 principles of, 3:44, 3:498–499 resolution achieved using, 3:500 scanner components, 3:498 signal production, 3:498–500 structural abnormalities evaluations, 3:500–501 T1 relaxation, 3:500–3:501 T2 relaxation, 3:500 vascular imaging using, 3:45 Magnetic resonance spectroscopy description of, 3:45 in research facilities, 3:282–283 Mahogany mouse, 3:620 Maintenance energy requirements for, 3:325–328 protein requirements for, 3:332 Major histocompatibility complex antigen processing, 4:18–20 CD1 genes, 4:20–21 dendritic cell expression of, 4:138 description of, 4:15 history of study, 1:9 molecules class I, 4:15, 4:17–18, 4:308 class II, 4:17–18, 4:123 description of, 4:120 overview of, 4:119–120 receptors, 4:170 Major urinary protein, 3:207 Malarial parasites, 3:145 Malathion, 3:346–3:347 Male(s) breeding age for, 3:239 breeding lifespan of, 3:261
Male(s) (continued) energy requirements for, 3:325 fertility of, 3:104, 3:111–112 retirement of, 3:261 Male reproductive accessory glands, 3:96 description of, 3:94–95 epididymis, 3:95–96 evaluation of, 3:110 excretory ducts, 3:95–96 genital organs, 3:18–19 illustration of, 3:95 penis, 3:96 testes, 3:95 urethra, 3:96 Malocclusion C57BL mice, 2:648 features, 2:691 MaLR. See Mammalian apparent LTR-retrotransposons Mammalian apparent LTR-retrotransposons features, 2:276 insertional mutagenesis, 2:272 Mammalian Phenotype Browser, 3:530 Mammalian reovirus cardiorespiratory system infection, 2:254 cell culture growth studies, 2:251–252 central nervous system infection, 2:253–254 control and prevention, 2:258 diagnosis, 2:257–258 endocrine system effects, 2:255–256 genome, 2:247 hepatobiliary system infection, 2:254–255 history of study, 2:245–246 host entry, 2:252–253 host range, 2:257 human infection, 2:724 immune response, 2:256–257 stability, 2:249 strains mutants, 2:250–251 reassortants, 2:250 serotypes, 2:249–250 structure, 2:247–249 transmission, 2:257 vaccination, 2:248 Mammary glands abnormalities in, 3:721 anatomy of, 3:3 development of, 3:719–720 embryogenesis of, 3:718 molecular signaling, 3:720–723 morphology of, 3:718–719 postnatal changes in, 3:720 postpubertal, 3:719 prenatal development of, 3:719–720 sexual dimorphism in, 3:723 Mandibularis lymph nodes, 3:21 Manganese, 3:336 Mannose-binding lectin, 3:191 Mannose-binding lectin-associated serine proteases, 3:191 Manose-6-phosphate, 3:127 MAP. See Mouse antibody production
352 Mapping studies, 3:648–650 Marginal zone absence of, in neonatal period, 4:81 definition of, 4:78 macrophages, 4:14, 4:81 Margo acutus, 3:13 Margo basalis, 3:13 Margo obtusus, 3:13 Margo plicatus, 3:16 mass1, 3:571, 3:580 Mass air displacement units, 3:299–302 Masseter, 3:5, 3:8 Mast cells in allergic asthma, 4:295 description of, 4:35 IgE-activated, 4:294–295 Mastitis, Pasteurellaceae, 2:485 Maternal fetal interface, 4:284 Mating description of, 3:101–102 hemizygous, 3:260 systems for mouse production, 3:252 timed, 3:111–112, 3:261–262 Matrix metalloproteinases -9, 1:310 description of, 3:196, 3:546–3:547 Matrix-assisted laser desorption/ionization, 3:174 Maturity, 3:524 MAV-1. See Mouse adenovirus type 1 MAV-2. See Mouse adenovirus type 2 Maxilloturbinates, 3:49 Maximal electroshock seizure threshold, 3:577 MBL. See Mouse Brain Library MC3r, 3:184 MC4r, 3:184, 3:620 MCS. See Multispecies conserved sequences mDC-SIGN, 4:14 Meals, 3:353 Mean cell hemoglobin, 3:141, 3:144 Mean cell hemoglobin concentration, 3:144 Mean cell volume, 3:141, 3:144 Mean corpuscular hemoglobin, 3:141, 3:144 Mean platelet volume, 3:156 Measuring Behavior conference, 3:530 Mebendazole, 2:559 Mechanical washing equipment, 3:302–304 Medetomidine, 3:461 Mediastinales caudales lymph nodes, 3:21 Mediastinales craniales lymph nodes, 3:21-22 Mediastinalis cranialis dexter, 3:10 Mediastinales medii lymph nodes, 3:21 Medical Research Council, 3:324 Medicated diet, 3:353 Medulla oblongata, 3:7 Medulla spinalis, 3:7 Medullary epithelial cells, 4:63, 4:65, 4:67 Megaesophagus features, 2:691 strain 129 mice, 2:635 Megakaryocytes in bone marrow, 3:154 DNA content of, 3:154
CUMULATIVE
Megakaryocytes (continued) GATA-1 deficient, 3:154–155 in primitive hematopoiesis, 3:159 in spleen, 3:162 Megakaryocytopoiesis splenic, 3:163 thrombopoietin effects, 3:154 Meibomian glands, 3:711 Meiosis, 3:93 Meiotic chromosomes. See also Synaptonemal complex air-dried preparation analysis, 1:156–157 applications, 1:155 banding, 1:156 cell sources, 1:155–156 fluorescent in situ hybridization, 1:158–159 meiosis overview, 1:155 post-meiotic chromosome analysis, 1:159–160 preparation, 1:156 Melanin, 3:605 Melanocortin receptor-4 description of, 3:620 knockout mouse phenotype, 1:307 mutant phenotyping, 1:251 Meloxicam, 3:480 Membrane attack complex, 4:8 Membrane cofactor protein, 3:190 Membrane-anchored immunoglobulin, 4:23, 4:83 Membrane-bound G protein, 3:124 Memory B cells, 4:91 Memory T cells, 4:90 Menadione, 3:340 Mercury, 3:346 Mesangial cell, 3:72 Mesenchyme, 4:65 Mesentericus caudalis lymph node, 3:22 Mesoduodenum, 3:14 Mesometrium, 3:21 Mesosalpinx sinister, 3:20 Mesothelium, 3:27 Mesovarium, 3:21 Metabolic disorders, 1:251 Metabolic rate, 3:66–67 Metabolism ambient temperature effects on, 3:68 description of, 3:66–67 domestication effects on, 3:75 post-eating increases in, 3:325 Metabolizable energy, 3:324, 3:330 Metastasis, 2:611–612 Metestrus, 3:100–101 Methacholine, 4:293 Methemoglobin, 3:144 Methionine and cysteine, 3:333 Method detection limit, 3:351 Methoxychlor, 3:346 Methoxyflurane, 3:179, 3:183 Methyl methanesulfonate, 1:231 Methyl parathion, 3:346 6-Methylpurine deoxyriboside, 3:743–744 Met-RANTES, 3:196
INDEX
Metronidazole Clostridium perfringens management, 2:357 Giardia muris management, 2:521 MGD. See Mouse Genome Database MHC. See Major histocompatibility complex Mhc class I region, 4:125–126, 4:130 class II molecules, 4:123, 4:130 class III region, 4:124–125, 4:130 comparative map of, 4:121–130 DM, 4:123 DO, 4:123 DP, 4:123 DQ, 4:123 DR, 4:123 H2-M4, 4:129 H2-M region, 4:128 H2-Q region, 4:126–127 H2-T region, 4:127–128 M5, 4:129 M6, 4:129 M region, 4:129–130 non–class II molecules, 4:123 overview of, 4:119–120 polymorphism of, 4:120 regions of, 4:120 sequencing of, 4:120–121 MHV. See Mouse hepatitis virus Mice. See also specific strain aging-related classifications of, 3:644–645 animal care staff observations of, 3:387 behavior of, 3:238 as disease model, 3:174–175 domestic hierarchies of, 3:238–239 genetically engineered, 3:175 handling of, 3:439–440 humans and, differences between, 3:556–557 identification methods, 3:265–266 life span of, 3:645, 3:650 microbial status of, 3:389 nocturnal activity of, 3:238, 3:524–525 physiological parameters for, 3:25 Microbial contamination adventitious agents description of, 3:735–736 prevention of, 3:737–739 cell banks used to prevent, 3:738–739 contaminant removal or inactivation, 3:739–740 endogenous retroviruses, 3:736–737 laboratory practices to prevent, 3:739 manufacturing practices to prevent, 3:739 murine leukemia virus, 3:737 operator-induced, 3:739 sources of, 3:736 viruses quality control testing for, 3:740 removal or inactivation of, 3:739–740 Microbiological monitoring, 3:427–428 Microbiological quality control overview of, 3:734–735 polymerase chain reaction considerations for, 3:747
CUMULATIVE
353
INDEX
Microbiological quality control (continued) description of, 3:742 mycoplasma, 3:744 validation of, 3:747 viral genomic sequences detected using, 3:747–748 testing for amount of, 3:741 antibody-production tests, 3:746–747 bacteria, 3:742–743 endotoxin, 3:742 indications for, 3:740–741 infectivity assays, 3:749 methods, 3:741–742 mycoplasma, 3:743–744 viruses, 3:744–749 MicroCT, 3:43–44, 3:494, 3:497 Microcytosis, 3:146 ß2-Microglobulin, 4:15, 4:17 Microhematocrit tube, 3:140 Microisolation caging, 3:247 MicroRNA, 1:109 Midazolam, 3:461–3:462 Middle, 3:624 Middle abdomen, 3:2 Milk collection of, 3:459 composition of, 3:329 Millar catheter-tip microtransducers, 3:36 Mineralization, 3:338 Minerals ashing process for, 3:336 deficiency of, 3:337 dystrophic calcinosis, 3:338 immunity affected by, 3:370–371 measurement of, 3:336 in natural-ingredient diets, 3:337 nephrocalcinosis, 3:337–338 nutritionally important, 3:336–337 requirements for, 3:336–337 Ministry of Health, Labor, and Welfare, 1:298 MINK, 4:216 Minute virus of mice clinical signs of, 2:96 control of, 2:101 description of, 3:735 diagnosis, 2:100 epizootiology, 2:96–97 genome features, 2:94–95 history of study, 2:93–94 pathology and pathogenesis of, 2:98–100 physicochemical properties, 2:95–96 prevention of, 2:101 replication, 2:95 research applications, 2:101 Mirex, 3:346 Mismatch repair, 4:162–164 Mismatchings, 3:266–267 Mites. See Fur mites Mitochondria, 4:217–218 Mitogen-activated protein kinase kinase 6, 4:113 Mitogen-activated protein kinase pathways, 4:216
Mitotic chromosomes banding applications, 1:146–147 C-banding, 1:149–150 DAPI banding, 1:149 fluorescent in situ hybridization, 1:153 G-banding, 1:148–149 historical perspective, 1:5 Q-banding, 1:148–149 R-banding, 1:149 RBG-banding, 1:149 cell sources and preparation, 1:147–148 principles, 1:153–154 spectral karyotyping applications, 1:146–147, 1:154 Mitotic mapping, 1:131 Mitral valve, 3:27 Mixed inbred strains, 1:84–85 Mixed leukocyte reaction, 4:138 Mixed noninbred strains, 1:85 M-mode echocardiography, 3:45–46 MMS. See Methyl methanesulfonate MMTV. See Mouse mammary tumor virus Mob, 3:624 Mobe1, 3:624 Modified hole board test, 1:249 Modifier genes breeding strategies for chemical mutagenesis screening dominant modifiers, 1:238 recessive modifiers, 1:238–239, 1:241 linkage mapping, 1:124–125 Moist heat sterilization, 3:362 MOLD/Rk mice, 3:651 Molds, 3:346 Moloney murine leukemia virus, 3:748 Monitoring behavior, 3:516 blood pressure electrocardiography. See Electrocardiography indwelling fluid-filled catheters, 3:33–34 invasive, 3:33–36 noninvasive, 3:32–33 radiotelemetry, 3:34–35 tail-cuff measurements, 3:32–33 tethering techniques, 3:33 transducer-tipped catheters, 3:35–36 during imaging, 3:492–493 pest, 3:428–429 pulmonary function tests, 3:53 research facility environment, 3:292–293 water, 3:420 Monocrystalline ion oxide nanoparticles, 3:502 Monocyte(s), 3:150–151, 4:33 Monocyte chemoattractant protein-1, 3:544, 4:296 Monogamous breeding system, 3:252–253, 3:257 Monogenic audiogenic seizures, 3:571–573 Monomeric red fluorescent protein, 3:506 Monoxenic animal, 3:218 MoPn. See Chlamydia muridarum
Morris water maze, 3:518, 3:520 Morula aggregation, 1:218 Mosaic populations, 1:75 moth1, 3:610 Motor neuron diseases, 3:678–679 Mouse adenovirus type 1 control and prevention, 2:61–62 diagnosis, 2:61 genetic susceptibility, 2:61 genome E1, 2:51–52 E3, 2:52–53 E4, 2:53 major late promoter, 2:53 structure, 2:51 history of study, 2:50–51 host range and prevalence, 2:61 immune response cell-mediated immunity, 2:59 humoral immunity, 2:60 innate immunity, 2:58–59 model, 2:60 infection in mouse age effects, 2:56 cell tropism, 2:57–58 E3 mutants, 2:58 E1A mutants, 2:58 inoculation route effects, 2:57 persistence, 2:56 infection in vitro E3 mutants, 2:55–56 E1A mutants, 2:55 kinetics of replication, 2:53–54 receptor, 2:54 physical properties, 2:51 Mouse adenovirus type 2 history of study, 2:50–51 infection in mouse, 2:60–61 physical properties, 2:51 Mouse antibody production. See also Antibody assay for, 3:400 mouse hepatitis virus diagnostics, 2:161 mousepox virus prevention, 2:89 Sendai virus prevention, 2:298 Mouse Brain Library, features, 1:242 Mouse cytomegalovirus atherosclerosis and, 3:552 classification, 2:3–4 control, 2:32 description of, 3:105, 3:391 diagnosis enzyme-linked immunosorbent assay, 2:32 polymerase chain reaction, 2:32 serology, 2:32 history of study, 2:2 host range, 2:12–13 human cytomegalovirus similarities adrenalitis, 2:24 atherosclerosis, 2:24 central nervous system infection, 2:24–25 clinical significance, 2:2–3, 2:17 description of, 4:307–308
354 Mouse cytomegalovirus (continued) genome, 2:4–5 hemopoiesis studies, 2:26 hepatitis, 2:21–22 interstitial pneumonitis, 2:20–21 intrauterine infection and congenital disease embryonic development effects, 2:19 epidemiology, 2:18 gonadal tissue infection, 2:19–20 hearing loss, 2:25–26 reproduction effects, 2:20 myocarditis, 2:22–24 retinitis, 2:25 immune response B cell response, 2:28–29 cytokines, 2:27 dendritic cell, 2:27 evasion, 2:30–32 immunosuppression induction, 2:26–27 macrophage, 2:27 natural killer cells, 2:28 T cell CD4+, 2:30 CD8+, 2:29–30 laboratory mouse infection, 2:11–12 life cycle morphogenesis, 2:6–7 replication, 2:5–6 viral entry, 2:5 mouse biological control, 1:46 natural history, 2:11 natural killer cells, 4:172–173 pathogenesis age effects, 2:13 dose effects, 2:13 inoculation route effects, 2:13 latency and reactivation, 2:16–17 resistance mechanisms acute infection, 2:14–15 cell culture studies, 2:15–16 chronic resistance, 2:15 propagation in vitro cell growth cycle effects, 2:9–10 centrifugal enhancement, 2:9 kinetics of replication, 2:9 multicapsid virion production, 2:10 non-murine cells, 2:9 nonpermissive murine cells, 2:9 permissive murine cells, 2:8–9 in vivo, 2:10–11 species distribution of cytomegalovirus, 2:3–4 strains, 2:7–8 transmission mode, 2:12 vaccines, 2:32–33 virion structure, 2:4–5 Mouse genome, 3:490 Mouse Genome Database description of, 4:120 historical perspective, 1:7 Laboratory Code in strain designation, 1:84 strain name registration, 1:81
CUMULATIVE
Mouse Genome Sequencing Consortium, 3:25 Mouse Genomics Informatics Database System, 3:647 Mouse hepatitis virus cell interactions, 2:148–149 classification, 2:146 control, 2:169–170 description of, 3:390-391, 3:393, 3:399, 3:402, 3:525 diagnosis enzyme-linked immunosorbent assay, 2:160–161 immunofluorescence assay, 2:160–161 immunohistochemistry, 2:167 mouse antibody production, 2:161 pathology, 2:162–165, 2:167 polymerase chain reaction, 2:161–162 serology, 2:161 duration of infection, 2:144–145 genome mutation and recombination, 2:148 history of study, 2:142–143 host range, 2:145–146, 2:149 human infection, 2:723 immune response B cells, 2:155, 2:157 experimental brain disease, 2:155–156 immunomodulation, 2:157–158 interferons, 2:155 passively acquired maternal immunity, 2:156–157 reinfection immunity, 2:157 T cells, 2:154–155, 2:157 vaccination immunity, 2:157 isolates, 2:142–143 isolation and propagation, 2:158–160 natural history, 2:143–144 pathogenesis enterotropic infection, 2:150 experimental encephalitis and demyelination, 2:151–154 experimental hepatitis, 2:151 respiratory infection, 2:150 replication, 2:148 research-confounding effects, 2:625 surveillance, 2:168–169 transmission, 2:145 virion structure, 2:146–148 Mouse jacket, 3:443, 3:448 Mouse mammary tumor virus description of, 3:391, 4:123 host range, 2:271 loci, 2:275 receptors, 2:271 research-confounding effects, 2:625 tumor pathogenesis, 2:595 Mouse minute virus. See Minute virus of mice Mouse norovirus, 3:391 Mouse parvovirus clinical signs of infection, 2:96 control of, 2:101 diagnosis, 2:100 epizootiology, 2:96–97
INDEX
Mouse parvovirus (continued) genome features, 2:94–95 history of study, 2:94 pathology and pathogenesis, 2:97–98 physicochemical properties, 2:95–96 prevention of, 2:101 replication, 2:95 research applications, 2:101 Mouse Phenome Database, 3:173, 3:183, 3:518, 3:524, 3:530, 3:632 Mouse Phenome Project, 2:627 Mouse pneumonitis virus, 3:391 Mouse polyoma virus cell interactions in culture cell transformation, 2:108 productive and nonproductive infections, 2:107–108 description of, 3:391 history of study, 2:106–107 natural history, 2:108–109 prospects for study, 2:129–130 receptors and uptake, 2:122–123 regulatory sequences, 2:121–122 structure, 2:113 susceptibility genetics in inbred strains, 2:123–126 transgenic mouse studies JC virus T antigen, 2:127 polyoma T antigens, 2:127–128 polyoma virus regulatory sequences in transgene expression, 2:128 SV40 large T antigen, 2:126–127 tumor antigens genetic interactions, 2:119–120 molecular interactions, 2:118–119 pathogenicity determinants, 2:120–121 structures and functions, 2:116–118 tumor induction cancer modeling anti-apoptotic responses, 2:111–112 early progression, 2:110–111 genomic instability and progression, 2:111 immune response, 2:112 initiation, 2:110 invasion and metastasis, 2:112–113 profile, 2:109 sites, 2:107 tissue interactions, 2:109–110 VP1 pathogenicity determinants, 2:113–116 recombinant proteins, 2:113 self-assembly, 2:113 VP2, 2:116 VP3, 2:116 Mouse rotavirus, 3:391 Mouse sarcoma virus, 3:748 Mouse thymic virus description of, 3:391 diagnosis, 2:34–35 history of study, 2:33 pathogenesis, 2:34 properties, 2:33–34 Mouse urinary protein, 3:71 Mouse urinary syndrome, 2:639
CUMULATIVE
355
INDEX
Mousepox clinical disease age effects, 2:73 patterns, 2:73 sexual dimorphism, 2:73 control depopulation and disinfection, 2:87 rederivation of mouse strains, 2:88 serological screening, 2:88 vaccination, 2:87–88 diagnosis clinical signs, 2:86–87 enzyme-linked immunosorbent assay, 2:87 pathology, 2:87 polymerase chain reaction, 2:87 serology, 2:87 virus isolation, 2:87 ectromelia virus species distribution, 2:69 enzootic mousepox, 2:86 epidemiology, 2:84–85 epizootic mousepox, 2:85–86 history of study, 2:68–69 host range species, 2:83–84 strain susceptibility, 2:84 immune response adaptive immunity, 2:81–82 innate immunity, 2:80–81 resistance genetics, 2:82–83 pathogenesis footpad infection and viral spread, 2:75–76 inoculation routes and mechanisms arthropod vectors, 2:74 feeding, 2:74 intracerebral inoculation, 2:75 intradermal inoculation and scarification, 2:73–74 intranasal inoculation, 2:74–75 intraperitoneal inoculation, 2:74 intrauterine infection, 2:75 lower respiratory tract inoculation, 2:75 pathology intestine, 2:79–80 liver, 2:78 lymphoid tissue, 2:79 resistant mouse strains, 2:80 skin, 2:77–78 spleen, 2:78–79 prevention mouse antibody production, 2:89 quarantine, 2:88–89 sentinel surveillance, 2:88 propagation chick embryo, 2:72 replication cycle, 2:71–72 tissue culture, 2:72–73 strains Hampstead strain, 2:70–71 Ishibashi strain, 2:71 Moscow strain, 2:71 NAV strain, 2:71 NIH-79 strain, 2:71
Mousepox (continued) taxonomy, 2:68–69 virion properties composition, 2:70 morphology, 2:69–70 stability, 2:70 structure, 2:70 Mouse-specific reagents, 3:175–176 MPD. See Mouse Phenome Database MptV. See Murine pneumotropic virus MPV. See Mouse parvovirus MRI. See Magnetic resonance imaging MRL.lpr mice, 4:244–246 mSIGNR2, 4:13–14 Msx2 homeobox signaling, 3:703 Msx2–NOG mice, 3:699 Msx2tm1 Rilm/Msx2tm1 Rilm mice, 3:704 99mTc, 3:503 MTV. See Mouse thymic virus Mucosa-associated lymphoid tissue, 4:69 Mucosal epithelia, 4:32 Mullerian inhibiting substance, 3:93 Multimodality imaging, 3:509 Multiple sclerosis, 4:308–309 Multiplex immunoassays, 3:176, 3:178–179 Multispecies conserved sequences, 1:105–106 MuLV. See Murine leukemia virus Municipal water, 3:241, 3:307 Murine activating receptor-1, 4:175 Murine leukemia virus common integration site, 2:274 description of, 3:737 detection of, 3:746 endogenous, 3:748–749 Fv1 in susceptibility, 2:273–274 host range, 2:271 inbred mouse strain distribution, 2:271–272 insertional mutagenesis, 2:272–273 Moloney, 3:748 receptors, 2:271 tumor pathogenesis, 2:595 Murine pneumotropic virus features, 2:128 propagation, 2:128–129 prospects for study, 2:129–130 tumor antigens, 2:129 Murine typhus features, 2:725 human risks, 2:725 Mus behavior, 1:32, 1:34–35 diseases and control, 1:43–46 geographic origins, 1:2 house mice origin, radiation, and reproductive incompatibility, 1:17, 1:19 natural history, 1:26–31 phylogenetic relationships evolutionary trees, 1:2, 1:20 prospects for study, 1:21 species, 1:19, 1:21 subgenera, 1:19 physiology, 1:31–32
Mus (continued) population dynamics, 1:37–42 reproduction, 1:35–37 taxonomy conventions for recognizing house mouse lineages and inbred strains, 1:19 species and subspecies field mice, 1:16 geographic ranges, 1:16–18, 1:26–28 house mice, 1:16–17, 1:19 subgenera Coelomys, 1:14 Mus, 1:15 Nannomys, 1:14 Pyromys, 1:14 wild strain polymorphisms, 1:3 Muscles. See also specific muscle of body, 3:4 of forelimb, 3:5 of head, 3:5 of hindlimb, 3:6–7 respiratory, 3:52 Mutagenesis. See also Chemical mutagenesis conditional mutagenesis strain maintenance, 1:71–72 gene trap mutagenesis, 1:110 gene-specific mutagenesis conditional mutagenesis, 1:264–265 gene targeting, 1:262–263 gene trapping, 1:263–264 inducible mutagenesis, 1:265 RNA interference, 1:265–266 targeted trapping, 1:264 transgenic mouse, 1:262 germ cell sensitivity, 1:226 insertional mutagenesis of embryonic stem cells, 1:285 mapping of mutant gene, 1:125–126 mutation frequency determination, 1:227–228 spontaneous mutation rate, 1:226 naming of mutant phenotypes, 1:91 phenotyping. See Phenotyping radiation as mutagen, 1:229 Mutagenic Insertion and Chromosome Engineering Resource, 3:621–3:622 MVM. See Minute virus of mice Myc signaling, 3:712 transgenic mice, 2:597, 2:599, 2:602–603 Mycobacterium phlei, 3:416 Mycobacterium tuberculosis, 3:416, 4:308 Mycoplasma sp. description of, 3:734–736 indirect assays for, 3:743 M. arthritides, 2:453–454, 3:392, 3:397 M. collis, 2:454 M. muris, 2:454 M. neurolyticum, 2:454 M. pulmonis clinical signs, 2:441
356 Mycoplasma sp. (continued) culture, 2:440–441 description of, 3:392, 3:397 diagnosis culture, 2:450–451 electron microscopy, 2:451 enzyme-linked immunosorbent assay, 2:450 general considerations, 2:449–450 histopathology, 2:451 immunohistochemistry, 2:451 polymerase chain reaction, 2:452 geographic distribution, 2:448 history of study, 2:438–439 host range, 2:448 pathogenesis disease expression factors, 2:444–445 host injury, 2:445–446 immune response, 2:446–448 pathology genital disease, 2:443 polyarthritis, 2:443–444 respiratory disease, 2:441–443 prevalence of infection, 2:448–449 properties, 2:439–440 strains, 2:440 transmission, 2:449 microbiological quality control testing for, 3:743–744 Mycotoxins, 3:347–348 MyD88, 4:113–114 Myeloblasts, 3:148 Myelocytomatosis oncogene, 3:712 Myeloid dendritic cells, 4:112 Myeloid differentiation factor 88, 4:3 Myeloid:erythroid ratio description of, 3:145 splenectomy effects on, 3:163 Myeloperoxidase, 3:191 Myobia musculi clinical features, 2:571 description of, 3:398 diagnosis, 2:571 host range, 2:571 life cycle, 2:569, 2:571 morphology, 2:569 pathobiology, 2:571 prevention and control, 2:572 treatment, 2:571–572 MYOC, 3:604–605 Myocardial calcification, 3:27 Myocardial infarction, 3:553–554 Myocarditis, 2:22–24 Myocardium, 3:27 Myocillin, 3:604–605 Myocoptes musculinus clinical features, 2:573 description of, 3:398 diagnosis, 2:573 host range, 2:573 life cycle, 2:573 morphology, 2:572–573 pathobiology, 2:573 prevention and control, 2:573–574 treatment, 2:573
CUMULATIVE
Myocytes, 3:29 Myoid cells, 3:95 Myosin heavy chain promoter, 3:38 Myostatin, 1:309
N 13N,
3:503 Na+/H+ exchanger, 3:575 Na+H+ exchanger regulator factor-1, 3:70 Nails, 3:714–716 Nalbuphine, 3:461–462 Naloxone, 3:462 Nannomys, 1:14 Nanog, 1:283 Nasal cavity, 3:49 Nasal-associated lymphoid tissue, 4:69 Nasopharynx, 3:49 Nasoturbinates, 3:49 National Committee for Clinical Laboratory Standards, 3:138 National Institute of Aging, 3:668 National Institutes of Health, 3:410 National Research Council nutrient guidelines, 3:323–324 vitamin guidelines, 3:342 National Sanitation Foundation International, 3:301 Natural alleles, 3:626–630 Natural cytotoxicity receptors, 4:175 Natural killer cell(s) activating receptors, 4:174–175 activation of, 4:174–176 autoimmune diseases and, 4:173 cytokine and chemokine production by, 4:171–172 cytotoxicity functions of, 4:171 dendritic cells and, 4:172 description of, 4:37–38 development of, 4:170–171 discovery of, 4:169–170 function of, 4:171–174 inhibitory receptors, 4:174 interleukin-2 and, 4:171 lactate dehydrogenase-elevating virus response, 2:223 lymphocytic choriomeningitis virus persistence role, 2:201 major histocompatibility-recognizing receptors, 4:262 morphology of, 4:171 murine cytomegalovirus immune response, 2:28 myocarditis role, 2:23 reproductive functions of, 4:173–174 Sendai virus response, 2:295 summary of, 4:176 surface markers of, 4:171 tissue distribution of, 4:171 tumor immunity applications of, 4:172 uterine, 4:173, 4:284 viral immunity applications of, 4:172–173 Natural killer cell receptors description of, 4:14–15, 4:125 inhibitory, 4:268
INDEX
Natural killer cell receptors (continued) Ly49, 4:268 Natural killer cell stimulatory factor, 3:193 Natural killer cell-committed precursors, 4:170 Natural killer T cells, 4:43 Natural-ingredient diets blending of, 3:358 description of, 3:239, 3:304–305, 3:351 extrusion of, 3:358–359 manufacturing of, 3:358–359 packaging of, 3:359 pelleting of, 3:358 phytoestrogens in, 3:349 raw materials used in, 3:358 storage of, 3:360, 3:423 Naturalistic observation, 3:516 Neck glands of, 3:8 regions of, 3:2 Necropsy areas for, 3:281–282 diagnostic, 3:394 fixatives, 3:483 phenotyping, 1:251–253 technique for, 3:482–483 Negative costimulatory molecules, 4:237 Negative feedback, 3:94 Negative selection anti-CD3/peptide central tolerance models, 4:209 cell death mechanisms during, 4:235 costimulatory molecules in, 4:215 endogenous antigen models, 4:209–213 mediators of, 4:213–218 overview of, 4:208–209 signal transducers in, 4:215–216 T cell receptor in, 4:215 transcription factors in, 4:216–217 Neomycin, Citrobacter rodentium infection management, 2:377 Neonate drug administration to, 3:453 euthanasia of, 3:482 Fc receptors, 4:6 Neoplasia, 3:153 Nephrectomy, 3:468 Nephrocalcinosis, 3:337–338 Nephrons, 3:70–71 Nerve growth factor, 3:677 Nerve growth factor receptor, 3:703 Nervous mouse, 3:678 Nervus phrenicus sinister, 3:12 Nests. See also Bedding materials used to build, 3:108, 3:306, 3:314 thermoregulatory uses of, 3:239 N-ethyl-N-nitrosourea, 3:596, 3:625 Neu, 2:597 Neural degenerations, 3:679 Neuroleptanesthetics, 3:462 Neurologic system, 3:388 Neuronal necrosis, 3:415 Neuropeptide Y, 3:184, 3:624 Neurotrophin signaling, 3:703
CUMULATIVE
357
INDEX
Neurotrophins, 1:283 Neurulation, 1:173, 1:175–176 Neutropenia, 3:149 Neutrophil(s) in aging mice, 3:162 band, 3:153 characteristics of, 3:148–150 definition of, 4:33 excitement effects on, 3:153 function of, 3:148 functions of, 4:33 granulocyte-colony stimulating factor effects on, 3:148–149 half-life of, 3:149 inflammation effects on, 3:153 kinetics of, 3:149 morphology of, 3:149–150 transcription factors that affect, 3:149 transit time for, 3:149 Neutrophilia, 3:149 Neutrophilins, 3:708 Nhe1, 3:575 NHEJ, 4:164–165 Niacinamide, 3:340 Nickel, 3:336 Niclosamide, 2:732 Nicotinamide, 3:340 Nicotinic acid, 3:340 Nidd5, 3:624 Niemann-Pick type C disease, 3:677–678 Nitrate, 3:345 Nitrile gloves, 3:314 Nitrite, 3:345 Nitrosamines, 3:345, 3:350–351 Nits, 3:398 NKG2D, 4:15, 4:173, 4:175 NKR-P1C, 4:175 Nociception, phenotyping, 1:250–251 Nod2, 4:114 NOD-scid mice, 4:278 Noise audiogenic seizures caused by, 3:292, 3:415 autoclave-induced, 3:415 control of, 3:415 definition of, 3:414 from individually ventilated cages, 3:298 high levels of, 3:414 physiologic effects of, 3:414 in research facility environment, 3:292 ultrasonic, 3:292 white, 3:415 Nonciliated bronchiolar cells, 3:51 Nonciliated secretory cells, 3:50 Noncommercial breeding, 3:237 Non-critical infection risks, 3:424 Nonfasting glucose, 3:655 Nongenomic responses, 3:125 Noninvasive imaging, 3:43–44 Nonopioid analgesics, 3:480 Nonshivering thermogenesis, 3:68 Nonsteroidal anti-inflammatory drugs, 3:480 Norepinephrine, 3:181 Norovirus, 3:391 Nosocomial infections, 3:427
Nostrils, 3:49 Notch1, 3:703 Notch2, 3:703 Notch signaling, 3:703, 3:714 NPC1, 3:677 Npepps, 1:313 Npy, 3:577 Nr3c1, 3:130–131 NR2E3, 3:607 Ntp, 3:579 Nuclear factor-kB B cell receptor activation of, 4:84 description of, 4:72, 4:216 T cell activation of, 4:236 Nuclear imaging definition of, 3:502 event localization in, 3:503 positron emission tomography, 3:504–3:505 radionuclides, 3:503 single photon, 3:502–504 single photon emission computed tomography, 3:504 summary of, 3:505 Nuclear receptor subfamily 5A1, 3:93 5′-Nucleotidase, 3:200 Nucleus colony definition of, 3:253 mismatchings in, 3:266–267 Nucleus colony segment, 3:236 Nude mice athymic, 4:277 bedding considerations for, 3:241, 3:306 hair fibers in, 3:702–703 production index for, 3:260 Nur77, 4:217 Nutrients. See also Diet; Feed autoclaving effects on, 3:362–3:363 classification of, 3:324 fats, 3:334–335 fatty acids, 3:334–335 fiber, 3:335 irradiation effects on, 3:365–366 laboratory testing of, 3:351 National Research Council guidelines, 3:323–324 overview of, 3:323–324 protein. See Protein vitamins. See Vitamin(s) Nutrition. See also Diet; Feed for breeding colony, 3:240 immune system affected by, 3:369 postoperative, 3:480 NZB mice, 3:74 NZM2410 strain, 4:249–250 NZO mouse, 3:626 NZW mice, 3:74 [NZW × NZB]F1 mice, 4:247–249
O 15O,
3:503 O antigen, 2:367–369 Obesity bariatric surgery for, 3:618 body mass index criteria for, 3:618
Obesity (continued) comorbidities associated with, 3:618 contributing factors, 3:618–619 diabetes and, 3:184–185 energy requirements and, 3:325 future research, 3:631–632 gene-environment interactions, 3:630–631 gene-gene epistasis, 3:630 genetic influences, 3:619–620 leptin and, 3:184, 3:618 low birth weight and, 3:631 maternal effects, 3:631 mouse models of chemically mutagenized, 3:625–626 congenic strain analysis, 3:628–629 diet-induced, 3:630–631 haplotype analysis, 3:627–628 knockout, 3:621–623 multigenic, 3:626–630 natural alleles, 3:626–630 recombinant congenic strain, 3:629–630 recombinant inbred strain, 3:629–630 spontaneous single gene mutations, 3:620–621 transgenic, 3:623–624 multifactorial etiology of, 3:618–619 N-ethyl-N-nitrosourea studies, 3:625 prevalence of, 3:618 quantitative trait loci, 3:619–620 visfatin in, 3:184 Obliquus externus abdominis, 3:4 ob/ob mice development of, 3:620, 3:665 energy requirements for, 3:325 Obq3, 3:624 Obq10, 3:624 Obturator externus, 3:7 Ochratoxin, 3:346, 3:348 OCT 4, 3:93 Octomitus pulcher, 2:525 Oculomotorius nerve, 3:7 Odontoblasts, 3:63 Oesophagus, 3:16 Office of Laboratory Animal Welfare, 3:410, 3:480–481 Olfactory bulb ablation, 3:468–469 Olfactory bulbectomy, 3:468–469 Oligodendrocyte progenitor cell, 1:286 Omega-3 fatty acids, 3:334–335 Omentum majus, 3:14, 3:16 Omentum minus, 3:16 One-tailed tests, 3:658–659 Onychocytes, 3:714 Oocyte fertilization of, 3:102 follicles and, 3:99 growth of, 3:99 preimplantation development of, 3:102 OPC. See Oligodendrocyte progenitor cell Open formula diet, 3:355 Opioid analgesics, 3:480 Optic nerve colobomas of, 3:598 development of, 3:599 Optic stalk, 3:599
358 Optical imaging, 3:506–508 Optical projection tomography, 1:205–206 Opticus nerve, 3:7 Oral cavity, 3:63 Orbital sinus bleeding, 3:454–3:455 Orchidectomy, 3:470 Ordinal statistics, 3:656–657 Orexin A, 3:184 Orexin B, 3:184 Organogenesis LTßR ligation during, 4:73 signaling mutations effect on, 4:75 Ornithine transcarbamoylase, 3:199 Ornithonyssus bacoti features, 2:733 host range, 2:733 human infestation, 2:733–735 Oropharynx, 3:49 Orphan mouse mutants description of, 3:573 Nhe1, 3:575–576 p35, 3:576 serotonin receptor, 3:575 stargazer, 3:573–574 weaver, 3:574–575 Os ilium, 3:4 Os ischii, 3:4 Os penis, 3:96 Os pubis, 3:4 Osmolality, 3:181 Osmotic minipumps, 3:474 Osmotic pumps for intraperitoneal administration, 3:451 for subcutaneous administration, 3:450 Ossa carpi, 3:4 Ossa metacarpi, 3:4 Ossa metatarsi, 3:4 Osteoblasts description of, 3:125 dexamethasone effects on, 3:131 estrogen effects on, 3:130 glucocorticoids effect on, 3:131 thyroid stimulating hormone effects on, 3:128 Osteoclasts activation of, 3:126 in bone remodeling, 3:126 calcitonin gene-related peptide effects on, 3:129 description of, 3:125 parathyroid hormone effects on, 3:129 Osteocytes in bone remodeling, 3:126 description of, 3:125 Osteopenia, 3:642 Osteoporosis C57BL mice, 2:648 description of, 3:642 Osteoprotegerin, 3:126, 3:194 Otitis, 2:485 Outbred strains authenticity of, 3:267 breeding, 1:73, 1:75, 3:254–258 definition, 1:83, 2:628 description of, 3:648
CUMULATIVE
Outbred strains (continued) genetic quality control for, 3:267 nomenclature, 1:84 sources and origins, 2:662–663 Out-of-specification results, 3:751–752 Ova, 3:459–460 Ovalbumin, 4:237, 4:292 Ovarian follicles. See Follicle Ovarian teratoma, 1:129 Ovarian transplantation, 3:113, 3:470–471 Ovariectomy, 3:470–471 Ovaries comparative embryology, 1:196–197 cryopreservation of tissue, 1:219–220, 1:235 Ovariohysterectomy, 3:471 Ovarium dextrum, 3:20–21 Ovarium sinistrum, 3:20 Ovary anatomy of, 3:97–3:98 development of, 3:93 excision of, 3:460 gonadotrophin response, 3:94 Over-the-needle catheters, 3:474 Oviduct anatomy of, 3:97–98 excision of, 3:460 sperm in, 3:102 Ovulation description of, 3:99–100 inducement of, 3:460 mating before, 3:102 Oxantel, 2:559 Oxygen consumption, 3:41 Oxygen tension, 3:69 Oxytocin, 3:203–204, 3:459 Oxytocin receptor, 3:203 Ozone, 3:372
P p25, 3:683 p35, 3:576, 3:683 p53, 2:601 p63, 3:713 p21, 4:254 PA-C10, 3:34 Packaging of diet description of, 3:359 irradiated diet, 3:364 Packed cell volume, 3:140–141, 3:144 PAI. See Pathogenicity island Paired Ig-like receptor-B, 4:267 Pancreas comparative embryology, 1:196 description of, 3:15–16, 3:64 Pancreatic islets -cells, 3:183 ß-cells, 3:184 insulin released from, 3:182–183 Paneth cells, 3:66 Panhypopituitary dwarf mutation, 3:651, 3:665 Pantothenic acid, 3:341 Papillae mammae, 3:3
INDEX
Papillary stalk, 3:695 Para-aortic splanchnopleura, 3:135, 3:160 Parabiosis, 3:469 Paracetamol, 3:480 Paracrine hormones, 3:124 Paraformaldehyde, 3:310 Paralogs, 1:103 Paramesonephric duct, 3:93 Paraoxonase, 3:186–187 Parasites colony animal testing for, 3:397–398 erythrocyte, 3:145 external, 3:398 fur mites. See Fur mites internal, 3:398 irradiation of, 3:364 medications for, 3:403 pinworms. See Pinworms types of, 3:397–398 Parathion, 3:346 Parathyroid glands, 1:193, 1:195 Parathyroid hormone description of, 3:128–129, 3:205 hypercalcemia caused by increased levels of, 3:208 Parathyroid hormone-like peptide, 3:720 Parathyroid hormone-related peptide, 3:128–129, 3:205 Parathyroidectomy, 3:469 Parkinson’s disease, 3:666–667, 3:682–683 Parotid gland, 3:63 Parotideus lymph nodes, 3:21 Parotidoauricularis, 3:5 Parotidoauricularis et glandula, 3:4 Pars abdominalis, 3:3 Pars abdominalis esophagei et lymphonodi gastrici, 3:15 Pars anterior prostatae, 3:19 Pars ascendens duodeni et pancreas, 3:15 Pars cardiaca, 3:16 Pars cardiaca tunicae mucosae, 3:16 Pars cardiaca ventriculi, 3:15 Pars cervicalis, 3:3 Pars cervicalis esophagei, 3:15 Pars cervicalis thymi, 3:8 Pars cervicalis trunci sympathici dextri et sinistri, 3:10 Pars descendens duodeni, 3:15 Pars epithelium stratificatum squamosum tunicae mucosae, 3:16 Pars fundica et pylorica ventriculi, 3:15 Pars fundica tunicae mucosae, 3:16 Pars inguinalis, 3:3 Pars membranacea urethrae, 3:19 Pars pylorica tunicae mucosae, 3:16 Pars pylorica ventriculae, 3:16 Pars scapularis musculi deltoidei, 3:4 Pars thoracica caudalis, 3:3 Pars thoracica cranialis, 3:3 Pars thoracica esophagei, 3:15 Pars thoracica trunci sympathici dextri et sinistri, 3:10 Parthenogenesis, 1:214 Partial seizures, 3:566 Parturition, 3:103
CUMULATIVE
359
INDEX
Parvoviruses, 3:391. See also Minute virus of mice Pasteurella pneumotropica, 3:392–393, 3:397 Pasteurellaceae classification, 2:471, 2:480 co-pathogens and opportunism, 2:483 culture, 2:480–481 diagnosis culture chemotaxonomic criteria, 2:494–495 growth conditions, 2:492–493 organs, 2:493 phenotypic identification, 2:493–494 enzyme-linked immunosorbent assay, 2:495–496 polymerase chain reaction, 2:495 environmental sensitivity, 2:474–475 eradication in control, 2:497 geographic distribution, 2:491 history of study Actinobacillus, 2:472–473 Haemophilus, 2:473–474 overview, 2:470–471 Pasteurella, 2:471–472 host range, 2:488–489 human infection potential, 2:489–491 latency, 2:484 morbidity and mortality, 2:481–482 pathogenesis, 2:482 pathology, 2:486–487 phenotypic characteristics Actinobacillus muris, 2:477 growth factor-dependent bacteria, 2:479–480 Haemophilus influenzaemurium, 2:477–479 overview, 2:474–475 Pasteurella pneumotropica, 2:475–477 prevalence of infection, 2:491 prevention, 2:497–499 primary pathogenicity, 2:482–483 research-confounding effects, 2:487–488 storage, 2:475 target tissues conjunctivitis, 2:484–485 mastitis, 2:485 otitis, 2:485 overview, 2:481 respiratory tract, 2:484 urogenital tract, 2:485–486 transmission, 2:491–492 treatment, 2:496 Pasteurization, 3:361 Patella, 3:4 Pathogenic autoimmunity, 4:250–251 Pathogenicity islands, 2:369, 3:228 Pathogen-specific molecular patterns, 4:110 Pathology cilia-associated respiratory bacillus, 2:456 control and prevention, 2:453 lactate dehydrogenase-elevating virus, 2:222 minute virus of mice, 2:98–100 mouse hepatitis virus, 2:162–165, 2:167
Pathology (continued) mouse parvovirus, 2:97–98 mousepox, 2:77–80 Mycoplasma pulmonis genital disease, 2:443 polyarthritis, 2:443–444 respiratory disease, 2:441–443 Pasteurellaceae, 2:486–487 Pneumocystis murina, 2:509 pneumonia virus of mice, 2:301–302 Sendai virus, 2:290–292 treatment, 2:452–453 tumors. See Tumor pathology, in genetically engineered mice Pax1, 4:65 Pax9, 4:65 Pbwg1, 3:624 PCBs, 3:346 PCR. See Polymerase chain reaction PD-1, 4:237, 4:267–268 Pde6b, 3:679 Pde6brd1, 3:598 Pdgfa null mice, 3:704 PD-L1, 4:237 PD-L2, 4:28 Pectineus, 3:7 Pectoralis ascendens, 3:6 Pectoralis majoris, 3:4 Pedigree card, 3:262–263 Pedigree ledger, 3:263, 3:265 Pedigree tree, 3:265 Pelage hair follicles, 3:694–696 Pellets diet autoclaving of, 3:356–357, 3:422 description of, 3:240, 3:353 hardness of, 3:355 illustration of, 3:445 manufacturing process of, 3:357 natural-ingredient diets, 3:358 quality of, 3:356 steam sterilization of, 3:422 summary of, 3:357 drug administration using, 3:450 Pelvis, 3:2 Penetrance linkage mapping, 1:124–125 mutations, 1:72 Penis, 3:18–19, 3:96 Pentobarbital, 3:462–463 Pentraxins, 3:190 Pentylenetetrazol neurological phenotyping, 1:250 seizure test, 3:566 Pepducins, 3:196 Peptidoglycan, 4:110 Peracetic acid isolator sterilization using, 3:223–224 in research facilities, 3:310 Perianal hair follicles, 3:697, 3:711 Pericardium, 3:12 Peripheral blood leukocytes, 4:283–284 Peripheral nervous system, 3:674 Peripheral tolerance, 4:146, 4:266 periplaneta americana, 3:429
Peristalsis, 3:65 Peritoneal cells, 3:459 Permanent identification methods, 3:441–443 Permethrin Myobia musculi management, 2:572 Polyplax serrata management, 2:568 “Perox” channel scattergram, 3:152 Peroxisome proliferator activated receptor gamma signaling, 3:712 Peroxisome proliferator-activated receptor-α human gene knock-in mouse, 1:308 knockout mouse phenotype, 1:308 Peroxymonosulfate, 3:426 Personal protective equipment functions of, 3:313 in research facilities, 3:313–314 Pest(s) bait stations for, 3:429 cockroaches, 3:429–430 control of, 3:361 escaped mice, 3:429 filth flies, 3:430–431 flying insects, 3:429 glue boards for, 3:429–430 house flies, 3:429 integrated management program for, 3:428–429 invertebrate, 3:429 light traps for, 3:429–430 monitoring for, 3:428–429 prevention programs for, 3:429–431 wild mice, 3:429 Pesticides, 3:345–347, 3:428 Pet-I, 3:676 Peyer’s patches B cell entry and exit from, 4:76–77 description of, 4:69 PFGE. See Pulsed-field gel electrophoresis PGC. See Primordial germ cells Phagocytes, 4:10 Phagocytosis, 4:19 Phalanges digitorum, 3:4 Pharmacodynamics definition, 1:290 studies overview, 1:290–291 Pharmacogenomics definition, 1:292 mouse study prospects, 1:314–315 Pharmacokinetics ADME processes, 1:291 definition, 1:291 Pharynx, 3:49 Phenols, 3:310 Phenotypes allergen exposure and, 3:62 breathing pattern variations based on, 3:53 studies of, 3:25 Phenotypic shift, 3:640 Phenotyping allergologic phenotypes, 1:247 behavioral phenotyping, 1:247–249 cardiovascular disease, 1:245–246 clinical chemical and biochemical assays, 1:246–247
360 Phenotyping (continued) dysmorphological phenotyping, 1:243–244 electrocardiography for, 3:38–39 embryonic stem cell mutagenesis, 1:256 eye defects, 1:244–245 immune system defects, 1:247 lung function, 1:245 magnetic resonance imaging for, 3:45 metabolic disorders, 1:251 necropsy and pathology, 1:251–253 neurological phenotyping, 1:249–250 nociception, 1:250–251 phenotype gap, 1:241 protocols, 1:241–242 standardization data collection, 1:241 phenome databases, 1:242–243 Phenylalanine and tyrosine, 3:333 Phenylhydrazine-induced hemolysis, 3:163 Pheromones description of, 3:94, 4:129 pest traps using, 3:430 reproductive responses, 1:54 seasonal production of, 3:108 social cues transmitted by, 3:109 Phoenix valves, 3:290 Phosphatases, 4:174, 4:238 Phosphatidylinositol 3-kinase, 4:200–203 Phosphodiesterases PDE7A, 1:311 PDE4A/B, 1:311 Phosphoinositide 3-kinases, 3:193, 4:234 Phospholipase C-τ, 4:233 Phosphorus daily requirements for, 3:336 measurement of, 3:207 nephrocalcinosis, 3:338 reference range for, 3:181 Photoplethysmography, 3:32 Phrenicus dexter et sinister, 3:10 Phylloquinone, 3:340, 3:369 Physiology, 3:26 Phytates, 3:338 Phytoestrogens bone development affected by, 3:350 definition of, 3:348 description of, 3:107, 3:304 in diets, 3:348–350 isoflavones, 3:348 lignans, 3:348–349 reproduction affected by, 3:350 research variables affected by, 3:349–350 significance of, 3:349 uterus effects, 3:350 Pigmentary glaucoma, 3:605–606 Pigmented feed, 3:305 Pilocarpine, 3:458 Pilosebaceous unit, 3:711 Pinealectomy, 3:470 Pinealis, 3:7
CUMULATIVE
Pinworms. See also Aspiculuris spp.; Syphacia spp. anthelmintic-treated drinking water for, 3:309 Aspiculuris, 3:398 chemical disinfectant of, 3:225 description of, 3:105 diagnostic testing for, 3:391, 3:394, 3:398 eggs, 3:394, 3:398 eradication costs, 3:390 medicated diets for, 3:353 Syphacia, 3:398 Piperazine, 3:403 Pirimiphos, 3:346 Pituitary gland description of, 3:77 hormones secreted by, 3:94 hypophysectomy, 3:466–467 Placental lactogens, 3:103 Placodes, 3:699 Plague, 1:39–42 Planck’s constant, 3:498 Plaque, atherosclerotic bone marrow transfer studies of, 3:549 characteristics of, 3:540–3:543 development of, 3:548 matrix metalloproteinase effects on, 3:548 Plasma cells, 4:91 Plasma profiles, 3:178–179 Plasma protein profiling, 3:174 Plasmablasts, 4:92–93 Plasmacytoid dendritic cells description of, 4:37, 4:76–77, 4:142–143 maturation of, 4:143 toll-like receptor signaling in, 4:114 Plasmacytoma, 1:8–9 Plasmodium berghei, 3:145 Platelet(s) accelerated production of, 3:156 aggregation of, 3:157 biology of, 3:154–155 clumps of, 3:152, 3:155 decreased number of, 3:156–157 definition of, 3:154 development of, 3:154–155 -granules of, 3:155 hemostatic functions of, 3:155 life span of, 3:155, 3:157 morphology of, 3:155 parameters of, 3:156–157 production of, 3:154 scatter diagrams for, 3:156 size of, 3:156 thrombopoietin effects on, 3:156 volume of, 3:156 Platelet count, 3:141, 3:156 Platelet crit, 3:156 Platelet derived growth factor A-chain, 3:599 Platelet derived growth factor signaling, 3:703 Platelet distribution width, 3:156 Plethysmograph, 3:54 Plethysmography barometric, 3:59–61 body, 3:54
INDEX
Plethysmography (continued) description of, 4:292 photoplethysmography, 3:59 whole body, 3:59–60 Pleural pressure, 3:52 Plexus pulmonalis, 3:10 Plexus trachealis, 3:11 Plica vena cavae caudalis, 3:12 PLNA. See Popliteal lymph node assay Pluripotent cells. See Embryonal carcinoma cell; Embryonic germ cell; Embryonic stem cell pmn, 3:677 PMP. See Purine nucleoside phosphorylase Pneumocystis spp. P. carinii antibiotic-infused water to prevent, 3:309 description of, 3:220 testing for, 3:392 P. murina animal models of infection, 2:513 diagnosis, 2:509 epidemiology, 2:508–509 history of study, 2:508 morphology, 2:508 pathology, 2:509 taxonomy, 2:508 treatment and control, 2:509–510 Pneumonia, acidophilic macrophage C57BL mice, 2:649 features, 2:694 strain 129 mice, 2:634–635 Pneumonia virus of mice age effects, 2:301 classification, 2:298 clinical features, 2:300 control of, 2:304 description of, 3:391 diagnosis, 2:303–304 genome, 2:299 history of study, 2:298 host range, 2:302 immune response, 2:302 pathology gross changes, 2:301 microscopic changes, 2:302 prevention of, 2:304 propagation cell culture, 2:300 eggs, 2:300 mice, 2:300 sex differences, 2:301 strains, 2:299 structure, 2:299 susceptibility of inbred strains, 2:301 target sites, 2:300–301 transmission, 2:303 Pneumotropic virus. See Murine pneumotropic virus Point mutagenesis, 4:280–281 Polycarbon biphenyls, 3:347 Polychromasia, 3:143 Polycystic kidneys, 3:74 Polycythemia, 3:147
CUMULATIVE
361
INDEX
Polydipsia, 3:77 Polyethylene restraint collar, 3:444 Polygamous breeding system, 3:252, 3:257–3:258 Poly-Ig receptor, 4:5–6 Polymerase chain reaction biological material testing using, 3:400 cardiovirus uses, 2:320 cilia-associated respiratory bacillus uses, 2:459 Clostridium piliforme uses, 2:354 Corynebacterium bovis uses, 2:401 genetic monitoring, 1:141 Helicobacter uses, 2:427 lactate dehydrogenase-elevating virus uses, 2:227 lymphocytic choriomeningitis virus uses, 2:204 mammalian reovirus uses, 2:258 microbiological quality control using considerations for, 3:747 description of, 3:742 mycoplasma, 3:744 validation of, 3:747 viral genomic sequences detected using, 3:747–748 minute virus of mice uses, 2:100 mouse parvovirus uses, 2:100 mousepox virus uses, 2:87, 2:161–162 murine cytomegalovirus uses, 2:32 Mycoplasma pulmonis uses, 2:452 Pasteurellaceae uses, 2:495 Sendai virus uses, 2:297 serologic testing using, 3:396–397 Polyoma virus. See Mouse polyoma virus; Murine pneumotropic virus Polyplax serrata clinical features, 2:567 diagnosis, 2:567–568 host range, 2:567 life cycle, 2:566 morphology, 2:566 pathobiology, 2:567 prevention and control, 2:568 treatment, 2:568 Polyubiquitination, 4:237 Polyuria, 3:77 Pons, 3:7 Popliteal lymph node assay, 1:304 Popliteus lymph node, 3:22 Porphyromonas gingivalis, 3:552 Portal vein catheterization, 3:476 Portio vaginalis uteri, 3:21 Positional cloning, overview, 1:109 Positive flow mass air displacement units, 3:300 Positive selection, 4:208–209 Positron emission tomography, 3:504–3:505 Postanesthetic care, 3:479–480 Postoperative care, 3:479–480 Potassium daily requirements for, 3:336 measurement of, 3:207 reference range for, 3:181
Pou5fl, 4:127 Power analysis, 3:658–660, 3:667 Poxviruses. See Mousepox Ppgb, 3:622 Praziquantel, 2:732 Preanesthetic medications, 3:460–461 Preclinical testing. See Drug discovery Predator response, 1:34–35 Predictive validation, 3:653 Pregnancy description of, 3:103 energy requirements during, 3:327, 3:329 euthanasia during, 3:468 handling during, 3:440 human handling effects on, 3:109 progesterone’s role in, 3:203 stages of, 1:185–186 Pregnant mare serum gonadotrophin, 3:226 Preimplantation development, 3:102 Prekallikrein, 3:157 Prenatal mortality, 1:37 Preputial glands, 3:711 Preputium, 3:18–19 Preputium clitoridis, 3:21 Presenilin-1, 3:683–684 Presenilin-2, 3:683–684 Pressure-volume curves, 3:55–56 Primary open angle glaucoma, 3:604–605 Primitive erythropoiesis description of, 3:135 erythropoietin’s function during, 3:159 Primitive hematopoiesis erythroid cells in, 3:158–159 macrophages in, 3:159 megakaryocytes in, 3:159 onset of, 3:158 Primordial follicles, 3:99 Primordial germ cells description of, 3:92 embryogenesis of, 1:171, 1:173 in genital system development, 3:92–93 Prions, 3:734 Prkdcscid, 4:278 Prkwnk, 1:311 Probucol, 3:539, 3:550 Procarbazine, 1:229–230 Procedure laboratories, 3:278–279 Production colony definition of, 3:236 of outbred stock, 3:256 sections of, 3:259 Production colony segment, 3:236, 3:259 Production index, 3:259 Production report, 3:263–264 Production sales ratio, 3:265 Proestrus description of, 3:100–101 timed mating, 3:111 Progesterone description of, 3:102, 3:203 reference ranges for, 3:181 Programmed death-1, 4:28
Programming, 3:631 Progressive motor neuronopathy, 3:677 Progressive myoclonus epilepsies, 3:569–571 Progressive neuronal degeneration, 3:576 Prolactin, 3:77, 3:181, 3:203 Prolactin receptors, 3:103, 3:203 Promoters, 1:90 Promyelocytes, 3:148 Pronator teres, 3:6 Pronuclear microinjection principles, 1:214–216 transgenic mouse preparation, 1:216 Proopiomelanocortin, 3:184 Proparacaine hydrochloride, 3:179, 3:455 Prostaglandins D2, 3:196 E1, 3:196–197 E2, 3:72 F2α, 3:103, 3:196, 3:203 H2, 3:196 renal, 3:71–72 Prostata, 3:19 Prostate gland, 3:96 Protein body collection of, 3:459 dietary protein conversion to, 3:325 dietary amino acids, 3:331–333 for breeding, 3:332 in commercial diets, 3:334 composition of, 3:331 conversion to body protein, 3:325 digestible energy and, ratio between, 3:332 digestion of, 3:66 excretion of, 3:72–73 food intake effects on, 3:367 infectious diseases and, 3:370 kidney damage induced by, 3:334 measurement of, 3:331 quality of, 3:331 reference range for, 3:181 requirements for, 3:331–332 restriction of, 3:368 total serum, 3:208 turnover of, 3:367 Protein inhibitor of activated STATs description of, 4:180–182 in vivo functions of, 4:188 Pias1-deficient mice, 4:188 Piasx-deficient mice, 4:189 Piasy-deficient mice, 4:188–189 Protein kinase Cd, 4:254 Protein tyrosine kinase, 4:254 Protein tyrosine phosphatases description of, 4:180, 4:189 in vivo functions of, 4:189 JAK-STAT pathway regulation by, 4:182–183, 4:189 Proteinuria, 3:72, 3:207 Proteoglycans, 4:35 Proteomics, 3:174
362 Proteus mirabilis classification, 2:378 clinical features, 2:378 diagnosis, 2:379 epizootiology, 2:378–379 history of study, 2:377–378 pathogenesis, 2:378 properties, 2:378 treatment and control, 2:379 Prothrombin time, 3:141, 3:157, 3:206 Protozoa. See also specific organisms comparison of murine pathogens, 2:519 taxonomy, 2:518–519 P-selectin glycoprotein ligand 1, 4:296 Pseudogenes, 1:104 Pseudomonas aeruginosa clinical features, 2:382 control, 2:382–383 description of, 3:308, 3:372, 3:392 diagnosis, 2:382 epizootiology, 2:382 history of study, 2:380–381 pathogenesis, 2:381–382 properties, 2:381 spread of, 3:453 in watering systems, 3:418, 3:420 Pseudopregnancy, 3:103, 3:112 Psoas major, 3:7, 3:14 Psoas minor, 3:7 Psorergates simplex clinical features, 2:575 diagnosis, 2:575 host range, 2:574 life cycle, 2:574 morphology, 2:574 pathobiology, 2:575 prevention and control, 2:575 treatment, 2:575 PTEN knockout mouse, 2:599-603 prostate cancer signaling, 2:599–601 Pteroylmonoglutamic acid, 3:341 Pth, 3:128–129 Pthlh, 3:129 Pthrp, 3:720 Ptp1b, 4:189 Ptpra, 3:622 PTZ. See Pentylenetetrazol PTZ-induced epileptic seizures, 3:577, 3:582 PU.1, 3:142, 3:159, 4:34 Puberty description of, 3:94 onset of, 3:239 pheromones effect on, 3:94 signs of, 3:94 Pulmo sinister, 3:12–13 Pulmonary arteries, 3:52 Pulmonary cells, 3:459 Pulmonary circulation, 3:51–52 Pulmonary dynamic compliance, 3:56 Pulmonary function tests and testing airway pressure, 3:56 alveolar fluid transport, 3:61 barometric plethysmography, 3:59–61 body plethysmography, 3:54
CUMULATIVE
Pulmonary function tests and testing (continued) contraction of isolated airway segments, 3:55 definition of, 3:53 diffusion capacity of lungs, 3:61 ex-vivo techniques, 3:55–56 forced oscillation technique, 3:59 gas dilution, 3:54–55 in-vivo techniques, 3:56–61 in mice, 3:55–61 monitoring uses of, 3:53 pressure-volume curves, 3:55–56 spirometry, 3:54 Pulmonary resistance, 3:56, 3:58, 3:60 Pulmonary valve, 3:27 Pulmonary veins, 3:27 Pulmonary venules, 3:52 Pulsed vacuum sterilization, 3:305 Pulsed-field gel electrophoresis, 2:428 Pups caesarean section delivery of, 3:251 cannibalism of, 3:109 cross-fostering of, 3:252 cryoanesthesia of, 3:464–465 drug administration in, 3:453 energy requirements of, 3:330 fostering of, 3:112 growth of, 3:109 handling of, 3:440 infanticide of, 3:109 resuscitation of, 3:467–468 thymectomy in, 3:472 Purified diet for carcinogenicity studies, 3:352 composition of, 3:351–352 description of, 3:337, 3:351–352 manufacturing of, 3:359 vitamin levels in, 3:342 Purine nucleoside phosphorylase, 1:311 Purkinje cells degeneration of, 3:678 description of, 3:27 PVM. See Pneumonia virus of mice Pylorus, 3:16 Pyridoxal-5’ phosphate, 3:369 Pyridoxine, 3:341 Pyrimethamine Sarcocystis muris management, 2:533 Toxoplasma gondii management, 2:536 Pyrogen test, 3:742 Pyromys, 1:14
Q Q wave, 3:37 Qa2, 4:126 Q-banding, 1:148–149 QRS interval, 3:37 QT interval, 3:37 QTL. See Quantitative trait loci Quadriceps, 3:4 Quality assurance in clinical chemistry, 3:182 health care staff for, 3:389
INDEX
Quality assurance (continued) overview of, 3:386–387 Quality assurance program agents to be tested, 3:390 cage washers, 3:416 colony animal testing bacterial agents, 3:397–398 enzyme-linked immunosorbent assay, 3:396 frequency of, 3:395 Helicobacter spp., 3:397 hemagglutination inhibition assays, 3:396 immunofluorescence antibody testing, 3:396 Mycoplasma arthritidis, 3:397 Mycoplasma pulmonis, 3:397 number of animals to be tested, 3:395 parasites, 3:397–398 Pasteurella pneumotropica, 3:397 serologic tests, 3:395–396 sick or deceased animals, 3:394 variations in results, 3:394–395 viral genetic material, 3:396–397 components of, 3:390 costs of, 3:390 customization of, 3:390 importance of, 3:389 quarantine. See Quarantine sentinel animal testing, 3:393–394 serology used by, 3:393 testing frequency, 3:390, 3:393 Quantitative loci analysis, 3:578 Quantitative trait loci description of, 3:598 drug response gene mapping, 1:305–306 history of study, 1:7 linkage mapping, 1:126–127 mapping, 3:626–627, 3:649–650 natural alleles identified by, 3:626–627 nomenclature, 1:93–94 obesity, 3:619–620 single nucleotide polymorphisms, 1:107–108 Quarantine housing for, 3:278–280, 3:312, 3:399 importance of, 3:398 isolation, 3:399 of newly delivered animals, 3:393 process involved in, 3:399 testing during, 3:399–400 Quaternary ammonium compounds, 3:309 Quats, 3:425 Quiescent phase, of estrous cycle, 3:100
R Rabies clinical signs, 2:723 diagnosis of, 2:723 reservoir and incidence, 2:722 treatment of, 2:723 Rac GTPases, 4:202 Radappertization, 3:363
CUMULATIVE
363
INDEX
Radfordia spp. clinical features, 2:575 diagnosis, 2:575 host range, 2:575 life cycle, 2:575 morphology, 2:575 pathobiology, 2:575 prevention and control, 2:575–576 R. affinis, 3:398 treatment, 2:575–576 Radiation hybrid mapping, 1:130 Radicidation, 3:363 Radio frequency transponders, 3:266 Radioallergosorbent test, 2:738 Radionuclides, 3:280–281, 3:503 Radiopharmacy, 3:283 Radiotelemetry, 3:34–35 Radiotracers, 3:505 Radius, 3:4 Radix pulmonum, 3:10–11 Radurization, 3:363 RAG-1, 4:48 RAG-2, 4:48 Rag1, 4:278–279 Rag2, 1:286, 4:278–279 rag-1/2, 4:61 RAG/τc, 4:279 Rami arteriae et venae pulmonalis, 3:13 Rami cardiaci nervorum vagorum, 3:11 Rami pulmonales, 3:10–11 Ramus cardiacus nerve vagi dextri et sinistri, 3:10 Ramus communicans cum nervos sympathico, 3:10–11 Ramus muscularis dorsalis dexter, 3:17 Ramus muscularis dorsalis sinister, 3:17 Ramus ovaricae sinistrae, 3:21 Ramus thymicus dextra, 3:9–11 Ramus thymicus sinister, 3:9 Ramus uterinus arteriae, 3:21 Random bred colony, 3:267 RANKL description of, 3:126 parathyroid hormone affected by, 3:129 Rap1, 4:199–200 RapL, 4:199–200, 4:203 Ras, 2:597, 2:599–600, 2:603 Rasgrp1, 4:255 RAST. See Radioallergosorbent test Rat bite fever clinical signs, 2:727–728 diagnosis, 2:728 pathogens, 2:727 reservoir and incidence, 2:727 transmission, 2:727 Raw materials biologicals, 3:738 microbial contamination considerations, 3:738 in natural-ingredient diet, 3:358 R-banding, 1:149 RBG-banding, 1:149 Rearing behaviors, 3:109 Receptor activator of NF-kB ligand, 4:71 Receptor editing, 4:48
Receptor recombinase, 4:38 Recessive allele, 1:57–58 Recombinant congenic strains features, 1:71, 1:84 gene mapping, 1:128 Recombinant inbred strains definition, 2:628 description of, 3:173 features, 1:71, 1:84 gene mapping, 1:127–128 historical perspective, 1:7 intercross lines, 1:128 Recombination, genome analysis, 1:108 Recombination signal sequences, 4:38, 4:44 Records breeding cage-level, 3:262–263 description of, 3:104 pedigree card, 3:262–263 pedigree ledger, 3:263, 3:265 pedigree tree, 3:265 production report, 3:263–264 of sick mice, 3:387 Rectal administration of drugs, 3:446–447 Rectum, 3:15, 3:18 Rectus femoris, 3:4, 3:6–3:7 Red blood cell. See Erythrocyte(s) Red blood cell count changes in, 3:146–148 description of, 3:141, 3:143–144 in young mice, 3:161 Red blood cell indices, 3:144 Red cell distribution width description of, 3:141, 3:143 hemorrhage effects on, 3:147 Red cell mass absolute, 3:147 absolute decreased, 3:147 decreases in, 3:147–148 increases in, 3:146–147 relative decreased, 3:147 Rederivation embryo harvesting, 1:220 shipping, 1:220 surgical embryo transfer, 1:220–221 in vitro culture, 1:220 Reference colony, 3:236 Reference intervals, 3:136 References ranges definition of, 3:180 list of, 3:181 variability, 3:180, 3:182 Regio abdominis lateralis, 3:2 Regio analis, 3:2 Regio antebrachii, 3:2 Regio apicis caudae, 3:2 Regio articulationis coxae, 3:2 Regio articulationis humeri, 3:2 Regio articulationis temporomandibularis, 3:2 Regio auricularis et auricula, 3:2 Regio axillaris, 3:2 Regio brachii, 3:2 Regio buccalis, 3:2
Regio carpi, 3:2 Regio clitoridis, 3:2 Regio clunis, 3:2 Regio colli dorsalis, 3:2 Regio colli ventralis, 3:2 Regio corporis caudae, 3:2 Regio costalis, 3:2 Regio cruris, 3:2 Regio cubiti, 3:2 Regio dorsalis nasi, 3:2 Regio femoris, 3:2 Regio frontalis, 3:2 Regio genus, 3:2 Regio glutea, 3:2 Regio hypochondriaca, 3:2 Regio infraorbitalis, 3:2 Regio inguinalis, 3:2 Regio intermandibularis, 3:2 Regio interscapularis, 3:2 Regio lateralis nasi, 3:2 Regio lumbalis, 3:2 Regio mammaria abdominalis, 3:2 Regio mammaria inguinalis, 3:2 Regio mammaria thoracica, 3:2 Regio mandibularis, 3:2 Regio manus, 3:2 Regio masseterica, 3:2 Regio mentalis, 3:2 Regio naris et apex nasi, 3:2 Regio occipitalis, 3:2 Regio olecrani, 3:2 Regio oralis, 3:2 Regio orbitalis, 3:2 Regio parietalis, 3:2 Regio parotidea, 3:2 Regio pedis, 3:2 Regio perinealis, 3:2 Regio plicae lateris, 3:2 Regio presternalis, 3:2 Regio pubica, 3:2 Regio radicis caudae, 3:2 Regio sacralis, 3:2 Regio scapularis, 3:2 Regio sternalis, 3:2 Regio subhyoidea, 3:2 Regio supraorbitalis, 3:2 Regio tarsi, 3:2 Regio temporalis, 3:2 Regio trachealis, 3:2 Regio tricipitalis, 3:2 Regio tuberis coxae, 3:2 Regio tuberis ischiadici, 3:2 Regio umbilicalis, 3:2 Regio vertebralis thoracis, 3:2 Regio vulvae, 3:2 Regio xiphoidea, 3:2 Regio zygomatica, 3:2 Regulator of complement activation, 3:190 Regulatory T cells, 4:42 Related inbred strain, definition, 2:628 Relative decreased red cell mass, 3:147 Relative humidity description of, 3:291 in diet, 3:361 Reliability, 3:521–522
364 Ren dexter, 3:14, 3:17 Ren sinister, 3:14, 3:17–18, 3:20 Renal artery catheterization, 3:477 Renal clearance, 3:73–74 Renal corpuscle, 3:70 Renilla luciferase, 3:506 Renin-angiotensin system, 3:71 Reoviridae. See Mammalian reovirus; Rotavirus Reovirus-3, 3:391 Replication, 1:108 Replication protein A, 4:161 Repolarization, 3:37 Reproduction. See also Breeding artificial insemination, 1:212, 3:227 breeding records, 3:104 colony for evaluation of, 3:110 health status of, 3:105 maintenance of, 3:104–105 troubleshooting the performance of, 3:109–110 energy necessary for, 3:105 environmental effects enrichment, 3:108 feed, 3:107–108 housing, 3:109 human interaction, 3:109 humidity, 3:107 light, 3:106, 3:238, 3:291 sound, 3:106 temperature, 3:106–107, 3:239 time of year, 3:108–109 vibration, 3:106 in vitro fertilization principles, 1:212–213 rederivation, 1:220–221 intracytoplasmic sperm injection principles, 1:213–214 somatic nuclear transfer, 1:221–222 steps, 1:214 transgenic mouse preparation, 1:216 natural killer cells’ role in, 4:173–174 nesting material effects on, 3:306 overview of, 3:103–104 phytoestrogen effects on, 3:350 timed mating, 3:111–112 Reproductive system illness signs, 3:388 necropsy evaluations, 3:483 ontogeny of, 3:92–94 Reproductive tract development of, 3:93 female anatomy of, 3:97–99 cervix, 3:98–99 description of, 3:97 evaluation of, 3:110 ovary, 3:97 oviduct, 3:97–98 uterus, 3:98 vagina, 3:99 male accessory glands, 3:96 description of, 3:94–95
CUMULATIVE
Reproductive tract (continued) epididymis, 3:95–96 evaluation of, 3:110 excretory ducts, 3:95–96 illustration of, 3:95 penis, 3:96 testes, 3:95 urethra, 3:96 necropsy evaluations, 3:483 Research facilities animal receipt and transport, 3:312–313 animal receiving area, 3:284 architecture of, 3:272–287 automatic watering systems in, 3:241, 3:285–286, 3:308 automation in, 3:285–287, 3:412 barrier in, 3:273–274, 3:311 bedding used in, 3:305–306 biological safety cabinets Class I, 3:300–301 Class II, 3:301, 3:313 Class III, 3:302 illustration of, 3:281–3:282 building, 3:272–273 cages in. See Cage chemicals used in alcohols, 3:310 chlorine dioxide, 3:309–310 description of, 3:309 disinfectants, 3:309 halogens, 3:309 paraformaldehyde, 3:310 peracetic acid, 3:310 quaternary ammonium compounds, 3:309 synthetic phenols, 3:310 vaporized hydrogen peroxide, 3:310 circulation patterns in, 3:274–3:275 corridor systems in, 3:274–3:275 drinking water acidification of, 3:308 additives in, 3:308 anthelmintics added to, 3:309 antibiotics added to, 3:309 bisphenol A contamination, 3:307 chlorine added to, 3:308–309, 3:372, 3:418–419 hydrochloric acid added to, 3:308, 3:418 processing of, 3:307–308 reverse osmosis of, 3:307 steam sterilization of, 3:308 ultraviolet disinfection of, 3:307–308 variations in, 3:306–307 environment of air quality in, 3:295 ammonia, 3:293 carbon dioxide concentrations, 3:293 enrichment of, 3:314 lighting, 3:291–292 macroenvironment, 3:288–293 microenvironment, 3:292–294 monitoring of, 3:292–293 noise, 3:292 overview of, 3:287–288
INDEX
Research facilities (continued) relative humidity, 3:291 temperature, 3:290–292 exclusion level in, 3:311 feed used in, 3:304–305 floor plan, 3:273–274 hazardous agent containment, 3:279–281 heating, ventilation, and air-conditioning system, 3:272–273, 3:288–290 holding rooms description of, 3:274–276 lighting in, 3:292 temperature in, 3:291 imaging facilities, 3:282–283 information resources, 3:272 isolators, 3:299 material transport in, 3:311–312 mechanical washing equipment in, 3:302–304 necropsy facilities, 3:281–282 operational issues for, 3:311–314 personal protective equipment used in, 3:313–314 personnel in, 3:312 plan of, 3:272 procedure laboratories, 3:278–279 quarantine facility, 3:278–280, 3:312 rack installation in, 3:276 radiopharmacy in, 3:283 receiving area, 3:284 room-level exclusion, 3:311 security considerations, 3:273, 3:411 site selection for, 3:272–273 soiled cage wash, 3:311–312 standard operating procedures in, 3:311 summary of, 3:314 support areas, 3:284 ventilated cage. See Ventilated cage vibration considerations, 3:273 washers used in, 3:302–303, 3:310 water delivery automated systems, 3:241, 3:285–286 water bottles, 3:241, 3:286–290 Residual volume, 3:53–3:54 Resistin, 3:184 Resorcylic acid, 3:348 Respirators, 3:313 Respiratory muscles, 3:52 Respiratory rates, 3:52, 3:57 Respiratory system breathing patterns, 3:52–53 description of, 4:292 illness signs, 3:388 lungs. See Lung(s) physiology of, 3:48 Respiratory tract conducting zone, 3:49 description of, 3:48–49 larynx, 3:49–50 lower, 3:50–51 nasal cavity, 3:49 pharynx, 3:49 trachea, 3:50 upper, 3:49–50
CUMULATIVE
365
INDEX
Respiratory transfer impedance, 3:59 Respiratory zone, 3:49–50 Restenosis, 3:554 Resting heart rate, 3:39–40 Restraint for blood collection, 3:454 devices used for, 3:443–444 forceps, 3:439, 3:443 objective of, 3:442 polyethylene collar, 3:444 Restricted flora, 3:218 Restriction fragment length polymorphism, 1:118 Reticulocytes blood smear count of, 3:145 characteristics of, 3:144–145 definition of, 3:144 flow cytometric scatter for, 3:145 formation of, 3:143 hemorrhage effects on, 3:147 lyse-resistant, 3:152 Retina angiogenesis of, 3:599 astrocyte migration, 3:599 in C57BL/6 mice, 3:600 cells of, 3:607 degeneration of apoptosis and, 3:607 modifiers of, 3:609–611 mouse models of, 3:679 overview of, 3:605 photoreceptor degeneration, 3:607 primary, 3:607, 3:609 retinal folds associated with, 3:609 description of, 3:596 development of, 3:605, 3:607–3:608 dysplasia of, 3:601 lamination disorders of, 3:600–3:601 progenitor cells, 3:607 synaptic network, 3:607 Retinal degeneration-1, 3:598 Retinal pigment epithelium, 3:609 Retinitis pigmentosa description of, 3:607 murine cytomegalovirus myocarditis model, 2:25 retinoblastoma, 3:675 Retinoic acid inducible gene-I, 4:114 Retinoic acid–early inducible-1, 4:173 Retinol, 3:339, 3:342 Retroelement, 2:269–270 Retrograde perfusion, 3:29–30 Retro-orbital injection, 3:452 Retrotransposon insertional mutagenesis, 2:272 types, 2:270 Retroviruses. See also specific viruses description of, 3:736–737 gene transfer vectors, 1:272–273 infectivity assays for, 3:749 lentivirus vectors, 1:273–274 oncoretrovirus vectors, 1:273 quantitative assays for, 3:749 Reverse cholesterol transport, 3:186 Reverse osmosis, 3:307, 3:372, 3:420
Reverse transcriptase-polymerase chain reaction, 3:400, 3:748 RFLP. See Restriction fragment length polymorphism Riboflavin, 3:340 Ribosomal DNA, 1:150 Rickettsialpox clinical signs, 2:724–725 control and prevention, 2:725 geographic distribution, 2:724 Rieger’s malformation, 3:597 Right atrium, 3:27 Right coronary artery, 3:28 Right ventricle anatomy of, 3:27 pressure measurements, 3:36 Ring3, 4:123 Ringworm. See Dermatophytosis RNA interference history of study, 1:8 overview, 1:109 pharmacogenomic study prospects, 1:315 small interfering RNA delivery, 1:265–266 Rodent jacket, 3:443, 3:448 Rodent restraint bag, 3:443–444 Rodentolepis spp. R. microstoma, 2:561 R. nana features and management, 2:559–560 human infection, 2:732 Romanowsky-type stain, 3:140 Room level bioexclusion facility, 3:243–245 Roquin, 4:237–238 rora, 3:676 Rotavirus age effects, 2:243–244 cell culture growth studies, 2:240–241 control and prevention, 2:245 description of, 3:391 diagnosis, 2:246 diarrhea mechanisms, 2:242 genome, 2:238 history of study, 2:236–237 host range, 2:244–245 immune response, 2:242–243 pathogenesis, 2:241–242 stability, 2:238–239 strains electropherotypes, 2:239 groups, 2:239 murine strains, 2:240 serotypes, 2:239–240 structure, 2:237–238 RPE65, 3:609 RT1-N, 4:128 Runt-related transcription factor 1, 4:268 RV. See Residual volume
S S cells, 3:64 S wave, 3:37 SAA, 3:208 Sacculus rotundus, 3:15 Saccus cecus ventriculi, 3:16
Salicylazo-sulfapyridine, 3:367 Saliva collection, 3:458–459 Salivary glands, 3:63 Salmonella spp. classification, 2:369–370 clinical features humans, 2:729 mice, 2:371–372 description of, 3:346, 3:353 diagnosis, 2:372–373 epizootiology, 2:371 history of study, 2:370 pathogenesis, 2:371 properties, 2:370–371 reservoir and incidence, 2:728–729 S. enterica, 3:392 S. typhimurium, 4:112 SAMP-8 mice, 3:652 Sampling, 3:179–180 Sanitation background, 3:423 cage sanitation area chemical storage in, 3:277 clean activity, 3:277 description of, 3:276–277 location of, 3:273–274 mechanical equipment in, 3:277 soiled activity, 3:277, 3:285 disinfectants. See Disinfectants microbiological monitoring, 3:427–428 Saphenous vein blood collection, 3:456 Saponins, 3:351 Sarcocystis muris cell biology, 2:532 clinical features, 2:532–533 diagnosis, 2:533 history of study, 2:532 life cycle, 2:532 prevention, 2:533 research implications, 2:533 treatment and control, 2:533 Sarcopenia, 3:660 SC. See Synaptonemal complex Scapula, 3:4 Scavenger receptors, 3:187 Schistosoma mansoni, 3:150 Schlemm’s canal, 3:602–603 SCID mice, 3:260 SCID/BG mice, 3:260 SCL, 3:159 Scleral spur, 3:602 Scn2a, 3:568 S-cone syndrome, 3:607 “Scruff-of-the-neck” handling method, 3:440 Seasons mouse populations fluctuations, 1:38–39 reproduction affected by, 3:108–109 Sebaceous glands, 3:711–712 Second messengers, 3:125 Secondary follicle, 3:99 Secretin, 3:63 Security, 3:273, 3:411 Segregating inbred strains breeding, 1:63–64 nomenclature, 1:87
366 Seizures audiogenic definition of, 3:578 description of, 3:292, 3:415 in epilepsy prone mice, 3:580–581 experimental epilepsy model use of, 3:578–581 Frings mice, 3:571 genetic studies of, 3:580 interstrain variability in, 3:578–579 long-term potentiation and, 3:579 monogenic, 3:571–573 neurochemical findings, 3:579–580 neuropathologic findings, 3:579 serotonin levels and, 3:580 sudden unexpected death in epilepsy and, 3:579 susceptibility to, 3:578–579 EL mouse model. See EL mouse epileptic characteristics of, 3:566 classification of, 3:566 electroconvulsive shock-induced, 3:576–577 generalized, 3:566 kainic acid-induced, 3:577–578 partial, 3:566 PTZ-induced, 3:577, 3:582 quantitative trait loci mapping of drug response genes, 1:305–306 Selamectin, 2:558 Selectins, 4:296 Selenium, 3:336, 3:346, 3:370–371 Self-tolerance, 4:42–43 Semi-critical infection risks, 3:424 Semimembranosus, 3:6–7 Seminal vesicles, 3:96 Seminiferous tubules, 3:95 Semipermanent identification methods, 3:441 Semirigid isolators, 3:220–221, 3:245, 3:300 Semitendinosus, 3:6–7 Sendai virus age effects, 2:288 classification, 2:282 clinical features, 2:287 control of, 2:298 description of, 3:391, 3:402 diagnosis, 2:297–298 genome, 2:282–283 geographic distribution, 2:296 history of study, 2:282 host range, 2:296 human infection, 2:724 immune response adaptive immunity, 2:295–296 innate immunity, 2:292–295 pathology gross changes, 2:290 microscopic changes, 2:290–292 prevention of, 2:298 propagation cell culture, 2:286–287 eggs, 2:286 mice, 2:285–286
CUMULATIVE
Sendai virus (continued) receptors, 2:283 sex differences, 2:288 strains, 2:284–285 structure, 2:283–284 susceptibility of inbred strains, 2:288–290 target sites, 2:287–288 transmission, 2:297 Senescence aging vs., 3:640, 3:642–644 biological organization and, 3:640–642 disease vs., 3:642–644, 3:668 hypermorphosis-related, 3:640–641 immunologic, 3:642 life span and, 3:655–656 long-lived treatment models of, 3:651 onset of, 3:640 phenotypic shift, 3:640 rate biomarker of, 3:653 replication of symptoms of, 3:652 short-lived treatment models of, 3:651–652 T-cell, 3:640–641 Senescence-accelerated mice, 2:639 Sentinel animals aerosol exposure, 3:393–394 bedding transfer, 3:394 contact, 3:394 quality assurance testing of, 3:393–394 quarantine of, 3:399–400 soiled bedding exposure, 3:393–394 Sequence tagged sites, 1:90 Serial analysis of gene expression, 3:605 Serine protein inhibitor, 3:609 Serologic tests and testing disadvantages of, 3:395–396 enzyme-linked immunosorbent assay, 3:396 hemagglutination inhibition assays, 3:396 immunofluorescence antibody testing, 3:396 Serotonin, 3:580 Serotonin receptor, 3:575 Serratus ventralis, 3:4 Sertoli cells description of, 3:95 in spermatogenesis, 3:96 Serum reference range for, 3:181 storage of, 3:187 viral infections, 3:735 Serum amyloid P, 4:252 Serum amyloid protein, 3:190 Severe combined immune deficiency description of, 4:183 mouse models, 4:278, 4:307 Sevoflurane, 3:464 Sex chromosomal, 3:92 definition of, 3:92 determination and differentiation of, 3:92–94 energy requirements based on, 3:325 gonadal, 3:92 Sex-linked allele, 1:58–59
INDEX
Sexual dimorphism adrenal gland, 2:631 definition, 2:630 kidney, 2:631 parotid gland, 2:632 pathology. See specific organisms submandibular salivary gland, 2:631–632 Sexual dysfunction, 3:583 Sexual maturation, 1:36 SH2-containing protein tyrosine phosphatase, 4:83 5(S)-HETE, 3:197 12(S)-HETE, 3:197 15(S)-HETE, 3:197 SHH. See Sonic Hedgehog Shh null mice, 3:704 Shigella sonnei, 3:228 SHIP, 4:263–264, 4:270 Shipping of animals, 3:399 of feed, 3:422 SHIRPA protocol, 1:248–249 Shoe covers, 3:313 Short consensus repeat units, 4:10 Short interspersed nuclear elements, 1:104 Short-lived models, 3:651–652 SHP-1, 4:263, 4:269–270 SHP-2, 4:28, 4:263 Shp1, 4:189 Shp2, 4:189 Sialogogues, 3:458 Sick mice animal care staff observations of, 3:387 illness signs, 3:387–3:388 initial observations and actions, 3:388–389 reporting of, 3:387–389 testing of, 3:394 Signal-to-noise, 3:491 Signal-to-noise ratio, 3:43 Silica dioxide, 3:363 Silicon, 3:336 Simple sequence length polymorphism, 1:118–119 SINEs. See Short interspersed nuclear elements Single nucleotide polymorphisms comparative genome analysis, 1:106 description of, 3:649 genetic monitoring, 1:140–142 haplotype mapping, 1:126 linkage mapping, 1:118–120 quantitative trait loci, 1:107–108 Single photon nuclear imaging, 3:502–504 Single-photon emission computed tomography, 3:43, 3:504, 3:509 Single-strand conformational polymorphism, 1:256 Sinoatrial node, 3:27 Sipper tubes, for water delivery, 3:419 Six1, 4:65 SJL/J mice development, 2:654 phenotype, 2:654 spontaneous diseases
CUMULATIVE
367
INDEX
SJL/J mice (continued) comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:655 nonneoplastic conditions, 2:654 pathogen susceptibility, 2:654–655 Sjogren’s syndrome, 3:191 SKAP-55, 4:199 Skeleton, 3:4 Skeleton capitis, 3:4 SKH-1 mice bedding considerations for, 3:241 production index for, 3:260 Skin barrier function of, 4:32 cornification of, 4:32 drug administration through, 3:447–449 epidermis, 3:712 functions of, 3:692, 4:32 illness signs, 3:388 interfollicular, 3:712–713 production of, 3:693 Skin appendages hair follicles. See Hair follicles illustration of, 3:693 nails, 3:714–716 sebaceous glands, 3:711–712 sweat glands, 3:713 volar pads, 3:714–718 Skin grafts, 3:472–473 Skull, 3:2 SKY. See Spectral karyotyping Sle1, 4:249–250 Sle2, 4:251 Sle3/5, 4:250–251 Slow wave epilepsy, 3:575 SLP-76, 4:199 Slp, 4:124 SLT. See Specific locus test Small interfering RNA. See RNA interference Small intestine description of, 3:65–66 necropsy evaluations, 3:483 Smears blood evaluation of, 3:140, 3:145 preparation of, 3:139 reticulocyte count on, 3:145 bone marrow, 3:139–3:140 SMN, 3:678 α-Smooth muscle actin, 3:720 Snell dwarf mutation, 3:660, 3:665 SNPs. See Single nucleotide polymorphisms SNT. See Somatic nuclear transfer Social behavior, 1:32–34 Social cues pheromone transmission of, 3:109 puberty affected by, 3:94 Socs5 transgenic mice, 4:188
Sodium daily requirements for, 3:336 measurement of, 3:207 reference range for, 3:181 Sodium hypochlorite, 3:309, 3:419–420, 3:426 Sodium lamps, 3:291 Soiled bedding, 3:277, 3:285 Soiled cage wash, 3:311–312 Somatic cell hybrids, 1:130 Somatic cells, 3:93 Somatic hypermutation cytidine deamination in, 4:161 description of, 4:94–96, 4:156–157 mismatch repair proteins in, 4:164 proteins involved in, 4:158–160 Somatic nuclear transfer fusion, 1:222 intracytoplasmic sperm injection, 1:221–222 Somatostatin, 3:183 Sonic hedgehog description of, 3:703, 3:705 knockout mouse phenotype, 1:313 Sorbitol dehydrogenase, 3:200 Sound effects on reproduction, 3:106 Soya oil, 3:335 S1P1, 4:202 SP-A, 4:14 SPA-1, 4:200 Spatial resolution, 3:490 SP-D, 4:14 Specific dynamic action, 3:325 Specific dynamic effect, 3:325 Specific intracellular adhesion molecule-3 grabbing non-integrin family, 4:13 Specific locus test, 1:227 Specific minimal energy cost, 3:67 Specific pathogen free breeding colony, 3:247–248, 3:660 description of, 3:218, 3:312, 3:389, 3:525 Specimen collection bile, 3:454 blood. See Blood collection bone marrow, 3:458 cerebrospinal fluid, 3:458 feces, 3:458 lacrimal fluid, 3:459 lymph, 3:460 milk, 3:459 ova, 3:459–460 peritoneal cells, 3:459 pulmonary cells, 3:459 saliva, 3:458–459 sperm, 3:460 urine, 3:458, 3:460 Spectral Doppler echocardiography, 3:46 Spectral karyotyping applications, 1:146–147, 1:154 principles, 1:153–154 Sperm collection of, 3:460 genistein effects on, 3:350 maturation of, 3:95 motility of, 3:102
Spermatids, 3:96–97 Spermatogenesis events necessary for, 3:96–97 hormonal control of, 3:97 Sertoli cells in, 3:95–96 Spermatogonia, 3:96 Spermiogenesis, 3:97 Sphincter ani externus, 3:18, 3:20 Sphincter colli superficialis, 3:4 Sphincter of Oddi, 3:64 Spi-B, 4:95 Spinal and bulbar muscular atrophy, 3:681 Spinal muscular atrophy, 3:678 Spindle cell tumor, 2:596 Spine, 1:197 Spinocerebellar ataxia, 3:680–681 Spirometry, 3:54 Spironucleus muris cell biology, 2:524 clinical features, 2:524 diagnosis, 2:524 life cycle, 2:523 morphology, 2:523 prevention, 2:524 research implications, 2:524 taxonomy, 2:523 treatment and control, 2:524 Spleen accelerated erythropoiesis in, 3:163 anatomy of, 4:77 dendritic cells, 4:147–148 description of, 4:69 development of, 4:75–76 erythropoiesis in, 3:163 granulopoiesis, 3:163 hematopoietic role of, 3:162–163 hematopoietic stress effects on, 3:163 megakaryocytes in, 3:162 megakaryocytopoiesis in, 3:163 red pulp of, 4:77 white pulp of, 4:77 Splenectomy, 3:162–163, 3:471 Splice variants functions, 1:103–104 nomenclature, 1:90 Spontaneous disease. See specific strains Sprague Dawley rats, 3:128 Sry, 3:92 SSCP. See Single-strand conformational polymorphism SSLP. See Simple sequence length polymorphism ST segment, 3:37 staggerer, 3:676 Stainless steel isolators, 3:219, 3:221 Standard operating procedures, 3:311, 3:411 Staphylococcus spp. clinical features, 2:391–394 culture, 2:390 diagnosis, 2:394–395 epizootiology, 2:394 properties, 2:390 S. aureus, 2:729–730, 3:392, 3:742 strains, 2:390–391 treatment and control, 2:382–383
368 Stargazer, 3:573–574 stargazer, 3:573 Static microisolator cages, 3:294–295 STATs description of, 4:180–181 in vivo functions of, 4:185 Stat5a- and Stat5b-deficient mice, 4:186 Stat1-deficient mice, 4:184 Stat2-deficient mice, 4:184 Stat3-deficient mice, 4:184, 4:186 Stat4-deficient mice, 4:186 Stat6-deficient mice, 4:186 Stat1S727A-mutant mice, 4:184 Stat3S727A-mutant mice, 4:184, 4:186 Steam sterilization chemical-based, 3:421 description of, 3:219, 3:421 of isolators, 3:225, 3:279 of soiled materials, 3:312 research facility use of, 3:303 safety concerns, 3:421 time-temperature relationships, 3:421 Stearoyl-CoA desaturase-1, 3:185, 3:711 Stem cell(s) aging and, 3:665–666 damage to, leukocytes affected by, 3:153–154 embryonic. See Embryonic stem cells hair follicle, 3:693, 3:709–710 hematopoietic, 3:641, 3:666, 4:33 Stem cell factor, 4:170 Sterility, 1:60–61 Sterilization by irradiation, 3:364–365 definition of, 3:424 dry heat, 3:362 ethylene oxide, 3:362, 3:422 moist heat, 3:362 of cage, 3:280–281 of diet, 3:304–305, 3:361 of drinking water, 3:308 of isolators chemical, 3:223–225 chlorine dioxide for, 3:224 definition of, 3:236–237 documentation of, 3:224 formaldehyde gas for, 3:224 hydrogen peroxide and peracetic acid for, 3:224 irradiation, 3:225 peracetic acid for, 3:223–224 steam, 3:225, 3:279 of surgical instruments, 3:465 pulsed vacuum, 3:305 steam. See Steam sterilization Sternohyoideus, 3:5, 3:8 Sternooccipitalis, 3:4–3:5 Sternothyroideus, 3:8 Sternum anticlinalis, 3:4 Steroid hormones description of, 3:125 mechanism of action, 3:125 Steroid receptor coactivator-1, 3:202
CUMULATIVE
Stomach anatomy of, 3:16, 3:64 catheterization of, 3:477–478 musculature of, 3:64 Storage of bedding, 3:423 of feed, 3:360–361 vitamin losses caused by, 3:343–344 Strain. See specific strain Strain 129 historical perspective, 2:633 nomenclature, 2:633–634 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:636 nonneoplastic conditions, 2:634–636 related strains, 2:636 Stratum basalis, 3:712 Stratum corneum, 3:712 Stratum granulosum, 3:712 Stratum lucidum, 3:712 Strbp, 3:622 Streptobacillus moniliformis classification, 2:383 clinical features, 2:384 control, 2:384 diagnosis, 2:384 epizootiology, 2:384 history of study, 2:383 properties, 2:383 Streptococcus spp,. clinical features, 2:396–398 control of, 2:399 culture, 2:396 diagnosis, 2:398–399 epizootiology, 2:398 group B, 3:392 prevention of, 2:399 properties, 2:395–396 S. fecalis, 3:228 strains, 2:396 Stress convulsions and, 3:582 corticosterone levels affected by, 3:182 leukocytes affected by, 3:153 Stress erythropoiesis, 3:142 Stromal cells, 3:153–154 Sts-1, 4:238 Sts-2, 4:238 STSs. See Sequence tagged sites Subcutaneous drug administration description of, 3:445 injection, 3:449–450 osmotic pumps, 3:450 pellets, 3:450 Subcutaneous radio frequency transponders, 3:266, 3:441–443 Subfertility, 3:105 Subiliacus lymph node, 3:22 Sublingual gland, 3:63
INDEX
Submandibular bleeding, 3:457–458 Submaxillary gland, 3:63 Subscapularis, 3:6 Substrain definition, 1:82 nomenclature, 1:83 Substrain, definition, 2:628 Sudden unexpected death in epilepsy, 3:579 Sulfaquinoxaline, 2:533 Sulfhemoglobin, 3:144 Sulfobromophthalein, 3:206 Sulfur, 3:336 Superantigens, 4:213 Superoxide dismutase-1, 3:679 Suppressors of cytokine signaling proteins Cis transgenic mice, 4:188 description of, 4:180–181 in vivo functions of, 4:187 SOCS3, 3:184 Socs5 transgenic mice, 4:188 Socs1-deficient mice, 4:186–187 Socs2-deficient mice, 4:188 Socs3-deficient mice, 4:187–188 Supraspinatus, 3:6 Surface-enhanced laser desorption/ionization platform time-of-flight mass spectroscopy, 3:174 Surfactant, 3:51 Surfactant proteins, 4:14 Surgical procedures adrenal demedullation, 3:466 adrenalectomy, 3:466 analgesia after, 3:480–481 anesthesia for. See Anesthesia aseptic technique, 3:465–466 hepatectomy, 3:466 hypophysectomy, 3:466–467 hysterectomy, 3:467–468 hysterotomy, 3:467–468 instrument sterilization, 3:465 intestinal loops, 3:468 laparotomy pack, 3:465 nephrectomy, 3:468 nutritional support after, 3:480 olfactory bulb ablation, 3:468–469 olfactory bulbectomy, 3:468–469 orchidectomy, 3:470 ovarian transplantation, 3:470–471 ovariectomy, 3:470–471 ovariohysterectomy, 3:471 parabiosis, 3:469 parathyroidectomy, 3:469 pinealectomy, 3:470 postoperative care, 3:479–480 preconditioning before, 3:444 skin grafts, 3:472–473 splenectomy, 3:471 survival surgery, 3:465–466 testicular biopsy, 3:470 thymectomy, 3:471–472 thyroidectomy, 3:472 vagotomy, 3:473
CUMULATIVE
369
INDEX
Surgical procedures (continued) vascular catheterization. See Vascular catheterization vasectomy, 3:473–474 Survival diet restriction effects on, 3:366–367 protein restriction effects on, 3:368 SV40, 2:126–127, 2:597–599 SV40-T, 3:38 Swallowing, 3:63 Sweat glands, 3:713 Swimming exercise stress test, 3:41 Swiss mice description of, 3:260 development, 2:657 phenotype, 2:657–658 sources and origins, 2:662–663 spontaneous diseases comparison between strains and stocks, 2:663–672 glossaries neoplasms, 2:682–690 nonneoplastic conditions, 2:672–681 neoplasia, 2:659–660 nonneoplastic conditions, 2:658–659 related strains, 2:660 Swiss Webster mice, 3:260 SWR/J mice, 3:77 SWXL-4 mice, 3:585 Syk kinases, 4:23 Symphysis pelvina, 3:18, 3:20 Synaptonemal complex chromatin preparation for analysis, 1:157–158 formation, 1:155 immunostaining of proteins, 1:158–159 staining of surface-spread chromatin, 1:158 structure, 1:157 Syndecan-1, 4:93 Synteny, 1:106 Synthetic phenols, 3:310 Syphacia spp. description of, 3:398 S. muris, 2:559 S. obvelata diagnosis, 2:555 differentiation from Aspiculuris tetraptera, 2:553 human infection, 2:732–733 life cycle, 2:554 morphology, 2:553–554 research-confounding effects, 2:556 treatment, 2:556–559 Systemic lupus erythematosus antigen clearance, 4:252–253 description of, 3:74, 3:191, 4:251–254 immune complex clearance, 4:252–253 murine models of BXSB.yaa, 4:246–247 description of, 4:244 MRL.lpr, 4:244–246 NZM2410, 4:249–250 [NZW × NZB]F1, 4:247–249
Systemic lupus erythematosus (continued) summary of, 4:255–256 overview of, 4:243–244
T T1 cells, 4:85 T2 cells, 4:85 T cell(s) γδ, 4:39 activation of adaptor molecules, 4:238 antigen recognition and, 4:221, 4:233 description of, 4:88–90 E3 ubiquitin ligases, 4:237–238 humoral factors in, 4:236 negative costimulatory molecules, 4:237 negative regulation of, 4:236–239 phosphatases, 4:238 processes involved in, 4:233–234 adaptive immunity, 4:144–145, 4:208 aging biomarker use of, 3:655 in aging mice, 3:162 anergic, 4:237 antigen receptor binding of, 4:224 antigen-specific, 4:68, 4:145 apoptosis of, 4:236 autoimmunity mediated by, 4:234–235 B cells and, 4:62, 4:91–96 CD4+, 3:371, 4:39–40 CD8+, 3:371, 4:39–40 dendritic cells and, 4:88–89, 4:145 dendritic epidermal, 4:308 development of, 4:38–39, 4:62, 4:282, 4:307 differentiation of, 4:88–90 effector, 4:201, 4:236 epidermal, 4:308 helper cytokines secreted by, 4:91–92 description of, 3:189, 4:40–41, 4:89–90 homeostasis of, 4:236 integrin expression on, 4:196 interleukin-5 production by, 3:192 knockout models, 4:197–198, 4:202, 4:204 lactate dehydrogenase-elevating virus response, 2:223–224 lymphocytic choriomeningitis virus response CD4+ cells, 2:200 CD8+ cells, 2:199–200 description of, 2:187–188 mammalian reovirus response, 2:256 memory, 3:640, 4:90 migration of description of, 4:47, 4:201–202 knockout models, 4:197–198, 4:202, 4:204 transgenic models, 4:197–198, 4:204 mouse adenovirus type 1 response, 2:59 mouse hepatitis virus response, 2:154–155, 2:157 mouse polyoma virus response, 2:112
T cell(s) (continued) mousepox response, 2:81–82 murine cytomegalovirus immune response CD4+ cells, 2:30 CD8+ cells, 2:29–30 myocarditis role, 2:23 naïve, 3:640 natural killer, 4:43 nuclear factor-kB activation in, 4:236 phenotypic differentiation of, 4:40–41 precursors of, 4:63 priming of, 4:89 regulatory, 4:42, 4:235, 4:277 response of, 4:145 self-reactive, 4:42 self-tolerance, 4:42–43 Sendai virus response, 2:295–296 senescence of, 3:640–641 Th cells cytokines secreted by, 4:91–92 description of, 4:40–41, 4:89–90, 4:145 thymic, 4:39, 4:62 tolerance of, 4:235–236 transgenic models, 4:197–198, 4:204 T cell adaptor molecule, 4:255 T cell coreceptors, 4:25–26 T cell inhibitory receptors, 4:268–269 T cell receptor αß, 4:38 γδ rearrangement, 4:39 antigen-binding complex, 4:24–25 in clonal deletion, 4:215 description of, 4:24–25, 4:196 negative selection mediated by, 4:215 signal transduction, 4:234 signaling, 4:196–201 signal-transducing complex, 4:25 stimulation of, 4:235–236 transgenics, 4:209–212 V-D-J rearrangement, 4:38, 4:61 T cell specific adaptor, 4:238 T cell zone, 4:82 T1 relaxation, 3:500–501 T2 relaxation, 3:500 T wave, 3:37 Taenia taeniaeformis, 2:561 Tail cuff blood pressure measurements using, 3:32–33, 3:555 illustration of, 3:448 Tail hair follicles, 3:697 Tail length, 3:655 Tail tattooing, 3:265, 3:441 Tail tendon collagen denaturation rate, 3:655 Tail vein blood collection from, 3:456–457 catheterization of, 3:475 injection into, 3:451 Tap 1/2, 4:123 TAPA, 4:24 Tapasin, 4:18, 4:125 Targeted trapping, 1:264 Tattooing, 3:265, 3:441
370 Tauopathies, 3:683 Tbee, 3:677 T-box 3 transcription factor, 3:722 Tbsp, 4:123 Tcptp, 4:189 Teeth, 3:63 Telemetry, 3:555 Telogen phase, of hair growth, 3:706, 3:708–709 Telomere, 1:153 TEM. See Trimethylenemelamine Temperature ambient behavioral adaptations, 3:107 breeding affected by, 3:106–107, 3:239 energy requirements affected by, 3:325 in holding rooms, 3:291 reproduction affected by, 3:106–107, 3:239 research facility environment, 3:290–291 body heart rate affected by, 3:37 maintenance of, during electrocardiography, 3:37 thermoneutrality zone, 3:67–68 Temperature gradient capillary electrophoresis, 1:257 Temporal resolution, 3:490–491 Temporalis, 3:5 Temporary identification methods, 3:440–441 Tendo musculi peronei longi, 3:6 Tendo musculi tricipitis surae, 3:6 Tensor fasciae latae, 3:6–7 Teres major, 3:4, 3:6 Terminal bronchiole, 3:50 Terminal deoxynucleotidyl transferase dUTP nick-end labeling cells, 4:213 Territoriality, house mice, 1:32–34 Testes anatomy of, 3:95 biopsy of, 3:470 Testing. See Behavioral test Testis dexter, 3:19 Testis sinister, 3:18 Testosterone functions of, 3:202 luteinizing hormone effects on, 3:202 reference ranges for, 3:181 Test-retest reliability, 3:522 Tetracycline Citrobacter rodentium infection management, 2:377 Clostridium piliforme infection management, 2:355 Corynebacterium bovis management, 2:401 Mycoplasma pulmonis infection management, 2:452 TFH cells, 4:90 TGCE. See Temperature gradient capillary electrophoresis Th cells, 4:40–41, 4:89–90, 4:145, 4:291 Th17 cells, 4:41–42
CUMULATIVE
Th2 type immune response, 3:62 Theiler’s murine encephalomyelitis virus antigenic properties, 2:315 biophysical properties, 2:312 clinical course intracerebral inoculation, 2:316–318 oral inoculation, 2:316 control and prevention, 2:320 description of, 3:391 diagnosis, 2:319–320 epizootiology, 2:318–319 genome, 2:312–313 history of study, 2:311–312 immune response, 2:318 persistence, 2:317–318 propagation, 2:315 receptors, 2:314–315 structure, 2:313–314 Thermographs, 3:413 Thermoneutrality zone, 3:67–68 Thermoregulation basal metabolic rate, 3:67 cold adaptation, 3:68–69 description of, 3:67–68, 3:290 fever, 3:69 heat adaptation, 3:69 oxygen tension effects on, 3:69 Thiamine, 3:340, 3:369 Thimet, 3:346 Thoracic cavity, 3:12 Thoracic vagotomy, 3:473 Threonine, 3:333 Thrombopoiesis, 3:154 Thrombopoietin description of, 3:154 platelet production affected by, 3:156 Thromboxane A2, 3:196 Thromboxane B2, 3:196 Thymectomy, 3:471–472 Thymic epithelial cells cortical, 4:208 description of, 4:62, 4:67 origin of, 4:65 Thymic stromal lymphopoietin, 4:145 Thymocytes bone marrow, 4:208 description of, 4:64 double negative, 4:208 from transgenic mice, 4:200 T cell-epithelium interactions during selection of, 4:67–68 Thymus anatomy of, 4:62 cortical epithelial cells of, 4:63, 4:65 corticomedullary junction of, 4:63 dendritic cells in, 4:139 description of, 1:195–196 development of, 4:64–67, 4:208 epithelium of, 4:63 medullary epithelial cells of, 4:63, 4:65 mesenchyme of, 4:65 structure of, 4:62–63 T cell development in, 4:62–64 Thymus-independent antigen, 4:270 Thyroid cartilage, 3:50
INDEX
Thyroid hormone receptors, 3:125 Thyroid hormones, 3:125 Thyroid stimulating hormone characteristics of, 3:204 description of, 3:128 reference ranges for, 3:181 Thyroidectomy, 3:472 Thyrotropin-releasing hormone, 3:204 Thyroxine, 3:181, 3:204–205 Tibia, 3:4, 3:7 Tibialis caudalis, 3:7 Tibialis cranialis, 3:6 Tidal volume, 3:52–53, 3:57 Tight wire hanging time, 3:655 Timed matings, 3:111–112, 3:261–262 Tin, 3:336 Tissue nonspecific alkaline phosphatase, 3:198 Tissue oxygen consumption, 3:41 Tissue-specific antigens, 4:216 201Tl, 3:503 TLC. See Total lung capacity TLR4, 4:110 TLRs. See Toll-like receptors TMEV. See Theiler’s murine encephalomyelitis virus TMP. See Trimethylpsoralen Tnsf1a, 4:279 Toe clipping, 3:265, 3:442 Tolerance B cell, 4:48, 4:61 dendritic cell mediation of, 4:145–146 peripheral, 4:146, 4:266 T cell, 4:42–43, 4:235–236 Toll/interleukin-1 receptor homology domain, 4:3, 4:110 Toll-like receptors characteristics of, 4:3 definition of, 4:110 description of, 4:110 innate responses via, 4:144 mutations, 2:625 Salmonella susceptibility role, 2:371 signaling pathway for, 4:113–114 TLR1, 4:3, 4:110–111 TLR2, 4:3, 4:110–111 TLR3, 4:5, 4:112 TLR4, 4:5, 4:110 TLR5, 4:5, 4:112 TLR6, 4:5, 4:110–111 TLR7, 4:5, 4:112, 4:114 TLR8, 4:5, 4:112 TLR9, 4:5, 4:112, 4:114 TLR10, 4:5 TLR11, 4:5, 4:112–113 types of, 4:3–5 Toltrazuril, 2:531 Tongue, 1:192 Topical administration of drugs, 3:447–449 Total body water, 3:76–3:77 Total lung capacity, 3:53–3:54, 3:57 Total plasma cholesterol, 3:188 Total viable count, 3:365 Total white cell count, 3:141
CUMULATIVE
371
INDEX
Toxicology carcinogenicity studies, 1:302–303 genotoxicity, 1:302 immunotoxicity studies, 1:304 local tolerance studies, 1:301–302 overview, 1:300–301 photo-safety studies, 1:303 reproduction toxicity, 1:302 toxicokinetics, 1:301 Toxoplasma gondii cell biology, 2:535–536 clinical features, 2:536 description of, 4:112 diagnosis, 2:533 history of study, 2:534–535 life cycle, 2:535 prevention, 2:536 research implications, 2:536, 2:538 treatment and control, 2:536 Trace elements, 3:336 Trachea, 3:10, 3:12–3:13, 3:50 Tracheobronchiales lymph nodes, 3:21 Transcription factors description of, 4:216–217 in erythrocyte development, 3:142 leukocytes affected by, 3:149 SCL, 3:159 Transducer-tipped catheters, 3:35–36 Transfer port, 3:221–3:222 Transferrin receptor, 4:8 Transforming growth factor-ß, 3:705, 4:31, 4:236 Transforming growth factor-ß1, 3:193–194 Transgenic mice breeding colonies of, 3:258–259 Cis, 4:188 cytokine expression, 4:293 drug candidate screening using, 3:175 drug target study examples, 1:306–313 historical perspective, 1:7–8 intracytoplasmic sperm injection, 1:216 mutagenesis, 1:262 obesity studies using, 3:623–624 preclinical toxicity testing models, 1:302–303 production of, 3:258–259, 3:623 pronuclear microinjection, 1:214–216 Socs5, 4:188 suppressors of cytokine signaling, 4:186–187 T cell migration, 4:197–198 thymocytes from, 4:200 transgene nomenclature, 1:92–93 viral delivery-based transgenesis, 1:216 Transgenics T cell receptor, 4:209–212 Vß, 4:209, 4:213 Transmissible spongiform encephalopathies, 3:734 Transmission electron microscopy, 3:746 Transplantation bone marrow, 3:453–454 description of, 4:309 kidney, 3:468 ovarian, 3:113, 3:470–471
Transporter associated with antigen processing proteins, 4:18–19 Transpulmonary pressure, 3:56 Transthoracic echocardiography, 3:555–556 Trapezius, 3:4–3:6 Traps, 3:429 Treadmill exercise stress test, 3:41–42 Treg cells, 4:42, 4:235 Tribromoethanol, 3:461–462 Triceps brachii, 3:4–3:6 Trichoecius romboutsi clinical features, 2:576–577 diagnosis, 2:577 host range, 2:576 life cycle, 2:576 morphology, 2:576 pathobiology, 2:576 prevention and control, 2:577 treatment, 2:577 Trichomonas muris cell biology, 2:525 clinical features, 2:525 diagnosis, 2:525 life cycle, 2:524–525 morphology, 2:524–525 prevention, 2:525 research implications, 2:525 taxonomy, 2:524 treatment and control, 2:525 Trichuris muris, 2:559 Tricuspid valve, 3:27 TRIF-dependent pathway, 4:114 Triglycerides measurement of, 3:188 reference range for, 3:181 Triiodothyronine, 3:125, 3:138, 3:204–205 Trimethoprim, Pneumocystis murina management, 2:509–510 Trimethylenemelamine, 1:230 Trimethylpsoralen, 1:256 Triplet repeat disease models, 3:680–682 Trisodium citrate, 3:139 Trithion, 3:346 Trochlearis nerve, 3:7 Trophectoderm cells, 3:102 Trpv5, 1:309–310 Truncus brachiocephalicus, 3:9–10, 3:12 Truncus pulmonalis, 3:9 Truncus vagi dorsalis et aorta, 3:12 Trypanosoma musculi cell biology, 2:526 clinical features, 2:526–527 diagnosis, 2:527 life cycle, 2:526 morphology, 2:526 prevention, 2:527 taxonomy, 2:525–526 treatment and control, 2:527 Trypsin, 3:66 Tryptophan, 3:72 Tshr, 3:128 tub, 3:610, 3:620 Tuba uterina dexter, 3:21 Tuba uterina sinister, 3:20 Tubby hearing-1A, 3:610
Tubby mutation, 3:610 Tubby-like protein genes, 3:610 Tubulus rectus, 3:95 Tulp1, 3:610 Tulp2, 3:610 Tulp3, 3:610 Tumor antigens. See Mouse polyoma virus Helicobacter hepaticus mechanisms, 2:415–416, 2:422 spindle cell tumor features, 2:596 susceptibility of mouse strains AKR strain, 2:639 Balb/c, 2:641–642 C57BL, 2:649–650 C3H, 2:645–646 DBA, 2:651–652 FVB/N, 2:653 glossary of neoplasms, 2:682–689 SJL/J, 2:654 A strain, 2:637–638 strain 129, 2:636 Swiss mice, 2:659–660 Tumor necrosis factor-α burst forming units-erythroid stimulated by, 3:142 characteristics of, 3:194 converting enzyme, 3:194 description of, 4:31 insulin resistance induced by, 3:184 Tumor necrosis factor-ß, 3:194, 4:31 Tumor necrosis factor immediate family, 4:75 Tumor necrosis factor receptors, 4:70–71, 4:217 Tumor necrosis factor-receptor superfamily, 3:194–195 description of, 4:70–71 signaling events, 4:71–72 Tumor necrosis factor-related activation-induced cytokines, 3:194 Tumor necrosis factor-related apoptosis–inducing ligand, 4:171, 4:217 Tumor pathology, in genetically engineered mice comparative human pathology accuracy, 2:582, 2:584, 2:612 digital imaging, 2:614 morphometrics, 2:615 prospects, 2:616–617 reporting of results, 2:613–614 spontaneous tumor surveillance, 2:615–616 validation, 2:612–613 experimental design, 2:587, 2:592, 2:594 gene targets, 2:591 historical perspective, 2:584 Internet resources, 2:591–592 nomenclature conventions, 2:594–595 oncogenic event considerations molecular alterations and microscopic structure, 2:601–603
372
CUMULATIVE
Tumor pathology, in genetically engineered mice (continued) spontaneous and carcinogen-induced tumors, 2:595–596 uniqueness of genetically engineered mouse tumors, 2:596–601 progression metastasis versus microinvasion, 2:611–612 sequential microscopic changes, 2:605–606, 2:608–611 signature phenotypes, 2:586 tissue context effects microscopic structure, 2:604–605 strain effects, 2:604–605 tumor biology, 2:603–604 weak oncogenes, 2:603 tissue-specific expression promoters, 2:592, 2:601–602 Tunica albuginea, 3:95 Tunnel washer, 3:302–303, 3:310, 3:415–417 Turbinates, 3:49 Two-dimensional gel electrophoresis, 3:174 Two-tailed tests, 3:658–659 Tyk2-deficient mice, 4:184 Tylosin Clostridium perfringens infection management, 2:357 Mycoplasma pulmonis infection management, 2:452 Type I errors, 3:658–659 Type II errors, 3:658–659 Tyrosinase, 3:604 Tyrosine kinase receptors, 3:677 Tyrosine phosphatase-1B, 3:622 Tyrp1b, 3:605 Tyzzer’s disease. See Clostridium spp., C. piliforme
U UBE3A, 3:569 Ubiquitin, 3:682 Ubiquitin protein ligase E6-AP, 3:568–569 Ubr1, 3:622 UCH-L1, 3:682–683 α2u-Goblin, 3:71 UL-16 binding protein–like transcript-1, 4:173 Ulcerative dermatitis, C57BL mice, 2:648–649 Ulna, 3:4 Ultrafiltered diet, 3:354 Ultrasmall superparamagnetic iron oxide particles, 3:502 Ultrasonic noise, 3:292 Ultrasound advantages of, 3:496 attenuation, 3:495–496 contrast-enhanced, 3:497–498 Doppler effect, 3:497 in utero imaging using, 3:496, 3:499–500
Ultrasound (continued) physiologic information measured using, 3:497 principles of, 3:45, 3:494–495 properties of, 3:495, 3:497 real-time imaging uses of, 3:496 transducers used in, 3:45 two-dimensional, 3:45 Ultraviolet disinfection, of drinking water, 3:307–308 Unconjugated bilirubin, 3:205 Uncoupling protein-2, 3:620 Undernutrition, 3:367–368 Unfolded protein response, 3:187 Unverricht-Lundborg disease, 3:570 Upper respiratory tract, 3:49–50 Uracil N-glycosylase, 4:161 Urea nitrogen, 3:181, 3:206–207 Urease positive bacteria, 3:230 Ureter dexter et lymphonodus aorticus dexter, 3:17 Ureter sinister, 3:18–3:19 Ureter sinister et lymphonodus aorticus sinister, 3:17 Ureter sinister et vena renalis sinistra, 3:14 Urethane, 3:31 Urethra, 3:20–21, 3:96 Urethral plug, 3:583 Urethralis, 3:19 Urethralis et radix penis, 3:18 Uridine phosphorylase-1, 1:311 Urinalysis, 3:207 Urinary bladder catheterization of, 3:479 volume of, 3:179 Urinary system, 3:388 Urine antidiuretic hormone effects on, 3:70 collection of, 3:179–180, 3:458, 3:460 constituents of, 3:71–3:73 nephrocalcinosis and, 3:338 protein excretion in, 3:72–73 specific gravity of, 3:207 strain differences in, 3:72 volume of, 3:72, 3:181 Urine concentrating ability, 3:655 Urogenital sinus, 3:93 U.S. Biologics Control Act, 3:732 Uterine natural killer cells, 4:173, 4:284 Uterotubal junction, 3:102 Uterus anatomy of, 3:98 corpus of, 3:98 phytoestrogen effects on, 3:350 receptivity of, 3:102–103 Uveoscleral outflow, 3:602
V Vß transgenics, 4:209, 4:213 Vaccination mousepox, 2:87–88, 2:157 murine cytomegalovirus, 2:32–33 Mycoplasma pulmonis, 2:453
INDEX
Vagina anatomy of, 3:20, 3:99 daidzein effects on, 3:350 estrous cycle stage identified by appearance of, 3:100–101 evaluation of, 3:110 genistein effects on, 3:350 phytoestrogen effects on, 3:350 puberty-related opening of, 3:94 Vaginae, 3:21 Vagotomy, 3:473 Vagus nerve, 3:7, 3:65 Valine, 3:333 Vanadium, 3:336 Vancomycin, 2:360 Vaporized hydrogen peroxide, 3:310, 3:426 Variable air volume systems, 3:288 Variable-formula diet, 3:355 Variant Creutzfeldt-Jakob disease, 3:734 Vascular cannulation, 3:452 Vascular catheterization arteries, 3:475 carotid artery, 3:476 catheters used in, 3:474 femoral artery, 3:476–477 femoral vein, 3:476–477 indications for, 3:474 jugular vein, 3:475 nonsurgical approach, 3:474 portal vein, 3:476 renal artery, 3:477 tail vein, 3:475 Vascular cell adhesion molecule-1, 4:81, 4:296 Vascular endothelial growth factor, 3:158, 3:599, 3:601 Vasectomy, 3:473–474 Vasopressin, 3:77, 3:204 Vasopressin receptors, 3:204 Vastus lateralis, 3:6 Vastus medialis, 3:7 Vav1, 4:199 VC. See Vital capacity V-D-J rearrangement, 4:38, 4:61, 4:155–156 Vdr, 3:129 Vena azygos sinister, 3:9 Vena cava caudalis, 3:9, 3:11–12, 3:16–18 Vena cava cranialis dextra, 3:8–11 Vena cava cranialis sinistra, 3:9–11 Vena epigastrica cranialis superficialis, 3:3 Vena jugularis externa dextra, 3:9 Vena ovarica sinistra, 3:21 Vena portae, 3:16 Vena subclavia dextra, 3:9 Vena testicularis dextra, 3:19 Vena thoracica interna dextra, 3:9 Venae ovaricae sinistrae, 3:21 Venae pulmonales, 3:9, 3:11 Venae uterinus sinistrae, 3:21 Venipuncture dorsal pedal, 3:456–457 lateral tail, 3:457 Venous system, 1:193–194 Ventilated cage air quality concerns, 3:295 blowers, 3:298
CUMULATIVE
373
INDEX
Ventilated cage (continued) considerations in using, 3:298 description of, 3:284 excessive ventilation, 3:297–298 individually, 3:295–299, 3:412 intracage supply/intracage exhaust, 3:295–296 intracage supply/perimeter capture, 3:295–296 sanitation considerations, 3:299 Ventilated racks, 3:412 Ventilation, 3:53 Ventriculus dexter, 3:9, 3:11 Ventriculus sinister, 3:9, 3:11 Vermin, 3:402 Vertebrae, 1:197 Vertebrae caudales, 3:4 Vertebrae cervicales, 3:4 Vertebrae lumbales, 3:4 Vertebrae sacrales, 3:4 Vertebrae thoracicae, 3:4 Vertical flow changing station, 3:303 Very low-density lipoprotein description of, 3:185–186 measurement of, 3:188 in mice, 3:537 Very low-density lipoprotein receptor, 3:187 Vesica fellea, 3:15–17 Vesica urina, 3:20 Vesica urinaria, 3:17–19, 3:21 Vesica urinaria et ligamenta, 3:15 Vestibule, 3:50 Vestibulocochlearis nerve, 3:7 Vestibulum vaginae, 3:21 Veterinary pathologist, 3:389 Vibration from individually ventilated cages, 3:298–299 reproduction affected by, 3:106 Vibrissae hair follicles, 3:694, 3:696 Villi, of small intestine, 3:65–66 Virus. See also specific virus in biological materials, 3:400–401 genomic sequences, 3:747–748 hemagglutination methods, 3:746 immunoassays for detecting, 3:746–747 isolation of, 3:744–746 microbiological quality control testing for, 3:744–749 polymerase chain reaction testing, 3:746–748 removal of, from biologicals, 3:739–740 reproduction in mice infected with, 3:105 safety testing for, 3:745 transmission electron microscopy detection of, 3:746 types of, 3:391, 3:733 validation studies for, 3:739 Virus-Serum-Toxin Act, 3:732 Visfatin, 3:184 Vital capacity, 3:53–54, 3:57 Vitamin(s) autoclaving effects on, 3:362–363 classification of, 3:339 deficiency diseases, 3:343–344
Vitamin(s) (continued) diet restriction effects on, 3:368–369 excess of, 3:345 extrusion, 3:343 fat-soluble, 3:339–340, 3:345, 3:365 immunity affected by, 3:370 irradiation effects on, 3:365 measurement of, 3:339, 3:342 National Research Council guidelines, 3:342 premixes, 3:343–344 in purified diets, 3:342 requirements, 3:342–343 stability of, 3:343–344 storage losses of, 3:343–344 toxicities, 3:345 water-soluble, 3:340–341 Vitamin A1, 3:339, 3:342, 3:345, 3:368 Vitamin A2, 3:339, 3:342, 3:345, 3:368 Vitamin B1 autoclaving effects on, 3:362 description of, 3:340, 3:342 Vitamin B2, 3:340, 3:342 Vitamin B3, 3:340 Vitamin B4, 3:340–341 Vitamin B5, 3:341 Vitamin B6, 3:341–342 Vitamin B7, 3:341 Vitamin B9, 3:341 Vitamin B12, 3:341–342 Vitamin C, 3:550–551 Vitamin D2, 3:339 Vitamin D3, 3:339, 3:342 Vitamin D receptor, 3:129, 3:714 Vitamin D response elements, 3:129 Vitamin E, 3:340, 3:342, 3:345, 3:368–370, 3:550 Vitamin H, 3:341 Vitamin K1, 3:340, 3:344 Vitamin K2, 3:340, 3:344 Vitamin K3, 3:340, 3:342–344, 3:362 Vivarium. See Research facilities VL30 elements, 2:276 Volar pads, 3:714–718 Volatile hazardous agents, 3:280 Voltage-gated calcium channels, 3:572–573 Voltage-gated K+ channel KCNA 1, 3:568 Voltage-gated Na+ channel SCN2A, 3:567–568 Vomeronasal organ comparative embryology, 1:197 description of, 3:49 Vomitoxin, 3:348
W W0.75, 3:324 Waardenburg syndrome, 3:679 Wasting disease, 2:194 Water absorption of, 3:66 access methods for, 3:241 body content of, 3:76–77 for breeding colony, 3:241
Water (continued) drinking acidification of, 3:308, 3:372, 3:418 additives in, 3:308 anthelmintics added to, 3:309 antibiotics added to, 3:309 automatic systems for, 3:241, 3:285–286 bisphenol A contamination, 3:307 chlorine added to, 3:308–309, 3:372, 3:418–419 contaminants in, 3:241 delivery of, 3:241, 3:286–290, 3:307 drug administration in, 3:444–445 hydrochloric acid added to, 3:308, 3:418 irradiation of, 3:372 mechanical filtration of, 3:372 microorganisms in, 3:371 monitoring of, 3:420 municipal sources of, 3:241, 3:307 ozone treatment of, 3:372 processing of, 3:307–308 quality of, 3:371–372, 3:418 restriction of, 3:371 reverse osmosis of, 3:307, 3:372 steam sterilization of, 3:308 sterilization of, 3:225, 3:308 ultraviolet disinfection of, 3:307–308 variations in, 3:306–307 functions of, 3:74–75 gelled, 3:419 intake of, 3:75–76 loss of, 3:76 monitoring of, 3:420 mouse models used to study, 3:75–76 quality of, 3:417 for room level bioexclusion facilities, 3:244–245 surface, 3:307 turnover of, 3:76 well, 3:307 Water balance, house mice, 1:32 Water to food ratio, 3:76 Watering systems automatic chlorine added to, 3:308–309 cleaning of, 3:420 description of, 3:241, 3:285–286, 3:419–420 flush systems, 3:419–420 pressure in, 3:420 recirculating systems, 3:419–420 steam autoclaving of, 3:420 bottles chlorine dissipation in, 3:308 description of, 3:241, 3:286, 3:419 illustration of, 3:287–290 inspection of, 3:419 research facility use of, 3:307 stoppers for, 3:307 sipper tubes, 3:419 Water-soluble vitamins, 3:340–341 Weaning, 3:239 weaver, 3:574–575, 3:676
374 Well water, 3:307 Wensinck’s glycerol broth, 3:420 Wg1, 3:624 What’s Wrong with My Mouse?, 3:519, 3:530 White blood cell count, 3:151 White noise, 3:415 Whitten effect, 1:54 Whole body plethysmography, 3:59–60 Wild-derived mice allelic diversity of, 3:650–651 definition of, 3:650 evolution of, 3:650 Wilms’ tumor homolog 1, 3:93 Wlds, 3:677 Wnt, 2:598–599, 2:603–604 Wnt glycoproteins, 4:67 Wnt signaling in hair follicle morphogenesis, 3:705 in mammary gland development, 3:722–723
CUMULATIVE
Wnt signaling (continued) in sebaceous gland development, 3:712 in volar pad development, 3:718 Wobbler mouse, 3:677 Wolffian duct, 3:70, 3:93 Wound healing, 3:655
X XCR1, 3:197 Xiap, 4:280 X-linked immunodeficiency, 4:307 XO oocyte, gene mapping, 1:131 X-ray computed tomography, 3:493–494 X-ray imaging, 3:493–494 Xylazine, 3:461
Y Yellow fever vaccine, 3:735 Yolk sac, 1:190–192
INDEX
Z Z score, 3:659 ZAP-70, 4:199, 4:234, 4:307 Zearalenone, 3:346, 3:348 Zigzag hairs, 3:695–696 Zinc, 3:336, 3:371 Zoonoses. See also specific diseases allergy. See Laboratory animal-associated allergy arthropod infestation, 2:733–735 bacterial disease, 2:725–730 bites, 2:735 definition, 2:720 dermatophytosis, 2:730–731 helminth disease, 2:732–744 rickettsial disease, 2:724–725 viruses, 2:720–724 Zygomaticus, 3:5
