Autoantibodies

AUTOANTIBODIES Editors" Dr. James B. Peter Specialty Laboratories, Inc. 2211 Michigan Avenue Santa Monica, California 90404-3900 U.S.A.

Prof. Yehuda Shoenfeld Department of Medicine Research Unit of Autoimmune Diseases Chaim Sheba Medical Center Tel-Hashomer 52621 Israel

1996 ELSEVIER Amsterdam

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ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands

Library of Congress Cataloging-in-Publication Data A u t o a n t i b o d i e s / editors, J a m e s B. Peter, Y e h u d a S h o e n f e l d . p. cm. I n c l u d e s b i b l i o g r a p h i c a l r e f e r e n c e s a n d index. ISBN 0-444-82383-2 i. A u t o a n t i b o d i e s . I. Peter, J a m e s B. II. S h o e n f e l d , Yehuda. [DNLM: i. A u t o a n t i b o d i e s . 2. A u t o i m m u n e D i s e a s e s -- e t i o l o g y . QW 575 A939 1996] QRI86.82.A95 1996 616.97'8--dc20 DNLM/DLC 96-13604 for L i b r a r y of C o n g r e s s CIP

ISBN 0-444-82383-2 01996 Elsevier Science B.V. All rights reserved.

No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher, Elsevier Science B.V., Copyright & Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands. Special regulations for readers in the U . S . A . - This publication has been registered with the Copyright Clearance Center Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01293. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside the U.S.A., should be referred to the copyright owner, Elsevier Science B.V., unless otherwise specified. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should be made. This book is printed on acid-free paper. Printed in the Netherlands

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Dedication This book is dedicated to our wives Joan C. Peter

Irit Shoenfeld

and children Deborah Peter Estes Joan Peter Noneman James B. Peter, Jr. Karen Peter Cane Christine Peter Gard Arthur L. Peter

Netta Shoenfeld Guy Shoenfeld Amir Shoenfeld

and in loving memory of Carl J. Peter, III

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List of Contributors Gyiirgy Abel, M.D., Ph.D. Department of Immunology Research Lahey-Hitchcock Clinic Burlington, MA 01805, USA Mahmoud Abu-Shakra, M.D. Rheumatic Diseases Unit Department of Medicine Ben-Gurion University Soroka Medical Centre Beer-Sheva, Israel Nisen Abuaf, M.D., Ph.D. Laboratoire Central d'Immunologie et d'H6matologie H6pital Saint-Antoine 75571 Paris Cedex 12, France Vincent Agnello, M.D. Department of Laboratory Medicine Lahey-Hitchcock Clinic Burlington, MA 01805, USA Alaa E.E. Ahmed, Ph.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA Donato Alarc6n-Segovia, M.D. Department of Immunology and Rheumatology Instituto Nacional de la Nutrici6n Tlalpan, Mexico, D.F. 14000 Mexico

Mustafa S. Atta, M.B., Ch.B., M.Sc., Ph.D. Division of Molecular and Clinical Immunology Department of Clinical Laboratory Sciences University Hospital Queen's Medical Centre Nottingham NG7 2UH, UK Douglas C. Aziz, M.D., Ph.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA Ehud Baharav, M.D. Research Laboratory of Clinical Immunology The Basil and Gerald Felsenstein Medical Research Center Beilinson Campus, Petach-Tiqva 49100 Israel Noori E. Barka, Ph.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA Robert M. Barr, Ph.D. St. John's Institute of Dermatology UMDS, St. Thomas's Hospital London SE1 7EH, UK Klaus Bendtzen, M.D., D.M.Sc. Institute for Inflammation Research RHIMA Center, Rigshospitalet DK-2200, Copenhagen N, Denmark

Aftab A. Ansari, Ph.D. Department of Pathology Winship Cancer Center Emory University School of Medicine Atlanta, GA 30322, USA

Jo H.M. Berden, M.D., Ph.D. Division of Nephrology Academic Hospital St. Radboud NL-6525 GA Nijmegen, The Netherlands

Stanley H. Appel, M.D. Department of Neurology Baylor College of Medicine Houston, TX 77030, USA

Eloisa Bonfa, M.D., Ph.D. Division of Rheumatology University of S~o Paulo S~o Paulo, Brazil

Gowthami Arepally, M.D. University of Pennsylvania School of Medicine Children's Hospital of Philadelphia Philadelphia, PA 19104, USA

Marie B0rretzen, M.Sc. Institute of Immunology and Rheumatology The National Hospital N-0172 Oslo, Norway

vii

Per Brandtzaeg, Ph.D. Laboratory for Immunohistochemistry and Immunopathology Institute of Pathology University of Oslo The National Hospital N-0027 Oslo, Norway Nathan Brot, Ph.D. Roche Institute of Molecular Biology Roche Center Nutley, NJ 07110, USA C. Lynne Burek, Ph.D. Departments of Pathology, Molecular Microbiology and Immunology Johns Hopkins Medical Institutions Baltimore, MD 21205-2196, USA Dan Buskila, M.D. Rheumatic Diseases Unit Department of Medicine Ben-Gurion University Soroka Medical Centre Beer-Sheva, Israel Ralph Butkowski Ph.D. INCSTAR Corporation Stillwater, MN 55455, USA Per Bygren, M.D. Department of Nephrology Lund University Hospital S-221 85 Lund, Sweden Antonio R. Cabral, M.D. Department of Immunology and Rheumatology Instituto Nacional de la Nutrici6n Tlalpan, Mexico, D.F. 14000 Mexico

Rieard Cervera, M.D. Unitat de Malalties and Autoimmunes Sist~matiques Hospital Clinic I Provincial de Barcelona 08036 Barcelona, Catalonia, Spain Aristidis Charonis, M.D., Ph.D. Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, MN 55082, USA Alain Chevailler, M.D. Laboratoire d'Immuno-Pathologie Centre Hospitalier Universitaire d'Angers Angers, Cedex 01, France Marco Cicardi, M.D. Clinica Medica III Istituto di Medicina Interna Universit~ di Milano Milan 20122, Italy Douglas B. Cines, M.D. Department of Pathology/Laboratory Medicine Hospital of the University of Pennsylvania Pennsylvania, PA 19104, USA Ross L. Coppel, M.D., Ph.D. Department of Microbiology Monash University Clayton, Victoria, 3168, Australia Joseph E. Craft, M.D. Section of Rheumatology Department of Internal Medicine Yale University School of Medicine New Haven, CT 06520-8031, USA Elena Csernok, Ph.D. Department of Rheumatology University of Ltibeck Ltibeck 23538, Germany

Margarida Castell, Ph.D. Unit of Physiology and Physiopathology Faculty of Pharmacy University of Barcelona Barcelona 08028, Spain

Charlotte Cunningham-Rundles, M.D., Ph.D. Departments of Medicine, Pediatrics and Biochemistry The Mount Sinai Medical Center New York, NY 10029-6574, USA

Carlo Catassi, M.D. Department of Pediatrics University of Ancona 60123 Ancona, Italy

Josep O. Dalmau, M.D., Ph.D. Department of Neurology Memorial Sloan Kettering Cancer Center New York, NY 10021, USA

viii

Alvin E. Davis lII, M.D. Division of Nephrology Children's Hospital Medical Center Cincinnati, OH 45229-3039, USA

Janet A. Fairley, M.D. Department of Dermatology Medical College of Wisconsin Milwaukee, WI 53226-0509, USA

Roger L. Dawkins, M.D., D.Sc. Department of Clinical Immunology Royal Perth Hospital Sir Charles Gairdner Hospital The Centre for Molecular Immunology and Instrumentation University of Western Australia Perth 6001, Western Australia, Australia

Pnina Fishman, Ph.D. Research Laboratory of Clinical Immunology The Basil and Gerald Felsenstein Medical Research Center Beilinson Campus, Petach-Tiqva 49100 Israel

Judah A. Denburg, M.D. Division of Clinical Immunology and Allergy McMaster University Hamilton, Ontario, L8N 3Z5 Canada Luis A. Diaz, M.D. Department of Dermatology Medical College of Wisconsin Milwaukee, WI 53226-0509, USA Hemmo A. Drexhage, M.D., Ph.D. Department of Immunology Erasmus University 3000 DR Rotterdam, The Netherlands Maryvonne Dueymes, M.D., Ph.D. Laboratoire d'Immunologie Centre Hospitalier R6gional et Universitaire Brest, Cedex, France Edward Dwyer, M.D. Department of Medicine Columbia University College of Physicians & Surgeons New York, NY 10032, USA Keith B. Elkon, M.D. The Hospital for Special Surgery Cornell University Medical Center New York, NY 10021, USA Agustin Espafia-Alonso, M.D. Department of Dermatology Medical College of Wisconsin Milwaukee, WI 53226-0509, USA

David M. Francis, B.Sc. St. John's Institute of Dermatology UMDS, St. Thomas's Hospital London SE1 7EH, UK Marvin J. Fritzler, M.D., Ph.D. Department of Medicine The University of Calgary Calgary, Alberta T2N 4N1 Canada Robert S. Fujinami, Ph.D. Department of Neurology University of Utah Salt Lake City, UT 84132, USA Henry M. Furneaux, Ph.D. Laboratory of Molecular Neuro-Oncology Program in Molecular Pharmacology and Therapeutics Memorial Sloan Kettering Cancer Center New York, NY 10021, USA Uri Galili Ph.D. Department of Microbiology and Immunology Medical College of Pennsylvania Philadelphia, PA 19129, USA Tamara S. Galloway, B.Sc., Ph.D. Division of Medicine University of Plymouth Drake Circus Plymouth PL4 8AA, UK Jacob George, M.D. Department of Medicine "B" Research Unit of Autoimmune Diseases Sheba Medical Center Sackier Faculty of Medicine Tel Aviv University Tel-Hashomer 52621, Israel

ix

M. Eric Gershwin, M.D. Division of Rheumatology, Allergy and Clinical Immunology School of Medicine University of California at Davis Davis, CA 95616, USA Azzudin E. Gharavi, M.D. Department of Medicine, Section of Rheumatology Louisiana State University Medical Center New Orleans, LA 70112-2822, USA Jean Guy Gilles, Ph.D. Katholieke Universiteit Leuven Center for Molecular and Vascular Biology 3000 Leuven, Belgium George J. Giudice, Ph.D. Department of Dermatology Medical College of Wisconsin Milwaukee, WI 53226-0509, USA Paul A. Gleeson, Ph.D. Department of Pathology and Immunology Monash University Medical School Melbourne, Victoria 3181, Australia Tom P. Gordon, Ph.D. Department of Clinical Immunology and

Centre for Transfusion Medicine and Immunology Flinders Medical Centre Bedford Park, South Australia 5042

Malcolm W. Greaves, M.D., Ph.D. St. John's Institute of Dermatology UMDS, St. Thomas's Hospital London SE1 7EH, UK Wolfgang L. Gross, M.D. Department of Rheumatology University of Ltibeck Ltibeck 23538

Morten Bagge Hansen, M.D. Institute for Inflammation Research RHIMA Center, Rigshospitalet DK-2200, Copenhagen N, Denmark Leonard C. Harrison M.D., D.Sc. Burnet Clinical Research Unit The Walter and Eliza Hall Institute of Medical Research Royal Melbourne Hospital Parkville, Victoria 3050 Australia Kip R. Hartman, M.D. Department of Hematology Walter Reed Army Institute of Research Washington, DC 20307, USA Thomas Hellmark, M.Sc. Department of Nephrology Lund University Hospital S-221 85 Lund, Sweden Martin Herrmann, Ph.D. Department of Medicine III Institute for Clinical Immunology and Rheumatology Friedrich-Alexander University of ~rlangen-Nurenberg Erlangen 91054, Germany Ahvie Herskowitz, M.D. Ischemia Research and Education Foundation San Francisco, California 94134 Michihiro Hide, M.D., Ph.D. Department of Dermatology Onomichi General Hospital Onomichi 722, Japan A. Hoek, M.D., Ph.D. Department of Immunology Erasmus University 3000 DR Rotterdam, The Netherlands

and

Rheumaklinik Bad Bramstedt GmbH Bad Bramstedt 24572, Germany

William A. Hagopian, M.D., Ph.D. R.H. Williams Laboratory Department of Medicine University of Washington Seattle, WA 98195-7110, USA

Peter N. Hollingsworth, D.Phil. Department of Clinical Immunology Royal Perth Hospital Sir Charles Gairdner Hospital The Centre for Molecular Immunology and Instrumentation University of Western Australia Perth 6001, Western Australia, Australia

Jean-Claude Homberg, M.D., Ph.D. Laboratoire Central d'Immunologie et d'H6matologie H6pital Saint-Antoine 75571 Paris Cedex 12, France Graham R. V. Hughes, M.D. Lupus Arthritis Research Unit The Rayne Institute St. Thomas' Hospital London SE1 7EH, UK Per Hultman, M.D., Ph.D. Department of Pathology I Link6ping University S-581 85 Link6ping Sweden Catherine Johanet, M.D. Laboratoire Central d'Immunologie et d'H6matologie H6pital Saint-Antoine 75571 Paris Cedex 12, France Joachim R. Kalden, M.D., Ph.D. Department of Medicine III Institute for Clinical Immunology and Rheumatology Friedrich-Alexander University of Erlangen-Nuremberg Erlangen 91054, Germany Cees G.M. Kallenberg, M.D. Department of Clinical Immunology University Hospital of Groningen 9700 RB Groningen, The Netherlands Christopher Karopoulos, B.Sc. (hons) Centre for Molecular Biology and Medicine Monash University Clayton, Victoria 3168, Australia Daniel L. Kaufman, Ph.D. Department of Molecular and Medical Pharmacology Brain Research and Molecular Biology Institute University of California, Los Angeles Los Angeles, CA 90095-1735, USA Michel D. Kazatchkine, M.D. INSERM U430 and Universit6 Pierre et Marie Curie H6pital Broussais 75674 Paris Cedex 14, France

Catherine L. Keech, B.Sc. Department of Clinical Immunology and Centre for Transfusion Medicine and Immunology Flinders Medical Centre Bedford Park, South Australia 5042, Australia Munther A. Khamashta, M.D., Ph.D. Lupus Arthritis Research Unit The Rayne Institute, St. Thomas' Hospital London SE1 7EH, UK Glenn B. Knight, Ph.D. Department of Molecular Biology Lahey-Hitchcock Clinic Burlington, MA 01805, USA Takao Koike, M.D. Department of Medicine II Hokkaido University School of Medicine Sapporo 060, Japan Konstantin N. Konstantinov, M.D., Ph.D. W.M. Keck Autoimmune Disease Center Department of Molecular and Experimental Medicine The Scripps Research Institute La Jolla, CA 92037, USA Romano G. Krueger, B.Sc. Department of Clinical Immunology Royal Perth Hospital Sir Charles Gairdner Hospital The Centre for Molecular Immunology and Instrumentation University of Western Australia Perth 6001, Western Australia, Australia Robert G. Lahita, M.D., Ph.D. Division of Rheumatology St. Luke' s/Roosevelt Hospital New York, NY 10019, USA Paul Le Goff, M.D. Department of Rheumatology Centre Hospitalier Rdgional et Universitaire Brest, Cedex, France Vanda A. Lennon, M.D., Ph.D. Neuroimmunology Laboratory Departments of Immunology, Neurology and Laboratory Medicine and Pathology Mayo Clinic Rochester, MN 55905, USA xi

Ake Lernmark, Ph.D. R.H. Williams Laboratory Department of Medicine University of Washington Seattle, WA 98195-7110, USA Peter S.C. Leung, Ph.D. Division of Rheumatology, Allergy and Clinical Immunology University of California at Davis School of Medicine Davis, CA 95616, USA Shuguang Li, M.D., Ph.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA Hans Link, M.D., Ph.D. Division of Neurology Karolinska Institute Huddinge University Hospital S- 141 86 Huddinge, Sweden Zhi Liu, Ph.D. Department of Dermatology Medical College of Wisconsin Milwaukee, WI 53226-0509, USA Luis Llorente, M.D. Department of Immunology and Rheumatology Instituto Nacional de la Nutricion Salvador Zubiran Tlalpan, Mexico, D.F. 14000, Mexico

lan R. Mackay, M.D. Centre for Molecular Biology and Medicine Monash University Victoria 3168, Australia Peter J. Maddison, M.D. Royal National Hospital for Rheumatic Diseases University of Bath Bath BA1 1RL, UK Mart Mannik, M.D. Department of Medicine, Division of Rheumatology University of Washington School of Medicine Seattle, WA 98195, USA Michael P. Manns, M.D. Department of Gastroenterology and Hepatology Zentrum Innere Medizin Medizinische Hochschule Hannover Hannover, Germany Raya Maran, M.D. Department of Medicine "B" Research Unit of Autoimmune Diseases Sheba Medical Center Sackler Faculty of Medicine Tel Aviv University Tel-Hashomer 52621, Israel Eric Martini, M.D. Laboratoire Central d'Immunologie et d'H6matologie H6pital Saint-Antoine 75571 Paris Cedex 12, France

C. Martin Lockwood, M.D. Department of Medicine University of Cambridge School of Clinical Medicine Addenbrooke' s Hospital Cambridge, CB2 2QQ UK

Jos6 M. Mascar6 Jr., M.D. Department of Dermatology Medical College of Wisconsin Milwaukee, WI 53226-0509, USA

Margalit Lorber, M.D. Institute of Clinical Immunology and Allergya Rambam Medical Center The B. Rappaport Faculty of Medicine Technion, Haifa, Israel

Eiji Matsuura, Ph.D. Immunology Laboratory Diagnostics Division Yamasa Corporation Choshi 288, Japan

Richard Lubin, Ph.D. Unit6 301 INSERM Institut de G6nEtique Mol6culaire 75010 Paris, France

Gale A. McCarty-Farid, M.D. Department of Health and Human Services Primary Care Center- GWU/FDA Washington, DC 20036, USA

xii

James McCluskey, M.D. Department of Clinical Immunology and Centre for Transfusion Medicine and Immunology Flinders Medical Centre Bedford Park, South Australia 5042 Neil John McHugh, M.D. Royal National Hospital for Rheumatic Diseases Department of Rheumatology Upper Borough Walls Bath BA1 1RF, UK Sandra M. McLachlan, Ph.D. Thyroid Molecular Biology Unit V.A. Medical Center San Francisco, CA 94121, USA

Marc Monestier, M.D., Ph.D. Department of Microbiology and Immunology Temple University School of Medicine Philadelphia, PA 19140, USA Giuseppe Montagnino, M.D. Divisione di Nefrologia e Dialisi Ospedale Maggiore, IRCCS 20122 Milan, Italy Luc Mouthon, M.D. INSERM U430 and Universit6 Pierre et Marie Curie H6pital Broussais 75674 Paris Cedex 14, France

Thomas A. Medsger, Jr., M.D. Division of Rheumatology/Clinical Immunology University of Pittsburgh School of Medicine Pittsburgh, PA 15261, USA

Sylviane Muller, Ph.D. Institut de Biologie Mol6culaire et Cellulaire CNRS UPR 9021 Immunochimie des Peptides et Virus 67000 Strasbourg, France

Ove J. Mellbye, M.D., Ph.D. Institute of Immunology and Rheumatology The National Hospital N-0172 Oslo, Norway

Loren Karp Murphy, M.A. Inflammatory Bowel Disease Center Cedars-Sinai Medical Center Los Angeles, CA 90048, USA

Ofer Merimsky, M.D. Department of Oncology Tel-Aviv Sourasky Medical Center Sackler Faculty of Medicines Tel Aviv University, Israel

Jacob B. Natvig, M.D., Ph.D. Institute of Immunology and Rheumatology The National Hospital N-0172 Oslo, Norway

Pier Luigi Meroni, M.D. Istituto di Medicina Interna, Malattie Infettive & Immunopatologia Universith degli Studi di Milano 20122 Milan, Italy Karl-Hermann Meyer zum Biischenfelde, M.D. Department of Internal Medicine Johannes Gutenberg University Mainz 55101 Mainz, Germany Frederick W. Miller, M.D., Ph.D. Molecular Immunology Laboratory Division of Cellular and Gene Therapies Center for Biologics Evaluation and Research Food and Drug Administration Bethesda, MD 20892, USA

Todd Nelson Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, MN 55082, USA David A. Neumann, Ph.D. ILSI Risk Science Institute Washington, DC 20036, USA Chester V. Oddis, M.D. Department of Medicine Division of Rheumatology and Clinical Immunology University of Pittsburgh Pittsburgh, PA 15213-3221, USA Emmanuel A. Ojo-Amaize, Ph.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA

xiii

Yutaka Okano, M.D. Department of Medicine Nippon Kokan Hospital Kawasaki 210, Japan

Basil Rapoport, M.D. Thyroid Molecular Biology Unit V.A. Medical Center San Francisco, CA 94121, USA

William Parker, Ph.D. Department of Surgery Duke University Medical Center Durham, NC 27110, USA

Jerome B. Rattner, Ph.D. Department of Medical Biochemistry The University of Calgary Calgary, Alberta T2N 4N1 Canada

Stanford L. Peng, B.A., B.S. Department of Biology and Section of Rheumatology Department of Internal Medicine Yale University School of Medicine New Haven, CT 06520-8031, USA Jeffrey L. Platt, M.D. Departments of Surgery, Immunology and Pediatrics Duke University Medical Center Durham, NC 27110, USA K. Michael Pollard, Ph.D. W.M. Keck Autoimmune Disease Center Department of Molecular and Experimental Medicine The Scripps Research Institute La Jolla, California 92037 Jerome B. Posner M.D. Department of Neurology Memorial Sloan Kettering Cancer Center New York, NY 10021, USA Richard J. Powell, D.M. Division of Molecular and Clinical Immunology Department of Clinical Laboratory Sciences University Hospital Queen's Medical Centre Nottingham NG7 2UH, UK Stephen C. Pummer, B.Sc. Department of Clinical Immunology Royal Perth Hospital Sir Charles Gairdner Hospital The Centre for Molecular Immunology and Instrumentation University of Western Australia Perth 6001, Western Australia, Australia

xiv

Westley H. Reeves, M.D. Departments of Medicine and Microbiology and Immunology Thurston Arthritis Research Center and UNC Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill Chapel Hill, NC 27599-7280, USA Hansotto Reiber, Ph.D. Neurochemisches Labor University of G6ttingen 37075 G6ttingen, Germany Morris Reichlin, M.D. Arthritis/Immunology Program Oklahoma Medical Research Foundation Department of Medicine, College of Medicine Oklahoma University Health Sciences Center Oklahoma City, OK 73104, USA Gilles Renier, M.D. Laboratoire d'Immuno-Pathologie Centre Hospitalier Universitaire d'Angers Angers, Cedex 01, France Manfred Renz, Ph.D. Institute of Immunology and Molecular Genetics D-76133 Karlsruhe, Germany Herminio Reyes, Ph.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA Dieter Roelcke, M.D. Ruprecht-Karls-University Heidelberg Institute for Immunology 69120 Heidelberg, Germany

Noel R. Rose, M.D., Ph.D. Departments of Pathology, Molecular Microbiology and Immunology Johns Hopkins Medical Institutions Baltimore, MD 21205-2196, USA Christian Ross, M.D. Institute for Inflammation Research RHIMA Center, Rigshospitalet DK-2200, Copenhagen N, Denmark Naomi F. Rothfield, M.D. Department of Medicine Division of Rheumatic Diseases University of Connecticut Health Center Farmington, CT 06030-1310, USA Merrill J. Rowley, Ph.D. Centre for Molecular Biology and Medicine Monash University Clayton, Victoria 3168, Australia

Robert S. Schmidli, M.B., Ch.B. Burnet Clinical Research Unit The Walter and Eliza Hall Institute of Medical Research Royal Melbourne Hospital Parkville, Victoria 3050 Australia R. Hal Scofield, M.D. Arthritis/Immunology Program Oklahoma Medical Research Foundation Department of Medicine, College of Medicine Oklahoma University Health Sciences Center Oklahoma City, OK 73104, USA Helge Scott, M.D. Laboratory for Immunohistochemistry and Immunopathology Institute of Pathology University of Oslo The National Hospital, Rikshospitalet N-0027 Oslo, Norway

Robert L. Rubin, Ph.D. W.M. Keck Autoimmune Disease Center Department of Molecular & Experimental Medicine The Scripps Research Institute La Jolla, CA 92037, USA

Hans Peter Seelig, M.D. Institute of Immunology and Molecular Genetics D-76133 Karlsruhe, Germany

Alejandro Ruiz-Argiielles, M.D. Department of Immunology and Rheumatology Instituto Nacional de la Nutricion Salvador Zubiran Tlalpan, Mexico, D.F. 14000, Mexico

Mhrten Segelmark, M.D., Ph.D. Department of Nephrology Lurid University Hospital S-221 85 Lund, Sweden

E. William St. Clair, M.D. Department of Medicine Division of Rheumatology, Allergy and Clinical Immunology Duke University Medical Center Durham, NC 27710, USA

Guy Serre, M.D., Ph.D. Department of Biology and Pathology of the Cell Purpan Medical School University of Toulouse 31059 Toulouse Cedex, France

Jean-Marie R. Saint-Remy, M.D., Ph.D. Katholieke Universiteit Leuven Center for Molecular and Vascular Biology 3000 Leuven, Belgium

GuoQiu Shen, M.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA

Minoru Satoh, M.D. Department of Medicine Thurston Arthritis Research Center and UNC Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill Chapel Hill, NC 27599-7280, USA

Yehuda Shoenfeld, M.D. Department of Medicine "B" Research Unit of Autoimmune Diseases Sheba Medical Center Sackier Faculty of Medicine Tel Aviv University Tel-Hashomer 52621, Israel

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Ruud J.T. Smeenk, Ph.D. Dept of Autoimmune Diseases, C.L.B. NL- 1066 CX Amsterdam, The Netherlands

Christine Stemmer, B.Sc. Institut de Biologie Mol6culaire et Cellulaire CNRS UPR 9021 Immunochimie des Peptides et Virus 67000 Strasbourg, France

R. Glenn Smith, M.D., Ph.D. Department of Neurology Baylor College of Medicine Houston, TX 77030, USA

Ann E. Stitzel, M.S. Department of Pediatrics SUNY Health Science Center at Syracuse Syracuse, NY 13210, USA

Josef S. Smolen, M.D. 2nd Department of Medicine Lainz Hospital Department of Rheumatology University of Vienna Vienna A- 1090 Austria

Lovorka Stojanov, M.D. Department of Medicine Thurston Arthritis Research Center and UNC Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill Chapel Hill, NC 27599-7280, USA

Enrique Roberto Soriano, M.D. Unidad de Reumatologia Hospital Italiano de Buenos Aires Gascon 450 (1181) Buenos Aires Argentina Thierry Soussi, Ph.D. Unit6 301 INSERM Institut de G6n6tique Mol6culaire 75010 Paris, France Joseph Sperling, Ph.D. Department of Organic Chemistry The Weizmann Institute of Science Rehovot 76100, Israel Ruth Sperling, Ph. D. Department of Genetics The Hebrew University of Jerusalem Jerusalem 91904, Israel Roger E. Spitzer, M.D. Department of Pediatrics SUNY Health Science Center at Syracuse Syracuse, NY 13210, USA Giinter Steiner, Ph.D. Ludwig Boltzmann-Institute for Rheumatology and Balneology Dept. of Rheumatology Vienna A- 1130, Austria

xvi

Morten Svenson, Ph.D. Institute for Inflammation Research RHIMA Center, Rigshospitalet DK-2200, Copenhagen N, Denmark Antonius J.G. Swaak, M.D., Ph.D. Department of Rheumatology Dr. Daniel den Hoed Clinic 3085 EA Rotterdam, The Netherlands Christof H. Szymkowiak, Ph.D. Rheumaklinik Bad Bramstedt GmbH Bad Bramstedt 24572, Germany Stephan R. Targan, M.D. Inflammatory Bowel Disease Center Cedars-Sinai Medical Center Los Angeles, CA 90048, USA Ira N. Targoff, M.D. University of Oklahoma Health Sciences Center Oklahoma Medical Research Foundation Department of Arthritis and Immunology Oklahoma City, OK 73104, USA Jeff W. Terryberry, B.S. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA Charles E. Thirkill, Ph.D. Ophthalmology Research University of California, Davis Medical Center Sacramento, CA 95816, USA

Keith M. Thompson, Ph.D. Institute of Immunology and Rheumatology The National Hospital N-0172 Oslo, Norway lan Todd, Ph.D. Division of Molecular and Clinical Immunology Department of Clinical Laboratory Sciences University Hospital Queen's Medical Centre Nottingham NG7 2UH, UK Ban-Hock Toh, M.B.B.S, D.Sc. Department of Pathology and Immunology Monash University Medical School Melbourne, Victoria 3181, Australia Ulrich Treichel, M.D. Department of Internal Medicine Johannes Gutenberg University Mainz 55101 Mainz, Germany Douglas A. Triplett, M.D. Department of Hematology Ball Memorial Hospital Muncie, IN 47303, USA Diana S. Trundle, Ph.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA George C. Tsokos, M.D. Department of Clinical Physiology Bldg. 40, Room 3078 Walter Reed Army Institute of Research Washington, DC 20307-5100, USA David Joseph Unsworth, Ph.D. Department of Clinical Immunology Southmead Hospital Bristol BS10 5ND, UK lan R. van Driel, Ph.D. Department of Pathology and Immunology Monash University Medical School Melbourne, Victoria 3181, Australia Dolores Vazquez-Abad, M.D. Department of Medicine Division of Rheumatic Diseases University of Connecticut Health Center Farmington, CT 06030-1310, USA

Angela Vincent, M.B., M.Sc., M.R.CPath. Department of Clinical Neurology Institute of Molecular Medicine John Radcliffe Hospital Oxford OX3 9DU, UK Christian Vincent, M.D. Department of Biology and Pathology of the Cell Purpan Medical School University of Toulouse 31059 Toulouse Cedex, France Robert Volp6, M.D. Professor Emeritus, Division of Endocrinology Department of Medicine University of Toronto Director, Endocrinology Research Laboratory Wellesley Hospital Toronto, Ontario M4Y 1J3 Canada Jingsong Wang, M.D. Department of Medicine Thurston Arthritis Research Center and UNC Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill Chapel Hill, NC 27599-7280, USA Herbert Weissbach, Ph.D. Roche Institute of Molecular Biology Roche Center Nutley, NJ 07110, USA Mark H. Wener, M.D. Department of Laboratory Medicine University of Washington School of Medicine Seattle, WA 98195, USA Senga Whittingham, Ph.D. Centre for Molecular Biology and Medicine Monash University Victoria 3168, Australia Jiirgen Wieslander, Ph.D. Wieslab AB S-233 70 Lund, Sweden Allan Wiik, M.D., D.Sc. Department of Autoimmunology Statens Seruminstitut DC-2300 Copenhagen 5, Denmark xvii

Terence J. Wilkin, M.D. University of Plymouth Division of Medicine Drake Circus Plymouth PL4 8AA, UK Hugh J. Willison, Ph.D., M.B.B.S. University of Glasgow Department of Neurology Southern General Hospital Glasgow G51 4TF, Scotland Martin A. Winer, Ph.D. Specialty Laboratories, Inc. Santa Monica, CA 90404-3900, USA

xviii

Nico M. Wulffraat, M.D., Ph.D. Department of Immunology University Hospital for Children 3501 CA Utrecht, The Netherlands Pierre Youinou, M.D., Ph.D. Laboratoire d'Immunologie Centre Hospitalier R6gional et Universitaire Brest, Cedex, France Ming-Hui Zhao, M.D., Ph.D. Department of Medicine University of Cambridge School of Clinical Medicine Addenbrooke' s Hospital Cambridge, CB2 2QQ UK

INTRODUCTION It started as an impromptu conversation between colleagues at an international conference. "What we need is something comprehensive, yet up-to-date about autoimmune diseases". "A compilation of what is substantiated and what is surmised." "The 'best guess' of the genuine experts on the real role of autoantibodies in human autoimmune conditions." Now, barely a year later, it is h e r e - A U T O A N T I B O D I E S - - a timely critical review of more than 100 autoantibodies by the leading experts in their respective fields and perspectives on the processes which induce, inhibit or otherwise affect these autoantibodies in humans. To produce an up-to-date book of this caliber so rapidly is a prodigious undertaking and a tribute to the enthusiastic cooperation of our authors throughout the world. The abbreviated timeline was necessary in order to keep pace with the advances in knowledge about autoimmunity. The widespread interest in the field is evidenced by the number of books published recently which are dedicated to the diverse aspects of autoimmunity: from molecular mimicry to clinical manifestations of specific disease entities unknown even 20 years ago. A U T O A N T I B O D I E S i s uniquely formatted to aid the researcher and/or clinician. Chapters on antibodies are presented in alphabetical order; each of these chapters is divided based on our template with sections on Historical Notes ' Autoantigen(s) 9Autoantibodies generally including methods of detection, pathogenetic role, factors in pathogenicity and genetics -- ' Clinical Utility with disease associations and frequencies 9and Conclusion. We have urged our authors to be brief and encouraged the use of tables and figures for concise communication. The references emphasize the latest literature; the reader can use these citations as a starting point for access to earlier works if desired. The summary table which forms the Appendix, entitled: Autoantibodies: Critical Characteristics, furnishes a capsulized overview of the autoantibodies discussed in the text. Segments from the chapter contributors were focused by the Editors to allow quick review of important features and topics by our readers. We are genuinely very grateful to our international cadre of authors who provided such incisive and insightful chapters on topics in which they are truly experts. We also want to acknowledge the efforts of our production team Linda Dearing, M.S., who refined the individual chapter organization and style in accordance with the template; Marion Logan, who directed all communications with authors and publisher; Maria Martinez, Mindy Shaffer, Rose Yesowitch and Paul Lomax who compiled the chapters and revisions, verified references and generally kept us going at our breakneck pace. We also want to thank Mr. Paul Taylor and Elsevier Science B.V. for their contributions to this publication. We sincerely hope that AUTOANTIBODIES will help both the novice and the expert in their efforts to understand and further our understanding of autoimmune disease.

James B. Peter, M.D., Ph.D. Yehuda Shoenfeld, M.D.

xix

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CONTENTS List of Contributors

vii

Introduction

xix

Foreword- The Uses of Autoantibodies N.R. Rose

xxvii

Acetylcholine Receptor Autoantibodies A. Vincent

Actin Autoantibodies J. George and Y. Shoenfeld

10

Affinity and Avidity of Autoantibodies A.E. Gharavi and H. Reiber

13

Alpha-galactosyl (Anti-Gal) Autoantibodies U. Galili

24

Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies P.J. Maddison

31

Aminoacyl-tRNA (other than Histidyl) Synthetase Autoantibodies I.N. Targoff

36

Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Diseases L.K. Murphy and S.R. Targan

47

Antineutrophil Cytoplasmic Autoantibodies with Specificity for Myeloperoxidase C. G.M. Kallenberg

53

Antineutrophil Cytoplasmic Autoantibodies with Specificity for Proteinase 3 W.L. Gross, E. Csernok and C.H. Szymkowiak

61

Antineutrophil Cytoplasmic Autoantibodies with Specificity other than PR3 and MPO (X-ANCA) M.-H. Zhao and C.M. Lockwood

68

Antinuclear Antibodies P.N. Hollingsworth, S.C. Pummer and R.L. Dawkins

74

Autoantibodies in Therapeutic Preparations of Human IgG (IVIg) L. Mouthon and M.D. Kazatchkine

91

Autoantibodies that Penetrate into Living Cells D. Alarc6n-Segovia, L. Llorente and A. Ru/z-Argiielles

96

Autoantibody Subclasses P. Youinou, R. Maran, Maryvonne Dueymes and Y. Shoenfeld

103

[32-Glycoprotein I Autoantibodies E. Matsuura and T. Koike

109

Beta-adrenergic Receptor (and other Hormone Receptor) Autoantibodies D. C. Aziz

115

Bromelain-treated Erythrocyte Autoantibodies A.R. Cabral and D. Alarc6n-Segovia

120

C1 Inhibitor Autoantibodies A.E. Davis III and M. Cicardi

126

C 1q Autoantibodies M.H. Wener and M. Mannik

132

Calcium Channel and Related Paraneoplastic Disease Autoantibodies V.A. Lennon

139

Calcium Channel Autoantibodies and Amyotrophic Lateral Sclerosis R.G. Smith and S.H. Appel

148 xxi

Centriole and Centrosome Autoantibodies J.B. Rattner and M.J. Fritzler Centromere Autoantibodies N.J. McHugh Chromo Autoantibodies E.R. Soriano and N.J. McHugh Coagulation Factor VIII Autoantibodies J. G. Gilles and J.-M.R. Saint-Remy Coagulation Factor (Excluding Factor VIII) Autoantibodies A.E.E. Ahmed Collagen Autoantibodies G. Q. Shen Cryoglobulins G. Montagnino Cryoglobulins Secondary to Hepatitis C Virus Infection G. Abel G.B. Knight and V. Agnello Cytokine Autoantibodies K. Bendtzen, M.B. Hansen, C. Ross and M. Svenson Cytoskeletal Autoantibodies M. Castell dsDNA Autoantibodies R.J.T. Smeenk, J.H.M. Berden and A.J.G. Swaak Endomysial Autoantibodies H. Scott and P. Brandtzaeg Endothelial Cell Autoantibodies P.L. Meroni and P. Youinou Fibrillarin Autoantibodies P. Hultman and K.M. Pollard Fibronectin Autoantibodies M.S. Atta, R.J. Powell and I. Todd 56-kd Nuclear Protein Autoantibodies R. Sperling and J. Sperling Filaggrin (Keratin) Autoantibodies G. Serre and C. Vincent Ganglioside Autoantibodies H.J. Willison Gliadin Antibodies C. Catassi Glomerular Basement Membrane Autoantibodies T. Hellmark, M. Segelmark, P. Bygren and J. Wieslander Glutamic Acid Decarboxylase Autoantibodies in Diabetes Mellitus R.S. Schmidli and L.C. Harrison Glutamic Acid Decarboxylase Autoantibodies in Stiff-man Syndrome D.L. Kaufrnan Glycolipid (Excluding Ganglioside) Autoantibodies M.A. Winer and J. W. Terryberry Golgi Apparatus Autoantibodies G. Renier, M.J. Fritzler and A. Chevailler Granulocyte-specific Antinuclear Antibodies A. Wiik

xxii

153 161 168 172 179 185 195 205 209 217 227 237 245 253 260 266 271 277 285 291 299 308 314 325 331

Heat Shock Protein Autoantibodies M.J. Rowley and C. Karopoulos Heparin-associated Autoantibodies G. Arepally and D.B. Cines Heterophile Antibodies R.L. Dawkins, S.C. Pummer, R.G. Krueger and P.N. Hollingsworth Hidden Autoantibodies M. Lorber, J. George and Y. Shoenfeld Histone (H2A-H2B)-DNA Autoantibodies R.L. Rubin Histone Autoantibodies other than (H2A-H2B)-DNA Autoantibodies C. Stemmer and S. Muller Hormone Nonpeptide Autoantibodies: Thyroid D. C. Aziz Hormone Peptide Autoantibodies D.S. Trundle Human Antimouse Antibodies J.R. Kalden Idiotypes and Anti-idiotypic Antibodies M. Abu-Shakra, D. Buskila and Y. Shoenfeld IgA Autoantibodies C. Cunningham-Rundles IgE Receptor Autoantibodies M. Hide, R.M. Barr, D.M. Francis and M.W. Greaves Insulin Autoantibodies T.S. Galloway and T.J. Wilkin Interferon-inducible Protein IFI 16 Autoantibodies H.P. Seelig and M. Renz Islet Cell Autoantibodies W.A. Hagopian and A. Lernmark Ku and Ki Autoantibodies W.H. Reeves, M. Satoh, L. Stojanov and J. Wang Liver Cytosol Antigen Type 1 Autoantibodies J.-C. Homberg, N. Abuaf C. Johanet and E. Martini Liver/Kidney Microsomal Autoantibodies M.P. Manns Liver Membrane Autoantibodies U. Treichel and K.-H. Meyer zum Biischenfelde Lupus Anticoagulant D.A. Triplett Lymphocytotoxic Autoantibodies A.J. G. Swaak Mi-2 Autoantibodies I.N. Targoff Mitochondrial Autoantibodies P.S.C. Leung, R.L. Coppel and M.E. Gershwin Mitotic Spindle Apparatus Autoantibodies J.B. Rattner and M.J. Fritzler Molecular Mimicry R.S. Fujinami

336 343 351 357 364 373 385 390 403 408 417 423 430 436 441 449 456 462 467 474 478 484 494 501 507

xxiii

Myelin-associated Glycoprotein Autoantibodies H. Link

513

Myelin Basic Protein Autoantibodies S. Li

520

Myocardial Autoantibodies A. Herskowitz, D.A. Neumann and A.A. Ansari

527

Natural Autoantibodies J. George and Y. Shoenfeld

534

Nephritic Factor Autoantibodies R.E. Spitzer, A.E. Stitzel and G.C. Tsokos

540

Neuronal Autoantibodies J.A. Denburg

546

Neuronal Nuclear Autoantibodies, Type 1 (Hu) H.M. Furneaux

551

Neutrophil Autoantibodies K.R. Hartman

555

Nuclear Envelope Protein Autoantibodies K.N. Konstantinov

561

Nucleolar Autoa'ntibodies M. Monestier

567

Nucleosome-specific Autoantibodies J.H.M. Berden and R.J.T. Smeenk

574

Other Autoantibodies to Nuclear Antigens H.P. Seelig

582

p53 Autoantibodies T. Soussi and R. Lubin

595

Parietal Cell Autoantibodies P.A. Gleeson, I.R. van Driel and B.-H. Toh

600

Pathogenic Mechanisms R. Cervera and Y. Shoenfeld

607

Perinuclear Factor (Profilaggrin) Autoantibodies P. Youinou, P. Le Goff and R. Maran

618

Phospholipid Autoantibodies -- Cardiolipin M.A. Khamashta and G.R.V. Hughes

624

Phospholipid Autoantibodies- Phosphatidylserine N.E. Barka

630

Platelet Autoantibodies A.E.E. Ahmed

635

PM-Scl Autoantibodies C. V. Oddis and I.N. Targoff

642

Proliferating Cell Nuclear Antigen Autoantibodies G.A. McCarty-Farid

651

Purkinje Cell Autoantibodies, Type 1 (Yo) J.O. Dalmau and J.B. Posner

655

RA-33 (Heterogeneous Nuclear Ribonucleoprotein Complex) Autoantibodies G. Steiner and J.S. Smolen

660

Recombinant Autoantigens E. W. St. Clair, M.D.

668

Red Cell Autoantibodies D. Roelcke

xxiv

677

Reticulin Autoantibodies D.J. Unsworth Retinal Autoantibodies C.E. Thirkill Retroviral Antibodies M. Herrmann and J.R. Kalden Rheumatoid Factors M. BOrretzen, O.J. Mellbye, K.M. Thompson and J.B. Natvig Ribosomal Autoantibodies E. Dwyer and R.G. Lahita Ribosomal P Protein Autoantibodies E. Bonfa, H. Weissbach, N. Brot and K.B. Elkon RNA Polymerase I-III Autoantibodies Y. Okano and T.A. Medsger Signal Recognition Particle Autoantibodies F. W. Miller Silicate and Silicone Antibodies G.-Q. Shen and E.A. Ojo-Amaize Skin Diseases Autoantibodies L.A. Diaz, A. EspaYta-Alonso, J.A. Fairley, G.J. Giudice, J.M. Mascar6 Jr. and Z. Liu Smooth Muscle Autoantibodies S. Whittingham and I.R. Mackay Spliceosomal snRNPs Autoantibodies S.L. Peng and J.E. Craft SS-A (Ro) Autoantibodies M. Reichlin and R.H. Scofield SS-B (La) Autoantibodies C.L. Keech, J. McCluskey and T.P. Gordon Steroid Cell Autoantibodies A. Hoek, N.M. Wulffraat and H.A. Drexhage Striational Autoantibodies H. Reyes Thyroglobulin Autoantibodies C.L. Burek and N.R. Rose Thyroid Peroxidase Autoantibodies B. Rapoport and S.M. McLachlan Thyrotropin Receptor Autoantibodies

R. Volp~ Topoisomerase-I (Scl-70) Autoantibodies D. Vazquez-Abad and N.F. Rothfield Tubular Basement Membrane Autoantibodies R. Butkowski, T. Nelson and A. Charonis Tyrosinase Autoantibodies P. Fishman, O. Merimsky, E. Baharav and Y. Shoenfeld Xenoreactive Human Natural Antibodies W. Parker and J.L. Platt

684 694 700 706 716 721 727 735 741 746 767 774 783 789 798 805 810 816 822 830 836 842 846

Appendix

853

Subject Index

873

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01996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

FOREWORD

THE USES OF A U T O A N T I B O D I E S

Noel R. Rose, M.D., Ph.D.

Departments of Pathology, Molecular Microbiology, Immunology, Johns Hopkins Medical Institutions, Baltimore, MD 21205-2196, USA

In introducing this first book devoted exclusively to autoantibodies, I cannot help but reflect upon the effect that the study of autoantibodies has had on the development of modern immunology. Studies of the generation of autoantibodies have shaped our current understanding of the basic mechanisms of immune regulation, while the detection of autoantibodies has profoundly influenced our advancing knowledge in clinical immunology and immunopathology.

The Importance of Autoantibodies In recent years, testing for autoantibodies has become the major responsibility of the immunology laboratory. In truth, one autoantibody, anticardiolipin has been the "work horse" of hospital immunology laboratories since time immemorial. Now the diagnostic immunology laboratory is more preoccupied with tests for antinuclear antibodies, rheumatoid factor, ANCA, and other autoantibodies associated with the more prevalent autoimmune conditions. The scope of this work illustrates the important role that the demonstration of autoantibodies has assumed in the diagnosis and monitoring of human disease. It has even become difficult to establish the diagnosis of many diseases, such as systemic lupus erythematosus or thyroiditis, in the absence of the relevant autoantibodies. It is, therefore, appropriate to consider the role that autoantibodies play in autoimmune disease.

Autoimmunity vs. Autoimmune Disease We define autoimmune disease as the pathologic sequel of an autoimmune response. Autoimmunity is signaled by the presence of self-reactive antibodies or self-reactive T cells. In practical terms, the demonstra-

tion of self-reactive T cells is still beyond the capability of most clinical laboratories. Fortunately, no human autoimmune diseases have yet been discovered in which self-reactive T-cells are found in the absence of autoantibodies. Because tests for autoantibodies are relatively easy to perform compared with cellular methods, the demonstration of autoantibodies is likely to remain the cornerstone of the diagnosis of autoimmunity in humans for the foreseeable future. From the standpoint of laboratory diagnosis, the sobering reality is that autoantibodies are relatively common in humans without autoimmune disease. If sufficiently sensitive methods are used, autoantibodies may well occur universally as a normal mechanism for purging the body of effete cell products. In other words, natural autoantibodies may be physiological. These considerations raise two important limitations in the uses of autoantibodies in clinical immunology. The first concern is that autoantibodies are commonly found in human serum in the absence of any discernible disease. Such (naturally occurring autoantibodies) are usually present in low titer, have relatively poor affinity for their corresponding antigen and largely belong to the IgM class. Such is not always the case, however; sometimes IgGs with reasonable binding affinities and elevated titers are present even in the absence of disease. The mere presence of autoantibodies (without appropriate clinical evidence) is rarely, if ever, the basis for diagnosis. The second limitation in the use of autoantibodies derives from the first one. Detection of autoantibodies generally requires some empirically defined threshold value. Only if the activities of autoantibodies are considerably above this threshold can they be deemed of clinical significance.

xxvii

Most autoantibodies are probably not the immediate cause of disease. They are best looked at as markers, rather than agents, of pathology. The question then arises of how to relate the presence of autoantibodies to autoimmune disease. Establishing an Autoimmune Disease

The presence of autoantibodies does not necessarily imply the presence of autoimmune disease. Establishing the causal role of autoimmunity demands additional information. This evidence may be direct, indirect or circumstantial (see Table 1). Direct information requires the demonstration that a self-reactive antibody is the immediate cause of injury or dysfunction. A number of such instances are well documented. Autoantibodies, for example, directly produce the autoimmune forms of hemolytic anemia, leukopenia and thrombocytopenia. Autoantibodies to receptors are clearly involved in the pathogenesis of Graves' hyperthyroidism and myasthenia gravis. There are a few reports that antibodies to hormones may produce corresponding deficiencies. The causal role of autoantibodies in disease can also be approached via the lesions of the disease. Autoantibodies may bind directly to basement membranes of the kidney in glomerulonephritis or localize on intercellular components of the skin in pemphigus and bullous pemphigoid. Such antibodies can sometimes be eluted and demonstrated to produce disease by transfer to an experimental animal. Antibodies may be present in the target organ in the form of immune complexes, as in lupus. It is the immune complex and not the antibody p e r s e which is pathogenetically important in these diseases. Demonstrating the pathogenetic potential of immune complexes is sometimes difficult. In the case of autoimmune diseases due to cellmediated immunity, direct evidence of causation is more difficult to educe. Recently, a model of autoimmune thyroiditis has been obtained in immunodeficient mice by implanting a fragment of human thyroid tissue under the kidney capsule, followed by injection of lymphocytes from patients. Indirect methods to prove the autoimmune etiology of a human disease are employed when it is not possible to demonstrate that autoantibodies directly cause the ~pathognomonic lesions. This strategy requires identification of the antigen target of the autoimmune response and isolation of the equivalent antigen from an experimental animal. The experimental animal can

xxviii

then be immunized with the candidate antigen to determine whether the typical lesions of the autoimmune disease are reproduced. This approach has been invaluable in establishing the autoimmune origin of chronic thyroiditis but, obviously, has some major limitations. Identifying the appropriate antigen can be a monumental task. The target antigen of autoantibodies may not be the antigen that initiates the autoimmune process. Finding an appropriate experimental animal is not easy. Susceptibility to autoimmune disease varies from species to species and from strain to strain. It may be necessary, for instance, to test many strains of mice, before an appropriate one can be identified. Finally, the lesions produced in experimental animals are rarely identical with those found in humans. In addition to the expected species differences, the human disease is often complex and involves more than one antigen. Once an appropriate animal model has been developed, however, it is possible to carry out more definitive experiments such as adoptive transfer of self-reactive T cells to delineate the pathogenetic mechanisms. Experimental immunization has proved to be more successful in reproducing the organ-localized autoimmune diseases (such as myasthenia gravis and thyroiditis) than the systemic ones. In the case of lupus, genetically induced models were developed in mice by selecting and breeding the occasional animals that develop the disease spontaneously. Other approaches involve perturbing normal regulatory functions of idiotypes, cytokines or thymic factors. In reality, most human autoimmune diseases are so defined on the basis of circumstantial evidence. Sometimes that evidence is merely the presence of autoantibodies in the absence of any other definable etiology. Since autoimmune diseases tend to occur in clusters, the presence of other, better defined autoimmune diseases in the same individual or other family member is supportive evidence of an autoimmune etiology. Most autoimmune diseases show statistically significant associations with particular HLA haplotypes. In the lesions of some experimentally induced autoimmune diseases, particular V-gene products are predominant in the T-cell receptor of infiltrating T cells. Therefore, a markedly shrewd V-gene usage suggests an autoimmune etiology. Antibodies as Diagnostic Tools

Establishing an autoimmune etiology of human disease is a difficult undertaking and there are rela-

tively few diseases for which direct or even indirect evidence is presently available. It is hazardous to depend upon circumstantial evidence. Nevertheless, these problems do not necessarily compromise the value of autoantibodies in the diagnosis of disease. In reality, the association of a particular disease with a particular autoantibody depends more upon statistical and epidemiological evidence than upon a cause-andeffect relationship. Autoantibodies may not be the cause of disease; they may not even contribute to disease, but may still be reliable biomarkers of

disease. The demonstration of autoantibodies together with informed interpretation of clinical findings is an essential first step in the diagnosis of many human diseases. In addition, autoantibodies are proving to be increasingly valuable for discriminating subgroups of patients that differ in prognosis or response to therapy. ANCA, for example, can be used to place patients into definable categories of vasculitides with differing clinical and pathological features. The present volume will surely improve the methodology and interpretation of autoantibody tests.

Table 1. Criteria of Human Autoimmune Disease I. DIRECT EVIDENCE A. Autoantibody-mediated 1. Circulating autoantibodies affecting function a. destruction or sequestration of a target cell b. interaction with receptor 1) stimulated function 2) impaired function c. interaction with hormones or enzymes 2. Localized autoantibodies a. demonstration of immunoglobulin and/or complement components at site of lesion b. ability to elute antibodies from lesions c. reproduction of lesions by immunoglobulin eluates 3. Localized immune complexes at site of lesion a. elution of antibody- antigen complex b. identification of antigen 4. Reproduction of disease by passive transfer a. maternal-fetal transfer b. transfer to experimental animals c. demonstration of in vitro injury to target cell B. Cell-mediated 1. Proliferation of T cells in vitro in response to selfantigen 2. Transfer of T cells to immunodeficient mice implanted with target organ 3. In vitro cytotoxicity of T cells with cells of target organ II.

INDIRECT EVIDENCE A. Reproduction of Autoimmune Disease by Experimental Immunization 1. Identification of initiating antigen 2. Immunization of susceptible, syngeneic host with analogous antigen

3. Production of characteristic lesions 4. Reaction of antibody or T cells with an analogous antigen or epitope B. Reproduction of Autoimmune Disease through Idiotype Network 1. Identification of disease-associated idiotype 2. Immunization of susceptible host with the idiotype 3. Production of characteristic lesions C. Spontaneous Models in Experimental Animals 1. Identification of disease in an experimental animal 2. Breeding and selection to increase disease frequency 3. Demonstration of self-reactive antibodies/T cells 4. Passive transfer/adoptive transfer of disease to syngeneic recipients D. Animal Models Produced by Dysregulation of the Immune System 1. Neonatal thymectomy 2. Irradiation with thymectomy 3. Cytokine-deficient homologous inbred animals 4. Transgenic animals with altered: a. cytokine production b. antigen expression c. co-stimulatory factor expression III. A. B. C. D.

CIRCUMSTANTIAL EVIDENCE Presence of Autoantibodies Association with Other Autoimmune Diseases Association with MHC Haplotype Lymphocyte Infiltration of Target Organ 1. Presence of germinal centers in lesions 2. Restricted V-gene usage of infiltrating T cells E. Favorable Response to Immunosuppression 1. Nonspecific 2. Specific (including oral tolerance)

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ACETYLCHOLINE RECEPTOR AUTOANTIBODIES Angela Vincent, M.B., M.Sc., M.R.CPath.

Department of Clinical Neurology, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK

H I S T O R I C A L NOTES Myasthenia gravis (MG) is a disorder in which autoantibodies to acetylcholine receptors (AChR) at the neuromuscular junction of skeletal muscle lead to AChR loss and muscle weakness (Table 1). MG was attributed to the presence of antibodies to muscle "endplate" protein as early as 1960 (Simpson, 1960), but the presence and role of antibodies to endplate acetylcholine receptors (anti-AChR) were not successfully demonstrated until the mid-seventies. Their identification relied on the discovery of a snake toxin, ~-bungarotoxin (~-BuTx), that binds specifically to muscle AChRs (Chang and Lee, 1962). Binding of 125I-cz-BuTx to denervated rat muscle extracts was shown to be inhibited by about 33% of MG sera (Almon et al., 1974) and binding of peroxidase-c~BuTx to human muscle endplates by 44% of MG sera (Bender et al., 1975). The limited success of these techniques probably reflects the relatively low level of

antibodies that interfere with the binding of 125I-o;BuTx to AChR in most human MG sera. The percentage of positive results is much improved with a technique based on immunoprecipitation of 125I-~BuTx-labeled AChR (Lindstrom et al., 1976) which is now the method of choice.

THE A U T O A N T I G E N Definition

The acetylcholine receptor is an oligomeric membrane ion channel protein (Claudio, 1989). Acetylcholine released from the motor nerve terminal binds to the two ~ subunits on the AChR and opens the central pore, allowing cations to diffuse down their electrochemical gradient into the muscle. The current generated represents the endplate potential (EPP) that initiates an action potential in the muscle fiber. In

Table 1. Acetylcholine Receptor Autoantibodies Overview

Antigen

Human acetylcholine receptor o~2,~, '~ or e, 5

Source

Human ischemic limb muscle or TE671 cells

Assay

Immunoprecipitation of 125I-~-Bungarotoxin-labeled AchR

Pathogenetic Role

Clinical response to plasma exchange; passive transfer to mice by injection of IgG; IgG and complement bound to endplates

Characteristics

IgG, all subclasses, high affinity, variable reactivity with different regions on AChR, including main immunogenic region, ~-BuTx binding sites and y subunit.

Incidence

85-90% in generalized MG; 50% in ocular MG; 0% in normal healthy individuals or most disease controls

False-positives

Infrequently found in thymoma without MG, amyotrophic lateral sclerosis, primary biliary cirrhosis, following penicillamine treatment, asymptomatic mothers of arthrogrypotic babies.

MG, anti-AChR lead to a reduction in the number of endplate AChRs, but because the EPP is normally above the threshold necessary for initiation of the action potential, a moderate reduction in AChR function can occur without leading to clinical weakness. The extent to which the EPP is above threshold varies considerably among species and can also differ among individual muscles, partly accounting for the varied clinical expression of MG. The anatomy and physiology of neuromuscular transmission is discussed in more detail elsewhere (Vincent and Wray, 1992).

Adult innervated muscle

Structure/Origin/Sources The AChR consists of five subunits (22, [~, ~, E surrounding a central pore (Figure 1, top). In normal adult muscle, the AChR is restricted to the endplate postsynaptic membrane (Figure 1, bottom); normal healthy human muscle is not a good source of AChR and only about 0.3 pmol/g of muscle can be obtained (Vincent and Newsom-Davis, 1985). In fetal muscle and in primary muscle cell cultures, the AChR is found throughout the myoblasts and myotubes. As developing muscle matures, preceded by innervation, the AChRs become restricted to the endplate region. Subsequently, the ~, subunit is replaced by an ~. In the human, the fetal form is replaced by the adult form by about 33 weeks gestation (Hesselmans et al., 1993); whereas, in rodents the switch takes place after birth. If denervation occurs in the adult, the AChR y subunit reappears along the surface of the muscle fiber (Figure 1, bottom) although the high density of AChRs at the endplate persists. Consequently, fetal and denervated muscle are a rich source of fetal-type AChR. The thymic medulla contains rare muscle-like cells, called 'myoid', that express fetal AChR. They are most common in the fetus and neonate but are also found in adult thymus. Their involvement in the etiology of MG is controversial (Vincent, 1994a). Human muscle AChR can be obtained from amputated limbs; partially denervated amputated muscle from patients with severe ischemia (particularly those with diabetic neuropathy associated with ischemia) is a good source of human fetal AChR, yielding up to 4 pmol/gram of (2-BuTx binding sites (Vincent and Newsom-Davis, 1985) or roughly the amount that can be obtained from fetal human muscle. A small amount of adult AChR will also be present. In partially denervated tissue, muscles with relatively short fibers, such as the gastrocnemius and soleus are

Figure 1. AChR distribution and structure in innervated adult (top) and denervated (bottom) muscle. After denervation (or in embryonic muscle) the epsilon subunit is replaced by a gamma subunit.

preferable to those with long fibers (e.g., sartorius), because the number of endplates/gram of muscle will be higher. Muscles from different amputations, some highly denervated, others containing mainly adult AChR, are homogenized and the crude membrane fraction washed once in buffer and extracted in an equal volume of 2% Triton X100 (Lindstrom et al., 1981; Vincent and Newsom-Davis, 1985). Phenylmethylsulphonyl fluoride and other protease inhibitors should be added. The detergent extracts can be mixed and stored at 4~ for one to two weeks or a t - 7 0 ~ for many months.

Purification/Commercial Sources The high affinity and specificity of this toxin ensures that the assay only measures antibodies to AChR. Consequently no purification of the antigen is required. The extract is labeled by addition of 2--3 nM 125I-t~-BuTx (specific activity should be around 300 Ci/mmol). This can be obtained from commercial sources such as Amersham International and New England Nuclear. An alternate source of AChR is a human rhabdomyosarcoma cell line, RD TE671 (Luther et al., 1989). This line which expresses fetal-type AChR at a level similar or greater than that in primary muscle cultures, is available commercially (ATCC) and in a kit containing TE671 cell extract prelabeled with 125It~-BuTx (RSR Ltd, Cardiff, CF2 7HE, UK). However, because these cells express only fetal AChR, antibodies specific for adult AChR will not be detected. This will probably lead to an increase in the number of negative results by about 7% (Somnier, 1994).

Sequence Similarity The genes for the AChR were first cloned and sequenced from the electric organs of certain fish, and cDNA sequences from many species including human are now available (Beeson et al., 1993). There is considerable sequence similarity between the individual subunits, and even more between analogous subunits of different species. However, the antigenic sites are sufficiently distinct to make it essential to use human or primate AChR as antigen for diagnostic immunoassays.

AUTOANTIBODIES Methods of Detection Anti-AChR binding to muscle acetylcholine receptor are detected by immunoprecipitation of 125I-t~-BuTxlabeled muscle extract (Lindstrom et al., 1976; 1981). Serum (1--5 pL) is added to an aliquot of labeled extract and after a suitable incubation period (2 hours at room temperature or overnight at 4~ antihuman IgG is added to precipitate the complexes. The precipitate is centrifuged at 5000 rpm for 3 min and the pellet washed without resuspension two to three times over a period of one hour, and then counted on a gamma counter. Control incubations with normal

healthy serum are run in parallel and the counts subtracted. Results are given in nmol of t~-BuTx binding sites precipitated per liter of serum. The cut-off value differs among laboratories, but results >0.5 nmol/L are generally considered positive. Values in normal healthy individuals are generally <0.2 nmol/L; values in neurological or immunological controls may be a little higher (0.2--0.5 nmol/L); and those in MG vary between 0 and >1000 nmol/L. Sera giving low-positive values (e.g., <2 nmol/L) can be retested to confirm their specificity by comparing binding to 125I-t~-BuTx-AChR with binding to AChR that has been presaturated with cold t~-BuTx to block specific binding sites (Vincent and Newsom-Davis, 1985). Although described, ELISA-type assays are not generally used due to the inefficiency with which the immobilized AChR binds antibodies and the relative lack of specificity. Assays based on binding of antibodies to cultured cell lines, such as C2 (Brooks et al., 1990) or TE671 (Martino et al., 1994) are potentially interesting in that they may also pick up antibodies to non-AChR determinants relevant in seronegative MG patients.

Pathogenetic Role Human Model. The poor overall correlation with clinical status suggested initially that anti-AChR might be secondary to the disease state rather than causative. However, several approaches indicate that MG is due to a serum factor that is almost certainly anti-AChR; plasma exchange, which reduces circulating antibodies, produces marked clinical improvement within a few days (Newsom-Davis et al., 1978). Injection of plasma immunoglobulins into mice produces neurophysiological evidence of MG (Toyka et al., 1977); AChRs at the neuromuscular junction are inversely correlated with IgG and complement binding to the postsynaptic membrane (Engel et al., 1987). Animal Models. There are no spontaneous models of MG in small laboratory animals, although dogs and cats develop a similar condition often associated with anti-AChR (Shelton et al., 1988). Experimental autoimmune MG (EAMG) can be induced in many species by immunization with adjuvants plus AChR affinity purified on Sepharose-t~-BuTx or related neurotoxins (Patrick and Lindstrom, 1973; Claudio, 1989). EAMG exhibits many of the key features of the human disease including fatigable weakness,

selective involvement of particular muscle groups, reduced AChR numbers and miniature EPPs and raised levels of anti-AChR. It can also be induced with less purified preparations and with purified AChR without adjuvant. EAMG was recently reviewed in detail (Vincent, 1994b). An important and relevant model of MG is the passive transfer of immunoglobulins from patients to experimental animals (Toyka et al., 1977). In spite of some limitations regarding the cross-reactivity of human autoantibodies with mouse or rat antigens, this approach has helped to demonstrate the role of autoantibodies in MG, in seronegative MG (Mossman et al., 1986; Burges et al., 1994), in the LambertEaton myasthenic syndrome and recently in some cases of acquired neuromyotonia (Shillito et al., 1995).

Factors Involved in Pathogenicity The antibodies probably act by three major mechanisms. First, they fix complement and cause lysis of the postsynaptic membrane with loss of AChR-containing membrane and release of AChR-antibodycomplement complexes into the synaptic cleft (Engel et al., 1987). Second, they can modulate AChR numbers by cross-linking and internalization of the protein (Drachman et al., 1978). Third, a small but variable proportion of antibodies bind directly to the ACh/c~-BuTx binding site and cause functional blockade (Burges et al., 1990). The latter is probably uncommon, but can be important in a minority of patients.

Isotypes/Subclasses Anti-AChR in MG are mainly IgG and are present in all subclasses although IgG 1 and IgG3 may predominate (Nielsen et al., 1985). Anti-AChR are high affinity, idiotypically heterogeneous and variable in antigenic specificity (Vincent et al., 1987). Very few MG sera bind to synthetic peptides representing AChR sequences and, in general, epitope mapping has been extremely disappointing. Conversely, antibodies raised against the denatured AChR subunits, do not bind to the intact molecule. These observations indicate that the autoantibody response is directed at the intact AChR molecule. The epitopes on the AChR can, however, be defined using monoclonal antibodies raised against purified AChR. Many of these bind to a complex of epitopes on each of the two ~ subunits

termed the main immunogenic region (MIR) (Tzartos et al., 1980; 1982) that includes the sequence ~67-76. Some monoclonal antibodies specific for fetal AChR bind to the recombinant 7 subunit on immunoblots, probably between 7120 and 7150 (Jacobson and Vincent, unpublished results). MG antibodies can be mapped indirectly by competition with monoclonal antibodies. A high but variable proportion of antiAChR in any serum compete with anti-MIR antibodies, confirming the importance of this region, but most sera also compete with monoclonal antibodies to other extracellular epitopes, particularly the fetalspecific one. These competition assays show that antibodies in the different subgroups of MG (see below) do not differ substantially in their reactivity with different regions (Heidenreich et al., 1988). A variable (usually small) proportion of antibodies bind to the ~-BuTx binding sites (around ~185-195) but these seldom interfere with antibody detection because of the coexistence of antibodies to other epitopes. A few sera, however, can displace the ~-BuTx from its binding sites on one or both of the AChR c~ subunits; this can lead to false-negative results if only high serum-to-AChR ratios are employed in the assay (Vincent and Newsom-Davis, 1985). The origin of the antibodies in MG is unknown. It is thought that the etiology of the disease may differ among the various subgroups (Vincent, 1994b). Although suggested (Schwimmbeck et al., 1989), an autoantibody with such high affinity is unlikely to have been induced by cross-reaction with an external antigen, particularly in view of the conformationdependency, heterogeneity and high specificity of antiAChR binding. On the other hand, it is possible that the anti-AChR that is detected in routine assays is secondary to determinant spreading, following a cross-reacting, lower-affinity response (Vincent, 1994b). About 15% of patients with typical acquired MG do not have detectable serum anti-AChR, yet they respond to plasma exchange and immunosuppression; injection of their immunoglobulin (Ig) into mice produces a neuromuscular transmission defect (Burges et al., 1994). The nature of the autoantibodies in these cases is unclear, but seronegative Ig reduces AChR function in cultured TE671 cells (Barrett-Jolley et al., 1994) without evidence of binding to the AChR (Vincent et al., 1987), suggesting antibodies bind to AChR determinants that are lost after extraction or, alternatively, bind to another muscle surface antigen that modulates AChR function. Evidence in favor of

the latter is the binding of seronegative MG Ig to cultured mouse muscle cells (Brooks et al., 1990).

CLINICAL UTILITY Disease Association Myasthenia gravis is a classic acquired autoimmune disorder. Patients can be divided into five main groups (Compston et al., 1980) distinguished by their age of onset, thymic pathology and HLA associations, absence of generalized symptoms or absence of antiAChR (seronegative MG). The etiology of the disease in these different subgroups is discussed elsewhere (Vincent, 1994a). The overall prevalence is about one in 10,000. Although the prevalence of young-onset patients is greatest, the incidence of patients presenting over the age of 40 is high. About 15% of MG patients present with purely ocular muscle involvement, usually complaining of diplopia and ptosis, and do not progress to generalized disease. These patients are more frequently seronegative (Sommer et al., 1995) (Figure 2). The remaining 85% may present with ocular or generalized symptoms. Cases with thymoma are relatively infrequent (about 15%) but they are always positive for anti-AChR and antibodies to skeletal muscle antigens (Figure 3). Increased concentrations of anti-AChR (>0.5 nmol/L in most laboratories) are found in 85--90% of patients with generalized MG and are absent from healthy controls and patients with other neurological or autoimmune disorders (Figure 2). Slight elevations of anti-AChR are also found in a few other conditions, usually associated with an increased risk of developing MG (Vincent and Newsom-Davis, 1985), e.g., cases of tardive dyskinesia, amyotrophic lateral sclerosis, polymyositis, primary biliary cirrhosis (Sundewall and Lefvert, 1990), rheumatoid arthritis treated with penicillamine and in thymoma without evidence of MG (Cuenoud et al., 1980). The amount of anti-AChR varies greatly among MG patients, and there is no clear correlation with disease severity (Figure 2). However, within an individual, serial antibody estimations correlate well with the clinical course of the disease (Newsom-Davis et al., 1978). A reduction of >50% of the initial value is often associated with marked clinical improvement; a positive result does not necessarily indicate active disease, and many patients in remission have antiAChR values above control values.

Fetal Development and Neonatal MG. The frequency of transplacental transfer of MG is not high. Babies born to about 10% of MG mothers have a transient neonatal form of MG that responds well to anticholinesterase therapy and usually remits within one month as maternal IgG disappears (Vernet-der Garabedian, 1994). Because the levels of anti-AChR in the fetus at birth are very similar to, or even higher than maternal levels (Vernet-der Garabedian et al., 1994; Lang and Vincent, unpublished observations, 1980), the lack of neonatal symptoms is surprising and suggests that the neonatal neuromuscular junction has a higher "safety factor," or that other factors such as circulating complement levels contribute to susceptibility to clinical disease. Very rarely, a form of arthrogryposis multiplex congenital (AMC) arises in offspring of MG mothers (Dinger and Prager, 1993). A recent case of AMC in successive fetuses of a mother who had never had symptoms of MG appears to be due to antibodies specific for a functional epitope on the fetal form of the AChR (Vincent et al., 1995). Whether "silent" antibodies of this kind exist in other mothers with fetal AMC or with other developmental problems is unknown.

CONCLUSION Measurement of anti-AChR is of importance in the diagnosis of MG; but their absence does not exclude the diagnosis. Their presence in patients without MG is rare, but usually associated with other disorders with increased susceptibility to MG. Fetal damage can occasionally be caused by antibodies specific for fetal AChR in the absence of maternal symptoms. The antibodies in MG are typically high affinity and heterogeneous and bind to a number of different epitopes on the AChR, all of which appear to be conformation-dependent. An important consideration is that the assay in current use can only detect such high affinity antibodies because of limitations in the AChR concentration. The issue of lower affinity antibodies, that might cross-react with other autoantigens or microbial antigens, has not been addressed. As things stand, anti-AChR are diagnostically useful and serial determination levels correlate well with disease within an individual. An important challenge for the future is to determine the target antigen in patients with undetectable anti-AChR, so that progress can be made toward the diagnosis and treatment of these patients. See also STRIATIoNAL AUTOANTIBODIES.

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O s s e r m a n Grade Figure 2. Anti-AChR in MG. Patients were divided according to their clinical classification at presentation. Serum anti-AChR was measured at presentation before thymectomy or immunosuppressive treatment was initiated. The cut-off value is 0.5 nmol/L. A high proportion of purely ocular (Osserman grade 1) patients are seronegative.

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REFERENCES Almon RR, Andrews CG, Appel SH. Serum globulin in myasthenia gravis: inhibition of alpha-bungarotoxin binding to acetylcholine receptors. Science 1974;186:55-57. Barrett-Jolley R, Byrne N, Vincent A, Newsom-Davis J. Plasma from patients with seronegative myasthenia gravis inhibit nAChR responses in TE671/ RD cell line. Pflugers Arch 1994;428:492-498. Beeson D, Brydson M, Betty M, Jeremiah S, Povey S, Vincent A, Newsom-Davis J. Primary structure of the human muscle acetylcholine receptor, cDNA cloning of the ], and ~ subunits. Eur J Biochem 1993;215:229-238. Bender AN, Ringel SP, Engel WK, Daniels MP, Vogel Z. Myasthenia gravis: a serum factor blocking acetylcholine receptors of the human neuromuscular junction. Lancet 1975; 1:607-609. Burges J, Wray DW, Pizzighella S, Hall Z, Vincent A. A myasthenia gravis plasma immunoglobulin reduces miniature endplate potentials at human endplates in vitro. Muscle Nerve 1990; 13:407-413. Burges J, Vincent A, Molenaar PC, Newsom-Davis J, Peers C, Wray D. Passive transfer of seronegative myasthenia gravis to mice. Muscle Nerve 1994; 17:1393--1400. Brooks EB, Pachner AR, Drachman DB, Kantor FS. A sensitive rosetting assay for detection of acetylcholine receptor antibodies using BC3H-1 cells: positive results in "antibodynegative" myasthenia gravis. J Neuroimmunol 1990;28:8393. Chang CE, Lee CY. Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action. Arch Int Pharmacodyn Ther 1962;144:241257. Claudio T. Molecular genetics of acetylcholine receptorchannels. In: Glover DM, Haines BD, eds. Frontiers in Molecular Neurobiology. Oxford: IRL Press, 1989:63-142. Compston DA, Vincent A, Newsom-Davis J, Batchelor JR. Clinical, pathological, HLA antigen and immunological evidence for disease heterogeneity in myasthenia gravis. Brain 1980;103:579--601. Cuenoud S, Feltkamp TE, Fulpius BW, Oosterhuis HJ. Antibodies to acetylcholine receptor in patients with thymoma but without myasthenia gravis. Neurology 1980;30:201-203. Dinger J, Prager B. Arthrogryposis multiplex in a newborn of a myasthenic mother-case report and literature. Neuromuscul Disord 1993;3:335--339. Drachman DB, Angus CW, Adams RN, Michelson JD, Hoffman GJ. Myasthenic antibodies cross-link acetylcholine receptors to accelerate degradation. N Engl J Med 1978;298: 1116-1122. Engel AG, Arahata K. The membrane attack complex of complement at the endplate in myasthenia gravis. Ann NY Acad Sci 1987;505:326-332. Heidenreich F, Vincent A, Newsom-Davis J. Differences in fine specificity of antiacetylcholine receptor antibodies between subgroups of spontaneous myasthenia gravis of recent onset, and of penicillamine induced myasthenia. Autoimmunity 1988;2:31-37.

Hesselmans LF, Jennekens FG, Van den Oord CJ, Veldman H, Vincent A. Development of innervation of skeletal muscle fibers in man: relation to acetylcholine receptors. Anat Record 1993;236: 553-562. Lindstrom J., Einarson B, Tzartos S. Production and assay of antibodies to acetylcholine receptors. Methods Enzymol 1981;74:432-460. Lindstrom JM, Seybold ME, Lennon VA, Whittingham S, Duane DD. Antibody to acetylcholine receptor in myasthenia gravis. Prevalence, clinical correlates and diagnostic value. Neurology 1976; 26:1054--1059. Luther MA, Schoepfer R, Whiting P, Casey B, Blatt Y, Montal M, Linstrom J. A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671. J Neurosci 1989;9:1082-- 1096. Martino G, Twaddle G, Brambilla E, Grimaldi LM. Detection of antiacetylcholine receptor antibody by an ELISA using human receptor from a rhabdomyosarcoma cell line. Acta Neurol Scand 1994;89:18-22. Mossman S, Vincent A, Newsom-Davis J. Myasthenia gravis without acetylcholine-receptor antibody: a distinct disease entity. Lancet 1986; 1:116-- 119. Newsom-Davis J, Pinching AJ, Vincent A, Wilson SG. Function of circulating antibody to acetylcholine receptor in myasthenia gravis:investigation by plasma exchange. Neurology 1978;28:266--272. Nielsen FC, Rodgaard A, Djurup R, Somnier F, Gammeltoft S. A triple antibody assay for the quantitation of plasma IgG subclass antibodies to acetylcholine receptors in patients with myasthenia gravis. J Immunol Methods 1985;83:249--258. Patrick J, Lindstrom J. Autoimmune response to acetylcholine receptor. Science 1973;180:871-872. Schwimmbeck PL, Dyrberg T, Drachman DB, Oldstone MA. Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus. J Clin Invest 1989;840:1174-1180. Shelton GD, Cardinet GH, Lindstrom JM. Canine and human myasthenia gravis autoantibodies recognize similar regions on the acetylcholine receptor. Neurology 1988;38:1417-- 1423. Shillito P, Molenaar PC, Vincent A, Leys K, Zheng W, Van den Berg RJ, Plomp JJ, Van Kempen GT, Wintzen AR, Van Dijk G, Newsom-Davis J. Acquired neuromyotonia: evidence for autoantibodies directed against K+ channels of peripheral nerves. Ann. Neurol 1995;in press. Simpson, JA. Myasthenia gravis: a new hypothesis. Scott Med J 1960;5:419-439. Sommer N, Melms A, Weller M, Dichgans J. Ocular myasthenia gravis. A critical review of clinical and pathophysiological aspects. Doc Opthalmol 1995; 84:309-333. Somnier FE. Antiacetylcholine receptor (AChR) antibodies measurement in myasthenia gravis: the use of cell line TE671 as a source of AChR antigen. J Neuroimmunol 1994;51:6368. Sundewall AC, Lefvert AK. Acetylcholine receptor antibodies in primary biliary cirrhosis: characterization of antigen and idiotypic specificity. Scand J Immunol 1990;31:477-484. Toyka KV, Drachman DB, Griffin DE, Pestronk A, Winkelstein

JA, Fishbeck KH, Kao I. Myasthenia gravis. Study of humoral immune mechanisms bv passive transfer to mice. N Engl J Med 1977;296: 125-131. Tzartos SJ, Lindstrom JM. Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits. Proc Natl Acad Sci USA 1980;77:755-759. Tzartos SJ, Seybold ME, Lindstrom JM. Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies. Proc Natl Acad Sci USA 1982;79:188--192. Vernet-der Garabedian B, Lacokova M, Eymard B, Morel E, Faltin M, Zajac J, Sadovsky O, Dommergues M, Tripon P, Bach J-F. Association of neonatal myasthenia gravis with antibodies against the fetal acetylcholine receptor. J Clin Invest 1994;94: 555-559. Vincent A. Aetiological factors in development of myasthenia gravis. Adv Neuroimmunol 1994a;4: 355-371.

Vincent A. Experimental autoimmune myasthenia gravis. In: Autoimmune Disease Models: A Guidebook. Academic Press Inc., 1994b:83. Vincent A, Newsom-Davis J. Acetylcholine receptor antibody as a diagnostic test for myasthenia gravis: results in 153 validated cases and 2967 diagnostic assays. J Neurol Neurosurg Psychiatry 1985;48: 1246-1252. Vincent A, Whiting PJ, Schluep M, Heidenreich F, Lang B, Roberts A, Willcox N, Newsom-Davis J. Antibody heterogeneity and specificity in myasthenia gravis. Ann N Y Acad Sci 1987;505:106-- 120. Vincent A, Wray, D. Neuromuscular transmission: basic and applied aspects. Oxford: Pergammon Press, 1992. Vincent A, Newland C, Brueton L, Beeson D, Riemersma S, Huson SM, Newsom-Davis J. Arthrogryposis multiplex congenital with maternal autoantibodies specific for a fetal antigen. Lancet 1995;346:24-25.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ACTIN AUTOANTIBODIES Jacob George, Ph.D. and Yehuda Shoenfeld, M.D.

Department of Medicine "B", Research Unit of Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel

H I S T O R I C A L

NOTES

Antiactin antibodies (AAA) were initially detected in sera being exarrfined for the presence of antinuclear antibodies (Johnson et al., 1965). The investigators noticed a distinct, hitherto undescribed immunofluorescent pattern of staining, consistent with the distribution of smooth muscle in the tissue samples taken from patients with chronic active hepatitis (CAH). Subsequent studies (Ironside et al., 1966) validated these observations and increased the spectrum of associated disorders to include infectious diseases (Holborow et al., 1973), rheumatic disorders (Lidman et al., 1976), malignancies (Whitehouse and Holborow, 1971) and numerous other conditions (Wilson et al., 1975). Initially considered specific for smooth muscle, these antibodies are now known to react with a diverse group of cells from skeletal muscle, glomeruli and other tissues (Toh et al., 1978). The nonselective nature of smooth muscle antibody reactions in patients with CAH implied a distinct but similar antigen serving as a common denominator. The antigen was soon characterized further by inhibition of smooth muscle antibodies in five sera from CAH patients after prior incubation with thrombosthenin, an actin component found in platelets (Gabbiani et al., 1973). This strongly suggested that some smooth muscle antibodies (SMA) are antiactin antibodies (AAA). Similar inhibition and a clear resemblance in staining pattern between the actin-antigen complex and smooth muscle antibodies provided unequivocal proof that AAA are actually SMA exhibiting actin specificity (Anderson et al., 1976; Bottazzo et al., 1976; Lidman et al., 1976).

THE

AUTOANTIGEN

Because it displays only minor structural diversity among different species, actin was traditionally considered nonimmunogenic. This theory was challenged by works showing enhanced immunogenicity by prior adjuvant processing, resulting in production of actin autoantibodies in animal models (Trenchev and Holborow, 1976). The cell's "backbone" (the cytoskeleton) consists of three basic filaments discerned and characterized by their width: -the microfilaments: 6 nm filaments containing mainly actin but also myosin, tropomyosin and alpha actinin. -the microtubules: 25 nm width, the principal component of which is tubulin. -intermediate filaments: 10 nm width heterogeneous group displaying specificities toward such diverse tissues as fibroblasts (the vimentin component), smooth muscle cells (desmin), epithelial cells (keratin), glial cells (glial fibrillary acidic protein) and neurons (neurofilaments). Smooth muscle antibody specificity for actin is proved only in patients with chronic active hepatitis; whereas, in viral infections such as infectious mononucleosis, viral hepatitis, measles and mumps, SMA are reactive with nonactin cytoplasmic constituents (Kurki et al., 1978; Toh et al., 1979).

A U T O A N T I B O D I E S

The presence of viral agents is apparently necessary

10

rats chronically injected with phalloidin to achieve an easy-to-interpret, reproducible and low-cost way to detect actin autoantibodies (Dighiero et al., 1990). RIA (Pederson et al., 1982), hemagglutination (Thorstensson et al., 1981) and affinity chromatography (Riisom et al., 1982) are also utilized. Determination of AAA titers by passive hemagglutination using actin-sensitized, tanned sheep erythrocytes is sensitive and relatively convenient, but is less specific for IgM than IgG antibodies. ELISA for the detection of IgG antibodies to Gactin is highly effective displaying 85% sensitivity for AAA detection in autoimmune CAH compared to 70--80% sensitivity using the traditional immunofluorescence (Bretherton et al., 1983) ELISA is an easy-to-perform method which can provide quantitative results. Overall, the sensitivity of ELISA for the detection of AAA is 60--70%, while the specificity is 94%.

but not sufficient for the production of AAA; tissue destruction caused by mechanisms other than viral damage does not consistently result in antibody formation. The most appealing theory holds that the viral-actin complex, acting as a hapten carrier, is responsible for the production of AAA. This theory gained substantial support by observations of antibody production following the use of several medications (such as the laxative oxyphenisatin) (Lidman et al., 1976). The occurrence of previous viral infection or medication use thus seems responsible for the appearance of AAA in low titers among normal individuals. On the other hand, CAH patients produce AAA in the persistent fashion typical of an abnormal immune reaction. As expected, HLA-DR3 and-B8 are highly prevalent in this selected group of patients (Fusconi et al., 1990). Occurrence of AAA was recently observed following treatment of chronic hepatitis B patients with gamma-interferon (Weber et al., 1994); the prognostic significance of pre-existent AAA for responsiveness to gamma-interferon in chronic HBV and chronic HCV infection is under investigation.

CLINICAL UTILITY

Disease Association Pathogenetic Role AAA are a subgroup of smooth muscle antibodies (SMA), displaying specificity towards the actin component of the cytoskeleton (Fagraeus and Norberg, 1978; Hawkins et al., 1979). Although SMA are found in low titer in sera of 3--18% of the general healthy population (Fagraeus and Norberg, 1978) and in a wide spectrum of chronic illnesses, only some of these SMA-positive sera exhibit actin specificity. AAA are found in sera of 52--85% of patients with autoimmune CAH and 22% of those with primary biliary cirrhosis (Hamlyn and Berg, 1980) (Table 1). AAA detected in autoimmune cholangiopathy are thought to reflect overlapping features of CAH and PBC (Ben-Ari et al., 1993). Although several investigators noted an increase in

The role of AAA in the etiology of tissue damage is not yet clear. However, the likelihood that these antibodies are responsible for deleterious effects seen in CAH is quite low, taking into account the antigen location within the cytoplasm rendering them inaccessible to the AAA.

Methods of Detection The method most frequently used to detect AAA was, until recently, indirect immunofluorescence with fibroblast cultures or rodent gastric muscle cells serving as substrates. This method was recently improved by the use of cryostat sections of liver from

Table 1. A Summary of the Prevalence of Actin Autoantibodies in Several Works Method

CAH

PBC

Work

Hemagglunination

54%

21%

Hamlyn and Berg, 1980

Immunofluorescence

55%

NC

Pederson et al., 1982

ELISA

85%

NC

Bretherton et al., 1993

ELISA

52%

44%

Dighiero et al., 1990

CAH: Chronic Active Hepatitis; PBC: Primary Biliary Cirrhosis; NC: Not Checked.

11

A A A titer during C A H "flare up" and a decrease following steroid treatment (Maggiore et al., 1993), others failed to observe similar results (Johnson et al., 1991). Reports of an association of S M A with certain malignant states (Whitehouse and Holborow, 1971) were also not confirmed.

CONCLUSION Autoantibodies to actin are a selected group of S M A

REFERENCES Anderson P, Small JV, Sobieszek A. Studies on the specificity of smooth-muscle antibodies. Clin Exp Immunol 1976;26: 57-66. Ben-Aft Z, Dhillon AP, Sherlock S. Autoimmune cholangiopathy: part of the spectrum of autoimmune chronic active hepatitis. Hepatology 1993;18:10-15. Bottazzo GF, Florin-Christensen A, Fairfax A, Swana G, Doniach D, Groeschel-Stewart U. Classification of smooth muscle autoantibodies detected by immunofluorescence. J Clin Pathol 1976;129:403-410. Bretherton L, Brown C, Pedersen JS, Toh BH, Clarke FM, Mackay IR, Gust ID. ELISA assay for IgG autoantibody to G-actin: comparison of chronic active hepatitis and acute viral hepatitis. Clin Exp Immunol 1983;51:611--616. Dighiero G, Lymberi P, Monot C, Abuaf N. Sera with high levels of antismooth muscle and antimitochondrial antibodies frequently bind to cytoskeleton proteins. Clin Exp Immunol 1990;82:52--56. Fagraeus A, Norberg R. Antiactin antibodies. Curr Top Microbiol Immunol 1978;82:1-13. Fusconi M, Cassani F, Zauli D, Lenzi M, Ballardini G, Volta U, Bianchi FB. Antiactin antibodies: a new test for an old problem. J Immunol Methods 1990;130:1--8. Gabbiani G, Ryan GB, Lamelin JP, Vassali P, Majno G, Bouvier CA, Cruchaud A, Luscher EF. Human smooth muscle antibody. Its identification as antiactin antibody and a study of its binding to "nonmuscular" cells. Am J Pathol 1973;72:473--488. Hamlyn AN, Berg PA. Hemagglutinating antiactin antibodies in acute and chronic liver disease. Gut 1980;21:311-317. Hawkins BR, O'Connor KJ, Dawkins RL, Dawkins B, Rodger B. Autoantibodies in an Australian population. I. Prevalence and persistence. J Clin Lab Immunol 1979;2:211-215. Holborow EJ, Hemsted EH, Mead SV. Smooth muscle autoantibodies in infectious mononucleosis. Br Med J 1973;3:323325. Ironside PN, De Boer WG, Nairn RC. Smooth muscle antibody in lupoid hepatitis. Lancet 1966;i:1210. Johnson GD, Holborow EJ, Glynn LE. Antibody to smooth muscle in patients with liver disease. Lancet 1965;ii:878--890. Johnson PJ, McFarlane IG, Eddleston AL. The natural course and heterogeneity of autoimmune-type chronic active 12

directed against the actin protein, a vital and abundant c o m p o n e n t of the cytoskeleton. A A A are increased in patients with autoimmune chronic active hepatitis and primary biliary cirrhosis. No other autoimmune, neoplastic and infectious conditions are associated with significantly increased titers of this autoantibody. The presence of A A A are unlikely to have an important role in etiology or pathogenesis of h u m a n A I - C A H or PBC. W h e t h e r A A A are by-products of other pathogenic mechanisms is yet to be established. See also SMOOTH MUSCLE AUTOANTIBODIES.

hepatitis. Semin Liver Dis 1991 ;11:187-- 196. Kurki P, Linder E, Miettinen A, Alfthan O. Smooth muscle antibodies of actin and nonactin specificity. Clin Immunol Immunopathol 1978;9:443--453. Lidman K, Biberfeld G, Fagraeus A, Norberg R, Torstensson R, Utter G, Carlson L, Luca J, Lindberg U. Anti actin specificity of human smooth muscle antibodies in chronic active hepatitis. Clin Exp Immunol 1976;24:266--272. Maggiore G, Veber F, Bernard O, Hadchouel M, Homberg JC, Alvarez F, Hadchouel P, Alagille D. Autoimmune hepatitis associated with antiactin antibodies in children and adolescents. J Pediatr Gastroenterol Nutr 1993;17:376-381. Pederson JS, Toh BH, Mackay IR, Tait BD, Gust ID, Kastelan A, Hadzic N. Segregation of autoantibody to cytoskeletal filaments, actin and intermediate filaments with two types of chronic active hepatitis. Clin Exp Immunol 1982;48:527--532. Riisom K, Diederichsen H, Andersen I. Reaction against liver cells by human antiactin antibodies purified by affinity chromatography. J Clin Pathol 1982;35:750--753. Thorstensson R, Utter G, Norberg R, Fagraeus A. A radioimmunoassay for determination of antiactin antibodies. J Immunol Methods 1981;45:15-26. Toh BH, Clarke FM, Ceredig R. Reaction of human smooth muscle antibody with skeletal muscle, cardiac muscle, and thymic myoid cells. Clin Immunol Immunopathol 1978;9: 28-36. Toh BH, Yildiz A, Sotelo J, Osung O, Holbororw EJ, Kanakoudi F, Small JV. Viral infections and IgM autoantibody to cytoplasmic intermediate filaments. Clin Exp Immunol 1979;37:76--82. Trenchev P, Holborow EJ. The specificity of antiactin serum. Immunology 1976;31:509--517. Weber P, Wiedmann KH, Klein R, Walter E, Blum HE, Berg PA. Induction of autoimmune phenomena in patients with chronic hepatitis B treated with gamma-interferon. J Hepatol 1994;20:321-328. Whitehouse JM, Holborow EJ. Smooth muscle antibody in malignant disease. Br Med J 1971;4:511--513. Whittingham S, Mackay IR, Irwin J. Autoimmune hepatitis, immunofluorescence reactions with cytoplasm of smooth muscle and renal glomerular cells. Lancet 1966;i:133--135. Wilson C, Elstein M, Eade OE, Wright R. Smooth-muscle antibodies in infertility. Lancet 1975;ii:1238--1239.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

AFFINITY AND AVIDITY OF A U T O A N T I B O D I E S Azzudin E. Gharavi, M.D. a and Hansotto Reiber, Ph.D. b

aDepartment of Medicine, Section of Rheumatology, Louisiana State University Medical Center, New Orleans, LA 70112-2822, USA; and bNeurochemisches Labor, University of G6ttingen, 37075 GOttingen, Germany

INTRODUCTION Antibody affinity is defined as the thermodynamic measurement of the degree of association between antibody and antigen. The strength of the association of a monovalent antigen binding site on an antibody and a single epitope on an antigen molecule is defined as "intrinsic affinity" (with microscopic equilibrium constant); the overall force that binds a multivalent antibody to a multivalent antigen (ligand) is referred to as "functional affinity" or more commonly as "avidity" (with macroscopic equilibrium constant) (Underwood, 1988). This chapter focuses on two aspects of affinity: (1)biological relevance of and biophysical methods for quantitation of antibody affinity, and (2)the role of affinity and avidity in autoimmune diseases.

BIOLOGICAL RELEVANCE OF AND BIOPHYSICAL METHODS FOR QUANTITATION OF ANTIBODY AFFINITY

Affinity of Antibodies In a primary immune response, antibodies may have low affinity; however, even without repeated immunization, the affinity of antibodies will increase (affinity maturation). The maturation of affinity in germination centers causes subsequent production and selection of B lymphocytes with affinities higher than that of the original virgin B cell (Berek et al., 1991; Nossal, 1992; Rolink and Melchers, 1991; Leanderson et al., 1992). A medium affinity represents a necessary base for cross-reactivities to establish an immunological network (Varela and Coutinho, 1991;

Perelson, 1989). Affinity measurement can provide useful information about the duration of infections which may have important applications in clinical immunology.

Biological Relevance of Affinity 1. Antigen binds to receptors in the membrane of the B lymphocyte and induces an antigen-driven immune response with B-cell proliferation and maturation of plasma cells. Both these functions are controlled by receptor occupancy which depends on affinity (with equilibrium constant, K) and antigen concentration, [A]. High occupancy (a large product of K . [A]) leads to paralysis of Bcell activity (Figure 1). 2. Antigen binds to the free antibodies formed in the plasma cells of a specific cell clone to form an antigen/antibody complex as a basic reaction to eliminate the foreign antigen. The antigen-dependent activation of secretion by a B cell automatically results in neutralization of whatever ligand (antigen, idiotype) led to its activation. So soluble and cell-bound V-regions have roughly opposite effects on the dynamics of the immunological network. 3. Immune networks are structured around matrices of connectivity, the elements of which are experimentally measurable molecular affinities (Varela and Coutinho, 1991). In mice, such connectivity matrices are described and can be divided into three different groups with high, minor and low reactivity. An important parameter is the network depth of a certain antibody which is measured by the number of components (antibodies, idiotypes) in the chain required to cycle

13

Maturation

Proliferation

sociated state (free single molecules). Reaction: k1

A+B Cumulative receptor occupancy (sensitivity) Small resting cells ( I ~-

Decay

", Activated cells

),( . . . . . .

Maturation

)<

)(-

) ~_____~.

Maturation Proliferation Paralysis and (decay) proliferation

F i g u r e 1. Influence of receptor occupancy (K a 9[A]), on B-cell activity (Varela and Coutinho, 1991). K a = association constant, [A] = antibody concentration.

back to the first component. Network depth is a nonlinear function of antibody affinities (Perelson, 1989). Successful regulatory interaction depends on concentration and affinities of the interacting molecules. It is the product K - [A] (antibody affinity, K, and antigen concentration, [A]), that is important. At high concentrations, low affinity interactions are significant; whereas, at low concentrations, only highaffinity interactions are important for regulation. Low affinity with low concentrations may exclude a biologically relevant complex formation. High affinity is necessary for high specificity of antibodies, but high affinity and high concentrations paralyze the regulation. Regarding network depth (performance of the immune system), an intermediate connectivity guarantees robustness of the network. If links are very difficult to make, naturally the network's depth is shallow, but at high connectivity, depth is predicted to be shallow again (Perelson, 1989; Varela and Coutinho, 1991). There is only a relatively narrow range of connectivity where enough depth can exist for the emergence of network properties and a normal immune status.

Biophysics of Affinity, Avidity, Cooperativity, Specificity Molecules which attract each other like an antigen (A) and an antibody (B) are found in an equilibrium between associated state (complex, AB) and dis-

14

~

AB

(1)

A single molecule in solution is in a steady flux between its bound and free state. The probability with which this molecule is found in bound or dissociated state in equilibrium can be characterized by the equilibrium constant, K. In thermodynamic terms, this equilibrium constant is described on the basis of chemical potentials and gain of free energy. Much more helpful for immunological purposes is the description in kinetic terms by which the equilibrium constant, K, is defined as a quotient of the rate constants for association, k 1 and for dissociation, k 2. In equilibrium of reaction (1), the rate of formation must be equal to the rate of dissociation: At equilibrium: k 1 [A].

[ g ] -- k 2 [ A N ]

( 2)

The corresponding equilibrium constant for association is defined by: Association constant: Ka

~

kl k2

~

~--

[AB] [A] [B]

[L/moll

(3)

Occasionally, the reciprocal dissociation constant, K d = 1/K a [mol/L] is used. The amount of bound antibody (concentration of complex, [AB]) in equilibrium -- the biologically most relevant p a r a m e t e r - is calculated with: [AB] = K a [A]-[B]

(4)

or

[AB]

= K a [A o - AB]

9[ g o - A B ]

( 5)

The concentration of a complex [AB] in equilibrium depends on the equilibrium constant (affinity) and the concentration of total antibody [B o] and antigen [Ao]. This coupled influence of affinity and antibody concentration is most important regarding the biological and methodological aspects of antibody affinity. Several attempts have been made to separately quantify both these parameters, affinity and concentration, but the heterogeneous nature of antibodies in sera makes this task extremely difficult.

Equations (1) through (5) describe the simple case if one (low molecular weight) molecule encounters a macromolecule. These equations are only relevant for description of intrinsic affinity or microscopic equilibrium constant.

Cooperativity The equations must be modified if several molecules (low molecular weight) bind to the macromolecule in a cooperative way, i.e., in such a way that the binding to one site influences the strength of binding at other sites. This cooperativity, which is a very important biochemical property of macromolecules which control enzymatic processes, provides an on-off character to some processes. In immunology, a different type of modification of intrinsic affinity becomes relevant.

Multivalency/Avidity Because viral epitopes are effectively multivalent, as are antibody molecules in vivo, their association needs a different, more general, equation for the equilibrium constant, which takes into account the effective antibody valency and effective virus valency (Underwood, 1988; Friguet et al., 1985). The maximal valency of IgG of two or of IgA of four may be realized, but for IgM, an effective valency does not exceed five. The effective valency of viruses with several thousands of identical antigenic subunits depends on the degree of steric hindrance between antibodies binding to adjacent epitopes. The empirically detectable overall association constant can be seen as a macroscopic equilibrium constant, K a, or, as previously defined, as functional affinity. These terms contribute to understanding of the more common term "avidity" which is somewhat imprecise. But this enhancing effect of multivalent interaction is of great biological importance, for example, in virus neutralization. m

Specificity The strength of the association of two macromolecules increases with the binding of an increasing number of complementary binding sites. The association constant increases with an increasing number of structurally coupled sites involved, mainly due to a decreased rate constant for dissociation (k 2 in equation [2]). This property of macromolecules extremely decreasing the

dissociation constant presents a new emergent property known as "specificity." So, specificity of an antibody for a certain antigen is defined by its higher affinity compared to the affinities to other antigens. Cross-reactivity is then only a question of avidity, always happening but recognized only if sufficiently strong to form a complex stable enough to be detected in the organism and in the analytical method. Subsequently, besides a high level of affinity or avidity, there must always be a probability for cross-reactions at a low level of affinity. As learned from immunological network theory, it is important to have crossreactivities of antibodies to establish a network.

Polyclonal Heterogeneity From a methodological point of view, the real situation in blood offers additional problems for a biophysically exact determination of avidity. The immune response is polyclonal, i.e., there are several different antibody species in blood with different avidity to different epitopes of a certain antigen (virus or bacteria). Depending on methods, only a part of the whole spectrum may be quantitated. The polyclonal heterogeneity represents the main analytical problem for quantitation of avidity and for interpretation of data.

Methods for Analysis of Affinity/Avidity Two primary objectives are used to help discriminate among the many approaches to quantitating affinity/ avidity of an antibody to a certain antigen: 1) to get an absolute ("true") value of the equilibrium constant; 2) to compare reactivity of sera with high and low affinity antibodies to a certain antigen. In regard to these objectives, it should be noted that: 1. The correct detection of an equilibrium constant needs several strongly restricting conditions, including 1) Restrictions to a monoclonal antibody: for a monoclonal antibody and an isolated epitope (antigen, hapten), a very sophisticated physical method, Surface Plasmon Resonance (Biosensor, Pharmacia), can be applied. 2) Straight line relationships after mathematical transformation: several mathematical transformations of the equations for the association constant have been introduced for a graphical representation of a linear function (Underwood,

15

1988; Friguet et al., 1985). It is advantageous to have a straight line relationship from which any deviations can be easily observed and interpreted.

r/c

Scatchard Plot and Klotz Plot The most commonly used approach in immunology is still the Scatchard plot (Underwood, 1988). Regarding the special case of one epitope/antigen (a) or one paratope/antibody (b) we find with the rearranged, transformed equation for the equilibrium constant (Underwood, 1988): a) Equation for antigen (with s = 1): (A ~ - A ) / B

o _ Ka (

n - (A o - A)

A

)

g d

Ao-n(Bo-B

-1+ )

Kd(8 ) A

with A o = total antigen concentration, A = free antigen concentration, B o - total antibody concentration, B = free antibody concentration, Bo(OD) = absorbance in ELISA for free antibody sites, n = B with n = antibody valency. A simplification of the mathematical equation of

16

antibo

.

I

r

s(n)

Figure 2. Typical form of a Scatchard plot. In case s = 1 (case (a) in text) or n = 1 (case (b) in text), the linear (dashed) line with constant slope is representative for the association constant, K a. The curved line represents a heterogeneous antibody population containing species of high (steep slope) and low (shallow slope) affinity. The meaning of r and c varies depending whether antigen or antibody is regarded (see text).

(7)

with B = free antibody, B o = total antibody, A o = total antigen, s = antigen valency, r = (B o - B)/A o, c = B. Cases with higher valencies than these need very special transformations to solve the problem (if possible at all) (Underwood, 1988). Real sera contain a mixture of high and low affinity antibodies representing additional restrictions for application of the Scatchard Plot. The application of case (a), with equation (6), or case (b), with equation (7), for a serum with high and low affinity antibodies is shown in Figure 2. Another convenient mathematical representation of the dissociation constant (K d) is the Klotz equation (Friguet et al., 1985):

Bo-B

(monovalent

(6)

(B ~ - B) / A ~ = Ka( s - ( B ~ - B) ) B A~

=1+

ideal line. - . . ~ N S i o p e = _Ka (low affinity antibody)

B~

with A = free antigen, Ao = total antigen, B o = total antibody, n = antibody valency, r = (A o - A)/B o, c = A. b) Equation for antibody (with antibody valency, n = 1):

Bo

- K a (high affinity antibody)

Klotz (and of Scatchard) may occur with total concentration of antigen A o > 10 x ( n - Bo), the total concentration of antibody sites. When A approaches A o, we get absorbances instead of concentrations: B~ Bo(OD ) - B(OD)

= 1 + ~Kd

(9)

A~

For these evaluations (Figure 3), the ELISA method needs B o > 10-l~ M and, hence, A o at least 10 -9 M. The fraction of free antibody will be large enough to ensure precise measurement only if K d is above about 10 -9 M. It is likely that complexes with dissociation constants down to 10-11 M could be analyzed using chemiluminescence detection replacing the final step of the ELISA. The reliability of the determined constants depends on the methods. Antigen and antibody have to be in solution to get a "true" equilibrium constant, which is different from the equilibrium constants detected if the antigen is bound to a solid phase (ELISA). One of the most reliable approaches is that in which the antigen/ antibody complexes are formed in solution and the unbound, free antibody is quantitated in an ELISA which is sensitive enough to analyze only a small

1.0

[UREA](M) oO

OM

2,5

.2 ,

4

A

(3

2

0.5.

6M 2 2;5

1.5

s=o ~o

26o

460

86o 1#oo 32bo

SERUM DILUTION

1.0 ~'UREA~ (M)

B

o 0

0

1

2

1/Ao

9

3

10-8M-1

Figure 3. Klotz plots of the binding of two different pairs of

antibody/antigen with impure mixtures of antibodies. 1/v represents the reciprocal fraction of bound antibody expressed in the measured absorbences Bo(OD)/Bo(OD) - B(OD). The concentrations of impure antibodies were 10-4 mg/mL (1) and 3 x 10-4 mg/mL (2) respectively. The dissociation constants KD are obtained from the slopes with 3.8 x 10-9 M (1) and 1.4 x 10.8 M (2). A o = total antigen, B = free antibody sites, Bo(OD) = absorbance in ELISA for total antibody sites, n = B o. B(OD) = absorbances in ELISA for free antibody sites, n = B.

fraction (< 10%) of the free antibody and thereby avoids any relevant interference with the equilibrium between bound antibody and free antibody (Friguet et al., 1985). 2. The comparison of two different sera to discriminate h i g h - a n d low-affinity i m m u n e response in a simultaneous analysis does not need an absolute value of the equilibrium constants. In this case, a wide spectrum of E L I S A methods may be sufficient, if for detection of affinity the influence of different concentrations is taken into account. A most reliable method is the serial dilution of serum samples in a duplicate series, followed by the binding of antibody to the antigen in solid phase (under equilibrium conditions) (Polanec et al., 1994; H e d m a n et al., 1989) (Figure 4). The subsequent dissociation of the complex with six molar urea in one series is compared with an undissociated second series. Most important for evaluation, the ratio of the titers at a

92

0 ul O

,,,

-4

96 98

0.5

2T5

5tO

' ~oo

~ ~ - ......;~o 200 4o0 SERUM DILUTION

~6oo a2'o0

Figure 4. EPR-evaluation (Hedman et al., 1989). Effect of urea on pooled acute-phase sera (A) and sera of pre-existing toxoplasma immunity (B). The serum pools were diluted serially and applied onto immobilized toxoplasma antigen; the antigen-bound antibodies were washed with the indicated concentrations of urea. Residual antibody-bound IgG was quantitated immunoenzymatically. Dashed line shows level of cutoff absorbance (A406 = 0.200). The endpoint titer of the acute phase IgG was 800 and with 6M urea was 60. The ratio of endpoint titers at 0.200 OD is given in %: EPR = 60:800 x 100 = 7.5% (Figure 4A). The EPR of the previously active disease was 50% (Figure 4B), indicating the higher affinity or lower dissociation rate in this group.

certain endpoint (fixed optical density, OD, 0.200) is used. This evaluation takes the varying concentrations into account suggesting that at a certain optical density in the same test system, almost the same concentration of antibodies is bound. Minimal effort is required to detect differences in affinity (Kasp et al., 1992). A single dilution of the serum sample is used to get an optical density in E L I S A of between 0.5 and 1 . 0 0 D (Kasp et al., 1992). Optical densities of untreated and dissociated complex (with 1 mol/L SCN) are compared. In this case, the concentration in different samples may vary by a factor of two due to the range of optical densities allowed in the ELISA.

17

Several other approaches have also been discussed (Underwood, 1988; Friguet et al., 1985).

AFFINITY AND AVIDITY IN AUTOIMMUNE

DISEASES Unlike conventional antibodies, the role of affinity and avidity in the function of autoantibodies is not clearly understood. How, if at all, autoantibody affinity and avidity are related to pathogenicity is controversial. The clinical relevance of avidity of autoantibodies is the subject of ongoing study.

Systemic Lupus Erythematosus (SLE) Antibodies to double-stranded DNA (anti-dsDNA) are the most important autoantibodies in systemic lupus erythematosus (SLE) and are associated with certain clinical complications, such as glomerulonephritis (ter Borg et al., 1990; Reeves et al., 1993). During the past three decades, many investigators evaluated the relationship between anti-dsDNA avidity and lupus glomerulonephritis. Mice susceptible to virus-induced immune complex diseases (Oldstone and Dixon, 1969) produce antibodies with lower affinity than do mice resistant to these diseases (Steward et al., 1973). Low-affinity antibodies are less efficient at immune elimination of antigens compared with high-affinity antibodies (Alpers et al., 1972); this is thought to result in the persistence of immune complexes in the circulation that may then be deposited in the tissue. This hypothesis was examined in New Zealand Black/White hybrid mice (NZB/W F 1) (Steward et al., 1975) that spontaneously develop a disease resembling SLE characterized by deposition of DNA/anti-dsDNA immune complexes in the kidneys (Helyer and Howie, 1963). Antibodies to DNA can be demonstrated in the sera of these animals; the concentrations are higher in females than in males and in both sexes they rise with increasing age (Steinberg et al., 1969). Female mice are much more severely affected and begin to die at about six months of age; within a year, 98% of the females are dead (Tonietti et al., 1970). The anti-dsDNA in these animals were measured by ammonium sulfate precipitation (Steward et al., 1975) with a modification from the conventional Farr assay (Farr, 1958; Pincus et al., 1969; Hughes, 1972). The avidity of anti-dsDNA antibodies was estimated by 1)measurement of the rate of dissociation of 125I-DNA/anti-dsDNA complexes in

18

the presence of 200- to 300-fold molar excess of unlabeled DNA; 2) construction of binding curves; and 3)inhibition studies and determination of the quantity of the deoxyadenosine 5'-monophosphate, which inhibited the binding of the test serum from 30% to 0 after incubation for 48 hours at 4~ The avidity of these antibodies increased for up to 32 weeks but, thereafter, fell to a low level; the females had lower avidity compared with males. These findings suggest the association of severe disease with high-titer anti-dsDNA of low avidity (Steward et al., 1975). Interestingly, failure to produce high avidity in these mice was not restricted to endogenous antigens, but the antibodies produced against foreign proteins such as human serum transferrin also had low avidity. Longitudinal studies showed that antibody avidity in female NZB/W F 1 mice increased from the age of 14 weeks up to 32 weeks of age and thereafter fell to a low level (Steward et al., 1975). Many investigators have studied the relationship between the titer and avidity of the anti-dsDNA and severity of disease in human SLE. Although there is a general agreement on the association of high-titer anti-dsDNA with disease activity and the presence of glomerulonephritis (GN), the role of antibody avidity remains highly controversial. In 46 patients with SLE assessed for the titer and avidity of anti-DNA antibodies adapting the established methods (Steward et al., 1975), 37 had kidney biopsies showing diffuse proliferative GN in 20, segmental GN in 3, membranous GN in 3 and isolated granular deposits of immunoglobulin in 11 patients (Tron and Bach, 1977). There was a clear association between high-titer antiDNA and the presence of GN; whereas, low- and high-affinity antibodies were present in all groups. However, low-affinity anti-dsDNA were more common in the sera of patients with glomerular changes than in those without them, but several patients had high-affinity anti-dsDNA and severe GN (Tron and Bach, 1977). In another 38 SLE patients, anti-dsDNA were of higher avidity in patients with lupus nephritis compared to those without lupus nephritis (Gershwin and Steinberg, 1974). Studies of the avidity of antidsDNA in serum, cryoprecipitate and IgG glomerular eluates from patients with SLE revealed low-avidity, anti-dsDNA in the serum of patients with GN which sharply contrasted with very high avidity (-~10-fold) anti-dsDNA in the IgG glomerular eluates from autopsy kidneys of patients with severe lupus GN (Winfield et al., 1977). Anti-dsDNA of intermediate

avidity were found in the sera of patients without nephritis. The avidity of anti-dsDNA in the cryoprecipitate was not different from that in the serum. These findings suggest that antibodies with very high avidity may form DNA/anti-dsDNA complexes with optimum potential for renal tissue injury; whereas, the low-affinity anti-dsDNA, perhaps with little or no deleterious effect, remain detectable in the circulation (Winfield et al., 1977). Further evaluation of the predictive values of anti-dsDNA for the disease activity in SLE showed that significant changes of anti-dsDNA levels, as detected by Farr assay (which detects only the high-avidity antibodies), had the best predictive value for disease exacerbation compared with Crithidia luciliae and ELISA that detect both high- and low-affinity antibodies. Seventy-two unselected SLE patients were followed up for the mean period of 18.5 months. Disease activity was assessed at least every three months, and anti-dsDNA levels by Farr, Crithidia and ELISA as well as C3 and C4 were determined every month. There were 33 disease exacerbations during the period of the study and 24 of them were preceded by a rise in the anti-dsDNA levels. Twenty-three of the 24 were detected by Farr assay, 12 by Crithidia and 17 by ELISA (ter Borg et al., 1990).

PEG Assay for Low-Affinity Anti-dsDNA. Radiolabeled DNA/anti-dsDNA complexes can also be precipitated by PEG (3.5% final concentration) rather than ammonium sulfate, which is used in the Farr assay (Riley et al., 1979). The high concentration of ammonium sulfate employed in the Farr assay dissociates complexes consisting primarily of low-avidity anti-dsDNA and, therefore, only higher avidity antibody populations are detected. By contrast, PEG precipitation is equally effective on all complexes, allowing detection of both low- and high- avidity antibodies. The PEG precipitation assay adds a new dimension to the evaluation of the avidity of antidsDNA and their clinical relevance, because in most previous studies, low-avidity antibodies not precipitated by the high-salt concentrations employed were eliminated. Therefore, such studies provided data on only high- and very high-avidity antibodies. The PEG precipitation technique was improved by addition of dextran sulfate to the test sera, thus preventing the non-anti-DNA-mediated DNA binding by LDL, which can produce false-positivity in normal human sera (Smeenk and Aarden, 1980). In a longitudinal study of the clinical relevance of

the anti-dsDNA avidity, 35 SLE patients who were positive for anti-dsDNA only by PEG a s s a y - that is, had only low-avidity anti-dsDNA in their circulation -- had a mild course of SLE with 26 exacerbations (8 minor, 18 major) and no renal involvement. PEG assay had little predictive value but a high specificity for the clinical exacerbations. In contrast, 14 patients positive by both Farr and PEG a s s a y s - that is, with high avidity anti-dsDNA had a severe course of disease with renal and cerebral involvement and 23 exacerbations (2 minor, 21 major). There was a clear correlation between rises in Farr assay and changes in Farr/PEG ratio with major exacerbations. Renal and cerebral exacerbations were associated with 10-fold or greater increase in Farr/PEG ratio (Nossent et al., 1989). In 17 patients with SLE and nephritis and 17 SLE patients with central nervous system involvement, anti-dsDNA were measured by PEG precipitation as well as the Farr assay (Smeenk et al., 1988). Patients with SLE nephritis demonstrated higher Farr/PEG ratios than those with CNS involvement (Smeenk et al., 1988). In a longitudinal study of 19 patients with SLE, high-avidity (Farr) as well as lowavidity (PEG) anti-dsDNA were measured (McGrath and Biundo, 1985). Changes in antibody titer and avidity were correlated with clinical manifestations over 3.5 years. Amounts of high- and low-avidity antibodies to dsDNA did not change independently, but rose and fell in a parallel and relatively fixed manner throughout the course of the disease (McGrath and Biundo, 1985). In yet another study, the relationship between the levels of high-avidity anti-dsDNA during the disease exacerbation and total IgG and IgM levels as well as the levels of antibody to recall antigens (tetanus and cytomegalovirus late antigen) was evaluated in 72 patients with SLE. The levels of high-affinity anti-dsDNA changed independently from the levels of total IgG and IgM, as well as the levels of antibodies to recall antigens (ter Borg et al., 1991). The authors concluded that the rise in anti-dsDNA prior to exacerbation of SLE is due to preferential activation of anti-dsDNA-specific B-cells and not merely a part of polyclonal B-cell hyperactivity; this confirmed the conclusion of a previously reported study showing that SLE autoantibodies are not due to polyclonal B-cell activation (Gharavi et al., 1988).

Farr Assay. The ongoing controversies concerning the role of anti-dsDNA affinity/avidity may be due chiefly to the lack of a suitable method of studying anti-dsDNA affinity. The Farr assay, which is specific

19

for high affinity antibodies, cannot detect low titer antibodies; routine ELISA, which is sensitive enough to detect low titer antibodies, cannot determine the affinity of the antibodies. A new method for determining the dissociation constant of antigen-antibody complexes using a gel-shift assay may provide valuable information regarding anti-dsDNA affinity (Stevens et al., 1994).

(Harris et al., 1985). There was no difference in avidity of aPL in the two groups (Qamar et al., 1990). In 20 SLE sera and 16 syphilis sera, 50% binding, the slope of the double-reciprocal plot and Friguet's binding constant (Kd) were used to compare aPL avidity (Levy et al., 1990). Kd=(

OD Blank - 1 ) x IugCL OD B l a n k - OD Test

Antiphospholipid Syndrome (APS) Antibodies to acidic phospholipids (aPL) are found in the sera of patients with autoimmune diseases (SLE and primary antiphospholipid syndrome), certain infections (syphilis and HIV) and some lymphoproliferative disorders, or they may be drug-induced (chlorpromazine and procainamide). In autoimmune diseases, these antibodies are associated with thrombosis, spontaneous abohion and other clinical complications collectively called the "Antiphospholipid Syndrome" (Sammaritano and Gharavi, 1992; Lockshin, 1994). Autoimmune aPL require a cofactor for binding to phospholipid (PL). This cofactor is gz-glycoprotein I (f32-GPI) a normal plasma protein which binds acidic PL and is a natural regulator of coagulation (McNeil et al., 1990; Galli et al., 1990; Gharavi, 1992). There is little or no binding of autoimmune aPL to PL in the absence of B2-GPI, and aPL are believed to bind to the PL-132-GPI complex or to a new epitope(s) on PL or f32-GPI which is exposed after the binding (McNeil et al., 1990; Sammaritano et al., 1992). The avidity of aPL and its clinical significance were evaluated in sera from 46 women with high-titer aPL (>35 GPL: IgG aPL International Standard) and a history of two or more spontaneous abortions compared with sera from 12 women with high IgG aPL who had successful pregnancies (Qamar et al., 1990). The aPL were measured by anticardiolipin (aCL) ELISA (Gharavi et al., 1987), and the avidity was evaluated by five different methods, including determination of the amount of serum (~L) yielding 50% binding, Friguet's binding constant (Friguet et al., 1985), slope of the double reciprocal plot (Costello and Green, 1988) and the decrease in IgG binding when washed with high-salt buffer (2 x PBS). It should be mentioned that binding of aPL to PL is very sensitive to increases in salt concentration. For example, in affinity chromatography, aPL are eluted from the cardiolipin (CL) or phosphatidylserine affinity columns by 0.5 molar NaC1 (McNeil et al., 1988) and from CL micelles by 1--1.5 molar KI 20

After absorption of positive sera by incubation with varying amounts of cardiolipin micelles (0 to 1000 pg/mL), the solution was allowed to reach equilibrium, and the supernatants were tested by ELISA for residual aCL activity. The aPL avidity was significantly higher in SLE sera than syphilis sera. However, these findings contradicted others, who found higher mean aPL avidity in 47 syphilis sera compared with 22 SLE sera (Costello and Green, 1988). This discrepancy may reflect the fact that 14 of the 22 SLE sera (but none of the syphilis sera) were negative for aPL (decreasing the mean aPL avidity of this group) and the fact that the aPL were detected by an unusual dot blot method rather than the conventional ELISA (Costello and Green, 1988). Furthermore, the ELISA (Levy et al., 1990) contained bovine 132-GPI in the diluent, which is known to enhance the binding of autoimmune aPL and to inhibit the binding of infection-induced aPL to PL (Matsuura et al., 1990; Gharavi et al., 1994). In an investigation of the requirement of (I]2-GPI) as a cofactor for aPL binding to phospholipid in 20 preparations of purified IgG aPL, there was an inverse relationship between aPL avidity and the degree of cofactor requirement (Sammaritano et al., 1992); this could explain the relatively low degree of cofactor dependency in high-avidity aPL induced in mice and rabbits following immunization with heterologous 132-GPI (Gharavi et al., 1992). There is no detectable binding of aPL to gz-GPI in the absence of PL (McNeil et al., 1990; Sammaritano et al., 1992; Gharavi et al., 1994), but some investigators report the binding of aPL to gzGPI coated to y-irradiated high-binding ELISA plates (Matsuura et al., 1994). However, even under these conditions, the binding has very low affinity and requires high density of antigen coated on the ELISA plate ("y-irradiated high binding" polystyrene plates must be used), as well as bivalent antibodies (whole IgG or F(ab')2). Fab' fragments of aPL antibodies (monovalent) have very little or no binding (Roubey et al., 1995).

Polymyositis The evaluation of class switch and affinity maturation of autoantibodies to histidyl-tRNA synthetase antibodies (anti-Jo 1) by ELISA binding-inhibition (Friguet et al., 1985) in a patient with polymyositis showed increasing affinity in the preclinical period and stable high affinity thereafter (Miller et al., 1990).

Systemic Vaseulitis (Wegener's Granulomatosis) With the fluid-phase inhibition approach, the titers of myeloperoxidase antibodies (anti-MPO) increased during relapse in 28 patients with vasculitis, but the affinity of these IgG autoantibodies remained low (Kokolina et al., 1994).

Anti-GBM Disease (Goodpasture's Syndrome) In animal models of antiglomerular basement membrane (anti-GBM) disease which is caused by autoantibodies against an epitope on the alpha 3 chain of type IV collagen, the degree of glomerular injury correlated with the functional affinity (avidity) of such antibodies (Unanue et al., 1966). However, serial serum samples from nine patients with anti-GBM disease showed no changes in affinity in seven patients and an apparent decrease in two patients (Marriott and Oliveira, 1994). By the time clinical manifestations occur, affinity maturation of anti-GBM antibodies may be complete and no further increase possible (Marriott and Oliveira, 1994).

Myasthenia Gravis Experimental autoimmune myasthenia gravis can be induced in Lewis rat by immunization with acetylcholine receptor (AChR) in complete Freund's adjuvant. Oral administration of AChR prior to immunization with AChR resulted in the prevention of clinical symptoms as well as decrease in the level and avidity of anti-AChR antibodies determined by KSCN elution ELISA method (Wang et al., 1995).

health controls was estimated in an ELISA method using sodium thiocyanate for the dissociation of antigen-antibody complexes (Kasp et al., 1992). Antibody affinity was markedly lower in patients with retinal vasculitis than in healthy subjects; low-affinity antibodies were prevalent in patients with acute retinal vasculitis and those with normal amounts of circulating immune complexes. An association of lowaffinity antibodies with normal levels of circulating immune complexes was postulated; defective regulation of antiretinal autoimmunity may have important pathogenic implications (Kasp et al., 1992).

CONCLUSION Numerous studies have failed to end the controversy and provide a clear understanding of the role of affinity and avidity in the pathogenicity of autoantibodies. However, one might speculate loosely that in non-organ-specific, immune-complex-mediated disorders like SLE, low-avidity autoantibodies may be equally as pathogenic as high-avidity antibodies; whereas, in organ-specific autoantibody-associated diseases such as Goodpasture's syndrome and myasthenia gravis the avidity of the autoantibody may play a more critical role. New concepts which describe the development of autoimmune diseases regard the product of concentration and avidity as well as the depth of the immune network as parameters most relevant for the characterization of the immune system (Varela and Coutinho, 1991). Based on these concepts, the immune system can be described in terms of emergent properties and their changes in a type of developmental process. Based on these considerations, the affinity of autoantibodies might be less important than the change from immune tolerance to an autoimmune disease. The type of integration of certain autoantibodyproducing B-cell clones in the network of the immune system might well determine whether or not certain stimuli result in a pathological, autoimmune state.

Retinal vasculitis

ACKNOWLEDGEMENTS

The functional affinity (avidity) of antiretinal Santigen in 48 patients with retinal vasculitis and 46

This work has been supported by National Institute of Health Grant AR32929.

21

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MD. Characteristics of high-titer IgG antiphospholipid antibody in systemic lupus erythematosus patients with and without fetal death. Arthritis Rheum 1990;33:501--504. Reeves WH, Satoh M, Wang J, Chou CH, Ajmani AK. Systemic lupus erythematosus. Antibodies to DNA, DNAbinding proteins, and histones. Rheum Dis Clin North Am 1993 ;20:1--28. Riley RL, McGrath H Jr, Taylor RP. Detection of low avidity anti-DNA antibodies in systemic lupus erythematosus. Arthritis Rheum 1979;22:219--225. Rolink A, Melchers F. Molecular and cellular origins of B lymphocyte diversity. Cell 1991;66:1081-1094. Roubey RA, Eisenberg RA, Harper MF, Winfield JB. "Anticardiolipin" autoantibodies recognize beta 2-glycoprotein I in the absence of phospholipid. Importance of Ag density and bivalent binding. J Immunol 1995; 154:954-960. Sammaritano LR, Gharavi AE. Antiphospholipid antibody syndrome. Clin Lab Med 1992;12:41--59. Sammaritano LR, Lockshin MD, Gharavi AE. Antiphospholipid antibodies differ in aPL cofactor requirement. Lupus 1992; 1: 83--90. Smeenk R, Aarden L. The use of polyethylene glycol precipitation to detect low-avidity anti-DNA antibodies in systemic lupus erythematosus. J Immunol Methods 1980;39:165-- 180. Smeenk RJ, Van Rooijen A, Swaak TJ. Dissociation studies of DNA/anti-DNA complexes in relation to anti-DNA avidity. J Immunol Methods 1988;109:27-35. Steinberg AD, Pincus T, Talal N. DNA-binding assay for detection of anti-DNA antibodies in NZB-NZW F1 mice. J Immunol 1969;102:788--790. Stevens SY, Swanson PC, Glick GD. Application of gel shift assay to study the affinity and specificity of anti-DNA antibodies. J Immunol Methods 1994;177:185--190. Steward MW, Petty RE, Soothill JF. Low affinity antibody- its possible immunopathologic significance. Int Arch Allergy Appl Immunol 1973;45:176--181. Steward MW, Katz FE, West NJ. The role of low affinity antibody in immune complex disease. The quantity of anti-

DNA antibodies in NZB/W F1 hybrid mice. Clin Exp Immunol 1975 ;21:121-- 130. ter Borg EJ, Horst G, Hummel EJ, Limburg PC, Kallenberg CG. Measurement of increases in anti-double-stranded DNA antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus. A long-term prospective study. Arthritis Rheum 1990;33:634--643. ter Borg EJ, Horst G, Hummel E, Limburg PC, Kallenberg CG. Rises in anti-double-stranded DNA antibody levels prior to exacerbations of systemic lupus erythematosus are not merely due to polyclonal B cell activation. Clin Immunol Immunopathol 1991;59:117-128. Tonietti G, Oldstone MB, Dixon FJ. The effect of induced chronic viral infections on the immunologic diseases of New Zealand mice. J Exp Med 1970;132:89-109. Tron F, Bach JF. Relationship between antibodies to native DNA and glomerulonephritis in systemic lupus erythematosus. Clin Exp Immunol 1977;28:426--432. Unanue ER, Dixon FJ, Lee S. Experimental glomerulonephritis. VIII. The in vivo fixation of heterologous nephrotoxic antibodies to, and their exchange among, tissues of the rat. Int Arch Allergy Appl Immunol 1966;29:140-150. Underwood PA. Measurement of the affinity of antiviral antibodies. In: Maramorosch K, Murphy FA, Shatkin AJ, eds. Advances in virus research. London: Academic Press, 1988;34:283--309. Varela FJ, Coutinho A. Second generation immune networks Immunol Today 1991;12:159--166. Wang ZY, Huang J, Olsson T, He B, Link H. B cell responses to acetylcholine receptor in rats orally tolerized against experimental autoimmune myasthenia gravis. J Neurol Sci 1995;128:167--174. Winfield JB, Faiferman I, Koffier D. Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. Association of high avidity antinative DNA antibodies with glomerulonephritis. J Clin Invest 1977;59:90-96.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ALPHA-GALACTOSYL (ANTI-GAL) AUTOANTIBODIES Uri Galili Ph.D.

Department of Microbiology and Immunology, Medical College of Pennsylvania, Philadelphia, PA 19129, USA

HISTORICAL NOTES

THE AUTOANTIGEN

Antibodies to ~-galactosyl (anti-Gal), the most abundant natural antibodies known to be present in humans (Galili et al., 1984), are produced as 1% of circulating immunoglobulins and interact specifically with the mammalian-produced carbohydrate epitope Gal~l3Gall31-4GlcNAc-R (termed the ~-galactosyl epitope) (Galili et al., 1985; 1987a). Anti-Gal were discovered in the course of studies on the specificity of antibodies which mediate the destruction of human senescent red cells. Anti-Gal comprise a large proportion of the several hundred IgG molecules that bind in vivo to normal human senescent red cells (Galili et al., 1986b), to red cells in ]3-thalassemia (Galili et al., 1983; 1984) and in sickle cell anemia patients (Galili et al, 1986a) and mediate the phagocytosis of these cells by macrophages. Increase in the serum titer of anti-Gal in Graves' disease (Etienne-Decerf et al., 1987), scleroderma (Gabrielli et al., 1991), Henoch Sch6nlein purpura (Davin et al., 1987), Chagas disease (Towbin et al., 1987; Avila et al., 1989; Almeida et al., 1991) and malaria (Ravindran et al., 1988) suggests that this antibody is involved in the pathology of these diseases. In addition, the unique pattern of distribution of anti-Gal and the ~-galactosyl epitope in mammals (Galili et al., 1987b; 1988b) and the studies of the biosynthesis of the ~-galactosyl epitope (Galili, 1993a) led to the understanding of the major contribution of anti-Gal to the rejection of xenografts in humans and primates (Galili, 1993b).

Nomenclature

24

Anti-Gal interact specifically with the mammalianproduced carbohydrate structure Galal-3Gal~l4GlcNAc-R (the c~-galoctosyl epitope).

Origin Expression of c~-galactosyl epitopes in various mammalian species can be measured by the binding of anti-Gal and the lectin Bandeiraea (Griffonia) simplicifolia IB4. This lectin displays specificity for the c~galactosyl epitope (Wood et al., 1979) similar to that of anti-Gal. Nucleated cells from nonprimate mammals (e.g., mouse, rat, dog, cow, pig, horse and sheep) express an abundance of ot-galactosyl epitopes (1 • 106 to 35 x 106 epitopes per nucleated cell) (Galili et al., 1988b). A similar expression of ~-galactosyl epitopes is found on prosimian (lemur) cells and on New World monkey cells (i.e., monkeys of South America). Importantly, this epitope is normally not found on cells of Old World monkeys (i.e., monkeys of Asia and Africa), apes or humans (Galili et al., 1988b). A similar distribution of ~-galactosyl epitopes is also found on red cells of various species (Galili et al., 1987b). The reciprocal distribution of anti-Gal and of ~galactosyl epitopes in mammals (Table 1) is the result of the differential activity of the glycosylation enzyme ~l,3galactosyltransferase (c~I,3GT). This enzyme synthesizes ~-galactosyl epitopes within the Golgi apparatus as follows:

Gall31-4GlcNAc-R + UDP-Gal

al,3GT

>

Galo~ 1-3Gal~31-4GlcNAc-R + UDP ~-galactosyl epitope

N-acetlyllactosamine

This enzyme is unique to mammals and is found to be active within the Golgi apparatus of nonprimate mammals and New World monkeys but not in Old World monkeys or humans (Galili et al., 1988b; Thall et al., 1991). Subsequent to its cloning, the gene was found not to be expressed in Old World monkeys, apes and humans (Joziasse et al., 1989; Larsen et al., 1989). In addition, the c~I,3GT gene in humans and apes contains frame-shift mutations which, in the event of gene transcription, would produce a truncated enzyme lacking catalytic activity (Larsen et al., 1990; Henion et al., 1994; Galili and Swanson, 1991). Comparative sequencing of a portion of the ~I,3GT pseudogene in Old World monkeys and apes suggests that this gene was inactivated in ancestral Old World primates 16--28 million years ago, after the divergence between monkeys and apes (Galili and Swanson, 1991; Joziasse et al., 1991). A possible scenario is that ancestral Old World primates were exposed to an infectious agent, endemic in the Old World, that expressed c~-galactosyl epitopes and was detrimental to primates. Such a pathogen could have exerted a powerful selective pressure for the evolution of ancestral monkeys and apes that suppressed c~galactosyl epitope expression by inactivation of the ~I,3GT gene. Loss of immune tolerance to this epitope would result in the production of anti-Gal antibodies as a means of defense against pathogens which express (x-galactosyl epitopes such as viruses (Repik et al., 1994), bacteria (Galili et al., 1988a) and protozoa (Couto et al., 1990). Presumably, the selective pressure exerted by such a putative pathogen was

geographically limited to the Old World, and thus, New World monkeys were not subjected to this evolutionary event.

THE AUTOANTIBODIES Terminology Anti-Gal are natural polyclonal antibodies that constitute approximately 1% of circulating IgG in humans, apes and Old World monkeys (Galili et al., 1984; 1987b). Anti-Gal are also found in the blood as IgM and IgA isotypes (Parker et al., 1994; Hamadeh et al., 1995). In body secretions such as saliva, colostrum, milk and bile, anti-Gal are found in large amounts, mostly as the IgA isotype (Hamadah et al., 1995). Anti-Gal can be isolated from normal human serum by affinity chromatography with coupled melibiose or with synthetic c~-galactosyl epitope (Galili et al., 1984; 1988b). The only known mammalian-produced carbohydrate epitope which interacts with anti-Gal is Gal~I-3Gal~31-4GlcNAc-R, i.e., the agalactosyl epitope on glycolipids (Galili et al., 1987a) and on glycoproteins (Galili, 1993a). No binding of these antibodies is observed with ~3-galactosyl, fucosyl, glucosyl, sialyl, mannosyl or N-acetylgalactosaminyl residues on glycolipids or glycoproteins. This highly restricted specificity of anti-Gal has been further confirmed by its interaction with synthetic carbohydrate chains linked to albumin (Weislander et al., 1990) or to silica beads (Galili, 1993a).

Table 1. Distribution of Anti-Gal and o~-Galactosyl Epitopes in Mammals

Species

o~-Galactosyl Epitope Expression

Anti-Gal Production

Nonprimate mammals Prosimians New World monkeys Old World monkeys Apes Humans

25

Methods of Detection The ~-galactosyl epitope is the major carbohydrate structure on rabbit red cells. Therefore the titer of anti-Gal can be readily determined by an indirect hemagglutination assay with these red cells, using rabbit antihuman Ig as a secondary antibody (Avila et al., 1989). In addition, anti-Gal activity in the serum can be determined in ELISA using mouse laminin as solid-phase antigen since this glycoprotein has 50-70 ~-galactosyl epitopes per molecule (Gabrielli et al., 1991; Galili et al., 1995).

Pathogenetic Role Anti-Gal are produced throughout life as a result of antigenic stimulation by environmental antigens such as gastrointestinal bacteria that have carbohydrate structures similar" to the o~-galactosyl epitope on their cell walls (Galili et al., 1988a). That a large proportion of human B lymphocytes are capable of producing this antibody is suggested by the finding that 1% of Epstein BaIT virus (EBV)-transformed B lymphocytes are capable of secreting anti-Gal in vitro (Galili et al., 1993). Analysis of the immunoglobulin heavy chain genes which encode for anti-Gal in EBVtransformed lymphocytes indicates that most of these genes are clustered within the VH3 family and that these genes undergo somatic mutations (Wang et al., 1995b). The affinity of anti-Gal differs from one individual to the other. Analysis of antibody affinity by equilibrium dialysis assay with radiolabeled-free c~-galactosyl epitopes ([3H]Galo~I-3Gal~I-4GlcNAc) indicates affinity ranges between 2 x 105 and 5 x 10 6 M -1 in most individuals (Galili et al., 1995; Wang et al., 1995a). A several-fold increase in anti-Gal affinity occurs as a result of the exposure of the immune system to o~-galactosyl epitopes on infectious agents such as Trypanosoma cruzi in Chagas disease (Avila et al., 1989; Almeida et al., 1991) or on porcine cells in patients undergoing xenotransplantation (Satake et al., 1994; Galili et al., 1995). In many elderly individuals, the affinity of anti-Gal is less than that of a younger population (Wang et al., 1995a). In addition to the specific interaction with ~galactosyl epitopes, anti-Gal from blood group A and O individuals can bind to the fucosylated form of this epitope (i.e., the blood group B antigen with the structure Galc~1-3 [Fuc(x 1-2] Gal~31-4GlcNAc-R) (Galili et al., 1987a). As much as 90% of the antiblood group

26

B antibody reactivity in blood group A and O individuals is, in fact, mediated by clones of anti-Gal that are capable of binding to the ~-galactosyl epitope whether or not it is fucosylated; whereas, only 10% of the anti-B antibodies bind to the blood group B antigen but not to the a-galactosyl epitope (Galili et al., 1987a; Galili, 1988b).

CLINICAL UTILITY

Disease Associations Anti-Gal and Red Cell Aging. The studies leading to the identification of anti-Gal in humans were originally aimed at the characterization of antibodies that mediate the removal of normal and pathologic human senescent red cells from the circulation. After circulating in humans for 120 days, cryptic autoantigens are exposed on the red cell membrane and bind several hundred IgG molecules that label those cells for phagocytosis by reticuloendothelial macrophages (Kay, 1975). To identify the specificity of these antibodies, senescent red cells were isolated based on their increased density. Subsequently, the possible carbohydrate specificity of these antibodies was investigated by attempts to elute the red cell-bound IgG molecules with various carbohydrates. A large proportion of the antibodies on senescent red cells was eluted by galactose and even more effectively by ~-galactosyl oligosaccharides such as melibiose (Galo~l-6Glc) or ~-methylgalactoside (Galili et al., 1986b). Furthermore, anti-Gal isolated from normal sera readily bound to senescent red cells depleted of autologous IgG but did not bind to young red cells (i.e., red cells of intermediate or low density). AntiGal bind in vivo to a large proportion of pathogenic red cells with intrinsic deformability defects, such as red cells from patients with ~-thalassemia or sickle cell anemia (Galili et al., 1983; 1984; 1986a). These findings led to the hypothesis that human red cells have on their surface cryptic ~-galactosyl epitopes that are exposed as the cell ages (Galili et al., 1988a). Senescent red cells, being more dense and thus less flexible than young red cells, are probably retained for longer periods in the small passages of the reticuloendothelial system in the spleen. At these sites the red cells are likely to be subjected to the activity of proteases present on the macrophages lining the passages. These enzymes remove glycoprotein molecules from the red cell membrane and expose cryptic

~x-galactosyl epitopes that are capable of binding antiGal (Galili, 1988a). In sickle cell anemia and in [3thalassemia, the intrinsic defects in the deformability of red cells are likely to result in the retention of young, abnormal red cells within the small sinuses of the reticuloendothelial system and the subsequent premature exposure of the cryptic ~-galactosyl epitopes. The binding of anti-Gal to these red cells greatly contributes to their early removal from the circulation (Galili et al., 1986a). There are approximately 2,000 cryptic cz-galactosyl epitopes per red cell (Galili et al., 1986a). Because of their small number per cell, it is difficult to isolate and characterize these epitopes on human red cells. Thus, the mechanism involved in their biosynthesis is not clear as yet. Furthermore, the occurrence of red cell cryptic epitopes of uncharacterized structure, which are different from c~-galactosyl epitopes, but which are capable of binding anti-Gal, cannot be excluded at present. The anti-Gal-mediated destruction of normal senescent red cells and of some pathologic red cells is one of several mechanisms which contribute to the removal of senescent red cells. Evidently, this mechanism does not exist in nonprimate mammals and New World monkeys, all of which lack antiGal. In humans, however, anti-Gal together with other autoantibodies (Kay et al., 1983; Sorette et al., 1991) mediate removal of senescent red cells.

thyrocytes from Graves' disease patients but not to normal human thyrocytes (Winand et al., 1994). Binding of anti-Gal increases the uptake of iodine and production of cAMP in Graves' disease thyrocytes, but not normal human thyrocytes (Winand et al., 1994). Graves' disease thyrocytes are also stimulated in vitro by autologous serum. However, specific removal of anti-Gal from the autologous serum causes a 50-80% decrease in the stimulatory effect of serum on autologous Graves' disease thyrocytes (Winand et al., 1994). Overall, these studies suggest that the thyrocytes in patients with Graves' disease aberrantly express agalactosyl epitopes on the TSH receptors. The possibility that these pathologic thyrocytes also express other epitopes of unknown structure which can bind anti-Gal cannot be excluded. This binding might contribute significantly to the continuous autoimmune stimulation of the thyroid gland and the resulting hyperthyroidism. The biosynthetic mechanism for the production of anti-Gal binding epitopes in Graves' disease and their exact structure are unknown due to the difficulties in obtaining sufficient pathologic tissue for such analyses. Hypothetically, the aberrant expression of these epitopes and the subsequent binding of anti-Gal might occur on a number of tissues in humans, resulting in a variety of autoimmune disorders (Galili, 1989).

Anti-Gal in Graves' Disease. Increased activity of anti-Gal in patients with Graves' disease (EtienneDecerf et al., 1987)suggests that ~-galactosyl epitopes may be aberrantly expressed on thyrocytes in these patients; thus, inducing the increased production of anti-Gal as previously postulated (Galili, 1989). TSH-like stimulation by anti-Gal can be demonstrated with porcine thyrocytes, because these cells express an abundance of ~-galactosyl epitopes on cell surface glycoproteins (Thall et al., 1991). In vitro incubation of porcine thyrocytes with anti-Gal causes increased synthesis of cAMP, increased uptake of iodine and a higher proliferation rate (Winand et al., 1993). When the human TSH receptor cDNA is transfected into mouse 3T3 fibroblasts (i.e., cells producing an abundance of ~-galactosyl epitopes), the expressed human TSH receptor glycoprotein has ~galactosyl epitopes on some of the carbohydrate chains and anti-Gal binding to these epitopes stimulates cAMP synthesis in the fibroblasts, similar to the stimulatory effect of TSH (Winand et al., 1993). In humans, anti-Gal bind in vitro to cultured

Anti-Gal in Xenotransplantation. The appearance of anti-Gal in ancestral Old World primates erected an immunological barrier to xenotransplantation of organs or tissues from nonprimate mammals into humans and Old World monkeys (Galili, 1993b). Anti-Gal readily bind in vivo to ~-galactosyl epitopes on the xenograft and induces graft rejection by various immune mechanisms. The hyperacute rejection of porcine or New World monkey organs transplanted into baboons is mediated by anti-Gal IgM antibodies that bind to ~galactosyl epitopes on the endothelial cells of the graft and activates the complement cascade which ultimately lyses these cells and causes the collapse of the vascular system of the graft (Collins et al., 1995). In addition, human antibodies which lyse porcine cells in vitro are largely anti-Gal IgM molecules (Good et al., 1992; Sandrin et al., 1993). Although hyperacute rejection may be prevented by inactivation of complement (Leventhal et al., 1993), xenograft destruction by anti-Gal will not be prevented because the IgG moiety of anti-Gal can mediate antibody-dependent cell cytotoxicity (ADCC)

27

via monocytes, macrophages and granulocytes (Galili, 1993b). In this process, the various killer cells adhere to the xenograft cells as a result of the interaction between the Fc portion of anti-Gal on these cells and the Fc receptors on the effector cells. Furthermore, the human immune system, once exposed to c~-galactosyl epitopes on the xenograft, produces anti-Gal antibodies with increased affinity as compared to the affinity of this antibody pretransplantation (Satake et al., 1994; Galili et al., 1995). Such antibodies are likely to be more effective than the pretransplantation anti-Gal in mediating the destruction of the xenograft by ADCC. These high affinity anti-Gal antibodies may be the result of preferential proliferation of the lymphoid clones producing highaffinity anti-Gal (Wang et al., 1995b). These findings further suggest that pretransplantation removal of antiGal from the recipient's serum, or neutralization of the antibody by free oligosaccharides, may only temporarily inhibit the detrimental effect of anti-Gal on the xenograft. An ultimate solution could be the development of pig strains in which the ~ I , 3 G T gene is inactive. The very recent studies which demon-

strated the successful breeding of mice with disrupted ~ I , 3 G T gene (i.e., knock-out mice for czl,3GT) suggest the feasibility of inactivating this gene in pigs (Thall et al., 1995). The future use of organs and tissues derived from such pigs possibly would eliminate the detrimental effect of anti-Gal in xenotransplantation.

REFERENCES

lander J. Antibodies to mouse laminin in patients with systemic sclerosis (scleroderma) recognize galactosyl (c~l-3)galactose epitopes. Clin Exp Immunol 1991;86:367--373. Galili U, Korkesh A, Kahane I, Rachmilewitz EA. Demonstration of a natural antigalactosyl IgG antibody on thalassemic red blood cells. Blood 1983;61:1258-1264. Galili U, Rachmilewitz EA, Peleg A, Flechner I. A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J Exp Med 1984; 160:1519-- 1531. Galili U, Macher BA, Buehler J, Shohet SB. Human natural anti-alpha-galactosyl IgG. II. The specific recognition of alpha(czl,3)-linked galactose residues. J Exp Med 1985;162: 573--582. Galili U, Clark MR, Shohet SB. Excessive binding of the natural anti-c~-galactosyl immunoglobulin G to sickle erythrocytes may contribute to extravascular cell destruction. J Clin Invest 1986a;77:27-33. Galili U, Flechner I, Knyszinski A, Danon D, Rachmilewitz EA. The natural anti-c~-galactosyl IgG on human normal senescent red blood cells. Br J Haematol 1986b;62:317-324. Galili U, Buehler J, Shohet SB, Macher BA. The human natural anti-Gal IgG. III. The subtlety of immune tolerance in man as demonstrated by crossreactivity between natural anti-Gal and anti-B antibodies. J Exp Med 1987a;165:693-704. Galili U, Clark MR, Shohet SB, Buehler J, Macher BA. Evolutionary relationship between the anti-Gal antibody and the Gal cz,l-3Gal epitope in primates. Proc Natl Acad Sci USA 1987b;84:1369--1373. Galili U. The natural anti-Gal antibody, the B-like antigen, and

Almeida IC, Milani SR, Gorin PA, Travassos LR. Complement mediated lysis of Trypanosoma cruzi trypomastigotes by human anti ~z-galactosyl antibodies. J Immunol 1991;146: 2394-2400. Avila JL, Rojas M, Galili U. Immunogenic Gal czl,3Gal carbohydrate epitopes are present on pathogenic American Trypanosoma and Leishmania. J Immunol 1989;142:2828-2834. Collins BH, Cotterell AH, McCurry KR, Alvarado CG, Magee JC, Parker W, Platt JL. Cardiac xenografts between primate species provide evidence for the importance of the c~-galactosyl determinant in hyperacute rejection. J Immunol 1995; 154:5500--5510. Couto AS, Concalves MF, Colli W, de Lederkremer RM. The N-linked carbohydrate chain of the 85-kilodalton glycoprotein from Trypanosoma cruzi trypomastigotes contains sialyl, fucosyl and galactosyl (~1-3) galactose units. Mol Biochem Parasitol 1990;39:101--107. Davin JC, Malaise M, Foidart JM, Mahieu P. Anti-~-galactosyl antibodies and immune complexes in children with HenochSchonlein purpura or IgA nephropathy. Kidney Int 1987;31: 1132--1139. Etienne-Decerf J, Malaise M, Mahieu P, Winand R. Elevated anti-c~-galactosyl antibody titers. A marker of progression in autoimmune thyroid disorders in endocrine ophthalmopathy? Acta Endocrinol 1987;115:67--74. Gabrielli A, Candela M, Ricciatti AM, Caniglia ML, Wies28

CONCLUSION Anti-Gal are natural antibodies unique to humans and Old World primates. These antibodies appeared in ancestral primates subsequent to the evolutionary suppression of ~-galactosyl epitope (Gal~I-3Gal~I4GlcNAc-R) expression. Anti-Gal contribute to the removal of senescent red cells from the circulation, stimulate thyrocytes in patients with Graves' disease by interaction with ligands aberrantly expressed on these cells and prevent the transplantation of xenografts from nonprimate mammals into humans. See also XENOREACTIVE HUMAN NATURAL ANTIBODIES.

human red cell aging. Blood Cells 1988a;14:205-220. Galili U. The two antibody specificities within human antiblood group B antibodies. Transfus Med Rev 1988b;2:112--121. Galili U, Mandrell RE, Hamadeh RM, Shohet SB, Griffiss JM. Interaction between human natural anti-~-galactosyl immunoglobulin G and bacteria of the human flora. Infect Immun 1988a;56:1730--1737. Galili U, Shohet SB, Kobrin E, Stults CL, Macher BA. Man, apes, and Old World monkeys differ from other mammals in the expression of c~-galactosyl epitopes on nucleated cells. J Biol Chem 1988b;263:17755--17762. Galili U. Abnormal expression of c~-galactosyl epitopes in man. A trigger for autoimmune processes? Lancet 1989;2:358--361. Galili U, Swanson K. Gene sequences suggest inactivation of ~-1,3-galactosyltransferase in catarrhines after the divergence of apes from monkeys. Proc Natl Acad Sci USA 1991;88: 7401--7404. Galili U. Evolution and pathophysiology of the human natural anti-~-galactosyl antibody. Springer Semin Immunopathol 1993a;15:155--171. Galili U. Interaction of the natural anti-Gal antibody with ~galactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol Today 1993b;14:480--482. Galili U, Anaraki F, Thall A, Hill-Black C, Radic M. One percent of human circulating B lymphocytes are capable of producing the natural anti-Gal antibody. Blood 1993;82: 2485--2493. Galili U, Tibell A, Samuelsson B, Rydberg L, Groth CG. Increased anti-Gal activity in diabetic patients transplanted with fetal porcine islet cell clusters. Transplantation 1995;59: 1549--1556. Good AH, Cooper DC, Malcolm AJ, Ippolito RM, Koren E, Neethling FA, Ye Y, Zuhdi N, Lamontage LR. Identification of carbohydrate structures which bind human anti porcine antibodies: implication for discordant xenografting in man. Transplant Proc 1992;24:559--562. Hamadeh RM, Galili U, Zhou P, Griffiss JM. Anti-~-galactosyl immunoglobulin A (IgA),-IgA, and IgM in human secretions. Clin Diagn Lab Immunol 1995;2:125--131. Henion TR, Macher BA, Anaraki F, Galili U. Defining the minimal size of catalytically active primate (~l,3galactosyltransferase: structure-function studies on the recombinant truncated enzyme. Glycobiology 1994;4:193--201. Joziasse DH, Shaper JH, Van den Eijnden DH, Van Tunen AH, Shaper NL. Bovine ~l-3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. J Biol Chem 1989;264:14290-- 14297. Joziasse DH, Shaper JH, Jabs EW, Shaper NL. Characterization of an c~1-3 galactosyltransferase homologue on human chromosome 12 that is organized as a processed pseudogene. J Biol Chem 1991 ;266:6991--6998. Kay MM. Mechanism of removal of red cells by macrophages in situ. Proc Natl Acad Sci USA 1975;72:3521-3525. Kay MM, Goodman SR, Sorensen K, Whitfield CF, Wong P, Zaki L, Rudolff V. Senescent cell antigen is immunologically related to band 3. Proc. Natl Acad Sci USA 1983;80:16311636.

Larsen RD, Rajan VP, Ruff M, Kukowska-Latallo J, Cummings RD, Lowe JB. Isolation of a cDNA encoding a murine UDPgalactose:13-D-galactosyl,4-N-acetyl-D-glucosaminide ~1,3-galactosyltransferase: expression cloning by gene transfer. Proc Natl Acad Sci USA 1989:86:8227--8231. Larsen RD, Rivera-Marrero CA, Ernst LK, Cummings RD, Lowe JB. Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:13-DGal(1,4)-D-GlcNAc-~(1,3)-galactosyltransferase cDNA. J Biol Chem 1990;265:7055--7062. Leventhal JR, Dalmaso AP, Cromwell JW, Platt JL, Manivel CJ, Bolman RM, Matas AJ. Prolongation of cardiac xenograft survival by depletion of complement. Transplantation 1993;55:857-865. Parker W, Bruno O, Holzknecht ZE, Platt JE. Characterization and affinity isolation of xenoreactive human natural antibodies. J Immunol 1994;153:3791-3803. Ravindran S, Satapathy AK, Das MK. Naturally-occurring antic~-galactosyl antibodies in human Plasmodium falciparum infections: a possible role for autoantibodies in malaria. Immunol Lett 1988;19:137--141. Repik PM, Strizki JM, Galili U. Differential host-dependent expression of alpha-galactosyl epitopes on viral glycoproteins: a study of eastern equine encephalitis virus as a model. J Gen Virol 1994;75:1177-1181. Sandrin MS, Vaughan HA, Dabkowski PL, McKenzi IF. Antipig IgM antibodies in human serum react predominantly with Gal(1-3)Gal epitopes. Proc Natl Acad Sci USA 1993;90: 11391--11395. Satake M, Kawagishi N, Rydberg L, Samuelsson BE, Tibell A, Groth CG, Moller E. Limited specificity of xenoantibodies in diabetic patients transplanted with fetal porcine islet cell clusters. Main antibody reactivity against m-linked galactosecontaining epitopes. Xenotransplantation 1994;1:89--101. Sorette MP, Galili U, Clark, MR. Comparison of serum antiband 3 and anti-Gal antibody binding to density separated human red blood cells. Blood 1991;77:628--636. Thall A, Etienne-Decerf J, Winand R, Galili U. The c~-galactosyl epitope on mammalian thyroid cells. Acta Endocrinol 1991; 124:692-699. Thall AD, Maly P, Lowe JB. Oocyte Gal~l-3 Gal epitopes implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are not required for fertilization in the mouse. J Biol Chem 1995;270:21437--21440. Towbin H, Rosenfelder G, Weislander J, Avila JL, Rojas M, Szarfman A, Esser K, Nowack H, Timple R. Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis and normal individuals recognize terminal galactosyl (~l-3)-galactose epitopes. J Exp Med 1987;166:419--432. Wang L, Anaraki F, Henion TR, Galili U. Variations in activity of the human natural anti-Gal antibody in young and elderly populations. J Gerontol (Med Sci) 1995a;50A:M227--M233. Wang L, Radic MZ, Galili U. Human anti-Gal heavy chain genes: preferential use of VH3 and the presence of somatic mutations. J Immunol 1995b;155:1276-1285. Weislander J, Mannson O, Kallin E, Gabrielli A, Nowack H, Timpl R. Specificity of human antibodies against Gal-~l-

29

3Gal carbohydrate epitope and distinction from natural antibodies reacting with Gal(c~l-2)Gal or Gal(c~l-4)Gal. Glycoconjugate J 1990;7:85-100. Winand RJ, Anaraki F, Etienne-Decerf J, Galili U. Xenogeneic thyroid-stimulating hormone-like activity of the human natural anti-Gal antibody. Interaction of anti-Gal with porcine thyrocytes and with recombinant human thyroid-stimulating hormone receptors expressed on mouse cells. J Immunol 1993;151:3923-3934.

30

Winand RJ, Winand-Devigne J, Meurisse M, Galili U. Specific stimulation of Graves' disease thyrocytes by the natural antiGal antibody from normal and autologous serum. J Immunol 1994; 153:1386-1395. Wood C, Kabat EA, Murphy LA, Goldstein LT. Immunochemical studies of the combining sites of two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia. Arch Biochem Biophys 1979;198:1-11.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

AMINOACYL-tRNA HISTIDYL (Jo-1) SYNTHETASE AUTOANTIBODIES Peter J. Maddison, M.D.

Royal National Hospital for Rheumatic Diseases, University of Bath, Bath BA1 1RL, UK

HISTORICAL NOTES Autoantibodies are commonly found in the sera of patients with myositis, and some are highly specific for this disorder (Reichlin and Arnett, 1984). Myositis-specific antibodies (MSA) are directed to a number of different nuclear and cytoplasmic antigens, some of which have been characterized in molecular terms. Each MSA defines a group of myositis patients with distinctive clinical features (Love et al., 1991). In 1980, a precipitating antibody identified by immunodiffusion in sera of patients with primary polymyositis was labeled "Jo-l" after the prototype patient (Nishikai and Reichlin, 1980). In 1983, antiJo-1 sera were shown to immunoprecipitate tRNA ais (Rosa et al., 1983); shortly thereafter confirmation of the antigen as histidyl-tRNA synthetase included its inhibition by anti-Jo-l-containing sera (Mathews and Bernstein, 1983). Subsequently, antibodies were detected to four other aminoacyl synthetases, threonyl(anti-PL-7), alanyl- (anti-PL12), isoleucyl- (anti-OJ) and glycyl-tRNA synthetase (anti-EJ).

THE AUTOANTIGEN Nomenclature Jo-1 is synonymous with histidyl tRNA synthetase, abbreviated to HRS. This cytoplasmic enzyme catalyzes the esterification of histidine to its cognate tRNA. Binding of anti-Jo-1 antibodies is localized to the cytoplasm of the various cell types examined (Nishikai et al., 1990; Shi et al., 1991); the antigen is entirely associated with the cytoplasmic fraction (Dang et al., 1986). HRS is present as a homodimer

within the cell; identical subunits of approximately 50 kd are each bound to tRNA.

Sequence Similarities The tRNA synthetases are present in all prokaryotes and eukaryotes, and synthetases for a particular amino acid show substantial sequence similarity among species. Of the two recognized families of synthetases, HRS belongs to class II (Carter, 1993). Human autoantibodies do not cross-react among different aminoacyl-tRNA synthetases. Furthermore, anti-Jo-1 sera only recognize HRS from higher eukaryotes and react with greatest affinity with human enzyme (Miller et al., 1990a). This is probably explained by the observation that the enzymatic core of the molecule is best preserved in evolution and that there are species-specific additions at the amino- and carboxytermini which in the human enzyme result in unique structures recognized by the autoantibodies. The sequence of the cDNA for human HRS is known (EMBL accession-Z1 1518) (Raben et al., 1992), and the HRS gene is on chromosome 5 along with genes for threonyl-, arginyl- and leucyl-tRNA synthetases. The putative structure for the HRS molecule possesses all three motifs which characterize class II synthetases (Figure 1). The major Jo-1 epitope, situated in the amino-terminal region of the human molecule, is probably a 32 amino acid region predicted to have a coiled-coil configuration. HRS from other animal species, e.g., the hamster, which are recognized by anti-Jo-1 sera have this configuration; whereas, yeast HRS which fails to react with HRS does not. There is no simple linear epitope within this region of the HRS molecule because overlapping synthetic hexapeptides of the HRS

31

major epitope

motifI

motif 2

1O0

sigl

motif 3

200

300

sig2

400

500

Figure 1. A schematic representation of histidyl-tRNA synthetase (Jo-1). The major epitope of Jo-1 is found in the amino terminal 60 amino acids.

molecule do not react with anti-Jo-1 sera (Miller et al., 1990b). Presumably, the major epitope is conformationally determined although most sera react well in immunoblotting and with recombinant protein (Raben et al., 1994).

AUTOANTIBODIES

Pathogenetic Role Autoantibodies to aminoacyl transferases including anti-Jo-1 are found almost exclusively in the serum of myositis patients (Biswas et al., 1987). This, together with evidence suggesting that the autoantibody response is antigen driven, including an association with class II MHC genes, suggests that the anti-Jo-1 response is linked in some way to the etiopathogenesis of myositis. Perhaps an environmental agent acting on a host with the appropriate genetic background induces cellular and humoral autoimmune phenomena and chronic muscle inflammation. The finding that levels of anti-Jo-1 vary in proportion to disease activity suggests that this immune response is linked to that which is responsible for myositis in these patients. However, there is no evidence for the direct involvement of these antibodies in the development of myositis. On the other hand, there is circumstantial evidence that immune complexes containing anti-Jo-1 might be involved in the pathogenesis of the associated interstitial pneumonitis as manifest by alveolar septal deposits of immunoglobulin and complement identified in the lung biopsy of a patient in whom the antibodies were identified in an isolated mixed cryoglobulin (Lambie and Quismorio, 1991). Speculation that the anti-Jo-1 response reflects a previous specific viral infection is based on HRS interaction with the genomic RNA of certain picornaviruses (Florentz et al., 1984) which are associated with myositis in epidemiologic and animal model

32

studies (Christensen et al., 1986; Cronin et al., 1988). Retrospective identification of a seasonal pattern of onset with weakness developing mainly in the Spring in anti-Jo-1 patients contrasts with the predominantly Autumnal onset in patients characterized serologically by antibodies to signal recognition particle (SRP) (Left et al., 1991). This observation is consistent with a viral etiology. However, anti-Jo-1 are also reported in rheumatoid patients with penicillamine-induced myositis, in whom both the clinical and serological features resolve when the drug is discontinued (Jenkins et al., 1993). The presence of anti-Jo-1 is not, however, an invariable phenomenon in penicillamineinduced myositis (Carroll et a1.,1987).

Factors in Pathogenesis The immune response to aminoacyl transferases is very selective. Only one specific synthetase is targeted by the antibodies in each patient's serum. Antibodies to Jo-1 block the function of HRS and react with a limited number of epitopes with a dominant epitope in the amino terminal of the molecule (see above). Consistent with an antigen-driven immune response, autoantibodies are predominantly IgG and mostly belong to the IgG1 heavy chain isotype. In one study, IgG 1 accounted on average for 94% of the total antiJo-1 with small contributions by IgG3 and IgM (Miller et al., 1990a). As yet there is no information about immunoglobulin gene usage in the anti-Jo-1 antibody response.

Genetics HRS antibodies are associated with HLA DRw52 haplotypes regardless of subtype in both caucasoid and black patients (Goldstein et al., 1990). In white myositis patients positive for anti-Jo-1 antibodies, HLA-DR3 is mostly present and more frequent than in the total myositis group (Arnett et al., 1981).

Examination of the HLA-DR3, DR5, DRw6 and DRw8 haplotypes which bear the DRw52 specificity suggests that a region of sequence similarity in the first hypervariable region corresponding to amino acids 9--13, which is situated in the floor of the peptide-binding groove of the putative three-dimensional structure of the class II MHC molecule, is the candidate epitope. Immunoglobulin allotypes might also predispose to development of anti-Jo-1 antibodies as is suggested by an increase in the allotype Gm3;5 in anti-Jo-1 positive patients; the combination of Gin3;5 and HLA-DR3 is greatly increased (92 versus 15% in controls) (Enz et al., 1992).

characterizing one antisynthetase from another (Targoff, 1990). Most sera positive for Jo-1 antibodies by immunodiffusion are also positive by immunoblotting but there are exceptions. For immunoblotting and immunoprecipitation, HeLa $3 cells are the most widely referenced source of antigen (Verheijen et al., 1993). Sensitive and quantitative ELISAs for anti-Jo- 1 are available (Biswas et al., 1987). Titers of anti-Jo-1 fluctuate with disease activity and can disappear with treatment and remission (Miller et al., 1990a).

Methods of Detection

Disease Associations

Anti-Jo-1 antibodies yield diffuse granular cytoplasmic staining on HEp-2 cells in indirect immunofluorescence. Sometimes there is additional nuclear fluorescence resulting from additional antibody specificities. Immunodiffusion confirming identity with a standard prototype (Centers for Disease Control, Atlanta, Georgia, USA) is still an effective method for detecting anti-Jo-1. Thymus acetone powder which is commercially available (Pelfreez Biological Inc, Rogers, Arkansas, USA) is a convenient source of antigen. Methods for enriching for aminoacyl transferases have been described (Deuscher, 1967). Recombinant Jo-1 has also been generated (Raben et al., 1994). ELISA is the most sensitive routinely available assay although sera which are negative in immunodiffusion but ELISA-positive are uncommon. For research purposes, immunoprecipitation of tRNA from in vivo 32p-labeled cell extracts is the best method of

About 30% of adults with myositis have antibodies to an aminoacyl transferase, and in at least 80% of cases the antibodies are directed to histidyl tRNA synthetase (HRS). These antibodies are rarely found in childhood myositis although they are reported (Chmiel et al., 1995). Anti-Jo-1 antibodies are almost exclusively found in patients with myositis. In one large review (Love et al., 1991), 54% had primary myositis, 40% had dermatomyositis and 6% had myositis in the setting of another connective tissue disease. The majority of anti-Jo-1 patients show rather a distinctive pattern of multisystem disease (Table 1), shared by patients with other synthetases, termed the "antisynthetase syndrome" (Marguerie et al., 1990; Love et al., 1991; Miller, 1993). The onset is often acute with prominent systemic features such as fever. Myositis is often severe although cases without clinical muscle involvement

CLINICAL UTILITY

Table 1. The "Antisynthetase Syndrome" Feature

Bernstein et al. (n = 19) (%)

Marguerie et al. (n = 29) (%)

Love et al. (n = 47) (%)

Myositis

90

83

100

Pneumonitis

79

79

89

Arthritis

56

90

94

Mechanic's hands

NR

NR

71

DM rash

11

38

54

Raynaud's

89

93

62

Sclerodactyly

20

72

NR

Calcinosis

NR

24

NR

Sicca syndrome

56

59

NR

33

are reported (Marguerie et al., 1990; Lopes-Lancis et al., 1991). Interstitial pneumonitis was a prominent clinical manifestation in the index patient (Wasicek et al., 1984) and in subsequent experience (Bernstein et al., 1984; Hochberg et al., 1984) is the next most common clinical feature after myositis in anti-Jo-1positive patients, being present in 50--90% compared to <10% of other patients with polymyositis or dermatomyositis. On the other hand, anti-Jo-1 are not markers of interstitial pneumonitis p e r se, because they are not detected in idiopathic interstitial lung disease or in patients with systemic sclerosis and fibrosing alveolitis (Turner-Stokes et al., 1990). Polyarthritis is also common. It is usually mild, primarily affecting the small joints of the hands, wrists, shoulders and knees, but can be associated with deformities of the fingers and is occasionally erosive (Oddis et al., 1990). Anti-Jo-1 antibodies associated with myositis were reported in a patient with rheumatoid arthritis (O'Neill and Maddison, 1993). "Mechanic's hands" is another feature of the antisynthetase syndrome. This distinctive cutaneous eruption, previously described in association with polymyositis (Stahl et al., 1979), is a hyperkeratotic, nonpruritic rash accompanied by fissuring, hyperpigmentation and scaling. Occurring on the palm and the lateral and palmar aspects of the fingers, the appearance resembles the callused hands of a mechanic. Histologically, there is marked hyperkeratosis, perivascular infiltration of lymphocytes and a variable degree of liquefactive degeneration at the dermoepidermal junction (Mitra et al., 1994).

Approximately one-third have a rash more typical of dermatomyositis and characteristically abnormal morphology of the nail fold capillaries. These changes can accompany "mechanic's hands". Other features include Raynaud's phenomenon, sclerodactyly, acroosteolysis and soft tissue calcinosis. Features of some patients overlap with systemic sclerosis, but hypertension and scleroderma kidney do not occur. When compared with the rest of the spectrum of idiopathic polymyositis, patients with anti-Jo-1 tend to have a severe disease with a tendency to relapse and a poorer prognosis (Love et al., 1991). The latter is probably related to the high frequency of interstitial lung disease, which is a poor prognostic indicator (Arsura and Greenberg, 1988). Although these patients generally respond to corticosteroids, early institution of a cytotoxic immunosuppressive agent is recommended; patients are more likely to respond to methotrexate than azathioprine (Joffe et al., 1993).

REFERENCES

Rheumatol 1987;14:995--1001. Carter CW Jr. Cognition, mechanisms, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu Rev Biochem 1993;62:715--748. Chmiel JF, Wessel HU, Targoff IN, Pachman LM. Pulmonary fibrosis and myositis in a child with anti-Jo-1 antibody. J Rheumatol 1995;22:762--765. Christensen ML, Pachman LM, Schneiderman R, Patel DC, Friedman JM. Prevalence of Coxsackie B virus antibodies in patients with juvenile dermatomyositis. Arthritis Rheum 1986;29:1365-1370. Cronin ME, Love LA, Miller FW, McClintock PR, Notkins AL, Plotz PH. The natural history of encephalomyocarditis virusinduced myositis and myocarditis in mice. Viral persistence demonstrated by in situ hybridization. J Exp Med 1988;168: 1639-1648. Dang CV, LaDuca FM, Bell WR. Histidyl-tRNA synthetase, the myositis Jo-1 antigen, is cytoplasmic and unassociated with

Arnett FC, Hirsch TJ, Bias WB, Nishikai M, Reichlin M. The Jo-1 antibody system in myositis: relationships to clinical features and HLA. J Rheumatol 1981;8:925--930. Arsura EL, Greenberg AS. Adverse impact of interstitial pulmonary fibrosis on prognosis in polymyositis and dermatomyositis. Semin Arthritis Rheum 1988;18:29--37. Bernstein RM, Morgan SH, Chapman J, Bunn CC, Mathews MB, Turner-Warwick M, Hughes GR. Anti-Jo-1 antibody: a marker for myositis with interstitial lung disease. Br Med J 1984;289:151-152. Biswas T, Miller FW, Takagaki Y, Plotz PH. An enzyme-linked immunosorbent assay for the detection and quantitation of anti-Jo-1 antibody in human serum. J Immunol Methods 1987;98:243--248. Carroll GJ, Will RK, Peter JB, Garlepp MJ, Dawkins RL. Penicillamine induced polymyositis and dermatomyositis. J

34

CONCLUSION Antibodies to Jo-1 are very closely linked to inflammatory muscle disease and identify an important subset of idiopathic polymyositis which is distinctive clinically and immunogenetically. Further understanding of this autoimmune response might give important clues to the etiopathogenesis of myositis in these patients and perhaps point to a specific viral etiology. See also AMINOACYL-TRNA (OTHER THAN HISTIDYL) SYNTHETASE AUTOANTIBODIES and SIGNAL RECOGNITION PARTICLE AUTOANTIBODIES.

the cytoskeletal framework. Exp Cell Res 1986; 164:261--266. Deuscher MP. Rat liver glutamyl ribonucleic acid synthetase. J Biol Chem 1967;242:1123--1131. Enz LA, Love LA, Targoff IN, Pandey JP. Association among Gm phenotypes, HLA alleles, and myositis-specific autoantibodies (MSA) in idiopathic inflammatory myopathy (IIM). Arthritis Rheum 1992;35:$52. Florentz C, Briand JP, Giege R. Possible functional role of viral tRNA-like structures. FEBS 1984;176:295--300. Goldstein R, Duvic M, Targoff IN, Reichlin M, McMenemy AM, Reveille JD, Warner NB, Pollack MS, Arnett FC. HLAD region genes associated with autoantibody responses to histidyl-transfer RNA synthetase (Jo-1) and other translationrelated factors in myositis. Arthritis Rheum 1990;33:12401248. Hochberg MC, Feldman D, Stevens MB, Arnett FC, Reichlin. Antibody to Jo-1 in polymyositis/dermatomyositis: association with interstitial pulmonary disease. J Rheumatol 1984; 11:663--665. Jenkins EA, Hull RG, Thomas AL. D-penicillamine and polymyositis: the significance of the anti-Jo-1 antibody. Br J Rheumatol 1993;32:1109-1110. Joffe MM, Love LA, Left RL, Fraser DD, Targoff IN, Hicks JE, Plotz PH, Miller FW. Drug therapy of the idiopathic inflammatory myopathies: predictors of response to prednisone, azathioprine, and methotrexate and a comparison of their efficacy. Am J Med 1993;94:379--387. Lambie PB, Quismorio FP Jr. Interstitial lung disease and cryoglobulinemia in polymyositis. J Rheumatol 1991;18: 468--469. Left RL, Burgess SH, Miller FW, Love LA, Targoff IN, Dalakas MC, Joffe MM, Plotz PH. Distinct seasonal patterns in the onset of adult idiopathic inflammatory myopathy in patients with anti-Jo-1 and antisignal recognition particle autoantibodies. Arthritis Rheum 1991 ;34:1391-- 1396. Lopes LA, Manero RF, Bello DS, Vila JM, Povar MJ, Perez D. Pulmonary fibrosis as a presentation form of the Jo-1 syndrome. An Med Interna 1991;8:393--394. Love LA, Left RL, Fraser DD, Targoff IN, Dalakas M, Plotz PH, Miller FW. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups. Medicine (Baltimore) 1991 ;70:360-374. Marguerie C, Bunn CC, Beynon HL, Bernstein RM, Hughes JM, So AK, Walport MJ. Polymyositis, pulmonary fibrosis and autoantibodies to aminoacyl-tRNA synthetase enzymes. Q J Med 1990;77:1019--1038. Mathews MB, Bernstein RM. Myositis autoantibody inhibits histidyl-tRNA synthetase: a model for autoimmunity. Nature 1983;304:177--179. Miller FW, Twitty SA, Biswas T, Plotz PH. Origin and regulation of a disease-specific autoantibody response. Antigenic epitopes, spectrotype stability, and isotype restriction of anti-Jo-1 autoantibodies. J Clin Invest 1990a;85:468-475. Miller FW, Waite KA, Biswas T, Plotz PH. The role of an autoantigen, histidyl-tRNA synthetase, in the induction and

maintenance of autoimmunity. Proc Nat Acad Sci USA 1990b;87:9933-9937. Miller FW. Myositis-specific autoantibodies. Touchstones for understanding the inflammatory myopathies. JAMA 1993; 270:1846-1849. Mitra D, Lovell CL, Macleod TI, Tan RS, Maddison PJ. Clinical and histological features of "mechanic's hands" in a patient with antibodies to Jo-1 - a case report. Clin Exp Dermatol 1994; 19:146-- 148. Nishikai M, Reichlin M. Heterogeneity of precipitating antibodies in polymyositis and dermatomyositis. Characterization of the Jo-1 antibody system. Arthritis Rheum 1980;23: 881--888. Nishikai M, Aoyagi S, Kanazawa N, Sato A. Indirect immunofluorescent staining pattern of myositis-autoantibody, the Jo1. Jap J Rheumatol 1990;2:245--250. Oddis CV, Medsger TA Jr, Cooperstein LA. A subluxing arthropathy associated with the anti-Jo-1 antibody in polymyositis/dermatomyositis. Arthritis Rheum 1990;33:16401645. O'Neill TW, Maddison PJ. Rheumatoid arthritis associated with myositis and anti-Jo-1 antibody. J Rheumatol 1993;20:141-143. Raben N, Borriello F, Amin J, Horowitz R, Fraser D, Plotz PH. Nucleic Acids Res 1992;20:1075--1081. Raben N, Nichols R, Dohlman J, McPhie P, Sridhar V, Hyde C, Left R, Plotz P. A motif in human histidyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigen epitope. J Biol Chem 1994 ;269:24277-24283. Reichlin M, Arnett FC Jr. Multiplicity of antibodies in myositis sera. Arthritis Rheum 1984;27:1150-1156. Rosa MD, Hendrick JP Jr, Lerner MR, Steitz JA, Reichlin M. A mammalian t-RNAHis-containing antigen is recognized by the polymyositis-specific antibody anti-Jo-1. Nucleic Acids Res 1983;11:853-870. Shi MH, Tsui FW, Rubin LA. Cellular localization of the target structures recognized by the anti-Jo-1 antibody: immunofluorescence studies on cultured human myoblasts. J Rheumatol 1991;18:252--258. Stahl NI, Klippel JH, Decker JL. A cutaneous lesion associated with myositis. Ann Intern Med 1979:91:577--579. Targoff IN. Autoantibodies to aminoacyl-transfer RNA synthetases for isoleucine and glycine. Two additional synthetases are antigenic in myositis. J Immunol 1990;144:1737--1743. Turner-Stokes L, Haslam P, Jones M, Dudeney C, Le Page S, Isenberg D. Autoantibody and idiotype profile of lung involvement in autoimmune rheumatic disease. Ann Rheum Dis 1990;49:160-162. Verheijen R, Salden M, van Venrooij WJ. Protein blotting. In: van Venrooij WJ, Maini RN, eds. Manual of Biological Markers of Diseases. The Netherlands: Kluwer Academic Publishers, 1993;A4:1-25. Wasicek CA, Reichlin M, Montes M, Raghu G. Polymyositis and interstitial lung disease in a patient with anti-Jo-1 prototype. Am J Med 1984;76:538--544.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

AMINOACYL-tRNA AUTOANTIBODIES

(OTHER

THAN

HISTIDYL)

SYNTHETASE

Ira N. Targoff, M.D.

University of Oklahoma Health Sciences Center, Veterans Affairs Medical Center, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA

HISTORICAL NOTES

Non-Jo-1 antisynthetases (aS) were found after recognition that the more common antibody, anti-Jo-l, was an aS. Originally described in 1980 as an immunodiffusion precipitin line strongly associated with polymyositis (PM), anti-Jo-1 was identified as antihistidyl-tRNA synthetase (HisRS) in 1983 (Mathews and Bernstein, 1983), based on immunoprecipitation (IP) of a specific tRNA (shown to be tRNA his) and specific inhibition of HisRS enzymatic activity. This led to testing of sera with unidentified precipitin lines that immunoprecipitated different tRNAs for the ability to inhibit other synthetases. The first non-Jo-1 aS was anti-PL-7 which was found to react with threonyltRNA synthetase (ThrRS), based on IP of a restricted set of tRNAs and a protein of compatible size, and specific inhibition of ThrRS but not other synthetases (Mathews et al., 1984). Serial testing for inhibition of each synthetase using sera that showed distinct, restricted sets of tRNAs by IP showed that anti-PL-12 reacted with alanyl-tRNA synthetase (AlaRS) (Bunn et al., 1986) and identified antibodies to glycyl-tRNA synthetase (GlyRS) ("anti-EJ") and isoleucyl-tRNA synthetase (IleRS) ("anti-OJ") (Figure 1) (Targoff, 1990). Unlike Jo-1, the non-Jo-1 aS were not reported as autoantibodies until they were known to be aS. A small number of other sera showed unique tRNAs by IP, but do not inhibit any synthetases. A remarkable feature of the aS is that antibodies to different members of the same enzyme family in different patients are associated with a similar clinical syndrome, characterized not only by myositis, but also by a markedly increased frequency of certain associated clinical features compared to other PM and

36

dermatomyositis (DM) patients. First noted with antiJo-1, each of the non-Jo-1 aS is individually associated with a similar syndrome that is referred to as the "antisynthetase syndrome".

THE AUTOANTIGEN Definition

Aminoacyl-tRNA synthetases are a set of cellular enzymes, each of which catalyzes the binding of one amino acid to its cognate tRNAs (formation of aminoacyl-tRNA) for incorporation into polypeptides (Mirande, 1991): R + tRNA R ~ R-tRNA R, in the presence of Mg ++ and enzyme, with ATP --+ AMP + PPi. The four primary non-Jo-1 antigenic synthetases are listed in Table 1. The letter designations were laboratory codes before the enzyme identifications (PL for precipitin line, "EJ" and "OJ" for prototype patients). One aminoacyl-tRNA synthetase, "gluprolyltRNA synthetase" (GluProRS), was found in Drosophila (and apparently all higher species) to serve as synthetase for both glutamic acid and proline (Cerini et al., 1991). The corresponding human protein, the 170 kd component of the synthetase complex, carries the two functions on separate parts of the molecule, each homologous to the respective independent bacterial enzymes. There are thus 19 synthetase polypeptides for the 20 amino acids. Two classes of synthetases have been distinguished (Moras, 1992). Those in Class II share up to three characteristic motifs and similarities in the positions and organization of structural features (motifs, helices, 13-sheet regions, etc.). These differ from the two short

Figure 1. Immunoprecipitation for nucleic acids from HeLa cells using antisynthetase sera. Immunoprecipitates were prepared by incubating protein A-Sepharose that was coated with patient sera with unlabeled HeLa cell extract, followed by phenol-extraction; they were then analyzed by 7M urea, 10% PAGE developed with silver stain. Sera used included: standard sera for anti-Jo-1, anti-PL-7, anti-PL-12, and anti-Sm; prototype sera from patients EJ and OJ (patient NJ also had anti-OJ); a serum with anti-Jo-1, anti-Ro/SSA, and anti-LA/SSB; and normal human serum (NHS). TNA - total nucleic acid. Each antisynthetase precipitates a recognizable set of tRNAs that can be distinguished from total tRNA and from other antisynthetases. (Targoff IN, 1990).

motifs and the three-dimensional structural features of the Class I synthetases. Classes also differ in the site of initial amino acid attachment on the tRNA. Despite their shared features and analogous activity, each must recognize the individual amino acid and its specific tRNAs, while excluding all others. The molecules

differ in size (Table 1, Figure 2), have very different primary sequences and are immunologically distinct. ThrRS, AlaRS, and GlyRS (and HisRS) are "Class II synthetases", and each is found free in the cytoplasm. IleRS is a Class I synthetase and is part of a multienzyme complex with synthetases for nine amino

37

Table 1. The Non-Jo-1 Antisynthetases Name

Antigen

PL-7

ThrRS

PL-12

AlaRS +tRNA ala

EJ OJ

Subunit MW

Frequency

Comment

80,000

<3 %

Reacts well by ID; IB usually negative

110,000

--3%

Reacts by CIE; direct IP of tRNA; lower frequency of myositis than other aS

GlyRS

75,000

<2%

Reacts well by IB but not ID; 1" DM

IleRS

150,000

<2 %

Does not react by IB or ID

170,000

<1%

Single molecule is synthetase for 2 amino acids; reacts only by IB - no inhibition

LysRS

70,000

<1%

Sera with antibody react by IB and inhibition

LeuRS

130,000

< 1%

Sera inhibit but do not blot

Multienzyme complex Other components of complex that may react: GluProRS

Figure 2. Immunoprecipitated proteins from 35S-labeled HeLa extract using anti-synthetase and anti-OJ sera (10% SDS-PAGE). a: AntiJo-1, anti PL-7, anti-PL-12 and anti-EJ all immunoprecipitate single proteins with molecular weights as in Table 1; all anti-OJ sera (prototype OJ serum, NJ serum, and sera A-G) immunoprecipitate the multienzyme complex of synthetases, that includes proteins a-j indicated at fight. Proteins a - h are synthetases (a = GluProRS; b = IleRS; c = LeuRS; d = MetRS; e = GlnRS; f = LysRS; g = ArgRS; h = AspRS) (Targoff IN et al., 1993). b: (next page). 38

Figure 2. b: Immunoprecipitation for nucleic acids using anti-OJ sera from HeLa extract (7M urea, 10% PAGE, developed with silver

stain). Sera are as in part A. Note that anti-OJ sera all precipitate the same set of tRNAs, despite the fact that other experiments show that some react with other components; for example, sera NJ, A, and F blot GluProRS, and serum F blots LysRS. Only serum E blots IleRS (see Figure 3). Serum B has co-existent anti-SS-A (Ro) and serum C anti-U1RNP (Targoff IN et al., 1993).

acids plus additional proteins (Mirande, 1991). All anti-OJ sera thus far reported show evidence of reaction with IleRS, but some also react with onethree other enzymes in the complex (LysRS, GluProRS, LeuRS and/or ArgRS) (Targoff et al., 1993) (Figure 3). Because the latter sera show the same tRNAs as other anti-OJ sera by IP (tRNA i~e) (Figure 2b) and always have anti-IleRS with their other activities, IleRS is considered the primary antigen for all anti-OJ sera described thus far, although one had a higher anti-LysRS than anti-IleRS titer. At present, any serum with the characteristic IP findings (the multienzyme complex and typical RNA) is considered to have anti-OJ (Figures 1 and 2). Although all aS immunoprecipitate specific tRNAs (Figure 1), most sera with antibodies to PL-7, OJ or EJ do not react with the tRNAs directly in the absence of the synthetases. The tRNAs are thought to precipitate due to their affinity for the synthetase antigen. However, almost all anti-PL-12 sera have

antibodies directed at tRNA ala, in addition to those directed at the enzyme itself, and precipitate tRNAalas in the absence of protein (Bunn et al., 1986; Bunn and Mathews, 1987) (Figure 4). All antisynthetases react well with native human antigen in IP. Anti-PL-7 sera did not react with ThrRS by immunoblot (IB), implying exclusive reaction with conformational epitopes (Dang et al., 1988). Nine of eleven anti-OJ sera failed to react with IleRS by IB; the evidence of reaction with IleRS derived from specific inhibition of the enzyme's function (Targoff et al., 1993). One serum in which anti-IleRS was detected by IB failed to inhibit IleRS. Of four sera in which antibodies to components of the complex other than IleRS were detected by IB, two in which antiLysRS was detected by IB also inhibited LysRS, but none in which anti-GluProRS was detected by IB inhibited either glutamic acid or proline charging. Five sera appeared to react with LeuRS by inhibition, but it was not detected by lB. Reaction of many anti-PL-

39

Figure 3. Immunoblot of anti-OJ sera against immunoprecipitates. Anti-OJ immunoprecipitates contain the multienzyme complex of synthetases, as in Figure 2a. The line marked "~IP" indicates which serum was used to prepare the immunoprecipitate to be used as antigen, the immunoblot was developed with the serum indicated in the line marked "\WB." Sera RH, GZ, JH, JS and GZ have anti-OJ by immunoprecipitation; NL = normal serum, Jo - anti-Jo-1 and P = protein stain. RH is itself negative by immunoblot, while GZ stains only a 140 kd protein that is not part of the complex (not seen in RH immunoprecipitates), thus representing a co-existing antibody. JH and JS stain components of the complex (seen in anti-OJ but not anti-Jo-1 immunoprecipitates). Comparison to the protein stain and an autoradiogram indicated that JH blotted bands a and f, and JS (serum E in Figure 2) band b (IleRS) (Targoff IN et al., 1993).

12 and all anti-EJ sera by IB suggests reaction with linear or renatured epitopes. Recombinant GlyRS is as sensitive as HeLa GlyRS in detecting anti-EJ by IB (Ge et al., 1994a); recombinant forms of other non-Jo1 synthetases have not been tested.

and the autoantibodies react with HEp-2 cell cytoplasm by indirect immunofluorescence. Mitochondria have their own forms of synthetases, but aS appear to react with the cytoplasmic forms.

Methods of Purification Origin, Sources, Organs, Tissue, Cells All cells must contain all synthetases to make proteins with all amino acids. HeLa cells are a good source of synthetases for IP and functional assays. Mammalian liver is a good tissue source for functional studies; calf liver and rabbit thymus can be used to test for anti-PL-7 by ID. The species specificity for non-Jo-1 aS is not well studied but at least includes bovine forms. The enzymes are predominantly cytoplasmic,

40

Purifications of mammalian forms of several synthetase antigens and the synthetase complex for biochemical studies are available (Mirande, 1991) using varying methods such as affinity for the tRNA (Pan et al., 1982). As an autoantigen, PL-7 (ThrRS) was substantially purified using biochemical and immunoaffinity methods (Targoff et al., 1988; Walker et al., 1989). IP results indicate that immunoaffinity purification is effective for other non-Jo-1 antigenic

Figure 4. Immunoprecipitation for nucleic acids from whole HeLa extract using anti-PL-12 sera (a-i). Lanes to the left of the total nucleic acid (TNA) lane used whole extract, and those to the right used deproteinized extract. Results with sera b--h and normal serum (NL) are shown with both extracts, "i" and "a" with deproteinized only (the whole-extract immunoprecipitate with a was lost). Immunoprecipitates were pheno-extracted and electrophoresed in 7M urea-10% PAGE, stained with silver stain. Almost all anti-PL-12 sera immunoprecipitate cognate tRNAs from both whole and deproteinized extracts, unlike most sera with other antisynthetases (or the anti-U1RNP in "b", "d", "g", anti-Sm in "d" and anti-Ro in "f"), which precipitate RNAs only from whole extract. Although no tRNA is seen with "c", this may be due to the low titer. Most anti-PL-12 sera immunoprecipitate less tRNA from deproteinized than from whole extract, such as sera "d", "e", "g" and "h", despite the availability of more tRNA demonstrated by "b" and "f' (Targoff IN and Arnett FC, 1990). synthetases; IP was used to enrich EJ (Ge et al., 1994a) and the synthetase complex (Targoff et al., 1993) and to purify IleRS (Nichols et al., 1995).

Commercial Sources Non-Jo-1 synthetase antigens are not available commercially.

Sequence Information cDNA cloning shows that: human ThrRS is 53% identical with the yeast form (Cruzen and Arfin, 1991); human GlyRS is 61.6% identical with silk moth (Ge et al., 1994a), and human IleRS has 53.5% sequence similarity with yeast (Nichols et al., 1995). ThrRS has all three Class II motifs, but GlyRS has

only 1 (motif 2). A GlyRS N-terminal helical region (amino acids 20--56) is very similar to a corresponding region of HisRS near its major epitope, despite the lack of cross-reaction of anti-EJ or anti-Jo-1. A similar sequence is found in the connecting region of GluProRS and in TrpRS. The B-cell epitopes of ThrRS, IleRS and AlaRS are not known. The tRNA ala epitope reacting with anti-PL-12 sera is a sequence of 7--9 bases including the anticodon loop (Bunn and Mathews, 1987). AntiPL-12 precipitates at least six different tRNAalas, but all have the IGC anticodon (part of the binding site) (Bunn and Mathews, 1987)" some tRNA ala forms are not precipitated. Using PCR-derived expressed fragments of GlyRS, all anti-EJ sera reacted with both Nterminal (amino acids 127--249) and C-terminal (385--685) regions (Ge et al., 1994b). The N-terminal

41

epitope appears to be different in position than the major HisRS N-terminal epitope. The C-terminal epitope might have a conformational aspect, because no subfragment reacted as well as the whole fragment.

AUTOANTIBODIES Pathogenetic Role Human Diseases. The role of non-Jo-1 aS in the pathogenesis of muscle, lung or other tissue injury is not known; that of anti-Jo-1 is also unknown, but they are likely to be similar. The correlation of anti-Jo-1 titer with disease activity suggests a possible role. aS can occur in either PM or DM, but if antibody is involved in myositis, it is more likely in DM. In DM, complement-mediated injury to small muscle blood vessels is the m~jor process (Targoff, 1993), but the reason for local complement activation is unknown. This picture is most consistently observed in juvenile DM, where aS are very rare, but this does not preclude a role for aS when present. The major mechanism for muscle injury in PM appears to be T-cell mediated, antigen-directed attack on muscle fibers (Plotz et al., 1995); complement deposition is not seen. Whatever the effects of aS, their production appears to relate to fundamental etiologic factors, rather than being secondary to muscle injury; the very high disease specificity, and their occurrence prior to or without detectable muscle involvement support this. They are often thought to be markers of the original inciting event, which may be viral or environmental (Bernstein, 1993). Animal Models. Mice with experimentally induced chronic graft vs. host disease produce antinuclear antibodies, including some associated with human autoimmune diseases such as anti-Sm. In one study (Gelpi et al., 1994), IP of a specific tRNA, with identical mobility to that of anti-OJ was apparently due to antibody to the synthetase complex. Seven of 10 mouse sera reacted by IB with a 76 kd protein consistent with LysRS (one of which weakly inhibited LysRS) and three weakly inhibited IleRS. The mice developed muscle disease, but it may relate to the GVH rather than the anti-OJ.

Genetics When considered as a group, aS are strongly associ-

42

ated with HLA DR3, with the supertypic marker DR52, and with DQAI*0501 or "0401, but the association with DR3 appears to be due to anti-Jo-1 (66-73% DR3) which dominates such analyses (Goldstein et al., 1990; Garlepp, 1993). Non-Jo-1 aS are still associated with DR52 (>90%), but not with DR3 (18--20%), although the small number of patients precludes definitive conclusions. Non-Jo-1 aS in more than one member of the same family has not been reported. Anti-PL-7 was found in a woman with Raynaud's and sclerodactyly for >10 years whose monozygotic twin was healthy without antibody (McHugh et al., 1994).

Factors Involved in Pathogenicity and Etiology Isotypes. In a study of one anti-PL-7 patient over time, samples during an initial disease episode had higher IgM than IgG, and all subsequent samples from the next year onward had more IgG than IgM, including during exacerbations (Walker et al., 1989). No other studies of relative isotype levels of non-Jo-1 aS are available.

ldiotypes. The finding of both anti-AlaRS and antitRNA ala in anti-PL-12 sera suggested a possible idiotype/anti-idiotype relationship. In support of this, anti-PL-12 sera absorbed with tRNA ala still precipitated AlaRS but not tRNA ala (suggesting that the antiAlaRS reacts with the tRNA-binding site, preventing IP of tRNA) (Bunn et al., 1986). It was hypothesized that aS might arise when tRNA-like structures on genomes of myositis-inducing viruses lead to antibodies whose binding site resembles the tRNAbinding site of the cognate synthetase, which also has affinity for the specific tRNAs (Plotz, 1983). Antiidiotypes might then bind to the synthetase. However, there is no direct evidence for this.

Epitopes. All auto-aS show IP of specific tRNAs and inhibition of enzyme function, properties that probably reflect binding to specific epitopes, suggesting a common mechanism of immunization for the auto-aS (Targoff, 1994). Indirect IP of tRNA by anti-PL-7, EJ and OJ implies sparing of the tRNA-binding site by at least a portion of the antibodies. Although anti-OJ does not react directly with tRNA lie, the tRNAs precipitated are predominantly tRNA ile, with little tRNA of other multienzyme complex components, suggesting that affinity of tRNA for enzyme is not enough for IP; auto-aS may enhance this affinity

(Targoff et al., 1993). These observations suggest that the immunogen may be an RNA-protein complex. Although anti-PL-12 sera react directly with tRNA ala, they precipitate more tRNA from whole extract than from deproteinized extract, suggesting that some tRNA ala is precipitated indirectly (Targoff and Arnett, 1990). Inhibition of enzyme activity suggests binding to functional sites, which tend to be conserved, possibly accounting for the lower frequency of inhibition by animal antisera. Inhibition of synthetases would interfere with protein synthesis, but this is not believed to be a mechanism for cell injury in vivo because of lack of access of antibody to enzymes.

Molecular Mimicry. Although frequently suggested, no significant evidence for molecular mimicry in the generation of non-Jo-1 aS has been found. Reports of tRNA-like structures on genomes of certain picornaviruses suggest that such structures on myositisinducing viruses may bind to cellular synthetases and produce an immunogenic complex (Mathews and Bernstein, 1983). Related viruses could theoretically have structures resembling different tRNAs, resulting in different aS in different patients, thus accounting for this unique feature of aS in myositis. However, there has been no evidence supporting this hypothesis. Reported shared sequences of bacterial HisRS and EMC virus (Walker and Jeffrey, 1988) were not found in human HisRS. Short, shared sequences of 6--7 amino acids between GlyRS and certain viruses were noted, but whether they are epitope regions is not known (Ge et al., 1994a). Low titer ELISA reactivity of one anti-PL-7 serum with muscle proteins was partially inhibited by purified ThrRS, suggesting some cross-reaction (Walker et al., 1989).

Polyclonal Activation. Elevated IgG was seen in 66% of anti-Jo-1, PL-7 or PL-12 patients (Marguerie et al., 1990), increased ESR in 79% and rheumatoid factor in 34%. Antbodies to SS-A (Ro) are more common with aS overall (Love et al., 1991) and were seen in 4 of 10 with anti-PL-12 (Targoff and Arnett, 1990); anti-U 1 RNP was found in three of 10. These associations may relate to MHC predisposition, connective tissue disease (CTD) overlap or other factors.

Cellular Autoimmunity. T-cell-mediated autoimmunity plays a central role in PM, but the antigen is unknown, and the relation of this to aS is unclear. Tcell receptor analysis of muscle infiltrating lymphocytes in anti-Jo-1 patients showed restricted clonality

for PM but not DM patients (Plotz et al., 1995); nonJo-1 aS patients have not been studied. This emphasizes that at least one aS can be associated with either the cell-mediated pathogenesis seen in PM or the humorally mediated DM. There are no studies of cellular immunity to non-Jo-1 synthetases reported.

Methods of Detection IP for nucleic acid and protein is currently the most sensitive and specific method for detection of the nonJo-1 aS. Each precipitates more than one tRNA band, giving a characteristic set of tRNAs by PAGE which, combined with the expected protein(s), unequivocally confirms specificity. Silver-stained as well as 32p_ labeled RNA gels can be used (Targoff et al., 1992; Targoff et al., 1993). Non-Jo- 1 aS precipitate intense protein bands from HeLa extract, increasing sensitivity. These tests are unfortunately complicated and time-consuming, decreasing their clinical utility. Most but not all anti-PL-7 sera are positive by ID against calf thymus extract (>90%), but availability of standard anti-PL-7 serum is limited by its low frequency. Counter-immunoelectrophoresis can detect anti-PL-7 (vs. rabbit thymus extract) and anti-PL-12 (vs. human spleen extract) (Marguerie et al., 1990). Antibodies to PL-12, OJ, or EJ have not been detected by ID against calf thymus extract. For anti-OJ, this may relate to the large size of the multienzyme complex, limiting diffusion. Lack of reaction of most anti-OJ sera with IleRS by IB makes this method unsuitable for detection. The anti-LysRS and anti-GluProRS in some anti-OJ sera do react by IB, but an extract enriched for the synthetase complex should be used (Targoff et al., 1993). Anti-EJ and often anti-PL-12 are positive by IB, but these are single bands that would need confirmation or purified antigen. Anti-PL-7 is often negative by IB (Dang et al., 1988), but at least one IB-positive serum was described (Walker et al., 1989). Because almost all sera with non-Jo-1 aS inhibit the antigenic enzyme, testing for this inhibition can be used to detect the antibodies (aminoacylation inhibition) (Targoff and Arnett, 1990; Marguerie et al., 1990). This is sensitive and specific, but less so than IP, in part due to nonspecific serum inhibition. It is easier and quicker than IP, but each antibody must be sought separately. ELISA for anti-PL-7 with purified antigen has been used (Targoff et al., 1988; Walker et al., 1989) but is not currently available. Recombinant human

43

GlyRS is an effective antigen to detect anti-EJ (Ge et al., 1994a), but is not yet commercially available. Recombinant forms of ThrRS and IleRS have not yet been tested as antigens and might not express the relevant epitopes as the antibodies often do not react by IB. Eventually, ELISA and/or IB with recombinant antigen is expected to be the most useful method for clinical detection of these antibodies.

CLINICAL UTILITY Application and Disease Associations The non-Jo-1 aS have much the same clinical implications as anti-Jo-1 (Miller, 1993). In a patient with evidence of muscle disease shown by muscle weakness, elevated CPK and compatible EMG, the finding of any aS would be extremely strong support for a diagnosis of PM or DM as opposed to other myopathies. In such a patient, the presence of an aS can establish the diagnosis without a muscle biopsy or despite a negative biopsy (seen in 10% of PM/DM) or an equivocal biopsy; this assumes an accurate antibody assay and exclusion of thyroid disease. Finding an aS is especially useful in PM, often a diagnosis of exclusion. The rash of DM makes diagnosis much easier, but an aS often adds confidence. These antibodies are of very low frequency even in myositis, and their absence is of no help in excluding PM and DM and should not be considered evidence against the diagnosis. A major role of aS is in defining a clinical subgroup of PM/DM, a picture marked by a high frequency of several clinical features, labeled the "antisynthetase syndrome" (Miller, 1993; Targoff, 1994). The major features of the syndrome are associated with all aS, including myositis, interstitial lung disease (ILD) (80%), arthritis/arthralgias (90%), Raynaud's phenomenon (60-90%) and mechanic's hands (70%) (Love et al., 1991; Marguerie et al., 1990). As in other CTDs, the interstitial lung disease can range in severity from subclinical to fatal (Targoff, 1994). The arthritis can lead to deformities similar to RA (Oddis et al., 1990). "Mechanic's hands" refers to a hyperkeratosis on the edges of the fingers resembling changes associated with manual labor. Some have found that sclerodactyly and sicca are also frequent (Marguerie et al., 1990). Compared with anti-Jo-1 patients, clinically significant myositis may be less frequent with anti-PL-12

44

and probably anti-OJ than with anti-Jo-l, but all nonJo-1 aS patients without myositis have had interstitial lung disease and/or arthritis (Targoff and Arnett, 1990; Friedman et al., 1994). Each non-Jo-1 aS has had a higher DM:PM ratio than anti-Jo-1; for example, five of six anti-EJ patients had DM in one study (Targoff et al., 1992). However, more patients with these antibodies must be studied to better distinguish these groups. The clinical picture may be confusing when myositis is absent, subclinical or mild, while other features of the syndrome are prominent. It may resemble other CTD syndromes (MCTD, undifferentiated CTD, lupus or overlap syndromes); finding an aS can help clarify the diagnosis. In patients with known myositis, the aS can alert the physician to look for other features of the syndrome. An aS-associated myositis is often more difficult to treat and control, and more likely to recur, than other myositis, especially that associated with anti-U1 RNP or anti-PM-Scl. Differences in responses to specific treatments are also seen (Joffe et al., 1993). Thus, aS testing may affect treatment decisions.

Patient Population aS are very rare in children. The few cases reported are atypical for juvenile DM, and resemble the antisynthetase syndrome (Rider et al., 1994). Females outnumber males overall by 2.7:1 (Love et al., 1991); nine of 10 anti-PL-12 patients were female in one study (Targoff and Arnett, 1990), and eight of 10 antiPL-12 or PL-7 patients in another (Marguerie et al., 1990). Based on small numbers, anti-PL-12 may be more common in African-American patients (seven of 10) (Targoff and Arnett, 1990) and anti-EJ in Asian patients (Targoff et al., 1992; Hirakata et al., 1992), but the aS are found in various ethnic groups. AntiPL-12 is considerably more common than anti-PL-7 in the U.S. Southwest, especially in a group from Houston (Targoff and Arnett, 1990), but anti-PL-7 was more common in the Northeast; these geographic differences in frequency could result from ethnic or genetic differences in the population or from endemic viruses or other regional environmental factors.

Frequency Non-Jo-1 aS are very uncommon, together accounting for 5--8% of adult myositis patients. The frequency may vary with referral patterns, since aS patients with

associated CTD features are more likely to be seen by rheumatologists than neurologists (especially when myositis is mild or absent). In the U.S., anti-PL-7 and anti-PL-12 (-2--3% of PM/DM) are slightly more common than anti-EJ and anti-OJ (-1--2%).

Correlation with Disease Activity Some studies have found a correlation of activity with anti-Jo-1 titer, and it is likely that other aS act similarly. As with anti-Jo-1, non-Jo-1 aS are always found in the earliest samples and generally persist throughout the disease course regardless of disease activity or therapy. Anti-PL-7 ELISA activity of one patient fell with treatment and rose before exacerbations (Walker et al., 1989). Persistent and apparently stable anti-EJ antibody was found over years in one patient (Targoff et al., 1992). There is no other information for nonJo-1 aS.

numbers of patients are needed to confirm these impressions. If one views the aS syndrome as a distinct entity that may have incomplete expression with only one or two manifestations (arthritis, ILD), then all aS, including anti-PL-12, have very high specificity for some form of this syndrome. The positive predictive value for myositis of a positive test for a non-Jo-1 aS parallels that of the specificity, because the specificity is so high, and the falsepositive rate of the currently used assays (IP) is so low. If ELISA is used in the future, false positives might become a problem, as the antibodies are exceedingly rare on a population basis. All non-Jo-1 aS have low sensitivity among myositis patients and among ILD patients. About 6 0 - 8 0 % of patients with PM/DM and ILD have aS, but the majority have antiJo-1, and each individual non-Jo-1 aS has low sensitivity.

Transplacental Transfer

CONCLUSION

No neonatal disease has been associated with aS. In one case, anti-PL-7-associated myositis developed during pregnancy with subsequent fetal loss and severe disease in the mother (Satoh et al., 1994).

The non-Jo-1 aS present one of the most puzzling pictures of autoantibodies in CTDs. Autoantibodies to different members of the same family of enzymes without cross-reaction or co-existence arise in different individuals who then go on to have the same unique syndrome, distinct from others with PM/DM or ILD. The antibodies are highly specific for this condition. The reasons for development of these antibodies has remained elusive, but this unusual picture suggests that they may hold clues to important etiologic mechanisms for these diseases. Despite their low frequency, aS can be very helpful clinically when found, because of their very high disease specificity. Current methods of detection limit their clinical utility, but recombinant forms of the proteins may allow more rapid and accessible testing in the near future. See also AMINOACYL-TRNA HISTIDYL (JO-1) SYNTHETASE AUTOANTIBODIES.

Sensitivity, Specificity Non-Jo-1 aS are not found in other forms of myopathy, indicating a very high specificity of aS for myositis among patients with myopathy. Among patients with autoimmune disease, the specificity of anti-PL-12 for clinically significant myositis is lower, in the 6 0 - 7 0 % range. Most reported patients with anti-PL-7 and anti-EJ have had myositis; their specificity is higher than anti-PL-12 among autoimmune disease patients. The specificity of anti-OJ may be closer to that of anti-PL-12 than anti-PL-7. Greater

REFERENCES Bernstein RM. Autoantibodies in myositis. Baillieres Clin Neurol 1993;2:599-615. Bunn CC, Bernstein RM, Mathews MB. Autoantibodies against alanyl-tRNA synthetase and tRNAAla coexist and are associated with myositis. J Exp Med 1986;163:1281-1291. Bunn CC, Mathews MB. Autoreactive epitope defined as the anticodon region of alanine transfer RNA. Science 1987;238: 1116--1119.

Cerini C, Kerjan P, Astier M, Gratecos D, Mirande M, Semeriva M. A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase. EMBO J 1991; 10:4266--4277. Cruzen ME, Arfin SM. Nucleotide and deduced amino acid sequence of human threonyltRNA synthetase reveals extensive homology to the Escherichia coli and yeast enzymes. J Biol Chem 1991;266:9919--9923. Dang CV, Tan EM, Traugh JA. Myositis autoantibody reactivity and catalytic function of threonyl-tRNA synthetase.

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FASEB J 1988;2:2376--2379. Friedman AW, Targoff IN, Arnett FC. Association of antibodies to aminoacyl-tRNA synthetases with lung disease without myositis. Arthritis Rheum 1994;37:$242. Garlepp MJ. Immunogenetics of inflammatory myopathies. Baillieres Clin Neurol 1993;2:579-597. Ge Q, Trieu EP, Targoff IN. Primary structure and functional expression of human Glycyl-tRNA synthetase, an autoantigen in myositis. J Biol Chem 1994a;269:28790-28797. Ge Q, Wu Y, Targoff IN. Analysis of epitope reactivity of autoantibodies to glycyl-tRNA synthetase. Arthritis Rheum 1994b;37:$351. Gelpi C, Martinez MA, Vidal S, Targoff IN, Rodriguez-Sanchez JL. Autoantibodies to transfer RNA-associated protein in a murine model of chronic graft versus host disease. J Immunol 1994;152:1989--1999. Goldstein R, Duvic M, Targoff IN, Reichlin M, McMenemy AM, Reveille JD, Warner NB, Pollack MS, Arnett FC. HLAD region genes associated with autoantibody responses to histidyl-transferRNA synthetase (JO-1) and other translationrelated factors in myositis. Arthritis Rheum 1990;33:12401248. Hirakata M, Mimori T, Akizuki M, Craft J, Hardin JA, Homma M. Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in Japanese patients with inflammatory muscle disease. Arthritis Rheum 1992;35:449--456. Joffe MM, Love LA, Left RL, Fraser DD, Targoff IN, Hicks JE, Plotz PH, Miller FW. Drug therapy of the idiopathic inflammatory myopathies: predictors of response to prednisone, azathioprine, and methotrexate and a comparison of their efficacy. Am J Med 1993;94:379--387. Love LA, Left RL, Fraser DD, Targoff IN, Dalakas M, Plotz PH, Miller FW. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups. Medicine 1991;70:360-374. Marguerie C, Bunn CC, Beynon HL, Bernstein RM, Hughes JM, So AK, Walport MJ. Polymyositis, pulmonary fibrosis and autoantibodies to aminoacyl-tRNA synthetase enzymes. Q J Med 1990;77:1019-1038. Mathews MB, Bernstein RM. Myositis autoantibody inhibits histidyl-tRNA synthetase: a model for autoimmunity. Nature 1983;304:177--179. Mathews MB, Reichlin M, Hughes GR, Bernstein RM. Antithreonyl-tRNA synthetase, a second myositis-related autoantibody. J Exp Med 1984;160:420-434. McHugh NJ, Harvey G, Whyte J, Dorsey K, Silman A. Autoantibodies segregate with disease in monozygotic twin pairs discordant for scleroderma: three further cases. Arthritis Rheum 1994;37:$261. Miller FW. Myositis-specific autoantibodies. Touchstones for understanding the inflammatory myopathies. JAMA 1993; 270:1846-1849. Mirande M. Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: structural domains and their implications. Prog Nucleic Acid Res Mol Biol 1991;40:95-142. Moras D. Structural and functional relationships between

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aminoacyl-tRNA synthetases. Trends Biochem Sci 1992;17: 59--164. Nichols RC, Raben N, Boerkoel CF, Plotz PH. Human isoleucyl-tRNA synthetase: sequence of the cDNA, alternative mRNA splicing, and the characteristics of an unusually long C-terminal extension. Gene 1995;155:299-304. Oddis CV, Medsger TA Jr, Cooperstein LA. A subluxing arthropathy associated with the anti-Jo-1 antibody in polymyositis/dermatomyositis. Arthritis Rheum 1990;33:1640-1645. Pan F, Lee HH, Pai SH, Lo KY. Purification and subunit structure studies of human placental threonyl-tRNA synthetase. Int J Pept Protein Res 1982;19:300--309. Plotz PH. Autoantibodies are anti-idiotype antibodies to antiviral antibodies. Lancet 1983;2:824--826. Plotz PH, Rider LG, Targoff IN, Raben N, O'Hanlon TP, Miller FW. Myositis: Immunologic contributions to understanding cause, pathogenesis, and therapy. Ann Intern Med 1995;122:715-724. Rider LG, Miller FW, Targoff IN, Sherry DD, Samayoa E, Lindahl M, Wener MH, Pachman LM, Plotz PH. A broadened spectrum of juvenile myositis. Myositis-specific autoantibodies in children. Arthritis Rheum 1994;37:1534-1538. Satoh M, Ajmani AK, Hirakata M, Suwa A, Winfield JB, Reeves WH. Onset of polymyositis with autoantibodies to threonyl-tRNA synthetase during pregnancy. J Rheumatol 1994;21:1564-- 1566. Targoff IN, Arnett FC, Reichlin M. Antibody to threonyltransfer RNA synthetase in myositis sera. Arthritis Rheum 1988;31:515--524. Targoff IN. Autoantibodies to aminoacyl-transfer RNA synthetases for isoleucine and glycine. Two additional synthetases are antigenic in myositis. J Immunol 1990; 144:1737-1743. Targoff IN, Arnett FC. Clinical manifestations in patients with antibody to PL-12 antigen (alanyl-tRNA synthetase). Am J Med 1990;88:241-251. Targoff IN, Trieu EP, Plotz PH, Miller FW. Antibodies to glycyl-transfer RNA synthetase in patients with myositis and interstitial lung disease. Arthritis Rheum 1992;35:821--830. Targoff IN. Humoral immunity in polymyositis/dermatomyositis. J Invest Dermatol 1993;100:S116--S123. Targoff IN, Trieu EP, Miller FW. Reaction of anti-OJ autoantibodies with components of the multienzyme complex of aminoacyl-tRNA synthetases in addition to isoleucyl-tRNA synthetase. J Clin Invest 1993;91:2556--2564. Targoff IN. Immune manifestations of inflammatory muscle disease. Rheum Dis Clin North Am 1994;20:857--880. Walker EJ, Jeffrey PD. Sequence homology between encephalomyocarditis virus protein VP1 and histidyl-tRNA synthetase supports a hypothesis of molecular mimicry in polymyositis. Med Hypotheses 1988;25:21--25. Walker EJ, Jeffrey PD, Webb J, Tymms KE. Polydermatomyositis with anti-PL-7 antibody: clinical and laboratory followup over a five year period. Clin Exp Rheumatol 1989;7:537-540.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES IN INFLAMMATORY BOWEL DISEASES Loren Karp Murphy, M.A. and Stephan R. Targan, M.D.

Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA

HISTORICAL NOTES

In the early 1980s, the presence of granulocytespecific antinuclear antibodies (a form of antineutrophil cytoplasmic antibodies; ANCA) was detected in the serum of patients with ulcerative colitis (Nielsen et al., 1983). By 1990, ANCA were reported in patients with Wegener's granulomatosis, polyarteritis nodosa, Churg-Strauss allergic granulomatosis, idiopathic crescentic glomerulonephritis, in addition to the inflammatory bowel diseases (IBD) (Jennette and Falk, 1994). The ANCA associated with Wegener's granulomatosis correlate directly with disease course and pathogenesis (van der Woude et al., 1985; Gross et al., 1991; Mulder et al., 1994). The ANCA associated with the inflammatory bowel diseases are proving useful as determinants of subgroups within IBD. Until very recently, the classic categorization of patients with IBD included Crohn's disease and ulcerative colitis (Podolsky, 1991).

THE AUTOANTIGENS Nomenclature

The nature of the antigens to which the P-ANCA associated with inflammatory bowel disease reacts is not yet known, and the topic is quite controversial. Examination of serum samples from P-ANCA-expressing patients for antimyeloperoxidase, antielastase and anticathepsin G activity shows that none of these are the target of the ulcerative colitis-related P-ANCA (Billing et al., 1995).

Through the use of electron microscopy, the antigen has been localized to the neutrophil nucleus, in association with the heterochromatin (Billing et al., 1995). This finding renders "ANCA" a misnomer, however, as no new nomenclature has been officially accepted, "ANCA" will continue to be used throughout this chapter. In further efforts to identify the nature of P-ANCA antigen recognition, P-ANCA sensitivity to DNase treatment was compared among patients with ulcerative colitis and other P-ANCA-expressing diseases (primary sclerosing cholangitis, type 1 autoimmune hepatitis). These experiments showed a loss of antigenic recognition after DNase digestion of neutrophils (Vidrich et al., in press). This finding supports the utility of subgrouping of disease for definition of distinct inflammatory processes. The loss of antigenic recognition was a dominant feature of the ulcerative colitis-specific P-ANCA. The same effect is not seen in the majority of sera from patients with primary sclerosing cholangitis or autoimmune hepatitis, thereby distinguishing the ulcerative colitis population. Progress in the effort to identify the antigen to which the P-ANCA of ulcerative colitis reacts is being made by the use of phage display combinatorial immunoglobulin libraries to produce clones of ulcerative colitis-related P-ANCA (Eggena et al., 1995). Attempts to define the antigen(s) to which ANCAs react using conventional technology have failed thus far. At present, phage display technology is being employed to clone and characterize the ulcerative colitis associated P-ANCA. Large Fab libraries are generated; antibodies of desired specificity can be selected from the library.

47

AUTOANTIBODIES Pathogenetic Role Whereas, the C-ANCA associated with Wegener's granulomatosis might be implicated in the pathogenic process, the role of P-ANCA in the mucosal inflammation of ulcerative colitis is not yet clear. B cells from disease-involved and uninvolved mucosa spontaneously produce P-ANCA in 60% of patients with ulcerative colitis, but not in normal subjects, those with diverticulitis, or active or inactive Crohn's disease. The overabundance of P-ANCA-secreting B cells in some UC mucosa could reflect their participation in a disease-related mucosal immune response that differs from the response in mucosa devoid of PANCA B-cell clones. Furthermore, P-ANCA were not produced by peripheral blood or mesenteric node lymphocytes from patients with ulcerative colitis. Furthermore, peripheral blood lymphocytes and mesenteric lymph node lymphocytes lacked spontaneous P-ANCA production, and P-ANCA production from peripheral blood lymphocytes was not inducible. The implication from these data is that P-ANCA may be induced in the mesenteric lymph nodes by antiCD40 and IL-4 generate a B cell specific response. B cells can function as antigen-presenting cells for Tcell activation. Following B-cell activation, T cells expressing activation markers proliferate and return to the mucosa, where local P-ANCA antigen (specific response) and other mucosal factors (nonspecific response, i.e., bacterial products) activate B cells and T cells to produce a local and poorly controlled T-cell response to P-ANCA antigen and a "spill over" of PANCA, allowing its detection in the peripheral blood. The cells then traffic back to the mucosa where local P-ANCA antigen (specific response) and other mucosal factors (nonspecific response) activate B cells and T cells to produce a local and poorly controlled T-cell response to P-ANCA antigen and a "spill over" of PANCA, allowing its detection in the peripheral blood. Thus, the site of disease-specific inflammation is also the site of homing by P-ANCA-producing B cells (Targan et al., 1995a).

Genetics P-ANCA are a marker of genetic susceptibility to ulcerative colitis. Early studies showed that the ulcerative colitis-associated P-ANCA was found not only in probands (68%), but also in family members

48

with no evidence of disease (15%) (Shanahan et al., 1992; Seibold et al., 1994). Other evidence of genetic susceptibility includes higher prevalence of disease in monozygotic twins (84%) versus dizygotic twins (18%) (Yang and Rotter, 1995), higher rates in firstdegree relatives (8--9%) versus environmental controls (5%) (Shanahan et al., 1992) and the association with human leukocyte antigens (HLA) (UC 41%; CD 27%). The ability to stratify patient subgroups based on the presence of ANCA and the HLA class II allele DR2 demonstrates that ulcerative colitis is a genetically heterogeneous disease (Yang et al., 1993) (Figure 1). Patients who do not express serum ANCA are associated with HLA class I allele DR4 (p = 0.004) (Yang et al., 1993). Because other studies reveal inconsistencies in the HLA associations in ulcerative colitis (Annese et al., 1995; Duerr et al., 1995), the cited differences are only tentative and might reflect variable study populations. Other data suggest subgroups based on presence/ absence of specific alleles of the intercellular adhesion molecule-1 (ICAM-1), which is required for T-cell activation; alleles of exon 4 and exon 6 of the ICAM1 gene differ in ANCA-positive versus ANCA-negative ulcerative colitis patients. In addition, in ulcerative colitis, ICAM-1, when stratified by ANCA expression is associated with different degrees or types of inflammation (ANCA-negative 16% versus ANCA-positive 6.6%; p = 0.047). (Yang et al., 1995). The gene for ICAM-1 is on the short arm of chromosome 19; investigators are focusing their efforts on this region in an attempt to elucidate the genetic links

Figure 1. HLA-DR2 allele frequency in ANCA-positive ulcerative colitis (UC) and ANCA-UC patients compared with ANCA-negative controls. ANCA-positive UC probands exhibited an increased frequency of DR2 (p = 0.0013), while ANCA-negative UC patients had the identical frequency of DR2 as controls.

in Crohn's disease and ulcerative colitis (Yang et al., 1995).

Methods of Detection Traditionally, ANCA are detected by the use of standard indirect immunofluorescence assays with microscopy to determine staining patterns. In ANCApositive ulcerative colitis patients, 72% of serum samples studied expressed IgG1. Using indirect immunofluorescence, these samples stained in a perinuclear pattern using alcohol-fixed neutrophils (Targan et al., 1995b). Enzyme-linked immunosorbent assay (ELISA) is also used with an indirect immunofluorescence procedure for confirmation (Targan et al., 1995a; Seibold et al., 1994). The use of the methods described below offer a far greater degree of objectivity in analysis and permit the determination of titers of these autoantibodies. Using ELISA for ANCA titer measurements is easy as it is simply provided in the read out. The lack of baseline and endpoint data inhibit the utility of IIF (Farrant, unpublished data; Targan et al., 1995a). For example, using formalinbased fixation in the indirect immunofluorescence assay could result in loss of reaction, or obscured interpretation to artifact-transformed staining, rendering it impossible to differentiate among C-ANCA and what is actually a positive P-ANCA. It has been previously shown that when neutrophils are fixed by non-alcohol-based reagents (e.g., paraformaldehyde or formalin), the perinuclear reaction obtained with myeloperoxidase- or elastase-reactive P-ANCA is abolished and converted to a more cytoplasmic reaction pattern (Vidrich et al., 1995). When using neutrophils as a substrate for enzyme immunoassay in the presence of anti-DNA antibodies, it is possible that any antinuclear antibody will be detected and counted as ANCA. Although some binding of ANA does occur (as seen in immunofluorescence), it is not a significant proportion of the total bound antibodies, since no correlation exists between titers of ANCA and titers of ANA (Targan et al., 1995a). In the ELISA system, use of a starting dilution at 1:100 eliminates low-titer sera which are usually scored as positive at 1:10 dilution in indirect immunofluorescence assays. Stratification on the basis of ANCA titer as determined by ELISA allows examination of more homogenous groups of patients based on ANCA production. As has been shown, high titer ANCA is positively associated with Type 1 autoimmune hepatitis (11,410 _+ 1875 (p < 0.001) as compared to lower

titers with primary sclerosing cholangitis (dot plots are published in reference Targan et al., 1995b). It is becoming increasingly apparent that patient stratification on the basis of ANCA titer is important since this parameter probably distinguishes between two populations of ulcerative colitis patients with ANCA, i.e., those with a high titer and those patients with a low titer. A fixed neutrophil ELISA is used to determine ANCA expression. Microtiter plates are coated with 2.5 x 105 neutrophils per well and treated with 100% methanol to fix the cells. Cells are incubated with bovine serum albumin (0.25%) in phosphate-buffered saline to block nonspecific antibody binding. Next, sera are added at 1:100 dilution to the bovine serum/ phosphate-buffered saline blocking buffer. Alkaline phosphatase conjugated goat F(ab') 2 antihuman immunoglobulin G (y-chain specific) antibody is then added at 1:1000 dilution to label neutrophil-bound antibody. Substrate solution containing p-nitrophenol phosphate is then added. Color development is allowed to proceed until absorbance at 405 nm in the positive control wells was 0.8--1.0 optical density units greater than that in blank wells. Results are expressed as percent of positive-standard binding. Positive is defined as >2 standard deviations above mean of control. Titers are also determined (Saxon et al., 1990). It is not possible at present to derive a standard, therefore, the use of titer measurements and results calculated at percent of positive control is the best result. Industry standards for detection of antibody are not based on a standard curve. Indirect immunofluorescence staining is performed on ANCA-expressing samples to determine the predominant staining pattern; i.e., perinuclear or cytoplasmic. Glass slides containing approximately 100,000 neutrophils per slide are prepared by cytocentrifugation. Slides are fixed in 100% methanol, air dried and stored a t - 2 0 ~ Coded sera are diluted (1:20). The reaction is visualized with fluoresceinlabeled F(ab')2 (y chain-specific) antibody. The slides are examined using an epifluorescence-equipped microscope (Saxon et al., 1990).

C L I N I C A L UTILITY

Application Traditionally, differentiating among the inflammatory bowel diseases has been very difficult, due to many

49

clinical similarities. Arriving at a diagnosis of either Crohn' s disease or ulcerative colitis was dependent on groupings of physical, endoscopic, radiologic, histopathologic and historical criteria. Diagnosis could literally take years, and in some patients could not be made at all. At the most fundamental level, presence of P-ANCA in the serum is now considered by many to be diagnostic for ulcerative colitis. The combination of a positive value in the ELISA and a perinuclear IIF staining pattern is 60% sensitive and 95% specific for ulcerative colitis. Disease Association

Serum ANCA associated with autoimmune disease are divided into three categories, as determined by staining patterns observed upon indirect immunofluorescence microscopy. For example, the ANCA typically associated with Wegener's granulomatosis, polyarteritis nodosa, idiopathic crescentic glomerulonephritis and pulmonary renal syndrome has a distinct granular cytoplasmic fluorescent staining pattern (C-ANCA). In addition to being commonly found in necrotizing vasculitis, type 1 autoimmune hepatitis and primary sclerosing cholangitis ANCA with perinuclear neutrophil staining patterns (P-ANCA) are used to identify subgroups of ulcerative colitis and Crohn's disease (Lesavre et al., 1993). Ulcerative colitis-associated PANCA recognize only neutrophils. Although the perinuclear nature of some ANCA reactions is an artifact of the alcohol fixation of neutrophils, which causes cytoplasmic granules to redistribute around the nucleus, the ulcerative colitis P-ANCA reaction has been found not to be similarly affected. The ulcerative colitis P-ANCA reaction is actually within the nucleus, localized primarily over the chromatin (Billing et al., 1995). Among patients with ulcerative colitis, serum P-ANCA are found in 60--80%; P-ANCA are also present in the serum of 10--20% of patients with Crohn's disease (Kallenberg et al., 1992). In patients with gastrointestinal complaints, P-ANCA are highly specific for ulcerative colitis, being absent in a variety of other colitides or diarrheal conditions (Duerr et al., 1991). Unlike the C-ANCA of Wegener' s granulomatosis, titers of P-ANCA do not correlate with disease activity or duration (Saxon et al., 1990; Duerr et al., 1991; Oudkerk Pool et al., 1993). Associations with HLA types and genetic markers further subdivide the group of patients with ulcerative colitis and Crohn's disease. Presence of serum P-ANCA has been used in

50

association with specific clinical findings to identify subgroups of patients with ulcerative colitis and Crohn's disease. For example, in ulcerative colitis, PANCA was found to be present in 100% of patients following ileo pouch anal anastomosis (Sandborn et al., 1995a). P-ANCA also has been associated with a severe and treatment-resistant disease course and leftsided disease distribution (Sandborn et al., 1995b). Young age at disease onset (26.7 + 12.2 years vs. 35.0 + 12.4 years) and requirement for surgery (PANCA-positive: 5.5 + 6.3; P-ANCA-negative 16.3 + 7.3) early in the course of ulcerative colitis are also associated with P-ANCA expression (Boerr et al., 1995). In Crohn's disease, presence of P-ANCA seems to represent a distinct subpopulation of patients with a number of traits generally considered to be characteristic of ulcerative colitis. One hundred percent of the population of Crohn's disease patients who express P-ANCA exhibit ulcerative colitis-like features. Although diagnosis of Crohn's disease was based on clinical, laboratory and histopathologic criteria, they also had typical characteristics of ulcerative colitis, such as left-sided colitis, crypt abscesses and predominantly superficial inflammation. These data suggest that Crohn's disease patients expressing P-ANCA represent a subpopulation of patients with an ulcerative colitis/Crohn's disease overlap syndrome (Vasiliauskas et al., 1995). Indeed, evidence from these recent studies suggests that PANCA in Crohn's disease may represent a distinct disease subgroup (Vasiliauskas et al., 1995).

CONCLUSION

The existence of subgroups of pathogenically distinct inflammatory bowel diseases, if confirmed by subclinical (genetic and/or immunologic markers) and clinical markers, might permit tailored therapeutic regimens, targeted at selected points in the pathogenic process. The reverse approach, i.e., matching clinical manifestations (including response to certain therapies) to define subgroups and suggest effective treatment has traditionally been employed with success; such correlation studies are underway in inflammatory bowel disease. Genetic and subclinical marker studies of ulcerative colitis and Crohn's disease will continue to elucidate the ongoing puzzles of disease manifestation and diagnosis. In inflammatory bowel diseases, standard therapy is directed against the final stages of

nonspecific inflammation. The treatment does not alter the natural history of relapse and remission of the disease and is based predominantly on anti-inflammatory properties. Stratification of patient subgroups by various markers hopefully will permit the creation of a cadre of homogeneous patient groups; this should expedite the process of investigation of novel therapeutic approaches because patients will have been preselected based on specific parameters associated a

priori with

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diagnosis of inflammatory bowel disease. Acta Pathol Microbiol Immunol Scand 1983;91:23--26. Podolsky DK. Inflammatory Bowel Disease. N Engl J Med 1991 ;325:1008--1016. Oudkerk Pool MO, Ellerbroek PM, Ridwan BU, Goldschmeding R, von Blomberg BM, Pena AS, Dolman KM, Bril H, Dekker W, Nauta JJ, Gans RO, Breed H, Meuwissen SG. Serum antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease are mainly associated with ulcerative colitis. A correlation study between perinuclear antineutrophil cytoplasmic autoantibodies and clinical parameters, medical, and surgical treatment. Gut 1993;34:46-50. Sandborn WJ, Landers CJ, Tremaine WJ, Targan SR. Antineutrophil cytoplasmic antibody correlates with chronic pouchitis after ileal pouch-anal anastomosis. Am J Gastroenterol 1995a;90:740-747. Sandborn WJ, Landers CJ, Steiner BL, Tremaine WJ, Targan SR. Unexpectedly high prevalence of antineutrophil cytoplasmic antibody in patients with treatment resistent, left-sided ulcerative colitis. Mayo Clin Proc 1995b;(in press). Saxon A, Shanahan F, Landers C, Ganz T, Targan SR. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. J Allergy Clin Immunol 1990;86:202-210. Seibold R, Slametschka D, Gregor M, Weber P. Neutrophil autoantibodies: a genetic marker in primary sclerosing cholangitis and ulcerative colitis. Gastroenterology 1994; 107: 532--536. Shanahan F, Duerr RH, Rotter JI, Yang H, Sutherland LR, McElree C, Landers CJ, Targan SR. Neutrophil autoantibodies in ulcerative colitis: familial aggregation and genetic heterogeneity. Gastroenterology 1992; 103:456-461. Targan SR, Landers CJ, Cobb L, MacDermott RP, Vidrich A. Perinuclear antineutrophil cytoplasmic antibodies are spontaneously produced by mucosal B cells of ulcerative colitis patients. J Immunol 1995a;155:3262--3267. Targan SR, Landers C, Vidrich A, Czaja AJ. High-titer antineutrophil cytoplasmic antibodies in type-1 autoimmune hepatitis. Gastroenterology 1995b; 108:1159-1166. Van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van Es IA, van der Giessen M, van der Hem GK, Teh TH. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 1995; 1:425. Vasiliauskas EA, Plevy SE, Ferguson DM, Vidrich A, Landers

Annese V, Piepoli A, Napolitano F, Perri F, Caruso N, Ciavarella G, Andriulli A. Are HLA and pANCA markers of genetic heterogeneity in ulcerative colitis? Gastroenterology 1995;108:A771. Billing P, Tahir S, Gagne G, Calfin B, Cobb L, Targan SR, Vidrich A. Nuclear localization of the ulcerative colitis specific perinuclear antineutrophil cytoplasmic antibody (pANCA) reactive antigen. Am J Pathol 1995;147:979-987. Boerr LA, Sambuelli AM, Katz S, Sole L, Gil A, Gonealves S, Negreira Bautista S, Camartino G, Bai JC, Pena AS. Clinical heterogeneity of ulcerative colitis in relation to frequency of pANCA activity. Gastroenterology 1995;108:A785. Duerr RH, Targan SR, Landers CJ, Sutherland LR, Shanahan F. Antineutrophil cytoplasmic antibodies in ulcerative colitis. Comparison with other colitides/diarrheal illnesses. Gastroenterology 1991; 100:1590-- 1596. Duerr RH, Neigut DA. Molecularly defined HLA-DR2 alleles in ulcerative colitis and antineutrophil cytoplasmic antibodypositive subgroup. Gastoenterology 1995; 108:423--427. Eggena M, Targan SR, Vidrich A, Braun J. Phage display cloning and characterization of an immunogenetic marker (pANCA) in ulcerative colitis. Gastroenterology 1995;108: A815. Gross WL, Csernok E, Schmitt WH. Antineutrophil cytoplasmic autoantibodies: immunobiological aspects. Klin Wochenschr 1991;69:558-566. Jennette JC, Falk RJ. The coming of age of serologic testing for antineutrophil cytoplasmic autoantibodies. Mayo Clin Proc 1994 ;69:908--910. Kallenberg CG, Mulder AH, Tervaert JW. Antineutrophil cytoplasmic antibodies: a still-growing class of autoantibodies in inflammatory disorders. Am J Med 1992;93:675--682. Lesavre P, Noel LH, Gayno S, Nusbaum P, Reumaux D, Erlinger S, Grunfeld JP, Halbwachs-Mercarelli L. Atypical autoantigen targets of perinuclear antineutrophil cytoplasm antibodies (P-ANCA): specificity and clinical associations. J Autoimmun 1993;6:185--195. Mulder AH, Broekroelofs J, Horst G, Limburg PC, Nelis GF, Kallenberg CG. Antineutrophil cytoplasmic antibodies (ANCA) in inflammatory bowel disease: characterization and clinical correlates. Clin Exp Immunol 1994;95:490-497. Nielsen H, Wiik A, Elmgreen J. Granulocyte specific antinuclear antibodies in ulcerative colitis. Aid in differential

likelihood of response. By the same token, subgroups of patients unlikely to respond will be eliminated from unnecessary and fruitless investigation. See also A N C A WITH SPECIFICITY FOR MYELOPEROXIDASE, A N C A WITH SPECIFICITY FOR PROTEINASE 3, A N C A WITH SPECIFICITY OTHER THAN PR3 AND M P O (X-ANCA) and GRANULOCYTE-SPECIFIC ANTINUCLEAR ANTIBODIES.

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C, Targan SR. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn's disease define a clinical subgroup. Gastroenterology 1995; 108:935. Vidrich A, Lee J, James E, Cobb L, Targan SR. Segregation of pANCA antigenic recognition by DNase treatment of neutrophils: ulcerative colitis, type 1 autoimmune hepatitis and primary sclerosing cholangitis. J Clin Immunol 1995;(in press). Yang H, Rotter JI, Toyoda H, Landers C, Tyran D, McElree CK, Targan SR. Ulcerative colitis: a genetically heterogenous disorder defined by genetic (HLA class II) and subclinical

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(antineutrophil cytoplasmic antibodies) markers. J Clin Invest 1993 ;92:1080-- 1084. Yang H, Rotter J. Genetics of inflammatory bowel disease. In: Targan S, Shanahan F, eds. Inflammatory Bowel Disease: From Bench to Bedside. Baltimore: Williams and Wilkins, 1995:32-64. Yang H, Vora DK, Targan SR, Toyoda H, Beaudet AL, Rotter JI. Intercellular adhesion molecule 1 gene associations with immunologic subsets of inflammatory bowel disease. Gastroenterology 1995; 109:440--448.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

A N T I N E U T R O P H I L C Y T O P L A S M I C A U T O A N T I B O D I E S WITH SPECIFICITY FOR M Y E L O P E R O X I D A S E Cees G.M. Kallenberg, M.D.

Department of Clinical Immunology, University Hospital of Groningen, 9700 RB Groningen, The Netherlands

HISTORICAL NOTES

THE AUTOANTIGEN

Although first described in 1982 in a few patients with segmental necrotizing glomerulonephritis (Davies et al., 1982), antineutrophil cytoplasmic antibodies (ANCA) were recognized in 1985 as a sensitive and specific marker for Wegener's granulomatosis (WG) (Van der Woude et al., 1985). ANCA detected by indirect immunofluorescence (IIF) on ethanol-fixed neutrophils produced a cytoplasmic fluorescence pattern with accentuation of the fluorescence intensity in the area within the nuclear lobes (Figure 1). The antigen recognized by most sera that produce such a cytoplasmic staining pattern (C-ANCA) is now known to be proteinase 3 (Ltidemann et al., 1990). During the routine screening of sera for the presence of ANCA, it became apparent that some sera produce a perinuclear to nuclear fluorescence pattern (P-ANCA) clearly different from the C-ANCA pattern) (Falk and Jennette, 1988) (Figure 1). During the First International Workshop on ANCA held in January, 1988 in Copenhagen, many P-ANCA-positive sera were shown to be directed against myeloperoxidase (MPO) (Falk and Jennette, 1989). During the same workshop, antibodies to human leukocyte elastase were also shown to produce a P-ANCA fluorescence pattern (Goldschmeding et al., 1989). The presence of antibodies to MPO (anti-MPO) is associated with idiopathic or vasculitis-associated necrotizing crescentic glomerulonephritis (Falk and Jennette, 1988). That anti-MPO might have pathogenic potential is supported by their capacity to further activate primed neutrophils in vitro (Falk et al., 1990) and by their presence in animals with experimentally induced glomerulonephritis (Brouwer et al., 1993) and vasculitis (Mathieson et al., 1992).

Definition

MPO, an enzyme from the azurophilic granules of neutrophils, catalyzes the peroxidation of chloride into hypochlorite according to the following equation: H20 2 + C1-+ H30 + ~ HOC1 + 2H20 (Weiss, 1989) Because of its green color, which is responsible for the color of purulent secretions, the enzyme was originally designated "verdoperoxidase" (Agnes, 1941). The functional significance of the MPO is twofold. First, the generation of hypochlorite is important for the intracellular killing of phagocytosed microorganisms within the phagosomes after phagosomelysosome fusion. Secondly, hypochlorite inactivates protease inhibitors and, as such, allows lytic enzymes released from neutrophils to degrade tissues and foreign material in the vicinity of neutrophils (Weiss, 1989). Deficiency of MPO, which occurs in one out of 2,000 to 4,000 individuals, generally does not result in severe infections or other clinical symptoms; apparently the enzyme is not of major importance for survival (Rosen et al., 1995). MPO is present in cells of the myeloid lineage from the stage of the promyelocyte and is abundantly present in neutrophils, constituting almost 5% of their total protein content. MPO, which has a molecular weight of approximately 140 kd, is a homodimer that can be split into two halves which still have enzymatic activity. These hemi-MPO monomers consist of a 59 kd s-chain and a 13.5 kd ~-chain which, together with some lower molecular weight degradation products, can be demonstrated by immunoblotting and immunoprecipitation (Figure 2). The highly cationic 53

Figure 1. Staining of cytoplasmic components of fixed neutrophils by indirect immunofluorescence using a serum sample from a patient with active Wegener's Granulomatosis and antibodies to proteinase 3. A characteristic granular pattern of fluorescence (CANCA) is seen (a). This fluorescence pattern is different from the perinuclear pattern that can be produced by serum samples from patients with anti-MPO antibodies (P-ANCA) (b). 54

suggests a restricted number of conformational epitopes reactive with human autoantibodies as shown by inhibition studies using mouse monoclonal and human autoantibodies. Recombinant MPO as expressed in hamster ovary cells (Moguilevsky et al., 1991) is enzymatically active and is recognized by MPOspecific, ANCA-positive sera.

Methods of Purification MPO can be isolated from human peripheral blood polymorphonuclear cells (PMN) (Merrill, 1980; Brouwer et al., 1994). Separated from whole blood on Lymphoprep density gradients, purified PMN (75 x 109) in 300 mL buffer (100 mM KC1, 3 mM NaC1, 1 mM ATP, 3.5 mM Mg C12, and 10 mM PIPES, pH 7.3) are disrupted by nitrogen cavitation. After removal of nuclei and unbroken cells by centrifugation at 500 x g for 10 min at 4~ the supernatant is collected and centrifuged for 30 min at 35,000 x g at 4~ The pellet which contains the mixed granule fractions is suspended in PBS --0.1% (v/v) Triton X100 and sonicated at 45 kHz for three periods of 10 seconds followed by centrifugation at 220,000 x g for 1 hr at 4~ The supernatant after dialysis against PBS, is applied to matrix gel orange A column from which proteinase 3 is purified by dye-ligand affinity chromatography; MPO in the flow through of the Matrix Gel Orange A is absorbed to a Con A Sepharose gel and eluted with o~-methyl-D mannoside. Eluted fractions with a ratio (OD 428 nm/280 nm) >0.7 are pooled and extensively dialyzed against sodium acetate buffer pH 4.7 containing 0.05% cetyltrimethyl ammonium bromide followed by further purification on a sephadex G150 gel. Fractions with ratio (OD 428 nm/280 rim) >0.8 are pooled. By gelelectrophoresis, this preparation shows only bands specific for MPO (at 15, 39 and 58 kd).

Figure 2. Immunoprecipitation of myeloperoxidase (MPO) by a mouse monoclonal antibody to MPO (left, lane a) and an MPO-antibody-positive serum (right, lane b). charge of MPO (isoelectric point higher than I l) may be relevant for its localization at anionic structures such as the glomerular basement membrane (GBM) (Brouwer et al., 1993). The 3-dimensional structure includes five central o~-helices which are surrounded by polypeptides (Zeng and Fenna, 1992). Although not yet fully characterized, mapping of the molecules

Commercial Sources MPO is also commercially available. Some commercial preparations may contain lactoferrin (Esnault et al., 1993).

THE AUTOANTIBODIES Terminology Most sera from patients with idiopathic or vasculitis-

55

associated necrotizing and crescentic glomerulonephritis (NCGN) yield a P-ANCA pattern which reflects the presence of antibodies to MPO (Falk and Jennette, 1988). However, P-ANCA are certainly not synonymous with anti-MPO; indeed, in one study only 12% (50/424) of P-ANCA-positive sera contained antibodies to MPO (Cohen Tervaert et al., 1990). Thus, the preferred terminology distinguishes antiMPO from P-ANCA, because the latter can be due to antibodies reactive with autoantigens other than MPO, e.g., lactoferrin (Kallenberg et al., 1992). Pathogenetic Role Human Model. In contrast to the multiple studies which generally suggest that concentrations of antibodies to proteinase 3 (anti-PR3) fluctuate in relation to disease activity in WG, the relation between disease activity and fluctuations in amounts of anti-MPO is not well established. In the few studies available, antiMPO do tend to fluctuate with changes in disease activity in some 70% of the patients (Cohen Tervaert et al., 1990; Kyndt et al., 1995). IgG preparations from anti-MPO-positive sera can further activate primed neutrophils to produce reactive oxygen species and to release lysosomal enzymes which also supports a pathogenic role of anti-MPO in the diseases with which they are associated (Falk et al., 1990). Priming of neutrophils with low doses of proinflammatory cytokines such as TNF-c~, results in surface expression of lysosomal enzymes, including MPO, and resultant accessibility to the corresponding antibodies. Binding of anti-MPO induces neutrophil activation only in the presence of the total IgG molecule, including the Fcfragment (Mulder et al., 1994). There are three Fcreceptors of IgG: Fc 7 RI (CD64), Fc 7 RII (CD 32), Fc 7 Rill (CD 16). Blocking of the second Fc-receptor (CD 32) on neutrophils by nonactivating monoclonal antibodies inhibits their activation by anti-MPO (Mulder et al., 1994). In addition, because neutrophil activation by anti-MPO only occurs when neutrophils adhere to a surface and not when they are kept in suspension, neutrophil activation in vivo might take place only at the surface of the endothelial cell. At sites of local inflammation, neutrophils may adhere to up-regulated endothelial cells that express adhesion molecules such as E-selectin and intercellular adhesion molecule-1 (ICAM-1). These adherent cells might be further activated by anti-MPO. Indeed, in vitro primed neutrophils in the presence of anti-MPO can lyse endothelial cells in culture (Savage et al.,

56

1992; Ewert et al., 1992). Animal Model. Injection of rabbit antirat MPO into rats and induction of an immune response to human MPO in rats, whether or not with cross-reactivity to rat MPO, do not cause lesions suggestive of vasculitis (Brouwer et al., 1993; Yang et al., 1994). However, the products of activated neutrophils such as MPO, H20 2 and lytic enzymes, when perfused into the left kidney of rats immunized with MPO, produce a pauciimmune NCGN similar to that of humans with antiMPO (Brouwer et al., 1993). This model shows that the immune response to MPO alone is not sufficient for the induction of vasculitis/glomerulonephritis but that a second signal, in particular one that results in neutrophil activation, is additionally required. Finally, a polyclonal autoimmune response including antibodies to MPO, among others, develops in BrownNorway rats treated with mercuric chloride; a necrotizing vasculitis, particularly involving the gut, develops in some animals possibly in conjunction with a microbial infection (Mathieson et al., 1992). Potential pathogenic roles of ANCA including MPO-ANCA are further discussed elsewhere (Kallenberg et al., 1994; 1995; Jennette, 1994). Genetics and Factors in Pathogenicity Little is known about the induction of anti-MPO. Only weak HLA-associations are described for the ANCAassociated diseases. In WG, persistent ANCA-positivity is associated with DR2; whereas, patients only transiently positive for A N C A are more likely to have DQ7 (Spencer et al., 1992). A decreased frequency of DR6/DR13 is reported in ANCA-associated vasculitis without any difference between anti-PR3- and antiMPO-positive patients (Hagen et al., 1995). Although these MHC class II associations point to antigenspecific T-cell involvement in the induction of ANCA, data about T-cell reactivity to MPO are not convincing (Mathieson and Oliveira, 1995). Whether exogenous antigens, by way of molecular mimicry or as superantigens, play a role in the induction of the MPO-directed autoimmune response, is unknown. Interestingly, several studies show, that although IgG1 and IgG3 subclasses are present, the IgG4 subclass of anti-MPO also is prominently present (Brouwer et al., 1991), as might be consistent with repeated antigenic stimulation of a T-cell dependent immune response. IgM- and IgA-class antibodies to MPO have not been studied in detail and do not seem to have clinical

utility. No consistent data are presently available concerning changes in epitope specificity, avidity or idiotypes that occur during the course of the disease in patients with anti-MPO. Methods of Detection

When ANCA are detected by indirect immunofluorescence on ethanol-fixed leukocytes (Wiik, 1989), the presence of lymphocytes in the preparation is important for enabling the distinction between antinuclear antibodies (ANA) which do stain lymphocytes and ANCA which do not stain lymphocytes. When both ANA and anti-MPO are present, the use of paraformaldehyde-fixed neutrophils may allow the distinction between ANA and anti-MPO as ANA will still stain nuclei; whereas, anti-MPO will produce a cytoplasmic staining pattern (Mulder et al., 1993). Anti-MPO generally produce a perinuclear to nuclear fluorescence pattern, but exceptions do occur. As a result every positive test for ANCA by IIF should be followed by antigen-specific assays, e.g., for antibodies to MPO. Among several ELISAs used to measure anti-MPO, a capture ELISA in which a monoclonal antibody to MPO is used to catch MPO from a crude extract of neutrophils (Cohen Tervaert et al., 1990) has merit, but at present, most laboratories use an ELISA in which the purified, commercially available antigen is directly coated according to standard procedures. It should again be noted that some of these commercial preparations are contaminated with lactoferrin (Esnault et al., 1993).

C L I N I C A L UTILITY Disease Association

First described in patients with necrotizing and crescentic glomerulonephritis (NCGN) without immune deposits (pauci-immune), the clinical spectrum associated with anti-MPO includes some patients with idiopathic NCGN without signs of extrarenal disease and others with NCGN associated with systemic vasculitis, either WG or a form of vasculitis in which small vessels are involved without granuloma formation (Falk and Jennette, 1988). The latter condition is called "microscopic polyangiitis (MPA)" according to the definitions for the primary vasculitides as formulated by the Chapel Hill Consensus Conference (Wiik, 1989) (Table 1). Indeed, anti-MPO are detectable in 65% of patients with idiopathic NCGN, 45% of patients with MPA and 10% of patients with WG (Table 2). Most of the remaining patients with the aforementioned diseases are positive for anti-PR3 (Table 2). In general, anti-MPO and anti-PR3 do not occur in the same patient concurrently. Antibodies to bactericidal-permeability increasing protein (BPI) can be found in patients with WG and MPA who are negative for anti-MPO and anti-PR3 and are especially common in patients with cystic fibrosis (Zhao et al., 1995). MPO antibodies are present in some 60% of patients with the Churg-Strauss syndrome characterized by a history of asthma, hypereosinophilia and systemic vasculitis (Cohen Tervaert et al., 1991; Guillerin et al., 1993).

Table 1. Classification of the Idiopathic Vasculitides as Proposed by an International Study Group at the Chapel Hill Consensus Conference on the Nomenclature of Systemic Vasculitis* Large vessel vasculitis 1. Giant cell (temporal) arteritis 2. Takayasuarteritis II.

Medium-sized vessel vasculitis 1. Polyarteritisnodosa 2. Kawasaki'sdisease

III.

Small vessel vasculitis Wegener's granulomatosis Churg-Strauss syndrome Microscopic polyangiitis Henoch Sch6nlein purpura Essential cryoglobulinemic vasculitis Cutaneous leukocytoclastic angiitis

*Adapted from: Jennette JC et al. Nomenclature of systemic vasculitides: the proposal of an international consensus conference. Arthritis Rheum 1994;37:187--92.

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Table 2. Disease Associations of Antiproteinase 3 Antibodies and Antimyeloperoxidase Antibodies* Disease entity

Sensitivity of antiproteinase 3 (%)

antimyeloperoxidase (%)

Wegener's granulomatosis

85

10

Microscopic polyangiitis

45

45

Idiopathic crescentic glomerulonephritis

25

65

Churg-Strauss syndrome

10

60

5

15

Polyarteritis nodosa *Data derived from the references cited in the text.

Antibody Frequencies Together with GBM antibodies, anti-MPO are detected in about 30-40% of patients with anti-GBM disease (Goodpasture syndrome) (Bosch, 1991). Patients with GBM disease and both GBM and MPO antibodies are generally somewhat older and have a better recovery of renal function than patients with anti-GBM disease in the absence of anti-MPO. Although reported in patients with classical polyarteritis nodosa (PAN), anti-MPO are uncommon in classical PAN as defined by the Chapel Hill Conference (Jennette, 1994), i.e., vasculitis restricted to arterial vessels (Kallenberg et al., 1994). Patients whose sera contain anti-MPO include those with well-established forms of vasculitis and a substantial group whose overlapping symptoms suggest one of the primary vasculitides, albeit in the absence of criteria for those diseases (Cohen Tervaert et al., 1990). Of these patients with what is designated "polyangiitis overlap syndrome," the percentage who will eventually develop one of the well-defined vasculitides is unknown. MPO antibodies are reported in 8% of patients with SLE. There is no evidence at present that patients with SLE and anti-MPO represent a distinct entity characterized by vasculitis. Rather, the presence of ANCA in SLE may be associated with a chronic inflammatory response manifest by arthritis, serositis and raised C-reactive protein (CRP) (Spronk et al., submitted). In patients with drug-induced LE, however, antiMPO are probably more common (50-100%), and may occur simultaneously with antielastase antibodies (H~issberger et al., 1990; Cambridge et al., 1994). However, only small numbers of patients have been studied. Anti-MPO are also found in some patients

58

who develop vasculitic-like lesions during treatment with thyrostatic drugs (Dolman et al., 1993). Finally, MPO antibodies occur incidentally in diseases such as rheumatoid arthritis and inflammatory bowel disease. Anti-MPO occurrence in systemic sclerosis is reportedly associated with scleroderma renal crisis (Endo et al., 1994), but this is not confirmed. It should be mentioned again that the positive predictive value of a positive ANCA test result by IIF in unselected patients is as low as <5% (Jennette and Falk, 1994). In the right clinical context, the predictive value is, however, >90%; 90% of biopsy-proven pauci-immune necrotizing and crescentic glomerulonephritis proved positive for anti-MPO (Jennette and Falk, 1994). The relation between anti-MPO and disease activity of various vasculitides is not well studied, however, the few data available, suggest that serum anti-MPO reflect disease activity in some 70% of the patients with primary vasculitides (Cohen Tervaert et al., 1990; Kyndt et al., 1995). Prospective studies are, however, badly needed. The effects of treatment on serum anti-MPO during follow-up are not established. Remissions induced with intravenous immunoglobulin in some patients with anti-MPO-associated vasculitis are accompanied by decreases in serum anti-MPO concentrations (Jayne et al., 1991).

CONCLUSION Anti-MPO are found in a substantial number of sera that produce a perinuclear fluorescence pattern (PANCA) on ethanol-fixed neutrophils. Many sera that produce a P-ANCA pattern by IIF do not, however, contain autoantibodies to MPO. Therefore, a positive

test for A N C A by IIF should be followed by antigenspecific assays, in particular for anti-MPO as generally performed by ELISA using purified, commercially available MPO. A test for anti-MPO is indicated in every patient suspected of vasculitis or glomerulonephritis of unknown origin. The presence of anti-MPO strongly suggests necrotizing vasculitis or idiopathic pauciimmune necrotizing and crescentic glomerulonephritis (NCGN). Anti-MPO are found in some 65% of patients with NCGN, 45% of those with microscopic polyangiitis, 60% of patients with Churg-Strauss

syndrome and 10% of patients with WG. Most of the patients with the aforementioned diseases who are negative for anti-MPO have antibodies to proteinase 3. With respect to the specificity of anti-MPO for the necrotizing vasculitides, it should be noted that the antibodies do occur in drug-induced LE and, occasionally, in certain connective tissue diseases. Several in vitro and in vivo data suggest that antiMPO are involved in the pathophysiology of the associated vasculitides. See also A N C A WITH SPECIFICITY FOR PROTEINASE 3 and ANCA WITH SPECIFICITY OTHER THAN PR3 AND MPO (X-ANCA).

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lupus. Clin Exp Immunol 1990;81:380-383. Jayne DR, Davies MJ, Fox CJ, Black CM, Lockwood CM. Treatment of systemic vasculitis with pooled intravenous immunoglobulin. Lancet 1991;337:1137-1139. Jennette JC. Pathogenic potential of antineutrophil cytoplasmic autoantibodies. Lab Invest 1994;70:135--137. Jennette JC, Falk RC. The coming of age of serologic testing for antineutrophil cytoplasmic autoantibodies. Mayo Clin Proc 1994;69:908-910. Kallenberg CG, Brouwer E, Mulder AH, Stegeman CA, Weening JJ, Tervaert JW. A N C A - pathophysiology revisited. Clin Exp Immunol 1995;100:1--3. Kallenberg CG, Brouwer E, Weening JJ, Tervaert JW. Antineutrophil cytoplasmic antibodies: current diagnostic and pathophysiological potential. Kidney Int 1994;46:1-15. Kallenberg CGM, Mulder AHL, Cohen Tervaert TW. Antineutrophil cytoplasmic antibodies: a skill growing class of autoantibodies in inflammatory disorders. Am J Med 1992; 93:675--682. Kyndt X, Reumaux D, Bataille, P, et al. Relationship between MPO-ANCA and disease activity in vasculitis. Clin Exp Immunol 1995;101(S1):67. Ltidemann J, Utecht B, Gross WL. Antineutrophil cytoplasm antibodies in Wegener's Granulomatosis recognize an elastinolytic enzyme. J Exp Med 1990;171: 357--362. Mathieson PW, Thiru S, Oliveira DB. Mercuric chloride-treated Brown Norway rats develop widespread tissue injury including necrotizing vasculitis. Lab Invest 1992;67:121-129. Mathieson PW, Oliveira DB. The role of cellular immunity in systemic vasculitis. Clin Exp Immunol 1995;100:183-185. Merrill DP. Purification of human myeloperoxidase by Concanavalin A-Sepharose affinity chromatography. Prep Biochem 1980;10:133--150. Moguilevsky N, Garcia-Quintana L, Jacquet A, Tournay C, Fabry L, Pierard L, Bollen A. Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines. Eur J Biochem 1991; 197:605-614. Mulder AH, Horst G, Limburg PC, Kallenberg CG. Activation of granulocyte by antineutrophil cytoplasmic antibodies (ANCA): a FcrRII-dependent process. Clin Exp Immunol

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1994;98:270-278. Mulder AHL, Horst G, van Leeuwen MA, Limburg PC, Kallenberg CG. Antineutrophil cytoplasmic antibodies in rheumatoid arthritis. Characterization and clinical correlates. Arthritis Rheum 1993;36:1054-- 1060. Rosen FS, Wedgewood RJP, Eibl M, et al. Primary immunodeficiency diseases. Report of a WHO Scientific Group. Clin Exp Immunol 1995;99:1--24. Savage CO, Pottinger BE, Gaskin G, Pusey CD, Pearson JD. Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity towards cultured endothelial cells. Am J Pathol 1992;141: 335-342. Spencer SJ, Burns A, Gaskin G, Pusey CD, Rees AJ. HLA class II specificities in vasculitis with antibodies to neutrophil cytoplasmic antigens. Kidney Int 1992;41:1059-1063. Spronk PE, Horst G, Huitema MG, Limburg PC, Cohen Tervaert JW, Kallenberg CGM. Antineutrophil cytoplasmic antibodies in systemic lupus erythematosus; a reflection of persistent subclinical disease activity? Submitted. Van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van Es LA, van der Giessen M, van der Hem GK, The TH. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 1985; 1:425--429. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989;320:365-376. Wiik A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. APMIS 1989; 97(Suppl): 12--13. Yang JJ, Jennette JC, Falk RJ. Immune complex glomerulonephritis is induced in rats immunized with heterologous myeloperoxidase. Clin Exp Immunol 1994;97:466--473. Zeng J, Fenna RE. X-ray crystal structure of caninine myeloperoxidase at a 3 Angstrom resolution. J Mol Biol 1992;226: 185--207. Zhao MH, Jones SJ, Lockwood CM. Bactericidal/permeabilityincreasing protein is an important antigen for antineutrophil cytoplasmic autoantibodies in vasculitis. Clin Exp Immunol 1995;99:49-56.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

A N T I N E U T R O P H I L C Y T O P L A S M I C A U T O A N T I B O D I E S WITH SPECIFICITY FOR P R O T E I N A S E 3 Wolfgang L. Gross, M.D. a'b, Elena Csernok, Ph.D. a and Christof H. Szymkowiak, Ph.D. b

aDepartment of Rheumatology, University of Liibeck, Liibeck 23538; and bRheumaklinik Bad Bramstedt GmbH, Bad Bramstedt 24572, Germany

HISTORICAL NOTES

Antineutrophil cytoplasmic antibodies (ANCA) that recognize proteinase 3 (PR3) are strongly associated with Wegener' s granulomatosis (WG) and are relevant to the immunopathogenesis of the associated vasculitis. The association of ANCA (now known to be a variety of antibodies including those directed against PR3) with vasculitis (itself a symptom rather than a diagnosis) was first described in 1982 (Davies et al., 1982). In 1985, WG was associated with the specific cytoplasmic indirect immunofluorescence (IIF) staining pattern now defined as C-ANCA (van der Woude et al., 1985); PR3 was identified as the antigen for this subset of ANCA (Ludemann et al., 1990). Presently, PR3-ANCA (and to a certain extent myeloperoxidase-specific ANCA (MPO-ANCA)) are the major subspecificities of ANCA of immunodiagnostic and immunopathogenic significance in vasculitis (Gross and Csernok, 1995). However, because of the strong association of PR3-ANCA and MPO-ANCA with WG, Churg-Strauss syndrome and microscopic polyangiitis, these diseases are separated from primary systemic vasculitides and termed ANCA-associated vasculitides (Jennette and Falk, 1993).

THE AUTOANTIGEN(S) Definition/Nomenclature

PR3, the main target antigen of c-ANCA, is a cationic protein (isoelectric point, pH 9.4) consisting of 228 amino acids residues and belonging to the trypsin family of serine proteases. Expressed only in primates and humans, PR3 has different functions, including

proteolysis of elastin, hemoglobin, fibronectin, laminin and collagen type IV, antimicrobial activities against C. albicans and E. coli, involvement in myeloid cell differentiation, and Napthol-ASD-Chloracetate cleavage (Gross and Csernok, 1995). PR3 is identical to other molecules described as AGP7 (azurophil granule protein) (Gabay et al., 1989) and myeloblastin (Bories et al., 1989). Origin/Sources

PR3 is found in MPO-positive granules of PMN and monocytes (Csernok et al., 1990), in human endothelial cells (HUVEC) (Mayet et al., 1993) and in two human cell lines: a promyelocyte cell line (HL-60) and a human kidney carcinoma cell line (SK-RC 11) (Muller-Berat et al., 1994). Currently, the only sources of native PR3 are azurophile granules of blood polymorphonuclear leukocytes (PMN) (Kao et al., 1988) and purulent sputum (Ballieux et al., 1993). A baculovirus system previously successful in the production of recombinant myeloperoxidase yielded a recombinant PR3 recognized by C-ANCA. Methods of Purification

There are two major protocols for the purification of PR3. The first is based on dye ligand (Orange A) affinity binding of PR3 and subsequent purification through cation exchange chromatography (Kao et al., 1988). This method yields pure, immunologically active PR3 from azurophile granules of blood neutrophils and from purulent sputum. Proteolytically active PR3 can be purified on a Bio-Rex 70 column that binds other proteases without binding PR3 (Leid et al., 1993). 61

Commercial Sources To date, PR3 is not commercially available, although ELISA for the detection of PR3-ANCA is distributed by several companies. A European Union study group was founded to standardize the preparation of antigen utilizing IIF and ELISA protocols (Hagen et al., 1993).

Sequence Information The cDNA sequence for PR3 was established (Bories et al., 1989). PR3 is located on chromosome 19 in a gene cluster together with azurocidin and neutrophil elastase (Zimmer et al., 1992). As shown by treatment of PR3 with heat, I]-mercaptoethanol and low pH, ANCA binding is dependent on the quaternary structure of the molecule, indicating that C-ANCA recognize conformational epitopes on PR3 (Ludemann et al., 1990). Surprisingly, binding is inhibited by incubating sera from ANCA-positive WG with 7mer synthetic peptides (Williams et al., 1994). This implies that linear epitopes of PR3 also are recognized by ANCA.

THE AUTOANTIBODIES Terminology Historically, "ANCA" referred to the cytoplasmic fluorescence of PMN observed in vasculitic conditions (predominantly renal vasculitis without further classification of the disease entity) without further description of staining pattern (Davies et al., 1982). A distinct fluorescence pattern termed anticytoplasmic antibodies (ACPA) was later associated with WG (Van der Woude et al., 1985). The term "ANCA" currently is used to delineate a whole spectrum of autoantibodies. C-ANCA denotes the "cytoplasmic" or "classic" ANCA staining pattern in IIF. The major fluorescence pattern not associated exclusively with vasculitis is the "perinuclear" (P-ANCA) staining pattern. Other, less well defined, fluorescence patterns are characterized as "atypical" ANCA (A-ANCA or X-ANCA).

Pathogenetic Role Human Disease. There is ample evidence that PR3ANCA plays a direct pathogenic role in the develop62

ment of WG. First, there is a good correlation between C-ANCA titers and disease activity during the course of WG (N611e et al., 1989). Second, ANCA titers correlate with the degree of activation of PMN in WG patients with renal involvement (Brouwer et al., 1994), although there is no correlation between the ability of ANCA-IgG to activate PMN and the number of activated PMN found in renal biopsies. Third, ANCA can activate PMN in vitro (Falk et al., 1990) with resultant degranulation and the release of toxic compounds. ANCA inhibit the irreversible binding of natural protease inhibitors such as ~l-antitrypsin to PR3, possibly allowing the proteolytic activity of PR3 to destroy endothelial cells (Dolman et al., 1993). Anti-PR3 and ~l-antitrypsin are associated with ANCA-associated vasculitides (Testa et al., 1993). This association is of clinical importance: patients with heterozygous ~l-antitrypsin deficiency should not receive any treatment that leads to a further decrease in ~l-antitrypsin (i.e., plasmapheresis). There is a genetic link between the occurrence of the PiZ ~l-antitrypsin variant and WG. Heterozygotes for the PiZ variant of the ~l-antitrypsin gene carry a greater risk of developing WG than the general population (Elzouki et al., 1994). In addition, phenotypes usually associated with a moderate or severe reduction of ~l-antitrypsin serum levels or with dysfunctional activity are found more often in individuals with PR3-ANCA than in the general population. However, none of the 31 sera with anti-PR3 antibodies investigated have low levels of c~l-antitrypsin (Savige et al., 1995). These observations support the recently updated ANCA-cytokine sequence model (Gross and Csernok, 1995).

Animal Models. Currently, there is a single mouse model for WG. Immunization of B ALB/C mice with purified ANCA of two patients with active WG led either to the death of the mice from multiple nonbacterial lung microabscesses or to the appearance of granuloma. In both conditions, ANCA antibodies were demonstrated (Shoenfeld, 1994). Genetics HLA class II genes are associated with vasculitis and may influence the duration of the associated autoimmune response. Patients with the DRw7, DR4 haplotype are significantly more likely to have transiently positive tests for ANCA than patients with other DRw7-bearing haplotypes, whereas patients with

DR2-bearing haplotypes are more likely to have persistently positive ANCA. Although V-domain antibody fragments specific for ANCA are present in the normal B-cell repertoire, there is most likely no polyclonal B-cell activation (Finnern et al., 1993). Factors in Pathogenicity Isotypes. Although first described in the IgG class, IgM ANCA (with and without associated IgG ANCA) are found in patients with pulmonary hemorrhage (Esnault et al., 1992). IgA ANCA occur in patients presenting with Henoch-Sch6nlein purpura (79%) and IgA nephropathy (3%) (Ronda et al., 1994). The role of cellular immunity in systemic vasculitis was recently reviewed (Mathieson and Oliveira, 1995). The predominance of IgG1 and IgG4 subclasses for PR3-ANCA is different from the distribution of IgG subclasses of ANA, but resembles that of anti-GMBautoantibodies. IgG4 is produced after recurrent stimulation with antigen. The predominance of IgG4 subclass ANCA may therefore suggest a chronic antigen stimulation of the immune system and an antigen-driven B-cell stimulation underlying the development and production of ANCA. The occurrence of granuloma and renal interstitial T-cell infiltration support the hypothesis concerning the autoreactive T-cell response. In WG, elevated concentrations of soluble IL-2 receptor (a marker for the activation of T-cells) correlate with disease activity. In addition, there is a growing body of evidence that autoantibody activity in autoimmune diseases might be regulated through idiotype-anti-idiotype antibody reactions; antiidiotypic antibodies were found in pooled human immunoglobulin preparations (Jayne et al., 1993). Clinical data describing a "flu-like" illness associated with WG and seasonal variations in the onset of WG (Raynauld et al., 1993) suggest that WG might be triggered by a previous infection. Coxsackie B3 virus, parvovirus B19 and S t a p h y l o c o c c u s aureus might be environmental triggers for the development of C-ANCA (Barrett et al., 1993). A variety of S. a u r e u s - e n c o d e d serine proteases show sequence similarities with PR3. An association of chronic nasal carriage of S. a u r e u s and high relapse rates in WG w~is also found (Stegeman et al., 1994). Methods of Detection The most common method for detection of ANCA is IIF on ethanol-fixed human granulocytes or on the

human leukemia cell line HL-60. Fixation of the cells by ethanol allows discrimination between different fluorescence patterns: C-ANCA, P-ANCA and XANCA (Wiik et al., 1993). The fine granular cytoplasmic ANCA is clearly distinguishable from the other ANCA staining patterns (Figure 1) and is highly specific for WG. Reliability of the IIF assays depends on the type of substrate employed, the source of cells, fixation, storage, incubation and washing steps. To circumvent these problems and to demonstrate a clear association between ANCA and the target antigens, an ELISA for different antigens should be performed. Differences in antigen preparations also may cause problems and attempts are underway for standardization of assay procedures (Hagen et al., 1993). Although standardization for IIF and ELISA are still underway, the following "gold standard" is recommended. First, IIF is used to determine the ANCA type (C-ANCA, P-ANCA or X-ANCA) and the ANCA-titer. Second, ELISA is necessary for the determination of subspecificities (PR3-ANCA-positive or-negative). There is a good correlation between IIF and ELISA for the detection of positive sera but only a minor correlation between C-ANCA titer and ELISA units. Between 80 and 90% of C-ANCApositive samples are also positive in ELISA and vice versa; 90% of PR3-positive ELISA samples are also C-ANCA-positive (Wieslander, 1991). ELISA detection of C-ANCA antigen (PR3) is also important because some sera showing a C-ANCA staining pattern are specific for MPO-ANCA (Segelmark et al., 1994). On the other hand, PR3-ANCA are also found in combination with a P-ANCA staining pattern (Falk and Jennette, 1988). The presence of C-ANCA (PR3ANCA) alone is not sufficient for the diagnosis of WG in the absence of histologic proof.

CLINICAL UTILITY Disease Associations ANCA are found in primary systemic vasculitis and connective tissue diseases but also in chronic inflammatory bowel disease and associated conditions, in some infections (e.g., HIV) and a few other conditions (Gross et al., 1993). C-ANCA with positive ELISA for PR3 antibodies are highly sensitive and specific for WG. WG in its fulminant forms or a "formes fruste" (in which the classic triad is never manifested) can be classified by the American College of Rheu-

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Figure 1. a: Fine granular cytoplasmic ANCA pattern as seen by IIF (C-ANCA). b: Perinuclear ANCA pattern (P-ANCA). e: Homogenous cytoplasmic pattern with staining of perinuclear zone of PMN (X-ANCA).

matology criteria (Leavitt et al., 1990) and by definitions developed at the Chapel Hill conference in 1992 (Jennette et al., 1994). Differentiation of primary

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systemic vasculitides from ANCA-associated vasculitides can be achieved using ANCA serology (Jennette et al., 1994). Despite the strong association

between PR3-ANCA and WG, there is a small percentage of patients with microscopic polyangiitis and about 30% of Churg-Strauss syndrome patients who are PR3-ANCA-positive (Gross et al., 1993). In patients with classic polyarteritis nodosa (n = 36), about 25% of the sera tested positive for C-ANCA, of which only 1/3 were also positive for PR3-ANCA (Hauschild et al., 1994). C-ANCA are also found in patients with invasive amoebiasis of whom 75% are PR3-ANCA-positive (Pudifin et al., 1994). These results wait for further confirmation. On the other hand, the detection of ANCA permits the recognition (and earlier treatment) of WG which presents itself first under the "disguise" of Tolosa-Hunt syndrome (Montecucco et al., 1993), "idiopathic" facial nerve paralysis (Macias et al., 1993), polyneuritis cranialis (Chakravarty and Scott, 1993), peripheral neuropathy (Chalk et al., 1993), polychondritis (Handrock and Gross, 1993), pulmonary hemorrhage following alveolar capillarities (Bosch et al., 1994) and necrotizing crescentic glomerulonephritis (Velosa et al., 1993) and in patients undergoing selective hemodialysis with renal failure of largely unknown origin (Weidemann et al., 1993). However, as described above, the presence of C-ANCA (PR3-ANCA) p e r se does not allow the diagnosis of WG, but is only a clue for further substantiating the diagnosis according to all accepted guidelines (Jennette et al., 1994; Leavitt et al., 1990). There is a good correlation between CANCA titers and disease activity during the course of WG. The specificity for C-ANCA in the diagnosis of WG is >90%, with the additional screening for PR3ANCA over 95% (N611e et al., 1989). The sensitivity is dependent on the phase and on the activity of the disease. In the initial inactive phase of WG, only 50% of the patients have C-ANCA while in the active generalization phase of WG, C-ANCA is detected in nearly 100% of the patients.

Effect of Therapy The mainstay of therapy in ANCA-associated vasculitides is with corticosteroids and immunosuppressive drugs. Such therapeutic schemes result in almost complete remission accompanied by low or undetectable levels of C-ANCA (Gross and Rasmussen, 1994). Also, high-dose IVIG treatment is beneficial for WG patients. In an uncontrolled study, 15 patients with ANCA-associated vasculitis were treated with IVIG screened for anti-idiotypic antibodies to PR3-ANCA. Approximately 40% of patients benefited from IVIG

treatment, although complete remission of disease activity did not occur (Richter et al., 1993). In another study, patients with WG (all PR3-ANCA positive) were treated with IVIG; ANCA levels rose initially in all patients, but later fell to levels substantially lower than pretreatment values, becoming undetectable in one patient (Rossi et al., 1991). Plasmapheresis is effective in some acute vasculitic conditions. Plasma exchange may be of benefit in combination with glucocorticoids and cytotoxic therapy in dialysis patients with necrotizing glomerulonephritis (Pusey et al., 1991) although this has not been confirmed on a controlled basis.

Sensitivity and Specificity The specificity for C-ANCA in the diagnosis of WG with the additional screening for PR3-ANCA by ELISA is over 95% (N611e et al., 1989; Hauschild et al., 1993). The sensitivity is dependent on the phase and on the activity of the disease. "False-positive" CANCA are reported in infective disorders such as HIV infections (Klaassen et al., 1992), endocarditis (Soto et al., 1994), pneumonia and infections in cystic fibrosis (Efthimiou et al., 1991). Furthermore, they are seen in monoclonal gammopathy (Esnault et al., 1990) and in a few cases of malignancy without signs of secondary vasculitis (Hauschild et al., 1993).

CONCLUSION The discovery of ANCA has improved the diagnostic procedure in patients with primary vasculitides and has led to a new diagnostic group: "ANCA-associated vasculitides". The clinical value of C-ANCA (PR3ANCA) testing in WG is now well established. Furthermore, it is suspected that the presence of ANCA is an important factor in the pathogenesis of these disease groups. Based on our current knowledge of ANCA, it can be concluded that C-ANCA with specificity for PR3 may be directly involved in the pathogenesis of ANCA-associated vasculitides and can serve as a useful marker for this disorder analogous to the role of ANA in connective tissue disease. See also ANCA IN INFLAMMATORY BOWEL DISEASES, ANCA WITH SPECIFICITY FOR MYELOPEROXIDASE, ANCA WITH SPECIFICITY OTHER THAN PR3 AND MPO (X-ANCA) and GRANULOCYTE-SPECIFIC ANTINUCLEAR ANTIBODIES.

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Klaassen RJ, Goldschmeding R, Dolman KM, Vlekke AB, Weigel HM, eeftink Schattenkerk JK, Mulder JW, Westedt ML, von dem Borne AE. Antineutrophil cytoplasmic autoantibodies in patients with symptomatic HIV infection. Clin Exp Immunol 1992;87:24-30. Leavitt RY, Fauci AS, Bloch DA, Michel BA, Hunder GE, Arend WP, Calabrese LH, Fries JF, Lie JT, Lightfoot RW Jr, Masi AT, McShane DJ, Mills JA, Stevens MB, Wallace SL, Zvailfler NJ. The American College of Rheumatology 1990 criteria for the classification of Wegener's granulomatosis. Arthritis Rheum 1990;33:1101-1107. Leid RW, Ballieux BE, van der Heijden I, Kleyburg-van der Kuer C, Hagen ED, van Es LA, van der Woude FJ, Daha MR. Cleavage and inactivation of human C 1 inhibitor by the human leukocyte proteinase, proteinase 3. Eur J Immunol 1993 ;23:2939-2944. Ludemann J, Utecht B, Gross WL. Antineutrophil cytoplasm antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme. J Exp Meal 1990;171:357--362. Macias JD, Wackym PA, McCabe BF. Early diagnosis of otologic Wegener's granulomatosis using the serologic marker C-ANCA. Ann Otol Rhinol Laryngol 1993;102:337-341. Mathieson PW, Oliveira DB. The role of cellular immunity in systemic vasculitis. Clin Exp Immunol 1995;100:183-185. Mayet WJ, Csernok E, Szymkowiak C, Gross WL, Meyer zum Bfischenfelde KH. Human endothelial cells express proteinase 3, the target antigen of anticytoplasmic antibodies in Wegener's granulomatosis. Blood 1993;82:1221-1229. Montecucco C, Caporali R, Pacchetti C, Turla M. Is TolosaHunt syndrome a limited form of Wegener's granulomatosis? Report of two cases with antineutrophil cytoplasmic antibodies. Br J Rheumatol 1993;32:640-641. Muller-Berat N, Minowada J, Tsuji-Takayama K, Drexler H, Lanotte M, Wieslander J, Wiik A. The phylogeny of proteinase 3/myeloblastin, the autoantigen in Wegener' s granulomatosis, and myeloperoxidase as shown by immunohistochemical studies on human leukemia cell lines. Clin Immunol Immunopathol 1994;70:51--59. NOlle B, Specks U, Ludemann J, Rohrbach MS, DeRemme RA, Gross WL. Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener granulomatosis. Ann Intern Med 1989; 111:28--40. Pudifin DJ, Duursma J, Gathiram V, Jackson TF. Invasive amoebiasis is associated with the development of antineutrophil cytoplasmic antibody. Clin Exp Immunol 1994; 97:48--51. Pusey CD, Rees AJ, Evans DJ, Peters DK, Lockwood CM. Plasma exchange in focal necrotizing glomerulonephritis without anti-GBM antibodies. Kidney Int 1991;40:757--763. Raynauld JP, Bloch DA, Fries JF. Seasonal variation in the onset of Wegener's granulomatosis, polyarteritis nodosa and giant cell arteritis. J Rheumatol 1993;20:1524-1526. Richter C, Schnabel A, Csernok E, Rheinhold-Keller E, Gross WL. Treatment of Wegener' s grnulomatosis with intravenous immunoglobulin. In: Gross WL, ed. ANCA-Associated

Vasculitides. London: Plenum Press, 1993:487--489. Ronda N, Esnault VL, Layward L, Sepe V, Allen A, Feehally J, Lockwood CM. Antineutrophil cytoplasm antibodies (ANCA) of IgA isotype in adult Henoch-Sch~Snlein purpura. Clin Exp Immunol 1994;95:49-55. Rossi F, Jayne DR, Lockwood CM, Kazatchkine MD. Antiidiotypes against neutrophil cytoplasmic antigen autoantibodies in normal human polyspecific IgG for therapeutic use and in the remission sera of patients with systemic vasculitis. Clin Exp Immunol 1991 ;83:298--303. Savige JA, Chang L, Cook L, Burdon J, Daskalakis M, Doery J. Alpha 1-antitrypsin deficiency and antiproteinase 3 antibodies in antineutrophil cytoplasmic antibody (ANCA)associated systemic vasculitis. Clin Exp Immunol 1995;100: 194-197. Segelmark M, Baslund B, Wieslander J. Some patients with antimyeloperoxidase autoantibodies have a C-ANCA pattern. Clin Exp Immunol 1994;96:458-465. Shoenfeld Y. Idiotypic induction of autoimmunity: a new aspect of the idiotypic network. FASEB J 1994;8:1296--1301. Soto A, Jorgensen C, Oksman F, Noel LH, Sany J. Endocarditis associated with ANCA. Clin Exp Rheumatol 1994;12:203-204. Stegeman CA, Tervaert JW, Sluiter WJ, Manson WL, de-Jong PE, Kallenberg CG. Association of chronic nasal carriage of Staphylococcus aureus and higher relapse rates in Wegener granulomatosis. Ann Intern Med 1994;120:12-- 17. Testa A, Audrain M, Baranger T, Sesboue R, Martin J, Esnauld VLM. Antineutrophil cytoplasm antibodies and o~-1 antitrypsin phenotype. Clin Exp Immunol 1993;93:16. Van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van Es LA, van der Giessen M, van der Hem GK, The TH. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 1985; 1:425--429. Velosa JA, Homburger HA, Holley KE. Prospective study of antineutrophil cytoplasmic autoantibody tests in the diagnosis of idiopathic necrotizing-crescentic glomerulonephritis and renal vasculitis. Mayo Clin Proc 1993;68:561-565. Weidemann S, Andrassy K, Ritz E. ANCA in haemodialysis patients. Nephrol Dial Transplant 1993;8:839-845. Wieslander J. How are antineutrophil cytoplasmic autoantibodies detected? Am J Kidney Dis 1991;18:154--158. Wiik A, Rasmussen N, Wieslander J. Methods to detect autoantibodies to neutrophilic granulocytes. Manual Biol Mark Dis 1993;A9:1--14. Williams RC Jr, Staud R, Malone CC, Payabyab J, Byres L, Underwood D. Epitopes on proteinase-3 recognized by antibodies from patients with Wegener's granulomatosis. J Immunol 1994;152:4722--4737. Zimmer M, Medcalf RL, Fink TM, Mattmann C, Lichter P, Jenne DE. Three human elastase-like genes coordinately expressed in the myelomonocyte lineage are organized as a single genetic locus on 19pter. Proc Natl Acad Sci USA 1992;89:8215-8219.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ANTINEUTROPHIL CYTOPLASMIC AUTOANTIBODIES WITH SPECIFICITY OTHER THAN PR3 AND MPO (X-ANCA) Ming-Hui Zhao, M.D., Ph.D. and C. Martin Lockwood, M.D.

Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK

HISTORICAL NOTES

Detection of anti~eutrophil cytoplasmic autoantibodies (ANCA) is a useful laboratory diagnostic test for certain small vessel vasculitides (van der Woude et al., 1985; Savage et al., 1987) and some nonvasculitic clinical syndromes, such as inflammatory bowel disease (IBD) (Seibold et al., 1992). ANCA can be identified using indirect immunofluorescence (IIF) techniques; this procedure produces two staining patterns: an uneven, granular staining of neutrophil and monocyte cytoplasm with interlobular accentuation (C-ANCA) and a perinuclear staining pattern (PANCA) (Rasmussen et al., 1989; Wiik and van der Woude, 1989). Proteinase 3 (PR3) (Goldschmeding et al., 1989) and myeloperoxidase (MPO) (Falk and Jennette, 1988) are the major C-ANCA and P-ANCA antigens, respectively. As a historical note, the term X-ANCA was first applied to the ANCA found in patients with inflammatory bowel disease (Hauschild et al., 1993). However, the concise definition of XANCA or atypical ANCA has yet to gain consensus among those involved in studies of ANCA. In this chapter, X-ANCA, are autoantibodies reacting with neutrophil antigens other than PR3 and MPO.

THE AUTOANTIGENS Nomenclature

Most of the autoantigens of the specificities subsumed under the term X-ANCA are found within azurophilic or primary granules of polymorphonuclear leukocytes (PMN) including: (1) the microbicidal protein: bacte68

ricidal/permeability-increasing protein (BPI) (Zhao et al., 1995a); (2) two neutral serine proteinases: elastase (Nassberger et al., 1989) and cathepsin G (HalbwachsMecarelli et al., 1992) and (3) ~-glucuronidase (Nassberger et al., 1992). Among the proteins of secondary granules, lactoferrin (Coremans et al., 1992) is an ANCA antigen. Lysozyme, also described as an ANCA antigen (Schmitt et al., 1993), is found in both primary and secondary granules. In addition, antibodies to human lysosomal-associated membrane protein 2 (h-lamp-2), a neutrophil granule membrane protein, were recently described in certain forms of glomerulonephritis (Kain et al., 1995). As well as these granule antigens, ~-enolase, a cytoplasmic antigen, was also reported as an ANCA antigen (Moodie et al., 1993). Origins, Sources/Methods of Purification

BPI. BPI, also termed Cap57 (Pereira et al., 1990), is a 55--57 kd cationic membrane-bound antimicrobial protein found only in the azurophilic granules of PMN. It is formed from two domains: the N-terminal part contains all known antimicrobial activity and the C-terminal part contains several potential transmembrane regions which may anchor the holo-protein in the granule membrane (Gray et al., 1989). Holo-BPI contains 487 amino acids: from residue 240--245 there is a potential cleavage site for elastase (Gray et al., 1989). This potential susceptibility to elastase cleavage may account for B PI not receiving appropriate recognition as an ANCA antigen. Failure to acidify buffer used during and after extraction of ANCA antigens allows optimum pH for elastase activity. Unless the neutral serine proteinase inhibitor PMSF is

added, cleavage of BPI can occur; thereby destroying antigenicity. Indeed, the antigenicity of BPI is threatened by two serine proteinases, both elastase and PR3 (Jones et al., 1995). BPI displays a striking cytotoxicity toward many species of gram-negative bacteria (Elsbach et al., 1994). This may be a consequence of the high affinity of the very basic N-terminal portion of the molecule for the negatively charged lipopolysaccharide (LPS) moieties uniquely found in the outer envelope of gram-negative bacteria which constitute an important part of free endotoxin when it is released from the bacterial cell wall. Hence, the reported action of BPI as an endotoxin-neutralizing protein (Ooi et al., 1991). BPI shares 44% amino acid sequence homology with LPS-binding protein (LBP), an acute-phase protein in plasma which is the other major plasma molecule binding to LPS (Schumann et al., 1990). The LBPLPS complex can activate monocytes via the CD14 molecule to release proinflammatory factors such as tumor necrosis factor ~ (TNF~) and interleukin 1 (ILl) which in turn can stimulate PMN to cause tissue damage. However, BPI has a contrasting effect on LPS (neutralizing) and a much higher affinity for LPS than LBP (over 75 times); thus, BPI can inhibit inflammation caused by LPS (Heumann et al., 1993). B PI is a major azurophilic protein and can be purified from PMN granules. Several biochemical methods are described in which gram-negative bacteria were used as the target cells to identify the presence of BPI in a bioassay (Weiss et al., 1978; Shafer et al., 1984; Mannion et al., 1989; Gabay et al., 1989). A simple, quick, robust immunobiochemical method to isolate B PI was recently described (Zhao et al., 1995a), which involved the use of a single cation exchange column (Mono-S, Pharmacia Sweden) at two different pH (3 and 8) and ELISA, incorporating a reference BPI antibodypositive serum as a probe for the presence of B PI, rather than a bioassay. The yield is about 0.7--1% of the total protein in the crude granule acid extract. Recombinant holo-BPI molecules and their N-terminal segments are expressed by both E. coli (Qi et al., 1994) and eukaryotic cells (Gazzano-Santoro et al., 1992); they are used extensively to study the bactericidal and LPS-neutralizing activities of the protein (Elsbach et al., 1994). However, whether recombinant BPI or the N-terminal fragments retain autoantigenicity requires further study. Neither native nor recombinant BPI are commercially available. Native BPI can be immunoblotted by autoantibodies under both

reducing and nonreducing conditions, which suggests that the epitopes on the BPI molecule adopt a linear configuration.

Other X-ANCA Antigens. Lactoferrin, elastase, cathepsin G, lysozyme, ]3-glucuronidase h-lamp-2 and c~-enolase are each reported as ANCA antigens. Lactoferrin, an iron-binding protein, has a single polypeptide chain of 692 residues which migrates at 78 kd on SDS-PAGE. Human lactoferrin, which is found in milk, tears and saliva, is also a prominent component of the specific granules of PMN (Iyer and Lonnerdal, 1993). Elastase and cathepsin G are the two prominent serine proteinases (besides PR3) found in human neutrophil azurophilic granules, which share amino acid sequence homology with the latter. All these migrate at 29 kd on SDS-PAGE. Lysozyme is a 14.5 kd microbicidal enzyme constituent of both azurophilic and specific granules. 13-glucuronidase is a 75 kd constituent of acid hydrolases in azurophilic granules and ~-enolase is a 48 kd cytosolic component of neutrophils. The h-lamp-2 in neutrophil granule membrane is a glycoprotein which can be demonstrated as a component in moieties with apparent molecular masses of 170 and 80--110 kd (gpl70/ 80--110) (Kain et al., 1995). The majority of these minor ANCA antigens are commercially available.

AUTOANTIBODIES

Methods of Detection BPI. Purified BPI is used in ELISA and immunoblotting to detect B PI autoantibodies. The autoantibodies against B PI were first described in patients with clinically suspected vasculitides: they comprised 45/100 double-negative samples (IIF positive, yet recognizing neither PR3 nor MPO) and 44/400 consecutive serum samples sent for routine ANCA assay (Zhao et al., 1995a). The specificity was confirmed by fluid-phase inhibition assay using purified BPI and validated by immunoblotting using a fresh acid extract of PMN granules. Review of the 89 BPI antibody-positive patients revealed a male dominance (M:F ratio 55:34), a mean age of 60.4 years and clinical diagnoses ranging from organ-limited to widespread, multisystem vasculitis. Subsequently both IgG and IgA isotype B PI autoantibodies were found in an index patient with bronchiectasis, cutaneous vasculitis and acute pulmonary infection with Pseudo-

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monas aeruginosa. After appropriate antibiotic treatment, the pulmonary infection and cutaneous vasculitis recovered and IgA BPI antibody levels fell. Recently, autoantibodies against B PI were found in a subgroup of patients with primary bronchiectasis (Zhao and Lockwood, unpublished data).

Other Antigens. Similarly, autoantibodies to lactoferrin, elastase and cathepsin G can be easily detected by ELISA, at a coating concentration of 1 lag/mL in 0.05 ~~ M bicarbonate buffer, pH 9.6.

CLINICAL UTILITY Disease Associations BPI. To explore the prevalence and clinical associations of these autoantibodies, sera from a variety of patients with different ANCA-associated diseases were screened for the presence of IgG and IgA B PI autoantibodies. The most striking finding was that 60/66 (91%) of samples from adult patients with cystic fibrosis (CF) were positive for IgG anti-BPI and 55/66 (83%) were positive for IgA anti-BPI antibodies (Zhao et al., 1995b); all the IgA anti-BPI-positive samples were also IgG anti-BPI-positive. None of 46 sera from normal blood donors were positive for IgG anti-BPI antibody and only 1/46 (2%) was IgA antiBPI antibody. In other ANCA-associated diseases, the IgG and IgA anti-BPI antibody-positive percentages were as listed in Table 1. Anti-BPI autoimmune responses are frequently found in CF patients with advanced pulmonary damage or with cutaneous vasculitis. The anti-BPI levels, especially of IgA isotype in patients with CF, are inversely correlated with decline of pulmonary function (IgA anti-BPI levels vs. % FEVI: R = -0.508; p < 0.0001). Anti-BPI antibody levels in CF patients with cutaneous vasculitis (6/66) are significantly higher than in CF patients without vasculitic complications (p < 0.05). Positive serum samples from CF patients with advanced pulmonary damage can be diluted to a titer of 1/12,800 for both IgG and IgA isotypes. The specificity for BPI in CF sera can be confirmed by (1) ELISA using purified BPI and antigen-free wells to control for nonspecific binding; (2) fluid phase inhibition assay using purified BPI; and (3) immunoblotting using fresh PMN granule acid extract to reveal that CF sera only bind the 55 kd band which represents B PI.

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Although frequently positive in ELISA, only 21/66 (32%) of the CF samples produced easily detectable homogeneous cytoplasmic staining by IIF on ethanolfixed PMN, 20/66 (30%) were borderline and the other 25/66 (38%) were negative. This might reflect B PI cleavage by neutral serine proteinases either during preparation of PMN slides using ethanol fixation or during processing of the assay (Jones et al., 1995). Steroids (Auerbach et al., 1985) and the nonsteroid anti-inflammatory drug, ibuprofen, were used to reduce morbidity and prevent further pulmonary damage (Konstan et al., 1995), in young CF patients; their efficacy may be attributable to reducing the effects of the autoimmune response to BPI. This finding also adds weight to the hypothesis that chronic pulmonary inflammation plays a key role in the destruction of pulmonary tissue in CF. The IgA ANCA correlation with decline in pulmonary function provides an opportunity to investigate their interrelationship with pulmonary mucosal immunity in CF. Anti-BPI activity in patients with primary bronchiectasis, inflammatory bowel disease (such as ulcerative colitis and Crohn's disease), autoimmune liver diseases, (such as primary sclerosing cholangitis and primary biliary cirrhosis) as well as CF suggests that such autoimmunity might play a pathogenetic role in the progressive sclerosis or fibrosis commonly found in these disorders. The high frequency of pulmonary infection with Pseudomonas aeruginosa in patients with CF and some patients with bronchiectasis suggests the need to study the role of molecular mimicry. Interference by anti-BPI antibodies with the bactericidal and LPS-neutralizing functions of BPI molecule is also a topic which may have major clinical relevance. The bactericidal activity of a crude acid extract of PMN towards susceptible E. coli corresponds closely to the B PI content and can be completely blocked by a goat anti-BPI, IgG-rich fraction but not by a preimmune fraction (Weiss et al., 1982). Later it was reported that one of two monoclonal BPI antibodies can block (50%) the bactericidal activity of BPI (Spitznagel et al., 1987). These findings suggest that some epitope(s) might be located in the functional area of the B PI molecule. Whether human autoantibodies against BPI might similarly block functions of BPI, allowing nonneutralized products such as LPS to cause tissue injury directly requires further investigation.

Table 1. IgG and IgA Anti-BPI in Normal and Disease Controls Controls

IgG (% positive)

IgA (% positive)

Total

Normal

0/46 (0)

1/46 (2%)

1/46 (2%)

HSP

1/22 (5%)*

3/22 (14%)

3/22 (14%)

IgAN

0/10 (0)

2/10 (20%)

2/10 (20%)

PR3+ve

10/41 (24%)

5/41 (12%)?

13/41 (32%)

MPO+ve

8/41 (20%)

4/41 (10%)[]

10/41 (24%)

UC

7/25 (28%)

3/25 (12%)

8/25 (32%)

CO

6/27 (22%)

4/27 (15%)

6/22 (22%)

PSC

5/28 (18%)

5/28 (18%) $

9/28 (32%)

PBC

4/20 (20%)

4/20 (20%) =

6/20 (30%)

AIH

0/9 (0)

0/9 (0)

0/9 (0)

Note: HSP = Henoch-Sch6nlein purpura; IgAN = IgA nephropathy; PR3+ve = PR3 antibody-positive vasculitis controls; MPO+ve = MPO antibody-positive vasculitis controls; UC = ulcerative colitis; CD = Crohn's disease; PSC = primary sclerosing cholangitis; PBC = primary biliary cirrhosis' AIH: autoimmune hepatitis. * Also positive for IgA; ? 2/5 also posinve for IgG; [] 2/4 also posinve for IgG; 2/3 also posinve for IgG; all 4 also positive for IgG; $ 1/5 also posinve for IgG; and 9 2/4 also posinve for IgG.

O t h e r M i n o r A n t i g e n s . Autoantibodies against lac-

toferrin are found in some rheumatic vasculitides including rheumatoid vasculitis, mixed connective tissue disease and some cases of SLE, particularly those with nephritis (Coremans et al., 1992; Mulder et al., 1993; Sinico et al., 1993). In general, antibodies against lactoferrin are found only in a minority of these cases. Antibodies against neutrophil elastase were reported in only 6/104 patients with systemic lupus erythematosus (SLE), of whom the four with the highest titer all had neurological disease (Nassberger et al., 1989; 1990). In another study, six sera from 1,102 P-ANCA-positive samples gathered over a 6year period were identified as having antielastase antibodies (Cohen Tervaert et al., 1993). Both lactoferrin and elastase are associated with a positive ANCA in patients receiving hydralazine or another immunostimulating drug (Nassberger et al., 1991). Commercial MPO can be contaminated with lactoferrin. A subgroup of patients with systemic vasculitis whose sera gave false anti-MPO reactivity on ELISA actually had antilactoferrin antibodies; such antilactoferrin specificity was also associated with antinuclear (histone) activity (Esnault et al., 1994). Recently,

antibodies against lactoferrin, as well as cathepsin G were identified in a subgroup of patients with inflammatory bowel disease (Peen et al., 1993). However, these ANCA specificities do not appear to correlate with disease activity. In patients with active necrotizing and crescentic glomerulonephritis (NCGN), autoantibodies against the neutrophil granule membrane protein, h-lamp-2 are reported to cross-react with a related membrane protein (gpl30) in glomerular endothelial cells (Kain et al., 1995). This crossreaction sheds light on a possible way in which ANCA with h-lamp-2 specificity may contribute to the pathogenesis of NCGN. Although lysozyme, ~-glucuronidase and ~-enolase are also reported as ANCA antigens, the significance of these less common ANCA specificities is unclear at present.

CONCLUSION BPI is an important antigen for ANCA in patients with a variety of vasculitides; however, a distinctive clinical pattern, such as that associated with anti-PR3 (Wegener's granulomatosis) or MPO (microscopic

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polyangiitis) has yet to emerge. BPI is also the target for autoantibodies in patients with cystic fibrosis. Anti-BPI antibodies producing characteristic IIF changes are found in some patients with primary bronchiectasis, inflammatory bowel disease or certain autoimmune liver diseases. The autoimmunity against BPI in these disorders might suggest a c o m m o n pathogenetic process, resulting in tissue damage including sclerosis or fibrosis which are commonly found in these diseases. The frequency and level of

anti-BPI antibodies in cystic fibrosis suggest that pulmonary infection, especially with P s e u d o m o n a s aeruginosa, may have an important role in the development of the autoimmune response via molecular mimicry. Other minor A N C A specificities require further study. See also A N C A IN INFLAMMATORY BOWEL DISEASES, A N C A WITH SPECIFICITY FOR MYELOPEROXIDASE, A N C A WITH SPECIFICITY FOR PROTEINASE 3 and GRANULOCYTE-SPECIFIC ANTINUCLEAR ANTIBODIES.

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Auerbach HS, Williams M, Kirkpatrick JA, Colten HR. Alternate-day prednisone reduces morbility and improves pulmonary function in cystic fibrosis. Lancet 1985:2:686-688. Cohen Tervaert JW, Mulder A, Stegeman C, Elema J, Huitema M, The H, Kallenberg C. The occurrence of autoantibodies to human leukocyte elastase in Wegener's granulomatosis and other inflammatory disorders. Ann Rheum Dis 1993;52: 115--120. Coremans IE, Hagen EC, Daha MR, van der Voort EA, Kleijburg-van der Keur C, Breedveld FC. Antilactoferrin antibodies in patients with arthritis are associated with vasculitis. Arthritis Rheum 1992;35:1466--1475. Elsbach P, Weiss J, Doerfler M, Shu C, Kohn F, Ammons WS, Kung AH, Meszaros KK, Parent JB. The bactericidal/permeability increasing protein of neutrophils is a potent antibacterial and antiendotoxin agent in vitro and in vivo. Prog Clin Biol Res 1994;388:41--51. Esnault VL, Short AK, Audrain MA, Jones SJ, Martin SJ, Skehel JM, Lockwood CM. Autoantibodies to lactoferrin and histone in systemic vasculitis identified by antimyeloperoxidase solid phase assays. Kidney Int 1994;46: 153--160. Falk RJ, Jennette JC. Antineutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 1988;318:1651--1657. Gabay JE, Scott RW, Campanelli D, Griffith J, Wilde C, Marra MN, Seeger, M, Nathan F. Antibiotic proteins of human polymorphonuclear leukocytes. Proc Natl Acad Sci USA 1989;86:5610-5614. Gazzano-Santoro H, Parent JB, Grinna L, Horwitz A, Parsons T, Theofan G, Elsbach P, Weiss J, Conlon PJ. High-affinity binding of the bactericida/permeability-increasing protein and a recombinant amino-terminal fragment to the lipid A region of lipopolysaccharide. Infect Immun 1992;60:4754-4761. Goldschmeding R, van der Schoot CE, ten Bokkel Huinik D, Hack CE, van der Ende ME, Kallenberg CG, von dem Borne AE. Wegener's granulomatosis autoantibodies identify a novel diisopropylflourophosphate binding protein in the lysosomes of normal human neutrophils. J Clin Invest 1989;84:1577-1587. Gray PW, Flaggs G, Leong SR, Grumina RJ, Weiss J, Ooi CE, 72

Arthritis Rheum 1993;36:1054--1060. Nassberger L, Jonsson H, Sjoholm AG, Sturfelt G, Heubner A. Circulating antielastase in systemic lupus erythematosus. Lancet 1989; 1:509. Nassberger L, Sjoholm AG, Jonsson H, Sturfelt G, Akesson A. Autoantibodies against neutrophil cytoplasm component in systemic lupus erythematosus and in hydralazine-induced lupus. Clin Exp Immunol 1990;81:380--383. Nassberger L, Johnansson AC, Bjorck S, Sjoholm AG. Antibodies to neutrophil granulocyte myeloperoxidase and elastase: autoimmune responses in glomerulonephritis due to hydralazine treatment. J Intern Med 1991;229:261--265. Nassberger L, Ljungh A, Schumacher G, Kollberg B. 13glucuronidase antibodies in ulcerative colitis. Lancet 1992; 340:734--735. Ooi CE, Weiss J, Doerfier ME, Elsbach P. Endotoxin-neutralizing properties of the 25 kD N-terminal fragment and a newly isolated 30 kD C-terminal fragment of the 55-60 kD bactericidal/permeability-increasing protein of human neutrophils. J Exp Med 1991;174:649-.655. Peen E, Almer S, Bodemar G, Ryden BO, Sjolin C, Tejle K, Skogh T. Antilactoferrin antibodies and other types of ANCA in ulcerative colitis, primary sclerosing cholangitis, and Crohn's disease. Gut 1993;34:56--62. Pereira HA, Spitznagel JK, Winton EF, Shafer WM, Martin LE, Guzman GS, Pohl J, Scott RW, Marra MN, Kinkade JM Jr. The oncogeny of a 57-kD cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population. Blood 1990;76:825--834. Qi SY, Li Y, O'Connor CD. The region around residue 115 of human bactericidal/permeability-increasing protein is not involved in lipopolysaccharide binding or bactericidal activity. Chemical synthesis and expression of a gene coding for the active domain and characterization of recombinant proteins. Biochem J 1994;298:711--718. Rasmussen N, Wiik A, Hoiter-Madsen M, Borregaard N, van der Woude FJ. Conclusion of the 1st International Workshop on ANCA, 1988. APMIS 1989;97(Suppl 6):27-29. Savage CO, Winearls CG, Jones S, Marshall PD, Lockwood CM. Prospective study of radioimmunoassay for antibodies against neutrophil cytoplasm in diagnosis of systemic vasculitis. Lancet 1987; 1:1389-- 1393. Schmitt WH, Csernok E, Flesch B K, Hauschild S, Gross WL. Autoantibodies directed against lysozyme: a new target antigen for antineutrophil cytoplasmic antibodies (ANCA). Adv Exp Med Biol 1993;336:267--272.

Schumann RR, Leong SR, Flaggs G, Gray PW, Wright SD, Mathison JC, Tobias PS, Ulevitch RJ. Structure and function of lipopolysaccharide binding protein. Science 1990;249: 1429-1431. Seibold F, Weber P, Klein R, Berg PA, Wiedmann KH. Clinical significance of antibodies against neutrophils in patients with inflammatory bowel disease and primary sclerosing cholangitis. Gut 1992;33:657--662. Shafer WM, Martin LE, Spitznagel JK. Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of isopropyl fluorophosphate. Infect Immun 1984; 45:29-35. Sinico RA, Pozzi C, Radice A, Tincani A, Li Vecchi M, Rota S, Comotti C, Ferrario F, Amico G. Clinical significance of antineutrophil cytoplasmic autoantibodies with specificity for lactoferrin in renal diseases. Am J Kidney Dis 1993;22:253260. Spitznagel JK, Pereira HA, Martin LE, Guzman GS, Shafer WM. A monoclonal antibody that inhibits the antimicrobial action of a 57 kD cationic protein of human polymorphonuclear leukocytes. J Immunol 1987;139:1291--1296. van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, van Es LA, van der Giessen M, van der Hen GK, The TH. Autoantibodies against neutrophils and monocytes: tool for diagnosis and a marker of disease activity in Wegener's granulomatosis. Lancet 1985;23:425-429. Weiss J, Elsbach P, Olsson I, Odeberg H. Purification and characterisation of a potent bactericidal and membrane active protein from the granules of human polymorphonuclear leukocytes. J Biol Chem 1978;253:2664--2672. Weiss J, Victor M, Stendahl O, Elsbach P. Killing of gramnegative bacteria by polymorphonuclear leucocytes: role of an O2-independent bactericidal system. J Clin Invest 1982; 69:959--970. Wiik A, van der Woude FJ. Antineutrophil cytoplasmic antibodies (ANCA): a historic review. APMIS 1989;97(Suppl 6):7. Zhao MH, Jones SJ, Lockwood CM. Bactericida/permeabilityincreasing protein (BPI) is an important antigen for antineutrophil cytoplasmic autoantibodies (ANCA) in vasculitis. Clin Exp Immunol 1995a;99:49--56. Zhao MH, Jayne DRW, Ardiles LG, Culley F, Hodson ME, Lockwood CM. Autoantibodies against bactericida/permeability-increasing protein (BPI) in patients with cystic fibrosis. Proceedings of the Sixth International ANCA Workshop. Paris: 1995b.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ANTINUCLEAR ANTIBODIES Peter N. Hollingsworth, D.Phil., Stephen C. Pummer, B.Sc. and Roger L. Dawkins, M.D., D.Sc.

Department of Clinical Immunology, Royal Perth Hospital, Sir Charles Gairdner Hospital, The Centre for Molecular Immunology and Instrumentation, University of Western Australia, Perth 6001, Western Australia, Australia

HISTORICAL NOTES

Antinuclear antibodies (ANA) were discovered when it was realized that the LE cell phenomenon was due to neutrophil phagocytosis of cell nuclei opsonized by autoantibodies (Hargraves et al., 1948). Testing serum on frozen sections of rodent tissue provided a more convenient and sensitive detection system for ANA (Holborow, 1957; Friou, 1993). Several patterns of reactivity, particularly homogeneous, speckled and nucleolar, as well as mixtures of these were soon recognized in the sera from different patients. These and other patterns were more readily recognized on monolayers of cultured cells with their large nuclei and nucleoli and with cells in various phases of the cell division cycle. This implied that autoantigens in various particles within the nucleus and the cytoplasm might be the target of antinuclear antibodies. Extraction and partial or complete biochemical identification of some of these target antigens has been achieved. Indeed antinuclear antibodies are commonly used as probes for purification of antigens by immunoprecipitation and affinity purification and to investigate the function of the antigens by in vitro inhibition of function. Antibodies that bind certain nuclear antigens were shown to be specific for diseases or subsets of disease. For example, high titers of anti-snRNP autoantibodies alone are sometimes considered a marker of mixed connective tissue disease; anticentromere antibodies a marker of CREST syndrome and antidouble-stranded DNA a marker of SLE. The indirect immunofluorescence test for ANA, with its capacity to detect one or several such antibodies does not have the diagnostic specificity of detection of individual

74

antibodies but has endured as a sensitive screening test for the presence of these ANA. ANA is one of the 11 modified A.R.A. criteria for the classification of SLE.

AUTOANTIGENS Standard Nomenclature

In contrast to tissue-specific antigens such as thyroid peroxidase and thyroglobulin, many nuclear and cytoplasmic antigens are common to all nucleated cells. Common and unifying characteristics of the nuclear antigens include evolutionary conservation and function in the cell cycle or in transcription and translation. Many of these antigens form functional particles (e.g., nucleosomes and spliceosomes) with other proteins and are often bound to nucleic acids. The individual nuclear antigens and cognate autoantibodies are named variously according to their cytological location (nucleolar, centromere), their appearance in indirect immunofluorescence (speckled, homogeneous), the name of the patient in whom the cognate autoantibody was first described (Sm, Ro, La), disease associations of the cognate autoantibody (SS-A, SS-B), the particle in which they occur (U1 snRNP) or chemistry (dsDNA) (Tables 1--7). In the tissue sections and cultured cells used in the immunofluorescence assay for ANA, the autoantigens are in their native location and form, undenatured or minimally denatured. Their tertiary structure, epitopes, glycosylation and association with other proteins, nucleic acids, particles and membranes are preserved. The antigens (except Ro/SS-A) are conserved, are not

Table 1. Initial Classification of Immunofluorescence on HEp-2 Cells, According to Location of Binding Interphase cells

Mitotic cells

Nuclear Nuclei Membrane N u c l e o l i Nuclear Homogeneous #1 Speckled #3 Membrane Nucleolar #6

Cytoplasm

Spindle

Chromosomes Nucleoplasm

n

+ +

+

Cytoplasmic

+

Spindle

--

+

#: AFCDC reference preparation number. *: important exceptions. No symbol: obscured or variable. tissue/organ specific and are, therefore, predicted to be available in all tissues from all vertebrates, or at least mammals. In systematic studies, liver, kidney and certain cultured cells lines from human, monkey, dog, various rodents including mouse, rat, rabbit and hamster and from chicken, were roughly equivalent as substrates for detection of ANA to conserved antigens (Harmon et al., 1984; Kozin et al., 1980). In contrast, Ro/SS-A was available in human, monkey, dog and guinea pig, but not mouse, rat, rabbit or hamster. Among cultured human and monkey cell lines, it was more abundant in epithelial cancer cell lines and kidney cells than in lymphoid cells (Harmon et al., 1984) (Table 8).

Fixation and Permeabilization. For tissue sections, air drying of the tissue on a slide immobilizes components adequately and chemical fixation is not usually needed. However, light fixation with acetone may improve tissue preservation and reduce leaching

of soluble antigens during processing. Cultured cells, grown in a monolayer on slides, have an intact cell membrane. Permeabilization of the cell membrane with a lipid solvent is necessary to ensure that antibodies can penetrate. Additional fixation is also used, and the optimal fixation may vary for different antigens. Fixatives and tissues were systematically evaluated (Harmon et al., 1984; Kozin et al., 1980) (Table 8). Although a range of fixatives and fixation times are adequate for most relevant antigens, Ro/SS-A is very susceptible to leaching and denaturation. Acetone alone for 1 to 10 rain is satisfactory for Ro/SS-A. Ethanol and methanol reduce nuclear immunofluorescence and permit translocation of Ro/SS-A to the cytoplasm and entire cellular area. These changes progress with time of fixation from 1 to 10 min. Paraformaldehyde with Triton-X preserves Ro/SS-A in the nucleus but does permit some to appear in the cytoplasm (Humbel, 1993).

Table 2. Homogeneous Nuclear Immunofluorescence Pattern

Figure

Particle

Antigen

Antibody Frequency % Health*

Diffuse # 1

Rim

Disease

0.5

1.1

1.2

Ref.

Chromatin

dsDNA

0

SLE 60

a

Chromatin

Histone

0

Drug-LE 95 SLE 60

b, c

Chromatin

Topo-1

0

PSS 15-70

a

Chromatin

dsDNA

0

SLE

a, d

*Topo-l: Topoisomerase-1 (Scl-70); SLE: Systemic Lupus Erythematosus; PSS: Progressive Systemic Sclerosis (Scleroderma); Drug-LE: Drug Induced SLE (Fritzler et al., 1985). a) Tan, 1989; b) Fritzler and Tan, 1978; c) Burlingame and Rubin, 1994; d) Senecal and Raymond, 1991

75

Table 3. Speckled Nuclear Immunofluorescence

Pattern

Figure

Particle

Antigen

Antibody Frequency % Health

Speckled #3

Ref.

Disease

2.7

Large sp

1.3

Matrix

hnRNP

1.0

MCTD 100 SLE, CTD

b

Coarse sp #4

1.4

UlsnRNP

U1 snRNP 70,33, 22

0.04

MCTD 100 SLE 25

a

U 1, 2, 4+6, 5 snRNP

Sm snRNP core 29, 28, 16

0.0

SLE 20

a

Ki 66,86

0.0

SLE l0

c

Coarse sp #5 Coarse sp Fine sp #2

1.5

SS-A/Ro et al.

SS-B/La48

0.04

SLE15 Sj 40

a

Finer sp #7

1.6, 1.7

SS-A/Ro

SS-A/Ro 60, 52

0.44

SLE 35 Sj 60

a

Finest, chr+ #9

1.8

Chromatin

Topo-1 (Scl 70)

0.0

PSS 15-70 CREST 7--21

a, d

Pleiomorphic

1.9

PCNA

Cyclin

0.0

SLE 2

e

CenPE (NSp2)

0.76

various

f

Fine & chr + sp 46 dots #8

1.10

Centromere

CENP 17, 80, 160

0.08

CREST 80 PBC15

g

5-- 10 dots

1.11

Nuclear body

Sp 100 (NSp 1)

0.76

Sj, PBC

f, h

Coiled body

Coilin p80

0.16

Sj, PBC

i

2--6 dots

chr +: chromosome immunofluorescence in mitotic cells; MCTD: Mixed Connective Tissue Disease; Sj: Sj6gren's syndrome. CREST: Calcinosis, Raynaud's phenomenon, Esophageal dysfunction, Sclerodactyly and Telangiectasia; PBC: primary biliary cirrhosis. a) Tan, 1989; b) Fritzler et al., 1984a; c) Francoeur et al., 1986; d) Kuwana et al., 1994); e) Fritzler et al., 1983; f) Fritzler et al., 1984b; g) Tan et al., 1980; h) Fuscini et al., 1991; i) Andrade et al., 1991.

T i s s u e Sections. Sections of frozen, unfixed rodent tissue are widely used, because the in situ antigens are undenatured and sections can be prepared inexpensively. Light fixation with acetone by dipping the slides with freshly cut sections into two changes of

acetone before air drying may improve preservation and reduce leakage of soluble antigen from the tissue during processing. If a composite block of tissue (including rat and m o u s e stomach and rat kidney, liver and heart) is used, A N A can be quantitated without

Table 4. Nuclear Membrane Immunofluorescence

Pattern

Homogeneous Punctate

Figure

1.12

Particle

Antibody Frequency % Health

Disease

Ref.

Inner membrane

Lamin A,C,B

0.0

Hepatitis, SLE, ACL, cytopenia

a

Pore complexes

gpl20

0.0

Polymyositis, rare

b

Lamin B/receptor ACL: anticardiolipin. a) Lassoued et al., 1988; b) Senecal and Raymond, 1991.

76

Antigen

Table 5. Predominantly Nucleolar Immunofluorescence Pattern

Figure

Particle

Antigen

Nucleolar #6 Homogeneous

1.13

Speckled Clumpy chr+

1.14

Dots

Antibody Frequency %

Ref.

Health

Disease

0.8

PSS 15

Preribosomes

Nucleolin (PM/Scl)

PM/PSS 50 PSS 3; PM 8

RNA polymerase

Poll Poll, 2, 3

PSS 5 PSS 2-43

a, c

U3 SnRNP

Fibrillarin

PSS 8

c,d

NOR

NOR-90

PSS

NOR: Nucleolar Organizing Region. PM: Polymyositis a) Reimer et al., 1988; b) Bluthner et al., 1992; c) Kuwana et al., 1994; d) Okano et al., 1992; e) Rodriguez-Sanchez, 1987.

serial dilution (Bonifacio et al., 1986), and other autoantibodies including parietal cell, mitochondrial and smooth muscle antibodies can be detected simultaneously. Sensitivity for detection of antibodies to conserved antigens is similar to that of cultured cells (Harmon et al., 1984). The detection limit for the W H O 6 6 / 2 3 3 h o m o g e n e o u s A N A standard is approxi-

mately two international units (IU) per mL (Table 9), (Bonifacio et al., 1986). This is similar to the limit of detection on HEp-2 cells (see below), despite the fact that the initial dilution and end point titers are usually two- to fourfold lower than on HEp-2. Mouse kidney may be the easiest substrate with which to exclude ANA, and, thereby, SLE (Molden et al., 1984).

Table 6. Cytoplasmic Immunofluorescence on HEp-2 Cells Pattern

Figure

Particle

Antigen

Antibody Frequency % Health

Cytoplasmic

Ref.

Disease

3.6

Fine granular Perinuclear # 10

1.15

tRNA synthetase

Jo-1

0.0

PM+ILD 70

Dense

1.16

Ribosome

Ribosomal P

0.0

SLE 10%

b

Coarse sp

1.17

Mitochondria

M2 (2-OADC)

1.0

PBC 97% CREST 15% CAH/PBC Benign PBC

c, d, e

M4 M8,9

d d

Coarse sp

other*

Eccentric sp

ER

b b

Polar

1.18

Golgi

Filamentous

1.19

Long short radial

Actin Tropomyosin Vimentin

1.0

CAH

b

Hepatitis B

Jo-l: Histidyl t-RNA synthetase. ER: Endoplasmic reticulum. OADC: Oxo Acid Dehydrogenase Complex. ILD: Interstitial Lung Disease. CAH: Chronic Active Hepatitis. * Lysosomes, Peroxisomes, Signal Recognition Protein. a) Saito et al., 1991; b) Humbel, 1993; c) Berg et al., 1982; d) Berg and Klein, 1989; e) Coppel et al., 1988.

77

Table 7. Mitotic Spindle Apparatus Immunofluorescence. Pattern

Figure

Panicle

Antigen

Antibody Frequency % Health

2 Dots

Centriole

Enolase 48

0.08

Ref.

Disease

Spindle fibers

1.20

MSA

Tubulin

0.16

Spindle Poles

1.21

NuMA

NuMA, 250

0.0

SLE, Sj, (rare)

c

Mid-body

1.22

MSA-2

0.0

PSS

d

MSA-3

0.16

Granules around metaphase plate

a) Rattner et al., 1991" b) Saito et al., 1991; c) Price et al., 1984" d) Fritzler et al., 1984b" e) McCarty et al., 1984.

Disadvantages are that solid tissues are poor in Ro/SS-A antigen, the nuclei and nucleoli are small and few cells are dividing. Some antibodies, which yield cell cycle-dependent patterns, will not be recognized. C u l t u r e d Cells. Monolayers of cultured cells, particularly HEp-2, a human laryngeal carcinoma cell line (American Type Culture Collection CCL-23), are

superseding rodent tissue. Nuclei and nucleoli are large, and dividing cells are plentiful so that antibody patterns are readily recognized. Fixation of the cells and permeabilization of the cell m e m b r a n e is necessary. Of several fixation methods (Harmon et al., 1984), acetone is the benchmark for preservation of Ro/SS-A. For A N A screening in general, two methods are r e c o m m e n d e d (Humbel, 1993). Methanol-acetone: Wash slides three times in cold phosphate-buffered

Table 8. Species, Tissues, Cultured Cells, and Fixatives Suitable for Detection of ANA to Conserved Nuclear Antigens, with Suitability for Ro/SS-A Shown as + or Tissue (Liver, Kidney) Human

Monkey Dog Rodents Guinea Pig Mouse Rat Rabbit Hamster

Cultured Cells +

+

Human Epithelial HEp-2, KB, HeLa*** Fibroblast Lymphoid WiL2**, Ramos Monkey Kidney-Vero

Fixatives

++ ++ ++

++

+ +

Mouse Myeloma Ehrlich Ascites

Hamster Kidney BHK-21

Chicken Unsuitable fixatives: Formalin; Periodate-Lysine-Paraformaldehyde; 2% Glutaraldehyde. * Paraformaldehyde 3%. ** EBV infected. *** Cancer cell lines: HEp-2 laryngeal, KB oropharyngeal, HeLa cervical. Based on Harmon et al., 1984; Kozin et al., 1980; Humbel, 1993.

78

None Acetone Ethanol Methanol Pf*/Triton-X

++ + +

Table 9. Fluorescence Intensity Scale Scale

Molden et al.

Fritzler et al.

Fluorescent Beads

IU/mL

4

brilliant

highest

100%

>30

3

bright

50%

30

2

clear

25%

15

1

weak*

pattern visible

0

none**

none

12.5% 0%

7.5 0

* "weak but reliably detectable". ** "none or insufficient for reliable detection".

saline; immerse in methanol a t - 2 0 ~ for 5 min; dip in two changes of acetone; air dry for 15 min; store in air-tight container a t - 2 0 ~ immediately before use, thaw quickly with a cool fan. P a r a f o r m a l d e h y d e Triton-X: Wash cells three times in cold PBS; fix in 3% paraformaldehyde in PBS for 15 min at room temperature; wash three times in PBS; immerse in Triton-X 100 0.2% in PBS for 5 min at room temperature; wash three times in PBS; use immediately. Methods of fixation of commercially supplied HEp-2 cells are usually not disclosed. They vary in capacity to detect antibodies, especially to Ro/SS-A (Figure 1). Transfection of HEp-2 cells with a full-length Ro60 clone and overexpression of the Ro in a subset of HEp-2 cells ensures detection and positive identification of antibodies to Ro/SS-A (Keech et al., 1994).

AUTOANTIBODIES

Terminology ANA has superseded antinuclear factors (ANF) as the preferred appellation for any or all autoantibodies reactive with cell nuclei. In diagnostic practice, ANA is taken to mean antinuclear antibodies demonstrable in s i t u by indirect immunofluorescence or immunoenzyme techniques. ANA may be classified according to patterns observable by indirect immunofluorescence. Binding of antibody to intracellular structures and particles produces the patterns. Inversely, the pattern predicts, imperfectly, the structure or particle and the proteins within it that bind the antibody (Tables 1--7) (Figure 1). With few exceptions, (anticentromere, anti-PCNA and anti Ro/SS-A on transfected cells, for example), however, immunofluorescence patterns do not provide definitive identification of antibodies (Figure 2).

Mixture of antibodies with or without mixed patterns are usual, particularly because screening dilutions can be difficult to discern; certain patterns are produced by more than one antibody, and subtle differences in patterns; for example, coarse vs. fine-speckled (antiU I-snRNP vs. anti-SS-B). Secondary testing is, therefore, necessary for identification of the specific autoantigen reactive with the autoantibodies. The role of the immunofluorescence ANA test is to select the sera in which such testing is necessary and to guide the selection of secondary tests (Homburger, 1995).

Pathogenetic Role Anti-Ro/SS-A bind to keratinocytes in a model system and are found in affected tissue in parotitis, glomerulonephritis and congenital heart block (Reichlin, 1993; Sontheimer et al., 1992). Anti-dsDNA bound to DNA and to nucleosomes can be found in the circulation and in kidney tissue of animals and humans with lupus glomerulonephritis (Sontheimer et al., 1992).

Genetics Prevalence of ANA, and of SLE, is increased in firstdegree relatives of SLE patients. ANA is also increased in their spouses and in their pet dogs (Jones et al., 1992). Therefore, genetic and environmental factors are implied. The association of alleles of the HLA DR locus in the major histocompatibility complex with particular antibodies to extractable nuclear antigens (Smolen et al., 1987) might be explained as an antigen-specific effect due to the polymorphism of these antigen-presenting molecules.

Pathogenetic Factors In sera of SLE patients, over 96% of ANA include

79

Figure 1.1. Nuclear: homogeneous, diffuse. Even immunofluorescence throughout all nuclei and in chromosomes of mitotic cells (anti-dsDNA, antihistone, anti-Scl-70).

Figure 1.2. Nuclear: homogeneous, rim. A thick rim of homogeneous immunofluorescence around the edge of the nucleus and in chromosomes (anti-dsDNA).

Figure 1.3. Nuclear: large speckled. Irregular large speckles, nucleoli negative, chromosomes negative (antinuclear matrix).

Figure 1.4. Nuclear: coarse speckled. Irregular large and

Figure 1.5. Nuclear: fine speckled, chromosomes negative (antiLa/SS-B).

80

smaller granules, nucleoli negative, chromosomes negative (anti-U 1 snRNP).

Figure 1.6. Nuclear: fine speckled, chromosome negative with cytoplasmic speckles (anti-Ro/SS-A).

Figure 1.7. Nuclear: Anti-Ro/SS-A on HEp-2 cells transfected with Ro/SS-A. Brilliant nuclear and nucleolar immunofluorescence in the cells overexpressing Ro/SS-A and weak in the remainder.

Figure 1.8. Nuclear: Very fine speckled~homogeneous immunofluorescence of nuclei and chromosomes, and sometimes nucleoli (anti-Scl-70).

Figure 1.9. Nuclear: pleiomorphic speckled. Various speckled

Figure 1.10. Nuclear: discrete even nuclear speckles or dots, in

patterns confined to nondividing (S-phase) cells. Dividing cells negative (anti-PCNA).

multiples of 46, in cell nuclei. Aligned with chromosomes in dividing cells (anticentromere).

Figure 1.11. Nuclear: 5-10 nuclear dots. NSpl or Multiple Nuclear Dot pattern.

Figure 1.12. Nuclear membrane: thin homogeneous ring. Immunofluorescence in nucleoplasm around, but not in, chromosomes in dividing cells (antilamin).

81

Figure 1.13. Nucleolar: homogeneous within nucleolus (antiPM/Scl).

Figure 1.14. Nucleolar: clumpy within nucleolus (antifibrillarin).

Figure 1.15. Cytoplasmic: fine granular, concentrated around the nucleus (anti-Jo-1/Histidyl tRNA synthetase).

Figure 1.16. Cytoplasmic" dense fine granular (antiribosomal).

Figure 1.17. Cytoplasmic: coarse streaked speckles (antimitochondria).

Figure 1.18. Cytoplasmic: Polar arrowhead-shaped structure (anti-Golgi).

82

Figure 1.191 Cytoplasmic: Filamentous.

Figure 1.20. Mitotic spindle Fibers: Mitotic Spindle Apparatus (MSA).

Figure 1.21. Spindle Poles: Nuclear mitotic apparatus (NuMA).

Figure 1.22. Midbody: Negative in interphase; patchy speckles in S and G2; speckled chromosomal in metaphase and confined to the cleavage furrow (midbody) in telophase (MSA-2).

30

20 C

z

10

000

the IgG isotypes; 35% include IgM and 16% IgA (Puritz et al., 1973). All examples of anti-SS-B include IgG, with much lower IgA in 50% and even lower IgM in 25% (Venables et al., 1983). These findings suggest that testing for IgG A N A will be sufficient to detect sera that contain ANA. In ANA, IgG1 and IgG3 predominate. In one study, IgG1 accounted for 55% of total antibody activity to native and denatured DNA, Sm, and histone; 84% to anti-SS-B and 92% to anti-RNP

oooo

I

B

I

C

HEp-2 Cell Supplier

I

A(Ro)

Figure 2. ANA values for 12 sera with anti-Ro (SAS) demonstrated by immunodiffusion but low or undetectable ANA immunofluorescence on HEp-2 cells from manufacturer A. Values determined on substrates from three manufacturers A, B and C are shown as well as results for cells transfected with and overexpressing a full-length Ro-60 clone, provided by manufacturer A. 83

(Rubin et al., 1986). The remainder was mainly IgG3. IgG2 constituted only 3--12% of anti-DNA, <5% of anti-SS-B in <10% of cases, and was not detectable in anti-RNP and anti-Sm. IgG4 was uncommon but did constitute some 10% of drug-induced antihistone, 5% of anti-dsDNA in SLE, and 5% of anti-RNP and antihistone antibodies. Anti Ro/SS-A was found to be restricted to IgG1 (Maran et al., 1993). Predominance of IgG1 and IgG3 in all these types of ANA suggests a common immunogenic process and is characteristic of responses driven by protein antigens. Methods of Detection

Technical details of immunofluorescence microscopy and the indirect immunofluorescence assay are widely available (Humbel, 1993). In brief, incident light fluorescence (el~ifluorescence) is used. In order to reduce the background staining and improve contrast, sera are diluted 1:10 for screening on tissue and 1:40 for screening on HEp-2 cells. Tissue sections should be 4 microns or less in thickness and should be airdried promptly after cutting and used immediately or

stored in air-tight containers a t - 2 0 ~ and thawed quickly immediately before use. Similarly HEp-2 cell slides should be kept in air-tight bags at 4~ until immediately before use. The tissue and cells should not be allowed to dry during processing. High-quality, fluorescein-conjugated anti-immunoglobulins are commercially available. Animal IgG antihuman IgG is now preferred for the fluorochrome conjugate (Humbel, 1993). Detection of IgM and IgA ANA will not detect more SLE or connective tissue diseases and may reduce specificity of the test (Molden et al., 1984; Puritz et al., 1973). Previously, antiimmunoglobulin, raised against gamma globulin (Cohn fraction II) was recommended (Holborow and Johnson, 1983) and widely used. This reacts mainly with IgG but also with IgM and IgA. If screening for IgM and IgA as well as IgG is required, then a blend of equal proportions of isotype-specific anti-IgG, antiIgM and anti-IgA is more efficient. Higher ratios of fluorochrome to antibody will increase immunofluorescence but also nonspecific immunofluorescence. Useful conjugates typically have molar ratios of fluorescein to protein of around 2. The optimum dilution should be determined by a checkerboard

Table 10. Frequency Distributions of ANA in Putatively Healthy Adult Donors and in Consecutive Diagnostic Referrals, Relating Titers, International Units and Percentiles Percentile

Diagnostic

HealthyDonors

WA

Alberta*

Western Australia (WA)

Female

Female

2500

106

106

91

91

63014

HEp-2

HEp-2

Rat

HEp-2

Rat

HEp-2/Rat

Titer

IU/mL

IU/mL

IU/mL

IU/mL

IU/mL

100th

>1/160

25

20

9

12

>30

99th

1/160

20

15

8

l0

>30

98th

1/80

12

10

8

8

30

95th

1/40

7

7

6

6

20

5

4

4

3

10

2

0

2

0

0

0

0

0

90th 85th

1/20

75th 65th

1/10

50th * Fritzler et al., 1985.

84

Female and Male

Male

titration (Holborow and Johnson, 1983). The diluted conjugate should be centrifuged at 10,000 rpm for 10 min before use to remove aggregates.

Reading of Immunofluorescence. The tasks of the reader are to estimate the immunofluorescence intensity as a measure of the amount of ANA and to recognize the pattern. Between 75 and 95% of diagnostic samples in a routine laboratory will have ANA values that can also be encountered in putatively healthy individuals (see below and Table 10). The lower end of the distribution of ANA in newly presenting SLE also overlaps the range found in health (see below and Figure 3). The first task then is to quantitate relatively low amounts of ANA precisely so that discrimination of health from SLE and other ANA-associated conditions is optimal. The ANA patterns most commonly found in putatively healthy individuals are mixed homogeneous and speckled, speckled and homogeneous (Fritzler et al., 1985). These patterns provide rather diffuse nuclear immunofluorescence, particularly when viewed at 100 x magnification. For quantitation of immunofluorescence intensity, the scale of 0, 1+, 2+, 3+, 4+ is commonly used (Molden et al., 1984; Fritzler et al., 1985) (Table 9). This scale spans the range from undetectable to 100 90-

-

Health

SLE

(196)

{45)

>.. 8 0 -

O c

70-

~r 6 0 0 L_

"

50-

=-

40-

_m 3 0 E 200 10-

I

<2 .5 2.5

5

7.5 ANA

10

15

20

30 >30

IU/ml

Figure 3. Cumulative frequency plots for ANA in healthy individuals (summed to the left) and for newly presenting SLE (summed to the fight). See also Table 11.

maximal perceived immunofluorescence intensity It has not been related to any independent standard of ANA activity. The use of fluorescent glass beads with relative intensities of 0%, 12.5%, 25%, 50% and 100%, to define 0, 1+, 2+, 3+ and 4+. This scale gives three doubling increments between 1+ and 4+. This is a discontinuous scale and the appropriate way to assign a score to sera lying between 0 and 1+ is not clear (Molden et al., 1984). Only two increments (0, 1+, 2+) describe the range from 0 to approximately 15 IU/mL within which lie the majority of ANA values encountered in health and the lower end of the range encountered in newly diagnosed SLE (see below and Table 10). We have found that the range 0 to 4+ equates to the range 0 to over 30 IU/mL as defined by the reference preparation WHO 66/233 (Johnson and Holborow, 1980) (Table 9). The use of immunofluorescent beads for that quantitation of immunofluorescence from 0 to 4+ has not been widely adopted (Molden et al., 1984). Rather, titration by serial 1 in 2 or 1 in 4 dilution is most commonly used. The end point is taken as 1+ (Fritzler et al., 1985) or as the last dilution in which ANA is detectable. Predictably, such titrations are imprecise (Hollingsworth et al., 1987) and inaccurate, with coefficients of variation (cv) of over 100% and up to seven doubling dilutions of difference in the results reported from different laboratories (Hollingsworth et al., 1987; Feltkamp, 1993). Use of standard sera as in other immunoassays improves precision and accuracy (Hollingsworth et al., 1987; Feltkamp, 1993). ANA within the range 0 - 3 0 IU/mL and particularly within the range 0--15 IU/mL can be estimated on rodent tissue (Table 11). Greater analytical sensitivity and precision can be achieved, detecting increments of 2.5 units or lower with a coefficient of variation 9% at 7 IU/mL (Bonafacio et al., 1986). Similar analytical sensitivity and precision can also be achieved by visual assessment of immunofluorescence intensity on HEp-2 cells, assisted by calibrator sera that have, in turn, been calibrated against the reference preparation WHO66/233 defining activity of homogeneous ANA in international units (IU/mL) (Figures 4--6). The coefficient of variation among assays at 7 IU/mL is 9%. The practical advantage of measuring ANA over the range 0--30 IU/mL in this way is that ANA can be quantitated in up to 98% of diagnostic samples without the necessity for serial dilution. Sera with ANA activity beyond this range can be prediluted in negative serum prior to assay, if required.

85

Table 11. Method of Quantitation of Nuclear Immunofluorescence on Rat Tissue Nuclear Immunofluorescence

Score

Approx. IU/mL

Heart

Liver

Kidney

+

0

0

0.5

1

+

0

0

1

2.5

+

0

+

2

5

+

+

0

2

5

+

+

+

3

7.5

+

+

+

4--10

10--30

At scores of 2 and 3, only the nuclei at the edge of the liver section are stained. Scores of 4 to 10 are judged on intensity in nuclei throughout the liver, assisted by calibrator sera.

Figure 5. ANA values for four sera included as masked controls in 50 consecutive routine assays.

Figure 4. A trial of quantitation of ANA by estimation of immunofluorescence intensity. The homogeneous ANA standard WHO66/233, with a value of 100 IU/ampule was diluted in negative serum to produce dilutions with target values increasing in increments of 2.5 from 0 to 25 U/mL. These dilutions were used as calibrators. They were also masked and assayed (71). A second serum with homogeneous ANA 175 IU/mL determined by end point titration versus WHO66/233 was also diluted to produce dilutions with target values in increments of 2.5 units from 0 to 25, masked and assayed simultaneously (1). A third set of dilutions of a serum with mixed speckled and homogeneous ANA that had been previously calibrated against WHO66/233 was similarly masked and assayed (n).

Patterns of Immunofluorescence. An initial classification can be achieved with either rodent tissue or cultured cells using 100 x magnification (Table 1).

86

The texture and location of i m m u n o f l u o r e s c e n c e and variation with cell cycle as shown on cultured cells are informative. M o r e patterns can be resolved on cultured cells with their large nuclei and cells in various phases of mitosis than on tissue sections. Further resolutions of patterns can be attempted at 400 • magnification (Tables 2--7) (Figure 1). Of the ten A F C D C reference preparations defining various types of A N A , only 1, 2 and 3 have patterns formally assigned, but patterns of the other seven preparations have been described ( M o l d e n et al., 1984) (Tables 1--7). There are not yet reference preparations for all of the A N A patterns.

!

_>320 -

Brl

-% 'A'W

160 -

(9 r

80-

DV

PATTERNS

<:: Z

.D***,

40-

<1::

o Homogeneous (H) . Speckled (S) [] Nucleolar (N) 9 Centromeric

<40

O Anti-SSA

00000000 O00iOOeO00 OOgO 9 9 9

-

VS+H E]S+N 9 No pattern designated

I

""i'

0

5

"

I

I ......

10

15

I

I

20

25

...........

I

'

30

>30

ANA units Figure 6. Comparison

of titration by doubling dilution with assay by assessment of immunofluorescence intensity for sera with ANA of various values and patterns.

100 -

100

-

g080UJ

80

70-

03

60-

-r" p-

50-

60

40-

40

3020100

20

== O

tO

II O)

-9

ANA

O~

O

i

A

0 CM

IU/ml

Figure 7. Observed predictive value of ANA for SLE in teaching hospital patients. Consecutive patients with ANA values falling in the ranges shown were selected and their case notes were examined to determine the number of ARA diagnostic criteria for SLE. The percentage with three or more such criteria other than ANA is plotted.

u~ V

LO

LO

O

U3

O

if)

O

O

,

I LO

I

I

I .CO "-"

I ,.'04

I t.D 04

A

r

~ I~.

ANA

~ ~--

IU/ml

Figure 8. Predictive value of ANA for anti-DNA and antiENA. Data for 100 consecutive diagnostic samples are shown, relating the percentage with anti-dsDNA >7 IU/mL and/or antiENA detected by immunodiffusion are plotted in relation to the ANA value.

87

CLINICAL UTILITY Laboratory test results can be used to confirm or exclude a diagnosis, to subclassify a disease and to monitor disease activity (Dawkins and Peter, 1980). Few, if any, tests will satisfy all these objectives.

Application The usual application of the immunofluorescence assay for ANA is a screening test for the presence of these antibodies and thereby as a screening test for SLE, as described above. The prevalence of ANA in certain autoimmune diseases is well established (Tables 2--7). Other conditions are also associated with increased ANA (Reichlin, 1993; Homburger, 1995)which increase with age and certain infectious diseases such as chronic abscesses, tuberculosis, subacute bacterial endocarditis and malaria. Many drugs increase ANA, particularly procainamide, hydralazine, isoniazid, chlorpromazine and beta-blockers. The diagnostic utility of ANA as a screening test for SLE can be simply illustrated (Figure 3). ANA values below 5 IU/mL exclude untreated SLE; values above 20 IU/mL exclude health. The diagnostic sensitivity (probability of positive ANA given SLE) falls and the positive predictive value (probability of SLE given positive ANA) rises as the decision threshold is increased (Dawkins and Peter, 1980). Clearly, there is no decision threshold which will absolutely separate health from SLE. The decision threshold selected depends on the purpose of the test. If ANA are used as a screening test for suspected SLE, a decision threshold around 7 IU/mL is most appropriate, and assay should be controlled so as to produce optimal precision at this decision threshold. Selection of a different decision threshold or an underestimate or overestimate of ANA around the decision threshold will have a large impact on the predictive value of the

REFERENCES Andrade LEC, Chan EKL, Raska I, Peebles CL, Roos G, Tan EM. Human antibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. J Exp Med 1991;173:1407-1419. Berg PA, Klein R, Lindenborn-Fotinos J, Kloppel W. ATpaseassociated antigen (M2): marker antigen for serological diagnosis of primary biliary cirrhosis. Lancet 1982;2:14231425. 88

test (Figure 3). The predictive value for SLE of several quantitative intervals of ANA concentrations can also be estimated from consecutive ANA results from laboratory records together with independent evaluation of hospital case notes to ascertain the prevalence of diagnostic criteria for SLE (Figure 7). The probability of SLE rises steeply as the results of ANA quantitation increase from 5 to >30 IU/mL in these patients of a teaching hospital. The same trend, if not the same probabilities, might be expected in patients from general practice where the a priori probability of SLE would be lower, but data are not available. Quantitation of ANA can also be useful as a predictor of the presence of anti-dsDNA and of antiENA including anti-snRNP/Sm, anti-SS-A and antiSS-B antibodies. In 100 consecutive diagnostic samples, the predictive value increases steeply as the ANA concentrations increase over a range from 0 - 3 0 IU/mL (Figure 8).

CONCLUSION The immunofluorescence assay for ANA can be configured and controlled so as to provide an efficient screening test for SLE and for antinuclear antibodies relevant to SLE and connective tissue disease. However, titration by serial dilutions is imprecise and has not been satisfactorily standardized. Small inaccuracies around the decision threshold will have a large impact on sensitivity, specificity and predictive value of the test. Use of standard sera for preparation of a standard curve improves precision at decision thresholds and greatly enhances the clinical utility of ANA testing. Improved analytical sensitivity and precision can be achieved by careful assessment of the intensity of immunofluorescence, assisted by calibrator sera, and results can be rendered in units based on the WHO standard for homogeneous ANA 66/233.

Berg PA, Klein R. Heterogeneity of antimitochondrial antibodies. Sem Liver Dis 1989;9:103--116. Bluthner M, Bautz FA. Cloning and characterization of the cDNA coding for a polymyositis-scleroderma overlap syndrome-related nucleolar 100-kd protein. J Exp Med 1992;176:973-980. Bonifacio E, Hollingsworth PN, Dawkins RL. Antinuclear antibody: precise and accurate quantitation without serial dilution. J Immunol Methods 1986;91:249--255. Burlingame RW, Rubin RL. Histones. In: Van Venrooij WJ,

Maini RN, eds. Manual of Biological Markers of Disease. The Netherlands: Kluwer Academic Publishers, 1994:1-28. Coppel RL, McNeilage LJ, Surh CD, Van De Water J, Spithill TW, Whittingham S, Gershwin ME. Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase. Immunology 1988;85:7317-7321. Dawkins RL, Peter JB. Laboratory tests in clinical immunology. Am J Med 1980;68:3--5. Feltkamp TEW. Standards and reference preparations. In: Van Venrooij WJ, Maini RN, eds. Manual of Biological Markers of Disease. The Netherlands: Kluwer Academic Publishers, 1993:A11/1-11. Francoeur A, Peebles CL, Gompper PT, Tan EM. Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity. J Immunol 1986; 136:1648-1653. Friou GJ. The early days of the antinuclear antibody story: where and how did it all start? Ann Med Interne (Paris) 1993;144:154-156. Fritzler MJ, Tan EM. Antibodies to histones in drug-induced and idiopathic lupus erythematosus. J Clin Invest 1978;62: 560--567. Fritzler MJ, McCarty GA, Ryan JP Kinsella TD. Clinical features of patients with antibodies directed against proliferating cell nuclear antigen. Arthritis Rheum 1983;26:140-- 145. Fritzler MJ, Ali R, Tan EM. Antibodies from patients with mixed connective tissue disease react with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) of the nuclear matrix. J Immunol 1984a;132:1216-1222. Fritzler MJ, Valencia DW, McCarty GA. Speckled pattern antinuclear antibodies resembling anticentromere antibodies. Arthritis Rheum 1984b;27:92-96. Fritzler MJ, Pauls JD, Kinsella TD, Bowen TJ. Antinuclear, anticytoplasmic, and anti-Sj6gren's syndrome antigen A (SSA/Ro) antibodies in female blood donors. Clin Immunol Immunopathol 1985;36:120-128. Fuscini M, Cassani F, Govohi M, Caselli A, Farabegoli F, Lenzi M, Ballardini G, Zauli D Bianchi FB. Antinuclear antibodies of primary biliary cirrhosis recognize 78-92 kd and 96-100 kd problems of nuclear bodies. Clin Exp Immunol 1991;83:291-297. Hargraves M, Richmond H, Morton R. Presentation of two bone marrow components, the tart cell and the LE cell. Mayo Clin Proc 1948;27:25-28. Harmon CE, Deng J, Peebles CL, Tan EM. The importance of tissue substrate in the SS-A/Ro antigen-antibody system. Arthritis Rheum 1984;27:166-173. Holborow EJ, Weir DM, Johnson GD. A serum factor in lupus erythematosus with affinity for tissue nuclei. Br Med J 1957;2:732. Holborow EJ, Johnson GD. Standardization in immunofluorescence. Ann NY Acad Sci 1983;420:62-64. Hollingsworth PN, Bonifacio E, Dawkins RL. Use of a standard curve improves precision and concordance of antinuclear antibody measurement. J Clin Lab Immunol 1987;22:197200. Homburger HA. Laboratory medicine and pathology: cascade testing for autoantibodies in connective tissue diseases. Mayo

Clin Proc 1995;70:183-184. Humbel RL. Detection of antinuclear antibodies by immunofluorescence. In: Van Venrooij WJ, Maini RN. Manual of Biological Markers of Disease. The Netherlands: Kluwer Academic Publishers, 1993:A2/1-16. Johnson GD, Holborow EJ. Standardisation of tests for antinuclear antibody [Letter]. Ann Rheum Dis 1980;39:529. Jones DRE, Hopkinson ND, Powell RJ. Autoantibodies in pet dogs owned by patients with systemic lupus erythematosus. Lancet 1992;339:1378--1380. Keech CL, McCluskey J, Gordon TP. Transfection and overexpression of the human 60-kd Ro/SS-A autoantigen in HEp2 cells. Clin Immunol Immunopathol 1994;73:146-151. Kozin F, Fowler M, Koethe SM. A comparison of the sensitivities and specificities of different substrates for the fluorescent antinuclear antibody test. Am Soc Clin Pathol 1980;74: 785-790. Kuwana M, Okano Y, Kaburaki J, Tojo T, Medsger TA Jr. Racial differences in the distribution of systemic sclerosisrelated serum antinuclear antibodies. Arthritis Rheum 1994 ;37:902--906. Lassoued K, Guilly M-N, Danon F, Andre C, Dhumeaux D, Clauvel A-P, Brouet J-C, Seligmann M, Courvalin JC. Antinuclear autoantibodies specific for lamins. Ann Intern Med 1988;108:829-833. Maran R, Dueymes M, Pennec YL, Casburn-Budd R. Predominance of IgG1 subclass of anti-Ro/SS-A, but not anti-La/SSB antibodies in primary Sj6gren's syndrome. J Autoimmun 1993;6:379--387. McCarty GA, Valencia D, Fritzler MJ. Antibody to the mitotic spindle apparatus Immunological characteristics and cytological studies. J Rheumatol 1984;11:213-218. Molden DP, Nakamura RM, Tan EM. Standardization of the immunofluorescence test for autoantibody to nuclear antigens (ANA): use of reference sera of defined antibody specificity. Am J Clin Pathol 1984;82:57-66. Okano Y, Steen VD, Medsger TA Jr. Autoantibody to U3 nucleolar ribonucleoprotein (fibrillarin) in patients with systemic sclerosis. Arthritis Rheum 1992;35:95--100. Price CM, McCarty GA, Pettijohn DE. NuMA protein is a human autoantigen. Arthritis Rheum 1984;27:774-779. Puritz EM, Yount WJ, Newell M, Utsinger PD. Immunoglobulin classes and IgG subclasses of human antinuclear antibodies. Clin Immunol Immunopathol 1973;2:98-113. Rattner JB, Martin L, Waisman DM, Johnstone SA, Fritzler MJ. Autoantibodies to the centrosome (centriole) react with determinants present in the glycolytic enzyme enolase. J Immunol 1991 ;146:2341-2344. Reichlin M. ANAs and antibodies to DNA: their use in clinical diagnosis. Bull Rheum Dis 1993;42:3--5. Reimer G, Steen VD, Penning CA, Medsger TA Jr, Tan EM. Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma). Arthritis Rheum 1988;31:525-532. Rodriguez-Sanchez JL, Gelpi C, Juarez C, Hardin JA. AntiNOR 90: a new autoantibody in scleroderma that recognizes a 90-kd component of the nucleolus-organizing region of chromatin. J Immunol 1987;139:2579-2584.

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Rubin RL, Tang F, Chan EKL, Pollard M, Tsay G, Tan EM. IgG subclasses of autoantibodies in systemic lupus erythematosus, Sj6gren's syndrome, and drug-induced autoimmunity. J Immunol 1986;137:2528-2534. Saito E, Yoshimoto Y, Oshima H, Yoshida H Kinoshita M. Fluorescent antibodies in polymyositis using cultured human skin fibroblasts: granular perinuclear cytoplasmic staining pattern by sera from patients with polymyositis and pulmonary fibrosis. J Rheumatol 1989;16:47-52. Senecal J-L, Raymond Y. Autoantibodies to DNA, lamins, and pore-complex proteins produce distinct peripheral fluorescent antinuclear antibody patterns on the HEp-2 substrate. Arthritis Rheum 1991;34:249-251. Smolen JS, Klippel JH, Penner E, Reichlin M, Steinberg AD, Chused TM, Scherak O, Graninger W, Hartter E, Zielinski CC, Wolff A, Davey RJ, Mann DL, Mayr WR. HLA-DR antigens in systemic lupus erythematosus: association with

90

specificity of autoantibody responses to nuclear antigens. Ann Rheum Dis 1987;46:457--462. Sontheimer RD, McCauliffe DP, Zappi E, Targoff IN. Antinuclear antibodies: clinical correlations and biologic significance. Adv Dermatol 1992;7:3--52. Tan EM, Rodnan GP, Garcia I, Moroi Y, Fritzler MJ, Peebles C. Diversity of antinuclear antibodies in progressive systemic sclerosis. Arthritis Rheum 1980;23:617-626. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol 1989;44:93--151. Venables PJ, Charles PJ, Buchanan RR, Yi T, Mumford PA, Schrieber L, Room GR, Maini RN. Quantitation and detection of isotypes of anti-SS-B antibodies by ELISA and Farr assays using affinity purified antigens. Arthritis Rheum 1983;26:146-155.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

AUTOANTIBODIES IN THERAPEUTIC PREPARATIONS OF HUMAN IgG (IVIg) Luc Mouthon, M.D. and Michel D. Kazatchkine, M.D. INSERM U430 and Universit6 Pierre et Marie Curie, H6pital Broussais, 75674 Paris Cedex 14, France

HISTORICAL NOTES Normal human polyspecific IgG for therapeutic use (intravenous immunoglobulin, IVIg) are preparations of intact normal IgG obtained from pools of plasma of a large number of healthy blood donors. The use of IVIg was initially restricted to the substitutive therapy for patients with primary immunoglobulin deficiencies. In the early 1980s, IVIg was first reported to increase platelet counts in children with immune thrombocytopenia associated with the Wiskott-Aldrich syndrome and in acute idiopathic thrombocytopenic purpura of childhood (Imbach et al., 1981). In the last 10 years, IVIg has increasingly been used in the treatment of a variety of autoimmune and systemic inflammatory conditions (Dwyer, 1992). Several mechanisms of action proposed in order to explain the immunoregulatory effects of infused IgG in autoimmune diseases include functional blockade of Fc receptors, inhibition of complement-mediated damage, modulation of cytokine production and selection of immune repertoires (Mouthon et al., 1994). A number of these mechanisms depend on V regiori-mediated interactions of IVIg with circulating molecules and with surface molecules on immunocompetent cells in the recipient (Kazatchkine et al., 1994).

THE AUTOANTIGENS Because IVIg originates from plasma of several thousand donors, the spectrum of IgG antibody reactivities to external antigens and self antigens is that expressed in normal human serum. The characterization of autoantibodies and antiautoantibodies in

IVIg thus directly pertains to that of natural IgG antibodies in human serum.

AUTOANTIBODIES Characteristics Autoantibodies of the IgM, IgG and IgA isotypes are present in normal serum (Avrameas, 1991). Natural autoantibodies react with a wide range of self antigens, including cellular components and soluble molecules. Natural autoantibodies are often polyreactive, express various degrees of affinity for self antigens and are encoded by V H genes in germ line configuration (Coutinho et al., 1995). Natural autoantibodies are "connected" through V regions in the sense that they are capable of recognizing and being recognized by other autoantibodies of the same individual. V regionmediated connectivity is observed within the IgM and IgG fractions of serum and between IgM and autologous IgG molecules (Adib et al., 1990; Hurez et al., 1993). Complementary interactions between IgM and autologous IgG contribute to downregulation of selfreactivity of IgG in whole serum. Natural IgG and IgM autoantibodies recognize a limited set of dominant self antigens. The repertoire of self-reactive antibodies to these dominant autoantigens is highly conserved among individuals and through aging (Mouthon et al., 1995a; 1995b; LacroixDesmazes et al., 1995). Methods of Detection The large number of reactivities with self antigens

91

that are present in IVIg is documented by immunoblotting IVIg with proteins in solubilized extracts of normal human tissues (Figure 1). Among the multiple antigens recognized by normal IgG in homologous/self tissues, IVIg predominantly reacts with a dominant set of 20 to 25 as yet uncharacterized protein bands (Mouthon et al., 1995b). The dominant autoreactivities present in IVIg are similar to those which are expressed in purified IgG of healthy donors (Figure 1). Individual antibody specificities detectable in IVIg by means of ELISA or functional assays include a large number of soluble and membrane-associated molecules. Some of these molecules represent phylogenetically conserved cellular components (e.g., cytoskeletal proteins and myoglobin); some of the molecules are targets for autoantibodies in autoimmune disease, e.g., thyroglobulin, DNA, intrinsic factor and coagulation factor VIII (Kazatchkine et al., 1994).

Finally, some of the autoantibodies present in IVIg are directed against functional molecules of the immune system (Table 1), e.g., antibodies reactive with the CD5 and CD4 molecules (Vassilev et al., !993; Hurez et al., 1994). Anti-CD5 specificity in IVIg might be relevant to the therapeutic modulation of autoimmunity in that the CD5 + subpopulation of B cells might represent a predominant source of autoantibody-producing cells (Casali and Notkins, 1989). Antibodies to human CD4 can also be documented in IVIg by immunochemical and functional approaches. Anti-CD4 antibodies affinity-purified from IVIg on human recombinant CD4 can inhibit the proliferative responses in conventional mixed lymphocyte reaction (MLR) as well as the in vitro infection of CD4 + T cells with HIV-1 (Hurez et al., 1994). These observations might be relevant to graft-versus-host disease (GVHD) in recipients of bone marrow allotransplants

Figure 1. Densitometric profile of reactivity of IVIg with liver antigens. The Figure depicts the reactivity profile of IVIg (SandoglobulinR) (full line) and the mean reactivity profile of purified IgG from 18 healthy adult male donors (i.e., the arithmetic mean of the 1,200 recorded intensities constitutive of the reactivity profile of each donor) (dotted line). The shaded area depicts background staining observed in the presence of antihuman IgG antibody alone. IgG was tested at 200 lag/mL. Migration distance and light absorption were expressed as arbitrary units. 92

Table 1. Reported Antibody Reactivities Present in IVIg Directed Against Functional Molecules of the Immune System v regions of immunoglobulins Idiotypic determinants of immunoglobulins Fcy Framework and variable determinants of the 13chain of the ~13 T-cell receptor Cytokines and cytokine receptors CD5 CD4 MHC class I-derived peptides Adhesion molecules

(Sullivan et al., 1990) or in autoimmune diseases that benefit from therapy with monoclonal anti-CD4 antibodies. IVIg preparations also contain antibodies reactive with a conserved peptide of class I molecules involved in the interaction between class I and the Tcell receptor. Affinity-purified antibodies to this peptide dose-dependently inhibit CD8-mediated HLA class I-restricted cellular cytotoxicity of T cells toward virus-infected targets (Kaveri et al., 1996). Antibodies to V regions (idiotypes) of human anticlass I and class II antibodies in IVIg (Atlas et al., 1993) might also be

relevant to the ability of immunoglobulin to prevent GVHD and to decrease the titers of cytotoxic antibodies in hyperimmunized patients with chronic renal failure (Glotz et al., 1993).

CLINICAL UTILITY The ability of IVIg to interact with idiotypes of autoantibodies is widely documented in the case of both disease-associated and natural autoantibodies.

Table 2. Disease-Associated Autoantibodies Inhibited by IVIg Coagulation factor VIII (antifactor VIII autoimmune disease) Cardiolipin (antiphospholipid syndrome) Antineutrophil cytoplasmic antigen (ANCA) (vasculitis) Thyroglobulin (autoimmune thyroiditis) DNA (SLE) Retinal S antigen Platelet (idiopathic thrombocytopenia) Erythroblast Intrinsic factor (autoimmune megaloblastic anemia) Neuroblastoma cells (108cc15 line) (Guillain-Barr6 and chronic inflammatory demyelinating neuropathy) Endothelial cell (vasculitis) C3 convertase C1 inhibitor (autoimmune C1 inhibitor deficiency) Acetylcholine receptor (myasthenia gravis) Mitochondrial antigens (primary biliary cirrhosis) HLA class I (alloimmunization)

93

The first evidence that IVIg contains anti-idiotypes against pathogenic autoantibodies came from the study of patients with coagulation factor VIII autoimmune disease treated with IVIg in whom the infusion of immunoglobulin resulted in a rapid and dramatic fall in autoantibody titer in plasma (Sultan et al., 1984). F(ab') 2 fragments of IVIg neutralize the functional activity of the autoantibodies in vitro. Factor VIII autoantibodies are selectively retained on affinity chromatography columns of F(ab') 2 fragments of IVIg coupled to Sepharose (Rossi et al., 1989). In addition, IVIg shares anti-idiotypic reactivities with mouse monoclonal Ab213 anti-idiotypes directed against idiotypes of factor VIII autoantibodies. IVIg can bind to or inhibit the functional activity of a wide range of autoantibodies of patients with autoimmune disease (Table 2). The potential relevance of finding complementary (idiotypic) interactions between V regions of IVIg and autoantibodies is suggested by the presence of anti-idiotypic antibodies directed against prerecovery autoantibodies in patients who spontaneously recover from autoimmune disease, e.g., in factor VIII autoimmune disease (Sultan et al., 1987) and in systemic vasculitis with ANCA autoantibodies (Rossi et al., 1991). Anti-idiotypes to autoantibodies in IVIg might directly contribute to the neutralization of circulating autoantibodies as well as to long-term modulation of autoantibody production by the corresponding B-cell clones (Rossi et al., 1989; Kazatchkine et al., 1994). Several sources contributing to the anti-idiotypic activity of IVIg against autoantibodies include donors who spontaneously recovered from an autoimmune disease, healthy individuals aged over 65 years and multiparous women whose plasma contains antiidiotypes in higher frequency than the plasma of younger donors and nulliparous women (Dietrich et al., 1992a). A relative increase in anti-idiotypes to autoantibodies in IVIg preparations can be obtained by affinity chromatography of IVIg on F(ab') 2 fragments of the IVIg preparation itself (Dietrich et al., 1992b).

The eluted subfraction, which is termed the "V region-connected" fraction of IVIg, exhibits a high content in autoantibodies compared with unfractionated IVIg and therefore contains high amounts of complementary (anti-idiotypic) autoantibodies (Dietrich et al., 1992b). Likewise, the IVIg content of V region-complementary pairs of antibodies (F[ab'] 2F[ab'] 2 dimers) increases with the number of donors in the pool (Dietrich et al., 1992c). In addition to idiotypic determinants on diseaseassociated autoantibodies, IVIg recognizes idiotypes on natural IgM and IgG autoantibodies of healthy individuals. Thus, IVIg interacts with V regions of natural polyreactive IgM as exemplified by autoreactive IgM monoclonal antibodies generated by EBVtransformed normal B lymphocytes as sources for natural autoantibodies (Rossi et al., 1990). Through its ability to react with natural IgM molecules that are components of the normal idiotypic network, IVIg may exert a selective control of the expression of the available antibody repertoire of a given individual.

REFERENCES

leukemia patients transfused with platelet concentrates. Blood 1993;81:538-542. Avrameas S. Natural autoantibodies: from "horror autotoxicus" to "gnothi seauton". Immunol Today 1991;12:154-159. Casali P, Notkins AL. CD5+ B lymphocytes, polyreactive antibodies and the human B-cell repertoire. Immunol Today 1989;10:364--368. Coutinho A, Kazatchkine MD, Avrameas S. Natural autoanti-

Adib M, Ragimbeau J, Avrameas S, Ternynck T. IgG autoantibody activity in normal mouse serum is controlled by IgM. J Immunol 1990;145:3807-3813. Atlas E, Freedman J, Blanchette V, Kazatchkine MD, Semple JW. Down regulation of the anti-HLA alloimmune response by variable region-reactive (anti-idiotypic) antibodies in

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CONCLUSION The finding of autoantibodies and anti-idiotypes to autoantibodies in IVIg substantiates data obtained in mouse and in man indicating that normal human serum IgG is largely composed of self-reactive antibodies. Perhaps the immunoregulatory properties of IVIg in autoimmune patients reflect the role of normal IgG in selecting repertoires and maintaining tolerance to self under physiological conditions. The beneficial effects of IVIg in autoimmune diseases might thus be in part dependent on the supply of regulatory IgG antibodies that normally contribute to the homeostasis of autoreactivity and might be absent or present in insufficient amounts in patients with autoimmune disorders. See also COAGULATION FACTOR VIII AUTOANTIBODIES, HIDDEN AUTOANTIBODIES and NATURAL AUTOANTIBODIES.

bodies. Curr Opin Immunol 1995;in press. Dietrich G, Algiman M, Sultan Y, Nydegger UE, Kazatchkine MD. Origin of antiidiotypic activity against antifactor VIII autoantibodies in pools of normal human immunoglobulin G (IVIg). Blood 1992a;79:2946--2951. Dietrich G, Kaveri SV, Kazatchkine MD. A V region-connected autoreactive subfraction of normal human serum immunoglobulin G. Eur J Immunol 1992b;22:1701-1706. Dietrich G, Kaveri SV, Kazatchkine MD. Modulation of autoimmunity by intravenous immune globulin through interaction with the function of the immune/idiotypic network. Clin Immunol Immunopathol 1992c;62:$73-$81. Dwyer JM. Manipulating the immune system with immune globulin. N Engl J Med 1992;326:107--116. Glotz D, Haymann JP, Sansonetti N, Francois A, MenoyoCalonge V, Bariety J, Druet P. Suppression of HLA-specific alloantibodies by high-dose intravenous immunoglobulins (IVIg). A potential tool for transplantation of immunized patients. Transplantation 1993;56:335--337. Hurez V, Kaveri SV, Kazatchkine MD. Expression and control of the natural autoreactive IgG repertoire in normal human serum. Eur J Immunol 1993;23:783--789. Hurez V, Kaveri SV, Mouhoub A, Dietrich G, Mani JC, Klatzmann, Kazatchkine M. Anti-CD4 activity of normal human immunoglobulins G for therapeutic use (intravenous immunoglobulin, IVIg). Therap Immunol 1994;1:269-278. Imbach P, Barandun S, d'Apuzzo V, Baumgartner C, Hirt A, Morell A, Rossi E, Schoni M, Vest M, Wagner HP. Highdose intravenous gamma globulin for idiopathic thrombocytopenic purpura in childhood. Lancet 1981; 1:1228-1231. Kaveri SV, Vassilev V, Hurez V, Lengagne R, Cot S, Pouletty PL, Glotz D, Kazatchkine MD. Antibodies of a conserved region of HLA class I molecules, capable of modulating CD8 T cell mediated function, are present in pooled normal immunoglobulin for therapeutic use. J Clin Invest 1996:In press. Kazatchkine MD, Dietrich G, Hurez V, Ronda N, Bellon B, Rossi F, Kaveri SV. V region-mediated selection of autoreactive repertoires by intravenous immunoglobulin (IVIg). Immunol Rev 1994;139:79--107. Lacroix-Desmazes S, Mouthon L, Coutinho A, Kazatchkine MD. Analysis of the natural human IgG antibody repertoire: life-long stability of reactivities towards self antigens contrasts with age-dependent diversification of reactivities against bacterial antigens. Eur J Immunol 1995;25:2598-2604. Mouthon L, Piketty C, Kazatchkine MD. Immunomodulation of

autoimmune and systemic inflammatory diseases with intravenous immunoglobulin. Vox Sang 1994;67:$53-$59. Mouthon L, Nicolas N, Lacroix-Desmazes S, Nobrega A, Barreau C, Kaveri SV, Coutinho A, Kazatchkine MD. Invariance and restriction towards a limited set of self antigens characterize neonatal IgM antibody repertoires and prevail in autoreactive repertoires of healthy adults. Proc Natl Acad Sci USA 1995a;92:3839-3843. Mouthon L, Haury M, Lacroix-Desmazes S, Barreau C, Coutinho A, Kazatchkine MD. Analysis of the normal human IgG antibody repertoire. Evidence that IgG autoantibodies of healthy adults recognize a limited and conserved set of protein antigens in homologous tissues. J Immunol 1995b; 154:5769--5778. Rossi F, Dietrich G, Kazatchkine MD. Anti-idiotypes against autoantibodies in normal immunoglobulins: evidence for network regulation of human autoimmune responses. Immunol Rev 1989;110:135-149. Rossi F, Guilbert B, Tonnelle C, Ternynck T, Fumoux F, Avrameas S, Kazatchkine MD. Idiotypic interactions between normal human polyspecific IgG and natural IgM antibodies. Eur J Immunol 1990;20:2089--2094. Rossi F, Jayne DR, Lockwood CM, Kazatchkine MD. Antiidiotypes against antineutrophil cytoplasmic antigen autoantibodies in normal human polyspecific IgG for therapeutic use and in the remission sera of patients with systemic vasculitis. Clin Exp Immunol 1991;83:298--303. Sullivan KM, Kopecky KG, Jocom J, Fisher L, Buckner CD, Meyers JD, Counts GW, Bowden RA, Peterson FB, Witherspoon RP, Budinger MD, Schwartz RS, Appelbaum FR, Clift RA, Hansen JA, Sanders JE, Thomas ED, Storb R. Immunomodulatory and antimicrobial efficacy of intravenous immunoglobulin in bone marrow transplantation. N Engl J Med 1990;323:705--709. Sultan Y, Kazatchkine MD, Maisonneuve P, Nydegger UE. Anti-idiotypic suppression of autoantibodies to Factor VIII (antihaemophilic factor) by high-dose intravenous gamma globulin. Lancet 1984;2:765--768. Sultan Y, Rossi F, Kazatchkine MD. Recovery from anti-VIII:C (antihemophilic factor) autoimmune disease is dependent on generation of antiidiotypes against anti-VIII:C autoantibodies. Proc Natl Acad Sci USA 1987;84:828--831. Vassilev T, Gelin C, Kaveri SV, Zilber MT, Boumsell L, Kazatchkine MD. Antibodies to the CD5 molecule in normal human immunoglobulins for therapeutic use (intravenous immunoglobulins, IVIg). Clin Exp Immunol 1993;92:369-372.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

A U T O A N T I B O D I E S THAT P E N E T R A T E INTO LIVING CELLS Donato Alarc6n-Segovia, M.D., Luis Llorente, M.D. and Alejandro Rufz-Argtielles, M.D.

Department of Immunology and Rheumatology, Instituto National de la Nutricion Salvador Zubiran, Mexico City; and Department of Immunology (A.R-A.), Laboratorios Clinicos de Puebla, Puebla, Mexico

HISTORICAL NOTES

Long-standing immunological dogma holds that functionally intact autoantibodies do not penetrate living cells (Benacerraf and Unanue, 1979). Such an impediment is thought to preserve the internal milieu of cells as an immunologically privileged site where autoantibodies, albeit present, could not reach their antigens. Scant observations suggested otherwise (reviewed by Alarc6n-Segovia and Rufz-Argtielles, 1980). For example, intravenously injected human gammaglobulin can be detected by immunofluorescence inside lymphoid, and reticuloendothelial cells and mice immunized with UV-irradiated DNA and subjected to whole body UV-irradiation show intranuclear immunoglobulin in epidermal cells. Also, antipurine or antipyrimidine antibodies, anti-RNA or antienzyme antibodies can alter the function of cells incubated in them. Common denominators of these studies include production in laboratory animals of the antibodies employed and involvement of primitive, immature, tumoral, chemically transformed, UV-irradiated or virus-infected eukaryotic cells. In 1978 fluorescein-tagged IgG antibodies to nuclear ribonucleoprotein (nRNP) from a patient with mixed connective tissue disease (MCTD) was shown to penetrate live human mononuclear cells (MNC) and migrate all the way to the nucleus (Alarc6n-Segovia et al., 1978). Those results contradicted the notion that nuclear staining found on direct immunofluorescent studies of skin or kidney biopsies from patients with MCTD or systemic lupus erythematosus (SLE) was a mere artifact due to entrance of the antibodies into cells already dead. The demonstration that live MNC from patients with MCTD, when incubated with goat

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antihuman IgG, reveal intranuclear antibody with a pattern akin to that given by anti-nRNP antibody in dead cells contradicted this (Alarc6n-Segovia et al., 1979a). The penetration of anti-nRNP antibodies was shown to occur via the Fc receptors of MNC (Alarc6n-Segovia et al., 1978) and to cause cell death and abrogation of suppressor cell function (Alarc6nSegovia et al., 1979b). Penetration of anti-nRNP into activated T cells arrested the cell cycle on its G0/G1 phases as determined by cell cytometry of MNC stained with intercalating dyes; whereas, anti-DNA, also shown to penetrate, permitted increase of nuclear RNA but not of DNA thus arresting the cell cycle in its G1A phase (Alarc6n-Segovia and Llorente, 1983). That this took place in vivo was evidenced by the presence of circulating T cells with a similar DNA block in patients with SLE having anti-DNA antibodies (Alarc6n-Segovia et al., 1982a; 1982b). Despite expectations of a profound impact on immunobiological thinking, the dogma prevailed and the data (Alarc6n-Segovia et al., 1978; 1979a; 1979b; 1982a; 1982b; Alarc6n-Segovia and Llorente, 1983) albeit published in prestigious journals and soon confirmed by others, were considered merely puzzling. Nevertheless, the original paper (Alarc6nSegovia et al., 1978) triggered the autoantibodyassisted study of the function of small nRNP and the discovery of their role in splicing (Steitz J, personal communication). The penetration of anti-nRNP into live cells was confirmed in keratinocytes (Galoppin and Saurat, 1981) instead of MNC; neither the absence of a cytotoxic effect nor the presence of Fc receptors seemed necessary for penetration to occur since few keratinocytes have Fc receptors.

Figure 1. Normal lymphocyte incubated with SLE IgG and stained with PAP method (Courtesy of Okudaira and Williams). Antilymphocytic antibodies were shown to penetrate into live T lymphocytes and locate in the cytoplasm as opposed to the nuclear localization of anti-RNP or anti-DNA (Okudaira et al., 1982). Blocking experiments with aggregated IgG did not impede the penetration of the antilymphocyte autoantibodies but pepsin digestion (i.e., removal of the Fc fragment) prevented penetration. Penetration of anti-DNA was confirmed using a monoclonal mouse antibody (Okudaira et al., 1987); treatment with DNase abrogated this reactivity, suggesting that binding occurred through cell membrane DNA. Further studies (Okudaira and Williams) on this subject met resistance and were never published. Figures 1 and 2 (Okudaira and Williams, personal communication) show penetration of human SLE IgG into normal lymphocytes.

show that some penetrate and some do not (Vlahakos et a1.,1992; Avrameas et al., unpublished observations). Similar unpublished (Llorente and Alarc6nSegovia) and published (Golan et al., 1993) observa-

AUTOANTIBODY PENETRATION OF CELLS

Autoantibodies The original observations on the entrance of autoantibodies into live cells were made with polyclonal antinRNP or anti-DNA (Alarc6n-Segovia et al., 1978; 1982b; Alarc6n-Segovia and Llorente, 1983). Subsequent studies using monoclonal antibodies to DNA

Figure 2. Electron microscopy of lead uranyl staining of SLE IgG inside a normal lymphocyte (courtesy of Okudaira and Williams).

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tions made with polyclonal anti-nRNP reflect physical properties or binding characteristics of specific idiotypes as yet unknown. With polyclonal autoantibodies, the likelihood of having an antibody with appropriate characteristics available to penetrate, even if others do not, might favor detection. In addition to these autoantibodies which were described before 1984, several others are now recognized to do so (Table 1). Most of the antigens with which these autoantibodies react are nuclear (nRNP, DNA, neuronal nuclear [Hu]); some may be nuclear or cytoplasmic (SS-A[Ro]) and others are cytoplasmic (proteinase 3). The cytoplasmic antigen(s) to which antilymphocytic antibodies were directed was not determined (Okudaira et al., 1982). In all instances described, the autoantibodies that penetrate living cells are of the IgG isotype. Diseases in which these penetrating autoantibodies occur include MCTD (anti-nRNP), SLE (anti-nRNP, anti-DNA, antilymphocyte, anti-Ro), primary Sj6gren's syndrome (anti-Ro), Wegener's granulomatosis and related vasculitides (antiproteinase 3), paraneoplastic subacute sensory neuronopathy and encephalomyelitis associated with small-cell lung cancer (antineuronal nuclear autoantibodies type 1, ANNA-l; anti-Hu) and chronic active hepatic disease (antiribosomal P protein). Penetrated Cells

Anti-nRNP penetrates live human blood T and B lymphocytes, as well as monocytes (Alarc6n-Segovia et al., 1978) and natural killer (NK) cells (Ma et al., 1991).

The proportion of live keratinocytes showing nuclear fluorescence when incubated with anti-RNP reached 70% but was only 9.5% upon incubation with anti-DNA (Galoppin and Saurat, 1981). This probably reflected properties of autoantibodies because the percentage of dead cells with nuclear staining was similar with both antibodies. The autoantibodies present in five "penetrating" sera yielded higher intranuclear fluorescence with human cell lines of epithelial origin (COLO-16, A-431 and HeLa) compared with keratinocytes (Golan et al., 1993). No penetration occurred into murine T- or B-cell lines. Four of the sera had anti-DNA activity while the other had anti-Ro and anti-La activities (Golan et al., 1993). Species differences could have accounted for this lack of penetration into murine T- or B- cell lines since human anti-nRNP may not also penetrate guinea pig cells (Iwatzuki et al., 1982). Murine monoclonal autoantibodies, however, can penetrate human MNC as they do into murine thymocytes (Okudaira et al., 1987). Infused, anti-Ro antibodies penetrate readily in vivo into epidermal cells of normal human skin grafted onto nude athymic mice whose own cells they did not penetrate (Lee et al., 1989). This could be because human anti-Ro do not usually bind murine Ro antigen (Lee et al., 1986). However, infusion of various antinuclear antibodies, including anti-Ro, into neonatal Balb/C mice or into their pregnant mothers yields widespread intranuclear deposition of antibodies in skin, liver, spleen and kidneys in the pups. (Herrera et al., 1988; Guzman-Enriquez et al., 1990). This apparent difference between in utero or newborn animals, as opposed to adults, may have a bearing on the pathogenesis of heartblock induced in humans in

Table 1. Autoantibodies Demonstrated to Penetrate Living Cells Antibody

References

Anti-nRNP

Alarc6n-Segovia et al., 1978; Gallopin and Saurat, 1981" Ma et al., 1987

Polyclonal anti-DNA

Alarc6n-Segovia et al., 1982b; Golan et al., 1993

Monoclonal anti-DNA

Okudaira et al., 1987; Vlahakos et al., 1992

Antilymphocytic

Okudaira et al., 1982

SS-A (Ro)

Herrera et al., 1988; Lee et al., 1989

Antiproteinase 3 (C-ANCA)

Csernok et al., 1993

Antisynaptosomal

Fabian, 1988

Antineuronal nuclear (anti-Hu)

Dalmau et al., 1991; Hormigo and Lieberman, 1994

Antiribosomal P protein

Reichlin, unpublished observations

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by anti-Ro but not in the mother (Bacman et al., 1994). Nuclei of murine glomerular mesangial cells, as well as rat hepatoma cells, can also be penetrated by murine anti-DNA that reaches their nuclei (Yanase et al., 1994). Nervous system cells are also penetrated by type IIa (anti-Hu) antineuronal nuclear autoantibodies which are found in patients with paraneoplastic neuronopathy and encephalomyelitis associated with smallcell carcinoma of the lung. These can reach the nuclei of most neurons, of some glial cells (Dalmau et al., 1991) and of rat cerebellar granule cells (Greenlee et al., 1993), as well as nuclei of the tumor (Dalmau et al., 1991; Hormigo and Lieberman, 1994). Both antiHu and control IgG enter the cytoplasm of Hu-positive and Hu-negative cells but, anti-Hu IgG appears within the nuclei more rapidly than does the irrelevant antibody (Hormigo and Lieberman, 1994). Neurons also accumulate antisynaptosomal antibody to a greater extent than irrelevant IgG (Fabian, 1988). Penetration of autoantibodies to proteinase 3 into neutrophils might be related to expression of the antigen on the cell (Csernok et al., 1993). utero

Penetration Mechanisms

The Fc receptor may be a portal of entry of autoantibodies (Alarc6n-Segovia et al., 1978) by receptormediated endocytosis. This mechanism, which internalizes IgG into the yolk-sac or intestinal epithelial cells through coated pits, occurs in the passive transfer of immunity in some species (Goldstein et al., 1979). Indicators of the role of Fc receptors in the penetration of autoantibodies include: 1) the number of cells penetrated by anti-nRNP is similar to that of Fc receptor-bearing MNC and parallels their presence in the various cell subpopulations; 2) incubation longer than an hour does not increase the percentage of cells with nuclear fluorescence; 3) when tested simultaneously for antibody penetration with fluorescent antinRNP and for Fc-gamma receptors by means of antibody-coated chicken erythrocytes, only cells that form rosettes have intranuclear fluorescence; 4) aggregated gammaglobulin and purified Fc fragments block the penetration of the anti-nRNP in a dose-dependent fashion; 5) F(ab')2 anti-nRNP, although capable of staining nuclei in rat kidney sections, does not stain nuclei of live cells; and 6) antibody penetration inhibits antibody-dependent cellular cytotoxicity, another Fc receptor mediated function (Llerena et al., 1981).

The Fc portion of autoantibodies is sometimes required for penetration (Okudaira et al., 1982), but F(ab')2 fragments can also enter under certain experimental conditions (Golan et al., 1993). An alternative pathway of autoantibody penetration is also apparent from a study (Galoppin and Saurat, 1981) which showed that the proportion of keratinocytes entered by anti-nRNP is larger than the proportion bearing Fc receptors (Galoppin and Saurat, 1981). This other pathway for penetration might be mediated by nuclear antigens bound to a cell surface receptor as described for DNA (Bennett et al., 1983), or surface expression of nuclear antigens as described for DNA (Bennett et al., 1986) and for nRNP (Ma et al., 1993). That both pathways may be operative is suggested by rapid (<1 hour), Fc-mediated penetration of antinRNP and, to lesser extent, control IgG; whereas, the penetration of anti-nRNP, but not control IgG, proceeded after the first hour and seemed to be mediated by expression of nRNP on the cell surface (Ma et al., 1991). Some autoantibodies that penetrate may readily find their antigen in the cytoplasm, but others need to traverse the nuclear membrane. Nuclear localization of autoantibodies is a temperature-dependent process (Alarc6n-Segovia et al., 1978; Yanase et al., 1994). It is also a specific process because different 125I-labeled monoclonal anti-DNA antibodies have different rates of entry and those not retaining anti-DNA activity after iodination enter poorly. As assessed by tracking monoclonal anti-DNA with colloidal gold-labeled protein A, immunoglobulin can be detected in endosomes and cytoplasm within 15 minutes, and a few molecules are already in nuclear condensed chromatin (Yanase et al., 1994). Monoclonal anti-DNA covalently linked to nanogold can be tracked to cell membranes, endosomes, Golgi vesicles, endoplasmic reticulum and to nuclear pores through which it is presumed to enter the nucleus (Yanase et al., 1994). An apparent requirement for an antibody to traverse the nuclear envelope seems to be the presence of the relevant antigen within the nucleus (Einck and Bustin, 1984). Thus, a goat-antihuman IgG can penetrate live cells previously penetrated by human autoantibodies (Alarc6n-Segovia et al., 1979a). Lactoferrin, an extracellular polypeptide, may act as its own messenger and gain entrance into the cell nucleus to activate transcription directly (He and Furmanski, 1995). It also has specific DNA and RNA binding properties and may bind to cell membrane DNA (Bennett et al., 1983). Whether autoantibodies

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use a mechanism similar to that of transferrin for trans-cytoplasmic movement to reach nucleic acids deserves investigation.

Effects of cell penetration by autoantibodies Penetration of anti-nRNP IgG into T-gamma cells can cause them protracted death (after 20 hours or more) shown by 51Cr release (Alarc6n-Segovia et al., 1979b). Cell death also occurs after penetration of antilymphocytic antibodies (Okudaira et al., 1982) and with anti-Hu antibodies (Greenlee et al., 1993), although the latter is disputed (Hormigo and Lieberman, 1994). Recent reanalysis of this question shows that polyclonal anti-DNA cause apoptosis upon penetration (Alarc6n-Segovia, et al., 1995; 1996). Some monoclonal anti-DNA antibodies cause apoptosis and others do not; internalization of polyclonal anti-nRNP can also cause apoptosis. The suppressor functions of T-gamma cells on B cells is abrogated by anti-nRNP antibodies (Alarc6nSegovia et al., 1979b). Intact anti-nRNP also blocks the progression of MNC or enriched T-gamma cells in the cell cycle as manifested by inhibition of thymidine incorporation after stimulation with phytohemagglutinin or concanavalin A, but not with pokeweed mitogen (Alarc6n-Segovia et al., 1982b). Anti-DNA antibodies caused a DNA block in the cell cycle of activated cells (Alarc6n-Segovia and Llorente, 1983). A DNA block was also found in the circulating MNC of SLE patients with active disease and serum antiDNA antibodies (Alarc6n-Segovia et al., 1982b). The intranuclear deposits of IgG found in glomerular cells of normal mice injected with murine monoclonal anti-DNA are associated with morphologic and functional abnormalities such as hypercellularity, epithelial foot process fusion, new fiber bundle formation within the mesangium suggesting new collagen synthesis and proteinuria (Vlahakos et al., 1992). That penetration of anti-nRNP can cause increased cellularity and collagen deposition is also suggested by increased production of IL-1 and IL-6

REFERENCES Alarc6n-Segovia D, Rufz-Argtielles A, Fishbein E. Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc receptors. Nature 1978;271:67--69. Alarc6n-Segovia D, Rufz-Argtielles A, Fishbein E. Antibody penetration into living cells. I. Intranuclear immunoglobulin in peripheral blood mononuclear cells in mixed connective lO0

by peripheral blood monocytes incubated with antinRNP (Okawa-Takatsuji et al., 1994). Penetration of antiribosomal P protein autoantibodies results in alteration of transmembrane calcium flux and decreased synthesis/secretion of apolipoprotein B with accumulation of cholesterol within cells (Alarc6n-Segovia et al., 1996).

CONCLUSION Now that the dogma that autoantibodies do not penetrate live cells has been vanquished, there are several theoretical and practical implications to the penetration of autoantibodies and the resultant alterations of cell functions and cellular damage. Recognition and continued study of these phenomena might markedly modify basic concepts of autoimmunity. Inasmuch as many autoantibodies may be germline gene-encoded natural autoantibodies, including some of IgG isotype, their capability of cell penetration may be part of a regulatory network. The possibility that autoantibodies regulate cell functions by interacting with specific, functionally important intracellular antigens is of enormous importance; its study poses a challenge to basic immunologists and cell biologists. The apoptosis caused by autoantibodies entering certain cells might contribute to the regulatory network as a mechanism for deleting clones that could cause autoimmune disease. It should be recognized that, for reasons still unknown, not all autoantibodies can penetrate cells and those that do penetrate do so in different amounts and at different rates. Also, not all cells can be penetrated by autoantibodies; some young cells might be penetrable; whereas, mature cells of the same type might not. The presence of the antigen inside the cell may be a requirement either for entrance and/or for permanence, as well as for functional effects or, at least, for laboratory detection that entrance has taken place. All this may have a meaning that we have yet to unravel. See also ISLET CELL AUTOANTIBODIES.

tissue disease and systemic lupus erythematosus. Clin Exp Immunol 1979a;35:364--375. Alarc6n-Segovia D, Rufz-Argtielles A, Llorente L. Antibody penetration into living cells. II. Antiribonucleoprotein IgG penetrates into T lymphocytes causing their deletion and the abrogation of suppressor function. J Immunol 1979b;122: 1855--1862. Alarc6n-Segovia D, Rufz-Argtielles A. Antibody penetration

into living cells: mechanisms and consequences. In: Larralde C, Willms K, Ortiz-Ortiz L, eds. Molecules, Cells, and Parasites in Immunology. New York: Academic Press, 1980:53--64. Alarc6n-Segovia D, Llorente L, Fishbein E, Diaz-Jouanen E. Abnormalities in the content of nucleic acids of peripheral blood mononuclear cells from patients with systemic lupus erythematosus. Arthritis Rheum 1982a;25:304-317. Alarc6n-Segovia D, Llorente L, Rufz-Argtielles A. Antibody penetration into living cells. III. Effect of antiribonucleoprotein IgG on the cell cycle of human peripheral blood mononuclear cells. Clin Immunol Immunopathol 1982b;23: 22-33. Alarc6n-Segovia D, Llorente L. Antibody penetration into living cells. IV. Different effects of antinative DNA and antiribonucleoprotein IgG on the cell cycle of activated T gamma cells. Clin Exp Immunol 1983;52:365--371. Alarc6n-Segovia D, Llorente L, Ru/z-Argtielles A, RichaudPatin Y, P6rez-Romano B. Penetration of anti-DNA antibodies into mononuclear cells (MNC) causes apoptosis. Arthritis Rheum 1995;38(suppl):S 182. Alarc6n-Segovia D, Llorente L, Rufz-Argtielles A. Broken dogma: Autoantibodies to intracellular constituents penetrate live cells, reach their antigen, alter function and cause death. Immunol Today 1996. Bacman S, Sterin-Borda L, Camusso JJ, Hubscher O, Arana R, Borda ES. Circulating antibodies against neurotransmitter receptor activities in children with congenital heart block and their mothers. FASEB J 1994;8:1170--1176. Benacerraf B, Unanue ER. Immunopathology. In: Benacerraf B and Unanue ER, eds. Textbook of Immunology. Baltimore: Williams & Wilkins Co., 1979:250. Bennett RM, Davis J, Campbell S, Portnoff S. Lactoferrin binds to cell membrane DNA. Association of surface DNA with an enriched population of B cells and monocytes. J Clin Invest 1983;71:611-618. Bennett RM, Davis J, Merritt M. Anti-DNA antibodies react with DNA expressed on the surface of monocytes and B lymphocytes. J Rheumatol 1986:13:679--685. Csernok E, Schmitt WH, Ernst M, Bainton DF, Gross WL. Membrane surface proteinase 3 expression and intracytoplasmic immunoglobulin on neutrophils from patients with ANCA-associated vasculitides. In: Gross WL, ed. ANCAAssociated Vasculitides: Immunological and Clinical Aspects. New York: Academic Press, 1993:45--50. Dalmau J, Furneaux HM, Rosenblum MK, Graus F, Posner JB. Detection of the anti-Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy. Neurology 1991; 41:1757--1764. Einck L, Bustin M. Functional histone antibody fragments traverse the nuclear envelope. J Cell Biol 1984;98:205--213. Fabian RH. Uptake of plasma IgG by CNS motoneurons: comparison of antineuronal and normal IgG. Neurology 1988;38:1775-1780. Galoppin L, Saurat JH. In vitro study of the binding of antiribonucleoprotein antibodies to the nucleus of isolated living keratinocytes. J Invest Dermatol 1981 ;76:264--267.

Golan TD, Gharavi AE, Elkon KB. Penetration of autoantibodies into living epithelial cells. J Invest Dermatol 1993; 100:316-322. Goldstein JL, Anderson RG, Brown MS. Coated pits, coated vesicles and receptor-mediated endocytosis. Nature 1979; 279:669--685. Greenlee JE, Parks TN, Jaeckle KA. Type IIa ('anti-Hu') antineuronal antibodies produce destruction of rat cerebellar granule neurons in vitro. Neurology 1993;43:2049--2054. Guzman-Enriquez L, Avalos-Diaz E, Herrera-Esparza R. Transplacental transfer of human antinuclear antibodies in mice by injection of lupus IgG in pregnant animals. J Rheumatol 1990;17:52--56. He J, Furmanski P. Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA. Nature 1995;373:721-724. Herrera R, Guzman L, Avalos E. Fate of human antinuclear antibodies from lupus erythematosus passively transferred to mice [Abstract]. Arthritis Rheum 1988;31:A16. Hormigo A, Lieberman F. Nuclear localization of anti-Hu antibody is not associated with in vitro cytotoxicity. J Neuroimmunol 1994;55:205--212. Iwatsuki K, Tagami H, Imaizumi S, Ginoza N, Yamada M. The speckled epidermal nuclear immunofluorescence of mixed connective tissue disease seems to develop as an in vitro phenomenon. Br J Dermatol 1982,107:653--657. Lee LA, Gaither KK, Coulter SN, Norris DA, Harley JB. Pattern of cutaneous immunoglobulin G deposition in subacute cutaneous lupus erythematosus is reproduced by infusing purified anti-Ro (SSA) autoantibodies into human skin-grafted mice. J Clin Invest 1989;83:1556-1562. Lee LA, Weston WL, Krueger GG, Eman M, Reichlin M, Steens JO, Surbrugg SK, Vasil A, Norris DA. An animal model of antibody binding in cutaneous lupus. Arthritis Rheum 1986;29:782--788. Llerena JM, Ruiz-Arguelles A, Alarcon-Segovia D, Llorente L, Diaz-Jouanen E. Antibody penetration into living cells. V. Interference between two fc receptor-mediated functions: antibody penetration and antibody-dependent cellular cytotoxicity. Immunology 1981 ;43:249--254. Ma J, Chapman GV, Chen SL, Penny R, Breit SN. Flow cytometry with crystal violet to detect intracytoplasmic fluorescence in viable human lymphocytes. Demonstration of antibody entering living cells. J Immunol Methods 1987; 104: 195--200. Ma J, Chapman GV, Chen SL, Melick G, Penny R, Breit SN. Antibody penetration of human lymphocytes by anti-RNP IgG. Clin Exp Immunol 1991;84:83--91. Ma J, King N, Chen SL, Penny R, Breit SN. Antibody penetration of viable human cells. II. Anti-RNP antibodies binding to RNP antigen expressed on cell surfaces, which may mediate the antibody internalization. Clin Exp Immunol 1993 ;93:396-404. Okawa-Takatsuji M, Aotsuka S, Uwatoko S, Sumiya N, Yokohari R. Enhanced synthesis of cytokines by peripheral blood monocytes cultured in the presence of autoantibodies against U l-ribonucleoprotein and/or negatively charged molecules: implication in the pathogenesis of pulmonary

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hypertension in mixed connective tissue disease (MCTD). Clin Exp Immunol 1994;98:427-433. Okudaira K, Searles RP, Tanimoto K, Horiuchi Y. Williams RC Jr. T lymphocyte interaction with immunoglobulin G antibody in systemic lupus erythematosus. J Clin Invest 1982; 69:1026-1038. Okudaira K, Yoshizawa H, Williams RC Jr. Monoclonal murine anti-DNA antibody interacts with living mononuclear cells. Arthritis Rheum 1987;30:669-678. Vlahakos D, Foster MH, Ucci AA, Barrett KJ, Datta SK,

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Madaio MP. Murine monoclonal anti-DNA antibodies penetrate cells, bind to nuclei, and induce glomerular proliferation and proteinuria in vivo. J Am Soc Nephrol 1992;2:1345--1354. Yanase K, Smith RM, Cizman B, Foster MH, Peachy LD, Jarett L, Madaio MP. A subgroup of murine monoclonal antideoxyribonucleic acid antibodies traverse the cytoplasm and enter the nucleus in a time- and temperature-dependent manner. Lab Invest 1994;71:52--60.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

AUTOANTIBODY SUBCLASSES Pierre Youinou, M.D., Ph.D. a, Raya Maran, M.D. b, Maryvonne Dueymes, M.D., Ph.D. a and Yehuda Shoenfeld, M.D. b

aLaboratoire d'Immunologie, Centre Hospitalier Rdgional et Universitaire, Brest, Cedex, France; and bDepartment of Medicine "B", Research Unit of Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel

HISTORICAL NOTES Widely varying affinities of antisera used for the detection of IgG subclasses originally led to controversy. Quantitation of IgG subclasses now employs monoclonal antibodies raised against human myelomas of each IgG subclass (Aucouturier et al., 1985; Jefferis et al., 1985). Testing with these subclassspecific mouse monoclonals shows that the distribution of antigen-specific subclasses of IgG antibodies does not simply reflect a consistent ratio of the serum concentrations of the individual subclasses (IgG1, IgG2, IgG3 and IgG4) (Jefferis et al., 1994) but rather the relative levels of antigen-specific IgG subclasses are now recognized to vary widely (Preud'homme, 1992). Hence, the distribution of autoantibody subclasses is being probed for insights into the role of the antigen itself in the autoimmune process (Jefferis and Kumararatne, 1990) and the pathogenicity of different antigen-specific IgG subclasses.

THE FINDINGS IgG Subclasses in Autoimmune Diseases Systemic Lupus Erythematosus. Anti-double-stranded DNA (dsDNA) antibodies are extensively investigated (Table 1) in systemic lupus erythematosus (SLE) and are consistently reported to be restricted to the IgG1 and IgG3 subclasses (Winkler et al., 1988). Serum concentrations of dsDNA antibodies of the IgG1 and IgG3 subclasses correlate with disease activity in general and with renal involvement in particular

(Devey et al., 1988). Seemingly tightly related to the clinical presentation of the disease (Dueymes et al., 1993), the rank order of dsDNA subclass frequency is IgG1, IgG3, IgG2 and IgG4 in SLE patients with myositis; IgG1, IgG2, IgG3 and IgG4 in those with renal complications; IgG3, IgG1, IgG2 and IgG4 in those with cutaneous lesions; and IgG1, IgG3, IgG2 and IgG4 in those with hematological complications. In fact, each patient's serum displays an individual isotype distribution that remains constant in longitudinal surveys, independent of anti-dsDNA fluctuations (Winkler et al., 1988). A skewed pattern of subclasses is documented for most autoantibodies commonly present in SLE, including anti-Sm (Eisenberg, et al., 1985), anti-RNP (Rubin et al., 1986), anti-Ro/SSA (Blanco et al., 1992) and antihistone activity (Rubin et al., 1986). In contrast, all subclasses of anticardiolipin antibodies (aCL) are detectable in SLE with prevalences ranging from 34% for IgG3 to 57% for IgG1 (Gharavi et al., 1988). Complications are slightly more frequent in patients with sera containing aCL of the IgG2 and IgG4 subclasses than in those with sera containing IgG1 and IgG3 cardiolipin antibodies. For example, five of six SLE patients with IgG2 and IgG4 cardiolipin antibodies had complications, e.g., thrombosis, fetal loss and thrombocytopenia, compared with 10 of 16 patients with IgG1 and IgG3 or IgG 1, IgG2, IgG3 and IgG4 cardiolipin antibodies (Gharavi et al., 1988).

Primary Antiphospholipid Syndrome. The distribution of cardiolipin antibodies among IgG subclasses in sera with and without anti-J32 glycoprotein I activity

103

Table 1. Predominant Autoantibody IgG Subclass(es) in Non-organ-specific Autoimmune Diseases Disease State

Targeted Autoantigen

Predominant IgG Subclass(es)

Reference

Systemic Lupus Erythematosus

ds DNA Sm U1-RNP Ro/SS-A Histone Cardiolipin

IgG1 + IgG3 IgG1 + IgG3 IgG 1 + IgG3 IgG1 + IgG3 IgG1 + IgG3 IgG 1 - IgG4

Zouali et al., 1984 Eisenberg et al., 1985 Rubin et al., 1985 Blanco et al., 1992 Rubin et al., 1986 Gharavi et al., 1988

Drug-induced Lupus

Histone

IgG1 - IgG4

Rubin et al., 1986

Antiphospholipid Syndrome

132-glycoprotein I

IgG4

Arvieux et al., 1994

SjOgren's Syndrome

Ro/SS-A La/SS-B

IgG1 IgG1 - IgG4

Maran et al., 1993 Maran et al., 1993

Rheumatoid Arthritis

Fc-gamma Type II collagen Stratum corneum

IgG1 + IgG4 IgG3 IgG1

Cohen et al., 1987 Watson et al., 1986 Vincent et al., 1986

Wegener's Granulomatosis

Proteinase-3

IgG1 + IgG4

Brouwer et al., 1991

Primary Biliary Cirrhosis

Pyruvate dehydrogenase

IgG1 - IgG3

Maran et al., 1994

differs in that the cardiolipin antibodies are mainly IgG2 and IgG3 in patients with a primary antiphospholipid syndrome (Arvieux et al., 1994).

IgG subclasses to putative self-components in RA include IgG3 to type II collagen (Watson et al., 1986) or IgG1 to keratin (Vincent et al., 1990).

Drug-Induced Lupus. In drug-induced lupus, antihis-

Primary Biliary Cirrhosis. A particular class of

tone antibodies are not restricted to IgG1 and IgG3 (as is the case in SLE) but are broadly distributed among all IgG subclasses (Rubin et al., 1986). In primary Sj6gren's syndrome (pSS), IgG1 predominates the response to Ro/SS-A; whereas, the antiLa/SS-B subclass pattern varies from one serum to another (Maran et al., 1993). Infants with neonatal lupus erythematosus also display high activities of anti-Ro/SS-A IgG1 (McCune et al., 1987). IgG2 and IgG3 anti-La/SS-B are more frequent in those pSS patients with extraglandular manifestations. Furthermore, IgG2 and IgG4 constitute more of the antiLa/SS-B response in HLA-DR3-positive individuals than in HLA-DR3-negative patients.

antimitochondrial antibodies, collectively termed antiMi2 antibodies, serves as a reliable marker for primary biliary cirrhosis. The pyruvate dehydrogenase (anti-PDH) antibodies belong not only to the IgG 1 and IgG3 subclasses, but also to the IgG2 and IgG4 (Maran et al., 1994). Using an indirect immunofluorescence technique, one group (Mahmud et al., 1990) showed that antireticulin antibody was of the IgG and IgA subclass. Increased IgG 1 and IgG4 antineutrophil cytoplasmic antibodies are found in Wegener's granulomatosis and related disorders (Brouwer et al., 1991). By contrast, in systemic vasculitis, antineutrophil cytoplasmic antibodies are predominantly of the IgG1 and IgG3 subclasses; interestingly, when the active and clinical remission IgG subclass distributions were compared, IgG3 ANCA activities had fallen and IgG2 were increased during remission (Jayne et al., 1991).

Rheumatoid Arthritis. IgG subclass distribution of rheumatoid factor (RF) presents conflicting results in patients with rheumatoid arthritis (RA). Early studies indicated IgG3 present in a high proportion of plasma cells from RA synovial membranes (Munthe and Natvig, 1972); whereas, in a later study, the most important subclasses of RF in sera from patients with RA were IgG1 and IgG4 (Cohen et al., 1987). Other selective distributions of autoantibody activities among 104

Organ-Specific Autoimmune Diseases In organ-specific autoimmune diseases (Table 2), there is also selective participation of IgG subclasses in a variety of autoantibodies; for example: IgG1 islet

Table 2. Predominant Autoantibody IgG Subclass(es) in Organ-Specific Autoimmune Diseases

Disease State

Targeted Autoantigen

Predominant IgG Subclass(es)

Reference

Insulin-dependent diabetes mellitus

Islet cell

IgG1

Omar et al., 1987

Myasthenia gravis

Acetylcholine receptor

IgG1 + IgG3

Nielsen et al., 1985

Infertility

Spermatozoa

IgG1 + IgG3

Haas and D'Cruz, 1991

Myxedema

Thyroglobulin

IgG1 + IgG4

Barra et al., 1986

Bullous pemphigoid

Basement membrane

IgG4

Bird et al., 1986

antibodies occur alone in patients with early insulindependent diabetes mellitus (Omar et al., 1987); IgG 1 and IgG3 antibodies to acetylcholine receptors occur in myasthenia gravis (Nielsen et al., 1985); IgG1 and IgG3 antibodies to spermatozoa are present in infertility (Haas and D'Cruz, 1991); and IgG1 associated with IgG4 antibodies to thyroglobulin are found in myxedema (Barra et al., 1986). Finally, a disproportionately high percentage of antibasement membrane antibodies are IgG4 in bullous pemphigoid (Bird et al., 1986).

THE METHODS

Various enzyme-linked immunosorbent assays are much more widely used than indirect immunofluorescence techniques for measuring antibody subclasses. The application of this method may be difficult, given that the antigen must bind to the plate. Indeed, negatively charged antigens bind very poorly to the polystyrene commonly used as a solid phase. For example, polysaccharides must be immobilized to such solid phases, e.g., by precoating with poly-Llysine or after covalent binding to poly-L-lysine, biotin or tyramine (Barra et al., 1988). Formerly controversial due to the widely varying affinities of the available monoclonal antibodies, quantitation of IgG subclasses is now quite reliable when thoroughly characterized monoclonals are used and when the results for total IgG (e.g., by nephelometry) are compared with the sum of the concentrations of the four IgG subclass assays. To control for varying performance of the subclass reagents in quantitation of antigen-specific IgG subclasses, the assays can be calibrated with sera affinity-depleted into subclass-specific fractions. The optimal working dilutions for each monoclonal are defined as those giving comparable optical densities for approximately

the same amounts of IgG subclasses. The subclass of the bound antibodies are determined by incubation with these monoclonals, followed by detection with antimouse IgG antibody. The latter reagent must be extensively absorbed with human IgG and coupled with peroxidase or alkaline phosphatase.

T H E CAUSES

There is compelling evidence that IgG antibody to certain antigens (defined by their biochemical properties and thymus dependency) is restricted in its distribution between the subclasses (Jefferis and Kumararatne, 1990; Preud'homme, 1992). Thus, IgG 1 and IgG3 are particularly involved in the response to proteins. In contrast, IgG2 is more frequently seen following immunization with polysaccharides, and IgG4 is frequently found in the sera of patients with hypersensitivities and chronic parasitic infections. Indeed, patients with filariasis usually generate vigorous IgG4 antibody responses to their infections (Ottesen et al., 1985). IgG4-specific assays effectively overcome the cross-reactivity of anti-Brugia malayi antibody with phosphocholine, because normal humans are generally incapable of mounting IgG4 responses to phosphocholine epitopes (Lal and Ottesen, 1988). Given the arguments to support the concept of antigen induction of the immune response, presentation is crucial in this phenomenon. This implies that the type and duration of stimulus affect the distribution of the IgG subclasses synthesized, in contrast, for example, to pokeweed mitogen which induces IgG plasma cells that have a subclass distribution similar to that found in normal serum. In addition, the fact that the 5' of the 7 heavy chain genes is 73, followed in the 3' direction by 71, 72 and 74 (Apiller and Hood, 1989), suggests that the switch from IgG goes over

105

the most proximal 7 heavy chain gene. Indeed, a switch of anti-dsDNA antibodies from predominantly IgG3 to predominantly IgG1 can occur in SLE. This indicates that, as a result of chronic immunization, the autoantibody response is antigen-driven. In this respect, it is interesting that anti-PDH IgG are restricted to IgG1 in the family members of patients with PBC, and that IgG subclass combinations are not found in these healthy relatives (Maran et al., 1994). Cytokines also have profound and pleiotropic effects on B-cell proliferation and differentiation (Kroemer and Martinez, 1991). Interleukin (IL)-4 facilitates IgE and IgG1 by normal and atopic donor mononuclear cells; whereas, IL-10 and transforming growth factor cooperate to favor IgA production. IL-5 together with lipopolysaccharides upregulate the synthesis of IgM and IgG3. Conversely, interferon (IFN) 7 and IL-6 act antagonistically in the induction of IgG 1, but add~'tively in that of IgG2 (Kawano et al., 1994). Immunoglobulin class switching is thus tightly related to the biochemical nature of the antigen and the way this is processed and presented to the B cells. Current information about the mechanism and regulation of class-switch recombination is limited (Lieber, 1991). However, this seems to be controlled by Tlymphocyte-derived cytokines, such as IL-4, IFN7 and transforming growth factor 13(Purkenson and Isakson, 1992). In fact, the majority of immune responses, whether "humoral" or "cellular," require the participation of helper T lymphocytes (Th). T cells markedly influence the character of the humoral response by directing the synthesis of specific IgG isotypes. Induction of Thl and Th2 responses might be a clue to the understanding of the immune response (Romagnani, 1992). Processed antigens originating from intracellular bacteria and viruses induce specific immune responses of the Thl type (resulting in the production of IL-2 and IFN7); whereas, soluble antigens as well as some helminth components promote the differentiation of Th cells into the Th2 phenotype (the absence of IFN7 and the presence of IL-4 seem to be critical). The nature of the B-cell activator, combined with cytokines produced by nonspecific, non-T-cells, e.g., macrophages, NK cells and polyclonally activated B-cells, may play an

additional role in the process leading to immunoglobulin isotype switching (Snapper and Mond, 1993). Cytokines are not sufficient stimulus so that the second signals, such as physical contact with activated T cells may be operating (Purkenson and Isakson, 1992). To summarize, the basis for these observations requires further clarifications.

REFERENCES

Arvieux J, Roussel B, Ponard D, Colomb MG. IgG2 subclass restriction of anti-~2 glycoprotein I antibodies in autoimmune patients. Clin Exp Immunol 1994;95:310-315. Aucouturier P, Mounir S, Preud'homme JL. Distribution of IgG

Hunkapiller T, Hood L. Diversity of the immunoglobulin gene superfamily. Adv Immunol 1989;44:1-63. 106

THE CONSEQUENCES The most severe manifestations of autoimmunity are associated with the IgG class; antigen-specific subclass distribution is probably significant. Indeed, IgG 1 and IgG3 fix complement more efficiently than IgG2; whereas, IgG4 does not activate it (Michaelsen et al., 1991). In addition, the affinity of IgG 1 and IgG3 for Fc- 7 receptor I, II and III is much higher than the other-two subclasses (Fridman et al., 1992). The biased restriction pattern of autoantibody subclass may thus be significant in terms of pathogenesis. Type III receptors for the Fc part of IgG1 and IgG3 are shed upon stimulation, and cell-free receptors can be demonstrated in the serum of patients with autoimmune diseases, such as RA, pSS and SLE (Lamour et al., 1995a; 1995b). The significance of this release is not fully understood, but an immunoregulatory role for these soluble receptors is under study.

CONCLUSION Increases of antigen-specific subclasses are found in some autoimmune diseases. Absence or decreases of antigen-specific IgG subclasses in such sera could reflect their deposition in tissue sites. Isotype restriction in autoimmune diseases might result from an unidentified environmental agent interacting with genetically determined abnormal immune response. Caution must be exercised in interpreting the results of IgG subclass-specific assays, given the methodological pitfalls in such measurements. Because current information about the fine mechanism and regulation of class-switch recombination is still limited, the basis for such observations requires further study.

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JP, Magnusson CG, Kubagawa H, Cooper M, Vartdal F, Vandvik B, Haaijman JJ, Makela O, Sarnesto A, Lando Z, Gergely J, RajnavOlgyi E, L~iszl6 G, Radl J, Molinaro G. Evaluation of monoclonal antibodies having specificity for human IgG subclasses. Results of an IUIS/WHO collaborative study. Immunol Lett 1985;10:223-252. Jefferis R, Kumararatne DS. Selective IgG subclass deficiency: quantitation and clinical relevance. Clin Exp Immunol 1990;81:357-367. Jefferis R, Pound J, Lund J, Goodall M. Effector mechanisms activated by human IgG subclass antibodies: clinical and molecular aspects. Ann Biol Clin 1994;52:57-65. Kawano Y, Noma T, Yata J. Regulation of human IgG subclass production by cytokines. IFN-gamma and IL-6 act antagonistically in the induction of human IgG1 but additively in the induction of IgG2. J Immunol 1994;153:4948-4958. Kroemer G, Martinez C. Cytokines and autoimmune disease. Clin Immunol Immunopathol 1991 ;61:275-295. Lal RB, Ottesen EA. Enhanced diagnostic specificity in human filariasis by IgG4 antibody assessment. J Infect Dis 1988; 158:1034--1037. Lamour A, Baron D, Le Goff P, Youinou P. Anti-Fc gamma receptor III antibodies correlate to the levels of cell-free Fc gamma receptor III in rheumatoid arthritis serum and synovial fluid. J Autoimmunity 1995a;8:249-265. Lamour A, Le Corre R, Pennec YL, Youinou P. Heterogeneity of neutrophil antibodies in patients with primary SjOgren's syndrome. Blood 1995b;in press. Lieber MR. Site-specific recombination in the immune system. FASEB J 1991 ;5:2934--2944. Mahmud T, Peakman M, Senaldi G, McWhirter A, Black CM, Vergani D. Antireticulin antibody in systemic sclerosis. Ann Rheum Dis 1990;49:177-180. Maran R, Dueymes M, Pennec YL, Casburn-Budd R, Shoenfeld Y, Youinou P. Predominance of IgG1 subclass of antiRo/SSA, but not anti-La/SSB antibodies in primary Sj6gren' s syndrome. J Autoimmunity 1993;6:379-387. Maran R, Dueymes M, Adler Y, Shoenfeld Y, Youinou P. Isotypic distribution of antipyruvate dehydrogenase antibodies in patients with primary biliary cirrhosis and their family members. J Clin Immunol 1994;14:323-326. McCune AB, Weston WL, Lee LA. Maternal and fetal outcome in neonatal lupus erythematosus. Ann Intern Med 1987;106: 518--523. Michaelsen TE, Garred P, Aase A. Human IgG subclass pattern of inducing complement-mediated cytolysis depends on antigen concentration and to a lesser extent, on epitope patchiness, antibody affinity and complement concentration. Eur J Immunol 1991;21:11--16. Munthe E, Natvig JB. Immunoglobulin classes, subclasses and complexes of IgG rheumatoid factor in rheumatoid plasma cells. Clin Exp Immunol 1972;12:55-70. Nielsen FC, Rodgaard A, Djurup R. A triple antibody assay for the quantitation of plasma IgG subclass antibodies to acetylcholine receptors in patients with myasthenia gravis. J Immunol Methods 1985;83:249-258. Omar MA, Srikanta S, Eisenbarth GS. Human islet cell antibodies: immunoglobulin class and subclass distribution

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defined by monoclonal antibodies. Diabetes Res 1987;4: 155--157. Ottesen EA, Skvaril F, Tripathy SP, Poindexter RW, Hussain R. Prominence of IgG4 in the IgG antibody response to human filariasis. J Immunol 1985;134:2707-2712. Preud'homme JL. Coversation with Jean-Louis Preud'homme. What is going to happen tomorrow with subpopulations of IgG (dosage, deficit, significance)? Ann Med Interne 1992; 143:319-321. Purkenson J, Isakson P. A two-signal model for regulation of immunoglobulin isotype switching. FASEB J 1992;6:3245-3252. Romagnani S. Induction of TH1 and TH2 responses: a key role for the 'natural' immune response? Immunol Today 1992; 13:379-381. Rubin RI, Tang FL, Lucas AH, Spiegelberg HL, Tan EM. IgG subclasses of antitetanus toxoid antibodies in adult and newborn normal subjects and in patients with systemic lupus erythematosus, Sj6gren's syndrome and drug-induced

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autoimmunity. J Immunol 1986;137:2522-2527. Snapper CM, Mond JJ. Towards a comprehensive view of immunoglobulin class switching. Immunol Today 1993;14: 15--17. Vincent C, Serre G, Basile J-P, Lestra HC, Girbal E, Sebbag M, Soleilhavoup J-P. Subclass distribution of IgG antibodies to the rat oesophagus S t r a t u m c o r n e u m (so-called antikeratin antibodies) in rheumatoid arthritis. Clin Exp Immunol 1990;81:83--89. Waston WC, Cremer MA, Wooley PH, Townes AS. Assessment of the potential pathogenicity of type II collagen autoantibodies in patients with rheumatoid arthritis. Evidence of restricted IgG3 subclass expression and activation of complement C5 to C5. Arthritis Rheum 1986;29:1316-1321. Winkler TH, Henschel TA, Kalies I, Baenkler HW, Skvaril F, Kalden JR. Constant isotype patterns of anti-dsDNA antibodies in patients with systemic lupus erythematosus. Clin Exp Immunol 1988;72:434-439.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

z-GLYCOPROTEIN I AUTOANTIBODIES Eiji Matsuura, Ph.D. a and Takao Koike, M.D. b

almmunology Laboratory, Diagnostics Division, Yamasa Corporation, Choshi 288; and bDepartment of Medicine II, Hokkaido University School of Medicine, Sapporo 060, Japan

HISTORICAL NOTES It is widely believed that antiphospholipid antibodies (aPL), a series of autoantibodies found in patients with antiphospholipid syndromes and often found in the sera of patients with systemic lupus erythematosus (SLE) and related connective tissue diseases, are immunoglobulins that react with anionic phospholipids (PLs) directly. Further, aPL are associated with thromboembolic manifestations (Harris et al., 1983; 1985; Hughes et al., 1986). Against this background, recent studies indicate that a 50 kd plasma protein first described in 1961 (Schultze et al., 1961), [32glycoprotein I (~2-GPI), is required for binding of aPL (McNeil et al., 1990; Galli et al., 1990; Matsuura et al., 1990; 1992). The conformational epitope for anti~2-GPI antibodies develops when I]2-GPI interacts with a lipid membrane composed of negatively charged PLs or when ~2-GPI is adsorbed on a polyoxygenated polystyrene plate (Matsuura et al., 1994).

THE AUTOANTIGENS Origin/Sources I]2-GPI is isolated by sequential perchloric acid extraction or cardiolipin affinity and ion-exchange chromatography. Because it also appears in the lipoprotein fractions on ultracentrifugation and activates lipoprotein lipase in vitro ~2-GPI is also designated apolipoprotein H. The complete amino acid sequence of human I]2-GPI as determined by peptide sequencing reveals a single polypeptide chain composed of 326 amino acid residues with five oligosaccharide attachment sites (Lozier et al., 1984). The

complete nucleotide sequence and the deduced amino acid sequence were established by cDNA cloning from a human hepatoma cell line (HEpG2) and sequencing (Matsuura et al., 1991). I]2-GPI is composed of five homologous motifs of approximately 60 amino acids which contain highly conserved cysteines, prolines and tryptophans (Lozier et al., 1984). The motif is characterized by a framework of four conserved half-cystine residues involved in the formation of two internal disulfide bridges. These repeating motifs were designated as short consensus repeats/ complement control protein repeats or as sushi domains (Kato and Enjoji, 1991) (Figure 1). However, the fifth domain (the carboxyl terminus) of ~2-GPI is a modified form which contains 82 amino acid residues and six half cystines. Purified human I]2-GPI is commercially available from Yamasa Corporation (Chiba, Japan), Diagnostica Stago (France) and elsewhere. Alternatively, a recombinant protein of human I]2-GPI can be successively produced by an expression system using baculovirus and insect cells, Sf9 cells (Igarashi et al., 1993). This expression system can provide large amounts of the recombinant I]2-GPI which is correctly processed and biologically active.

Sequence Information ~2-GPI binds to various kinds of negatively charged substances such as phospholipids (PLs) and lipoproteins, and inhibits the intrinsic blood coagulation pathway (Schousboe, 1985), prothrombinase activity (Nimpf et al., 1986) and ADP-dependent platelet aggregation (Nimpf et al., 1987). ~2-GPI binds to solid-phase PLs via a particular region in the fifth domain, C 281KNKEKKCzs8 (Hunt and Krilis, 1994;

109

Figure 1. Amino acid sequence and location of disulfide bonds in human ]32-GPI.

Matsuura et al., 1995) Both the primary sequence and the tertiary structure of the region are involved in binding to PLs (Matsuura et al., 1995). Autoantibodies against ~2-GPI from sera of patients or from NZW x BXSB (W/B) F1 mice, an animal model of antiphospholipid syndrome (APS) (Hashimoto et al., 1992), recognize a conformational epitope developed when ~32-GPI interacts with a lipid membrane composed of negatively charged PLs or when ]32-GPI is absorbed on a polyoxygenated polystyrene plate (Matsuura et al., 1994) (Figure 2). 110

THE AUTOANTIBODIES Terminology Antiphospholipid antibodies (aPL) include a wide family of circulating immunoglobulins, mainly IgG isotype, which include anticardiolipin antibodies (aCL) and lupus anticoagulant (LA). These antibodies recognize complexes of negatively charged PLs, a variety of plasma proteins including ~2-GPI and prothrombin and an indirect conformational epitope

Figure 2. Each drawing represents a solid-phase antigen for antibody detection in ELISA: (I) cardiolipin coated on a plain polystyrene plate; (II) ~2-GPI complexed with the cardiolipin-coated plate and altered structure of [32-GPI suitable for anti-~2-GPI antibody detection; (III) ~2-GPI on a plain polystyrene plate and the native structure of ~2-GPI; and (IV) [32-GPIadsorbed on an oxygenated polystyrene plate and altered structure on [32-GPI suitable for anti-[32-GPI antibody detection.

appearing on the altered structure of ~2-GPI but not PLs directly. Thus, at least some antibodies classified as "aPL" are in fact "anti-I]z-GPI antibodies." Further, these anti-~z-GPI antibodies seem to be the dominant autoantibody type in aPL-positive sera of those patients with APS studied to date.

Pathogenetic Role Animal Model. Several autoantibodies, circulating immune complexes and lupus nephritis are present in NZW x BXSB (W/B) F1 male mice with systemic lupus-like disease (Hang et al., 1981; Berden et al., 1983). Myocardial infarction is the hallmark of the W/B F1 male mice; the incidence of myocardial infarction in these mice increases with age and more than 80% of male W/B F1 mice with myocardial infarction have small multiple infarctions in the right ventricular free wall and arterial, posterior and septa ventricular walls (Yoshida et al., 1987). The affected coronary arteries have intimal thickening with cellular components and frequent recanalization or an eosinophil thrombus-like substance. Thrombocytopenia is a frequent finding of unexplained mechanism in patients with aPL. Anti-]32-GPI autoantibodies are raised in W/B F1 male mice. W/B F1 male mice produce autoantibodies against CL and the titer of aCL increases with age (Hashimoto et al., 1992). These antibodies were detected by the conventional aCL ELISA (Koike et al., 1984) and alternatively could be detected by appropriate ELISA for anti-[32-GPI detection, using ~2-GPI complexed with solid-phase CL (Matsuura et al., 1992) or using ~2-GPI coated on a polyoxygenated polystyrene plate as an antigen

(Matsuura et al., 1994). WB F1 mice also develop thrombocytopenia with age; platelet-associated antibodies and circulating antiplatelet antibodies are also present.

Human Disease. Membranes of activated platelets are an important source of negatively charged phospholipids, e.g., phosphatidylserine, providing a catalytic surface for blood coagulation. Factor Xa and thrombin are generated by the tenase and prothrombinase complexes, respectively, via the catalytic surface of the activated platelets and procoagulant microparticles shed by the activation of platelets (Shi et al., 1993). [32-GPI inhibits the generation of factor Xa by the activation of platelets and aCL (i.e. anti-132-GPI antibodies) interfere with this inhibition (Shi et al. 1993). ~2-GPI affects the generation of factor Xa rather than its binding to platelet-derived microparticles, and aCL (anti-[32-GPI antibodies) interfere with this inhibition (Nomura et al., 1994). On the other hand, the anticoagulant effect of aCL is dependent on the presence of [32-GPI. Thus, although there are discrepancies about the function of ]32-GPI and aCL (anti-]32-GPI antibodies) in the generation of factor Xa, activated platelets may be a dominant target of [32-GPI and anti-132-GPI antibodies. Oxidative modification of LDL is thought to play a central role in atherogenesis (Palinski et al., 1989). Antibodies against epitopes of oxidized LDL recognize materials in atherosclerotic lesions but not in normal arteries (Salonen et al., 1992). aCL cross react with oxidized LDL (Vaarala et al., 1993). There is an interaction between oxidized plasma lipoproteins, [32-

111

GPI and anti-[32-GPI antibodies in that ~2-GPI specifically binds to oxidized lipoproteins (i.e., oxidized VLDL, oxidized LDL, or oxidized HDL) and anti-~zGPI antibodies sequentially bind to the ~2-GPI complexed with oxidized lipoproteins. These findings strongly suggest that oxidized lipoproteins are sequentially targeted by ~32-GPI and anti-[32-GPI antibodies and that this immunoreaction is involved not only in the metabolism of oxidized plasma lipoproteins but also in atherosclerotic events in APS. Monoclonal Antibodies. Two monoclonal aCL, i.e.,

anti-~2-GPI autoantibodies, namely WB-CAL-1 (IgG2a ~c) and WB-CAL-3 (IgG2b ~:) established from 4month-old WIB F1 male mice (Hashimoto et al., 1992), induce thrombosis in vivo when injected into mice (unpublished observation). Sequence analysis of V~j and V K genes of these pathogenic anti-[32-GPI antibodies shows Via558 and VK21 or VK23 genes (Kita et al., 1994). Although WB-CAL-1 antibody has an 86.6% homology with WB-CAL-3 antibody, these clones are not thought to be closely related clones because, there were no restricted DII and Jii genes among these two antibodies. These findings suggest the possibility that the use of IgVIa genes in the pathogenic antibodies might be limited. WB-CA-1 shared a 91.5% homology to the known germ-line V H gene, 43Y, suggesting that this anti-[32-GPI clone with pathogenic capacity is generated by somatic mutations. There is, however, no evidence that 43Y gene is the origin of WB-CAL-1 VII gene. A comparison of the Via gene encoding the pathogenic anti-[32-GPI antibodies with the genetically related germline VII gene should shed light on mechanisms underlying these events in subjects with autoimmune disease. Five human monoclonal antibodies (IgM) against [32-GPI established from peripheral blood lymphocytes of three British patients with APS by sequential Epstein-Barr virus transformation and cell fusion techniques (Ichikawa et al., 1994) bind both to the solid-phase complexes of ~2-GPI and negatively

charged PLs and to ~2-GPI adsorbed on a polyoxygenated polystyrene surface. These monoclonal antibodies obtained from W/B F1 mice and from APS patients have similar characteristics to those found in the sera from APS patients. Methods of Detection

Detection of anti-[32-GPI antibodies in patient sera is important in the diagnosis of APS. As previously described, such pathogenic anti-~z-GPI autoantibodies recognize an induced or cryptic epitope of ~2-GPI which appears only when ~32-GPI binds to a lipid membrane composed of negatively charged PL or when ~32-GPI is adsorbed onto a polyoxygenated polystyrene plate. Anti-~z-GPI antibodies can routinely be detected by ELISA using two types of antigen solid phase, i.e., (1) 132-GPI associated with a CLcoated polystyrene plate; and, (2) ~2-GPI adsorbed on a polyoxygenated polystyrene plate (Table 1). In assay using a CL-coated plate, control assay without [32-GPI is of particular importance and antibody binding must be compared in the absence and/or the presence of ~2-GPI (e.g., 15 pg/mL) to evaluate the 132-GPI-dependency of antibodies. Antibodies specifically or nonspecifically reactive to PLs in sample fluids may bind to a CL-coated plate directly, even if the plate is previously and/or simultaneously treated with ~2-GPI. Direct binding of aPL to a CLcoated plate is decreased in a dose-dependent fashion by the addition of [32-GPI but is not completely abolished even by addition of a large excess of [32GPI (more than 1.0 mg/mL). By comparison, the anti~2-GPI ELISA using a polyoxygenated plate is superior. Procedures are simple and false-positive results for aPL are negligible. Standardization of calibration materials (monoclonal anti-~2-GPI antibodies) and of assay protocols is under continuous consideration by the ISTH SSC Subcommittee Meeting for Phospholipid-dependent Antibodies/Lupus Anticoagulant.

Table 1. Reactivity of Autoantibodies Raised in APS Patients to Solid-phase Antigens ~2-GPI Adsorbed on Polystyrene Plates

CL-Coated Plates

Nontreated

Polyoxygenated

Without ~2-GPI

With ~2-GPI

With FCS

Anti-132-GPI

No

Yes

No

Yes

Varies

aCL

No

No

Yes

Varies

Varies

Autoantibodies

112

CLINICAL UTILITY

CONCLUSION

A subgroup of patients with systemic lupus erythematosus (SLE) with antibodies against PLs and a constellation of features which can include stroke, venous thrombosis, recurrent abortion and thrombocytopenia were identified as having the "antiphospholipid syndrome (APS)" (Harris et al., 1983; 1985; Hughes et al., 1986). APS is seen frequently in patients with SLE, but other patients not suffering from defined SLE can also exhibit the characteristic features. Many of these patients have some clinical or serologic features of SLE, but fail to fulfill four of the 1982 revised criteria for the classification of SLE. These patients are considered to suffer from a lupus-like illness or "probable" SLE. Other patients with aPL (and/or anti-132-GPI antibodies) and thrombosis or recurrent abortion do not have any of the typical clinical or serological features of SLE or any other defined connective tissue diseases; these patients are defined as having "primary" APS.

Because antiphospholipid antibodies (aPL) are associated with thromboembolic manifestations, the term "antiphospholipid syndrome (APS)" seems appropriate to define this set of pathologic features. At least some antibodies detected as aPL, especially aCL, recognize a cryptic epitope on the Ig2-glycoprotein I (~2-GPI) molecule, and the epitope appears only when 132-GPI interacts with lipid membranes composed of negatively charged phospholipids (PLs) or when [32-GPI is adsorbed on a polyoxygenated polystyrene plate. Although the pathogenic roles of 132-GPI and anti-132GPI are still poorly understood, the detection of anti132-GPI autoantibodies is a clear diagnostic sign of APS. See also LuPus ANTICOAGULANT, PHOSPHOLIPID AUTOANTIBODIES- CARDIOLIPIN and PHOSPHOLIPID AUTOANTIBODIES- PHOSPHATIDYLSERINE.

REFERENCES

diolipin antibodies from patients with the antiphospholipid syndrome. Arthritis Rheum 1994;37:1453--1461. Igarashi M, Matsuura E, Igarashi Y, Nagae H, Matsuura Y, Ichikawa K, Yasuda T, Voelker DR, Koike T. Expression of anticardiolipin cofactor, human 132-glycoprotein I, by a recombinant baculovirus/insect cell system. Clin Exp Immunol 1993;93:19--25. Kato H, Enjyoji K. Amino acid sequence and location of the disulfide bonds in bovine 132-glycoprotein I: the presence of five Sushi domains. Biochemistry 1991;30:11687-11694. Kita Y, Sumida T, Iwamoto I, Yoshida S, Koike T. V gene analysis of anticardiolipin antibodies from (NZW x BXSB) F1 mice. Immunology 1994;82:494--501. Koike T, Sueishi, H, Funaki H, Tomioka H, Yoshida S. Antiphospholipid antibodies and biological false positive serological test for syphilis in patients with systemic lupus erythematosus. Clin Exp Immunol 1984;56:193--199. Lozier J, Takahashi N, Putman FW. Complete amino acid sequence of human plasma 132-glycoprotein I. Proc Natl Acad.Sci USA 1984;81:3640-3644. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: 132-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA. 1990;87:4120-4124. Matsuura E, Igarashi, Y, Fujimoto M, Ichikawa K, Koike T. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990;336:177-178. Matsuura E, Igarashi M, Igarashi Y, NagaeH, Ichikawa K, Yasuda T, Koike T. Molecular definition of human 132glycoprotein I (132-GPI) by cDNA cloning and interspecies

Berden JH, Hang LM, McConahey PJ, Dixon FJ. Analysis of vascular lesions in murine SLE. I. Association with serological abnormalities. J Immunol 1983;130:1699-1705. Galli M, Comfurius P, Maassen C, Hemker HC, de Baets MH, van Breda-Vriesman PJ, Barbui T, Zwaal RF, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990;335:1544-1547. Hang LM, Izui S, Dixon FJ. (NZW x BXSB) F1 hybrid, a model of acute lupus and coronary vascular disease with myocardial infarction. J Exp Med 1981;154:216--221. Hashimoto Y, Kawamura M, Ichikawa K, Suzuki T, Sumida T, Yoshida S, Matsuura E, Ikehara S, Koike T. Anticardiolipin antibodies in NZW x BXSB F1 mice: a model of antiphospholipid syndrome. J Immunol 1992;149:1063-1068. Harris EN, Gharavi AE, Boey ML,. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;2:1211-1214. Harris EN, Gharavi AE, Hughes GR. Antiphospholipid antibodies. Clin Rheum Dis 1985;11:591--609. Hughes GR, Harris NN, Gharavi AE. The anticardiolipin syndrome. J Rheumatol 1986;13:486--489. Hunt JE, Krilis SA. The fifth domain of 132-glycoprotein I contains a phospholipid binding site (Cys281-Cys288) and a region recognized by anticardiolipin antibodies. J Immunol 1994;152;653-659. Ichikawa K, Khamashta MA, Koike T, Matsuura E, Hughes GR. 132-Glycoprotein I reactivity of monoclonal anticar-

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differences of ]]2-GPI in alteration of anticardiolipin binding. Int Immunol 1991;3:1217-1221. Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Suzuki T, Sumida T, Yasuda T, Koike T. Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor. J Immunol 1992; 148: 3885-3891. Matsuura E, Igarashi Y, Yasuda T, Triplett DA, Koike T. Anticardiolipin antibodies recognize ~2-glycoprotein I structure altered by interacting with an oxygen-modified solid phase surface. J Exp Med 1994;179:457--462. Matsuura E, Igarashi M, Igarashi Y, Katahira T, Nagae H, Ichikawa K, Triplett DA, Koike T. Molecular studies on phospholipid-binding sites and cryptic epitopes appearing on ~2-glycoprotein I structure recognized by anticardiolipin antibodies. Lupus 1995;4:S13-S17. Nimpf J, Bevers EM, Bomans PH, Till U, Wurm H, Kostner GM, Zwaal RF. Prothrombinase activity of human platelets is inhibited by ~2-glycoprotein I. Biochim Biophys Acta 1986;884:142--149. Nimpf J, Wurm H, Kostner GM. ]]2-glycoprotein I (apo-H) inhibits the release reaction of human platelets during ADPinduced aggregation. Atherosclerosis 1987;63:109-114. Nomura S, Fukuhara S, Komiyama Y, Takahashi H, Matsuura E, Nakagaki T, Funatsu A, Sugo T, Matsuda M, Koike T. 132glycoprotein I and anticardiolipin antibody influence factor Xa generation but not factor Xa binding to platelet-derived microparticles. Thromb Haemost 1994;71:526--527. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC,

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Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci USA 1989;86: 1372--1376. Salonen JT, Yla-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssonen K, Palinski W, Witztum JL. Autoantibody against oxidized LDL and progression of carotid atherosclerosis. Lancet 1992:339:883--887. Schousboe I. ]32-glycoprotein I: a plasma inhibitor of the contact activation of the intrinsic blood coagulation pathway. Blood 1985 ;66:1086-- 1091. Schultze HE, Heide K, Haupt H. Uber ein bisher unbekanntes Niedermolikulars [3z-globulin des Humanserums. Naturwissenschaften 1961;48:719--724. Shi W, Chong B H, Hogg PJ, Chesterman CN. Anticardiolipin antibodies block the inhibition by [32-glycoprotein I of the factor Xa generating activity of platelets. Thromb Haemost 1993;70:342--345. Vaarala O, Alfthan G, Jauhiainen M, Leirisalo-Repo M, Aho K, Palosuo T. Crossreaction between antibodies to oxidized lowdensity lipoprotein and to cardiolipin in systemic lupus erythematosus. Lancet 1993;341:923--924. Yoshida H, Fujiwara H, Fujiwara T, Ikehara S, Hamashima Y. Quantitative analysis of myocardial infarction in (NZW x BXSB) F1 hybrid mice with systemic lupus erythematosus and small coronary artery disease. Am J Pathol 1987;129: 477--485.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

BETA-ADRENERGIC RECEPTOR (AND OTHER HORMONE RECEPTOR) AUTOANTIBODIES Douglas C. Aziz, M.D., Ph.D.

Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES Dilated cardiomyopathy is believed to be a sequela to acute viral myocarditis. As a direct extension of this, the possibility of an etiology related to a heart-reactive circulating immunoglobulin was investigated. In patients with dilated cardiomyopathy, immunofluorescent staining demonstrates various components of the myocardial cell, including the ADP/ATP carrier, myosin heavy chain, laminin and the beta-adrenergic receptor (Abelman, 1988; Maisch et al., 1982; 1983; 1987; Schultheiss, 1989; Rose et al., 1987; Wolff et al., 1989). Antibodies to myolemma are detectable in patients with postmyocarditic cardiomyopathy; interfibrillary autoantibodies are detectable in patients with primary, alcoholic and postmyocarditic dilated cardiomyopathy and myosin and fibrillary autoantibodies are found in patients with these cardiomyopathies as well as other secondary cardiomyopathies (Rada et al., 1992). Reduced responsiveness of the failing myocardium to inotropic beta-agonists was evidence of a decrease in the number of cardiac beta-receptors in the myocardium of these patients and an autoimmune cause was sought (Fowler et al., 1986; Limas et al., 1989a; Murphee and Saffitz, 1989). A substantial proportion of patients with dilated cardiomyopathy were found to have circulating autoantibodies directed against the beta- 1-adrenoceptor.

AUTOANTIGENS The human heart contains both beta-l-adrenoceptors and a significant number of beta-2-adrenoceptors, both of which are mechanistically involved in the positive inotropic and chronotropic effects of beta-adrenergic

agonists. Beta-3-adrenoceptors are described, but their exact physiological function is not yet known (Emorine et al., 1994).

AUTOANTIBODIES Method of Detection

The prevalence and pathogenetic role in specific disease states of the autoantibodies to beta-adrenoceptors is dependent on the assay used for their detection. Ligand-binding inhibition, isoproterenol-sensitive adenylate cyclase activity, enzyme-linked immunoassay of beta-adrenoceptors peptides and immunoblot are assays used to detect these autoantibodies (Limas and Limas, 1992). In the ligand-binding inhibition assay, cardiac membranes obtained from homogenized myocytes isolated from experimental animals, usually rat, are preincubated with and without the patient's serum, before incubation with tritiated dihydroalprenolol. The number of beta-adrenoceptors are determined from Scatchard plots of the binding data. Nonspecific binding is determined by adding propranolol, which is subtracted from the total binding. Binding in the absence of serum is taken to be 100%. Inhibition of adenylate cyclase may occur at a postreceptor step and can be missed in assays that utilize ligand-binding inhibition (Limas and Limas, 1992). The cardiac adenylate cyclase activity assay measures the ability of beta-adrenoceptors in homogenized myocyte preparations to convert radiolabeled adenosine triphosphate to radiolabeled cyclic adenosine monophosphate (Limas and Limas, 1991). The effect of patient sera on basal, isoproterenol- and NaF115

stimulated adenylate cyclase activities is measured. In the enzyme-linked immunoassay (ELISA), peptide sequences specific for beta-l-adrenoceptors (HWWRAESDEARRCYNDPKCCDFVTNR) and for beta-2-adrenoceptors (HWYRATHQEAINCYANETCCDFFTNQ) and common to beta-l- and beta-2adrenoceptors (CONFGNFWCEFWT) are incubated with affinity-purified antibodies and detected with biotinylated-antihuman rabbit antibody which reacts with strepavidin-peroxidase and chromogenic substrate (Magnusson et al., 1990). Although this assay will differentiate antibodies to beta-1- from beta-2-adrenoceptor, it will not detect antibodies that inhibit adenylate cyclase activity. In an immunoblot format, antigen is prepared from Escherichia coli expressing human beta-1-adrenoceptors, electrophoresed in polyacrylamide-SDS gel and transferred to nitrocellulose where affinity-purified antibodies react ~vith the recombinant antigens (Magnusson et al., 1990).

CLINICAL UTILITY Disease Association Chronic Heart Failure. In chronic heart failure, the number of both beta-l- and beta-2-adrenoceptors is reduced. Beta-l-adrenoceptors are decreased in all forms of chronic heart failure; whereas, beta-2-adrenoceptors are decreased in mitral valve disease, tetralogy of Fallot and end-stage ischemic cardiomyopathy. In dilated cardiomyopathy, and possibly in aortic valve disease, beta-1-adrenoceptors are decreased, but beta2-adrenoceptors appear unaltered (Brodde, 1991). Using the ligand-binding inhibition assay, beta-receptor autoantibodies were detectable in 30--40% of patients with dilated cardiomyopathy (Limas et al., 1989a; Limas and Limas, 1992). About 15% of patients with other cardiac diseases and none of the control patients had elevated autoantibodies. In particular, ischemic cardiomyopathy is associated with the presence of autoantibodies in 22% and alcoholic cardiomyopathy in 25% (Limas and Limas, 1992). Using the ELISA assay, which differentiates antibodies reactive with beta-l- from beta-2-adrenoceptors, the highest reactivity is toward the second extracellular loop of the beta-1 peptide, with very little reactivity towards the beta-2 peptide (Magnusson et al., 1990). Inhibition of isoproterenol-sensitive adenylate cyclase activity is demonstrable in 52% of 116

patients with dilated cardiomyopathy, but basal and NaF-stimulated activities are unaffected (Limas et al., 1990b).

Dilated Cardiomyopathies. Affected family members with familial idiopathic dilated cardiomyopathy have very high frequency of beta-adrenoceptor autoantibodies (62%) and unaffected members to a lesser extent (29%) (Graber et al., 1986; Limas and Limas, 1993). The higher-than-expected frequency of autoantibodies in unaffected members suggests that the autoantibodies can be detected presumably before the syndrome is clinically manifest. Antibodies to beta-adrenoceptor cause a positive chronotropic response to beating cultured rat myocytes, which are subject to beta-adrenoceptor blockade (Wallukat et al., 1991). Paradoxically, beta-blockers have beneficial effects in the treatment of dilated cardiomyopathy, which can be partially explained by their ability to displace the antibody (Waagstein et al., 1975; Engelmeir et al., 1985; Chatterjee, 1989; Adampoulos et al., 1995). Antibody-bound receptors do not recirculate after internalization in endocytosis, in contrast to isoproterenol-mediated endocytosis in which the receptor recirculates to the plasma membrane, thus avoiding permanent downregulation (Limas and Limas, 1991; Limas et al., 1991). After cardiac transplantation, there is a prompt decline in the titer of beta-adrenoceptor autoantibodies (Limas et al., 1989b). Patients with dilated cardiomyopathy have a significant prevalence of HLA-DR4 antigen (40% versus 24% in normal subjects). Of the patients with HLA-DR4 and dilated cardiomyopathy, 60-80% have beta-adrenoceptor antibodies, in contrast to only 22--25% of HLA-DR4-negative patients (Limas et al., 1990a). This association is most specific for the ligand-binding inhibition and adenylate cyclase activity assays (Limas et al., 1990a). Most of these HLA-DR4-negative patients with dilated cardiomyopathy are HLA-DR1. None of the patients with autoantibodies have HLA-DR3 antigen; whereas, 37% of patients who do not have autoantibodies have HLA-DR3. Linkage analysis of RFLP polymorphisms shows an association with HLA-DR-beta and HLADQ-alpha haplotypes (Limas et al., 1994). Chagas Cardiomyopathy. Chronic infection with Trypanosoma cruzi, a major health problem in South America, sometimes leads to Chagas' disease, a persistent, inflammatory cardiomyopathy that often

results in congestive heart failure and death. Experimental Chagas' disease in mice is associated with autoantibody production to cardiac myosin, desmin (Tobbetts et al., 1994) and beta-adrenergic and cholinergic receptors (Gorelik et al., 1990). An autoantibody that cross-reacts with a T. cruzi ribosomal protein is identified in 30% of Chagas' disease patients (Bonfa et al., 1993). Antilaminin antibodies are detectable in 50% of Chagas patients compared to 5% of controls and the highest titers are detected in the group with the most severe symptoms (Milei et al., 1993). Antibodies to both beta-l- and beta-2-adrenoceptors are detected in Chagasic patients. Chagasic IgG inhibits dihydroalprenolol binding to cardiac beta-1- and

splenic beta-2-adrenoceptors 1988).

(Sterin-Borda et al.,

CONCLUSION In summary, beta-adrenoceptor autoantibodies are detectable in patients with dilated cardiomyopathy, particularly in affected patients with familial idiopathic dilated cardiomyopathy (62%). The high rate of detection in alcoholic (25%) and ischemic (22%) cardiomyopathies preclude use of beta-adrenoceptor autoantibody testing as a predictive indicator of dilated cardiomyopathy.

Table 1. Diseases Associated with Other Receptor Autoantibodies

Autoantibody

Associated Disease (Frequency)

Frequency in Health or Other Diseases (Frequency)

References

Asialoglycoprotein Receptor

Autoimmune hepatitis (76%)

Viral hepatitis (11%) Other chronic hepatitis (8%)

Treichel et al., 1994

Glutamate Receptor

Rasmussen's encephalitis* (75%) Epilepsy (0%) CNS lysis (0%) Trauma (0%)

Rogers et al., 1994

Interleukin- 1a Receptor

Schnitzlers' syndrome** (66%)

Saurat et al., 1991

IgE Receptor

Chronic urticaria (68%)

[32-adrenergic Receptor

Asthma (40%)

~2-adrenergic Receptor

Myasthenia gravis (18%)

* **

Normal (18%)

Hide et al., 1993 Normal (5%)

Turki and Ligget, 1995; Wallukat and Wollenberger, 1991 Eng et al., 1992

Childhood epilepsy, hemiplegia, dementia and cerebral inflammation Urticaria, macroglobulinemia

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Brodde OE. Pathophysiology of the beta-adrenoceptor system in chronic heart failure: consequences for treatment with agonists, partial agonists or antagonists? Eur Heart J 1991; 12:$54--$62. Chatterjee K. Potential use of third-generation beta-blockers in heart failure. J Cardiovasc Pharmacol 1989;14:$22-$27. Emorine L, Blin N, Strosberg AD. The human beta 3-adrenoceptor: the search for a physiological function. Trends Pharmacol Sci 1994;15:3-7. Eng H, Magnusson Y, Matell G, Lefvert AK, Saponja R, Hoebeke J. Beta 2-adrenergic receptor antibodies in myashenia gravis. J Autoimmun 1992;5:213--227. Engelmeier RS, O'Connel JB, Walsh R, Rad N, Scanlon PJ, Gunnar RM. Improvement in symptoms and exercise tolerance by meoprolol in patients with dilated cardiomyo-

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pathy: a double-blind, randomized, placebo-controled trial. Circulation 1985;72:536--546. Fowler MB, Laser JA, Hopkins GL, Minobe W, Bristow MR. Assessment of the beta-adrenergic receptor pathway in the intact failing human heart: progressive receptor downregulation and subsensitivity to agonist response. Circulation 1986;74:1290-1302. Gorelik G, Genaro AM, Sterin-Borda L, Gonzales Cappa S, Borda ES. Antibodies bind and activate beta-adrenergic and cholinergic lymphocyte receptors in Chagas' disease. Clin Immunol Immunpathol 1990;55:221-236. Graber HL, Unverferth DV, Baker PB, Ryan JM, Baba N, Wooley CF. Evolution of a hereditary cardiac conduction and muscle disorder: a study involving a family with six generations affected. Circulation 1986;74:21-35. Hide M, Francis DM, Grattan CE, Hakimi J, Kochan JP, Greaves MW. Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria. N Engl J Med 1993;328:1599-1604. Limas CJ, Limas C, Goldenberg I. Intracellular distribution of adrenoreceptors in the failing human myocardium. Am Heart J 1989a;117:1310-1316. Limas CJ, Goldenberg IF, Limas C. Autoantibodies against beta-adrenoceptors in human idiopathic dilated cardiomyopathy. Circ Res 1989b;64:97-103. Limas CJ, Limas C, Kubo SH, Olivari MT. Anti-beta-receptor antibodies in human dilated cardiomyopathy and correlation with HLA-DR antigens. Am J Cardiol 1990a;65;483--487. Limas CJ, Goldenberg IF, Limas C. Influence of anti-betareceptor antibodies on cardiac adenylate cyclase in patients with idiopathic dilated cardiomyopathy. Am Heart J 1990b; 119:1322-1328. Limas CJ, Limas C. Beta-adrenoceptor antibodies and genetics in dilated cardiomyopathy- an overview and review. Eur Heart J 1991;12:S175-S177. Limas CJ, Goldenberg IF, Limas C. Effect of antireceptor antibodies in dilated cardiomyopathy on the cycling of cardiac beta receptors. Am Heart J 1991;122:108--114. Limas CJ, Limas C. HLA-DR antigen linkage of anti-j3 receptor antibodies in idiopathic dilated and ischaemic cardiomyopathy. Br Heart J 1992;67:402--405. Limas CJ, Limas C. Immune-mediated modulation of betaadrenoceptor function in human dilated cardiomyopathy. Clin Immunol Immunopathol 1993;68:204--207. Limas C, Limas CJ, Boudoulas H, Bair R, Graber H, Sparks L, Wooley CF. Anti-beta-receptor antibodies in familial cardiomyopathy: correlation with HLA-DR and HLA-DQ gene polymorphisms. Am Heart J 1994;127:382-386. Magnusson Y, Marullo S, Hoyer S, Waagstein F, Andersson B, Vahlne A, Guillet JG, Strosberg AD, Hjalmarson A, Hoebeke J. Mapping of a functional autoimmune epitope on the ]31adrenergic receptor in patients with idiopathic dilated cardiomyopathy. J Clin Invest 1990;86:1658-1663. Maisch B, Trostel-Soeder R, Stechemesser E, Berg PA, Kochsiek K. Diagnostic relevance of humoral and cellmediated immune reactions in patients with acute viral myocarditis. Clin Exp Immunol 1982;48:533--545. Maisch B, Deeg P, Liebau G, Kochsiek K. Diagnostic relevance

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of humoral and cytotoxic immune reactions in primary and secondary dilated cardiomyopathy. Am J Cardiol 1983;52: 1072-1078. Maisch B, Wedeking U, Kochsiek K. Quantitative assessment of antilaminin antibodies in myocarditis and perimyocarditis. Eur Heart J 1987;8:$233-$235. Milei J, S~nchez J, Storino R, Yu ZX, Denduchis B, Ferrans VJ. Antibodies to laminin and immunohistochemical localization of laminin in chronic chagasic cardiomyopathy: a review. Mol Cell Biochem 1993;129:161--170. Murphree SS, Saffitz JE. Distribution of beta-adrenergic receptors in failing human myocardium. Implications for mechanisms of downregulation. Circulation 1989;79:12141225.

Rada T, Okruhlicov~ I~, Mono~fkov~ R, Slez~k J, (~izm~rov~ E, Rydzi S. Levels of autoantibodies against beta-adrenergic receptors and mitochondrial antigens in sera of cardiomyopathic patients estimated by ELISA method. Basic Res Cardiol 1992;87:80-86. Rogers SW, Andrews PI, Gahring LC, Whisenand T, Cauley K, Crain B, Hughes TE, Heinemann SF, McNamara JO. Autoantibodies to glutamate receptor GluR3 in Rasmussen' s encephalitis. Science 1994;265:648--651. Rose NR, Beisel KW, Herskowitz A, Neu N, Wolfgram LJ, Alvarez FL, Traystman MD, Craig SW. Cardiac myosin and autoimmune myocarditis. Ciba Found Symp 1987;129:3--24. Saurat J-H, Schifferli J, Steiger G, Dayer J-M, Didierjean L. Anti-interleukin- 1~ autoantibodies in humans: characterization, isotype distribution, and receptor-binding inhibitionhigher frequency in Schnitzler's syndrome (urticaria and macroglobulinemia). J Allergy Clin Immunol 1991 ;88:244256. Schultheiss HP. The significance of autoantibodies against the ADP/ATP carrier for the pathogenesis of myocarditis and dilated cardiomyopathy- clinical and experimental data. Springer Semin Immunopathol 1989; 11:15-30. Sterin-Borda L, Perez Leiros C, Wald M, Cremaschi G, Borda E. Antibodies to ~1 and [32 adrenoreceptors in Chagas' disease. Clin Exp Immunol 1988;74:349--354. Tobbetts RS, McCormick TS, Rowland EC, Miller SD, Engman DM. Cardiac antigen-specific autoantibody production is associated with cardiomyopathy in Trypanosoma cruz# infected mice. J Immunol 1994;152:1493--1499. Treichel U, McFarlane BM, Seki T, Krawitt EL, Alessi N, Stickel F, McFarlane IG, Kiyosawa K, Furuta S, Freni MA, et al. Demographics of antiasialoglycoprotein receptor autoantibodies in autoimmune hepatitis. Gastroenterology 1994;107:799-804. Turki J, Liggett SB. Receptor-specific functional properties of beta 2-adrenergic receptor autoantibodies in asthma. Am J Respir Cell Mol Biol 1995;12:531-539. Waagstein F, Hjalmarson A, Varnauskas E, Wallentin I. Effect of chronic beta-adrenergic receptor blockade in congestive cardiomyopathy. Br Heart J 1975;37:1022-1036. Wallukat G, Wollenberger A. Autoantibodies to [32-adrenergic receptors with antiadrenergic activity from patients with allergic asthma. J Allergy Clin Immunol 1991;88:581-587. Wallukat G, Morwinski M, Kowal K, F6rster A, Boewer V,

Wollenberger A. Autoantibodies against the ~-adrenergic receptor in human myocarditis and dilated cardiomyopathy: 13-adrenergic agonism without desensitization. Eur Heart J 1991;12:S178--S181.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

BROMELAIN-TREATED ERYTHROCYTE AUTOANTIBODIES Antonio R. Cabral, M.D. and Donato Alarc6n-Segovia, M.D.

Department of Immunology and Rheumatology, Instituto Nacional de la Nutrici6n, Tlalpan, Mexico, D.F. 14000, Mexico

HISTORICAL NOTES For almost a century it has been known that normal human and mouse sera contain "pan-agglutinins" able to react with autologous protease or neuraminidasetreated erythrocytes (Grabar, 1975). In 1966, peritoneal cells from untreated healthy mice were shown to produce IgM antibodies directed to sheep red blood cells (Bussard, 1966). Normal and autoimmune murine spleen cells secrete antibodies that react with antigens buried in the erythrocyte membrane, but are exposed by the proteolytic enzyme bromelain (Cunningham, 1974). Subsequently, trimethylammonium (TMA)-containing compounds were found to prevent the lysis of bromelain-treated mouse red blood cells (BrMRBC) caused by New Zealand black (NZB) mice-derived monoclonal IgM antierythrocyte antibodies. Later studies show that the lytic activity of BrMRBC may also be abolished by phosphatidylcholine (PTC) and that these autoantibodies bind to and can be eluted from PTC attached to an insoluble matrix (Cox and Hardy, 1985).

THE AUTOANTIGEN(S) Definition/Characteristics Phosphatidylcholine, also known as lecithin, is a TMA-containing zwitterionic phospholipid constituent of most cell membranes. Phospholipids comprising the human erythrocyte membrane are asymmetrically arranged in bilayer leaflets (Op den Kamp, 1979). Together with sphingomyelin, PTC is a key component of the exoplasmic leaflet of mammalian erythrocyte membranes, while the cytoplasmic leaflet

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is rich in phosphatidylethanolamine and phosphatidylserine (Op den Kamp, 1979). Although the translocation rate of PTC over the bilayer is fast (half time = 13--24 h), the asymmetric distribution of PTC is uniformly maintained throughout the life span (120 days) of the erythrocyte. Like other phospholipids, phosphatidylcholine has a glycerol backbone with two primary hydroxyl group one of which is esterified to phosphatidic acid and the other to two fatty acid nonpolar chains and in turn esterified to the positively charged amino alcohol head group choline. Mild alkaline hydrolysis of PTC yields the fatty acids as soaps but leaves intact the glycerol-phosphoric acid/ alcohol portion of the molecule as phosphorylcholine (PRC), also a TMA-containing molecule.

AUTOANTIBODIES Over 20 years ago, murine B-cell populations were shown to contain large numbers of cells that produce IgM able to lyse BrMRBC, but not untreated RBC (Cunningham, 1974). The recognized epitope on BrMRBC was identified as the head group of phosphatidylcholine (Serban et al., 1981; Cox and Hardy, 1985) with extensive cross-reactivity with other phospholipids (Arnold and Haughton, 1992). For instance, 11 hybridomas from normal (BALB/c) mouse B cells that produced antibodies against BrMRBC (anti-BrMRBC) were developed (Kawaguchi, 1987). All of these reacted with PTC liposomes; two had cross-reactivity with cardiolipin (CL), phosphatidylglycerol and sphingomyelin. Most antibodies to PTC/BrMRBC (anti-PTC/ BrMRBC) are IgM polyreactive and produced mainly, but not exclusively, by the Ly-1 + (CD5 in humans)

subpopulation of B cells (Pennell et al., 1989; Andrew et al., 1990), whose primary responsibility is the production of IgM natural autoantibodies (Hayakawa et al., 1984). These B cells utilize unmutated VH/V L gene pairs (Reininger et al., 1988; Poncet et al., 1990; Arnold and Haughton, 1992) to encode PTC antibodies (aPTC), bear multivalent (likely self) antigens constitutively bound to their surface Ig receptors (Carmack et al., 1990) and appear to be under the restricted control of interleukin-5 both in vitro (Wetzel, 1990) and in vivo (Tominaga et al., 1991). Two variable region combinations, VHll/VK9 and VH12/VK4 account for greater than 80% of the antiPTC/BrMRBC repertoire (Reininger et al., 1988; Mercolino et al., 1989; Conger et al., 1991). Monoclonal antibodies of the Vnl 1/VK9 type bind TMA and PRC much more avidly than the Vnl2/VK4 type antibodies; both groups lyse BrMRBC with similar efficiencies (Conger et al., 1991), but none is polyreactive. In contrast, anti-BrMRBC belonging to the VnJ558 family show different degrees of multireactivity against anionic and cationic antigens (Conger et al., 1992). These VHJ558 monoclonals use an identical V z gene and show a 62% identity at the nucleotide level, with the anti-BrMRBC CH12 V z lymphomaderived multireactive monoclonal antibody (Mercolino et al., 1986). These data are consistent with the idea that the BrMRBC specific repertoire, is germline in sequence and that every known mechanism, except somatic mutation, may be used in generating diversity in this antibody repertoire (Conger et al., 1991; Arnold and Haughton, 1992). Together with CL, phosphatidylserine and phosphatidic acid, PTC is one of the small molecules recognized by human polyreactive natural autoantibodies (Avrameas, 1991). In this vein, human IgM aPTC has been recognized for several years, but information regarding aPTC as antibodies to bromelain-treated normal human erythrocytes (BrHRBC) is scant. Most humans have serum IgM antibodies that react with PTC and a variety of other anionic and cationic antigens including BrHRBC (Cabiedes et al., unpublished observations). Polyreactive human natural autoantibodies use a restricted portion of the V n germline repertoire with few somatic mutations and their reactivity with PTC is not known (Sanz et al., 1989). Although direct experimental evidence is limited, the presence of polyreactive natural autoantibodies in normal healthy individuals suggests important physiologic function(s) (Avrameas, 1991) including a role in:

(1) establishing a regulatory, idiotype network; (2) providing a platform for antigen-specific immune responses via somatic mutations and affinity maturation; (3) creating a first line of defense against microbial infections; and (4) clearing metabolic and catabolic cell products and senescent cells from the organism (Avrameas, 1991). A natural autoantibody directed to alpha-galactosyl may be directly involved in the removal of effete human red cells. If the in vitro storage of erythrocytes can be equated to their in vivo aging, the binding of aPTC to stored erythrocytes (Jenkins and Marsh, 1961; Cabral et al., 1990a) may argue that PTC is one of the "hidden" antigens of the erythrocyte membrane that are exposed as they age, consistent with a role for aPTC in the elimination of senescent erythrocytes (Clark, 1988). Murine natural aPTC bind to a TMAlike group on aged autologous erythrocytes (Serban et al., 1981; Conger et al., 1989). Phosphatidylcholine may be exposed in vivo through the effect of serum sialidase, sialidase-like molecules, and/or proteolytic enzymes that desialate or liberate the sialoglycoprotein of the erythrocyte membrane that covers PTC (Cook et al., 1960; Clark, 1988). Bromelain has this effect and has long been utilized to detect allo- and autoantibodies against RBC (Pirofsky and Magnum, 1959). In pathologic states, PTC may be exposed after IgG antierythrocyte antibodies bind to surface molecules with subsequent aPTC binding to its exposed autoantigen (Connor et al., 1989).

Pathogenetic Role Most normal mice have natural IgM antibodies that react with and lyse isologous BrMRBC in the presence of complement (Cunningham, 1974; Lord and Dutton, 1975). Included among other abnormalities of NZB mice are lymphocyte abnormalities, excessive proliferation early in life, spontaneous B-cell hyperproduction of IgM, early depletion of bone marrow B precursors, elevated numbers of CD5 + B cells, hyperploid CD5 + B cells, decreased autologous mixed lymphocyte reaction and decreased responsiveness to IL-2 (Whitmer et al., 1994). These abnormalities in NZB lead to the spontaneous development of autoimmune hemolytic anemia at 10-12 months of age (Table 1) (Yoshida et al., 1990; Hentati et al., 1994). One NZB autoantibody reacts against species-specific, naturally exposed surface antigen (antigen X) and another is directed to an antigen revealed by bromelain. Antigen X is proposed as the primary target 121

Table 1. LymphocyteAbnormalities in NZB Mice B Cells

T Cells Excessive proliferation early in life.

Decreased autologous mixed lymphocyte reaction.

Increased numbers of colony-forming units.

Decreased responsiveness to IL-2.

Spontaneous hypersecretion of IgM.

Hyper-responsive cytolytic activity.

Early depletion of bone marrow B precursor cells.

Accelerated proliferation and differentiation.

Elevated numbers of CD5+ B cells.

Age-dependent increase in t~[3TCR+CD4--CD8- cells.

Hyperdiploid CD5+ B cells.

Altered CD4:CD8 subset ratios.

Adapted from Whitmer et al., 1994.

of hemolytic autoantibodies (Linder "and Edington, 1972). Serum antibodies and those eluted from NZB red cells (Coombs' antibodies) react against RBC as well as with actin, myoglobin, myosin, tubulin, spectrin, DNA and other antigens by ELISA and immunoblotting (Hentati et al., 1994). These reactivities follow two different periods of autoantibody production: the first is characterized by a fluctuating high level of IgM and stable level of IgG natural autoantibodies. The second period of antibody production is characterized by a rise of IgG natural autoantibodies in parallel with IgG anti-RBC antibodies other than aPTC. Other authors found that NZB mice have antierythrocyte antibodies that do not react against BrMRBC and are encoded mainly by the VHJ558 gene with high somatic diversification (Reininger et al., 1990; Caulfield and Stanko, 1992; Whitmer et al., 1994). Taken together, these results are in agreement with the notion that IgM aPTC must act in parallel with other autoantibodies to cause hemolytic anemia (Linder and Edington, 1972; Cabral et al., 1990a; Whitmer et al., 1994). Though long hypothesized, a pathogenetic role for aPTC in the causation of human hemolytic anemia has not actually been demonstrated. In a patient with Coombs'-positive autoimmune hemolytic anemia and high titers of circulating IgM aPTC, but no other evidence of SLE, the antibody was also detected on the patient's erythrocytes at the time of active hemolysis, but not during remission (Cabral et al., 1990a). Normal human fresh untreated erythrocytes bound almost none of the affinity-purified aPTC, but normal BrHRBC did. Significant binding to in vitro-aged normal human RBC also occurred, and addition of complement to normal BrHRBC coated with purified IgM aPTC resulted in lysis. The antibody reacted by

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ELISA with PTC, CL, PRC and synthetic PTC of small fatty acid chain lengths, this suggests that the epitope recognized by these IgM aPTC is the amino alcohol polar head group of phospholipids, perhaps via a TMA moiety. This is akin to that described for routine monoclonal anti-BrMRBC of the unmutated VH11/VK9 and VH12/VK4 type (Conger et al., 1991). The IgM aPTC ,from some patients with primary antiphospholipid syndrome (Villarreal et al., 1991) bears the SA 1 idiotype which is related to the unmutated germline gene encoded 16/6 idiotype present in other natural autoantibodies (Shoenfeld et al., 1989).

Methods of Detection

The method most frequently utilized to detect antibodies to bromelain-treated erythrocytes is the complement-dependent plaque test (Arnold and Haughton, 1992). Other methods include a rapid and sensitive (but infrequently utilized) ELISA with fixed target cells as antigens (Klinman and Steinberg, 1987). Flow cytometry can also be used to detect anti-BrHRBC (Cabral et al., 1990a). A rapid and sensitive ELISA allows the detection of serum antibodies to 1-alphaphosphatidylcholine (bovine brain, Sigma, St. Louis, Mo. USA) (Cabral et al., 1990a; Cabral et al., 1990b).

CLINICAL UTILITY Disease Association

Long-standing idiopathic autoimmune hemolytic anemia can be associated with high titers of erythrocyte-bound serum IgM aL antibodies (Cabral et al.,

1990a; del Papa et al., 1992). Among 18 patients with idiopathic autoimmune hemolytic anemia, the same type of IgM polyreactive aPTC was detected in four (Guzm~in et al., 1994). The human association may have its animal model in the hemolytic anemia of the NZB mouse (Oken et al., 1973). Approximately 60% of systemic lupus erythematosus (SLE) patients with hemolytic anemia (with or without thrombocytopenia) have serum IgM aPTC (Cabral et al., 1990b). Patients with hemolytic anemia and false-positive serological tests for syphilis can also have antibodies that react with stored as well as with enzyme-treated red cells (Jenkins and Marsh, 1961). In a patient with lymphoma, history of hemolytic anemia and a strongly positive VDRL, a serum monoclonal IgM was found to react with both CL and PTC which are present with cholesterol in the VDRL antigen (Cooper et al., 1974). Another IgM monoclonal antibody reactive with sheep erythrocytes (that spontaneously express PTC on their surface) was produced by an Epstein-Barr virusimmortalized cell line from a patient with chronic lymphocytic leukemia (Crescenzi et al., 1988). The hemolytic anemia of patients with hairy cell leukemias can also be associated with aPTC (Domingo et al., 1992).

REFERENCES Andrew EM, Annis W, Kahan M, Maini RN. CD5 (Ly-1)negative, conventional splenic B cells make a substantial contribution to the bromelain plaque-forming cell response in CBA and BW mice. Immunology 1990;69:515--518. Arnold LW, Haughton G. Autoantibodies to phosphatidylcholine. The murine antibromelain RBC response. Ann N Y Acad Sci 1992;651:354--359. Avrameas S. Natural autoantibodies: from 'horror autotoxicus' to 'gnoti seuton'. Immunol Today 1991;12:154--159. Bussard AE. Antibody formation in non immune peritoneal cells after incubation in gum containing antigen. Science 1966;153:887-888. Cabral AR, C,abiedes J, Alarc6n-Segovia D. Hemolytic anemia related to an IgM autoantibody to phosphatidylcholine that binds in vitro to stored and to bromelain-treated erythrocytes. J Autoimmun 1990a;3:773-787. Cabral AR, Cabiedes J, Alarc6n-Segovia D. Hemolytic anemia (HA) in systemic lupus erythematosus (SLE) associates strongly with IgM antiphosphatidylcholine antibodies (aPTC) [Abstract]. Clin Exp Rheumatol 1990b;8:212.

CONCLUSION Over 20 years ago, murine B-cell populations were found to contain large numbers of cells that produce IgM able to lyse BrMRBC, but not untreated RBC. The epitope on BrMRBC is the head group of phosphatidylcholine with extensive cross-reactivity with other phospholipids and trimethylammonium-containing molecules. B cells with IgM of this specificity bind synthetic fluorescent liposomes and fluorescent aPTC antibodies. In mice, the majority of these IgM PTC-producing cells derive from the Ly-1 B-cell lineage and use primarily two VH/V L gene families, V H11 and VH12. These genes are unmutated and have restricted VDJ recombinations. Although specialized function is unproved, anti-PTC/BrRBC natural autoantibodies are postulated to serve as a first line of defense against microbial infections and/or as "transporteurs" to rid metabolic and catabolic cell products including senescent erythrocytes. IgM antiPTC/BrMRBC along with other natural autoantibodies, cause autoimmune hemolytic anemia to NZB mice. In humans, IgM aPTC also appear to be germline gene-encoded natural autoantibodies; these IgM anti-PTC/BrHRBC are associated with Coombs'positive autoimmune hemolytic anemia, either idiopathic or secondary to SLE or to hematologic malignancies. See also ALPHA-GALACTOSYL (ANTI-GAL) AUTOANTIBODIES.

Carmack CE, Shinton SA, Hayakawa K, Hardy RR. Rearrangement and selection of VH11 in the Ly-1 B cell lineage. J Exp Med 1990;172:371--374. Caulfield MJ, Stanko D. A pathogenic monoclonal antibody, G8, is characteristic of antierythrocyte autoantibodies from Coomb's-positive NZB mice. J Immunol 1992;148:2068-2073. Clark MR. Senescence of red blood cells: progress and problems. Physiol Review 1988;68:503--554. Conger JD, Sage HJ, Kawaguchi S, Corley R. Properties of murine antibodies from different V region families specific for bromelain-treated mouse erythrocytes. J Immunol 1991; 146:1216--1219. Conger JD, Sage HJ, Corley RB. Diversity in the available repertoire of murine antibodies reactive with bromelaintreated isologous erythrocytes. J Immunol 1989;143:40444052. Conger JD, Sage HJ, Corley RB. Correlation of antibody multireactivity with variable region primary structure among murine antierythrocyte autoantibodies. Eur J Immunol 1992;22:783-790. Connor JC, Bucana C, Fidler IJ, Schroit AJ. Differentiation-

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dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation. Proc Natl Acad Sci 1989;86:3184-3188. Cook GMW, Heard DH, Seaman GVF. A sialomucopeptide liberated by trypsin from the human erythrocyte. Nature 1960;11:1011--1012. Cooper MR, Cohen HJ, Huntley CC, Waite BM, Spees L, Spurr CL. A monoclonal IgM with antibody-like specificity for phospholipids in a patient with lymphoma. Blood 1974;43: 493--504. Cox KO, Hardy SJ. Autoantibodies against mouse bromelinmodified RBC are specifically inhibited by a common membrane phospholipid phosphatidylcholine. Immunology 1985;55:263--269. Crescenzi M, Napolitano M, Carboniari M, Antonelli A, Petrinelli P, Gaetano C, Fiorilli M. Establishment of a new Epstein-Barr virus-immortalized cell line from chronic lymphocytic leukemia with trisomy of chromosome 12 that produces monoclonal IgM against a sheep RBC antigen. Blood 1988;71:9-12. Cunningham AJ. Large numbers of cells in normal mice produce antibody components of isologous erythrocytes. Nature 1974;252:749--751. Del Papa N, Meroni PL, Barcellini W, Borghi MO, Fain C, Khamashtma M, Tincani A, Balestrieri G, Hughes GR. Antiphospholipid antibodies cross-reacting with erythrocyte membranes. A case report. Clin Exp Rheumatol 1992;10: 395--399. Domingo A, Crespo N, Desevilla AF, Domenech P, Jordan C, Callis M. Hairy cell leukemia and autoimmune hemolytic anemia. Leukemia 1992;6:606--607. Grabar P. Hypothesis: auto-antibodies and immunological theories. An analytical review. Clin Immunol Immunopathol 1975;4:453--466. Guzm~n J, Cabral AR, Cabiedes J, Pita-Ramffez L, Alarc6nSegovia D. Antiphospholipid antibodies in patients with idiopathic autoimmune haemolytic anemia. Autoimmunity 1994; 18:52--56. Hayakawa K, Hardy R, Honda M, Herzenberg LA, Steinberg AD. Ly-1 B cells: functionally distinct lymphocytes that secrete IgM autoantibodies. Proc Natl Acad Sci USA 1984;81:2494--2498. Hentati B, Payelle-Brogard B, Jouanne C, Avrameas S, Ternynck T. Natural autoantibodies are involved in the haemolytic anaemia of NZB mice. J Autoimmunity 1994;7:425439. Jenkins WJ, Marsh WL. Autoimmune haemolytic anemia. Three cases with antibodies specifically active against stored red cells. Lancet 1961;i:16-18. Kawaguchi S. Phospholipid epitopes for mouse antibodies against bromelain-treated mouse erythrocytes. Immunology 1987;62:11-16. Klinman DM, Steinberg AD. Novel ELISA and ELISA-spot assays used to quantitate B cells and serum antibodies specific for T cell and bromelated mouse red blood cell autoantigens. J Immunol Methods 1987;24:157--154. Linder E, Edington TS. Antigenic specificity of antierythrocyte

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autoantibody responses by NZB mice: identification and partial characterization of two erythrocyte surface autoantigens. J Immunol 1972;108:1615-1623. Lord EM, Dutton RW. The properties of plaque-forming cells from autoimmune and normal strains of mice with specificity for autologous erythrocyte antigens. J Immunol 1975;115: 1199--1205. Mercolino TJ, Locke AL, Afshari A, Sasser D, Travis WW, Arnold LW, Haughton G. Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline. J Exp Med 1989;169: 1869--1877. Mercolino TJ, Arnold LW, Haughton G. Phosphatidyl choline is recognized by a series of Ly-1 + murine B cell lymphomas specific for erythrocyte membranes. J Exp Med 1986;163: 155--165. Oken MM, Griffiths RW, Williams RC, Reimann BEF. Possible NZB syndrome in man. Arch Intern Med 1973;132:237--240. Op den Kamp JAF. Lipid asymmetry in membranes. Ann Rev Biochem 1979;48:47-71. Pennell CA, Sheehan KM, Brodeur PH, Clarke SH. Organization and expression of V n gene families preferentially expressed by Ly-l+ (CD5) B cells. Eur J Immunol 1989;19: 2115--2121. Pirofsky B, Magnum ME. Use of bromelin to demonstrate erythrocyte antibodies. Proc Soc Exp Biol Med 1959;101: 49--52. Poncet P, Huetz F, Marcos MA, Andrade L. All VH11 genes expressed in peritoneal lymphocytes encode anti-bromelaintreated mouse red blood cell autoantibodies but other V n gene families contribute to this specificity. Eur J Immunol 1990;20:1583--1589. Reininger L, Kaushik A, Izui S, Jaton JC. A member of a new V H gene family encodes antibromelinized mouse red cell autoantibodies. Eur J Immunol 1988; 18:1521-1526. Reininger L, Shibata T, Ozaki S, Shirai T, Jaton JC, Izui S. Variable region sequences of pathogenic antimouse red blood cell autoantibodies from autoimmune NZB mice. Eur J Immunol 1990;20:771--777. Sanz I, Casali P, Thomas JW, Notkins AL, Capra JD. Nucleotide sequences of eight human natural autoantibody V H regions reveals apparent restricted use of V H families. J Immunol 1989;142:4054--4061. Serban D, Pages JM, Bussard AE, Witz IP. The participation of trimethylammonium in the mouse erythrocyte epitope recognized by monoclonal autoantibodies. Immunol Lett 1981;3:315--319. Shoenfeld Y, Tepliziki HA, Mendelovic S, Blank M, Mozes E, Isenberg DA. The role of human anti-DNA idiotype 16/6 in autoimmunity. Clin Immunol Immunopathol 1989;51:313315. Tominaga A, Takaki S, Koyama N, Katoh S, Matsumoto R, Migita M, Hitoshi Y, Yamauchi S, Kanai Y, Miyazaki J-I, Usuku G, Yamamura K-I, Takatsu K. Transgenic mice expressing a B cell growth and differentiation factor gene (interleukin 5) develop eosinophilia and autoantibody production. J Exp Med 1991;173:429--437. Villarreal GM, Alarc6n-Segovia D, Villa AR, Cabral AR,

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

C1 INHIBITOR AUTOANTIBODIES Alvin E. Davis III, M.D. a and Marco Cicardi, M.D. b

aDivision of Nephrology, Children's Hospital Medical Center, Cincinnati, OH 45229-3039, USA; and bClinica Medica III, Instituto di Medicina Interna, Universita di Milano, Milan 20122, Italy

HISTORICAL NOTES

THE AUTOANTIGEN

C1 inhibitor (C1 INH) is the only inhibitor of the complement proteases C lr and C ls and is a major inhibitor of the kinin-generating system proteases factor XIIa and kallikrein. The recognition that autoantibodies to C1 INH (anti-C1 INH) may exist and that they may lead to an acquired secondary dysfunction of C1 INH is recent. Genetic deficiency or dysfunction of C1 INH results in hereditary angioedema (HAE), a disease characterized by recurrent subcutaneous or mucosal edema (Donaldson and Evans, 1963). In 1972, an acquired form of C1 INH deficiency was characterized (Caldwell et al., 1972). This syndrome, acquired angioedema (AAE), is most commonly associated with lymphoproliferative disorders, but also is observed either alone or in association with a variety of other conditions (Gelfand et al., 1979). A second form of acquired deficiency (type 2 AAE) which is mediated by autoantibody directed toward C1 INH was recognized in 1986 and characterized by an IgG autoantibody in the serum of an otherwise healthy 62year-old man with a recent onset of angioedema (Jackson et al., 1986) (Table 1). Two subsequent studies reported very similar findings in an additional five patients (Alsenz et al., 1987; Malbran et al., 1988). A total of only 22 well-documented patients are described in the literature (Jackson et al., 1986; Alsenz et al., 1987; Malbran et al., 1988; Cicardi et al., 1993; Mandle et al., 1994) (Table 1).

Definition

126

C1 INH is a member of the serpin family of serine proteinase inhibitors, which includes numerous other protease inhibitors, such as ~l-antitrypsin, antithrombin and plasminogen activator inhibitor I (Carrell et al., 1987). The family is defined by sequence similarity and contains members such as ovalbumin and thyroxin-binding globulin that do not have inhibitory functions. Inhibitory serpins, including C1 INH, share a similar three-dimensional structure and mechanism of action. Interaction of target protease with the reactive center results in formation of a denaturantstable 1:1 inhibitor:protease complex without cleavage of the reactive center peptide bond. The complex likely is a tetrahedral intermediate (Matheson et al., 1991). C1 INH, by virtue of its ability to inactivate Clr, C l s, factor XIIa and kallikrein, is a primary regulator of both the complement and kinin-forming systems (Schapira et al., 1985; Davis, 1988). Deficiency or dysfunction of C1 INH, therefore, results in unregulated activation of both systems.

Origin/Sources C1 INH is obtained primarily from plasma, but it also can be transiently expressed in mammalian cell lines, such as COS cells (SV40 transformed African green monkey kidney cells) (Eldering et al., 1988; Aulak et al., 1993). The recombinant protein, therefore, is obtained only in relatively small quantities. No studies specifically compared the autoantibody reactivity of

Table 1. Autoimmune C1 Inhibitor Deficiency Autoantibody

C 1 INH in Plasma

References

Clonality mono

cleaved

Jackson et al., 1986

cleaved

Alsenz et al., 1987

Age Sex Associated Disease Isotype

L-Chain

62

M

none

IgG

40

M

none

IgG1

50 M

none

IgG1

9

cleaved

Alsenz et al., 1987

9

M

none

IgA

mono

cleaved

Malbran et al., 1988

9

M

none

IgA

mono

cleaved

Malbran et al., 1988

9

M

none

IgG

mono

cleaved

Malbran et al., 1988

59

F

leiomyoma, uterus; benign cecal tumor IgG

9

9

Spath et al., 1989

50 F

leiomyoma, skin

IgG/IgA

9

cleaved

Zuraw and Altman, 1991

59

M

previous myocardial infarction, 1" BP

IgG4

9

cleaved

Donaldson et al., 1992

70

M

previous myocardial infarction, 1" BP

IgG/IgA

cleaved

Boyar et al., 1993

39 M

none

IgG

oligo

partially cleaved

Mandle et al., 1994

55

M

*M-component

IgM

mono

cleaved

Cicardi et al., 1993

41

F

echinococcus granulosus

IgG

mono

cleaved

Cicardi et al., 1993

56

M

none

IgG

cleaved

Cicardi et al., 1993

67

M

*M-component

IgM

mono

partially cleaved

Cicardi et al., 1993

54

M

*M-component

IgA

oligo

cleaved

Cicardi et al., 1993

53

M

Waldenstr6m's macroglobulinemia

IgG

9

cleaved

Cicardi et al, 1993

64 F

breast cancer

IgA

mono

cleaved

Cicardi et al., 1993

72 M

none

IgG

9

cleaved

Cicardi et al., 1993

40

F

none

IgA

9

9

Cicardi, unpublished

66 F

none

IgG

9

9

Cicardi unpublished

70 M

none

IgM

9

~

Cicardi, unpublished

~c,7

*M-component reactive with C 1 inhibitor.

the native C1 INH with that of the recombinant protein. However, no differences are noted in terms of functional activity or of reactivity with several polyclonal and monoclonal antibodies. C1 INH is present in plasma at concentrations of approximately 150 lag/mL. The primary site of synthesis is the liver, although several other cell types, including monocytes, fibroblasts and endothelial cells are capable of synthesizing C1 INH, at least in vitro (Davis, 1988). Although it is possible that synthesis by these cell types plays a role in vivo at sites of inflammation no direct data are available.

Methods of Purification/Commercial Sources Several standard methods for isolation of C1 INH from plasma are reported, all of which are effective (Nilsson and Wiman, 1982; Sim and Reboul, 1981; Harrison, 1983; Pilatte et al., 1989). For large-scale preparations, the method which consists of sequential chromatographic steps on D E A E Sephadex, Sepharose 6B and hydroxylapatite is preferred (Harrison, 1983). For small-scale preparation, including isolation of recombinant protein from tissue culture supernatant, a method which takes advantage of the fact that C1 INH is one of the few plasma proteins (the other

127

major one being IgA) that bind specifically to the lectin, jacalin, is convenient (Pillate et al., 1989). Isolated human C 1 INH can be obtained commercially from Advanced Research Technologies, Calbiochem and Sigma.

Epitopes The epitopes recognized by only a few autoantibodies have been mapped (Donaldson and Davis, 1993; Mandle et al., 1994). The available data suggest that most very likely recognize an epitope or epitopes at or near the reactive center. Similarly, none of the epitopes recognized by available polyclonal or monoclonal antibodies have been precisely mapped. Several monoclonals recognize conformational determinants that are expressed only on the complexed or reactive, center-cleaved inhibitor, but not on the native protein (de Agostini et al., 1985; Nuijens et al., 1989).

AUTOANTIBODIES Pathogenetic Role The autoantibodies studied appear to prevent the inactivation of target proteases by C1 INH and in most instances, bind to the immobilized C1 INH. Most, if not all, of the autoantibodies enhance cleavage of C1 INH, probably at the reactive center, by target protease (Table 1). Addition of autoantibody either to normal serum or to the isolated protein with target protease results in loss of C1 INH activity accompanied by its cleavage to a 96 kd product (Alsenz et al., 1987; Malbran et al., 1988; Jackson et al., 1989). In one study, the cleavage was confirmed to be at the reactive center (Alsenz et al., 1989). A likely mechanism for this enhanced cleavage is that the antibody alters the conformation of the inhibitor so that it is converted to a substrate of target proteases. A small amount of activation of target protease then would result in depletion of intact active C1 INH. It also is possible that the autoantibody might react with the protease-inhibitor complex and destabilize the complex with release of active protease and cleaved inhibitor. The limited available data are more consistent with the former mechanism in that reactivity with preformed protease-inhibitor complex is minimal or absent (Alsenz et al., 1987; Donaldson et al., 1992). However, in at least some patients, the autoantibody interferes with complex formation without leading to

128

C1 INH cleavage (Cicardi et al., unpublished data). This suggests that different autoantibodies may be directed toward different epitopes. The correlation of the levels of free autoantibody with the reduction in C1 INH function, C4 levels and development of symptoms is not perfect. This likely is at least partially related to the lack of availability of common standards for quantitation of antibodies and of their anti-C 1 INH activities. Both the in vivo and in vitro data are consistent with the interpretation that the autoantibody mediates the decrease in C1 INH function and is therefore responsible for the development of symptoms. No animal model for either acquired or hereditary deficiency has been described.

Factors Involved in Pathogenicity Although the majority are IgG, both IgM and IgA autoantibodies have been observed. Two patients have been described with the coexistence of an IgG and IgA autoantibody (Zuraw and Altman, 1991; Boyar et al., 1993). The preponderance of the data suggests, however, that the antibodies are of limited heterogeneity: some consist of a single subclass, some of a single light chain and others appear oligoclonal or monoclonal on electrophoresis (Table 1). Interestingly, several patients have readily detectable monoclonal immunoglobulins, some of which appear to represent the autoantibody, while others clearly do not (Table 1). For example, one patient with Waldenstr6m's macroglobulinemia expressed an IgG autoantibody (Cicardi et al., 1993). None of the other patients with identifiable M-components have developed clinically apparent lymphoproliferative disease. Aside from these observations, no other factors that might be related to the development of the autoantibody are described. There are no obvious associations with other diseases, and patients with autoantibodies to C1 INH do not develop other autoantibodies or other signs of autoimmunity. Conversely, patients with other autoimmune diseases do not develop antibodies to C 1 INH. Likewise, with a single exception, patients with HAE treated with C1 INH infusions do not develop autoantibodies (Donaldson and Davis, unpublished data).

Methods of Detection In general, two kinds of assays, one of which is functional and one of which demonstrates immunoglobulin binding to C1 INH, are used to identify these

autoantibodies. In the first, serum or isolated immunoglobulin containing the autoantibody is used to interfere with C1 INH-mediated inhibition of turnover of synthetic substrate by a target protease (Jackson et al., 1986; Donaldson et al., 1992; Cicardi et al., 1993). The other assay is a solid-phase ELISA in which a plastic plate is coated with C1 INH and then is incubated with autoantibody-containing sample (Alsenz et al., 1987; Donaldson et al., 1992; Cicardi et al., 1993; Mandle et al., 1994). Immunoglobulin binding to the C1 INH then is detected with appropriate enzymelabeled anti-immunoglobulin antibodies. Complete analysis of patient samples obviously requires both types of assays in order to demonstrate both the functional effect and the direct binding of immunoglobulin to the C1 INH molecule.

CLINICAL UTILITY Disease Association

Biochemically, autoimmune C1 INH deficiency (type 2 AAE) is characterized by decreased levels of C1 INH functional activity together with variable decreases in C1 INH antigen. Usually, the level of antigen is higher than would be expected from the functional level and may even be within the normal range. This is due to the presence, in nearly all patients, of circulating nonfunctional C 1 INH that has been cleaved at its reactive center (Alsenz et al., 1987; Malbran et al., 1988; Jackson et al., 1989). As a result of the complement activation that occurs secondary to the reduction in C1 INH function, C4 (and C2) are decreased (Table 2). Unlike hereditary angioedema, C lq levels are decreased as they are in acquired C1 INH deficiency that is not autoantibody mediated (type 1 AAE). The explanation for this decrease is unclear; in one instance, it clearly was shown that the autoantibody-C1 INH complex did not

activate complement (Jackson et al., 1986). Most of the biochemical characteristics of hereditary and acquired deficiency are shared. Each has diminished C1 INH function, and may have either normal or decreased antigenic levels. Hereditary deficiency with a dysfunctional C1 INH (type 2 AAE) may have levels of C1 INH antigen that are higher than the functional levels, as may acquired deficiency (due to the presence of the circulating cleaved inhibitor). C4 and C2 levels also are decreased in both. C lq levels, however, are not decreased in HAE, but are virtually always decreased in both types of AAE. Only one patient with autoantibody-mediated AAE and a persistently normal C lq level has been described (Cicardi et al., 1993). Definitive identification of autoantibody-mediated C1 INH deficiency (type 2 AAE) and its distinction from type 1 AAE requires analyses to identify directly the autoantibody. Type 1 AAE can be distinguished only by the absence of autoantibody and by its relatively frequent association with lymphoproliferative disorders and possibly with other malignancies (Gelfand et al., 1979). However, the fact that several patients with autoantibody-mediated C1 INH deficiency express readily detectable monoclonal immunoglobulins, and that one patient has Waldenstr6m's macroglobulinemia suggests that there probably is overlap between the two disorders, or that they may be variants of the same basic defect (Table 1). In any case, any patient with acquired C1 INH deficiency, with or without autoantibody, should be monitored for the development of malignancy. Unfortunately, most therapy for autoimmune C1 INH deficiency is less than satisfactory (Table 3). Unlike HAE, therapy with attenuated androgens is seldom effective. Presumably, synthesis cannot be enhanced to a great enough extent to be able to keep up with autoantibody-mediated consumption. Twelve patients have been treated with either epsilon amino caproic acid (EACA) or tranexamic acid, both of which are inhibitors of plasmin activation. This

Table 2. Autoantibody to C1 INH and Complement Levels in Angioedema

C 1 INH Antigen

C 1 INH Function

C4

HAE type 1

$

$

HAE type 2

N1 or 1"

AAE type 1 AAE type 2

C2

Clq

Autoantibody

,l,

N1

Absent

,l,

$

Absent

N1 or ,J,

$

$

N1 $

N1 or ,J,

$

$

+

Present

Absent

129

Table 3. Therapy of Autoimmune C1 INH Deficiency (Number of Patients) Effective

Partially Effective

Ineffective

Danazol/Stanazolol

1

3

8

Eaca/Tranexamic Acid

8

1

3

C1 INH infusion- acute

5

4

1

Corticosteroids

1

1

2

Cyclophosphamide

2

0

0

approach has been effective or partially effective in the majority. However, although this therapy results in significant improvement, complete cessation of symptoms or "cure" has not been achieved. There is very little experience with therapies directed specifically toward suppression of autoantibody production. Corticosteroids, in the limited number of patients in whom they have been used, have not been promising. The only patient with an apparent response was treated with corticosteroids combined with azathioprine (Jackson et al., 1986). One patient was treated with plasmapheresis followed by two pulses of cyclophosphamide (1 gm each, approximately three weeks apart) (Donaldson et al., 1992). This was followed by complete normalization of biochemical abnormalities and resolution of clinical symptoms. The patient with Waldenstr6m's macroglobulinemia (Table 1) was treated with cyclophosphamide (100 mg/day for one year) (Cicardi, unpublished data). Following cessation of therapy (three years), his biochemical (C1 INH, C4, autoantibody and immuno-

globulin levels) and clinical parameters all remained normal. One other factor related to therapy should be noted. Although most patients do not have a clinically apparent associated disease, among those who do, specific therapy of the associated disease has appeared to result in improvement or resolution of symptoms.

REFERENCES

Caldwell JR, Ruddy S, Schur PH, Austen KF. Acquired C1 inhibitor deficiency in lymphosarcoma. Clin Immunol Immunopathol 1972;1:39--52. Carrell RW, Pemberton PA, Boswell DR. The serpins: evolution and adaptation in a family of protease inhibitors. Cold Spring Harb Symp Quant Biol 1987;52:527-535. Cicardi M, Bisiani G, Cugno M, Spath P, Agostoni A. Autoimmune C1 inhibitor deficiency: report of eight patients. Am J Med 1993;95:169-175. Davis AE 3rd. C 1 inhibitor and hereditary angioneurotic edema. Annu Rev Immunol 1988;6:595--628. de Agostini A, Schapira M, Wachtfogel YT, Colman RW, Carrel S. Human plasma kallikrein and C1 inhibitor form a complex possessing an epitope that is not detectable on the parent molecules: demonstration using a monoclonal antibody. Proc Natl Acad Sci USA 1985;82:5190--5198. Donaldson VH, Evans RR. A biochemical abnormality in

Alsenz J, Bork K, Loos M. Autoantibody-mediated acquired deficiency of C1 inhibitor. N Engl J Med 1987;316:1360-1366. Alsenz J, Lambris JD, Bork K, Loos M. Acquired C1 inhibitor (CI-INH) deficiency type II. Replacement therapy with C1INH and analysis of patients' C I-INH and anti-C1 INH autoantibodies. J Clin Invest 1989;83:1794-1799. Aulak KS, Eldering E, Hack CE, Lubber YP, Harrison RA, Mast A, Cicardi M, David E 3d. A hinge region mutation in C 1-inhibitor (Ala436 - Thr) results in nonsubstrate-like behavior and in polymerization of the molecule. J Biol Chem 1993;268:18088-18194. Boyar A, Zuraw BL, Beall G. Immunoadsorption in acquired angioedema: a therapeutic misadventure. Clin Immunol Immunopathol 1993;66:181-183.

130

CONCLUSION Although unusual, autoimmune C1 INH deficiency presents several interesting biologic problems. These include the apparent association with evidence for monoclonal B lymphocyte activation and possibly with lymphoproliferative disorders, the lack of other autoantibodies and the absence of autoantibodies to C1 INH in other autoimmune diseases. Further analysis of these characteristics could provide important information related to the development of autoimmunity and the relationship of autoimmunity to lymphoproliferative disease.

hereditary angioneurotic edema. Am J Med 1963;35:37--44. Donaldson VH, Bernstein DI, Wagner CJ, Mitchell B H, Scinto J, Bernstein IL. Angioneurotic edema with acquired C1inhibitor deficiency and autoantibody to C 1-inhibitor: response to plasmapheresis and cytotoxic therapy. J Lab Clin Med 1992; 119:397-406. Donaldson VH, Davis AE. Evidence that an autoantibody to C 1 inhibitor is directed to the reactive center residue of normal Cl-inhibitor. Mol Immunol 1993;30(Suppl 1):9. Eldering E, Nuijens JH, Hack CE. Expression of functional human C1 inhibitor in COS cells. J Biol Chem 1988;263: 11776-11779. Gelfand JA, boss GR, Conley CL, Reinhart R, Frank MM. Acquired C1 esterase inhibitor deficiency and angioedema: a review. Medicine 1979;58:321--328. Harrison RA. Human C1 inhibitor: improved isolation and preliminary structural characterization. Biochemistry 1983; 22:5001-5007. Jackson J, Sim RB, Whelan A, Feighery C. An IgG autoantibody which inactivates Cl-inhibitor. Nature 1986:323:722724. Jackson J, Sim RB, Whaley K, Feighery C. Autoantibody facilitated cleavage of C 1-inhibitor in autoimmune angioedema. J Clin Invest 1989;83:698-707. Malbran A, Hammer CH, Frank MM, Fries LF. Acquired angioedema: observations on the mechanism of action of autoantibodies directed against C1 esterase inhibitor. J

Allergy Clin Immunol 1988;6:1199--1204. Mandle R, Baron C, Roux E, Sundel R, Gelfand J, Aulak K, Davis AE 3rd, Rosen FS, Bing DH. Acquired C1 inhibitor deficiency as a result of an autoantibody to the reactive center region of C1 inhibitor. J Immunol 1994;152:46804685. Matheson NR, van Halbeek H, Travis J. Evidence for a tetrahedral intermediate complex during serpin-proteinase interactions. J Biol Chem 1991 ;266:13489-13491. Nilsson T, Wiman B. Purification and characterization of human Cl-esterase inhibitor. Biochim Biophys Acta 1982: 705:271--276. Nuijens JH, Eerenberg-Belmer AM, Huijbregts CC, Schreuder WO, Felt Bersma RJ, Abbink JJ, Thijs LG, Hack CE. Proteolytic inactivation of plasma C1 inhibitor in sepsis. J Clin Invest 1989;84:443--450. Pilatte Y, Hammer CH, Frank MM, Fries LF. A new simplified procedure for C1 inhibitor purification. A novel use for jacalin-agarose. J Immunol Methods 1989;120:37--43. Schapira M, de Agostini A, Schifferli JA, Colman RW. Biochemistry and pathophysiology of human C1 inhibitor: current issues. Complement 1985;2:111-126. Sim RB, Reboul A. Preparation and properties of human C1 inhibitor. Methods Enzymol 1981;80:43-54. Zuraw BL, Altman LC. Acute consumption of C1 inhibitor in a patient with acquired C 1-inhibitor deficiency syndrome. J Allergy Clin Immunol 1991 ;88:908-918.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

Clq AUTOANTIBODIES Mark H. Wener, M.D. and Mart Mannik, M.D.

Department of Laboratory Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA

HISTORICAL NOTES

THE AUTOANTIGEN

Binding of monomeric IgG to Clq was first described in 1971 in sera'from patients with systemic lupus erythematosus (SLE) with hypocomplementemia (Agnello et al., 1971). In 1978 precipitation of C 1q by monomeric IgG was documented in the hypocomplementemic urticarial vasculitis syndrome (HUVS); the C lq-binding IgG was thought to represent small immune complexes (Marder et al., 1978). In the late 1970s and early 1980s, sera of some patients With SLE were found to contain monomeric (7S) IgG that bound to solid-phase Clq in tests for presumptive circulating immune complexes. The clinical activity of SLE correlated better with the C 1q solid-phase assay than with the fluid-phase C1 q-binding assay for circulating immune complexes (Abrass et al., 1980). The monomeric IgG binding by C 1q was also associated with proliferative forms of lupus nephritis (Wener et al., 1987). The nature of the Clq-binding 7S IgG was clarified in the late 1980s when monomeric IgG from SLE sera was found to bind to solid-phase but not fluidphase Clq (Uwatoko and Mannik, 1988; Antes et al., 1988). The binding was to the collagen-like region (CLR) rather than to the globular heads of C lq and was due to antibodies to C lq (anti-Clq) which persisted in 1.0 M NaC1; whereas, the binding of immune complexes was inhibited by 1.0 M NaC1. Sera from selected SLE patients are now known to contain IgG that bind CLR; the binding can not be accounted for by DNA-containing immune complexes. As expected for antibody-related binding, purified F(ab')2 fragments of C lq-binding IgG bind to the CLR (Uwatoko and Mannik, 1988).

Definition

132

C lq, a cationic (pi=9.3), 410--460 kd glycoprotein, binds to the Fc portions of immunoglobulins in immune complexes to initiate complement activation via the classical pathway (Reid, 1982). In high resolution electron micrographs, the molecule has a shape likened to a bouquet of six tulips, with the "stems" consisting largely of N-terminal CLRs with repeating amino acid sequence (gly-x-y), and the "blossoms" consisting of six C-terminal globular protein domains. C1 q consists of a total of eighteen 22--29 kd polypeptide chains with six copies each of the A, B and C polypeptides which are assembled as six disulfidelinked A-B dimers and three disulfide-linked C-C dimers. The structurally similar A, B and C peptides are encoded on the short arm of human chromosome 1 (Loos and Colomb, 1993). In the circulation, C1 q exists primarily in the form of C1, a calcium-dependent complex with two molecules each of the C1 esterases, Clr and Cls, which bind to the CLR of one molecule of C lq. The usual serum concentration of C lq is 60--200 ~g/mL. C lq serves as a recognition and regulatory protein for the complement cascade. Complement activation by the C 1-dependent classical pathway is initiated by the binding of multiple Fc regions of IgG or IgM to the globular regions of C 1q, which causes a steric change in the CLR of Clq and activates the zymogen Clr and C ls esterases. Monoclonal antibodies with different epitope specificities react with one or more neoepitopes created on C1 q when immune complexes bind to the globular head of Clq (Golan et al., 1982). Immune complexes or aggregated immunoglobulins

are not unique in binding to C lq, because DNA, Creactive protein and other substances also bind. C lq is related to the collectin family of proteins, each of which contains collagen-like domains adjacent to lectin-binding domains. The collectins include mannan-binding protein, lung surfactant proteins A and D, and bovine conglutinin (Loos and Colomb, 1993). Isolation/Purification/Sources

Effective approaches for isolation of C lq include differential salt solubility, ion exchange chromatography followed by size-exclusion gel chromatography or affinity isolation based on binding to IgG or DNA (Reid, 1982). Purified Clq is also available from several companies. No matter what the origin of C l q, its purity should be verified, because contamination with IgG and other plasma constituents is common. Removal of IgG can be achieved by passage over affinity columns of anti-IgG or staphylococcal protein A or protein G. C lq can also be purified by affinity chromatography on concanavalin A agarose and elution with alpha-methylglucopyranoside. To screen for the functional activity of C lq during isolation, IgG-coated latex particles (rheumatoid factor reagents) can be used to document binding to solid-phase and aggregated IgG. The biological activity of isolated C lq can be determined quantitatively and with sensitivity by measuring C1 hemolytic complement activity. C lq protein concentration can be determined by quantifying C lq antigen by nephelometry, radial immunodiffusion or similar immunochemical means, or by measuring absorbance using the extinction coefficient E 1 % cm " - 6.8 at 280 nm. Enzymatic digestion of C lq allows preparation of its two major functional regions. The globular region (MW --39 kd) is prepared by digesting the CLR with collagenase. The CLR (MW 176 kd) is prepared by removal of the globular head of C 1q by limited pepsin digestion (Figure 1), followed by gel chromatography (Reid, 1976). Under reducing conditions on SDSPAGEanalysis, the CLR polypeptides are approximately 16 kd and 14 kd. To prove that the enzymatic digestion is adequate, the size of the peptides should be determined by SDS-PAGE. The absence of binding of aggregated IgG and loss of hemolytic complement activity verify that the globular heads of C lq are destroyed. Antibodies specific for either the CLR (anti-CLR) or the globular regions of C lq can also be used to verify the purity of the C lq digests (Cook et al., 1994). CLR concentration can be quantified in

Figure 1. Schematic drawing of the structure of C lq and antiCLR. The F(ab') 2 antigen-binding portion of anti-CLR binds to epitopes in the collagen-like region of C lq. In contrast, the globular heads of C lq are the binding sites for the Fc portions of IgG in immune complexes. The globular heads are removed by limited pepsin digestion, as indicated by the dotted line.

solution using the extinction c o e f f i c i e n t El%cm -- 2.1 at 275 nm. The epitopes recognized by autoantibodies to CLR of C lq (anti-CLR and anti-C lq) are unknown. Sera from hypocomplementemic urticarial vasculitis patients with anti-CLR, as detected by ELISA, reacted in immunoblots with the reduced B and C chains of intact C lq and some reacted with the A-B or C-C dimers from the CLR of C lq. In contrast, tested sera of patients with SLE and anti-CLR did not react with CLR in immunoblots, and reacted only weakly with the A chain of intact Clq (M~trtensson et al., 1992). There was no clear correlation between the intensity of reactivity by immunoblot and the quantity of antiCLR by ELISA. In other studies, however, anti-CLRcontaining sera from patients with HUVS or SLE did not react with reduced or unreduced CLR fragments by immunoblot (Wisnieski and Jones, 1992a). Although some data suggest that sera from patients with HUVS and SLE recognized the same epitope(s), the epitope(s) remains unknown and may be conformational.

THE A U T O A N T I B O D I E S Methods of Detection

In ELISAs typically used to detect autoantibodies to the CLR of C lq, sufficient concentrations of CLR (1--10 ~g/mL concentration) are used to coat the solid-

9

133

phase in order to optimize binding of anti-CLR, because binding acidities differ among sera and tend to be lower in SLE than in HUVS (Mhrtensson et al., 1992). After coating, plates are blocked with BSA or another suitable blocking protein and then incubated with serum (typically diluted 1:50 to 1:200) in phosphate-buffered normal saline with 0.05--0.5% Tween20 to minimize nonspecific adsorption of IgG. Appropriate dilutions of enzyme-linked F(ab')2 fragments of antibodies to human IgG, IgA or IgM are added, incubated and then reacted with substrate for color development and recording of absorbance. A knownpositive serum is used at various dilutions to generate a standard curve. Positive controls should include at least one positive serum, and negative controls should include normal serum as well as aggregated IgG as a surrogate immune complex. To detect nonspecific binding to the wells, diluted sera should be routinely assayed on wells that are blocked, but not coated with antigen. An alternative assay is based on the finding that the binding of aggregated IgG to the globular heads of

C lq is totally eliminated in the presence of 1.0 M NaC1; whereas, anti-CLR binding to C lq is retained. The usual C lq solid-phase assay for immune complexes is performed but 1.0 M NaC1 is substituted for 0.15 M NaC1 during serum incubations and washes (Siegert et al., 1990). The high-salt Clq-binding modification is sufficient to exclude the binding of aggregated IgG, as surrogate immune complexes, to the globular head of C lq. Whether other potential autoantibodies, for example, to the globular head of C lq, can also be detected by this technique is unknown. Because high molecular weight IgG and IgA in serum can bind to C lq in the presence of 1.0 M NaC1, some immune complexes as well as anti-Clq might be detected by this technique (Siegert et al., 1990). Hence, the method is less rigorous and less definitive than use of CLR as a target antigen. In the review of disease associations below, results using the 1.0 M NaC1 modification are designated as "antiC lq," and results using CLR as "anti-CLR". Anti-C lq production as assessed by the ELISPOT technique allows assay for production of antibodies at

Table 1. Selected Studies of Prevalence of Anti-Clq in Sera from Patients with SLE Method

Patient Selection

Binding to Clq in 1.0 M NaC1

Prevalence

Comments

Reference

Positive tests for solid-phase 15 of 15 C 1q binding

Suggests that C 1q-binding IgG was not an immune complex

Uwatoko et al., 1984

Binding of monomeric IgG to Clq

Patients with renal biopsies

*Class IV 11/14 (79%) Class Vc or Vd 6/11 (54%)

Binding of IgG to Clq is associated with proliferative glomerulonephritis*

Wener et al., 1987

IgG binding to CLR

Nephrology and rheumatology patients with high prevalence of nephritis

31/68 (46%)

IgG binding to CLR

Unselected patients

7/20 (35%)

IgG anti-CLR correlate highly (r=0.94) with C lq solid-phase immune complex assay

Menzel et al., 1991

IgG binding to CLR

University hospital and clinics

48/174 (28%.)

Some patients with mild or inactive disease.

Wisnieski and Jones 1992a

Binding of IgG or IgA to C lq in 1.0 M NaC1

University rheumatology, nephrology and private clinics

IgG: 57/169 (34%) IgA: 8/169 ( 5 % )

With nephritis 83% positive, Siegert et al., without nephritis 21% positive 1990, 1992a, (p=0.0001). Younger patients 1993a more likely to have anti-CLR

Binding of IgG or IgA to C lq in 1.0 M NaC1

University center

IgG: 10/60 (17%) IgA: 6/60 (10%)

No correlation with antibodies to human type II collagen

Wener et al., 1989

Cook et al., 1994

*Class IV renal biopsy lesions by WHO criteria have diffuse proliferative lupus nephritis. Class Vc and Vd renal biopsy lesions by WHO criteria have membranous and proliferative glomerulonephritis.

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the single cell level and holds promise for study of the short-term kinetics of anti-Clq production in response to therapy (R6nnelid et al., 1994).

CLINICAL UTILITY Disease Associations SLE. Among SLE patients, the frequency of IgG antiClq in different series ranges from 17% to 46%, depending on the methods used and patient selection (Table 1). In contrast, the prevalence of IgA antibodies to C lq is low in patients with SLE. Anti-Clq are generally found with increased frequency among patients with lupus nephritis, compared with patients with nonrenal lupus (Wener et al., 1989; Siegert et al., 1993b). Furthermore, serial testing shows that increasing amounts of IgG anti-Clq predict renal flares in SLE patients; an increase in serum IgG anti-Clq has a sensitivity of 71%, specificity of 92%, positive predictive value of 50% and negative predictive value of 97% for the development of renal involvement over three years of follow-up (Siegert et al., 1993b). Elevated serum titers of anti-C1 q tend to be associated with proliferative forms of lupus glomerulonephritis (Siegert et al., 1993b; Wener et al., 1989) and subendothelial deposits of immune complexes (Wener et al., 1989). Concentrations of anti-Clq are only weakly correlated with anti-dsDNA or complement. Hypocomplementemic Urticarial Vasculitis Syndrome. Antibodies to CLR are closely associated with the HUVS, a relatively rare condition that shares features and may coexist with SLE. Virtually all patients with active HUVS or SLE with coexisting HUVS have IgG anti-CLR in their serum; indeed the presence of anti-CLR is considered a diagnostic component of HUVS (Wisnieski and Jones, 1992a; Wisnieski et al., 1995). Most patients with active HUVS have substantial elevations of anti-CLR, although in occasional patients the elevations may be only moderate. Patients with HUVS tend to have the largest amount of IgG anti-CLR of any diagnostic group. Rheumatoid Arthritis (RA) and Other Rheumatic Diseases. Anti-Clq is found in 5% of patients with uncomplicated RA, and most of those patients have IgG or very low titers of IgA anti-Clq. In contrast,

77% of patients with rheumatoid vasculitis and Felty syndrome have serum anti-Clq. Interestingly, IgG anti-Clq is the dominant form of anti-Clq among patients with Felty syndrome (IgG in 76% of patients, IgA in 29%); whereas IgA anti-Clq predominates among patients with rheumatoid vasculitis (IgG in 32%, IgA in 68% of patients) (Siegert et al., 1992a). Other rheumatic diseases with serum IgG anti-CLR include 13% of primary Sj6gren's syndrome (Wener et al., 1989). Among patients with the HLA B27related spondyloarthropathies 7.5% of 40 patients with ankylosing spondylitis had IgG anti-C1 q and 20% had IgA anti-Clq (Siegert et al., 1992a), but in another study none of 19 patients with ankylosing spondylitis and none of 12 patients with Reiter's syndrome had IgG anti-CLR (Wisnieski and Jones, 1992b). Serum anti-Clq was found in 11 of 26 (42%) patients with polyarteritis nodosa, 16 of 17 (94%) with mixed connective tissue disease, but none of 10 patients with systemic sclerosis (Siegert et al., 1992a). Anti-CLR was not found in 21 patients with polymyositis or dermatomyositis (Wisnieski and Jones, 1992b). In light of the overlap of MCTD with systemic sclerosis and polymyositis, these findings merit further investigation. Renal Diseases. Autoantibodies to C1 q are found in a high proportion of patients with membranoproliferative glomerulonephritis (MPGN) (Strife et al., 1989; Siegert et al., 1992a). Anti-Clq are found in 73% of sera from patients with MPGN type I, which is characterized by subendothelial immune deposits in glomerular basement membranes (Siegert et al., 1992a). Among patients with MPGN type II (subendothelial and subepithelial electron-dense deposits) or MPGN type III (only subepithelial electron-dense deposits), only 40--45% of patients have anti-CLR. Results of serial anti-CLR do not parallel the disease course in MPGN patients (Strife et al., 1989). Patients with other renal diseases may also have anti-Clq. Two of six patients with membranous glomerulonephritis, three of six patients with focal glomerulosclerosis, and one of two patients with minimal change glomerulonephropathy had anti-Clq (Siegert et al., 1992a). In contrast, none of 11 patients with IgA nephropathy had anti-Clq. Of 11 patients with disease associated with antibodies to glomerular basement membranes, seven (64%) had anti-Clq: two had only IgG anti-Clq, three with only IgA anti-Clq and two with both (Coremans et al., 1992). Changes in the amounts of anti-Clq

135

tended to parallel changes in anti-GBM with treatment, but cross-inhibition studies suggested that antiClq and anti-GBM do not cross react. These crossinhibition experiments, using fluid-phase C lq and fluid-phase GBM components to inhibit binding to solid-phase Clq and GBM, are difficult to interpret in light of the fact that lupus-associated anti-CLR react with solid-phase but not fluid-phase C lq or CLR.

glomerular basement membrane. Aggregation of C 1qbound complexes could be enhanced by anti-CLR, leading to larger, longer-lasting, and possibly more pathogenic immune deposits (Wener et al., 1989; Uwatoko et a1.,1991). Spontaneous murine lupus models may not be useful in defining the role of antiCLR, because at least some forms of murine lupus lack anti-CLR (Uwatoko et al., 1995).

Healthy Individuals and Others. Neither IgG nor IgA anti-Clq is specific for any single diagnosis. Furthermore, normal individuals may have anti-Clq. As is true for many other autoantibodies, increased levels of anti-Clq are normally found in older individuals, and occasionally in younger normal subjects. Whereas, only about 5% of randomly selected individuals in the 40-69 age ranges have IgG antiC lq above the usual upper limit of a blood bank donor reference population, 18% of septuagenarians have elevated amounts. Furthermore, elevated antiC lq in younger individuals are almost always minimally elevated above the upper limit of normal, but in elderly normal individuals anti-Clq may be elevated substantially above the upper limit of normal (Siegert et al., 1993a). IgG isolated from patients with positive serum assays for IgG anti-CLR sometimes fail to show anti-CLR activity, suggesting that nonspecific binding can cause false-positive results (Wisnieski and Jones, 1992b). Antibodies to CLR and type III human collagen, as well as bovine collagens, were detected in a patient undergoing repeated injections of bovine collagen as a cosmetic treatment (Trautinger et al., 1991). These cross-reactive antibodies are probably unrelated to the spontaneous autoantibodies developing in autoimmune patients.

Factors in Pathogenicity

Pathogenetic Role The pathogenetic role of anti-Clq remains uncertain. The association of anti-CLR with lupus nephritis, especially proliferative forms of lupus nephritis associated with subendothelial immune deposits, supports the possibility that anti-CLR is pathogenic. Anti-CLR might also be associated with subendothelial deposits among patients with MPGN, because anti-CLR is most frequently found in patients with type I MPGN which is characterized by the presence of these deposits. Anti-CLR could contribute to the formation and/or persistence of subendothelial immune deposits by promoting aggregation of different C lq-containing immune complexes in the renal

136

Anti-Clq antibodies from SLE patients probably do not influence complement activation directly, either in vitro or in vivo (Siegert et al., 1992b). IgG anti-CLR in both HUVS and SLE sera are predominantly the IgG2 isotype, although all patients also have IgG1, IgG3 and/or IgG4 antibodies (Wisnieski and Jones, 1992a; Prada and Strife, 1992). In contrast, IgG antiCLR from patients with MPGN was predominantly IgG3 (Prada and Strife, 1992). In other studies, isolated anti-CLR from SLE patients shows the same subclass distribution as normal IgG (IgG1 > IgG2 > IgG3 > IgG4) (Uwatoko and Mannik 1989). As discussed above, the predominant class of anti-Clq in patients with rheumatoid vasculitis is IgA (Siegert et al., 1992a). Whether the different isotopes of anti-C 1q could affect complement activation differently is unknown. Since autoantibodies to collagen are found in patients with autoimmune diseases, and because some monoclonal antibodies react with collagens and the collagen-like region of C lq, the possibility that autoantibodies to CLR cross-react with collagens was examined. In one study, no cross-reactivity and no correlation between antibodies to human type II collagen and anti-CLR was found in patients with SLE or RA (Cook et al., 1994). Although patients with HUVS may have severe pulmonary disease, antiCLR-containing sera from patients with HUVS did not react with the pulmonary surfactant proteins A or D, which are collectins with collagen-like regions nor with type IV collagen (Wisnieski et al., 1995). Anti-Clq/anti-CLR might reflect a response to a neoantigen expressed on C lq when C lq binds to immune complexes (Antes et al., 1988; Siegert et al., 1992a). The linkage of anti-Clq with a variety of immune complex-associated diseases makes that possibility attractive. Indeed, some murine monoclonal antibodies preferentially recognize C lq neoantigens that arise after C lq binds to immune complexes (Golan et al., 1982).

CONCLUSION IgG autoantibodies to the collagen-like region (CLR) of C lq are diagnostic markers for hypocomplementemic urticarial vasculitis syndrome. IgG anti-CLR are also frequent in patients with SLE, where they are associated with proliferative forms of glomerulonephritis. In SLE patients, anti-CLR, analogous to measurement of anti-dsDNA, is a useful marker for the progression of renal disease and for assessing

REFERENCES Abrass CK, Nies KM, Louie JS, Border WA, Glassock RJ. Correlation and predictive accuracy of circulating immune complexes with disease activity in patients with systemic lupus erythematosus. Arthritis Rheum 1980;23:273-282. Agnello V, Koffier D, Eisenberg JW, Winchester RJ, Kundel HG. C lq precipitins in the sera of patients with systemic lupus erythematosus and other hypocomplementemic states: characterization of high and low molecular weight types. J Exp Med 1971;134:$228. Antes U, Heinz H-P, Loos M. Evidence for the presence of autoantibodies to the collagen-like portion of C 1q in systemic lupus erythematosus. Arthritis Rheum 1988;31:457--464. Cook AD, Rowley MJ, Wines B D, Mackay IR. Antibodies to the collagen-like region of C lq and type II collagen are independent non-cross-reactive populations in systemic lupus erythematosus and rheumatoid arthritis. J Autoimmun 1994;7:369-378. Coremans IE, Daha MR, van der Voort EA, Muizert Y, Halma C, Breedveld FC. Antibodies against C lq in anti-glomerular basement membrane nephritis. Clin Exp Immunol 1992;87: 256--260. Golan MD, Burger R, Loos M. Conformational changes in C 1q after binding to immune complexes: detection of neoantigens with monoclonal antibodies. J Immunol 1982;129:445--447. Loos M, Colomb M. C1, the first component of complement: structure-function-relationship of C lq and collectins (MBP, SP-A, SP-D, congluinin), Cl-esterases (Clr and Cls and C1inhibitor in health and disease. Behring Inst Mitt 1993:1--5. Marder RJ, Burch FX, Schmid FR, Zeiss CR, Gewurz H. Low molecular weight Clq-precipitins in hypocomplemetemic vasculitis-urticaria syndrome: partial purification and characterization as immunoglobulin. J Immunol 1978; 121:613-618. Mgtrtensson U, Sj6holm AG, Sturfelt G, Truedsson L, Laurell A-B. Western blot analysis of human IgG reactive with the collagenous portion of Clq: evidence of distinct binding specificities. Scand J Immunol 1992;35:735-744. Menzel JE, Scherak O, Kolarz G, Gamerith F, Youngchaiyud U. A method to differentiate between anti-C 1q antibodies and C lq-binding immune complexes using collagenase-digested solid phase Clq. J Immunol Methods 1991;138:165--171. Prada AE, Strife CF. IgG subclass restriction of autoantibody

disease activity. Although present frequently in patients with membranoproliferative glomerulonephritis, especially type I, and in patients with anti-GBM disease, anti-CLR may not have a significant diagnostic or prognostic role in those conditions. IgA antiC1 q is frequent in patients with rheumatoid vasculitis, but its diagnostic role in that condition remains unclear. The pathogenic role of autoantibodies to C 1q is uncertain. See also COLLAGEN AUTOANTIBODIES.

to solid-phase C lq in membranoproliferative and lupus glomerulonephritis. Clin Immunol Immunopathol 1992;63: 84-88. Reid KBM. Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement. Biochem J 1976; 155: 5--17. Reid KB. C 1q. Methods Enzymol 1982; 82(Part A):319--324. R6nnelid J, Huang YH, Norrlander T, Rogberg S, Nilsson B, Gustafsson R, Klareskog L. Short-term kinetics of the humoral anti-Clq response in SLE using the ELISPOT method: fast decline in production in response to steroids. Scand J Immunol 1994:40:243-250. Siegert CE, Daha MR, van der Voort EA, Breedveld FC. IgG and IgA antibodies to the collagen-like region of C lq in rheumatoid vasculitis. Arthritis Rheum 1990;33:1646-1654. Siegert CE, Daha MR, Halma C, van der Voort EA, Breedveld FC. IgG and IgA autoantibodies to C 1q in systemic and renal diseases. Clin Exp Rheumatol 1992a; 10:19--23. Siegert CE, Daha MR, Lobatto S, van der Voort EA, Breedveld FC. IgG autoantibodies to C lq do not detectably influence complement activation in vivo and in vitro in systemic lupus erythematosus. Immunol Res 1992b; 11:91--97. Siegert CE, Daha MR, Swaak AJ, van der Voort EA, Breedveld FC. The relationship between serum titers of autoantibodies to C 1q and age in the general population and in patients with systemic lupus erythematosus. Clin Immunol Immunopathol 1993a;67(3 Pt 1):204--209. Siegert CE, Daha MR, Tseng CM, Coremans IE, van Es LA, Breedveld FC. Predictive value of IgG autoantibodies against C lq for nephritis in systemic lupus erythematosus. Ann Rheum Dis 1993b;52:851--856. Strife CF, Leahy AE, West CD. Antibody to a cryptic, solid phase C lq antigen in membranoproliferative nephritis. Kidney Int 1989;35:836-842. Trautinger F, Kokoschka EM, Menzel EJ. Antibody formation against human collagen and C lq in response to a bovine collagen implant. Arch Dermatol Res 1991;283:395-399. Uwatoko S, Mannik M. Low molecular weight Clq-binding immunoglobulin G in patients with systemic lupus erythematosus consists of autoantibodies to the collagen-like region of Clq. J Clin Invest 1988;82:816-824. Uwatoko S, Mannik M. IgG subclasses of antibodies to the collagen-like region of C lq in patients with systemic lupus 137

erythematosus. Arthritis Rheum 1989;32:1601-- 1603. Uwatoko S, Gauthier VJ, Mannik M. Autoantibodies to the collagen-like region of C 1q deposit in glomeruli via C 1q in immune deposits. Clin Immunol Immunopathol 1991 ;61 (2 Pt 1):268-273. Uwatoko S, Mannik M, Oppliger IR, Okawa-Takatsuji M, Aotsuka S, Yokohari R, Seki G, Taniguchi S, Suzuki K, Kurokawa K. C 1q binding immunoglobulin G in MRL/1 mice consists of immune complexes containing antibodies to DNA. Clin Immunol Immunopathol 1995;75:140-146. Wener MH, Mannik M, Schwartz MM, Lewis EJ. Relationship between renal pathology and size of circulating immune complexes in patients with systemic lupus erythematosus. Medicine (Baltimore) 1987;66:85-97. Wener MH, Uwatoko S, Mannik M. Antibodies to the collagenlike region of C lq in sera of patients with autoimmune

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rheumatic diseases. Arthritis Rheum 1989;32:544--551. Wisnieski JJ, Baer AN, Christensen J, Cupps TR, Flagg DN, Jones JV, Katzenstein PL, McFadden ER, McMillen JJ, Pick MA, et al. Hypocomplementemic urticarial vaseulitis syndrome. Clinical and serologic findings in 18 patients. Medicine (Baltimore) 1995;74:24--41. Wisnieski JJ, Jones SM. Comparison of autoantibodies to the collagen-like region of C lq in hypocomplementemic urticarial vasculitis syndrome and systemic lupus erythematOSUS. J Immunol 1992a;148:1396--1403. Wisnieski JJ, Jones SM. IgG autoantibody to the collagen-like region of C lq in hypocomplementemic urticarial vasculitis syndrome, systemic lupus erythematosus, and six other musculoskeletal or rheumatic diseases. J Rheumatol 1992b; 19:884--888.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CALCIUM CHANNEL AND RELATED PARANEOPLASTIC DISEASE AUTOANTIBODIES Vanda A. Lennon, M.D., Ph.D.

Neuroimmunology Laboratory, Departments of Immunology, Neurology and Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA

HISTORICAL NOTES In the late 1970s IgG autoantibodies were demonstrated to be the effectors of neurologic dysfunction in the Lambert-Eaton myasthenic syndrome (LES; Denys et al., 1979; Lang et al., 1981). The majority of patients with this presynaptic disorder of neuromuscular transmission have a lung carcinoma of small cell type (Lambert et al., 1961). The development of radioligands specific for different subtypes of high voltage-activated neuronal calcium (Ca 2+) channels revealed a diversity of anti-Ca 2+ channel autoantibodies in sera of patients with LES, but their pathogenicity is not yet proven (Sher et al., 1989; Lennon and Lambert, 1989; Lennon et al., 1995). It has recently been recognized that Ca 2+ channel binding autoantibodies and a marker autoantibody directed against neuronal nuclear or cytoplasmic antigens often coexist in sera of patients with a variety of encephalomyeloradiculoneuropathies associated with carcinoma of lung, breast and ovary (O'Suilleabhain et al., 1994; Lennon et al., 1995). Organ-specific antineuronal autoantibodies were first recognized as a serological marker of paraneoplastic autoimmunity in patients with sensory neuropathy associated with lung carcinoma. Complement fixation assays identified an autoantigen that was concentrated in grey matter of brain and dorsal root ganglia (Wilkinson, 1964). The autoantibodies were IgG, and indirect immunofluorescence revealed that they bound selectively to neurons, and were not species-specific (Wilkinson and Zeromski, 1965). In most neurons the pattern of immunostaining was consistent with autoantibodies known today as type-1

antineuronal nuclear autoantibodies (ANNA-1 or antiHu). However, the original authors mistook the large brightly stained nuclei of cerebral cortical neurons and their unstained nucleoli (Figure 1) for stained cytoplasm with unstained nuclei. Furthermore, the characteristic ANNA-1 immunostaining pattern in cerebellar Purkinje cells (Lennon, 1994b) was not initially observed (Wilkinson and Zeromski, 1965), presumably because the sera were not diluted sufficiently and the tissue sections were too thin (4 ja) and not fixed optimally (Altermatt et al., 1991). These earlier observations on ANNA-1 were clarified and extended in 1985 (Graus et al., 1985). After paraneoplastic autoimmune serology was introduced to clinical practice, ANNA-1 (Hu) autoantibodies were recognized in adult patients with a spectrum of inflammatory central and peripheral nervous system disorders associated with small cell lung carcinoma (SCLC), including gastrointestinal dysmotilities (Lennon et al., 1991; Lennon, 1994a). ANNA-1 were also found in several pediatric neurologic disorders, with and without neuroblastoma, and sometimes with Ca 2+ channel binding autoantibodies (Miller et al., 1994; Fisher and Singer, 1995). In 1988, superficially similar but antigenically and oncologically distinct antineuronal nuclear autoantibodies (ANNA-2 or anti-Ri) were identified (Budde-Steffen et al., 1988). ANNA-2 are reported in patients with midbrain, cerebellar and spinal cord disorders associated with breast carcinoma and SCLC (Luque et al., 1991; Lennon, 1994a). In 1983, the first paraneoplastic autoantibodies and their selective binding to the cytoplasm of neurons (type 1 anti-Purkinje cell cytoplasmic autoantibodies, PCA-1, or anti-Yo) were

139

Figure 1. Photomicrographs reproduced from Wilkinson and Zeromski, 1965 (Brain, 88:529-538) with permission; upper, plate LVIII (Fig. 1) and lower, plate LIX (Fig. 3). Guinea pig occipital lobe (unfixed frozen sections, 4 ja thick) treated with serum (neat or 1:4 dilution) from patient E.T. with sensory neuronopathy associated with lung carcinoma (a), or a normal subject (b), followed by fluorescein-conjugated antihuman IgG. a: the selective and intense staining of neuronal nuclei, unstained nucleoli and faint granular staining of the cytoplasm, was unchanged by prior absorption with kidney tissue but was removed by absorbing with brain tissue. These immunofluorescence characteristics are consistent with the ANNA-1 (or anti-Hu) specificity (Lennon, 1994b).

described in two patients with subacute cerebellar degeneration associated with ovarian carcinoma (Greenlee and Brashear, 1983). Autoantibodies of PCA-1 specificity were subsequently recognized as: (1) restricted to female patients; the clinical presentation usually cerebellar ataxia, but sometimes a peripheral neuropathy, and (2) a marker of an underlying m~llerian or breast carcinoma (Hetzel et al., 1990;

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Lennon, 1994a). Another breast carcinoma-related IgG, specific for the cytoplasmic antigen amphiphysin (a synaptic vesicle protein), was described in 1993 in patients with an atypical form of stiff-man syndrome (Folli et al., 1993). Organ-specific autoantibodies that serve clinically as markers of paraneoplastic neurologic autoimmune responses provide valuable insight into the immunobiology of human cancer. The relative

infrequency of these spontaneous antibody responses presumably reflects genetic determinants of the individual patient's immune responsiveness.

THE AUTOANTIGENS Tumor antigens that stimulate immune responses giving rise to paraneoplastic autoantibodies, both neuron or muscle-specific and non-organ-specific, appear to act as a co-immunogenic macromolecular complex of nuclear, cytoplasmic and plasma membrane proteins (Lennon, 1994a).

Plasma Membrane Autoantigens Ca z+ Channel Proteins. High-voltage activated Ca 2+ channels are integral plasma membrane proteins with multiple subunits. The amino acid sequence of the ion-translocating c~1 subunit is the basis of a molecular classification system. Biophysical and pharmacological properties define the following subtypes of neuronal Ca 2+ channels: P and Q (class A), N (class B), L (class D) and R (class E) (Lennon et al., 1995). Of current serological interest are P or Q-type Ca 2§ channels, which control acetylcholine release at the neuromuscular junction and are also involved in central neurotransmission, and N-type channels, which are involved in cerebrocortical, cerebellar, spinal and autonomic neurotransmission. Both are found in small cell lung carcinomas (Lennon et al., 1995). Solubilized P/Q and N-type Ca 2+ channels are identifiable immunologically by their respective high affinity binding of 125I-labelled c0-conopeptides MvIIC and GvIA (Lennon et al., 1995). For clinical assays, optimal sensitivity and specificity is obtained using native Ca 2§ channel proteins extracted from synaptic membranes of human brain in the nonionic detergent digitonin (Lennon, 1990; Lennon et al., 1993; Lennon et al., 1995; Griesmann and Lennon, 1996). Recombinant proteins corresponding respectively to part of a human neuronal Ca 2+ channel's 13 subunit (Rosenfeld et al., 1993) and a noncovalently associated synaptic vesicle protein, synaptotagmin (Takamori et al., 1995), have been reported to detect autoantibodies in sera of LES patients by immunoblot assays, but prospective clinical experience is lacking.

Nicotinic Acetylcholine Receptors (AChR) of Muscle Type. The skeletal muscle disorder myasthe-

nia gravis (MG) is sometimes a paraneoplastic accompaniment of thymic epithelial tumors (Griesmann and Lennon, 1996). Extracellular segments of AChR proteins in the postsynaptic membrane are the target of pathogenic antibody interactions. AChR proteins solubilized from human muscle (AChR) in the nonionic detergent Triton-X-100 and complexed with the high affinity ligand 125I-labelled o~-bungarotoxin detect, in 90% of patients who have MG, both pathogenic and marker autoantibodies; the latter may be directed against intramembranous/cytoplasmic epitopes. This antigen preparation also detects marker autoantibodies in 5-10% of patients with paraneoplastic neurologic disorders associated with primary lung cancer (Lennon, 1994c; Griesmann and Lennon, 1996). Antigens prepared from subprimate muscle (Lennon and Griesmann, 1989) or in grossly denatured form (Sano et al., 1991) are not suitable for clinical assays.

Nuclear Autoantigens Hu. The antigens recognized by type 1 antineuronal nuclear autoantibodies (ANNA-l, or anti-Hu) are expressed in all neurons of the central and peripheral nervous system, more in the nucleus than in the cytoplasm but not in the nucleolus (Altermatt et al., 1991), and in SCLC (Kiers et al., 1991). Immunostaining of tissue substrates is enhanced by brief fixation in 4% neutral phosphate-buffered formalin (Altermatt et al., 1991). In immunoblot analyses ANNA-1 autoantibodies bind to neuronal and SCLC proteins of relative molecular mass 35 to 40 kd (Dalmau et al., 1990). These antigens are highly conserved RNA-binding proteins encoded by a family of genes that includes HuD, HuC and Hel-N1 (Szabo et al., 1991; Manley et al., 1994; Dropcho et al., 1994).

Ri. The antigens recognized by type 2 antineuronal nuclear autoantibodies (ANNA-2, or anti-Ri) are restricted to neurons of the central nervous system and are also concentrated in the nucleus, and expressed in the cytoplasm but not in the nucleolus (Graus et al., 1993). These antigens are expressed in SCLC cells (Lennon, 1994b) and presumably also in some breast carcinomas. Immunoblotting defines two major neuronal proteins of relative molecular masses 55 and 80 kd (Budde-Steffen et al., 1988). A gene ("Nova") encodes the smaller of these highly conserved proteins (Buckanovich et al., 1993); in the developing mouse 141

nervous system, alternatively spliced products of Nova, thought to be RNA-binding proteins, are restricted to motor neurons.

Cytoplasmic Autoantigens Yo. Purkinje cell cytoplasmic antigen, type 1, the major antigen recognized by autoantibodies of PCA-1 (or anti-Yo) specificity is a hydrophilic cytoplasmic protein of relative molecular mass, 52 to 62 kd (Cunningham et al., 1986; Sakai et al., 1990). This antigen is not species-specific (Smith et al., 1988), is not restricted to Purkinje neurons, is preserved by brief fixation in 4% formalin (Altermatt et al., 1991) and is expressed in most large neurons, in Schwann cells (Altermatt et al., 1991) and in at least some breast and ovarian carcinoma cells (Furneaux et al., 1990). In Purkinje neurons the PCA-1 antigen is bound to ribosomes, endoplasmic reticulum and transGolgi membranes (Rodriguez et al., 1988). Amino acid sequences deduced for the 52 kd antigen (443 residues; Sakai et al., 1990) and the 62 kd antigen (Fathallah-Shaykh et al., 1991) are identical except for residues 368--382 and an additional 67 residues at the N-terminus of the 62 kd protein. Three other protein antigens, 34, 58 and 40 kd, are also reported to contribute to the PCA-1 specificity (discussed in legend to Figure 2, Lennon, 1994b).

Amphiphysin. The antigenic protein is associated with the cytoplasmic face of synaptic vesicle membranes. In immunoblots of brain-derived membranes it has a relative molecular mass -128 kd (Folli et al., 1993). A dominant epitope maps to the C-terminal region of human amphiphysin (David et al., 1994).

Contractile, Cytoskeletal and Non-Organ-Specific Autoantigens. Antistriational autoantibodies (StrAb) are characteristically found as paraneoplastic marker autoantibodies in patients with MG associated with thymoma (Strauss et al., 1965). They are also found in patients with paraneoplastic neurologic disorders associated with primary lung cancer (Lennon, 1994c; Griesmann and Lennon, 1996). Skeletal muscle antigens identified as targets of StrAb include actin, ~actinin, myosin (Williams and Lennon, 1986), titin (Williams et al., 1992) and ryanodine receptor (Mygland et al., 1994). For clinical purposes StrAb are :reliably detected by enzyme immunoassay using a crude mixture of muscle proteins (Griesmann and Lennon, 1996). Autoantibodies reactive with nonorgan-specific autoantigens including nuclei, smooth muscle and mitochondria are more frequent in patients with cancer than in those without cancer (Whitehouse, 1973; Imai et al., 1992; Lennon, 1994c). They are detected by indirect immunofluorescence using a panel of tissue sections as substrate.

Table 1. Oncologic and Neurologic Associations of Paraneoplastic Neuronal/Muscle Autoantibodies Carcinoma

Most Frequently Recognized Autoantibodies

Most Common Neurologic Presentations

Lung, small cell

N-type Ca2+ channel P/Q-type Ca2+ channel AChR binding/striational Hu (ANNA-1) Unclassifieda Ri (ANNA-2)b

Neuropathy (sensory/sensorimotor > autonomic >> motor) Encephalomyeloradiculopathy Lambert-Eaton myasthenic syndromec Intestinal dysmotility

Breast

N or P/Q-type Ca2+ channel Yo (PCA-1) Amphiphysin Ri (ANNA-2)

Neuropathy Subacute cerebellar ataxia Midbrain encephalitis Myelopathy

Ovary, endometrium, Fallopian tube, serous surface papillary adenocarcinoma

PCA-1 N-type Ca2+ channel P/Q-type Ca2+ channel Ri (ANNA-2)

Subacute cerebellar ataxia Encephalomyeloradiculopathy Neuropathy

aSeveral IgG neuronal nuclear and cytoplasmic autoantibodies (as yet unclassified) are identifiable by indirect immunofluorescence as small cell lung carcinoma-related in patients with assorted paraneoplastic neurologic presentations (VA Lennon, unpublished observations), bRi (ANNA-2) not yet documented with LES or intestinal dysmotility. CHu (ANNA-l) rare unless coexisting signs of neuropathy.

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Table 2. Clinical Correlations of P/Q and N-type Ca2+. Channel Binding Autoantibodies P/Q-type Ca2+ channel binding autoantibodies:

N-type calcium channel binding autoantibodies:

9 Confirm a diagnosis of LES (95% positive).

9 Justify a targeted search for lung cancer (small cell, squamous or adenocarcinoma) in patients with LES or an acquired neuropathy. Of LES patients with lung cancer, 73% are positive; without lung cancer, 36% are positive.

9 Distinguish LES from MG.

9 Aid diagnosis of autoimmune cerebellar ataxia in adults and children.

9 Justify a targeted search for cancer (lung, breast, or ovarian) in patients with a complex neurologic presentation, particularly in those with known risk factors for cancer (e.g., tobacco abuse; past or family history of lung, breast or ovarian cancer).

9 Distinguish autoimmune from hereditary neuropathies.

9 Justify a targeted search for cancer in patients who present with limbic encephalitis (lung cancer), cerebellar ataxia or neuropathy (lung, breast or ovarian cancer).

9 Justify a targeted search for cancer (lung, breast or ovarian) in patients with a complex neurologic presentation, particularly in those with known risk factors for cancer (e.g., tobacco abuse; past or family history of lung, breast or ovarian cancer).

9 Distinguish autoimmune paraneoplastic neuropathies from neuropathies attributed to chemotherapeutic toxicity (e.g., cis-platinum).

9 Distinguish autoimmune neurologic complications of cancer from metastatic complications.

9 Distinguish autoimmune neurologic complications of cancer from metastatic complications.

9 Distinguish autoimmune paraneoplastic neuropathies from neuropathies attributed to chemotherapeutic toxicity (e.g.,

cis-platinum).

AUTOANTIBODIES Paraneoplastic autoantibodies are a manifestation of a patient's genetically determined immune responses against membranous, cytoplasmic and nuclear antigens of a tumor, usually a carcinoma of lung (SCLC, or less often adenocarcinoma or squamous cell), ovary (or related mtillerian tissue) or breast. Because metastasis is limited in seropositive patients (Hetzel et al., 1990; Galanis et al., 1996), the tumors are notoriously difficult to find. Because patients predisposed to have cancer may have more than one neoplasm, the search for a carcinoma predicted by finding a characteristic autoantibody (Table 1) should continue if an unrelated neoplasm is initially found (Lucchinetti et al., 1994b).

CLINICAL U T I L I T Y Ca 2+ Channel Binding Autoantibodies A positive result for Ca 2+ channel binding autoantibodies of P/Q- or N-type (Table 2) is consistent with neurologic autoimmunity and suggests that a neoplasm

might be present (Lennon et al., 1995). These autoantibodies can coexist with A N N A - l , ANNA-2 or PCA-1 (Table 1, Table 3). Except for patients with sporadic cerebellar ataxia (Miller et al., 1994) and a minority with amyotrophic lateral sclerosis (Lennon et al., 1995), P/Q-type and N-type Ca 2+ channel binding autoantibodies are found in <3% of healthy subjects, or patients with control autoimmune or neurologic diseases. L-type Ca 2§ channel binding autoantibodies were reported in 75% of patients with sporadic amyotrophic lateral sclerosis (Smith et al., 1992), but prospective experience in a clinical setting is lacking for these autoantibodies. Either P/Q or N-type autoantibodies (or both) are found in 28% of patients with SCLC without neurological problems (Table 2) and are significantly more frequent in patients lacking distant metastases (Galanis et al., 1996). The diagnosis of LES depends on fulfillment of electromyographic criteria. On clinical grounds, this diagnosis is made most readily in patients with classic symptoms of proximal muscle weakness, dry mouth and, in males, erectile impotence. However, oculobulbar weakness can give a mistaken impression of MG. Because <5% of MG patients have P/Q-type

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Table 3. Clinical Correlations of Hu (ANNA-I), Ri (ANNA-2) and Yo (PCA-1) Autoantibodies

Hu (ANNA-l) autoantibodies:

Ri (ANNA-2) autoantibodies:

Yo (PCA-1) autoantibodies:

9 Not found in healthy subjects.

9 Not found in healthy subjects; found in <2% of patients who have cancer without evidence of neurologic dysfunction.

9 Not found in healthy subjects.

9 Females predominate 2:1; usual age >45 years,

9 Females predominate (61%); usual age >35 years.

9 >99% female; usual age >40 years.

9 Neurologic presentations include neuropathies (sensory/sensorimotor > autonomic >> motor), limbic encephalitis, subacute cerebellar ataxia, myelopathy or radiculopathy,

9 Neurologic presentations include idbrain, brain stem, cerebellar and/or spinal cord dysfunction; less often sensory, sensorimotor or motor neuropathy. Ocular psoclonus-myoclonus or jaw spasm may be prominent.

9 Usual neurologic presentation is subacute cerebellar ataxia; other neurologic signs often coexist; 6% present with sensory, ensorimotor or motor neuropathy.

9 SCLC (usually limited to the chest) is found in 83% of adult patients with adequate follow-up; 13% of these patients have an unrelated neoplasm (antecedent or coexisting). SCLC is sometimes detectable only by magnetic resonance imaging (Kimmel et al., 1988) or autopsy.

9 A marker of breast carcinoma or SCLC more often than gynecologic carcinoma (Lennon, 1994a).

9 Carcinoma (gynecologic > breast) found in-90% with adequate follow-up. Primary lung carcinoma not documented. Carcinomas are typically limited in metastatic spread. Serum CA-125 (ovarian cancer antigen) may or may not be elevated.

Tends to decline progressively in the course of cancer chemotherapy, with accompanying neurologic stabilization or striking improvement (Hunter et al., 1995).

9 Exploratory laparotomy recommended if no imaging evidence of cancer (as for a "second look" in management of ovarian carcinoma).

9 Early marker of a spectrum of paraneoplastic intestinal dysmotilities associated with SCLC (Lucchinetti et al., 1994a); obviates unnecessary abdominal surgery; found in children with intestinal dysmotility, cerebellar ataxia, and autoimmune encephalitides, with and without evident cancer (neuroblastoma). 9 Found in <10% of patients with uncomplicated SCLC; uncommon in SCLC-related neurologic disorders without evidence of a neuropathy, including patients with Lambert-Eaton myasthenic syndrome and/or cerebellar ataxia; precedes the finding of SCLC in 55% of patients.

9 Far less common than Hu (ANNA- 1) or Yo (PCA- 1).

Found in <2% of ovarian carcinoma patients without evidence of neurologic dysfunction.

9 May be accompanied CaZ+channel-binding autoantibodies.

9 May be accompanied by Ca 2+ channel binding autoantibodies.

May be accompanied by Ca2+ channel binding autoantibodies.

Ca 2+ channel binding autoantibodies, whereas 95% of LES patients are positive (Lennon et al., 1995), the assay for P/Q-type channel autoantibodies is very useful for distinguishing LES from MG. Coexistence of M G and LES is not yet d o c u m e n t e d convincingly by electrophysiologic criteria, but 13% of LES patients have A C h R binding or striational autoantibodies (Table 4) (Lennon, 1994c). A value above 100 p m o l / L for P/Q-type Ca 2+ channel binding autoantibodies in a patient who lacks obvious clinical signs of LES may

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justify electromyographic testing of n e u r o m u s c u l a r transmission for a defect characteristic of LES. These patients are often tobacco abusers with c o m p l e x neurologic findings that may include sensorimotor or autonomic neuropathy or encephalopathy/cerebellar ataxia. Tests for Ca 2+ channel binding autoantibodies may be negative in i m m u n o s u p p r e s s e d patients. The primary neurologic (and oncologic) diagnosis will be further obscured if the patient develops a superimposed steroid m y o p a t h y (Lennon, 1994c).

Table 4. Clinical Correlations for Acetylcholine Receptor Binding Autoantibodies and Striational Autoantibodies AChR binding autoantibodies:

Striational autoantibodies:

9 Aid diagnosis of MG, including subclinical MG in patients with thymoma.

9 Aid diagnosis of MG in patients seronegative for AChR binding autoantibodies; aid diagnosis of thymoma, especially with age of MG onset < 45 years.

9 Complementthe test for P/Q-type C a 2+ channel binding autoantibodies as a diagnostic aid for LES.

9 Complementthe test for P/Q-type C a 2+ channel binding autoantibodies as a diagnostic aid for LES.

9 Complementthe test for N-type C a 2+ channel binding autoantibodies as a diagnostic aid for primary lung carcinoma.

9 Complementthe test for N-type C a 2+ channel binding autoantibodies as a diagnostic aid for primary lung cancer.

9 Monitorimmunologic progress after tumor ablation/ chemotherapy.

Neuronal Nuclear and Cytoplasmic Autoantibodies Using strictly defined staining criteria coupled with serological absorption steps to exclude non-organspecific autoantibodies (Lennon, 1994b), the indirect immunofluorescence technique in most clinical circumstances can identify IgG autoantibodies of PCA-1 (Yo) specificity unambiguously, and can distinguish ANNA-1 (Hu) and ANNA-2 (Ri) specificities from each other and from non-organ-specific antinuclear antibody. A composite frozen section substrate (cerebellum, gut and kidney) provides central and peripheral (myenteric plexus) neurons and nonneural cells; after preliminary absorption with a nonneural tissue to reduce interference by non-organspecific autoantibodies, fewer than 10% of sera require further absorption to identify neuron-specific autoantibodies, and <10% of those will require immunoblot analysis (Lennon, 1994b). Although immunoblot assays with recombinant protein antigens (e.g., HuD for ANNA-l, Nova for ANNA-2) (Dalmau and Posner, 1994) have been recommended for routine clinical testing or for confirmation of immunohistochemical or immunofluorescence techniques, it is our experience that immunoblot assays do not offer superior sensitivity in a clinical setting, but they are useful in the uncommon situation when interfering non-organ-specific autoantibodies preclude interpretation of the immunofluorescence assay. For practical purposes, the exquisite specificity of single recombinant protein antigens (or purified native antigens) is a disadvantage because it precludes detection of alternative antineuronal autoantibodies (i.e., those not specifically requested by the physician, and those "as yet

Monitor immunologic progress after tumor ablation/ chemotherapy; monitor tumor recurrence after treatment of thymoma (Cikes et al., 1988).

unclassified"; Table 1). The various antineuronal autoantibodies that are currently identifiable by the immunofluorescence technique, are extremely useful in targeting the search for cancer (Table 3).

CONCLUSION Neuron or muscle-restricted autoantibodies are useful diagnostically as markers of paraneoplastic neurologic autoimmunity, and in some cases as specific tumor markers. The neoplasms are often occult and are commonly carcinomas of lung (small cell type), breast or ovary. Seropositive patients usually present with a subacute neurologic syndrome. Detection of certain marker autoantibodies in the serum or cerebrospinal fluid (ANNA-l, ANNA-2, PCA-1, antiamphiphysin, P/Q and N-type Ca 2+ channel binding autoantibodies) offers the prospect of earlier diagnosis and treatment of an underlying carcinoma. P/Q-type Ca 2+ channel binding autoantibodies are found in highest frequency and titer in patients with the Lambert-Eaton myasthenic syndrome (95% seropositive), but a pathogenic role is not yet definitively established. Acetylcholine receptor binding autoantibodies and striational autoantibodies are also valuable markers of primary lung carcinoma (small cell, squamous cell or adenocarcinoma) in patients with a paraneoplastic neurologic presentation, and as such complement N-type Ca 2+ channel binding autoantibodies. None of these autoantibody assays are recommended as a general screening test for cancer. However, novel strategies for early diagnosis and treatment of cancer can be anticipated as a dividend of future research based on immunobio-

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logical insights provided by investigating patients with paraneoplastic autoimmunity. See also NEURONAL

NUCLEAR AUTOANTIBODIES, TYPE 1 (Hu) and PURKINJE CELL AUTOANTIBODIES, TYPE 1 (Yo).

REFERENCES

Furneaux HM, Rosenblum MK, Dalmau J, Wong E, Woodruff P, Graus F, Posner JB. Selective expression of Purkinje-cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration. N Engl J Med 1990;322:1844-- 1851. Galanis E, Frytak S, Rowland KM, Lennon VA. Prevalence and titers of paraneoplastic antineuronal autoantibodies in the course of small cell lung carcinoma and platinum associated neuropathy. J Clin Oncol 1996;(In press). Graus F, Cordon-Cardo C, Posner JB. Neuronal antinuclear antibody in sensory neuropathy from lung cancer. Neurology 1985;35:538-543. Graus F, Rowe G, Fueyo J, Darnell RB, Dalmau J. The neuronal nuclear antigen recognized by the human anti-Ri autoantibody is expressed in central but not peripheral nervous system neurons. Neurosci Lett 1993; 150:212-214. Greenlee JE, Brashear HR. Antibodies to cerebellar Purkinje cells in patients with paraneoplastic cerebellar degeneration and ovarian carcinoma. Ann Neurol 1983;14:609--613. Griesmann GE, Lennon VA. Detection of autoantibodies in myasthenia gravis and Lambert-Eaton myasthenic syndrome. In: Rose ER, ed. Manual of Clinical Laboratory Immunology, 5th Edition. Washington, DC: America Society of Microbiology, 1996;(In press). Hartzel DJ, Stanhope CR, O'Neill BP, Lennon VA. Gynecologic cancer in patients with subacute cerebellar degeneration predicted by anti-Purkinje cell antibodies and limited in metastatic volume. Mayo Clin Proc 1990;65:1558-1563. Hunter SF, Parisi JE, Mastovich SL, Power W, Delahoussaye B, Duncan PR, Lennon VA. Chronic progressive paraneoplastic syndrome with prominent brainstem and spinal cord involvement, associated with type-2 antineuronal nuclear antibodies (ANNA-2) and breast carcinoma. J Neuropathol Exp Neurol 1995;54:464. Imai H, Ochs RL, Kiyosawa K, Furuta S, Nakamura RM, Tan EM. Nucleolar antigens and autoantibodies in hepatocellular carcinoma and other malignancies. Am J Pathol 1992;140: 859--870. Kiers L, Altermatt HJ, Lennon VA. Paraneoplastic antineuronal nuclear IgG autoantibodies (type I) localize antigen in small cell lung carcinoma. Mayo Clin Proc 1991;66:1209-1216. Kimmel DW, O'Neill BP, Lennon VA. Subacute sensory neuropathy associated with small cell lung carcinoma: diagnosis aided by autoimmune serology. Mayo Clin Proc 1988;63:29-32. Lambert EH, Rooke EG, Eaton LM, Hodgson CH. Myasthenic syndrome occasionally associated with bronchial neoplasm: neurophysiologic studies. In: Viets HR, Schwab RS, eds. Thymectomy for Myasthenia Gravis; a Record of Experiences at the Massachusetts General Hospital. Springfield, IL: Charles C. Thomas, 1960;362-410. Lambert EH, Lennon VA. Selected IgG rapidly induces Lambert-Eaton myasthenic syndrome in mice: complement independence and EMG abnormalities. Muscle Nerve 1988;11:1133-1145.

Altermatt HJ, Rodriguez M, Scheithauer BW, Lennon VA. Paraneoplastic anti-Purkinje and type-1 antineuronal nuclear autoantibodies bind selectively to central, peripheral, and autonomic nervous system cells. Lab Invest 1991;65:412-420. Buckanovich RJ, Posner JB, Darnell RB. Nova, the paraneoplastic Ri antigen, is homologous to an RNA-binding protein and is specifically expressed in the developing motor system. Neuron 1993;11:657-672. Budde-Steffen C, Anderson NE, Rosenblum MK, Graus F, Ford D, Synek BJ, Wray SH, Posner JB. An antineuronal autoantibody in paraneoplastic opsoclonus. Ann Neurol 1988;23: 528-531. Cikes N, Momoi MY, Williams CL, Howard FM Jr, Hoagland HC, Whittingham S, Lennon VA. Striational autoantibodies: quantitative detection by enzyme immunoassay in myasthenia gravis, thymoma, and recipients of D-penicillamine and allogeneic bone marrow. Mayo Clin Proc 1988;63:474--481. Cunningham J, Graus F, Anderson N, Posner JB. Partial characterization of the Purkinje cell antigens in paraneoplastic cerebellar degeneration. Neurology 1986;36:1163--1168. Dalmau J, Furneaux HM, Gralla RJ, Kris MG, Posner JB. Detection of the anti-Hu antibody in the serum of patients with small cell lung c a n c e r - A quantitative western blot analysis. Ann Neurol 1990;27:544-552. Dalmau J, Posner J. Neurologic paraneoplastic antibodies (antiYo; anti-Hu; anti-Ri): the case for a nomenclature based on antibody and antigen specificity. Neurology 1994;44:2241-2246. David C, Solimena S, De Camilli P. Autoimmunity in stiff-man syndrome with breast cancer is targeted to the C-terminal region of human amphiphysin, a protein similar to the yeast proteins, Rvs167 and Rvsl61. FEBS Lett 1994;351;73--79. Denys EH, Dau PC, Hofmann WW, Hussain F. Passive transfer of the myasthenic syndrome. A preliminary report. Acta Neurol Scand 1979;60:$205. Dropcho EJ, King PH. Autoantibodies against the Hel-N1 RNA-binding protein among patients with lung carcinoma: an association with type 1 antineuronal nuclear antibodies. Ann Neurol 1994;36:200-205. Fathallah-Shaykh H, Wolf S, Wong E, Posner JB, Furneaux HM. Cloning of a leucine-zipper protein recognized by the sera of patients with antibody-associated paraneoplastic cerebellar degeneration. Proc Natl Acad Sci USA 1991;88: 3451--3454. Fisher PG, Singer HS. Paraneoplastic opsoclonus. Neurology 1995;45:1421. Folli F, Solimena M, Cofiell R, Austoni M, Tallini G, Fassetta G, Bates D, Carlidge N, Bottazzo GF, Piccolo G, De Camilli P. Autoantibodies to a synaptic protein in three women with the stiff-man syndrome and breast cancer. N Engl J Med 1993;328:546--551.

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Lang B, Newsom-Davis J, Wray D, Vincent A, Murray N. Autoimmune aetiology for myasthenic (Eaton-Lambert) syndrome. Lancet 1981 ;2:224-226. Lennon VA. Reactivity of Lambert-Eaton autoantibodies with voltage-gated calcium channel complex of human cerebral cortex. Ann Neurol 1990;28:281. Lennon VA. Paraneoplastic autoantibodies: the case for a descriptive generic nomenclature. Neurology 1994;44: 2236--2240. Lennon VA. The case for a.descriptive generic nomenclature: clarification of immunostaining criteria for PCA-1, ANNA-1 and ANNA-2 autoantibodies. Neurology 1994;44:2412-2415. Lennon VA. Serological diagnosis of myasthenia gravis and the Lambert-Eaton myasthenic syndrome. In: Lisak RP, ed. Handbook of Myasthenia Gravis and Myasthenic Syndromes. New York: Marcel Dekker, 1994;149-164. Lennon VA, Kryzer TJ, Greismann GE, O'Suilleabhain PE, Windebank AJ, Woppmann A, Miljanich GP, Lambert EH. Calcium-channel antibodies in Lambert-Eaton syndrome and other paraneoplastic syndromes. N Engl J Med 1995;332: 1467--1474. Lennon VA, Lambert EH. Autoantibodies bind solubilized calcium channel-omega-conotoxin complexes from small cell lung carcinoma: a diagnostic aid for Lambert-Eaton myasthenic syndrome. Mayo Clin Proc 1989;64:1498-1504. Lennon VA, Sas DF, Busk MF, Scheithauer B, Malagelada J-R, Camilleri M, Miller LJ. Enteric neuronal autoantibodies in pseudo obstruction with small-cell lung carcinoma. Gastroenterology 1991;100:137-142. Lucchinetti CF, Camilleri M, Lennon VA. Gastrointestinal dysmotility spectrum in patients seropositive for paraneoplastic type 1 antineuronal nuclear autoantibodies. Clin Autonom Res. 1994;4:206. Luchinetti CF, Kimmel DW, Lennon VA. Clinical, oncological and serological profiles of patients seropositive for type 1 antineuronal nuclear antibody (ANNA-l, a.k.a. "anti-Hu"). Neurology 1994;44:A156. Luque FA, Furneaux HM, Ferziger R, Rosenblum MK, Wray SH, Schold SC Jr, Glantz MJ, Jaeckle KA, Biran H, Lesser M, Paulsen WA, River ME, Posner JB. Anti-ri: an antibody associated with paraneoplastic opsoclonus and breast cancer. Ann Neurol 1991 ;29:241--251. Manley G, Sillevis Smitt PA, Dalmau J, Posner JB. Molecular characterization of the HU paraneoplastic encephalitis-related proteins in small cell lung cancer tissues, and analysis of the major immunogenic sites. Neurology 1994;44:A156--A157. Miller VS, Iannaccone ST, Lennon VA. Progressive cerebellar ataxia in a child lacking evident neoplasm with seropositivity for type 1 antineuronal nuclear antibody (ANNA-1 ["antiHu"]) and anti-N-type voltage-gated Ca 2+ channel (VGCC) antibody. Neurology 1994;44:A209--A210. Mygland A, Aarli JA, Matre R, Gilhus NE. Ryanodine receptor antibodies related to severity of thymoma associated myasthenia gravis. J Neurol Neurosurg Psychiatry 1994;57:843-846. O'Suilleabhain PE, Lambert EH, Lennon VA. Anti-voltagegated calcium channel antibodies in women with paraneoplastic cerebellar degeneration (PCD) and seropositivity for type 1 anti-Purkinje cell antibody (PCAbl+). Neurology

1994;44:A157. Roberts A, Perera S, Lang B, Vincent A, Newsom-Davis J. Paraneoplastic myasthenic syndrome IgG inhibits 45Ca 2+ flux in a human small cell carcinoma line. Nature 1985;317:737-739. Rodriguez M, Truh LI, O'Neill BP, Lennon VA. Autoimmune paraneoplastic cerebellar degeneration: ultrastructural localization of antibody-binding sites in Purkinje cells. Neurology 1988;38:1380-1386. Rosenfeld MR, Wong E, Dalmau J, Manley G, Posner JB, Sher E, Furneaux HM. Cloning and characterization of a LambertEaton myasthenic syndrome antigen. Ann Neurol 1993;33: 113--120. Sakai K, Mitchell DJ, Tsukamoto T, Steinman L. Isolation of a complementary DNA clone encoding an autoantigen recognized by an antineuronal cell antibody from a patient with paraneoplastic cerebellar degeneration. Ann Neurol 1990;28:692-698. Sano M, Lennon VA. Enzyme immunoassay of antihuman acetylcholine receptor autoantibodies in patients with myasthenia gravis reveals correlation with striational autoantibodies. Neurology 1993;43:573--578. Sano M, McCormick DJ, Talib S, Griesmann GE, Lennon VA. Identification of three extended antibody-binding segments in recombinant human muscle acetylcholine receptor alpha subunit extracellular domain 1-210. Intl Immunol 1991;3: 983-989. Sher E, Pandiella A, Clement F. Voltage-operated calcium channels in small cell lung carcinoma cell lines: pharmacological, functional, and immunological properties. Cancer Res 1990;50:3892--3896. Smith JL, Finley JC, Lennon VA. Autoantibodies in paraneoplastic cerebellar degeneration bind to cytoplasmic antigens of Purkinje cells in humans, rats and mice and are of multiple immunoglobulin classes. J Neuroimmunol 1988;18: 37--48. Strauss AJ, van der Geld HW, Kemp PG Jr, Exum ED, Goodman HC. Immunological concomitants of myasthenia gravis. Ann N Y Acad Sci 1965;124:744-766. Szabo A, Dalmau J, Manley G, Rosenfeld M, Wong E, Henson J, Posner JB, Furneaux HM. HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to Elav and Sex-lethal. Cell 1991 ;67:325--333. Takamori M, Takahashi M, Yasukawa Y, Iwasa K, Nemoto Y, Suenaga A, Nagataki S, Nakamura T. Antibodies to recombinant synaptotagmin and calcium channel subtypes in LambertEaton myasthenic syndrome. J Neurol Sci 1995;133:95--101. Whitehouse JM, Circulating antibodies in human malignant disease. Br J Cancer 1973 28:S170-S174. Wlkinson PC. Serological findings in carcinomatous neuropathy. Lancet 1964;June 13:1301-- 1303. Williams CL, Hay JE, Huiatt T, Lennon VA. Paraneoplastic IgG striational autoantibodies produced by clonal thymic B cells and in serum of patients with myasthenia gravis and thymoma react with titin. Lab Invest 1992;66:331-336. Williams CL, Lennon VA. Thymic B-lymphocyte clones from patients with myasthenia gravis secrete monoclonal striational autoantibodies reacting with myosin, alpha-actinin or actin. J Exp Med 1986;164:1043-1059.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CALCIUM CHANNEL AUTOANTIBODIES AND AMYOTROPHIC LATERAL SCLEROSIS R. Glenn Smith, M.D., Ph.D. and Stanley H. Appel, M.D.

Department of Neurology, Baylor College of Medicine, Houston, TX 77030, USA

HISTORICAL NOTES

Sporadic amyotrophic lateral sclerosis (sALS) is a fatal disease of unknown etiology in which early loss of spinal cord and brain motoneurons produces progressive limb, facial and respiratory muscle weakness, spasticity and difficulty swallowing (Haverkamp et al., 1995). While different genetic defects have been identified in some of the 5-10% of patients with the familial form of ALS (fALS) (Rosen et al., 1993; Figlewicz et al., 1994), immune system alterations (Appel et al., 1986; Appel and Stefani, 1991) and histochemical identification of activated T cells, microglia and immunoglobulin/complement deposits at neuromuscular junctions, in spinal cords and in motor cortices of patients with sALS (Appel and Stefani, 1991; Smith et al., 1993; Engelhardt et al., 1993) suggest alterations typically observed in autoimmune disease. Electrophysiologic evidence of neuromuscular dysfunction is observed in 50% of patients with ALS (Killian et al., 1994), and passive transfer of immunoglobulin fractions from such patients to Balb/c mice increases both Ca 2+ entry into motoneurons (Engelhardt et al., 1995) and spontaneous miniature end plate potential frequency measured at neuromuscular junctions (Appel et al., 1991). The mechanism for these effects involves antibody-mediated changes in voltage-gated calcium channel (VGCC) function. sALS immunoglobulin fractions increase mean open time in P-type VGCCs from cerebellar Purkinje cells (Llinas et al., 1993) and in N-type VGCCs expressed from mouse brain message in frog oocytes (Mosier et al., 1994). sALS immunoglobulin effects are best

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studied on skeletal muscle L-type VGCCs, where altered channel function is observed following immunoglobulin addition to extracellular facing epitopes (Delbono et al., 1991; Magnelli et al., 1993). Altered ligand-binding kinetics are observed in skeletal muscle L-type VGCCs from patients with sALS but not fALS (Smith et al., 1995).

THE AUTOANTIGEN While many different VGCCs are identified by their distinct electrophysiologic and neurotoxic peptidebinding properties (Scott et al., 1991; Spedding and Paoletti, 1992; Hillyard et al., 1992; Sather et al., 1993; Stea et al., 1994), all VGCCs share structures that may render them immunologically similar. The ionophore-forming ~1 subunits of most VGCCs have been sequenced from human DNA. L-, N-, R- and Qtype ~1 subunits consist of four motifs, each comprised of six membrane-spanning sequences and variably sized cytoplasmic and extracellular domains (Mori et al., 1991; Schneider et al., 1994; Soldatov, 1992; Williams et al., 1994). Sequence homologies of >70% are reported among different L-type VGCCs (Biel et al., 1991; Hui et al., 1991), and there is 70% homology among non-L-type VGCCs (Zhang et al., 1993; Lennon et al., 1995). These ionophores, modulated in their function by other subunits, are important for initiating synaptic neurotransmitter release, for membrane voltage sensing and for regulating processes important to developmental cell death. Antibodies directed against such channels may thus have multiple effects, including actions on cell survival.

AUTOANTIBODIES Methods of Detection As previously mentioned, circulating antibodies in sera from most patients with ALS alter function of several different VGCC types in vitro. However, actual binding of these autoantibodies to VGCCs is detected biochemically in only two systems: 1) in purified L-type VGCC isolated from rabbit skeletal muscle (Smith et al., 1992), and 2) in detergent solubilized P/Q- and N-type VGCC fractions obtained from human brain (Lennon et al., 1995). Using as antigen L-type VGCC complex purified to homogeneity from rabbit skeletal muscle (Schneider et al., 1992), 75% of assayed sALS patient sera contain IgG reacting with L-type VGCC in ELISA; sera from fALS patients are not reactive in this assay (Smith et al., 1992). In immunoblots, IgG fractions isolated from sALS patient sera by ammonium sulfate precipitation and ion exchange chromatography react selectively with the (Z 1 subunit of this purified L-type VGCC complex, without demonstrable binding to other (t~2, [3, 7 or ~i) subunits (Kimura et al., 1994). Purification of larger amounts of isolated ~1 subunit may one day make this assay clinically useful (Nyormoi et al., 1994). Instead, using as antigen peptide toxin prelabelled VGCCs solubilized from autopsied human cortex or cerebellum, immunoprecipitation assays (Lennon and Lambert, 1989) detect anti-P/Qtype VGCC antibodies in 22% of tested ALS sera; 50% of these reactive sera also bind N-type VGCCs (Lennon et al., 1995). The presence in individual patient sera of immunoglobulins that block Ca 2+ flux through L-type VGCCs, augment Ca 2+ flux through non-L-type VGCCs, and bind VGCCs in ELISA and immunoblot, suggest relevant antibody interactions with multiple VGCC types through common epitopes (Mosier et al., 1995). Similar conclusions are suggested from immunoprecipitation assay data (Lennon et al., 1995). The greater sensitivity of functional assays for detecting VGCC antibodies may reflect the presence of antibodies to functionally important channel pore sites with resultant false-negative results by ELISA. Pathogenetic Role Correlation of serum L-type VGCC antibody titers in ELISA with disease progression rate (Smith et al., 1992) provides circumstantial evidence for a patholo-

gic role. Better evidence for IgG-mediated motoneuron injury is obtained in vitro using a motoneuronneuroblastoma cell line which expresses binding sites for L-, N-, and P-type VGCCs. Following differentiation with dibutyryl cAMP and removal of cAMP insensitive cells with aphidocolin, 40--60% of treated VSC 4.1 cells are killed within three days by 95% of tested ALS IgG fractions (Smith et al., 1994). Cell loss is preceded by transient Ca 2+ entry into affected cells (Colom et al., 1994) and is attenuated by extracellular calcium buffering with EGTA, by cell pretreatment with peptide blockers of N- or P-type VGCCs or by ALS immunoglobulin preincubation with purified whole VGCC or VGCC t~1 subunit prior to cell addition (Smith et al., 1994). In vivo, ALS immunoglobulins injected into mice produce ultrastructural changes specific for motoneurons, including increased intracellular calcium and evidence of cell injury (Engelhardt et al., 1995).

CLINICAL UTILITY Disease The observation in ALS of motor excitatory postsynaptic potential decrement following low frequency stimulation is reminiscent of similar findings observed in autoimmune diseases such as myasthenia gravis and Lambert-Eaton myasthenic syndrome (LEMS) (Lambert, 1966; Oh, 1989). While only rarely do patients with nonparaneoplastic myasthenia gravis have circulating VGCC antibodies (Smith et al., 1992; Lennon et al., 1995), more than 90% of patients with LEMS have antibodies directed against P/Q-type VGCCs (Lennon et al., 1995) that may be important to disease pathogenesis (Fukunaga et al., 1983; Fukuoko et al., 1987; Kim and Neher, 1988; Engel, 1991). It is likely that these antibodies are different from those observed in patients with ALS, as <50% of LEMS patient sera produce immediate functional changes in L-type VGCC activity (Delbono, personal communication). Reaction of LEMS patient sera with purified L-type VGCC in ELISA correlates with ~ as well as (z 1 subunit binding on immunoblot (Smith et al., 1992; Kimura et al., 1994), and [3 subunit crossreactive antibodies are detected in 43% of LEMS sera tested against a fetal brain expression library (Rosenfeld et al., 1993). Further, pancreatic ~-cell, anti-L-type VGCC antibodies with novel electrophysiologic properties are

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present in diabetes mellitus; (Juntti-Berggren et al., 1993). Antibodies to P-, Q- and/or N-type VGCCs are described in 54% of patients with paraneoplastic encephalomyelopathies and in 15% of patients with small-cell or ovarian carcinoma without paraneoplastic disease (Lennon et al., 1995). Their significance to these disease processes has yet to be determined. At present, the diagnostic utility of these findings is limited. The inherent difficulty of VGCC purification prevents its simple use in clinical testing and until ALS IgG-binding epitopes on the VGCC are identified, more aggressive diagnostic applications will be delayed. However, the utility of these findings as research tools with which to identify potential mechanisms of cell injury and to create more clinically relevant models of disease pathogenesis has been significant. It is now possible to suggest potential mechanisms of cell injury resulting from such antibodies and to create more defined animal disease models based on in vitro cell models. As newer technologies make it easier to identify both antibodybinding epitopes and antibody functions in vitro, diagnostic and therapeutic interventions based on these insights should become possible.

REFERENCES Appel SH, Engelhardt JI, Garcia J, Stefani E. Immunoglobulins from animal models of motor neuron disease and human amyotrophic lateral sclerosis passively transfer physiologic abnormalities to the neuromuscular junction. Proc Natl Acad Sci USA 1991;88:647--651. Appel SH, Stockton-Appel V, Stewart SS, Kerman RH. Amyotrophic lateral sclerosis. Associated clinical disorders and immunological evaluations. Arch Neurol 1986:43;234-- 238. Appel SH, Stefani E. Amyotrophic lateral sclerosis: etiology and pathogenesis. In: Appel SH, ed. Current Neurology. Vol 11. St Louis: Mosby-Year Book, 1991:287--310. Biel M, Hullin R, Freundner S, Singer D, Dascal N, Flockerzi V, Hofman F. Tissue-specific expression of high-voltageactivated dihydropyridine-sensitive L-type calcium channels. Eur J Biochem 1991;200:81-88. Colom LV, Alexianu ME, Smith RG, Appel SH. Amyotrophic lateral sclerosis immunoglobulins increase intracellular calcium in a motoneuron cell line [Abstract]. Soc Neurosci 1994;20:1649. Delbono O, Garcia J, Appel SH, Stefani E. Calcium current and charge movement of mammalian muscle. Action of amyotrophic lateral sclerosis immunoglobulins. J Physiol 1991;444: 723-742.

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CONCLUSION Antibodies to L-type voltage-gated calcium channels are detectable in a significant number of patients with sALS. These autoantibodies are likely cross-reactive with epitopes common to other VGCCs that may be relevant to disease pathogenesis. While their role in disease is still unclear, their potential importance is suggested by in vitro studies. Clinical use of detection assays for VGCC autoantibodies in ALS is currently limited by the complexity of VGCC purification. Identification of the specific epitope(s) involved should help establish the relevance of the antibodies to ALS pathogenesis and both simplify and increase specificity of autoantibody detection assays.

ACKNOWLEDGEMENTS Supported by grants from the Muscular Dystrophy Association, the NIH, and Cephalon, Inc.

Engel AG. Review of evidence for loss of motor nerve terminal calcium channels in Lambert-Eaton myasthenic syndrome. Ann NY Acad Sci 1991;63:246--258. Engelhardt JI, Tajti J, Appel SH. Lymphocytic infiltrates in the spinal cord in amyotrophic lateral sclerosis. Arch Neurol 1993:50;30-36. Engelhardt JI, Siklos L, Kouves L, Smith RG, Appel SH. Antibodies to calcium channels from ALS patients passively transferred to mice selectively increase intracellular calcium and induce ultrastructural changes in motoneurons. Synapse 1995 ;in press. Figlewicz DA, Krizus A, Martinoli MG, Meininger V, Dib M, Rouleau GA, Julien JP. Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis. Human Mol Genet 1994;3:1757-1761. Fukunaga H, Engel AG, Lang B, Newsom-Davis J, Vincent A. Passive transfer of Lambert-Eaton myasthenic syndrome with IgG from man to mouse depletes the presynaptic membrane active zones. Proc Natl Acad Sci USA 1983;80:7636--7640. Fukuoka T, Engel AG, Lang B, Newsom-Davis J, Prior C, Wray DW. Lambert-Eaton myasthenic syndrome. I. Early morphologic effects of IgG on the presynaptic membrane active zones. Ann Neurol 1987;22:193-199. Haverkamp LJ, Appel V, Appel SH. Natural history of amyotrophic lateral sclerosis in a database population. Validation

of a scoring system and a model for survival prediction. Brain 1995;118(Pt 3):707--719. Hillyard DR, Monje VD, Mintz IM, Bean BP, Nadasdi L, Ramachandran J, Miljanich G, Azimi-Noonooz A, McIntosh JM, Cruz LJ, et al. A new Conus peptide ligand for mammalian presynaptic Ca 2+ channels. Neuron 1992;9:69--77. Hui A, Ellinor PT, Krizanova O, Wang JJ, Diebold RJ, Schwartz A. Molecular cloning of multiple subtypes of a novel rat brain isoform of the alpha 1 subunit of the voltage-dependent calcium channel. Neuron 1991;7:35--44. Juntti-Berggren L, Larsson O, Rorsman P, Ammfilfi C, Bokvist K, W~hlander K, Nicotera P, Dypbukt J, Orrenius S, Hallberg A, Berggren P-O. Increased activity of L-type Ca 2+ channels to serum from patients with type I diabetes. Science 1993 ;261:86-90. Killian J, Wilfong AA, Burnett L, Appel SH, Boland D. Decremental motor responses to repetitive nerve stimulation in ALS. Muscle Nerve 1994;17:747--754. Kim YI, Neher E. IgG from patients with Lambert-Eaton myasthenic syndrome blocks voltage-dependent calcium channels. Science 1988;239:405-408. Kimura F, Smith RG, Delbono O, Nyormoi O, Schneider T, Nastainczyk W, Hofmann F, Stefani E, Appel SH. Amyotrophic lateral sclerosis patient antibodies label Ca 2+ channel alpha 1 subunit. Ann Neurol 1994;35:164-171. Lambert EH. Defects of neuromuscular transmission in syndromes other than myasthenia gravis. Ann NY Acad Sci 1966;135:367-384. Lennon VA, Lambert EH. Autoantibodies bind solubilized calcium channel-omega-conotoxin complexes from small cell lung carcinoma: a diagnostic aid for Lambert-Eaton myasthenic syndrome. Mayo Clin Proc 1989;64:1498-1504. Lennon V, Kryzer TJ, Griesmann GE, O'Suilleabhain PE, Windebonk AJ, Woppman A, Miljanich GP, Lambert EH. Calcium 2+channel antibodies in the Lambert-Eaton syndrome and other paraneoplastic syndromes. N Engl J Med 1995; 332:1467--1474. Llinas R, Sugimori M, Cherksey BD, Smith RG, Delbono O, Stefani E, Appel SH. IgG from amyotrophic lateral sclerosis patients increases current through P-type calcium channels in mammalian cerebellar Purkinje cells and in isolated channel protein in lipid bilayer. Proc Natl Acad Sci USA 1993;90: 11743--11747. Magnelli V, Sawada T, Delbono O, Smith RG, Appel SH, Stefani E. The action of amyotrophic lateral sclerosis immunoglobulins on mammalian single skeletal muscle Ca 2+ channels. J Physiol 1993;461:103--118. Mosier DR, Delbono O, Mayer P, et al. Amyotrophic lateral sclerosis IgG enhances brain calcium currents expressed in Xenopus oocytes [Abstract]. Soc Neurosci 1994;20:620. Mosier DR, Baldelli P, Delbono O, Smith RG, Alexianu ME, Appel SH, Stefani E. Amyotrophic lateral sclerosis immunoglobulins increase Ca 2+ currents in a hybrid motoneuron cell line. Ann Neurol 1995;37:102--109. Nyormoi O, Schneider T, Smith RG. A large scale preparative gel electrophoresis separation of (~1 and (~2 subunits of the voltage-gated Ca 2+ channel from rabbit skeletal muscle. Electrophoresis 1994; 15:1186-- 1190.

Oh SJ. Diverse electrophysiological spectrum of the LambertEaton myasthenic syndrome. Muscle Nerve 1989;12:464-469. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng H-X, Rahmani Z, Krizus A, McKenna-Yasek D, Cayabyab A, Gaston SM, Berger R, Tanzi RE, Halperin JJ, Herzfeldt B, Van den Bergh R, Hung W-Y, Bird T, Deng G, Mulder DW, Smyth C, Laing NG, Sorian E, Pericak-Vance MA, Haines J, Rouleau GA, Gusella JS, Horvitz HR, Brown Jr. RH. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993;362: 59--62. Rosenfeld MR, Wong E, Dalmau J, Manley G, Posner JB, Sher E, Furneaux HM. Cloning and characterization of a LambertEaton myasthenic syndrome antigen. Ann Neurol 1993;33: 113-120. Sather WA, Tanabe T, Zhang JF, Moil Y, Adams ME, Tsien RW. Distinctive biophysical and pharmacological properties of class A (BI) calcium channel alpha 1 subunits. Neuron 1993;11:291-303. Schneider T, Regulla S, Nastaincyk W, Hofman F. In: Longstaff A, Revest P, eds. Methods in Molecular Biology; Vol 13. Protocols in Molecular Neurobiology. Totowa NJ: Humana Press, 1992:273-286. Schneider T, Wei X, Olcese R, Constantin JL, Neely A, Palade P, Perez-Reyes E, Quin N, Zhou J, Crawford GD, et al. Molecular analysis and functional expression of the human type E-neuronal Ca 2+ channel alpha 1 subunit. Receptors Channels 1994;2:255--270. Scott RH, Pearson HA, Dolphin AC. Aspects of vertebrate neuronal voltage-activated calcium currents and their regulation. Prog Neurobiol 1991;36:485-520. Smith RG, Hamilton S, Hofman F, Schneider T, Nastainczyk W, Birnbaumer L, Stefani E, Appel SH. Serum antibodies to L-type calcium channels in patients with amyotrophic lateral sclerosis. N Engl J Med 1992;327:1721-1728. Smith RG, Engelhardt JI, Tajti J, Appel SH. Experimental immune-mediated motor neuron diseases: models for human ALS. Brain Res Bull 1993;30:373--380. Smith RG, Alexianu ME, Crawford G, Nyormoi O, Stefani E, Appel SH. Cytotoxicity of immunoglobulins from amyotrophic lateral sclerosis patients on a hybrid motoneuron cell line. Proc Natl Acad Sci USA 1994;91:3393--3397. Smith RG, Kimura F, Harati Y, McKinley K, Stefani E, Appel SH. Altered muscle calcium channel binding kinetics in autoimmune motoneuron diseases. Muscle Nerve 1995;18: 620--627. Spedding M, Paoletti R. Classification of calciu/n channels and the sites of action of drugs modifying channel function. Pharmacol Rev 1992;44:363-376. Stea A, Tomlinson WJ, Soong TW, Bourinet E, Dubel SJ, Vincent SR, Snutch TP. Localization and functional properties of a rat brain ~1A calcium channel reflect similarities to neuronal Q- and P-type channels. Proc Natl Acad Sci USA 1994 ;91:10576-- 10580. Williams ME, Marubio LM, Deal CR, HansM, Brust Pf, Philipson LH, Miller RJ, Johnson EC, Harpold MM, Ellis

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CENTRIOLE AND CENTROSOME AUTOANTIBODIES Jerome B. Rattner, Ph.D. a and Marvin J. Fritzler, M.D., Ph.D. b

Departments of aMedical Biochemistry and bMedicine, The University of Calgary, Calgary, Alberta T2N 4N1, Canada

HISTORICAL NOTES Centrioles and centrosomes, organelles in the cytoplasm of interphase cells, are located adjacent to the

nuclear membrane in most interphase mammalian cells. In metaphase, they "anchor" and establish opposite poles of the mitotic spindle (Figures 1 and 2). The term "centriole" is derived from the concept

Figure 1. Human autoimmune serum reactive with centrosome antigens (a,e) produces an amorphous staining pattern, b,d counterstained for DNA with DAPI (4,6-diamidino-2-phenylindole). Reactive regions can be seen adjacent to the nucleus at interphase (a arrows,b) and at the poles of the mitotic spindle at cell division (e arrows,d). The antigen detected by this antibody is present throughout the cell cycle. N=nucleus C=chromosomes. 153

that this organelle forms the anatomic "center" of the cell. In 1901, the centrosome was first described as a "polar corpuscle" containing centrioles (Boveri, 1901). The centrosome is the major microtubule organizing center (MTOC).

THE AUTOANTIGEN(S)

Definition/Characteristics A centriole is made up of two orthogonally oriented cylinders. The walls of each cylinder are composed of nine blades, each of which is composed of a triplet array of microtubules (Figure 3). A centriole which gives rise to a cilia or flagella is called a "basal body." The centrosome or MTOC found in animal cells surrounds and includes the centriole. In electron micrographs the~ is composed of amorph-

ous, electron-dense material and vesicles aggregated about the centriole (Figure 3). The centrosome and centrioles replicate during the cell cycle and organize the spindle poles at cell division and the cytoskeleton during interphase. At the completion of cell division, each daughter cell inherits one centrosome containing one pair of centrioles composed of two structurally mature centrioles (Figure 3A). During the G-1 period of the cell cycle, the two centrioles separate by a short distance. These centrioles are now called parent centrioles. Concurrent with the entry of the cell into the S-phase of the cell cycle, each parent centriole gives rise to a daughter centriole. The daughter grows outward from the wall of the parent and is positioned at one end and at right angles to the long axis of the parent (Figure 3B). The cell now contains two pairs of centrioles, each with one parent and one daughter centriole. The daughter is considered immature

Figure 2. Human autoimmune serum reactive with the centriole produces a small defined foci of staining, a,e: reacted with human autoimmune sera, b,d: counterstained for DNA with DAPI. N=nucleus C=chromosomes. The antibodies present in this serum react only with the interphase centrosome (a arrows) and not the centrosomes at the spindle poles (e arrows). 154

G-1 and then with each of the parent centrioles at late G-1. The centrosomal material migrates to the spindle poles with the centrioles at the onset of cell division (Figure 1c,d). In most cell types, the number of centrosome/ centrioles detected by indirect immunofluorescence (IIF) correlates with the stage of the cell cycle. Early G-1 is characterized by one centrosome; in late G-1 as well as in S and G-2, two centrosomes are usually seen in close proximity to the nucleus, and at cell division one centrosome complex is present at each pole. Supernumerary centrosomes in some tissue culture cell lines (Figure 4a,b) often aggregate into large or multiple complexes (Figure 4c) and at cell division can give rise to multipolar spindles. A number of centrosome antigens are identified with human autoimmune sera (Table 1). There has not been a comprehensive study to determine which of these are the most common antigenic targets. Other potential autoantigenic centriole/centrosome components can also be identified with monoclonal or polyclonal animal sera (Table 2).

AUTOANTIBODIES

Methods of Detection

Figure 3. a. Electron micrographs of early G-1 centrosome consisting of two mature centrioles in a right angle orientation. The walls of the centriole are formed by nine blades each composed of three microtubules. The blades are seen in crosssection in the upper centriole (small arrow) and in longitudinal section in the lower centriole (large arrow). The area of the centrosome is denoted by the curved arrows, b. Two pairs of centrioles as seen in an S-phase cell. Each pair is composed of a fully formed parent (P) seen here in oblique section and an immature daughter (D). Note the abundant microtubules, filaments, vesicles and amorphous material in the surrounding centrosome region.

because it does not attain its full length until the completion of the cell cycle. At cell division one centriole pair is found at each spindle pole. The centrosomal material is partitioned with the centrioles being associated first with the single centriole pair at

The preservation of centrosomes/centrioles in tissues and tissue culture cells can be accomplished by using standard fixatives such as methanol, acetone and paraformaldehyde. Techniques to obtain relatively pure preparations of centrosomes are available (Mitchison and Kirschner, 1994; Borens et al., 1987; Komesli et al., 1989) but are not widely applied to the identification of human autoantigens (Figure 4d). As identified on conventional HEp-2 substrates, the typical staining pattern of antibodies to centrosomes (anticentrosome) consists of one or two bright dots adjacent to the nuclear membrane in interphase cells and one bright dot at each of the poles of the mitotic spindle in metaphase cells (Figure 1). The presence of some anticentrosome can be confirmed by immunoblotting using purified or recombinant enolase as the target protein (Rattner et al., 1991), but there are no systematic studies of the sensitivity and specificity of the antienolase activity for the centrosome antigenic system. The number of different proteins in the centriole and centrosome (Table 1) makes it likely that several centriole/centrosome proteins are targets of the human autoantibody response.

155

When the presence of anticentrosome is suspected, antibodies to antigens of the mitotic spindle apparatus including antibodies to the nuclear mitotic spindle apparatus (NuMA) should be excluded, so as not to be confused with anticentrosome. Based on IIF staining patterns, centrosome antibodies produce a large spherical dot (Figure 1); whereas, the reactivity of centriole antibodies produce smaller, discrete dots (Figure 2). By comparison, N u M A antibodies stain the centrosome region and also react with antigens that extend out into the mitotic spindle apparatus. Because not all centrosome/centriole antigens are represented throughout the cell cycle, some human anticentrosome/centriole stain only interphase or mitotic centrosomes (compare Figures 1 and 2).

CLINICAL UTILITY Disease Associations

To date, only a few studies describe the presence of anticentrosome/centriole in human sera (Table 1). In the largest study of sera from 264 patients with the scleroderma spectrum of diseases (SSD) (Sato et al., 1994), anticentrosome/centriole were found in one (0.4%) secondary Raynaud's phenomenon (RP) patient but not in patients who met classification criteria for mixed connective tissue disease or primary RP. In another study, anticentriole were reported in 4/80 (5%) patients with SSD, including one patient with RP and telangiectasia, one with CREST syndrome,

Figure 4. Some tissue culture cells have supernumerary centrioles, a: Metaphase cells with three centrosomes at one pole (arrows) as detected with human autoimmune serum, b: The same cell counterstained with DAPI illustrating the central chromosomal mass. e: In interphase, cells with supernumerary centrosomes, anticentrosome antibodies often reveal a large irregular area of reactivity (arrows). d: It is possible to isolate centrosomes from cells and immobilize them on glass slides for reaction with human anticentrosome serum (d arrows denote centrosomes). 156

Table 1. Diseases Associated with Centrosome and Centriole Autoantibodies Disease

Antigenic Target

Reference(s)

Raynaud's phenomenon

centriole, enolase

Sato et al., 1994; Rattner et al., 1991

Raynaud's phenomenon, telangiectasia

centriole

Tuffanelli et al., 1983

Raynaud's phenomenon, arthralgia

centriole

Moroi et al., 1983

Hyperthyroidism, Raynaud's phenomenon, telangiectasia**

enolase

Rattner et al., 1991

Systemic sclerosis

centriole

Tuffanelli et al., 1983; Moroi et al., 1983; Osborn et al., 1986

Systemic sclerosis

centrosome

Calarco-Gillam et al., 1983

Diffuse scleroderma

centriole

Tuffanelli et al., 1983

CREST syndrome

centriole

Tuffanelli et al., 1983

CREST syndrome

centrosome 110--115 kd protein

Sager et al., 1989

Arthralgia

centrosome 190 kd protein

Balczon and West, 1991

Mycoplasma infection, cerebellar dysfunction centriole

Cimolai et al., 1994

Antibody to centrosome Patient initially reported to have hyperthyroidism and myalgia. Over five years of follow-up, the patient developed Raynaud's phenomenon and telangiectasia. Abbreviation: CREST: Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia

and two with diffuse scleroderma (Tuffanelli et al., 1983). Another report (Moroi et al., 1983) identified autoantibodies in 2/100 SSD patients; one patient had systemic sclerosis (SSc) and the other had RP and arthralgia. Of two other patients with SSD and anticentriole antibodies, one had SSc (Osborn et al., 1986) and one had RP (Rattner et al., 1991). Therefore, most patients with anticentrosome/centriole described to date have had RP and features of systemic sclerosis. The exceptions are two patients with Mycoplasma pneumoniae infections complicated by cerebellar dysfunction (Cimolai et al., 1994) and one patient with hyperthyroidism (Rattner et al., 1991) who later developed RP and telangiectasia over a five-year follow-up (unpublished observation). It is well known that the clinical features of systemic sclerosis may take 15--20 years to develop and long-term follow-up of patients is often required before a definitive diagnosis is known. On the other hand, the clinical associations of anticentrosome/centriole should be viewed with caution, because so few patients have been reported. Although anticentriole are reported in normal rabbits (Turkeson et al., 1982), only two patients (0.08%) with centriole autoantibodies were

identified in a study of 2,500 female Red Cross blood donors (Fritzler et al., 1985). Therefore, "naturally occurring" centriole autoantibodies in man are rare. Rabbit antibodies to enolase produce a centriole pattern of staining as do human centriole antibodies affinity purified with enolase (Rattner et al., 1991). Furthermore, sera from patients with anticentriole reacted with purified enolase in immunoblots. Whether the anticentriole-positive sera of patients with M. pneumoniae infection complicated by cerebellar dysfunction (Cimolai et al., 1994) react with neuronal enolase is unknown. There is no published evidence that centrosome/ centriole antibodies fluctuate in concert with disease activity or that therapeutic modalities affect their concentrations or epitope specificity. Centrosome/centriole antibodies can antedate clinical features of systemic sclerosis by several years (Rattner et al., 1991); studies of antibody affinity are not documented. There is no evidence that these antibodies have an adverse effect on pregnancy outcome or that the transplacental transfer of the antibodies have a deleterious effect on the fetus.

157

Table 2. Proteins Located at the Centrosome Protein/Molecular Mass

Species Distribution

Characteristics

Suggested Functions

Protein homologue of MAP 1-B

Mammalian cells

Located in the region around centrioles

Microtubule nucleating activity

CSPc~

Human melanoma cell line

Identified with mAb to residue 21--31 of hTGF-~

cAMP receptor

Cow, rat, vertebrates

Centrosome, basal bodies, ER, Golgi complex, same behavior and location as MAP2 in mouse neurons

MT organizer

14/17 kd Ag

Mammals

Centrin/calt gractin (20 kd), 165 kd protein (62/64 kd doublet)

Tetraselmis, Chlamydomonas, Polytomella, mammals, K37

Contractile protein implicated in MTOC localization; involved in MT nucleation Modification of centrosome for spindle formation

cells

50% homologous to Saccharomyces CDC31; 45--85% homologous to calmodulin; common epitope with 165 kd protein in PtK2 cells; Ca2+-sensitive; increased expression at mitosis also in cytoplasm

p34 cdc2 (34 kd)

Saccharomyces, Schizosaccharomyces, human

Associated with centrosome and kinetochore only during cell division

Centrosomin A (35 kd)

PtK cells

Identified with Ab to centrosomal fraction of PtK cells

AKAP 350 (35 kd)

K37 cells

Binds cAMP-dependent protein kinase type II

Centractin (42.6 kd)

MDCK cells, human

50--70% homologous to actin; human and MDCK centractin identical; also in cytoplasm

43 kd protein (43 kd and 56 kd)

HeLa cells, mouse, hamster, sea urchin, insects

Identified with MPM-13 Ab; highly conserved throughout evolution

Tektin A, B and C (46--57 kd)

Sea urchin, hamster, human, pig

Associated with centrioles and basal bodies throughout the cell cycle.

Centriole stability; MT assembly

y-Tubulin (50 kd)

Aspergillus, human, mouse, Schizosaccharomyces, Drosophila, Xenopus

A universal component of centrosome

Cell-cycle-dependent MT nucleation and nuclear division

50/51 kd protein

Sea urchin, mammals

Homologous to EFI-~, binds GTP and present only during cell division

MT nucleation

Nuf-2 (53 kd yeast; 73 kd mammals)

Saccharomyces, mammals

Related to myosin

Centrosome separation

Human (A, B 1), chicken (B2),

Present during cell division

Activation/deactivation of cdc2 kinase

Cyclins A, B 1 and B2 (60, 54 and 51 kd)

Drosophila

Part of dynactin complex; links centrosome to actin or other proteins

(continued)

Table 2. (continrted) ProteidMolecular Mass

Species Distribution

Characteristics

Suggested Functions

DMAP 60 (60 kd), DMAP 90 (90 kd)

Dmsophila

Increased staining at mitosis

Both form a centrosomal protein complex

Bx 63 Ag (661185 kd)

Drosophila

A t the centrosome only during mitosis

hsp72, hsp73 (72 and 73 kd)

HeLa cells

Only centrosomal during mitosis

CTR 26 1 1 Ag (74/170 kd)

MDCK

At centrosome during mitosis and minus end of interphase Mts; identified with Ab to centrosomes of K37 cell line

SPB components (80/90/110 kd)

Saccharomyces

Located at SPB (80 kd close to minus ends of Mts)

NSPI (85 kd)

Saccharornycrs, M D C K cells

Homologous to intermediate filaments and Ca2'-binding proteins; also located at nuclear membrane

Cell and nuclear division in Saccharornyces

Purine nucleoside phosphorplase (90 kd)

Human. mammals, birds. protozoa

Centrioles, basal bodies

Centriole duplication and MT assembly

Cytocentrin (102 kd)

Mammals

Phosphorylated and display a cell-cycle-dependent distribution

Spindle organization

Mitotin (125 kd)

Kangaroo rat. human

Mitosis; centrosomes. spindle: late mitosis: midbody interphase; nucleoplasm

CTR532 Ag (170 kd)

Paran~rciurn,mammals

Complex distribution associated with centrosome during ~nterphase.Identified with Ab to centrosome K37 cell line; associated with non-MT component of spindle

Human. cow, plants, mammals

Also located at kinetochore and metaphase plate

CTR56 (200 kd)

Human cells

Related to myosin I1

C H 0 3 Ag (225 kd)

PtKl cells. sea urchin, mammals, Dich~nstelliunr

Identified with antiphosphoprotein Ab CH03, with MPM-i and MPM-2 in sea urchin present at interphase centrosome

MT nucleation and transport of centrosomal proteins to spindle poles

CONCLUSION

centriole antibodies can antedate the clinical diagnosis of scleroderma by several years. See also MITOTIC

Centriole and c e n t r o s o m e antibodies are rare. They occur in approximately 0.1% of female blood donors and in 0.4--5% of SSD sera. Like anticentromere,

SPINDLE APPARATUS AUTOANTIBODIES and OTHER AUTOANTIBODIES TO NUCLEAR ANTIGENS.

REFERENCES

Mitchison T, Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature 1994;312:503--506. Moroi Y, Murata I, Takeuchi A. Human centriole autoantibody in patients with scleroderma and Raynaud's phenomenon. Clin Immunol Immunopathol 1983;29:381-390. Osborn TG, Ryerse JS, Bauer NE, Urhahn JM. Anticentriole antibody in a patient with progressive systemic sclerosis. Arthritis Rheum 1986:29:142-- 146. Rattner JB, Martin L, Waisman DM, Johnstone SA, Fritzler MJ. Autoantibodies to the centrosome (centriole) react with determinants present in the glycolytic enzyme enolase. J Immunol 1991 ;146:2341-2344. Sager PR, Rothfield NL, Oliver JM, Berlin RD. A novel mitotic spindle pole component that originates from the cytoplasm during prophase. J Cell Biol 1989;103:1--77. Sato S, Fujimoto M, Ihn H, Takehara K. Antibodies to centromere and centriole in scleroderma spectrum disorders. Dermatology 1994; 189:23--26. Tuffanelli DL, McKeon F, Kleinsmith DM, Burnham TK, Kirschner M. Anticentromere and anticentriole antibodies in the scleroderma spectrum. Arch Dermatol 1983;119:560-566. Turksen K, Aubin JE, Kalnins VI. Identification of a centrioleassociated protein by antibodies present in normal rabbit sera. Nature 1982;298:763-765.

Balczon R, West K. The identification of mammalian centrosomal antigens using human autoimmune anticentrosome antisera. Cell Motil Cytoskel 1991;20:121--135. Borens M, Paintrand M, Berges J, Marty MC, Karsenti E. Structure and chemical characterization of isolated centrosomes. Cell Motil Cytoskel 1987;8:238-249. Boveri T. fQber die natur der centrosomen. Jena Z Med Naturw 1901;28:1-220. Calarco-Gillam PD, Siebert MC, Hubble R, Mitchison T, Kirschner M. Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolarmaterial. Cell 1983;35:621--629. Cimolai N, Mah D, Roland E. Anticentriolar autoantibodies in children with central nervous system manifestations of Mycoplasma Pneumoniae infection. J Neurol Neurosurg Psychiatry 1994;57:638--639. Fritzler MJ, Pauls JD, Kinsella TD, Bowen TJ. Antinuclear, anticytoplasmic and anti-SjOgren's syndrome antigen-A (SSA/Ro) antibodies in female blood donors. Clin Immunol Immunopathol 1985;36:120-128. Komesli S, Tournier F, Paintrand M, Margolis RL, Job D, Borens M. Mass isolation of calf thymus centrosomes: identification of a specific conformation. J Cell Biol 1989; 109:2869--2878.

160

<01996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CENTROMERE AUTOANTIBODIES Neil John McHugh, M.D.

Department of Rheumatology, Royal National Hospital for Rheumatic Diseases, Upper Borough Walls, Bath BA1 1RF, UK

HISTORICAL NOTES Autoantibodies in human sera that stain the centromere region of cells were first described in 1980 (Moroi et al., 1980). The detection of anticentromere antibodies (ACA) by indirect immunofluorescence became easier with the use of rapidly dividing tissue culture cell lines such as HEp-2 cells (Figure 1). Several early studies confirmed that ACA were most often found in sera from patients with a variant of scleroderma (systemic sclerosis) characterized by limited skin involvement which was termed the "CREST" syndrome. Immunoblotting of chromosomal

extracts revealed the three major polypeptide antigens recognized by ACA, which were designated CENP A, CENP B and CENP C (Earnshaw et al., 1986) (Figure 2). The cDNA clones were isolated for CENP-B (Earnshaw et al., 1987b) and CENP-C (Saitoh et al., 1992), and CENP-A was purified to homogeneity from bovine sperm (Palmer et al., 1991). CENP-B, the first sequence-specific DNA-binding protein to be characterized within centromeric heterochromatin, bound a consensus sequence within (x-satellite DNA termed the "CENP-B box" (Muro et al., 1992). Heterologous and monoclonal antibodies to specific CENP antigens are useful probes for understanding

Figure 1. ACA stain a pattern of discrete dots in interphase cells that line up on metaphase plate in dividing cells. 161

ACA recognized components of the kinetochore, it now seems that centromere antigens are mostly centromeric and located apart from the kinetochore (Pluta et al., 1990). Therefore, the antibodies are best termed "anticentromere" rather than "antikinetochore" antibodies. The three main centromere antigens are CENP-A (19 kd), CENP-B (80 kd) and CENP-C (140 kd) (Earnshaw et al., 1986). Antibodies to the three CENPs (CENTromere-associated proteins) usually occur together in the same serum, but individually are termed anti-CENP-A, anti-CENP-B or anti-CENP-C. Three further CENP proteins (CENP-D, CENP-E and CENP-F) are recognized less often by autoimmune sera (Rattner et al., 1993).

Native versus Recombinant Performance

THE AUTOANTIGEN(S)

Centromere antigens are insoluble, tightly bound nuclear matrix proteins which are not easily purified for immunodiagnostic purposes. However, recombinant proteins derived from cloned CENP-B are used with some success and at least one epitope at the carboxyl-terminal end of CENP-B is recognized by virtually all sera with ACA. A carboxyl-terminal 147 amino acid fragment fused to [3-galactosidase in an ELISA for detecting anti-CENP-B antibodies in connective tissue disease populations has a sensitivity of 94--98% and a specificity of 94--95% compared to ACA detected by indirect immunofluorescence (Vazquez-Abad et al., 1994; Whyte et al., 1995). The false-positive results are most likely due to bacterial contaminants because such sera recognize lower molecular weight bands but not CENP-B on immunoblotting the CENP-B preparation (Whyte et al., 1995). A smaller 60 kd carboxyl-terminal fragment of CENPB fused to glutathionine S transferase was 100% sensitive in detecting 81 sera with ACA (Verheijen et al., 1992). Eukaryotic systems used to express CENPB (Stahnke et al., 1994) have the potential advantages of retaining epitopes dependent on posttranslational modifications without bacterial contamination products.

Definition/Standard Nomenclature

Characteristics

The centromere is the primary constriction site of eukaryotic chromosomes where sister chromatids appear most tightly paired. A trilaminar disk structure at the surface of the centromere, called the kinetochore, is the site of microtubule attachment for dividing cells. Although early studies suggested that

The centromere is responsible for the coordinated segregation of chromosomes to dividing cells in mitosis and meiosis. Centromere and kinetochore structure and the possible role of centromere-associated proteins in the cell cycle are the subject of recent reviews (Pluta et al., 1990; Earnshaw and Tomkiel,

Figure 2. K562 cell polypeptides from a nuclear-enriched extract recognized by sera with ACA after separation on a 12.5% SDS-polyacrylamide gel and transfer to nitrocellulose. CENP-A, CENP-B and CENP-C are recognized by two sera with ACA in lanes 1 and 2. CENP-C is recognized by the same sera in lanes 3 and 4 where an anti-IgM conjugate has been substituted for an anti-IgG conjugate used in lanes 1 and 2.

the events controlling cell mitosis (Earnshaw and Tomkiel, 1992). CENP-B fusion proteins are now used in solid-phase assays for the detection of ACA.

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1992). Centromere antigens found in several mammalian cells appear to be highly conserved (e.g., Drosophila and Leishmania tropica) (McNeilage et al., 1986), as expected from their key regulatory roles in eukaryotic cells. In addition to the three major centromere proteins (CENPs A, B and C) recognized by most sera with ACA (Earnshaw et al., 1986), a fourth protein, CENP-D, recognized by a minority of sera with ACA, is likely to be the human homologue of a protein that regulates chromosome condensation (RCC1) and entry into mitosis (Bischoff et al., 1990). Centromere proteins A--D are localized to the centromere region throughout the cell cycle. Two higher molecular weight proteins, CENP-E and CENP-F, transiently associate with the outer surface of the kinetochore in a cell cycle-dependent manner (Rattner et al., 1993). Some features of the major centromere antigens are well defined (Table 1). Unlike CENP-B and CENP-C, CENP-A has not been cloned but was purified from bull sperm and appears to be a centromerespecific histone (H3) variant (Palmer et al., 1991). CENP-B is located within the heterochromatin underneath the kinetochore (Pluta et al., 1990). Centromeric heterochromatin is composed of o~-satellite DNA (highly polymorphic repetitive sequences of DNA). CENP-B binds to selected o~-satellite sequences that contain a 17 base pair sequence termed the "CENP-B box" (Muro et al., 1992). CENP-B probably forms a dimer that binds DNA and has an important role in regulating higher order chromatin structure within the centromere (Yoda et al., 1992). CENP-C is less well studied but is a component of the inner kinetochore plate, and appears necessary for stabilizing the kinetochore and microtubule attachments prior to onset of anaphase (Tomkiel et al., 1994).

Methods of Purification CENP-A shows sequence similarity to histone H3 (Palmer et al., 1991). CENP-B and CENP-C have not been purified to homogeneity. However, CENP-B was purified 15,000-fold from a HeLa cell nuclear extract using an oligonucleotide affinity column, based on the interaction between CENP-B and its recognition site (the CENP-B box) on alphoid DNA (Muro et al., 1992).

Sequence Information CENP-B contains a DNA-binding domain within 158 residues of the amino terminus that localizes CENP-B to the centromere, and a dimerizing domain within a 20 kd fragment that can be cleaved from the carboxyl terminus (Pluta et al., 1992; Yoda et al., 1992). There are three possible hinge regions, two long acidic domains containing glutamic and aspartic residues and a proline-rich domain sensitive to proteases. CENP-B might function as a "solenoid breaker" and contribute in some unknown manner to the tertiary structure of heterochromatin. The gene encoding for CENP-B has been mapped to chromosome 20p13 by fluorescence in situ hybridization (Seki et al., 1994). CENP-C encodes for a hydrophilic and highly basic protein with a predicted molecular size of 107 kd, which may be polymorphic and has two possible sites for phosphorylation (Saitoh et al., 1992).

AUTOANTIBODIES Pathogenetic Role There is no direct evidence that ACA have a patho-

Table 1. The Major Centromere Autoantigens CENP-A

CENP-B

CENP-C

Cell location

Nucleosomal

Centromeric heterochromatin

Inner kinetochore plate

Method of isolation

Biochemically purified

Genomic and cDNA clones

cDNA clone

Predicted function

Modified core histone (H3)

Higher order chromosomal folding*

Stabilization of kinetochore

Multi-domain; two highly acidic regions; forms dimer

Polymorphism; hydrophilic, basic sites for phosphorylation

IgG (mainly IgG 1)

Mainly IgM

Features of clone and predicted protein sequence Class of Ig autoantibody

IgG

*Binds CENP-B box in s-satellite DNA.

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genetic role. Unlike other autoantibodies associated with scleroderma that are detected in murine models, there are no instructive animal models for ACA. However, the profound influence of the antibodies on mitosis when injected into eukaryotic cells may be more relevant to understanding the role of CENP antigens in cell cycling. Microinjection of ACA into cells disrupts mitosis, depending on the stage of the cell cycle in which the antibody is injected (Earnshaw and Tomkiel, 1992). Two critical stages were identified at two and three hours prior to metaphase before which time injection of ACA disrupted kinetochore assembly and progression through mitosis and meiosis. Injection of antiCENP-C cells during interphase causes a transient metaphase arrest (Tomkiel et al., 1994).

Genetics Chromosomal Abnormalities and ACA. An increase in chromosomal abnormalities has long been observed in patients with SSc. Patients with ACA and antiCENP-C antibodies, in particular, have an increased frequency of aneuploidy (Jabs et al., 1993) that might be the result of nondisjunction secondary to centromeric dysfunction. MHC Class II Associations with ACA. HLA associations with SSc are stronger when autoantibodydefined subsets of disease are analyzed separately, e.g., the associations between ACA and the broad phenotypes defined by HLA-DR1,-DR4 and-DRw8 (Genth et al., 1990). Similar HLA-DRB 1 associations as well as an association with HLA-DR5 are found in groups of patients from different ethnic backgrounds (Reveille et al., 1992b), including stronger associations with HLA DQw7 and DQw5. Analysis of genotype sequences revealed a polar glycine or tyrosine residue at position 26 of the DQB1 first domain which was always present in patients with ACA instead of a hydrophobic leucine residue. A recent family study suggests that a non-HLA-DQB 1:26:L allele is necessary but not sufficient for the generation of ACA (McHugh et al., 1994). Family Studies. A recent family study of systemic sclerosis (SSc) included 13 patients with ACA and underlined the close association of ACA with expression of disease (McHugh et al., 1994). Unaffected relatives, including one nonidentical twin sister of a proband with ACA, tested negative for ACA. Two

164

sisters in one family had ACA and SSc. One set of identical twins also had SSc and ACA. All patients with ACA recognized CENP-A, CENP-B and CENP-C.

Factors in Pathogenicity and Etiology lsotypes and Subclasses of ACA. ACA, usually present in high titer, are mostly of the IgG subclass, skewed toward the IgG1 isotype (Eisenberg et al., 1989), suggesting an antigen-driven process. However, most techniques favor detection of IgG antibodies, especially when a protein-A is used as a coupling reagent in immunoblotting assays. Whereas, CENP-A and CENP-B are recognized by IgG antibodies, CENP-C is predominately recognized by IgM antibodies (McHugh et al., 1988) (Figure 2). Curiously, IgM anti-CENP antibodies persist over time, perhaps suggesting continuous exposure of antigen in a T-cell independent manner. The profile of immunoglobulin reactivity with the other CENP antigens is similar. CENP Epitopes. There are at least five epitopes on CENP-B, one of which is shared with CENP-A and one with CENP-C (Earnshaw et al., 1987a). At least two epitopes on CENP-B and usually all CENPs are recognized by most sera with ACA, indicating a polyclonal immune response. A major epitope at the carboxyl terminus of CENP-B is recognized by virtually all sera with ACA, including the 60 Cterminal amino acid segment that does not contain stretches of highly acidic residues (Verheijen et al., 1992). As the CENPs show no primary sequence similarity with one another, the epitopes shared among the CENPs are probably constituted by posttranslational modifications or may be formed by conformational rather than linear determinants. Methods of Detection The usual method for detection of ACA is indirect immunofluorescence using a tissue culture cell line. Commercial slides with fixed HEp-2 cells should include at least a few cells in the later stages of mitosis. ACA characteristically give a discrete speckled appearance on cells in interphase and recognize paired dots of sister chromatids that assemble at the metaphase plate (Figure 1). Immunoblotting methods are necessary for characterization of reactivity to the individual CENP antigens (Figure 2). Crude nuclear extracts of tissue cell lines are adequate and may have advantages for

ACA in SSc. Early studies identified ACA as a marker for a variant of SSc termed the "CREST" (Calcinosis, Raynaud's phenomenon, Esophageal involvement, Sclerodactyly, Telangiectasia) syndrome. However, subgroups of SSc are best distinguished on the basis of the distribution of skin involvement. ACA are associated with a limited cutaneous form of SSc (lc SSc) in which skin involvement is limited to the skin distal to forearms, the lower legs and sometimes the face and neck. ACA are very rarely found (if at all) in a diffuse cutaneous form of SSc (dc SSc) where there is involvement of the trunk or areas proximal to the sites noted above. The major relevance of the division is that the prognosis is significantly better in lc SSc. Virtually all of these patients have Raynaud's phenomenon; other clinical features can include digital ischemic lesions, calcinosis and the absence of renal involvement. Pulmonary hypertension, a late clinical consequence, has the same frequency whether patients with lc SSc are positive or negative for ACA (Steen et al., 1988).

enabling detection of additional autoantibodies in occasional sera that recognize other intracellular autoantigens. Chromosomal preparations can reduce background autoreactivity. Immunoblotting methods using protein A will not be as sensitive for detecting anti-CENP-C antibodies as enzymatic detection methods that include anti-IgM conjugates. ELISAs, using fusion proteins of CENP-B, are very useful for the detection of ACA, because almost all sera with ACA recognize a carboxyl terminal fragment (Vazquez-Abad et al., 1994; Whyte et al., 1995; Verheijen et al., 1992). False-positive results can occur because of antibodies to bacterial contaminants. ELISA permits more precise quantitation of anti-CENP-B, but whether this will be of practical value is doubtful. The convenience of an ELISA allows more rapid screening for ACA, and positive results can be confirmed by other techniques.

CLINICAL UTILITY

ACA in Raynaud's Phenomenon. Raynaud's phenomenon is very often the first symptom of SSc and can precede other manifestations by several years. Therefore, the detection of ACA in such patients may be of prognostic significance. In one study of 77 patients with Raynaud's phenomenon, the presence of either antitopoisomerase I antibodies or ACA was associated with a 163-fold increased chance of developing a connective tissue disease (Weiner et al., 1991). Community-based studies of the significance of ACA in Raynaud's phenomenon are needed.

Disease Association The most common setting for finding ACA is in patients with a limited cutaneous form of Ssc (Table 2). Individuals positive for ACA in routine laboratory screening have more diverse associations, and ACA are rarely present in "normal" individuals (Lee et al., 1993). ACA are occasionally present in a number of other autoimmune rheumatic conditions apart from SSc, including rheumatoid arthritis, systemic lupus erythematosus and primary Sjrgren's syndrome.

Table 2. Clinical Features of Patients with Anticentromere Antibodies from Selected Large Series Number (male: female)

Age (median)

Disease duration

CREST Partial Raynaud's Limited SSc (%) (%) CREST (%) (%)

Diffuse SSc (%)

Other

Genth et al., 1990

63 (6:57)

51

8.0

4.8

Vlachoyiannopoulus et al., 1993

41 (1:40)

50.6

12.8

Earnshaw et al., 1986 39 (1:38)

50.8 (mean)

2 months to 32 yrs.

Steen et al., 1988"** 86 (7:79)

55.2 (estimated)

9.6 (mean)

30.7

31.8

38.4

79.7

57.9

1.6

56.9*

90

64

0

36**

100 97

99

*36.5% had rheumatoid arthritis and 20.4% had SjOgren's. **5% had rheumatoid arthritis and 17% had Sjrgren's. ***Patients studied on basis of diagnosis of systemic sclerosis.

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ACA in Primary Biliary Cirrhosis. SSc and primary biliary cirrhosis often overlap and may represent different parts of the spectrum of autoimmune connective tissue disease. ACA are found in about 10--20% of sera from patients with primary biliary cirrhosis. The presence of ACA in patients with primary biliary cirrhosis identifies those with features of SSc such as Raynaud's phenomenon and sclerodactyly (McHugh et al., 1990). The profile of autoreactivity to the CENP-antigens is the same as in lc SSc.

Gender and Race Associations with A CA. The female predominance in patients with ACA is remarkable, even more so than in SSc in general (Table 2). In studies not cited here, all individuals with ACA were female: in others, males accounted for less than 5% of patients with ACA. Evidence that ACA are not as frequent in American Blacks (Reveille et al., 1992a) or Thai patients (McNeilage et al., 1989) as in North American or Australian Caucasians is not explained by differences in the HLA background, but such studies may suffer from referral bias.

CONCLUSION ACA recognize a family of proteins that remain in the centromere region of eukaryotic cells throughout the cell cycle. The three main centromere proteins (CENP-A, CENP-B and CENP-C) localize to separate parts of the centromeric heterochromatin and adjacent kinetochore and together form targets for a polyclonal autoantibody response. Antibodies to the CENPs are proving useful probes for understanding the mechanisms that regulate higher order chromosome structure and cell division. ACA identify a group of patients with a limited cutaneous form of systemic sclerosis who are predominantly female and who may have a better long-term prognosis than patients without these autoantibodies. Certain MHC class II associations with ACA may bear on the genetic basis for autoimmunity and/or disease susceptibility. See also TOPOISOMERASE-I (SCL-70). AUTOANTIBODIES.

REFERENCES Bischoff FR, Maier G, Tilz G, Ponstingi H. A 47-kDa human nuclear protein recognized by antikinetochore auto immune sera is homologous with the protein encoded by RCC1, a gene implicated in onset of chromosome condensation. Proc Natl Acad Sci USA 1990;87:8617--8621. Earnshaw W, Bordwell B, Marino C, Rothfield N. Three human chromosomal auto antigens are recognized by sera from patients with anticentromere antibodies. J Clin Invest 1986; 77:426--430. Earnshaw WC, Machlin PS, Bordwell BL, Rothfield NF, Cleveland DW. Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B. Immunology 1987a;84: 4979--4983. Earnshaw WC, Sullivan KF, Machlin PS, Cooke CA, Kaiser DA, Pollard TD, Rothfield NF, Cleveland DW. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. J Cell Biol 1987b;104:817-829. Earnshaw WC, Tomkiel JE. Centromere and kinetochore structure. Curr Opin Cell Biol 1992;4:86--93. Eisenberg RA, Earnshaw WC, Bordwell BJ, Craven SY, Cheek R, Rothfield NF. Isotype analysis of the anti-CENP-B anticentromere autoantibody: evidence for restricted clonality. Arthritis Rheum 1989;32:1315--1318. Genth E, Mierau R, Genetzky P, von Muhlen CA, Kaufmann S, von Wilmowsky H, Meurer T, Krieg T, Pollman H-J, Hartl PW. Immunogenetic associations of scleroderma-related antinuclear antibodies. Arthritis Rheum 1990;33:657-665. Jabs EW, Tuck-Muller CM, Anhalt GJ, Earnshaw W, Wise RA,

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Wigley F. Cytogenetic survey in systemic sclerosis: Correlation of aneuploidy with the presence of anticentromere antibodies. Cytogenet Cell Genet 1993;63:169-175. Lee S-L, Tsay GJ, Tsai R-T. Anticentromere antibodies in subjects with no apparent connective tissue disease. Ann Rheum Dis 1993;52:586--589. McHugh NJ, James IE, Maddison PJ. Differential isotype recognition of two centromere associated polypeptides by immunoblotting in connective tissue disease. Clin Exp Immunol 1988;72:457-464. McHugh NJ, James IE, Kairburn K, Maddison PJ. Autoantibodies to mitochondrial and centromere antigens in primary biliary cirrhosis and systemic sclerosis. Clin Exp Immunol 1990;81:244-249. McHugh NJ, Whyte J, Artlett C, Briggs DC, Stephens CO, Olsen NJ, Gusseva NG, Maddison PJ, Black CM, Welsh K. Anticentromere antibodies in systemic sclerosis patients and their relatives: a serological and HLA study. Clin Exp Immunol 1994;96:267--274. McNeilage LJ, Whittingham S, McHugh N, Barnett AJ. A highly conserved 72,000 dalton centromeric antigen reactive with auto antibodies from patients with progressive systemic sclerosis. J Immunol 1986;137:2541--2547. McNeilage LJ, Youngchaiyud U, Whittingham S. Racial differences in antinuclear antibody patterns and clinical manifestations of scleroderma. Arthritis Rheum 1989;32:54-60. Moroi Y, Peebles C, Fritzler MJ, Steigerwald J, Tan EM.

Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci USA 1980;77:1627-1631. Muro Y, Masumoto H, Yoda K, Nozaki N, Ohashi M, Okazaki T. Centromere protein B assembles human centromeric asatellite DNA at the 17-bp sequence, CENP-B box. J Cell Biol 1992;116:585-596. Palmer DK, O'Day K, Le Trong K, Charbonneau K, Margolis L. Purification of the centromeric protein CENP-A and demonstration that it is a centromere specific histone. Proc Natl Acad Sci USA 1991 ;88:3734--3738. Pluta AF, Cooke CA, Earnshaw WC. Structure of the human centromere at metaphase. TIBS 1990;15:181--185. Pluta AF, Saitoh N, Goldberg I, Earnshaw WC. Identification of a sub domain of CENP-B that is necessary and sufficient for localization to the human centromere. J Cell Biol 1992; 116:1081-1093. Rattner JB, Rao A, Fritzler MJ, Valencia DW, Yen TJ. CENP-F is a ca. 400 kDa kinetochore protein that exhibits a cell-cycle dependent localization. Cell Motil Cytoskeleton 1993;26: 214-226. Reveille JD, Durban E, Goldstein R, Moreda R, Arnett FC. Racial differences in the frequencies of scleroderma-related autoantibodies. Arthritis Rheum 1992a;35:21 6-218. Reveille JD, Owerbach D, Goldstein R, Moreda R, Isern RA, Arnett FC. Association of polar amino acids at position 26 of the HLA-DQB1 first domain with the anticentromere autoantibody response in systemic sclerosis (scleroderma). J Clin Invest 1992b;89:1208-1213. Saitoh H, Tomkiel J, Cooke CA, Ratrie H, III, Maurer M, Rothfield NF, Earnshaw WC. CENP-C, an auto antigen in scleroderma, is a component of the human inner kinetochore plate. Cell 1992;70:115-125. Seki N, Saito T, Kitagawa K, Masumoto H, Okazaki T, Hori TA. Mapping of the human centromere protein B gene (CENPB) to chromosome 20p13 by fluorescence in situ hybridization. Genomics 1994;24:187-188. Stahnke G, Meier E, Scanarini M, Northemann W. Eukaryotic

expression of recombinant human centromere autoantigen and its use in a novel ELISA for diagnosis of CREST syndrome. J Autoimmun 1994;7:107--118. Steen VD, Powell VD, Medsger TA, Jr. Clinical correlations and prognosis based on serum autoantibodies in patients with systemic sclerosis. Arthritis Rheum 1988;31:196-203. Tomkiel J, Cooke CA, Saitoh H, Bernat RL, Earnshaw WC. CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase. J Cell Biol 1994; 125:531-545. Verheijen R, De Jong BAW, Obery6 EHH, Van Venrooij WJ. Molecular cloning of a major CENP-B epitope and its use for the detection of anticentromere autoantibodies. Mol Biol Rep 1992;16:49--59. Vlachoyiannopoulos PG, Drosos AA, Wiik A, Moutsopoulus HM. Patients with anticentromere antibodies, clinical features, diagnosis and evolution. Brit J Rheumatol 1993:32: 297-301. Vazquez-Abad D, Wallace S, Senecal J-L, Joyal F, Roussin A, Earnshaw WC, Rothfield N. Anticentromere autoantibodies: evaluation of an ELISA using recombinant fusion protein CENP-B as antigen. Arthritis Rheum 1994;37:248--252. Weiner E, Hilldebrandt S, Senecal J-L, Daniels L, Noell S, Joyal K, Roussin A, Earnshaw W, Rothfield NF. Prognostic significance of anticentromere antibodies and antitopoisomerase 1 antibodies in Raynaud's disease. A prospective study. Arthritis Rheum 1991 ;34:68-77. Whyte J, Soriano E, Earnshaw WC, McHugh NJ. Frequency of autoantibodies to a major epitope on the carboxyl terminal fragment of CENP-B in patients with autoimmune disease. Brit J Rheumatol 1995;34:407-412. Yoda K, Kitagawa K, Masumoto H, Muro Y, Okazaki T. A human centromere protein, CENP-B, has a DNA binding domain containing four potential (x helices at the NH2 terminus, which is separable from dimerizing activity. J Cell Biol 1992;119:1413--1427.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CHROMO AUTOANTIBODIES Enrique Roberto Soriano, M.D. a and Neil John McHugh, M.D. b

aUnidad de Reumatologia, Hospital Italiano de Buenos Aires, Gascon 450 (1181), Buenos Aires, Argentina; and bDepartment of Rheumatology, Royal National Hospital for Rheumatic Diseases, Upper Borough Walls, Bath BA1 1RF, UK

HISTORICAL NOTES Chromo autoantibodies (anti-chromo) were initially described as part of a study characterizing the proteins recognized by sera from 18 patients with anticentromere antibodies (Guldner et al., 1984). On immunoblotting a protein fraction enriched in HeLa chromosomal proteins, all 18 sera recognized a 19.5 kd protein, later known as CENP-A (Earnshaw and Rothfield, 1985). Five of the 18 anticentromereantibodies (ACA)-positive sera recognized additional 23 kd and 25.5 kd polypeptides. Whereas, affinitypurified anti-CENP-A bound the centromere region on reprobing chromosome spreads, neither the anti-23 kd nor the anti-25.5 kd localized to this region. The authors concluded that the 23 kd and 25.5 kd polypeptides were not confined to the centromere and were targets for a separate immune response. Subsequent work confirmed that anti-chromo were found in a small percentage of sera with ACA but recognized separate determinants (Earnshaw et al., 1986; McHugh et al., 1988; Soriano et al., 1994). Recently, antichromo were shown to recognize a 37-amino acid motif termed the "chromo domain" shared by proteins that are likely to regulate chromatin folding and gene transcription (Saunders et al., 1993). The name "chromo antibodies" was derived from this interaction.

AUTOANTIGENS Method of Purification

The autoantigens recognized by anti-chromo are a

168

group of chromosomal antigens with apparent molecular mass of between 23 and 26 kd as determined by immunoblotting (Guldner et al., 1984; Earnshaw and Rothfield, 1985; Earnshaw et al., 1986; McHugh et al., 1988; Soriano et al., 1994) (Figure 1). The two major bands seen by immunoblotting nuclear extracts and chromosomal preparations with autoimmune sera are designated p23 and p25 (Saunders et al., 1993). Experiments using affinity-purified antibodies show that p23 and p25 share at least one cross-reactive epitope with each other but not with any centromere antigens (Soriano et al., 1994). However, the p23 and p26 proteins appear to be distinct and not different modification states of a single polypeptide (Saunders et al., 1993). Immunolocalization. The precise location of the p23 and p25 antigens in human cells is unknown. Affinitypurified antibodies against the p23 and p25 antigens usually do not recognize the centromere or any other distinct cellular structures when used to reprobe fixed cells by indirect immunofluorescence. Affinity-purified anti-p23 antibodies gave a weak reaction with whole chromosomes (Guldner et al., 1984). More recently, affinity-purified anti-p26 antibodies (but not anti-p23 antibodies) were reported to stain the pericentromeric heterochromatin in mouse and human metaphase chromosomes (Nicol and Jeppesen, 1994). Staining of the human chromosomes was dependent on preculturing the cells with the DNA methylation inhibitor 5-azacytidine prior to the preparation of the metaphases. Such treatment allows decondensation or "stretching" of the heterochromatin which may be important for antigen accessibility.

F i g u r e 1. K562 cell polypeptides recognized by sera with anti-

chromo antibodies after separation on a 12.5% SDS-polyacrylamide gel and transfer to nitrocellulose. Lane 1, normal control serum. Lane 2, anticentromere control. Lane 3, antitopoisomerase I control. Lane 4, anticentromere serum with anti-chromo antibodies that recognize p23 and p26 polypeptides.

Heterochromatin. Heterochromatin, a specialized type of chromatin, is characterized by low levels of gene transcription. The activity of genes translocated in close proximity to heterochromatin are modified by a phenomenon called "position effect variegation" which is well studied in Drosophila. Heterochromatinassociated proteins such as heterochromatin protein 1 (HP1) may be responsible for this phenomenon (Eisenberg et al., 1992). The chromo antigens p23 and p26 are probably associated with centromeric heterochromatin in human cells, although their chromosomal distribution and relationship to the centromere autoantigens is still unknown. Recombinant Autoantigens and Sequence Information. A human expression library screened with anti-chromo yielded cDNA clones encoding a 25 kd

chromosomal protein (Saunders et al., 1993). These anti-chromo are directed against a human homologue of the Drosophila melanogaster HP1, designated HP1 hsa (Saunders et al., 1993). Comparison of the sequence of HPlhSa with that of Drosophila HP1 revealed close sequence similarity (71% amino acid identity at the amino-terminal domain) (Figure 2). A cDNA clone for p23 isolated from a human expression library is 53% similar in amino acid sequence to HP1, and 100% similar to mouse modifier protein (Furuta et al. 1994). The anti-chromo autoimmune response to HP1 hsa is directed solely against the 56-amino-terminal region of the protein (Saunders et a1.,1993). Within the amino-terminal region is a 37 amino acid motif that is termed the HP1/Pc box (Saunders et al., 1993) or chromo (ehromatin modification organizer) domain (Paro and Hogness, 1991). Shared by several chromosomal proteins that produce stable alterations in chromatin structure which may be responsible for suppressing gene expression, the HP1/Pc box includes a second Drosophila protein, the Polycomb (Pc) gene product that is involved in downregulation of homeotic genes (Paro and Hogness, 1991). The function of the HP1/Pc box is not known. Anti-chromo recognize a highly conserved autoepitope: The antigenic target for anti-chromo appears to be highly conserved. HP1 is a heterochromatinassociated protein first identified on chromosomes of Drosophila larvae (James and Elgin, 1986). Antichromo recognize a 26 kd protein (likely to be p25) in nuclear extracts from human, mouse and Chinese hamster ovary cells (Nicol and Jeppesen, 1994) and a 23 kd protein (likely to be p23) in nuclear extracts from human and mouse cells (Nicol and Jeppesen, 1994). Furthermore, anti-chromo recognized heterochromatic regions of Drosophila polytene chromosomes, and a fusion protein containing residues 2--95 "Chromo" domain of Drosophila melanogasterHPI

YAVEKII DRRVRKGKVEYYLKWKGYPETENTWEPENN

III

IIII

II III II III

Illllb

YVVEKVLDRRVVKGQVEYLLKWKGFSEEHNTWEPEKN

Homologous region within amino terminus of Homo sapiens HPI hsa

F i g u r e 2. Homology between the conserved HP1/Pc box of

Drosophila melanogaster HP1 and the equivalent region of cloned p25 (HPlhSa). Amino acid identities are indicated by colons (Saunders et al., 1993).

169

of Drosophila HP1 which included the chromo domain (Saunders et al., 1993). No autoepitopes are found outside the HP1/Pc box region of the protein. There appears to be at least one epitope shared between p23 and p25 but there may be one epitope unique to p25 (Soriano et al., 1994; Saunders et al., 1993; Nicol and Jeppesen, 1994).

AUTOANTIBODIES Terminology The anti-p23 and anti-p25 autoantibodies are termed "anti-chromo antibodies" because they recognize a region of HP1 that contains the "chromo domain," which is shared by several chromosomal proteins (Saunders et al., 1993). Unlike other autoantibodies which recognize multiple epitopes along an individual autoantigen, anti-chromo recognize only a limited region of the protein (Saunders et al., 1993). The autoantibodies may prove useful probes for isolating other human proteins containing the chromo domain (HP1/Pc motif). Anti-chromo are predominantly of the immunoglobulin G isotype (Soriano et al., 1994). In serum samples from one patient studied longitudinally, the presence of ACA preceded the appearance of antichromo (Soriano et al., 1994). Immunization experiments are needed to investigate whether there is an ordered development of the respective autoantibodies. Whether anti-chromo inhibit the function of their cognate autoantigens is unknown. Methods of Detection Immunoblotting nuclear extracts or chromosomal preparations using autoimmune sera is the usual method for detecting anti-chromo (Soriano et al., 1994). Cloning of p25 (Saunders et al., 1993) and p23 (Furuta et al., 1994) will allow the development of solid-phase recombinant autoantigen EL1SAs for detecting anti-chromo in the near future.

CLINICAL UTILITY Disease Association Anti-chromo are found in about 10--15% of sera from patients with ACA (Soriano et al., 1994) and are

170

typically not found in the absence of ACA. Therefore, it is not surprising that anti-chromo have much the same clinical associations as for ACA. Most patients have a limited cutaneous form of systemic sclerosis (formerly called CREST: Calcinosis, Raynaud's phenomenon, Esophageal involvement, Sclerodactyly and Telangiectasia). Neither anti-chromo nor ACA are found in the diffuse cutaneous form of systemic sclerosis. Patients with limited systemic sclerosis are typically older women with a long-standing history of Raynaud's phenomenon. On physical examination there is usually skin thickening of the digits (sclerodactyly) and occasionally of the hands and forearms distal to the elbows, and there may be multiple digital and facial telangiectasia. Esophageal hypomotility is very frequent, but often asymptomatic. Pulmonary hypertension is the most severe internal organ involvement and is sometimes the cause of death. Although clinical findings were not described in the first five patients reported with anti-chromo, all fulfilled criteria for systemic sclerosis (Masi et al., 1980; Guldner et al., 1984). In four patients with antichromo, two had the "CREST" form of systemic sclerosis, one had systemic lupus erythematosus and "non-CREST" scleroderma, and one had Sj6gren's syndrome and Raynaud's phenomenon (Saunders et al., 1993). In another three patients with anti-chromo, all had limited cutaneous systemic sclerosis with disease duration of fourteen, nine, and three years, respectively (Soriano et al., 1994). Two of the three had an erosive arthritis, were seropositive for rheumatoid factor and fulfilled criteria for rheumatoid arthritis (Arnett et al., 1988). In contrast, only three of the other 29 patients with ACA but without anti-chromo fulfilled criteria for rheumatoid arthritis. Therefore, anti-chromo antibodies may identify an overlap syndrome of limited scleroderma and rheumatoid arthritis, but larger numbers are needed to confirm this finding.

CONCLUSION A group of proteins with an apparent molecular mass between 23 and 26 kd are recognized by 10--15% of sera containing ACA. The two main autoantigens, termed p23 and p25 are likely to be heterochromatinassociated proteins. The cloned p25 antigen (HP1 hsa) has sequence similarity to a heterochromatin protein of Drosophila HP1. The autoantibodies recognize a conserved structural motif (the HP1/Pc box or the

" c h r o m o " domain) shared by Drosophila HP1 and a n u m b e r of other c h r o m o s o m a l proteins that may be responsible for suppressing gene transcription. The precise location and function of the p23 and p25 autoantigens and their relationship to centromere-

associated autoantigens are uncertain. Detected exclusively in sera containing ACA, anti-chromo are found mainly in sera from patients with limited cutaneous systemic sclerosis. See also CENTROMERE AUTOANTIBODIES.

REFERENCES

melanogaster and its gene. Mol Cell Biol 1986;6:3862-3872. Masi AT, Rodnan GP, Medsger TA Jr, Altman RD, D'Agelo WA, Fries JF, LeRoy EC, Kirsner AB, MacKenzie AH, McShane DJ, Myers AR, Sharp GC. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 1980;23:581--590. McHugh NJ, James IE, Maddison PJ. Differential isotype recognition of two centromere associated polypeptides by immunoblotting in connective tissue disease. Clin Exp Immunol 1988;72:457--464. Nicol L, Jeppesen P. Human autoimmune sera recognize a conserved 26 kD protein associated with mammalian heterochromatin that is homologous to heterochromatin protein 1 of Drosophila. Chromosome Res 1994:2:245-253. Paro R, Hogness D. The polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. Proc Natl Acad Sci USA 1991:88:263-267. Saunders WS, Chue C, Goebl M, Craig C, Clark RF, Powers JA, Eissenberg JC, Elgin SC, Rothfield NF, Earnshaw WC. Molecular cloning of a human homologue of Drosophila heterochromatin protein HP1 using anticentromere autoantibodies with anti-chromo specificity. J Cell Sci 1993;104: 573--582. Soriano E, Whyte J, McHugh NJ. Frequency and clinical associations of anti-chromo antibodies in connective tissue disease. Ann Rheum Dis 1994;53:666--670.

Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315--324. Earnshaw W, Bordwell B, Marino C, Rothfield N. Three human chromosomal autoantigens are recognized by sera from patients with anticentromere antibodies. J Clin Invest 1986; 77:426--430. Earnshaw WC, Rothfield N. Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma 1985;91:313-321. Eissenberg JC, Morris GD, Reuter G, Hartnett T. The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation. Genetics 1992; 131:345-352. Furuta K, Chan EK, Kiyosawa K, Tan EM. Molecular identity of human heterochromatin associated autoantigen p23. Arthritis Rheum 1994;37:S 170. Guldner HH, Lakomek HJ, Bautz FA. Human anticentromere sera recognize a 19.5 kd nonhistone chromosomal protein from HeLa cells. Clin Exp Immunol 1984;58:13--20. James TC, Elgin SC. Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila

171

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

COAGULATION FACTOR VIII AUTOANTIBODIES Jean Guy Gilles, Ph.D. and Jean-Marie R. Saint-Remy, M.D., Ph.D.

Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, 3000 Leuven, Belgium

HISTORICAL NOTES

Native Versus Recombinant FVIII Molecules

The detection of autoantibodies that interfere with the function of factor VIII (FVIII), although a rare event, with an annual incidence of 1/5 million people (Kessler, 1991), is nevertheless, the most common cause of antibody-mediated inhibition of the coagulation system. A large survey by the International Committee of Thrombosis and Hemostasis which covered the last 10 years and the experience of 118 physicians, identified 215 nonhemophilic patients with spontaneous FVIII:c inhibitors (Green and Lechner, 1981). In this group, 46% had no identifiable associated illness; the risk of developing autoantibodies increased with age: 17% of patients with autoantibodies were between 50 and 60 years old and 48% were older than 60. Auto-anti-FVIII antibodies disappear spontaneously in one-third but can persist for years and result in severe life-threatening bleeding episodes. Several therapies are used to reduce the production of these antibodies, but this goal remains difficult to achieve.

The functional properties of native and recombinant forms of FVIII are indistinguishable, as measured by coagulation assay systems (Langdeil et al., 1953) or by conversion of a synthetic substrate in a chromogenic assay (Svendsen et al., 1984). Preliminary indications suggest that the two forms of the molecule might be distinct in terms of reactivity with specific antibodies; this applies both to full-length recombinant molecules and to truncated molecules (see below). Immunoassays carried out with different types of FVIII might, therefore, generate different results. Origin and Sources Human FVIII, a 330 kd glycoprotein produced by the liver, can be purified from plasma using different methodologies. Recombinant molecules are produced by culture of mammalian cells transfected with fulllength or truncated forms of DNA. Two types of cells are used: Chinese hamster ovary cells and baby hamster kidney cells (Kaufman et al., 1988).

THE AUTOANTIGENS

Methods of Purification

Definition and Function

FVIII is obtained from multidonor plasma pools by cryoprecipitation (Smith, 1986) followed by removal of contaminant proteins, including filtration, chromatography on ion-exchangers or affinity sorbents (Burnouf et al., 1991) and/or immunoaffinity. For immunoaffinity purification, FVIII cryoprecipitate is passed over a column of immobilized mouse monoclonal antibodies to FVIII or to von Willebrand factor (vWF); the product is then recovered by elution and passed through an ion-exchange column to remove contaminant murine immunoglobulins. The degree of

FVIII, a critical glycoprotein participant in the coagulation pathway, is a cofactor to FIXa in the enzymatic cleavage of FX in presence of negatively charged phospholipids and calcium. FXa converts prothrombin into thrombin, which in turn converts fibrinogen into fibrin. Absence of FVIII or production of an abnormal molecule results in severe bleeding episodes characteristic of hemophilia A, an X-linked hereditary deficiency affecting one in 10,000 males.

172

purity of plasma-derived FVIII differs greatly from one preparation to the other, and is usually expressed in IU/mg of total protein. Full-length recombinant FVIII, either co-expressed with vWF or not, is currently produced by transfected mammalian cells. Following the demonstration that the B domain of FVIII is not essential for the function of the molecule (Pittman et al., 1994), several truncated molecules were constructed with complete or partial deletion of the B domain or its replacement by a synthetic peptide.

Commercial Sources of FVIII FVIII preparations for clinical use include plasmaderived FVIII with degrees of purity varying from intermediate to high or very high (affinity-purified product) and specific activities prior to addition of albumin of 10, >100 and >2,000 international units (IU) FVIII/mg protein, respectively. One IU corresponds to the activity of FVIII in 1 mL of normal plasma. Recombinant FVIII has an activity >2,000 IU FVIII/mg protein before albumin addition.

Sequence Information Native FVIII is a single polypeptide chain of 2,332 amino acids (amino acids), whose complete sequence is established (Vehar et al., 1984). The cloning of FVIII (Gitchier et al., 1984) and its expression by a number of mammalian cell lines (Wood et al., 1984; Toole et al., 1984) reveal a single open reading frame which yields a 2,351 amino acids single chain precursor from which a 19 amino acids signal peptide is released upon translocation into the lumen of the endoplasmic reticulum, in which posttranslational transformations, including glycosylations occur. The FVIII molecule is heavily glycosylated, reaching a MW of 330 kd in its complete form. Of the three domains (A, B and C) of FVIII, domain A is made of two segments, A1 (amino acids 1 to 329) and A2 (amino acids 380 to 711), separated by an acidic region that is essential for the function of the molecule (Figure 1), A1 and A2 constitute the FVIII heavy chain, with a MW of 92 kd. The light chain is made of 3 segments, namely A3 (amino acids 1649 to 2091), C 1 and C2, with a total MW of 80 kd. The heavy and light chains are linked together by a 948 amino acids B domain, which contains 80% of the glycosylation sites of the molecule. FVIII, therefore, presents two types of internal homologies. The

first is made of the triplication of the A segments, which show a sequence similarity of +30% to one another. The second is made of the duplication of the C segment at the carboxy-terminal end of the molecule. The overall FVIII structure is therefore arranged in a particular linear order, namely A1-A2-BA3-C1-C2. This structure should be kept in mind when interpreting the results of antibody-binding assays, because some antibodies could bind to two or more epitopes of the FVIII molecule. The circulating form of FVIII is in fact represented by heterodimer obtained by proteolytic cleavage of the precursor. Such proteolysis is carried out by thrombin or by the activated form of FX in a positive feed-back loop. Three major cleavage sites located in FVIII are essential for its activation, one in the acidic region between the A1 and A2 domains (amino acids Arg 372), a second at the carboxy-terminal end of the A2 domain (amino acids Arg 740) and a third at the carboxy-terminal end of a light chain acidic region (amino acids 1689) located at the junction of the B domain with A3 (Toole et al., 1984; Gitschier et al., 1984; Eaton et al., 1986). The activated form of FVIII found in circulation consists, therefore, of heterotrimer (Lamphear and Fay, 1992; Fay and Smudzin, 1992). Further cleavage by thrombin, FXa or activated protein C inactivates the molecule (Vehar and Davie, 1980) (Figure 1). The fact that FVIII circulates in different forms with possibly distinct antigenic properties should be borne in mind for the interpretation of immunoassays, because the relative proportion of each of these forms, e.g., the degree of FVIII activation could influence the binding of specific antibodies.

AUTOANTIBODIES Definition The two main categories of antibodies toward FVIII (anti-FVIII) include those that do and those that do not inhibit its function. Thus, the terms "inhibitor" or "functional antibody" should be restricted to antibodies showing a partial or complete inhibition of the function of FVIII in coagulation assays or in chromogenic assays. Functional antibodies to anti-FVIII are further divided into subcategories, type I or type II as defined by differences in the kinetics of FVIII activation. Type I antibodies completely inhibit FVIII function in a dose-dependent manner; whereas, type II

173

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Figure 1. Different structures of FVIII molecule are represented in this Figure. The native FVIII with the potential enzyme cleavage sites indicated by arrows (Th: thrombin; APC: activated protein C" Xa: activated factor X; ?: unknown enzyme); the heterotrimer plasma circulating FVIII and finally the activated and inactivated forms.

antibodies do not, even when used at high concentrations (Gawryl and Hoyer, 1982). This functional distinction is further substantiated by the relative affinity of antibodies; type I inhibitors are of high affinity in contrast to type II inhibitors. The second category of anti-FVIII consists of antibodies which do not neutralize the function of FVIII, although they are specific for the molecule (Gawryl and Hoyer, 1982).

Pathogenetic Role Inhibitors or functional antibodies can obviously interfere with the coagulation cascade by considerably slowing the rate at which FX is converted to its activated form, FXa, an essential step in the generation of thrombin and fibrin. The mechanism by which these antibodies inhibit the function of FVIII is heterogeneous. Antibodies can bind sites of FVIII that

174

are directly involved in the proteolytic cleavage required for full activation of the molecule, therefore precluding enzyme activity. Antibodies can interfere with the binding of FVIII to vWF and therefore reduce the half-life of circulating FVIII, which is normally protected from cleavage by vWF. In addition, antibodies can alter the 3-D structure of FVIII in such a way as to render it more resistant to enzyme activation. Anti-FVIII that do not neutralize the function of the molecule might accelerate the clearance rate from the circulation by increasing the uptake of FVIII-Ig complexes by phagocytic cells, such as liver Kupffer cells. Formal proof of this mechanism is lacking.

Genetics No information is available yet on the genetics of the

anti-FVIII immune response. Attempts to identify preferential usage of certain MHC have failed (Hoyer, 1991), probably because of the large size of the molecule and the great number of possible epitopes. Likewise, no information is available on the use of particular V H regions for antibody formation or of possible restriction in T-cell receptor usage.

Factors Involved in Pathogenicity and Etiology Autoantibodies to FVIII are not usually clonal but rather are produced by a small number of clones (Gilles et al., unpublished data). Isotype determination by immunoblotting shows an overrepresentation of the IgG4 subclass, frequently in association with IgG1; whereas, IgG3 is rarely detected (Kavanagh et al., 1981; Fulcher et al., 1987). ELISA permitted the identification of some IgG2 reactive with FVIII (Gilles et al., 1993). Kappa light chain predominance probably reflects its usual proportion in the general repertoire of antibodies. FVIII autoantibodies can be associated with benign or malignant lymphoproliferative disorders and paraproteinemias (Castaldi and Penny, 1970; Glueck and Hong, 1965), or with plasma cell dyscrasias, including IgA myeloma and IgM macroglobulinemia (Hultin, 1991). These autoantibodies are usually type II inhibitors, i.e., antibodies of relatively low affinity and that do not fully neutralize FVIII function. Selected regions of FVIII are preferentially recognized by anti-FVIII. Deletion mutants of the FVIII gene coding for single FVIII domains confirm previous observations that located the major epitopes of FVIII at the carboxy-terminal end of the C2 domain of the FVIII light chain and at the amino-terminal end of the A2 domain of the heavy chain (Scandella et al., 1989). A panel of mouse monoclonal antibodies to ten nonoverlapping epitopes identified several other binding sites for human antibodies, which are spread over the entire molecule, including one additional region of interest that mapped to the A3 domain of the light chain (Gilles et al., 1993). This region contains a cluster of acidic aminoacids and a cleavage site critical for FVIII activation.

Methods of Detection Antibodies to functional epitopes of the molecule are currently evaluated in vitro by their capacity to inhibit the procoagulant activity of FVIII. Quantitation is obtained by mixing dilutions of the plasma-containing

inhibitors with a fixed amount of FVIII. The number of inhibitor units per mL of plasma is then calculated as the reciprocal of the plasma dilution that neutralizes FVIII. Most usual laboratory methods are the "new Oxford" method (Rizza and Briggs, 1973), in which the source of FVIII is a plasma-concentrate, and the "Bethesda" method (Kasper et al., 1975), which utilizes pooled normal plasma as source of FVIII and provides results in Bethesda Units. FVIII inhibitors can also be evaluated with a chromogenic assay, in which thrombin-activated FVIII acts as a cofactor to FIXa in the conversion of colorless substrate by FXa (Svendsen et al., 1984); the concentration of anti-FVIII-specific antibodies is therefore inversely proportional to the optical density measured in a spectrophotometer. Results are quantitated by calculating the reciprocal of the sample dilution that inhibits 50% of the color formation as compared to that obtained with a standard FVIII solution (Gilles et al., 1993). Antibodies directed not only towards nonfunctional sites, but also to some of the functional sites, can be detected by direct binding to insolubilized FVIII. This can be carried out by reacting antibodies with FVIII blotted on nitrocellulose sheets after electrophoretic separation or by direct binding to polystyrene plates coated with FVIII. Variants of this latter assay include insolubilization of fragments of FVIII obtained by recombinant DNA technology or digestion, or a capture assay using plates coated with a monoclonal antibody to either FVIII itself or to vWF. Antibodies reactive with soluble FVIII, either fulllength FVIII or fragments, can also be assessed by immunoprecipitation of the conjugate (Scandella et al., 1992), by inhibition of the agglutination of latex particles coated with FVIII (Gilles and Saint-Remy, 1994) or by inhibition of antibody binding to FVIIIcoated plates (Gilles et al., 1993). Such immunoassays have the advantage of avoiding alteration of the 3-D conformation of FVIII, and therefore reduce the risk of false-negative results due to FVIII insolubilization. However, care should be taken in the choice of FVIII for nonfunctional assay systems. Moreover, the significant sequence similarity between different segments of FVIII render it possible to have antibodies that react with multiple epitopes of the molecule. The significance of these similarities remains to be established for assay systems using polyclonal antiFVIII antibodies. They are, however, of prime importance for interpreting results obtained with monoclonal antibodies, such as in epitope mapping studies.

175

CLINICAL UTILITY

Table 1. Frequency of Disease Association with FVIII Auto-

Application

No associated disease

46%

Autoimmune disease

18%

Any unexplained bleeding episode with prolonged clotting time should prompt a search for anti-FVIII, the cause of the most common antibody-mediated coagulation disorder. In about 50% of patients, the appearance of such antibodies is not associated with a specific pathology (see below). The current assays are sensitive enough to detect clinically relevant antibodies that interfere with the procoagulant function of the molecule. Assays detecting antibodies towards nonfunctional parts of FVIII need extensive validation prior to use on a regular basis, because up to 17% of normal healthy individuals have detectable noninhibitory anti-FVIII in their plasma (Algiman et al., 1992). Moreover, more than 90% of such individuals have circulating anti-FVIII in the form of complexes with anti-idiotypic antibodies (Gilles et al., 1993). These anti-idiotypic antibodies have not only the capacity to inhibit the binding of anti-FVIII to FVIII, but could participate in the regulation of the production of such anti-FVIII. Although a subclassification of anti-FVIII according to their functional properties may not be clinically useful at present, further delineation of the heterogeneity of anti-FVIII is warranted, because of the multiple ways by which an antibody could interfere with the function or metabolism of FVIII.

Disease Associations Apart from an increased incidence of anti-FVIII with age, there is no described difference in occurrence for gender or race (Hultin, 1991).

Antibody Frequencies in Diseases A general correlation between titers of inhibitory FVIII antibodies and disease activity is well established when there is a n associated autoimmune disease, namely in +18% of the cases (Green and Lechner, 1981). The treatment of patients with autoantibodies to FVIII remains difficult and nonspecific, whatever the possible underlying cause. Current therapies include, as for other autoimmune diseases, corticosteroids and cyclophosphamide alone (Green et al., 1993; Berrut et al., 1994) or in combination with infusions of large doses of FVIII concentrates (Lian et al., 1989).

176

antibodies

Postpartum state

7%

Malignancies

7%

Drug reaction

5%

Intravenous infusion of immunoglobulins is sometimes added (Sultan et al., 1984; Lionnet et al., 1990). No information is available on the possible transplacental transfer of such autoantibodies, probably due to the fact that the majority of the cases occur after childbearing age. The 1981 study of the International Committee of Thrombosis and Hemostasis (Green and Lechner, 1981) is the first attempt to establish the clinical associations of auto-anti-FVIII antibodies (Table 1). Acquired hemophilia of unknown origin, the most important cause of morbidity, occurs most frequently in the absence of associated diseases. Of the accompanying autoimmune diseases, systemic lupus erythematosus and rheumatoid arthritis are most frequently reported. Auto-anti-FVIII are sometimes associated with antinuclear antibodies (Marwaha et al., 1991). Penicillin derivatives (Hultin, 1991) and interferon therapy (Stricker et al., 1994; Castenskiold et al., 1994) can induce these autoantibodies. FVIII inhibiters are also described after pregnancy (Green, 1991; Hauser et al., 1995) and in association with plasma cell dyscrasias, such as myeloma (Loftus and Arnold, 1994) and Waldenstr6m's macroglobulinemia. Isolated cases of amyloidosis (Glueck et al., 1988) with an IgM paraprotein or lymphocytic leukemia are also described.

Sensitivity. Assays evaluating the capacity of FVIII autoantibodies to interfere with the procoagulant function of FVIII are most reliable. Assays measuring the total production of anti-FVIII require further validation to establish, if possible, normal cut-off values.

CONCLUSION Testing for autoantibodies towards FVIII should be carried out in cases in which an unexpectedly pro-

longed coagulation time is observed, even in the absence of overt associated diseases or predisposing conditions such as the postpartum period. The immediate pathological consequences of the presence of such antibodies can indeed be disastrous. The assay systems in which the neutralizing effect of antibodies on the procoagulant activity of FVIII is determined are simple to use and provide reliable results. The detection of antibodies that do not neutralize the activity of FVIII requires further validation, namely,

a better delineation between normal and pathological values. Possible interactions between antibodies and FVIII are numerous, and it is likely that antibodies will soon be subclassified according to their specificity and/or in vivo effects. Lastly, the antigenic properties of FVIII preparations, plasma-derived or of recombinant origin, may significantly differ, a factor which should be taken into account for in vitro assays. See also COAGULATION FACTOR (EXCLUDING FACTOR VIII) AUTOANTIBODIES.

REFERENCES

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Smith A, inventor. Verfahren zur Herstellung eines hochgereinigten Anti-hamophilie-Faktors. Lecarsa SA, European patent 0238701, 1986. Stricker RB, Barlogie B, Kiprov DD. Acquired factor VIII inhibitor associated with chronic interferon-alpha-therapy. J Rheumatol 1994;21:350--352. Sultan Y, Kazatchkine MD, Maisonneuve P, Nydegger UE. Anti-idiotypic suppression of autoantibodies to factor VIII (antihaemophilic factor) by high-dose intravenous gammaglobulin. Lancet 1984;2:765--768. Svendsen L, Brogli M, Lindeberg G, Stocker K. Differentiation of thrombin- and factor Xa-related amidolytic activity in plasma by means of synthetic thrombin inhibitor. Thrombosis Res 1984;34:457--462. Toole JJ, Knopf JL, Wozney JM, Sultzman LA, Buecker JL, Pittman DD, Kaufman RJ, Brown E, Shoemaker C, Orr EC, et al. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 1984;312:342--347. Vehar GA, Davie EW. Preparation and purification of bovine factor VIII (antihemophilic factor). Biochemistry 1980;19: 401--410. Vehar GA, Keyt B, Eaton D, Rodriguez H, O'Brien DP, Rotblat F, Opperman H, Keck R, Wood KI, Harkins RN, et al. Structure of human factor VIII. Nature 1984;312:337--342. Wood WI, Capon DJ, Simonsen C, Eaton DL, Gitschier J, Keyt B, Seeburg PH, Smith DH, Hollingshead P, Wion KL, et al. Expression of active human factor VIII from recombinant DNA clones. Nature 1984;312:330--337.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

COAGULATION FACTOR (EXCLUDING FACTOR VIII) AUTOANTIBODIES Alaa E.E. Ahmed, M.Sc., Ph.D.

Specialty Laboratories Inc., Santa Monica, CA 90404, USA

HISTORICAL NOTES Coagulation factor autoantibodies are pathological circulating autoantibodies that directly inhibit clotting factors, resulting in a deficiency of clotting activity. These autoantibodies are rarely encountered in noncongenitally deficient patients (Kunkel, 1992). Circulating anticoagulants that included autoantibodies were first reviewed in 1961 (Margolius et al., 1961). Since then, knowledge of the underlying mechanisms for such autoimmunity and the modes of action of these autoantibodies has increased dramatically.

THE AUTOANTIGEN(S) The coagulation mechanism consists of a cascade of linked proteolytic reactions in which zymogens are converted into serine (trypsin-like) proteases, ultimately leading to the formation of a fibrin clot. This complex series of reactions is arbitrarily divided into three distinct pathways (Furie and Furie, 1992): extrinsic (Factor VII, Tissue Factor), intrinsic (Factors VIII, IX, XI, XII) and common (Factors II, V, X). Coagulation proteins and their common names and functions are listed (Table 1); the Roman numeral system is most widely used and preferred. Methods of purification for these coagulation factors are well established, and monoclonal and polyclonal antibodies against the proteins are available commercially. Factor II (Prothrombin). Human Factor II is a

plasma zymogen with a molecular weight of 72 kd and a pI of 4.7. Factor II cDNA consists of 2005 bp, encoding most of a 43 amino acid leader sequence, a propiece of eight amino acids and a mature protein of

579 amino acids (Furie and Furie, 1988). Factor II assembles with the activated forms of Factor V, Factor X and phospholipid to form a catalytic unit known as the prothrombinase complex (Mann et al., 1982). In the presence of calcium ions, the complex cleaves membrane-associated Factor II (prothrombin) into thrombin, which is then released into the soluble phase (Furie and Furie, 1988). Factor V. Factor V is a large, asymmetric, single

chain 330 kd plasma glycoprotein. Analysis of the amino acid sequence reveals that the central 50% of the molecule contains 25 of the 37 asparagine-linked potential glycosylation sites (Mann et al., 1988). Factor V was once called labial factor due to its instability and susceptibility to proteolytic degradation. About 20% of the total Factor V in blood is located in platelets (Mann et al., 1988). This stored form of Factor V is secreted upon platelet activation and plays a significant role in normal hemostasis. As part of the prothrombinase complex, Factor V is an essential component for the rapid conversion of prothrombin to thrombin. Factor VII. Factor VII is a 56 kd single chain,

vitamin K-dependent zymogen component of the extrinsic pathway of blood coagulation. It differs from other blood coagulation proenzymes in that the zymogen form itself expresses significant enzyme activity (Mann et al., 1988). Factor X converts Factor VII to its active form (VIIa), a two-chain enzyme that, in complex with tissue factor, feeds back to activate Factor X. Factor VII contains ten gamma-carboxyglutamic acid residues, a characteristic feature of vitamin K-dependent proteins critical for calcium ion binding and interaction with cell membranes (Mann et

179

Table 1. Proteins of the Clotting System Factor

Common Name/Function

I

Fibrinogen

II

Prothrombin

lip

V

Ac-globulin

lq

VII

Prothrombin conversion accelerator

VIII

Antihemophilic factor

xq

IX

Christmas factor

xq

X

Stuart-Power factor

XI

Thromboplastin antecedent

4

XII

Hageman factor

6

XIII

Profibrinoligase

6p, lq

vWF

von Willebrand factor

al., 1988). Factor VII also contains two epidermal growth factor (EGF)-like domains containing a single beta-hydroxyaspartic acid (Furie and Furie, 1988). Factor IX. Factor IX is a vitamin K-dependent single

chain glycoprotein comprised of 415 amino acids. It is composed of gamma-carboxylglutamic acid domain, two EGF-like domains, an activation peptide and a catalytic domain (Furie and Furie, 1988). Factor IX is converted to its active form by Factor XI.

Chromosome Location 4q

13q

13

12p

enzyme upon contact with negatively charged surfaces (solid-phase autoactivation). Factor XII is capable of activating the fibrinolytic system, generating kinins and initiating blood coagulation through the activation of Factor XI (Wachtfogel et al., 1993). Factor XIII. Factor XIII is the precursor of a plasma

gen with a molecular weight of 56 kd. It acquires serine protease activity upon cleavage by activated Factor V (Va), which is an essential requirement for the assembly of the prothrombinase complex (Mann et al., 1988).

and platelet coagulation enzyme. Activated Factor XIII (XIIIa) is not a serine protease but a transglutaminase, which catalyzes the formation of cross links between glutamine and lysine residues in fibrin I and fibrin II (Hassouna, 1993). While plasma Factor XIII is a tetramer and platelet Factor XIII is a dimer, they share an identical function. In blood, Factor XIII is activated by thrombin in the presence of calcium ions. Factor XIII also catalyzes the cross-linking of fibrin and ~-2-antiplasmin (Hassouna, 1993).

Factor XI. This 160 kd zymogen contains two

von Willebrand Factor (vWF). vWF is a plasma

identical disulfide-linked chains. Upon activation by Factor XII or thrombin, two peptide bonds are cleaved to generate two heavy chain-light chain homodimers linked through the heavy chains by a disulfide bond. The catalytic domain is located on the light chain, and the heavy chain contains the recognition site for Factor IX and a binding site for high molecular weight kininogen (Natio and Fujikawa, 1991).

glycoprotein with multiple functions in normal hemostasis. In addition to serving as a transport protein for Factor VIII, vWF serves as a linker molecule between platelets and subendothelial components following vascular injury (Furie and Furie, 1988). This interaction leads to platelet aggregation and subsequent hemostatic plug formation (Furie and Furie, 1992). The primary translation product is a prepolypeptide of 370 kd, which is subject to posttranslational modifications, including proteolytic processing, glycosylation, sulfation, polymerization and disulfide bond formation. The plasma protein is a disulfide-linked multi-

Factor X. Factor X is a vitamin K-dependent zymo-

Factor XII. Factor XII, the first component of the

intrinsic pathway, is converted from a single-chain zymogen of 80 kd into a two-chain disulfide-linked

180

meric protein composed of 260 kd subunits generated from the primary translation product. Protein sequence analysis of the mature plasma subunit and a comparison to the cDNA-derived amino acid sequence indicate that all proteolytic processing occurs at the NHz-terminus. The mature vWF subunit contains 13 potential N-linked glycosylation sites. The vWF binding site for Factor VIII is located on the amino terminal portion of the disulfide-linked dimers (Wise et al., 1988).

THE AUTOANTIBODIES

Coagulation factor autoantibodies are associated with autoimmune diseases, lymphoid malignancies, and pregnancy, as well as advanced age (Schapiro and Siegel, 1991). Factor autoantibodies are rare in the pediatric population, except in cases where a severe congenital factor deficiency has been treated with factor replacement. IgG is the predominant isotype, with the IgG4 subclass as most common. Kappa light chains are more common than lambda light chains in circulating IgG anticoagulants (Armitage et al., 1994). They are usually nonprecipitating antibodies and are present in serum as well as plasma with longer stability than the clotting factors themselves. Unlike lupus anticoagulant, most acquired inhibitors specifically neutralize only one clotting factor, and most are species-specific (Schapiro and Siegel, 1991). Factor II Autoantibodies. Anti-Factor II (prothrombin) are extremely rare and have been seen in patients with systemic lupus erythematosus or after exposure to topical bovine thrombin (Baudo et al., 1990). A clear distinction between a specific anti-Factor II and a lupus anticoagulant has yet to be made. Some patients with antithrombin anticoagulant do not exhibit clotting deficiencies (Baudo et al., 1990). Factor V Autoantibodies. Anti-Factor V are considered rare; only 26 case reports are extant (Suehisa et al., 1995). In only one case did the patient suffer from a congenital Factor V deficiency; autoantibodies developed after plasma transfusion (Suehisa et al., 1995). However, the mechanism for the autoimmune reaction against Factor V in other cases is poorly understood and the responsive epitope on Factor V is not fully clarified. The autoantibody often arises after surgery with exposure to bovine thrombin or fibrin glue, blood transfusion or administration of aminogly-

coside antibiotics or cephalosporins (Israels and Israels, 1994). However, surgery, transfusion and antibiotics might only be triggers in the development of anti-Factor V, since most of the patients to whom these treatments are applied do not develop such autoantibodies. In two reports, the autoantibodies were directed against the light chain of Factor V. The development of anti-Factor V was transient in 18 of 25 previously reported cases; the remaining seven patients died of hemorrhage. Three of these seven cases were complicated with autoimmune disease, rheumatoid arthritis and bullous pemphigoid with and without Hashimoto's thyroiditis (Suehisa et al., 1995). Factor VII Autoantibodies. Factor VII deficiency is a rare hereditary coagulation disorder with various clinical manifestations. Only three cases of acquired Factor VII deficiency caused by an autoantibody were reported in association with a probable carcinoma of the lung, aplastic anemia and liposarcoma (de Raucourt et al., 1994). In one case, the antibody was determined to be of the IgG class. Factor IX Autoantibodies. Anti-Factor IX are the most prevalent, arising in 2.5--16% of hemophilia B patients (Roberts and Eberst, 1993) and occurring in the laboratory at about 10--20% of the frequency of anti-Factor VIII. Autoantibodies are of restricted polyclonal origin, consisting predominantly of the IgG4 subclass and occasionally IgG1 and IgG2 (Orstavik and Miller, 1988) with kappa light chains. Two types of anti-Factor IX are recognized: alloantibodies produced by hemophilia B patients and autoantibodies produced in nonhemophiliacs (Roberts and Eberst, 1993). A number of factors appear to affect the development of anti-Factor IX, including severity of hemophilia, age, genetic predisposition and antigenicity of factor replacement therapy (Aledort, 1994). Factor X Autoantibodies. Anti-Factor X are extremely rare. They were first described in two patients with leprosy. However, anti-Factor X have not actually been found in their circulation (Bick, 1992). Eleven patients have been described who experienced the sudden onset of bleeding due to Factor X deficiency arising after an acute respiratory infection or unknown cause; anti-Factor X was clearly demonstrated in three of the patients (Rao et al., 1994). Factor Xl Autoantibodies. Anti-Factor XI have been

181

reported in 17 patients, mostly in association with autoimmune disease (Schapiro and Siegel, 1991). One case was reported in association with pneumonia resulting from adenovirus (Bick, 1992). Factor XII Autoantibodies. Anti-Factor XII have been noted in systemic lupus erythematosus, Waldenstrom's macroglobulinemia and glomerulonephritis (Bick, 1992). Factor XIII Autoantibodies. Acquired Factor XIII deficiency is a severe coagulopathy characterized by the development of circulating autoantibodies that severely impair Factor XIII mediated fibrin-crosslinking. Only 18 patients with anti-Factor XIII have been described (Tosetto et al., 1995). The autoantibodies show a considerable heterogeneity, as they interfere either with thrombin-mediated Factor XIII activation, Factor XIII activity, or the Factor XIII binding site on fibrin (Tosetto et al., 1995). Recently, a new type of autoantibody directed against a hitherto unknown fibrin binding site on Factor XIII has also been described (Tosetto et al., 1995; Fukue et al., 1992). Thrombin Autoantibodies. Thrombin autoantibodies are not frequent and are usually observed with bleeding diseases (Sic et al., 1991). The development of such autoantibodies is usually associated with autoimmune disorders or a crossed immunization with bovine thrombin (Zhender and Leung, 1990) and with severe arterial thrombotic disease (Arnaud et al., 1994). von Willebrand (vWF) Factor Autoantibodies. Circulating autoantibodies, usually IgG, directed against vWF may be associated with yon Willebrand's disease in patients with benign monoclonal gammopathy, macroglobulinemia, multiple myeloma, lymphoproliferative diseases or autoimmune disorders such as SLE and scleroderma (Bick, 1992). The major mechanism proposed to explain the pathogenesis of the acquired deficiency of vWF is that antibodies inactivate immunologic or functional sites of vWF or form a complex, thereby inducing a rapid clearance of vWF from circulation (Jakway, 1992). In one case, an IgM autoantibody capable of inhibiting the binding of vWF to collagen resulted in the absence of functional vWF and a concomitant hemorrhagic tendency (van Genderen et al., 1994).

182

Methods of Detection Screening Methods. That an autoantibody (inhibitor) might be present should be suspected when a patient with no prior history of abnormal bleeding has an unexplained hemorrhage or when a hemophiliac fails to respond to replacement therapy. Alternatively, a prolonged result with laboratory coagulation screening tests such as prothrombin time (PT) or activated partial thromboplastin time (APTT) might suggest the presence of an autoantibody. The simplest screening test for autoantibodies involves incubation of equal amounts of normal plasma and test plasma at 37~ and performing an APTT at various times. A highaffinity autoantibody can show APTT inhibition after a short period of incubation, but a low-affinity autoantibody might not be detected until the mixture incubates for long periods (> 1 hour). Autoantibodies to Factors VIII, IX or XI do not affect the PT, but high affinity autoantibodies to Factor V may affect both the PT and APTT (Kaspar and Ewing, 1986). A different screening method, suitable for detection of autoantibodies to Factors V, VII, X or XI employs agarose gel mixed with citrated normal plasma. Dilutions of patient plasma are added to wells in the gel and incubated at room temperature for 16--20 hours. The gel is then immersed in calcium chloride, which permits fibrin formation throughout the gel. A clear ring around the well indicates the presence of an autoantibody (Kaspar and Ewing, 1986). Quantitative Methods. Three of the standardized methods developed for the quantification of the most common coagulation factor autoantibody (Factor VIII) can be modified to quantitate autoantibodies to Factor IX or Factor XI. The "old Oxford method" employs Factor VIII in the range of 10--20 units/mL diluted 1:10 with patient plasma and incubated for 1 hour at 37~ residual Factor VIII levels are measured using a specific Factor VIII assay. An autoantibody unit is defined as the amount of antibody that inhibits 0.75 units of Factor VIII. In the "new Oxford method," the incubation period is extended to four hours to allow more interaction of the antigen and antibody, and the antibody unit is defined as the amount of antibody that will inhibit 0.5 units of Factor VIII. The third method, known as "the Bethesda method," incubates equal volumes of normal plasma and diluted patient plasma for 2 hours at 37~ The control consists of normal plasma and imidazole buffer. The residual Factor VIII is measured by a specific Factor VIII

assay. The ratio between the test plasma and control is calculated. An autoantibody unit is defined as the amount of antibody that will inhibit a 0.5 units of Factor VIII (Armitage et al., 1994).

In vivo Detection of Autoantibodies. A shortened half-life of an infused clotting factor can indicate the presence of an autoantibody. An infusion of the factor concentrate to raise the plasma level to around 50% of normal values is followed by sampling the patient at different time intervals after the completion of the infusion (Kaspar and Ewing, 1986).

REFERENCES Aledort L. Inhibitors in hemophilia patients: current status and management. Am J Hematol 1994;47:208-217. Armitage JB, Hernandez JA, Kaplan HS. Laboratory assessment of circulating anticoagulants. Clin Lab Med 1994;14:795-812. Arnaud E, Lafay M, Gaussem P, Piccard V, Jandrot-Perrus M, Aiach M, Rendu F. An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation. Blood 1994;84:1843--1850. Baudo F, Redaelli R, Pezzetti L, Caimi TM, Busnach G, Perrino L, de Cataldo F. Prothrombin-antibody coexistent with lupus anticoagulant (LA): clinical study and immunochemical characterization. Thromb Res 1990;57:279--287. Bick RL. Acquired circulating anticoagulants. In: Bick RL, editor. Disorders of Thrombosis and Hemostasis: Clinical and Laboratory Practice. Chicago: ASCP Press, 1992:223-232. de Raucourt E, Dumont MD, Tourani JM, Hubsch JP, Riquet M, Fischer AM. Acquired factor VII deficiency associated with pleural liposarcoma. Blood Coagul Fibrinolysis 1994;5: 833--836. Fukue H, Anderson K, McPhedran P, Clyne L, McDonagh J. A unique factor XIII inhibitor to a fibrin-binding site on factor XIIIA. Blood 1992;79:65--74. Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988;53:505--518. Furie B, Furie BC. Molecular and cellular biology of blood coagulation. N Eng J Med 1992;326:800-806. Hassouna HI. Laboratory evaluation of hemostatic disorders. Hematol Oncol Clin North Am 1993;7:1161-1249. Israels SJ, Israels ED. Development of antibodies to bovine and human factor V in two children after exposure to topical bovine thrombin. Am J Pediatr Hematol Oncol 1994;16: 249--254. Jakway JL. Acquired von Willebrand's disease in malignancy.

CONCLUSION Although coagulation factor autoantibodies are rare events in clinical practice, suspicion should arise when an unexplained bleeding diathesis occurs in a patient, or screening tests such as PT or A P T T exhibit prolonged results without any known cause. The triggering mechanism of autoimmunity against coagulation factors is still a matter of debate. Both screening and confirmatory methods for the detection and quantitation of the autoantibodies are available and can be carried out in an experienced laboratory. The study of coagulation factors autoantibodies will undoubtedly continue to grow in significance and complexity, demanding ever greater laboratory expertise and competence.

Semin Thromb Hemost 1992;18:434--439. Kaspar CK, Ewing NP. Acquired inhibitors of plasma coagulation factors. J Med Technol 1986;3:431-439. Kunkel LA. Acquired circulating anticoagulants. Hematol Oncol Clin North Am 1992;6:1341-1357. Mann KG, Nesheim ME, Tracy PB, Hibbard LS, Bloom JS. Assembly of the prothrombinase complex. Biophys J 1982; 37:106--107. Mann KG, Jenny RJ, Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes. Annu Rev Biochem 1988;57:915--956. Margolius A, Jackson DP, Ratnoff OD. Circulating anticoagulants: a study of 40 cases and a review of the literature. Medicine 1961 ;40:145-156. Naito K, Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces. J Biol Chem 1991;226:7353--7358. Orstavik KH, Miller CH. IgG subclass identification of inhibitors to factor IX in haemophilia B patients. Br J Haematol 1988;68:451--454. Rao LV, Zivelin A, Iturbe I, Rapaport SI. Antibody-induced acute factor X deficiency: clinical manifestations and properties of the antibody. Thromb Hemost 1994;72:363-371. Roberts HR, Eberst ME. Current management of hemophilia B. Hematol Oncol Clin North Am 1993;7:1269--1280. Schapiro SS, Siegel JF. Hemorrhagic disorders associated with circulating inhibitors. In: Ratnoff OD, Forbes CD, editors. Disorders of Hemostasis. Philadelphia: WB Saunders, 1991:245-260. Sie P, Bezeaud A, Dupouy D, Archipoff G, Freyssinet JM, Dugoujon JM, Serre G, Guillin MC, Boneu B. An acquired antithrombin autoantibody directed toward the catalytic center of the enzyme. J Clin Invest 1991;88:290-296. Suehisa E, Toku M, Akita N, Fushima R, Takano T, Tada H, Iwatani Y, Amino N. Study on an antibody against F1F2

183

fragment of human factor V in a patient with Hashimoto's disease and bullous pemphigoid. Thromb Res 1995;77:63--68. Tosetto A, Rodeghiero F, Gatto E, Manotti C, Poli T. An acquired hemorrhagic disorder of fibrin crosslinking due to IgG antibodies to FXIII, successfully treated with FXIII replacement and cyclophosphamide. Am J Hematol 1995;48: 34-39. van Genderen PJ, Vink T, Michiels JJ, van't Veer MB, Sixma JJ, van Vliet HH. Acquired von Willebrand disease caused by an autoantibody selectively inhibiting the binding of von Willebrand factor to collagen. Blood 1994;84:3378-3384.

184

Wachtfogel YT, DeLa Cadena RA, Colman RW. Structural biology, cellular interactions and pathophysiology of the contact system. Thromb Res 1993;72:1--21. Wise RJ, Pittman DD, Handin RI, Kaufman RJ, Orkin SH. The propeptide of von Willebrand factor independently mediates the assembly of von Willebrand multimers. Cell 1988;52: 229-236. Zhender JL, Leung LL. Development of antibodies to thrombin and factor V with recurrent bleeding in a patient exposed to topical bovine thrombin. Blood 1990;76:2011--2016.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

COLLAGEN AUTOANTIBODIES GuoQiu Shen, M.D.

Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES The word collagen is a 19th century French neologism meant to designate the constituent of connective tissue that produces glue (Eastoe, 1967; van der Rest and Garrone, 1991). In the 1940s, investigators revealed that collagen alterations occurred in patients with systemic lupus erythematosus (SLE), scleroderma, dermatomyositis and periarteritis nodosa. Therefore, these conditions were defined as collagen diseases (Klemperer et al., 1942). By the end of the 1960s, the basic structure of all native collagens present in connective tissues such as bone, cartilage, skin, tendons, ligaments, synovial spaces and the vitreous gel of the eye were defined as consisting of triple helix, polypeptide chains composed of repeating glycine, proline and hydroxyproline residues (Morgan, 1990; Alberts et al., 1983).

THE AUTOANTIGENS

IV collagen, a 7S region and noncollagen (NC1) domains. But type VIII and X collagens form regular hexagonal lattices in the basement membrane of the corneal endothelium (Descemet's membrane). Type VI forms beaded filaments; this collagen is found in placenta, ligaments, skin, cornea, cartilage and intervertebral discs. Type VII participates in anchoring fibrils of epidermal-dermal junction (van der Rest and Garrone, 1991) (Figure 1).

Procollagen. Procollagen differs from collagen by a characteristic N-terminal peptide extension. Figure 2 shows the structure of type I procollagen (Byers, 1990; Mundlos and Spranger, 1991). Procollagen is a better immunogen than collagen and a large proportion of the antibodies in anticollagen-positive sera are specific for the procollagen extension. Collagenase digestion of pc~-chain or procollagen is a convenient way to obtain the immunologically active procollagen peptide. Complete cleavage of all intrachain disulfide bonds in pro-s- and p~-chain abolished serological reactivity (Byers, 1990).

Origin/Sources/Structure Methods of Purification The main structural proteins of the connective tissues in the body are collagens (Table 1), of which at least 14 genetically distinct types have so far been described (van der Rest and Garrone, 1991) (Figure 1). The fibrils found in most connective tissues (including bone, skin, cartilage, blood vessels, synovial membrane, liver and other tissues) are made up of allotypes of fibrillar collagens (types I, II, III, V and XI). Types IX and XIII collagens, which are found in cartilage, skin and tendons, contain interrupted triple helical domains and large NHz-terminal domains. Sheet basement membranes such as blood vessel, kidney, lung, eye and skin consists of triple helix type

Several methods are used to purify collagens, including extraction by NaC1 solutions (Anesey et al., 1975; Moro and Smith, 1977), acetic acid (Borel and Randoux, 1985; Timpl et al., 1978; Bazin and Delaunay, 1976) and other nondenaturing agents (Borel and Randoux, 1985; Timpl et al., 1978; Bazin and Delaunay, 1976), enzyme digestion, precipitation and chromatography.

Collagen Type II Preparation. Most experiments use native type II collagen prepared by limited pepsin digestion of bovine sternal cartilage and purified as

185

Table 1. Collagen Contents of some Tissues Tissue Liver

Collagen (g/100 g dry weight)* 3.9

Lung

10

Aorta

12--24

Ligamentum nuchae

17.0

Cartilage

46.1-63.7

Cornea

68.1

Skin

7.19

Achilles tendon

86.0

Whole cortical bone

22.8

Mineral-free cortical bone

88.0

*Values for ligamentum nuchae, cartilage and bone from bovine tissue; remainder from human tissues.

described (Borel and Randoux, 1985; Grant et al., 1988; Miller, 1972). Sternal cartilage is homogenized and extracted with 0.5 M acetic acid at 4~ for 48 h. After centrifugation, the pellets are digested with pepsin at a 1:50 ratio (e.g., add 40 mg pepsin per 2 g collagen extract) in 0.5 M acetic acid and incubated at 4~ for 12 h with stirring. Subsequent fractionation of the pepsin digests demonstrates that cartilaginous tissues contain several quantitatively minor collagens (IX, X and XI) in addition to type II collagen. These collagens can be separated from type II collagen and from each other by differential salt precipitation at acid pH. The precipitate is collected, dissolved in 2 M urea, 0.05 M Tris, pH 8.6. The extract is loaded on a DE-52 anion exchange collum which has been equilibrated with 2 M urea 0.05 M Tris, pH 8.6. The unbound fractions are pooled and concentrated by an Amicon ultrafiltration cell (membrane cutoff 10 kd).

Sequence Information Epitopes The genes for the major fibrillar collagens I, II and III have 52 to 54 exons. The exons code for the large triple-helical domain of the proteins and have a distinctive 54-base pair motif. Other exons are 108 base pairs (twice 54), and one is 162 base pairs (3 times of 54). The genes are widely dispersed in the genome, collagen IV c~l and c~2 on chromosome 13q34 and collagen VI c~l and {~2 on 21q22.3 (Cole, 1994). In the common collagens, the c~ chains each contain about 1050 amino acid residues and the molecule is 300 nm long.

AUTOANTIBODIES Pathogenetic Role

Commercial Sources Purified and crude collagens are available commercially from many sources (Accurate Chemical & Scientific Corp., Westbury, NY; Chemicon International Inc., Temecula, CA; Fisher Scientific, Tustin, CA; Heyltex Corporation, Houston, TX; ICN Biochemicals, Costa Mesa, CA; Sera-Lab Ltd., Sussex, England; Sigma Chemical Company, St. Louis, MO; Southern Biotechnology Assoc. Inc., Birminghan, AL; Telios Pharm. Inc., San Diego, CA; Vmrd Inc., Pullman, WA; Worthington Biochem. Corp., Freehold, NJ).

186

The hypothesis that autoimmunity to type II collagen is instrumental in the pathogenesis of rheumatoid arthritis is supported by several lines of investigation: (1) Antibodies to collagen II are detected in the serum, synovial fluid and cartilage of RA patients. These antibodies consist primarily of complementfixing IgG isotypes and are capable of binding to homologous cartilage and of converting C5 to C5a (Terato et al., 1990; Morgan, 1990; Clague, 1989). (2) Susceptible strains of rodents and nonhuman primates immunized with type II collagen produce high titers of autoreactive antibodies and develop an erosive

Figure 1. Molecular structure and supramolecular assemblies of collagens. This figure combines schematic scale representations and electron microscope micrographs of molecules and aggregates of various collagen types. AF: anchoring fibrils; BM: basement membrane; CF: collagen fibrils; GAG: glycosaminoglycan; NCI: noncollagen domain 1; NC4: noncollagen domain 4.7S is the domain of antiparallel interaction of type IV collage triple helices to form a tetramer (spider).

polyarthritis (Table 2) (Trentham et al., 1977; Durie et al., 1994; Courtenay et al., 1980; Cathcart et al.,

1986; Kerwar and Oronsky, 1988; Staines and Wooley, 1994). (3) The pathogenic potential of purified

187

Radiolabeled human antitype II collagen IgG accumulated in the peripheral joints of mice. Also cell-mediated immunity to collagen is important in RA animal models. Cellular studies have revealed that a longer-term arthritis results from passive transfer of spleen and lymph node cells isolated from rats with CIA (Trentham et al., 1978). Cells reactive with both native and denatured collagen are capable of inducing the disease (Poole et al., 1988).

Factors in Pathogenesis

Figure 2. Structure of procollagen type I molecule.

human anticollagen II antibodies is demonstrated by their ability to passively transfer arthritis into mice (Wooley et al., 1984a; Kerwar and Oronsky, 1988; Staines and Wooley, 1994). Animal Models. Collagen-induced arthritis (CIA) has been demonstrated to resemble human rheumatoid arthritis (RA) sufficiently to now be recognized as an important experimental tool (Trentham, et al., 1977; Durie et al., 1994). CIA can be induced in several species including primates by immunization with heterologous type-II collagen which is isolated from articular cartilage in a heterologous species (Durie et al., 1994; Courtenay et al., 1980; Cathcart et al., 1986). CIA is an acute disease and involves synovitis, periostitis, pannus and erosions. Other types of collagen or denatured collagen II were not able to induce arthritis; other types of collagens such as type I and III are able to induce an immune reaction in rats and mice but are incapable of producing CIA (Stuart et al., 1984). Passive immunization with monoclonal antibodies against collagen II suggests that no single epitope presented by collagen II is sufficient to induce arthritis; rather, several antibody species epitopes must be present simultaneously to induce the full arthritic response (Terato et al., 1992). Mice intravenously injected with the serum IgG fraction from a patients with seronegative rheumatoid-like arthritis which contained a high antitype II collagen antibody titer were susceptible to type II collagen-induced arthritis. Purified human antitype II collagen immunoglobulin injected into the knee joints of mice was shown to induce a mild, transient, inflammatory arthritis which was observed in 20--25% of the animals (Table 3).

188

Anticollagen antibodies are mainly of the IgG class, though IgM and IgA antibodies to collagens occur in some patients in association with the IgG antibodies. IgG1 and IgG3 are the predomint subclasses of IgG for both native and denatured type II collagen. Both these subclasses are potent fixers of complement (Collins et al., 1988; Morgan, 1990). Antibodies to collagen II occur more commonly very early in the disease and later disappear in most patients (Morgan, 1990).

Methods of Detection A number of investigators, using a variety of methods, such as passive hemagglutination, radioimmunoassay (RIA), immunofluorescence and enzyme-linked immunosorbent assay (ELISA) demonstrate antibodies that react with various types of collagen presenting in serum and synovial fluid (Lotz and Vaughan, 1988; Terato et al., 1990). There are discrepancies in the reported incidence and specificity of anticollagen antibodies which are attributed to differences in the sensitivities of the assays, different sources and concentrations of antigens (Table 2). ELISA and RIA are both highly sensitive techniques not subject to interference by nonantibody proteins (Clague, 1989; Beard et al., 1979). The RIA showed a greater sensitivity (54/75 RA patients for DCII, 41/75 for NCII) than either passive hemoagglutination (28/75 for NCII) or IFA (31/75) (Clague et al., 1983). ELISA offers several advantages: the assay does not require radioactive materials and performance time is shorter (4 h) than RIA (more than 24 h) (Terato et al., 1990). Most studies for detection of anticollagen antibodies utilize ELISA assays. Recently, a solid-phase enzyme-linked immunospot (ELISPOT) assay was developed which is performed like ELISA, where living cells in cell culture medium

Table 2. Anticollagen Antibodies Authors

Methods and Antigen Concentrations

Results

Michael et al., 1974

hemagglutination; hdCI

100 RA: 60% (+)

Andriopoulos et al., 1976

hemagglutination; hnCI, II, III; hdCI, II, III.

1 l0 RA: 97%(+) hnCI and hdCI; 94%(+) hnCII, 96%(+) hdCII; 85%(+) hnCIII. 50 nhS: 6%.

Menzei et al., 1978

Radioimmunoassay (RIA); hnCI and hdCI

27 RA SF: 30%(+) hnCI; 74%(+) hdCI

Smolen et al., 1978

hemagglutination; hnCI.

20 thromboangiitis obliterans: 35% hnCI. 34 nhS: 0%.

Ebringer et al., 1981

ELISA (hnCII 1 pg/mL coating) IFA (using fetal cartilage)

10 relapsing polychondritis: 60%(+) hnCII & 60% (+) IFA. 50RA: 80%(+) for hnCII. 260 RA: 2% (+) for IFA. 21 NHS 0%.

Mackel et al., 1982

ELISA; murine nCI, IV. 5 lag/mL

22 scleroderma: 54%(+) for nCIV, 86%(+) for nCI. 30nHS: 0%.

Black et al., 1983

ELISA; hnCI, II, III, IV, V.

106 SSc compare with 43 nHS" hnCI, II, IV Ab(s) sig. higher; hnCII,V Ab(S) no sig.

Adar et al., 1983

RIA; hcI, III

39 thromboangiitis obliterans: 44% hnCI or hnCIII; 20 nHS" 0%.

Clague et al., 1983

RIA; passive hemoagglutination and IFA; bnCII, bdCII.

75 RA; RIA: 72% dCII, 55% nCII; passive hemoagglutination: 37% nCII; IFA: 41%. 10 nHS: 0%.

Jasin HE, 1985

RIA; bnCII; bdCII.

30 RA Serum: 13% nCII, 30% dCII; 13 hHS: 0%; 17 RA SF: 53% nCII, 71% dCII. 13 OA SF: 12% nCII and dCII.

Petty et al., 1986

ELISA; bnCIV, bdCIV and hnCIV, hdCIV. 50 ~tg/mL.

20 JRA: 10% bnCIV, 5% bdCIV, 0% hnCIV, 5% hdCIV; 25 RA: 8%, 16%, 4%, 52%; 20 SLE: 15%, 15%, 45%, 40%" 27 PPS: 11%, 26%, 7%, 41%; 20 JDM: 15%, 25%, 0%, 20%; 20 MTCD: 20%, 25%, 10%, 20%. 30 nHS" 6%, 6%, 0%, 6%.

Watson et al., 1986

ELISA; hnCII. 1 ~tg/mL

9 RA: Anti-hnCII IgG subclass: IgG1 6%(+), IgG2 0%, IgG3 92%(+), IgG4 2%(+)

Morgan et al., 1987

ELISA; bcI, II, IX and XI. 10 ~tg/mL

76 RA, 12% bnCI, 4% bnCIX, 12% bnCXI; 36% bdCI, 93% bdCII, 12% bdCIX, 40% bdCXI. 90 bHS: 1%. bdI, II, IX, XI.

Collins et al., 1988

ELISA; bnCII, bdCII, l0 ~tg/mL.

81 RA: Anti-bnCII IgG subclass: G1 70%, G2 12%, G3 84%, G4 6%; Anti-bdCII IgG subclass: G1 86%, G2 23%, G3 86%, G4 6%. 50 nHS" 0%.

Gabrielli et al., 1988

ELISA; mouse CIV. 1 ~tg/mL

12 SLE: 8%(+); 5 MCTD: 0%; 48 PRP: 21% (+); 40 SSc: 68%(+). 38 NHS" 0%.

Morgan et al., 1988

ELISA; bnCII,XI; bdCII, XI 10 lag/mL. IB: a(II), a l(XI), a2(XI), a3(XI).

46 RA: ELISA: 89%(+) for bnCII, 17%(+) for bdCII; 87%(+) for bnCXI, 59%(+) for bdCXI. IB: 89% for a(II), 7% for al(XI), 35% for a2(XI), 80% for a3(XI)

(continued)

G~ ~D

Table 2. Continued

Authors

Methods and Antigen Concentrations

Results

Morgan et al., 1989

ELISA; bnCI, II, IX and XI; bdCI, II, IX, XI. l0 ~g/mL.

15 RA: 100%(+) for bnCII, 100%(+) for bdCII, 20%(+) for bnCI, 27%(+) for bdCI; 27%(+) for bnCIX and bdCX; 33%(+) for bnCIX, 80%(+) bdCIX.

Tarkowski, et al., 1989

ELISPOT (using SF B cells) and ELISA; rat nCI, II. 10 ~g/mL coating.

13 RA with RF: 92%(+) B cell secreted anti-nCII Ab; 14 RA RF negative 64%(+) B cell secreted anti-nCII Ab. Serum anti-nCII no sig. compare with 10 nHS, 7%(+) nCI.

Terato et al., 1990

ELISA; native hnCII, bnCII,cCII. 5 ~tg/mL

202 RA: 23% hCII, nCII 21%, cCII 17%; 26 RP: hCII 11%, 13% cCII; 19 OA" 1% ahCII, nCII, cCII; 54 gout: hCII and bCII 0%, cCII 4%" 200 nHS: hCII 0%, bCII 4%, cCII 1%.

Moreland et al., 1991

Radioimmunoassay; hnCI, II, III, IV, V, VI. l0 ~tg/mL.

20 SLE: 85% for CI, 60% CII, 44% CIII, 85% CIV, 70% CV, 15% CVI; 20 vasculitis: 20% CI, 35% CII, 0% CIII, 55% CIV, 15% CV, 0% CVI. 9 NHS: 0%.

Kobayashi et al., 1992

ELISA; hnCII, III. 5 ~g/mL

38 Kawasaki's disease: 18% CIII, 0% CII; 25 JRA: 20% CII, 4% CIII; 7 SLE and 1 MCTD: 0% CII, CIII. 114 NHS" 0%.

Morgan et al., 1993

ELISA; bnCII, bdCII. 10 jag/mL

79 early RA: anti-nCII: IgM and IgA 0%, IgG 3.8%. anti-dCII: IgM 10%, 0% IgA, 4% IgG.

Ronnelid et al., 1994.

ELISPOT; rat nCII. l0 ~g/mL.

31 RA: 52% for anti-CII-reactive cell in SF, 0% in blood cell.

CCI and CCII: chicken collagen I and II; CI to CXI: collagen I to XI; NCI to NCXI: native collagen I to XI; DCI to DCXI: denatured collagen I to XI; hnCI to hnCXI: human native collagen I to XI; hdCI to hdCXI: human denatured collagen I to XI; BNCI to BNXI: bovine native collagen I to XI; BDCI to BDCXI: bovine denatured collagen I to XI; CCII: chicken collagen II; RA: rheumatoid arthritis; RP: relapsing polychondritis; SSc: systemic sclerosis; SLE: systemic lupus erythematosus; MCTD: mixed connective tissue disease; PRP: primary Raynaud's phenomenon; OA: Osteoarthritis; NHS: normal healthy serum; SF: Synovial fluid; %: positive; ELISPOT: Detecting B cell secreted antibodies; sig.: significantly.

Table 3. Passive Transfer of Arthritis to Mice by Human Anti-type II Collagen Antibody Injected Intravenously Mouse Strain

IgG Preparation

Clinical Arthritis Incidence

Percent (%)

DBA/1J

hH Anti-CII

5/21

24

B 10.Q

hH Anti-CII

2/8

25

B 10.RIII

hH Anti-CII

1/5

20

B 10.M

hH Anti-CII

0/3

0

DBA/1J

RA IgG

0/4

0

B 10.Q

RA IgG

0/4

0

B 10.RIII

RA IgG

0/4

0

DBA/1J

Normal Human IgG

0/8

0

B 10.RIII

Normal Human IgG

0/4

0

are incubated on collagen II precoated plates. Only antibodies produced during the culture period and the antibody-producing B cells/plasma cells adhere to the plates. The reactions are determined as in a conventional ELISA (Tarkowski et al., 1989).

CLINICAL UTILITY Application Antibodies to collagens may be useful as markers of cartilage destruction in some patients. Changes in serum antibodies to collagens in individual patients may provide an early indication of renewed cartilage destruction in previously affected joints or in newly affected joints before it is clinically evident, thus allowing early appropriate treatment (Morgan, 1990; Morgan et al., 1993). In relapsing polychondritis (RPC), a human condition involving an inflammatory erosion and destruction of many of the hyaline and elastic tissues, the frequency of serum antibodies to both human native collagen II and fetal cartilage (by IFA) was 60%; these antibodies may play an important role in the pathogenesis of cartilage destruction in RPC (Ebringer et al., 1981).

Disease Association Humoral- and cell-mediated immunity to collagen is described in patients with rheumatoid arthritis (RA),

SLE, progressive systemic sclerosis (SSc), relapsing polychondritis, thromboangiitis obliterans (Buerger's disease) and other inflammatory conditions. Antibodies to native and denatured collagens, in particular to types I, II, III, IV, V, IX and XI, are reported in a number of diseases including RA, juvenile RA, SLE, SSc, relapsing polychondritis, mixed connective tissue disease (MCTD), primary Raynaud's phenomenon, ankylosing spondylitis, osteoarthritis, psoriatic arthritis and Kawasaki disease (Table 2). Most reports that the incidence (frequency) of anticollagen antibodies in RA patients sera is about 30-70% (positive for antibodies to native or denatured collagen II). Anticollagen antibodies were higher in synovial fluid relative to the total IgG than they were in simultaneously obtained blood serum from RA patients (Menzel et al., 1978). Anticollagen antibodies were eluted from cartilage samples of 69% (nine of 13) RA patients and likely contribute to the pathogenesis of joint injury (Terato et al., 1990). Most collagen autoantibodies in RA sera cross-reacted with all the heterologous type II collagens tested (Terato et al., 1990). Anticollagen I and II antibody secreting cells have been detected in rheumatoid synovia (Tarkowski et al., 1989). Antibodies are detected in juvenile RA, but there is no report of transplacental transfer to the neonate (Lotz and Vaughan, 1988; Terato et al., 1990; Lawrence et al., 1993). In SLE patients, autoantibodies to collagen IV were detected in 85%, collagen V in 70% and to collagens I and II in 15--35%. Autoantibodies

191

to type IV and V are involved in the immune response and may perpetuate vascular damage (Petty et al., 1986; Moreland et al., 1991). About 35--44% of patients with thromboangiitis obliterans (Buerger's disease) have antibodies to type I and IV collagens and 77% of patients have cell-mediated sensitivity to these two collagens (Smolen et al., 1978; Adar et al., 1983). In patients with scleroderma, antibodies against type I and IV collagens are elevated to 86 and 68%, respectively; only 12% of these patients display cellular immunity to collagen IV. Autoantibodies to basement membrane and interstitial collagens may participate in the pathogenesis of scleroderma (Mackel et al., 1982; Gabrielli et al., 1988; Black et al., 1983; Petty et al., 1986).

correlated with RA or other disease (Elson, 1993; Delustro et al., 1990; Frey et al., 1994; Zeide, 1986; Cooperman et al., 1985). Two quite unrelated proteins containing collagenlike sequences are the C lq subcomponent of the complement and acetylcholinesterase. In SLE, autoantibodies to the collagen-like portion of C lq could cross-react with collagen (Antes et al., 1988). Antibodies detected to denatured type XI collagen (~3 chain) may only be antibodies raised to the biochemically similar type II collagen (c~l chain); type XI collagen itself may not be immunogenic (Burgeson et al., 1979; Morgan et al., 1988).

Injectable Collagen and Autoimmune Disease

Rheumatoid arthritis is associated with HLA-DR4 type; increased cellular and humoral responses to collagen have been linked to DR4. In 105 patients with RA, there are no significant associations between any HLA antigens (A,B or DR) and a high antibody titer to native collagen, but significant associations between HLA antigens and high antibody titers to denatured collagen. Those patients with DR4 and DRw53 had high titers and those patients with DR2 or A3;B7;DR2 had low titers. Also A2 and DR4 together were the best markers for high antibody titers to denatured collagen II (Rowley et al., 1990; Stastny, 1978; Dyer et al., 1982; Wooley et al., 1984b).

The most widely characterized collagen devices are injectable collagens used for the correction of softtissue contour irregularities. The collagen implant (ZCI) consists of greater than 95% collagen I and the remainder of type III collagen. It is a weak immunogen in humans to which approximately 3% of the population develops hypersensitivity to the initial skin challenge with injectable collagen; most of the reactions occur within the first 72 hours. In addition, approximately 1% of subjects develop localized hypersensitivity response. Approximately 100 cases of alleged autoimmune disease out of 500,000 injections have been reported in the US and Canada, including 11 cases of polymyositis/dermatomyositis (PM/DM).

Effects of Oral Administration of Type V Oral administration of native type II--V collagen ameliorates two animal models of rheumatoid arthritis induced by collagen II. Of 60 patients with severe active rheumatoid arthritis, 28 patients who received chicken type II--V collagens for 3 months had a decrease in the number of swollen and tender joints, four patients had complete remission of the disease; 31 patients who received a placebo experienced no significant improvement (Trentham et al., 1993).

False-Positive Reaction, Cross-Reaction About 5% normal population have antibodies to bovine denatured collagen I indicating a presensitization, presumably due to dietary exposure. However, antibodies to denatured collagen I are not necessarily

192

Genetics

CONCLUSION Collagen is the most common protein in the animal world. Of the many collagenous structures involved in autoimmune disease, sites rich in basement membrane are especially prone to immunologically mediated injury. In rodents and monkeys, immunization with type II collagen produces collagen antibodies which, in turn, induce arthritis in these animals. Collageninduced arthritis in mice can be passively transferred with immunoglobulin concentrate from immunized donors to nonimmunized recipients. RIA and ELISA are the most commonly used methods for detecting collagen autoantibodies and the immunochemical properties of the collagen and precollagen antigens. Numerous investigations into the pathogenesis of autoimmune rheumatic diseases such as mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), rheumatoid arthritis (RA) and vasculi-

tides detected autoantibodies to collagen. In RA patients, antibodies to cartilage collagens can be present even after disease of long duration; and antibodies to native and denatured collagen II may be associated with severe disease. Changes of serum

anticollagen antibody levels in individual patients may provide an early indication of tissue destruction and monitor treatment efficacy (Morgan, 1990; M o r g a n et al., 1993; Clague, 1989).

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CRYOGLOBULINS Giuseppe Montagnino, M.D.

Divisione di Nefrologia e Dialisi, Ospedale Maggiore, IRCCS, 20122 Milan, Italy

H I S T O R I C A L NOTES Cryoglobulins are cold-precipitating immunoglobulins that spontaneously form insoluble aggregates when exposed to temperatures below 25~ They are classified into three main types based on the immunochemical characterization (Brouet et al., 1974) (Table 1).

THE AUTOANTIGENS Cryoprecipitation can be caused by intrinsic characteristics of both the monoclonal and the polyclonal immunoglobulin component, as well as the interactions among the individual components of the cryoprecipitate. Structural modifications of the variable portions of the H and L chains (Middaugh and Litman, 1987), reduced concentrations of sialic acid, reduced amounts of galactose in the Fc portion of the IgG (Tomana et al., 1988) and the presence of Nlinked glycosylation sites in the CH3 domain have each been demonstrated to contribute to immunoglobulin cold insolubility. The acquisition or loss of

charged amino acid residues as a result of somatic mutations in autoantibodies during the course of autoimmune responses also has been suggested as a possible cause of pathogenic, cryogenerating autoantibodies (Shlomchik et al., 1990). Nonspecific Fc-Fc interactions are a possible mechanism of self-aggregation for some immunoglobulins (Gyotoku et al., 1987). Other interactions are specific and involve the classical rheumatoid factor (RF) reaction between the cryoprecipitable IgM and the Fc portion of the corresponding IgG. Since the IgM component is an antiglobulin, these observations suggest that, at least in type II and III essential mixed cryoglobulinemia (EMC), the autoantigen is the immunoglobulin itself (mainly IgG). Origin Hepatitis B surface antigen (HBsAg) or hepatitis B surface antibody (HBsAb) is present in the cryoprecipitate, and anti-HBsAg reactivity of the cryoglobulin IgG can be isolated from EMC patients (Geltner et al., 1980). However, of 63 patients reported by different investigators, only two had HBsAg in their sera and

Table 1. Cryoglobulins Composition

Characteristics

Disease Association

Type I

Presence of a single monoclonal Ig (usually IgG, less frequently IgM or IgA, or even Bence-Jones proteins).

Self-association through the Fc portion of the molecule

Lymphoproliferative disorders: myeloma, Sjtigren's syndrome, Waldenstr6m's macroglobulinemia

Type II (mixed)

Composed of a monoclonal component (usually IgM, less frequently IgG or IgA) and by a polyclonal Ig (usually of the IgG isotype),

The monoclonal component has a rheumatoidfactor (RF) activity againstthe Fc portion of the polyclonal Ig.

Autoimmune diseases, chronic infections, essential forms.

Type III

Mixed polyclonal Ig of all isotypes

RF activity of one of the polyclonal components.

Autoimmune diseases, chronic infections, essential forms.

195

two others had HBsAb in serum or cryoprecipitate. In a multicenter Italian study (Tarantino et al., 1986) only four of 91 EMC patients were HBsAg-positive and HBsAg was found in the sera of only two of 19 patients from whom liver biopsies were taken. Epstein-Barr virus was considered as the possible viral agent underlying Type II EMC (Fiorini et al., 1988). More recent data suggest an association between hepatitis C and Type II cryoglobulinemia (Dammacco and Sansonno, 1992). Anti-HTLV-I activity was found in the plasma and washed cryoproteins from a patient with Type II essential cryoglobulinemia (EC) and a patient with Type I EC. The first patient showed specific antibodies to HTLV-I gag p19 and gag-precursor p55, while anti-p55 reactivity was detected in the second patient (Perl et al., 1991). In addition to core proteins, the env products gp46 and gp68 also were precipitated by sera from both the Type I and Type II EC patients. Four other Type II EC patients also showed reactivity with gag and/or env proteins. Cryoprotein-free serum samples were not reactive with HTLV-I proteins. Immunoblot and immunoprecipitation assays showed that reverse transcription and HTLV-I-related retroviral proteins might be involved in the pathogenesis of some subsets of EC. Moreover, striking amino acid homologies between certain retroviral gag proteins and human autoantigens suggest that the natural targets of HTLV-I reactive antibodies in these EC patients may be endogenous retroviral sequences (Haul et al., 1989). More recently, sera from seven EMC patients with active renal involvement reacted with a 50 kd kidney antigen, suggesting the presence of circulating glomerular-specific autoantibodies that might contribute to the induction of glomerulonephritis in EMC by forming immunocomplexes in situ (Dolcher et al., 1994).

THE AUTOANTIBODIES Pathogenetic Role

While F(ab') 2 fragments from cryoglobulins with no rheumatoid factor activity can precipitate by themselves at low temperatures, F(ab') 2 fragments from cryoglobulins with antiglobulin activity require the corresponding antigen for cold precipitation. Cryoprecipitable IgM from six patients with EMC had variable affinity for the Fc fragment of normal human IgG (Johnston and Abraham, 1979). Moreover, the

196

molar concentrations of Fc required for the reaction with the corresponding IgM were higher than those sufficient for the reaction with the intact IgG, and the reactivity with the Fc fragment was much weaker than with the intact molecule or with nonaggregated or partially reduced y H chains. The antigenic determinant is located in or near the hinge region of the IgG or within the amino-terminal portion of the IInd constant region of the y H chains. The absence of reactivity with fragments that lack an intact Cy2-Cy3 junction (i.e., pFc', Fc' or rabbit pF(acb)2 ) is one possible explanation for these results (Sasso et al., 1988) and the C3/2-Cy3 cleft is a likely site for binding of RF (Oppliger et al., 1987; Sasso et al., 1988). On the other hand, some IgM RF have specificities to allotypes of human IgG. The antigenic site related to or involving the allotype Gml occurs on the pFc' fragment and is located away from the Cy2-Cy3 interface area, close to the tail end of the Cy3 domain (Deisenhofer, 1981). The presence of glycosylation sites in the CH3 domain might influence the binding between the cryoimmunoglobulins. However, the binding of human monoclonal RF from patients with EMC is influenced by the isotype of the Fc fragment of the IgG and not by the extent of glycosylation (Newkirk et al., 1990). Enrichment of some IgG isotypes could be a factor in cryoprecipitability. Such isotype enrichment occurs both in experimental murine models of cryoglobulinemia (MRL-lpr mice) and in man, but the mechanisms accounting for this are less clear. Animal Models. The cryoprecipitable RF of MRL-Ipr mice show a marked enrichment in IgG3 (Shibata et al., 1992), as do nonautoimmune mice after polyclonal B-cell stimulation by bacterial lipopolysaccharides or infection with malaria parasites (Abdelmoula et al., 1989). Sera from lpr strains of mice react equally well with IgG1, IgG2a and IgG2b, but less well with the IgG3 subclass. V region sequences utilized for RF from MRL-lpr mice could be more cryogenic than those from other strains of mice resulting from extensive mutations in V regions of their RF and/or could be related to differences in fine specificity of RF (Shibata et al., 1992). The Cy3 constant region plays a direct role in cryoglobulin generation: cryoglobulin activity is gained after an immunoglobulin class switch of murine antibodies (mAb) from IgM to IgG3, but lost after a class switch from IgG3 to IgG1. Human Disease. The RF from cryoglobulinemia

patients bind preferentially to IgG1 and/or IgG2 isotypes, although a small subset binds equally well to IgG1, 2 and 3 (Newkirk et al., 1987; Sasso et al., 1988). Monoclonal RF from EMC patients bind equally well to Fc fragments from polyclonal and monoclonal IgG1, 2 and 4; hybridoma-generated RF from rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients bind poorly to Fc preparations from monoclonal IgG1, but better to normal and RA-derived polyclonal Fc preparations (Newkirk et al., 1990). The amino acid sequence of the Fc fragment appears to be important in determining binding; since each monoclonal RF displays a fine specificity of Fc isotype binding, there might be multiple RF binding sites on the Fc.

Genetics The majority of rheumatoid factors from unrelated individuals express cross-reactive idiotypes (CRI) and use selected V K genes (Radoux et al., 1986). Most monoclonal IgM RF share two primary sequencedependent CRI, corresponding to the second complementarity-determining region (CDR: heavy chain hypervariable region) and the third CDR of the ~: L chain. In contrast to the recurrent CRI on IgM RF L chains, the H chain idiotypes are extremely private. These results suggest either that RF H chains are encoded by a number of different V n and D n (diversity regions of H chain of immunoglobulin) genes or that IgM RF H chain sequences reflect an unusually high degree of somatic mutation in a limited number of V H genes (Chen et al., 1985). Therefore, monoclonal IgM RF synthesis is idiotypically stable in mixed cryoglobulinemia, suggesting that RF variable regions are not subject to somatic mutations during the course of the disease (Pasquali et al., 1989). Cryoglobulin IgM RF are derived from a limited set of germ-line genes (Radoux et al., 1986; Newkirk et al., 1987); IgM RF specificity for the Cy2-C73 interface is encoded in the germ line. It is likely that any alleged immunoregulatory role displayed by some IgM RF is germ-line encoded and not dependent upon random somatic mutations of their genes. However, it is possible that some RF result from somatic mutation of antibodies whose germ-line encoded specificities are directed against an antigen unrelated to their autoimmune specificity. Such DNA specificity arises in human myeloma proteins (Davidson et al., 1987). A monoclonal cryoglobulin IgM with reactivity against determinants shared by red blood cells and

immunoglobulins (usually carbohydrate determinants present both on the surface of erythrocytes and in the constant domains of the immunoglobulins) has also been reported (Farhangi and Merlini, 1986). These data might explain the "double reactivity" present in the IgM component of the cryocomplex. Similarities between mixed cryoglobulins and malignant B-cell chronic lymphocytic leukemia (B-cell CLL) have also been described. CLL B-cells display the CD5 antigen on their surface and rearrange the same V K gene as monoclonal RF secreting cells with high frequency (Kipps et al., 1987). However, the majority of peripheral blood monoclonal IgM RFsecreting cells from EMC patients are CD5-negative (Pasquali et al., 1991). Although the sequence of a monoclonal RF K variable region belongs to the human V K m group, as does that of B-cell CLL, these two diseases differ in CD5 membrane expression. This suggests either a different B-cell origin or differences in activation of the cells. These results indicate that clonal expansion of IgM RF-secreting B-cells occurs in EMC.

Clonal Expansion About 7-10% of monoclonal IgM are cryoglobulins (Duggan and Schattner, 1986). Flow cytometry and immunoglobulin gene rearrangement analysis of peripheral blood lymphocytes indicate clonal B-cell expansion of cells in the production of RF in EMC patients (Perl et al., 1989). Clonal expansion of B cells also was detected using DNA probes specific for the C~c, Cja and JH genes in 4/12 EMC patients, two of whom also showed specific expansions of ja heavy and ~: light chain-bearing cells. These data are in accordance with the presence of a solitary B-cell clone producing a monoclonal antibody with a unique idiotype. However, the clonally expanded B-cell populations detected by immunoglobulin gene rearrangements might not represent directly the cryoglobulin-producing B-cell clones, but might undergo further light chain rearrangements and class switch and represent the precursor of the cryoglobulinproducing cells (Perl et al., 1989).

Anti-Idiotypes The classical concept of reactivity between the Fab fragment of the RF and the Fc component of the corresponding autoantigenic IgG was challenged when IgM cryoglobulins endowed with a double reactivity

197

were isolated from 11 EMC patients (Geltner et al., 1980). These cryoglobulins possessed a classical RF reactivity against the Fc fragment and an anti-idiotypic reactivity against the F(ab') 2 fragment of both autologous and isologous IgG. Moreover, 4/5 of these IgG had anti-HBsAg reactivity. The reaction of the IgM with the corresponding IgG was inhibited by the addition of the putative HBsAg, suggesting that the antigen binding site of the IgG was also reactive with the IgM antiglobulins and that the last had to be considered as an anti-idiotype. This double reactivity could also explain the increased binding affinity of the complex and its reduced solubility in the cold. Alternatively, IgM fractions of cryoglobulins might preferentially bind to autologous cryoglobulin IgG (Renversez et al., 1984; 1986). However, rabbit antibodies that recognize epitopes on F(ab') 2 exposed by pepsin digestion were unable to detect idiotypic interactions between cryo IgM and the F(ab') 2 fragment of IgGs of 10 mixed cryoglobulins (Stone et al., 1988). Finally, IgM fractions that bind Fc fragments are unable to bind the intact cryoglobulin IgG from which these fragments are obtained (Renversez et al., 1984). These data suggest that either different antibody specificities are present within a monoclonal IgM population of molecules, or that some IgM fractions contain antibodies directed to hidden determinants of IgG exposed by pepsin digestion rather than antiidiotypes. The existence of homobodies (anti-idiotypes that are "internal images" of the antigen) and epibodies against a cross-reactive structure shared by epitopes and idiotopes on anti-idiotypes against them was demonstrated (Bona et al., 1984) (Figure 1). But how might an RF also be an epibody? Cells infected by certain viruses (HSV, CMV, VZV) can express Fc receptors (FcR) for IgG on their surfaces (Johansson et al., 1986). According to the network theory, antibodies against these FcR (c~-FcR) induce anti-idiotypic antibodies (~-~-FcR) which can be the "internal image" of FcR and are therefore epibodies directed both against the F(ab') 2 portion of ~-FcR (anti-idiotypic activity) and against the Fc portion of the IgG, as the internal image of the nominal antigen (antiepitope reactivity). Indeed, RF from 13 EMC patients bear the internal image of the Fc binding region of staphylococcal protein A (Oppliger et al., 1987). The binding site similarities between RFs and microbial Fc binding proteins suggest conformational similarities between the antigen-binding site of RF and the Fc binding-sites of these microbial structures (Nardella et 198

pl b

Abg

P2

G

17-38

ANTIGEN

_~

lr~uctosar~ W binding,

pl a

Abl =

A48 Id " 1~2 -~ 6 and A2 -~1 fructosan binding

Figure 1. Dual function of a monoclonal antibody carrying the

internal image of the antigen (Ab213). Ab 1: antiepitope antibody; Ab2: anti-idiotype antibody; p: paratope; I: idiotope; e: epitope (Adapted from Bona et al., 1984). al., 1985). If the RF binding site conformationally resembles Fc binding structures on microbial agents, then RF could arise as internal image anti-idiotypic antibodies in the course of an immune response to infections (Oppliger et al., 1987). Methods of Detection

The presence of different antibody specificities within a monoclonal IgM population challenges the monoclonality of these immunoglobulins. Isoelectrofocusing (IEF) analysis of 18 EMC IgG showed that 10/18 had a very limited number of bands (Renversez et al., 1986), possibly the result of clonal restriction occurring during an anti-idiotypic reaction against paratopes of IgM deriving from a single cellular clone. In that study, the monoclonal nature of the IgM was confirmed by IEF analysis for all patients. However, other studies established that cryoglobulin IgM from 8/18 Type II EMC patients were polyclonal (Montagnino, 1988). The IgG from these patients were all polyclonal and their isoelectric points were slightly more alkaline as compared to normal IgG. Type II cryoglobulins composed of microheterogeneous cryoglobulins containing oligoclonal IgM or a mixture of polyclonal and monoclonal IgM also have been observed (Tissot et al., 1994); these are called Type II--III cryoglobulins. In two other cases of EMC, the

lu-chain area of IgM appeared as a mixture of polyoligoclonal sets of lu-chains. The definition of Type II--IIIvariant was suggested, due to the presence of immunoglobulins of different isotypes.

CLINICAL UTILITY In EC, a chronic antigenic stimulation such as a chronic viral infection elicits antibody production against the responsible viruses, with subsequent induction of RF and/or auto-anti-idiotypic antibodies via the idiotypic network (either of the IgG or IgM isotype). Intrinsic characteristics and/or the antiglobulin reactivity of the RF is responsible for cryoprecipitation with eventual damage to the kidney by the cryoprecipitating immunocomplexes. By the time cryogenic immune complexes form, the nominal antigen is lost from the circulation; only the RF and/or the auto-anti-idiotypic reaction between the cryoprecipitable immunoglobulins remains. Alternatively, the IgG component of the cryoprecipitate, displaying autoantigenic reactivity against self components present within the glomerulus, first binds to the nominal antigen and only subsequently to its corresponding RF IgM (Dolcher et al., 1994). RFs might therefore contribute to immune complex formation, reacting with the immunoglobulins already bound to renal antigens. However, it is not known whether IgG binding to the glomerular antigen is artifactual. In experimental models, the ability to form cryoprecipitating immunocomplexes correlates with the induction of glomerulonephritis: injection of a cryoprecipitating murine RF (mRF) of the IgG3 class in normal mice can induce both peripheral vasculitis and glomerulonephritis (Gyotoku et al., 1987). In contrast, a hybrid molecule (composed of the ],3 chain of the original monoclonal immunoglobulin and a different light chain) retaining the original cryoprecipitating ability but lacking rheumatoid factor activity also can induce glomerulonephritis, but not peripheral vasculitis (Reininger et al., 1990). In this experimental model, both RF activity and cryoprecipitability are essential for the development of full-blown EMC. Modulation of autoantibody production through a perturbation of the idiotype/anti-idiotype network also occurs after bacterial infections. Anti-idiotypic antibodies to a human RF of a patient with mixed cryoglobulinemia occurs in association with pneumococcal bacteremia (Abe et al., 1984), and anti-idiotypic serum from mice and rabbits immunized with human RF

reacts with a cell wall peptidoglycan preparation from group A Streptococcus pyogenes (Johnson et al., 1985). In this view, persistent viral infection and consequent continuous B-cell stimulation and hypersecretion of polyclonal immunoglobulins can lead to Type III cryoglobulinemia in some patients. Possibly in response to hepatitis C infection of mononuclear cells, transformation of polyclonal to oligoclonal and finally to monoclonal IgM can occur, progressively leading to type II EC (Tissot et al., 1994). The monoclonal nature of these anti-idiotypic IgM could be due to their origin from restricted clones of B lymphocytes, as already described in rheumatoid arthritis patients. Indeed, EBV-inducible IgM RF-producing precursor B lymphocytes belong to a particular B lymphocyte subset which forms rosettes with mouse red blood cells (Fong et al., 1983). This subset increases in patients with rheumatoid arthritis (Room et al., 1982) and B lymphocytes with similar properties account for up to 48% of peripheral blood B cells in rheumatoid arthritis patients (Plater-Zyberk et al., 1985). An expanded clone of cells expressing the mRF idiotype is present even in the early stages of EMC (Ono et al., 1987).

Antibody Correlation with Disease Activity Morphological features of renal lesions are essentially characterized by endocapillary proliferation, varying from focal to diffuse. In most cases, the main feature is membranoproliferative glomerulonephritis (MPGN) with a "double contour" appearance of capillary loops. One of the most specific findings is the presence of numerous, large amorphous thrombi lying on the endothelial side of the glomerular basement membrane and occluding the capillary loop lumina. These deposits reflect the degree of severity of renal disease and indicate the presence of monoclonal immunoglobulins in the cryoprecipitate. Electron microscopy shows that intraluminal and subendothelial deposits are made up of a fibrillar or crystalloid material, which appears as tubular units in cross-sections and as parallel fibrils in longitudinal sections (Feiner and Gallo, 1977) (Figure 2). Antiglobulin activity similar to that of cryoprecipitable IgM occurs in renal tissue from patients with the most severe histological changes (Maggiore et al., 1982). The cross-reacting idiotype present in circulating cryoglobulin IgM RF is also detectable on the immunoglobulins found in the renal biopsies of 11/13 patients with EMC; in idiotype-positive biopsies, 199

Figure 2. Crystalloid deposits (tubular structures) surrounded by amorphous material due to the degradation of the deposits (magnification • preincubation with autologous serum rheumatoid factor almost completely blocked the binding of the corresponding antibody (Sinico et al., 1988). Two mechanisms have been proposed for the induction of vasculitis and glomerular lesions. Both mechanisms are operative in EMC and induce different histological expressions of the disease. The first is a mechanism of aggregation and precipitation within the vascular lumen induced by various specific factors such as increased protein concentration, interaction with fibronectin and modification of pH. This leads to acute endoluminal precipitation of cryoglobulins and induces the appearance of endoluminal thrombi. The second mechanism is immunocomplex mediated and is characterized by slow subendothelial deposition of complexes. This slower, more chronic deposition of cryoglobulins along the capillary walls as a consequence of their immunocomplex nature might be responsible for subendothelial deposition and induction of exudative MPGN (D'Amico et al., 1984). These two mechanisms may coexist, and it is possible that the aggregation and precipitation of cryoglobulins in the kidney may not only directly cause tissue damage, but also enhance in situ assembly of the IgM 200

and IgG which circulate uncomplexed. On the other hand, the generation of "wire loop" glomerular lesions by IgG3 RF and anti-DNA monoclonal cryoglobulins occurs in the absence of immune complex formation (Lemoine et al., 1992), suggesting that autoantibodies with cryoglobulin activity might participate in the pathogenesis of lupus nephritis, independent of their immunological specificities. This supports the concept that murine IgG3 cryoglobulins, due to their spontaneous aggregation, behave differently from mixed IgG-IgM cryoprecipitates. Moreover, these murine IgG3 cryoglobulins fail to show any crystalloid features, as observed for MC.

Therapeutic Approaches Both the development of cryoglobulins and of tissue lesions can be modulated with a monoclonal antiidiotypic antibody (Izui et al., 1993). The cryoprecipitation of the specific mAb is completely and specifically inhibited in vitro by the anti-idiotypic mAb (Spertini et al., 1989). This may be due to either modification of the electrostatic equilibrium of the mAb due to interactions with the anti-idiotype, or

modification of the spatial conformation of the antibody caused by binding of the anti-idiotype. Pretreatment of mice with the anti-idiotype antibody completely protected them against the development of skin and glomerular lesions. In addition to the anti-idiotype approach, noncryoprecipitable mAb might specifically inhibit cryoprecipitation. In fact, excess amounts of noncryogenerating mAb inhibit the cryoprecipitation of cryogenerating IgG3 mAb. The lack of inhibitory effect by F(ab')2 fragments from noncryoprecipitating mAb suggest that the observed inhibition is not caused by specific immunological interactions between cryogenerating and noncryogenerating IgG3 mAbs. Similar results are observed in rabbits undergoing cycles of immunization with Micrococcus lysodeikticus, in whom the spontaneous appearance of anti-idiotypic antibodies coincides with the disappearance of specific antimicrococcal clonotypes (Brown and Rodkey, 1979), and in experimental SLE induced by immunization of naive mice with an anti-DNA idiotype, where mice treated with specific anti-idiotypic antibody conjugated to a toxin-saporin showed a significant decrease of clinical manifestations (Blank et al., 1994). The already reported modifications of clinical activity after the appearance of specific anti-idiotypic antibodies (Abdou et al., 1981; Geha, 1982; Abe et al., 1984) point to a similar mechanism. The use of high dose intravenous immunoglobulins (IVIg) containing anti-idiotypic antibodies to recurrent anti-DNA idiotypes in SLE has an inhibitory effect in vitro to the anti-DNA-secreting cells (Silvestris et al., 1994). Although plasmapheresis reduces the levels of serum cryoglobulins, prompt reaccumulation of the cryoglobulin often occurs. Alternatively, treatment with IFN-~ resulted in sustained clinical and immunologic improvement in seven patients with EMC (Bonomo et al., 1987). Likewise, treatment with IFNdramatically reduced serum cryoglobulin levels and symptoms of cryoglobulinemia in a patient with MC and transfusion-associated hepatitis C without improving the signs of chronic hepatitis (Knox et al., 1991), suggesting a direct IFN effect on cryoglobulin synthesis. High doses of IFN suppress mitogen-induced immunoglobulin production in normal mononuclear cells in vitro through inhibition of late stages of B-cell differentiation (Peters et al., 1986). However, cryoglobulinemia can occur as a complication of IFN therapy for hematologic malignancies (Roy and Newland, 1988).

CONCLUSION Mixed cryoglobulins and RF are a common phenomenon, associated with a large number of infectious and autoimmune disorders and can also be found at low titers in normal individuals. Their appearance, along with RF, can be considered as a normal event in the immune clearance of antigen-antibody complexes. Cryoglobulins and RF at low levels rarely produce any symptoms, and no direct role of cryoglobulins has been established in the genesis of the various visceral injuries found in patients with primary infections. Only at consistently high titers do cryoglobulins assume a relevant role in the induction of systemic disease. Whether the derangement of immune regulation that results from an increased production of cryoglobulins is due exclusively to persistent production of RFs or also to the onset of idiotype/anti-idiotype interactions is unknown. RF produced in normal individuals and RF associated with autoimmune diseases differ in idiotype expression, as determined by mAbs to structural antigen determinants (Fong et al., 1986). The persistent production of autoantibodies, long after the eliciting agent has disappeared from the circulation and the induction of RF with peculiar characteristics and the possibility of auto-anti-idiotypic reactions suggest that, in some cases, the anti-idiotypic network may have failed in its downregulating activity. In these cases, the production of autoantibodies, instead of attenuating and eventually turning off the immune reaction, perpetuates it through the production of complexes with intrinsic pathogenic characteristics. Auto-anti-idiotypic IgG directed to circulating or cell-bound paraproteins occur in patients with B-cell lymphoproliferative disorders and acquired angioedema (Geha et al., 1985). However, the unlikelihood of the simultaneous presence of idiotypes and anti-idiotypes in the circulation has suggested the concept of an oscillatory activation of antigen-stimulated clones and anti-idiotypic ones (Kelsoe and Cerny, 1979). In experimental models (Brown and Rodkey, 1979) and humans (Geha, 1982; Abdou et al., 1981; Abe et al., 1984), the appearance of antiidiotypic antibodies coincides with the disappearance of specific antiantigen clonotypes. Thus, in the course of monoclonal B-cell disorders such as Type II EMC, idiotype production is likely sustained and presumably capable of suppressing the emergence of auto-antiidiotypic clones (Stone et al., 1988). This may be due to the development of tolerance to high concentrations 201

of idiotypes in anti-idiotypic B cells or through the acquisition of anti-idiotypic-specific suppressor T cells (Bona and Paul, 1988). However, the hypothesis that, after a viral infection, some cryoglobulin IgM RF might behave like epibodies reintroduces the possibility that reactivity between the cryoprecipitable immunoglobulins might be regulated via the network theory.

Whether E M C has to be considered as a syndrome characterized exclusively by the presence of classical RF with the peculiar characteristic of cryoprecipitation or as a syndrome in which idiotype/anti-idiotype interactions also play a significant role remains to be elucidated. See also CRYOGLOBULINS SECONDARY TO HEPATITIS C VIRUS INFECTION.

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Cryoglobulinemia induced by monoclonal immunoglobulin G rheumatoid factor derived from autoimmune MRL/MpJIpr/lpr mice. J Immunol 1987;138:3785-3792. Izui S, Bemey T, Shibata T, Fulpius T. IgG3 cryoglobulins in autoimmune MRL-lpr/plr mice: immunopathogenesis, therapeutic approaches and relevance to similar human diseases. Ann Rheum Dis 1993;52:$48-$54. Johansson PJH, Schroder AK, Nardella FA, Mannik M, Christensen P. Interaction between herpes simplex type Iinduced Fc receptor and human and rabbit immunoglobulin G (IgG) domains. Immunology 1986;58:251-255. Johnson PM, Phua KK, Evans HB. An idiotypic complementarity between rheumatoid factor and antipeptidoglycon antibodies? Clin Exp Immunol 1985;61:373--378. Johnston SL, Abraham GN. Studies of human anti-IgM antiIgG cryoglobulins. I. Patterns of reactivity with autologous and isologous human IgG and its subunits. Immunology 1979;36:671-683. Kelsoe G, Cemy J. Reciprocal expansions of idiotypic and antiidiotypic clones following antigen stimulation. Nature 1979;279:333--334. Kipps TJ, Fong S, Tomhave E, Chen PP, Goldfien RD, Carson DA. High-frequency expression of a conserved kappa lightchain variable-region gene in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 1987;84:2916--2920. Knox TA, Hillyer CD, Kaplan MM, Berkman EM. Mixed cryoglobulinemia responsive to Interferon-alpha. Am J Med 1991;91:554--555. Lemoine R, Bemey T, Shibata T, Fulpius T, Gyotoku Y, Shimada H, Sawada S, Izui S. Induction of "wire loop" lesions by murine monoclonal IgG3 cryoglobulins. Kidney Int 1992;41:65--72. Maggiore Q, Bartolomeo F, L'Abbate A, Misefari V, Montorano C, Caccamo A, di Belgiojoso GB, Tarantino A, Colasanti G. Glomerular localization of circulating antiglobulin activity in essential mixed cryoglobulinemia. Kidney Int 1982;21:387--394. Maul GG, Jimenez SA, Riggs E, Ziemnicka-Kotula D. Determination of an epitope of the diffuse systemic sclerosis marker antigen DNA topoisomerase. I: sequence similarity with retroviral p30 gag protein suggests a possible cause for autoimmunity in systemic sclerosis. Proc Natl Acad Sci USA 1989;86:8492-8496. Middaugh CR, Litman GW. Atypical glycosylation of an IgG monoclonal cryoimmunoglobulin. J Biol Chem 1987;262: 3671-3673. Montagnino G. Reappraisal of the clinical expression of mixed cryoglobulinemia. Springer Semin Immunopathol 1988;10:1-19. Nardella FA, Teller DC, Barber CV, Mannik M. IgG rheumatoid factors and staphylococcal protein A bind to a common molecular site on IgG. J Exp Med 1985; 162:1811-1824. Newkirk MM, Mageed RA, Jefferis R, Chen PP, Capra JD. Complete amino acid sequences of variable regions of two human IgM rheumatoid factors, BOR and KAS of the WA idiotypic family, reveal restricted use of heavy and light chain variable and joining region gene segments. J Exp Med 1987;166:55064.

Newkirk MM, Lemmo A, Rauch J. Importance of the IgG isotype, not the state of glycosylation, in determining human rheumatoid factor binding. Arthritis Rheum 1990;33:800900. Ono M, Winearls CG, Amos N, Grennan D, Gharavi A, Peters DK, Sissons JG. Monoclonal antibodies to restricted and cross-reactive idiotopes on monoclonal rheumatoid factors and their recognition of idiotope-positive cells. Eur J Immunol 1987;17:343--349. Oppliger IR, Nardella FA, Stone GC, Mannik M. Human rheumatoid factors bear the internal image of the Fc binding region of staphylococcal protein A. J Exp Med 1987;166: 702-710. Pasquali JL, Martin T, Knapp AM, Levallois H, Ferradji A. Monoclonal rheumatoid factor secreting cells in a patient with mixed cryoglobulinemia Homogeneity and stability of the idiotypic production and in vitro idiotypic suppression. J Immunol 1989;143:1826--1831. Pasquali JL, Waltzinger C, Kuntz JL, Knapp AM, Levallois H. The majority of peripheral blood monoclonal IgM secreting cells are CD5 negative in three patients with essential mixed cryoglobulinemia. Blood 1991 ;77:1761--1765. Perl A, Gorevic PD, Ryan DH, Condemi JJ, Ruszkowski RJ, Abraham GN. Clonal B-cell expansions in patients with essential mixed cryoglobulinaemia. Clin Exp Immunol 1989;76:54--60. Perl A, Gorevic PD, Condemi JJ, Papsidero L, Poiesz BJ, Abraham GN. Antibodies to retroviral proteins and reverse transcriptase activity in patients with essential cryoglobulinemia. Arthritis Rheum 1991 ;34:1313-- 1318. Peters M, Ambrus JL, Zheleznyak A, Walling D, H0ofnagle JH. Effect of interferon-alpha on immunoglobulin synthesis by human B cells. J Immunol 1986;137:3153--3157. Plater-Zyberk C, Maini RN, Lam K, Kennedy TD, Janossy G. A rheumatoid arthritis B cell subset expresses a phenotype similar to that in chronic lymphocytic leukemia. Arthritis Rheum 1985;28:971--976. Radoux V, Chen PP, Sorge JA, Carson DA. A conserved human germline V Kappa gene directly encodes rheumatoid factor light chains. J Exp Med 1986;164:2119--2124. Reininger L, Bemey T, Shibata T, Spertini F, Merino R, Izui S. Cryoglobulinemia induced by a murine IgG3 rheumatoid factor: skin vasculitis and glomerulonephritis arise from distinct pathogenetic mechanisms. Proc Natl Acad Sci USA 1990;87:10038-10042. Renversez JC, Roussel S, Vallee MJ, Brighouse G, Lambert PH. Idiotypic interactions in type II mixed cryoglobulins. Rev Fr Tranfus Immunohematol 1984;6:737--755. Renversez JC, Roussel S, Valle MJ, Lambert PH. Human type II mixed cryoglobulins as a model of idiotypic interactions. In: Ponticelli C, Minetti L, D'Amico G, eds. Antiglobulins, Cryoglobulins and Glomerulonephritis. Boston: Lancaster, 1986:147-160. Room GR, Plater-Zyberk C, Clarke MF, Maini RN. B-lymphocyte subpopulation which forms rosettes with mouse erythrocytes increased in rheumatoid arthritis. Rheumatol Int 1982;2:175-178. Roy V, Newland AC. Raynaud's phenomenon and cryoglobu-

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linemia associated with the use of recombinant-human alphainterferon. Lancet 1988;1:944--945. Sasso EH, Barber CV, Nardella FA, Yount WJ, Mannik M. Antigenic specificities of human monoclonal and polyclonal IgM rheumatoid factors. The C gamma 2-C gamma 3 interface region contains the major determinants. J Immunol 1988;140:3098-3107. Shibata T, Berney T, Spertini F, Izui S. Rheumatoid factors in mice bearing the Ipr or gld mutation. Selective production of rheumatoid factor cryoglobulins in MRL/MPJ-lpr/lpr mice. Clin Exp Immunol 1992;87:190-- 195. Shlomchik M, Mascelli M, Shan H, Radic MZ, Pisetsky D, Marshak-Rothstein A, Weigert M. Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation. J Exp Med 1990; 171:265-292. Silvestris F, Cafforio P, Dammacco F. Pathogenic anti-DNA idiotype-reactive IgG in intravenous immunoglobulin preparations. Clin Exp Immunol 1994;97:19-25. Sinico RA, Winearls CG, Sabadini E, Fornasieri A, Castiglione A, D'Amico G. Identification of glomerular immune deposits in cryoglobulinemia glomerulonephritis. Kidney Int 1988;34: 109-116.

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Spertini F, Donati Y, Welle I, Izui S, Lambert PH. Prevention of murine cryoglobulinemia and associated pathology by monoclonal anti-idiotypic antibody. J Immunol 1989;143: 2508-2513. Stone GC, Nardella FA, Oppliger IR, Mannik M. Absence of auto-anti-idiotypic activity between the IgM and IgG fractions of human mixed cryoglobulins. J Immunol 1988;140: 3114-3119. Tarantino A, Montagnino G, Baldassari A. Prognostic factors in essential mixed cryoglobulinemia nephropathy. In: Ponticelli C, Minetti L, D'Amico G, eds. Antiglobulins, Cryoglobulins and Glomerulonephritis. Boston: Kluwer Academic Publishers, 1986:219--225. Tissot JD, Schifferli JA, Hochstrasser DF, Pasquali C, Spertini F, Clement F, Frutiger S, Paquet N, Hughes GJ, Schneider P. Two-dimensional polyacrylamide gel electrophoresis analysis of cryoglobulins and identification of an IgM-associated peptide. J Immunol Methods 1994;173:63--75. Tomana M, Schrohenloher RE, Koopman WJ, Alarcon GS, Paul WA. Abnormal glycosylation of serum IgG from patients with chronic inflammatory diseases. Arthritis Rheum 1988;31:333--338.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CRYOGLOBULINS SECONDARY TO HEPATITIS C VIRUS INFECTION Gy6rgy Abel, M.D., Ph.D. b, Glenn B. Knight, Ph.D. c and Vincent Agnello, M.D. a

aDepartment of Laboratory Medicine, bDepartment of Immunology Research and CDepartment of Molecular Biology, Lahey-Hitchcock Clinic, Burlington, MA 01805, USA

HISTORICAL NOTES

Essential mixed cryoglobulinemia (EMC) is a disease of unknown etiology characterized by cold precipitable serum immunoglobulins that contain rheumatoid factors (RF). There are two types of mixed cryoglobulins: type II contains polyclonal IgG and a monoclonal IgM RF (mRF), while in type III both the IgG and IgM RF are polyclonal. Clinically, cryoglobulins are classified either as essential, i.e., no primary disease process can be identified, or as secondary to autoimmunity, infection or malignancy. The manifestations of the disease range from a benign cutaneous vasculitis to life-threatening severe vasculitis of vital organs. The high frequency of hepatocellular pathology in patients with essential mixed cryoglobulinemia suggested the involvement of hepatotropic viruses in the pathogenesis of the disease (Realdi et al., 1974). Although initially considered a possible cause (Levo et al., 1977), hepatitis B virus (HBV) infection is found in very few patients with EMC (Monti et al., 1995). The discovery and molecular cloning of hepatitis C virus (HCV), the major pathogen causing posttransfusion and sporadic non-A, non-B hepatitis, led to investigations of this virus in EMC. A strong association of HCV with mixed cryoglobulinemia is now established (Agnello and Romain, 1996). The specific concentration of HCV in type II cryoglobulins that have monoclonal rheumatoid factor (mRF) exhibiting the WA cross-idiotype (XId) suggests a direct role for the virus in the pathogenesis of EMC (Agnello et al., 1992). Cross-idiotypes of mRF were characterized more than 10 years before hepatotropic viruses were implicated in the disease (Kunkel et al., 1973). The WA XId that occurs in 80% of

mRF isolated from type II cryoglobulins is an antigen in the combining site of the antibody involving both heavy and light chains (Agnello and Barnes, 1986). Of the remaining 20% of mRF from type II cryoglobulins, 7% are PO WA XId-positive and 13% do not type as either WA or PO (Agnello et al., 1996). No clinical differences have been reported for patients with type II cryoglobulinemia with or without HCV infection associated with non-WA mRF compared to the WA mRF positive patients with these conditions.

THE AUTOANTIGENS

The antigen(s) that elicits production of the WA XId mRF and other mRF in type II cryoglobulins associated with HCV infection is not known. 1. Because type II and type III mixed cryoglobulins occur in a variety of infections, cryoglobulinemia might result from chronic stimulation of the immune system by complexes consisting of IgG, which becomes autoantigenic when complexed with an antigen of an infectious agent. For example, the long-term stimulation by HCV infection might result in the chronic production of HCV-IgG complexes that activate the proliferation of RFproducing cells (Agnello, 1995a); such HCV-IgG complexes are demonstrable in chronic HCV hepatitis (Thomssen et al., 1993). 2. Alternatively, the specification of WA XId mRF might be for an autoantigen other than IgG, such as an antigen of serum lipoproteins known to be associated with the virus. Indeed, HCV can be precipitated from serum with anti-[3 lipoprotein (Thomssen et al., 1993; Thomssen et al., 1992).

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Complex formation with lipoproteins is not unique to HCV; vesicular stomatitis virus is known to form complexes with VLDL (Mills et al., 1979). 3. A third possible antigenic stimulus for WA XId mRF production might be an HCV antigen. The genome of HCV encodes three putative structural proteins, the RNA-binding nucleocapsid protein and the envelope glycoproteins gp33 (El) and gp72 (E2) at the 5' end. The genome also encodes nonstructural proteins encoded by five regions, NS 1-NS5 (Houghton et al., 1991; Vandoom, 1994). Although nonreactive with the recombinant HCV nucleocapsid protein c22-3 and nonreactive with all of the available recombinant nonstructural proteins (5-1-1, c100-3, c33-c and c200) (Agnello et al., 1992), WA XId mRF has yet to be tested against the HCV envelope proteins E1 and E2.

THE AUTOANTIBODY Pathogenetic Role

The predominant autoantibody in type II mixed cryoglobulinemia associated with HCV infection is the WA XId-bearing mRF. Immunochemical, protein sequence and molecular genetic studies show that WA tuRF are products of germline genes; most are encoded with little somatic mutation by germline VrdIIb and Vr~l genes (Gorevic and Frangione 1991) and also VH3 genes (Knight et al., 1993). The cellular and molecular mechanisms for the development of WA XId-positive mRF-producing B cells in HCV-infected patients are unknown. Because CD5-positive B cells expressing germline light and heavy chain genes are considered prone to autoantibody production and malignant transformation (Kipps et al., 1987), the autoantigen complex involving HCV is suggested to stimulate directly a population of WA XId +, CD5 + B cells (B-la) in the liver to form lymphoid nodules. Such WA XId + B-la cells might initially produce WA IgM without RF activity, but with continued antigen stimulation, somatic mutation is postulated to result in development of RF activity, loss of the CD5 marker and the conversion of the CD5-, WA mRF + B cells to plasma cells in the bone marrow and spleen (Agnello, 1995b). Based on the "lymphoplasmacytoid" appearance of cells infiltrating the liver portal tracts and bone marrow, type II cryoglobulinemia is considered a

206

manifestation of a low-grade lymphoma (Monteverde et al., 1988; Pozzato et al., 1994). Only a small number of patients develop frank malignancy, however, (Gorevic and Frangione 1991; Brouet et al., 1974) and the progression of the restricted, HCVdriven benign proliferation of a B-cell subset in EMC to frank lymphoma may be the result of a second process involving multiple and stepwise mutations (Agnello, 1995b). Methods of Detection

The mRF in type II cryoglobulinemia can be isolated by column chromatography as previously described (Agnello and Barnes, 1986) but performed in neutral buffer at 37~ (Agnello V, unpublished observation). The primary structure of WA XId mRF is known from protein sequencing (Andrews and Capra 1981; Newkirk et al., 1987); the genes involved were deduced initially from protein structure and later by gene sequencing of cloned cells (Knight et al., 1993; Pascual et al., 1990) and directly from the mRF cells in the peripheral blood (Crouzier et al., 1995). AntiWA XId reagents are not commercially available, but polyclonal reagents are readily produced (Kunkel et al., 1973; Bonagura et al., 1982).

CLINICAL UTILITY Disease Association

Although production of polyclonal RF is common in chronic immune complex diseases such as rheumatoid arthritis, systemic lupus erythematosus and subacute bacterial endocarditis, the production of mRF is extremely rare. WA mRF is present only in mixed cryoglobulinemia and is associated with HCV infection in 84% of EMC cases. Cryoglobulin levels do not correlate with the progression of the disease. Preliminary studies suggest that quantitation of WA XId § B cells may be a measure of disease activity (Agnello V et al., unpublished observation). It has been proposed that in chronic hepatitis C there is a progression of polyclonal RF to type III cryoglobulinemia, and thence to type II cryoglobulinemia over a period of 20 years is proposed (Lunel et al., 1994). If this hypothesis is proved, then detection of WA XId among RF in infected patients may provide early identification of those at risk for major complication of the disease. No controlled clinical study of this approach is available.

Effect of Therapy The traditional treatment for type II cryoglobulinemia prednisone, immunosuppressive drugs and plasmapheresis, has been ineffective in inducing long-term remission. Interferon-oc is now clearly the drug of choice for treatment of this disease (Agnello and Romain, 1996). Neither glucocorticoids nor other immunosuppressives can be used as primary drugs in the therapy of EMC due to their potential for enhancing viral replication. However, these drugs may still have a role in the therapy combined with antiviral drugs in cases in which large production of mRF continues after HCV suppression (Agnello and Romain, 1996). For example, if the mRF-production and the benign monoclonal proliferation initiated by the viral infection continues due to stimulation by irrelevant complexes of IgG or if the proliferation be-

REFERENCES Agnello V, Barnes JL. Human rheumatoid factors crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains. J Exp Med 1986; 164:1809--1814. Agnello V, Chung RT, Kaplan LM. A role for hepatitis C virus in type II cryoglobulinemia. N Engl J Med 1992;327:1490-1495. Agnello V. Mixed cryoglobulinemia secondary to hepatitis C virus infection. Hosp Pract 1995a;30:35--42. Agnello V. The aetiology of mixed cryoglobulinemia associated with hepatitis C infection. Scand J Immunol 1995b;42:179-184. Agnello V, Romain PL. Mixed cryoglobulinemia associated with hepatitis C virus infection. Rheum Dis Clin North Am 1996;(in press). Agnello V, Zhang QX, Abel G, Knight G. The association of hepatitis C virus infection with monoclonal rheumatoid factors bearing the WA cross-idiotype: implications for the etiopathogenesis and therapy of mixed cryoglobulinemia. Clin Exp Rheumatol 1996;(in press). Andrews DW, Capra JD. Complete amino acid sequence of variable regions of two human monoclonal antigamma globulins of the WA cross-idiotypic group: suggestion that the J segments are involved in the structural correlate of the idiotype. Proc Natl Acad Sci USA 1981;78:3799-3805. Bonagura VR, Kunkel HG, Pernis B. Cellular localization of rheumatoid factor idiotypes. J Clin Invest 1982;69:13561362. Brouet JC, Clauvel JP, Danon F, Klein M, Seligmann M. Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 1974;57:775--788. Casato M, Lagana B, Antonelli G, Dianzani F, Bonomo L.

comes malignant, eradication of the virus alone may no longer be sufficient, and the process may become malignant. Interferon-~ is known to have both antiproliferative and antiviral actions, and both activities may be involved in the effectiveness of the drug in the therapy of patients with EMC (Casato et al., 1991; Ferri et al., 1993).

CONCLUSION A better understanding of the pathogenesis of HCVassociated type II cryoglobulinemia combined with and identification of markers for transition to malignant transformation might suggest new, more effective strategies of therapy using antiviral agents and immunosuppressive drugs. See also CRYOGLOBULINS.

Long-term results of therapy with interferon-c~ for type II essential mixed cryoglobulinemia. Blood 1991;78:3142--3147. Crouzier R, Martin T, Pasquali JL. Monoclonal IgM rheumatoid factor secreted by CD5-negative B cells during mixed cryoglobulinemia. Evidence for somatic mutations and intraclonal diversity of the expressed VH region gene. J Immunol 1995;154:413--421. Ferri C, Marzo E, Longombardo G, Lombardini F, La Civita L, Vanacore R, Liberati AM, Gerli R, Greco F, Moretti A, Monti M, Gentilini P, Bombardieri S, Zignego AL. Interferon-oc in mixed cryoglobulinemia patients: a randomized, crossover controlled trial. Blood 1993;81:1132--1136. Gorevic PD, Frangione B. Mixed cryoglobulinemia crossreactive idiotypes: implications for the relationship of EMC to rheumatic and lymphoproliferative disease. Semin Hematol 1991;28:79--94. Houghton M, Weiner A, Han J, Kuo G, Choo QL. Molecular biology of the hepatitis C viruses: implications for diagnosis, development, and control of viral disease. Hepatology 1991;14:381--388. Kipps TJ, Fong S, Tomhave E, Chen PP, Goldfien RD, Carson DA. High-frequency expression of a conserved light chain variable region gene in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 1987;84:2916--2920. Knight GB, Agnello V, Bonagura V, Barnes JL, Panka DJ, Zhang Q-X. Human rheumatoid factor cross idiotypes. IV. Studies on WA XId-positive IgM without rheumatoid factor activity provide evidence that the WA XId is not unique to rheumatoid factors and is distinct from the 17.109 and G6 Xlds. J Exp Med 1993;178:1903--1911. Kunkel HG, Agnello V, Joslin FG, Winchester RJ, Capra JD. Cross-idiotypic specificity among monoclonal IgM proteins with anti-gamma-globulin activity. J Exp Med 1973;137: 331-342.

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Levo Y, Gorevic PD, Kassab HJ, Zucker-Franklin D, Franklin EC. Association between hepatitis B and essential mixed cryoglobulinemia. N Engl J Med 1977;296:1501--1504. Lunel F, Musset L, Cacoub P, Frangeul L, Cresta P, Perrin M, Grippon P, Hoang C, Piette JC, Huraux JM, Opolon P. Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology 1994;106:1291-1300. Mills BJ, Beepe DP, Cooper NR. Antibody-independent neutralization of vesicular stomatitis virus by human complement: II. Formation of VSV-lipoprotein complexes in human serum and complement-dependent viral lysis. J Immunol 1979; 123:2518-2524. Monteverde A, Rivano MT, Allegra GC, Monteverde AI, Zingrossi P, Boglioni P, Gobbi M, Falini B, Bordin G, Pileri S. Essential mixed cryoglobulinemia, type II: a manifestation of d low-grade malignant lymphoma? Clinical-morphological study of 12 cases with special references to immunohistochemical findings in liver frozen sections. Acta Haematol (Basel) 1988;79:20--25. Monti G, Galli M, Invernizzi F, Pioltelli P, Saccardo F, Monteverde A, Pietrogrande M, Renoldi P, Bombardieri S, Bordin G, Candela M, Ferri C, Gabrielli A, Mazzaro C, Migliaresi S, Mussini C, Ossi E, Quintiliani L, Tirri G, Vacca A, Italian Group for the Study of Cryoglobulinaemias. Cryoglobulinaemias: a multicentre study of the early clinical and laboratory manifestations of primary and secondary disease. Q J Med 1995;88:115-126. Newkirk MM, Mageed RA, Jefferis R, Chen PP, Capra JD.

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Complete amino acid sequences of variable regions of two human IgM rheumatoid factors, BOR and KAS of the Wa idiotypic family, reveal restricted use of heavy and light chain variable and joining region gene segments. J Exp Med 1987;166:550--564. Pascual V, Randen I, Thompson K, Sioud M, Forre O, Natvig J, Capra JD. The complete nucleotide sequencing of heavy chain variable regions of six monospecific rheumatoid factors derived from Epstein-Barr virus transformed B cells isolated from the synovial tissue of patients with rheumatoid arthritis. J Clin Invest 1990;86:1320--1328. Pozzato G, Mazzaro C, Crovatto M, Modolo ML, Ceselli S, Mazzi G, Sulfaro S, Franzin F, Tulissi P, Moretti M, Santini GF. Low grade malignant lymphoma, hepatitis C virus infection, and mixed cryoglobulinemia. Blood 1994;84: 3047--3053. Realdi G, Alberti A, Rigoli A, Tremolada, F. Immune-complexes and Australia antigen in cryoglobulinemic sera. Z Immunitatsforsch Exp Klin Immunol 1974;147:114--126. Thomssen R, Bonk S, Propfe C, Heerman KH, Kochel HG, Uy A. Association of hepatitis C virus in human sera due to the binding of I]-lipoproteins. Med Microbiol Immunol (Berlin) 1992;181:293--300. Thomssen R, Bonk S, Thiele A. Density heterogeneities of hepatitis C virus in human sera due to the binding of I]lipoproteins and immunoglobulins. Med Microbiol Immunol (Berlin) 1993;182:329--334. Vandoorn LJ. Review: molecular biology of the hepatitis C virus. J Med Virol 1994;43:345--351.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CYTOKINE AUTOANTIBODIES Klaus Bendtzen, M.D., D.M.Sc., Morten Bagge Hansen, M.D., Christian Ross, M.D. and Morten Svenson, Ph.D.

Institute for Inflammation Research, RHIMA Center, Rigshospitalet, DK-2200, Copenhagen N, Denmark

HISTORICAL NOTES Autoantibodies to IFN~ Cytokine antibodies were first reported as cases of neutralizing antibodies to interferon alpha (IFN~) in patients with Varicella-zoster (Pozzetto et al., 1984) and hepatitis virus infections, in patients with autoimmune (Prtimmer et al., 1989) and neoplastic diseases (Prtimmer et al., 1991) and in a patient with chronic graft-versus-host disease (Mogensen et al., 1981; Panem et al., 1982; Trown et al., 1983; Panem, 1984; Pozzetto et al., 1984; Prtimmer et al.; 1989; 1991; 1994; Ikeda et al., 1991). In some cases, antibodies that bind other IFN species are found in patients with infectious diseases (Caruso et al., 1990; Viani et al., 1991). Low levels of IgG and IgM capable of neutralizing IFNcx, IFN~ and IFNy in vitro are also detectable in the blood of healthy individuals (Caruso et al., 1990; Ross et al., 1990). However, as in the case of antibodies to IFN in patients, these antibodies were not demonstrated to bind in a specific manner nor with any appreciable affinity. Recently, pharmaceutical preparations of normal human IgG were found to contain specific and high-avidity antibodies which suppressed the antiviral effect of IFNc~ through saturable binding to Fab; these autoantibodies have now been demonstrated in sera of normal individuals (Ross et al., 1995).

Autoantibodies to Interleukin (IL)-I~ and IL-1[~ and IL-6 Naturally occurring autoantibodies to IL-1 a were first reported in 1989 by demonstration of direct binding of IgG from normal individuals to radiolabeled human recombinant IL-1 ~ and by IgG-mediated competitive

interference with IL-I~ binding to cellular IL-1 receptors (Svenson et al., 1989). At the same time, IgG from patients with rheumatoid arthritis were found to interfere in vitro with the biological effect of IL-1 ~, and sera from these patients interfered with the effect of both IL-I~ and IL-1]3 (Suzuki et al., 1989). Subsequently, sera of normal individuals and of patients with various immunoinflammatory disorders as well as pharmaceutical preparations of human IgG were found to contain autoantibodies that bind to ILlc~ with high avidity (Svenson et al., 1990; 1992; 1993; Suzuki et al., 1990; 1991; Gallay et al., 1991; Hansen et al., 1991a; 1994; Mae et al., 1991; Saurat et al., 1991; Sunder-Plassmann et al., 1991; Satoh et al., 1994). Natural autoantibodies to IL-6 were found in sera of normal individuals in 1991; these IgG molecules bound with high avidity and in a saturable manner to radiolabeled human IL-6 and interfered specifically with an ELISA for IL-6 (Hansen et al., 1991b). Since then, the presence of naturally occurring autoantibodies to IL-6 and similar antibodies in patients with immunoinflammatory and fibrotic diseases was confirmed (Takemura et al., 1992; Svenson et al., 1993; Suzuki et al., 1994; Hansen et al., 1993; 1995b; 1995c).

Autoantibodies to IL-10, LIF and Other Cytokines Recently, human pharmaceutic IgG were found to bind to homodimeric recombinant IL-10, but not to monomeric IL-10 (Bendtzen et al., 1994; 1995). In contrast, 50 normal sera tested negative for binding to IL-IO, suggesting that IL-10 antibodies are rarely present in normal individuals or, perhaps more likely, that these antibodies are inaccessible for detection in serum because they are blocked by IL-10 and/or other serum factors. Serum IgG and IgM from 80% of

209

normal individuals bind to a glycosylated form of recombinant human leukemia inhibitory factor (LIF) (Bendtzen et al., 1995). These antibodies do not bind nonglycosylated LIF, suggesting that the carbohydrate moiety of LIF is crucial for antibody binding. Antibodies to other cytokines such as IL-2, IL-8, IFN7, tumor necrosis factor (TNF)-cz and soluble TNF receptors are also found in normal and diseased individuals (Fomsgaard et al., 1989; Ross et al., 1990; Sylvester et al., 1992; Heilig et al., 1993; Tiberio et al., 1993) and IgE antibodies to TNFo~, TNF[3, IFN 7 and IL-4 have been reported in sera of AIDS patients (Pedersen et al., 1991). Some of these antibodies, however, cannot be regarded as autoantibodies, in that they bind the relevant mediator(s) with low avidities and in some cases bind only to cytokines denatured by adsorption to nitrocellulose membranes or plastic surfaces (Hansen et al., 1992; Svenson et al., 1993).

THE AUTOANTIGENS Definition and Nomenclature

Cytokines are polypeptide or glycopeptide signaling molecules that act at extremely low concentrations (picomolar and femtomolar levels) as regulators of cell growth and essential mediators of infectious and immunoinflammatory reactions. Most cytokines act locally, but some clinically important cytokines also act systemically as pleiotropic hormones with overlapping and potentially dangerous functions (immunoinflammatory hormones) (Baron et al., 1992; Bendtzen, 1994; Dinarello, 1995). The production and functions of cytokines are tightly regulated by cytokines themselves and by several other factors. Thus, several recombinant cytokines bind to antibodies (Bendtzen et al., 1990; 1995). The most extensively studied cytokines that bind to IgG are IL-1 c~, IL-6 and IFNc~. The antibodies bind selectively and with high avidity to both recombinant and native forms of the cytokines and neutralize their activities in vitro and, possibly, in vivo. In addition, IgG and IgM bind in a saturable manner to a glycosylated form of recombinant human LIF; pharmaceutical preparations of human IgG bind to dimeric but not monomeric IL-10. Origin/Structure/Sequence Information

IFNc~. Composed of a group of at least 20 subtypes 210

of 16--27 kd glycoproteins, IFN~ is produced by virus-infected leukocytes interfering with the replication of many viruses (Baron et al., 1992). Produced by antigen- or mitogen-activated monocytes/macrophages (M~)) and T- and B-lymphocytes, the IFN~ group of cytokines has potentially important immunoregulatory functions along with antiproliferative effects on many cell types (Table 1). The importance of IFN~, along with IFN~, as physiological antiviral agents is highlighted by experiments with gene-disrupted ("knockout") mice lacking the IFN~z/[3 receptor; these mice become highly susceptible to viral infections. IL-I~. IL-lc~, a 17 kd protein synthesized by a number of cell types (Table 1), is part of the IL-1 family of cytokines: IL- 1~, IL- 113 and IL- 1 receptor antagonist (IL-lra). The first two are highly inflammatory cytokines which affect nearly every cell type in the body; whereas, IL-lra functions as a specific receptor antagonist (Bendtzen, 1994; Dinarello, 1995). IL-1 cz is usually absent from the circulation or present only at low concentrations. During infection and inflammation, however, substantial amounts of IL- 1c~ may be found in the blood, perhaps released from dying cells. IL-1 cz may also be found in a biologically active form on the surface of several cells, particularly on macrophages (M~)) and B-lymphocytes, i.e., "professional" antigen-presenting cells. It is believed that IL-lc~ has important functions as an intracellular and/or membrane-associated mediator of immunoinflammatory reactions and as an autocrine activator of T-helper-2 (Th2) lymphocytes. IL-lcz, like IL-113, is highly inflammatory when given to humans, and both mediators are implicated in the pathogenesis of autoimmune diseases such as rheumatoid arthritis and insulin-dependent diabetes mellitus as well as in complications to severe infectious and traumatic conditions (Bendtzen, 1994; Dinarello, 1995). IL-6. IL-6, a 21--28 kd glycoprotein (Table 1), is, like IL-I~, a multifunctional cytokine produced by many cell types. This cytokine, which participates in hematopoiesis and the terminal differentiation of activated B-lymphocytes into antibody-producing cells, is of central importance in acute-phase responses during infections and other immunoinflammatory reactions (Akira et al., 1993). IL-6 gene-disrupted mice develop normally, albeit with impairment of certain immune functions and acute-phase responses. On the other hand, transgenic

mice with systemic overexpression of IL-6 develop massive plasmacytosis or plasmacytoma, increased polyclonal IgG 1 and autoantibodies production as well as i m m u n e complex nephritis. Clinical IL-6 measurements suggest the involvement of IL-6 in the pathogenesis of many diseases, including multiple myeloma, Castleman's disease, glomerulonephritis, autoi m m u n e diseases and certain neurologic disorders. In

addition, patients with certain leukemias and autoi m m u n e disorders improve after administration of neutralizing IL-6 antibodies.

IL-10. IL-10, a 35--40 kd homodimeric protein, has profound effects on cells involved in the immune response (Mosmann, 1994) (Table 1). IL-10 is produced primarily by M~ and T-lymphocytes, (TH2 cells in

Table 1. Cytokines to Which Autoantibodies Have Been Demonstrated Cytokine

MW/kd

Producers

Major Functions

IFNc~

16--27

virus-infected leukocytes B and T cells M~)

Activates: M~), NK and B cells + other cells MHC I and MHC II modulation antiviral activity antiproliferative and antitumor effects

IL-I~

17

M~, dendritic- and Langerhans' cells B and T cells (TH2) NK cells neutrophils endothelial and epithelial cells neuronal cells astrocytes mesangial cells fibroblasts synovial cells keratinocytes smooth muscle cells

Activates: T, B and NK cells (synergism with IL-2 and IFN~) eosinophils (degranulation) endothelial cells and smooth muscle cells nerve cells, adipocytes, hepatocytes chondrocytes, osteoclasts and fibroblasts thyrocytes and pancreatic ]3 cells (low conc.) Cytotoxic: melanocytes and pancreatic 13cells In vivo effects: fever, anorexia, slow-wave sleep, neuropeptide prod. acute-phase protein induction insulin, ACTH, cortisol induction

IL-6

21--28

M~

as LIF and IL-1 (few exceptions) Induces: maturation of megakaryocytes Stimulates: hepatocytes (acute-phase proteins) Shortens: GO in hematopoietic progenitor cells Promotes: Ig secretion by activated B cells

T cells (TH2) fibroblasts hepatocytes endothelial cells neuronal cells cardiac myxoma cells thyrocytes and pancreatic islet cells various neoplastic cells IL-10

18 (x2)

M~ B and T cells (TH0, TH2) (delayed production) mast cells keratinocytes

LIF

46--90

bone marrow stromal cells T cells M~ astrocytes fibroblasts

Coactivates: thymocytes (with IL-2 and/or IL-4) B cells: MHC II, viability, Ig secretion mast cells (growth) Inhibits (through inhibition of IL-12 ?) IFNy production by Thl cells M~), cytotoxic T cells and NK cells

as IL-6 Promotes survival and growth of." sensory neurons Activates growth of." embryonic stem cells and megakaryocytes hepatocytes, fibroblasts, osteoblasts, pre-adipocytes, myoblasts, endothelial cells AIDS Kaposi sarcoma cells

211

particular). This cytokine is a potent suppressor of M~), chiefly because it counteracts the stimulatory functions of IFNy, e.g., induction of MHC class II expression and cytokine synthesis. IL-10, therefore, inhibits antigen presentation and, indirectly, T-lymphocyte functions. The cytokines whose production are most affected by IL-10 are those originating from TH 1 cells. Interestingly, two herpes viruses have acquired an IL-10 gene: an equine herpes virus and the EpsteinBarr virus (EBV). Thus, analysis of the coding sequence of the IL-10 gene reveals that it is highly homologous to the EBV open reading frame BCRF1, and that the EBV-derived polypeptide, viral IL-10, has the same biological activities as IL-10. IL-10 is expressed by some neoplastic cells, including primary B-cell tumors as well as basal cell and bronchogenic carcinomas. IL-10 suppresses the functions of cytotoxic T cells and natural killer (NK) cells, and it is a potent inhibitor of tumor cytotoxicity by human M~) (Mosmann, 1994). IL-10 gene disrupted "knock-out" mice develop chronic enterocolitis, suggesting a role of IL-10 as an immunoregulator in the intestinal tract. LIF. LIF, a 46--90 kd variously glycosylated protein, is produced by several cell types involved in hematopoiesis, nerve functions and immunoinflammatory reactions (Table 1); it shares a signal transducing receptor component termed gpl30 with other cytokines, such as IL-6. In adult life, LIF can influence M~) and platelet formation, osteoblast, endothelial cell and neuronal functions, calcium and lipid metabolism and the production of acute-phase proteins (Patterson, 1994). All of these effects appear to be exerted by direct actions through specific receptor complexes on the various target cells. Gene-targeted mice with overexpression of LIF develop excess bone formation, behavioral disorders, wasting and death. Female mice lacking a functional LIF gene are infertile because of a uterine failure which prevents implantation of the blastocyst; male mice are fertile.

such as IL-10 and LIF, is not understood. However, the Fab fragments of the respective autoantibodies bind in a saturable manner to the above-mentioned cytokines with exquisite specificity and with remarkably high avidities (Table 2). Indeed, the autoantibodies to IL-lc~ and IL-6 are the single most important binding proteins of these cytokines in normal human serum. Although measurable in human IgG preparations, autoantibodies to IFNo~ and IL-10 are difficult to detect in normal sera, most likely because they are present in serum in a saturated form complexed with their respective cytokines, or because of the presence of other inhibitory factors. Techniques to separate the pre-existing immune complexes in serum should ideally preserve autoantibodies binding to the cytokine(s); separate determination of autoantibodies and cytokine is a considerable challenge because of the high-avidity binding. Why and how high-affinity autoantibodies are induced to some cytokines and not to others is unknown. Also obscure is whether these in vitro neutralizing autoantibodies also neutralize their respective cytokines in vivo, or whether they exhibit carrier or cytokine-protective functions (Bendtzen et al., 1990). Clearly, however, in vivo circulating cytokines are stabilized by cytokine-binding proteins (including in vitro neutralizing monoclonal antibodies) in the form of cytokine-IgG complexes (Klein and Brailly, 1995). The longer in vivo half-life of these complexes provides a pharmacokinetic explanation as to why some cytokines (e.g., IL-6) accumulate in individuals treated with anti-IL-6 antibodies. The presence of preexisting autoantibodies to IL-6 and other cytokines is clearly of great clinical interest, with regard to cytokine therapy and administration of hyperimmune and/or normal human IgG. Autoantibodies to IL-1 o~are particularly interesting as natural immunomodulators, because IL-lo~ is an important co-stimulator of activated T cells (particularly antigen-presenting cells) and because IL-lo~ is probably an autocrine growth factor for TH2 cells. By binding to soluble as well as membrane-associated ILl o~ and inhibiting the bioactivity of both forms of the cytokine (Svenson et al., 1992), autoantibodies to IL-lo~ should affect the function of IL-1 o~responsive T cells.

THE AUTOANTIBODIES Methods of Detection Pathogenetic Role The biological role of specific autoantibodies to ILl c~, IL-6 and IFNo~, and possibly to other cytokines

212

Interpretation of clinical studies of autoantibodies to cytokines is hampered by significant methodological problems. Low levels of autoantibodies to a particular

cytokine are not necessarily a contributing factor to disease development and might simply reflect increased consumption of autoantibodies in conjunction with increased local or systemic production of the relevant cytokine(s) during active inflammation. Aside from their putative pathogenic role, autoantibodies to cytokines in biological fluids are also important as potential in vitro inhibitors of biochemical and biological assays for various cytokines. Methods used for the detection of antibodies to cytokines include antiviral neutralization assays, other cytokine bioassays, immunometric assays and various blotting techniques. Serum antibodies, however, do not always bind soluble polypeptides in a specific manner or, indeed, with any appreciable affinity (Hansen et al., 1992; Svenson et al., 1993). For example, immunoblotting techniques and immunometric assays may show some degree of specificity even though the binding of antibodies to ligand is weak and topographically unassociated with the specific binding sites of the antibodies (Figure 1). Although these techniques can be used for screening purposes, demonstration of ligand binding to the Fab fragments of the immunoglobulins, combined with saturation binding analysis and demonstration of cross-binding to the native cytokine, is necessary to verify the presence of specific autoantibodies to a given cytokine.

CLINICAL UTILITY

Disease Association The prevalence of autoantibodies to IL-1 a in immunoinflammatory disorders varies considerably (Bendtzen et al., 1995). There is an increased prevalence of highavidity autoantibodies to IL-6 in patients with rheumatoid arthritis and systemic sclerosis (Hansen et al., 1991b; 1993; 1995a; Takemura et al., 1992; Suzuki et al., 1994). By contrast, autoantibodies to IL-I~ are absent in certain immunoinflammatory diseases, including Crohn's disease of the gut and atopic diseases (Bendtzen et al., 1995). The presence of these autoantibodies signals a poor survival in patients with alcoholic cirrhosis (Homann et al., submitted). The increased prevalence of these autoantibodies in patients with Sdzary syndrome (the leukemic stage of cutaneous T-cell lymphoma) compared with patients with the tumor or plaque stages of the disease might promote dissemination of this disease by neutralizing the induction of adhesion molecules in the skin. There are as yet no data on the presence of autoantibodies to IL-10 and LIF in patients.

Effect of Therapy Because of its antiviral, antitumor and immunoregula-

Table 2. Autoantibodies to Cytokines in Healthy Adults1 IFN~ aAb

IL-1~ aAb

IL-6 aAb

Frequency in normal sera2

10%

30--75%

10--20%

Increased frequency with age

no

yes

no

Increased frequency in males

no

yes

no

Predominant Ig class

IgG 1

IgG4,2,1

IgG 1

Block receptor binding

yes

yes

yes

Block bioactivity in vitro

yes

yes

yes

Kd

<10-10 M

<10-11 M

<10-10 M

Max binding in 1 ml serum

9

30 ng

300 ng

Bind with Fab

yes

yes

yes

Miscellaneous

cross-reacts with IFN[3

Ligand binding

lAb to other cytokines such as IL-2, IL-8, IL-10, LIF, TNF~ and IFNy still have to be formally proven as true autoantibodies. 2Detected by double antibody precipitation and protein G binding of untreated serum (preexisting cytokine-Ig complexes may go undetected).

213

Figure 1. Cytokine antibodies. Specific and nonspecific reactions in ELISA designed to test for autoantibodies to a cytokine. Only case 1 detects specific antibody to the cytokine. The same principle applies to detection of (auto)antibodies by other immunometric and immunoblotting techniques. tory effects, IFNo~ has been given to patients with a variety of diseases with varying clinical responses (Baron et al., 1992). The development of neutralizing antibodies in some of these patients may result in therapeutic failure (Antonelli et al., 1991; Bocci, 1991). It is, therefore, of considerable clinical importance to determine prior to therapy whether highaffinity and in vitro neutralizing autoantibodies to IFNo~ are present and establish whether IFNo~ therapy will augment the production of these autoantibodies to the detriment of the patient's therapeutic outcome. The relatively high levels of IFNo~-specific IgG in hyperimmune and normal IgG preparations may be relevant to treatment of a number of immunoinflammatory conditions with high-dose IgG (Ross et al., 1995). Whether autoantibodies to IFNo~ in such preparations neutralize the effects of IFNo~ in vivo and hence contribute to a positive therapeutic outcome is unknown. On the other hand, IgG preparations containing autoantibodies to IFNo~ might have untoward effects in patients given IgG to combat infection, particularly if the infection is of viral origin. Because IL-1cz associated with the cytoplasmic membrane of antigen-presenting cells appears to be 214

particularly important in the triggering of T cells, the administration of IgG containing neutralizing autoantibodies to membrane IL-lo~ might contribute to prompt immunosuppression in vivo. Also, IgG1 and IgG2 autoantibodies to IL-1 oc could trigger cytotoxic processes directed against both IL-lo~-producing and IL- 1o~-responding cells, with resultant rapid decrease in the number of circulating T and B cells.

CONCLUSION Naturally occurring, specific, high-avidity autoantibodies to IFNo~, IL-lo~ and IL-6, and perhaps to ILl0, LIF and a few other cytokines interfere with biological and immunometric assays for these cytokines in vitro. Although these autoantibodies might neutralize cytokines in vivo, some cytokine antibodies function as carriers and, paradoxically, prolong cytokine functions in vivo. Inappropriate production of autoantibodies to cytokines could be pathogenetically involved in infectious and other immunoinflammatory diseases, including infections in patients with classical autoimmune diseases. Cytokine autoantibodies may

also contribute to the anti-inflammatory and i m m u n o suppressive effects of h u m a n high-dose I g G therapy. Viral diseases treated with h u m a n I g G preparations m i g h t be negatively influenced by cytokine-specific I g G contained in these preparations, particularly autoantibodies directed against I F N ~ .

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ACKNOWLEDGEMENTS Supported by the Danish Medical Research Council, the Danish Cancer Society and the Danish B iotechnology Program.

affinity IgG autoantibodies to IL-6 in sera of normal individuals are competitive inhibitors of IL-6 in vitro. Cytokine 1993;5:72-80. Hansen MB, Svenson M, Abell K, Varming K, Nielsen HP, Bertelsen A, Bendtzen K. Sex- and age-dependency of IgG autoantibodies against IL-lalpha in healthy humans. Eur J Clin Invest 1994;24:212-218. Hansen MB, Andersen V, Rohde K, et al. Cytokine autoantibodies in rheumatoid arthritis. Scand J Rheumatol 1995a;in press. Hansen MB, Svenson M, Abell K, Yasukawa K, Diamant M, Bendtzen K. Influence of interleukin-6 (IL-6) autoantibodies on IL-6 binding to cellular receptors. Eur J Immunol 1995b; 25:348--354. Hansen MB, Svenson M, Diamant M, Ross C, Bendtzen K. Interleukin-6 (IL-6) autoantibodies and blood IL-6 measurements. Blood 1995c;85:1145. Heilig B, Fiehn C, Brockhaus M, Gallati H, Pezzutto A, Hunstein W. Evaluation of soluble tumor necrosis factor (TNF) receptors and TNF receptor antibodies in patients with systemic lupus erythematodes, progressive systemic sclerosis, and mixed connective tissue disease. J Clin Immunol 1993; 13:321-328. Ikeda Y, Toda G, Hashimoto N, Umeda N, Miyake K, Yamanaka M, Kurokowa K. Naturally occurring anti-interferonalpha 2a antibodies in patients with acute viral hepatitis. Clin Exp Immunol 1991;85:80-84. Klein B, Brailly H. Cytokine-binding proteins: stimulating antagonists. Immunol Today 1995; 16:216-220. Mae N, Liberato DJ, Chizzonite R, Satoh H. Identification of high-affinity anti-IL-lc~ autoantibodies in normal human serum as an interfering substance in a sensitive enzymelinked immunosorbent assay for IL-1 alpha. Lymphokine Cytokine Res 1991 ;10:61--68. Mogensen KE, Daubas PH, Gresser I, Sereni D, Varet B. Metropol in essential tremor. Lancet 1981 ;2:1227--1228. Mosmann TR. Properties and functions of interleukin-10. Adv Immunol 1994;56:1--26. Panem S, Check IJ, Henriksen D, Vilcek J. Antibodies to ~interferon in a patient with systemic lupus erythematosus. J Immunol 1982;129:1--3. Panem S. Antibodies to interferon in man. Interferon 1984;2: 175-183. Patterson PH. Leukemia inhibitory factor, a cytokine at the interface between neurobiology and immunology. Proc Natl Acad Sci USA 1994;91:7833-7835. Pedersen M, Permin H, Bindslev-Jensen C, Bendtzen K, Norn S. Cytokine-induced histamine release from basophils of

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AIDS patients. Interaction between cytokines and specific IgE antibodies. Allergy 1991;46:129--134. Pozzetto B, Mogensen KE, Tovey MG, Gresser I. Characteristics of autoantibodies to human interferon in a patient with Varicella-zoster disease. J Infect Dis 1984;150:707-713. Priimmer O, Seyfarth C, Scherbaum WA, Drees N, Porzsolt F. Interferon-cz antibodies in autoimmune diseases. J Interferon Res 1989;9(Suppl) 1:$67-$74. Prfimmer O, Frickhofen N, Digel W, Heimpel H, Porzsolt F. Spontaneous interferon-cz antibodies in a patient with pure red cell aplasia and recurrent cutaneous carcinomas. Ann Hematol 1991;62:76--80. Priimmer O, Bunjes D, Wiesneth M, Arnold R, Porzsolt F, Heimpel H. High-titre interferon-cz antibodies in a patient with chronic graft-versus-host disease after allogeneic bone marrow transplantation. Bone Marrow Transplant 1994; 14: 483--486. Ross C, Hansen MB, Schyberg T, Berg K. Autoantibodies to crude human leucocyte interferon (IFN), native human IFN, recombinant human IFN-cz 2b and human IFN-7 in healthy blood donors. Clin Exp Immunol 1990;82:57--62. Ross C, Svenson M, Hansen MB, Vejlsgaard GL, Bendtzen K. High avidity IFN-neutralizing antibodies in pharmaceutically prepared human IgG. J Clin Invest 1995;95:1974--1978. Satoh H, Chizzonite R, Ostrowski C, Ni Wu G, Kim H, Fayer B, Mae N, Nadeau R, Liberato DJ. Characterization of antiIL-1cz autoantibodies in the sera from healthy humans. Immunopharmacology 1994;27:107-- 118. Saurat J-H, Schifferli J, Steiger G, Dayer J-M, Didierjean L. Anti-interleukin- 1cz autoantibodies in humans: characterization, isotype distribution, and receptor-binding inhibitionhigher frequency in Schnitzler's syndrome (urticaria and macroglobulinemia). J Allergy Clin Immunol 1991;88: 244-256. Sunder-Plassmann G, Sedlacek PL, Sunder-Plassmann R, Derfler K, Swoboda K, Fabrizii V, Hirschl MM, Balcke P. Anti-interleukin- 1cz autoantibodies in hemodialysis patients. Kidney Int 1991;40:787--791. Suzuki H, Akama T, Okane M, Kono I, Matsui Y, Yamane K, Kashiwagi H. Interleukin-1-inhibitory IgG in sera from some patients with rheumatoid arthritis. Arthritis Rheum 1989;32: 1528--1538. Suzuki H, Kamimura J, Ayabe T, Kashiwagi H. Demonstration of neutralizing autoantibodies against IL-lcz in sera from

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patients with rheumatoid arthritis. J Immunol 1990;145: 2140-2146. Suzuki H, Ayabe T, Kamimura J, Kashiwagi H. Anti-IL-lcz autoantibodies in patients with rheumatic diseases and in healthy subjects. Clin Exp Immunol 1991 ;85:407-412. Suzuki H, Takemura H, Yoshizaki K, Koishihara Y, ohsugi Y, Okano A, Akiyama Y, Tojo T, Kishimoto T, Kashiwagi H. IL-6-anti-IL-6 autoantibody complexes with IL-6 activity in sera from some patients with systemic sclerosis. J Immunol 1994;152:935--942. Svenson M, Poulsen LK, Fomsgaard A, Bendtzen K. IgG autoantibodies against interleukin l cz in sera of normal individuals. Scand J Immunol 1989;29:489--492. Svenson M, Hansen MB, Bendtzen K. Distribution and characterization of autoantibodies to interleukin 1cz in normal human sera. Scand J Immunol 1990;32:695--701. Svenson M, Hansen MB, Kayser L, Rasmussen AK, Reimert CM, Bendtzen K. Effects of human anti-IL- 1cz autoantibodies on receptor binding and biological activities of IL-1. Cytokine 1992;4:125-133. Svenson M, Hansen MB, Bendtzen K. Binding of cytokines to pharmaceutically prepared human immunoglobulin. J Clin Invest 1993;92:2533-2539. Sylvester I, Yoshimura T, Sticherling M, Schroder JM, Ceska M, Peichl P, Leonard EJ. Neutrophil attractant protein-1immunoglobulin G immune complexes and free anti-NAP-1 antibody in normal human serum. J Clin Invest 1992;90: 471-481. Takemura H, Suzuki H, Yoshizaki K, Ogata A, Yuhara T, Akama T, Kashiwagi H. Anti-interleukin-6 autoantibodies in rheumatic diseases. Increased frequency in the sera of patients with systemic sclerosis. Arthritis Rheum 1992;35: 940-943. Tiberio L, Caruso A, Pozzi A, Rivoltini L, Morelli D, Monte E, Balsari A. The detection and biological activity of human antibodies to IL-2 in normal donors. Scand J Immunol 1993;38:472--476. Trown PW, Kramer MJ, Dennin RA Jr, Connell EV, Palleroni AV, Quesada J, Gutterman JU. Antibodies to human leucocyte interferons in cancer patients. Lancet 1983;1:81--84. Viani E, Flamminio G, Caruso A, Foresti I, De Francesco M, Pollara P, Balsari A, Turano A. Purification of natural human IFN-7 antibodies. Immunol Lett 1991 ;30:53--58.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

CYTOSKELETAL AUTOANTIBODIES Margarida Castell, Ph.D.

Unit of Physiology and Physiopathology, Faculty of Pharmacy, University of Barcelona, Barcelona 08028, Spain

HISTORICAL NOTES Antiactin antibodies, the first anticytoskeletal autoantibodies described, were initially noted in liver diseases (Johnson et al., 1965; Gabbiani et al., 1973). Antitubulin antibodies were first described in infectious mononucleosis (Whitehouse et al., 1974), and antibodies to intermediate filaments of the cytoskeleton were first described in the sera of patients with a variety of conditions (Kurki et al., 1977).

THE AUTOANTIGENS Definition Anticytoskeletal antibodies comprise immunoglobulins that react with any of the proteins that constitute the cell skeleton. Anticytoskeletal antibodies have many specificities, including antiactin, antitubulin and antiintermediate filament antibodies. The cytoskeleton is formed by a complex network of filamentous proteins distributed throughout the cytoplasm (Lazarides, 1980). On the basis of morphological, biochemical and immunological criteria, three categories of fibers are recognized: microfilaments, microtubules and intermediate filaments (Table 1). Microfilaments. Microfilaments, rootlike structures formed from the protein actin, are organized in bundles and sheets arranged in parallel arrays on the internal face of the plasma membrane. Highly conserved in evolution, actin filaments are present in all eukaryote cells. Purified actin is a monomer called Gactin, which in the presence of ATP, polymerizes to F-actin. About half the microfilaments are composed of heteropolymers, in which actin is linked mainly to myosin (Weber and Osborn, 1985).

Microtubules. Larger than microfilaments, microtubules consist of tubulin, a highly conserved protein present in almost all eukaryote cells in amounts lower than actin. Microtubules are arranged like individual filaments that irradiate from centrioles of the centrosome to the cytoplasmic membrane (Weber and Osborn, 1985). Intermediate Filaments. Intermediate filaments (IF) derive their name from their diameter that is between those of actin filaments and microtubules. Review of the structure, dynamics and function of IF reveals interesting characteristics relevant to normal cell biology and disease (Fuchs and Weber, 1994). IF compose a heterogeneous superfamily, with cell-type specificity and often complex patterns of expression (Lazarides, 1980). IF are fibrous proteins, which are laterally associated and overlapped, forming highly resistant filaments. IF proteins are likely to share a common secondary structure (Fuchs and Weber, 1994). The nearly 50 known human IF proteins are grouped into five different types on the basis of differences in rod-domain sequences, end-domain sequence organization and gene structure. Based on continuing discoveries of new IF proteins, the size of the IF superfamily continues to increase. Type I and Type II IF Proteins. This group comprises keratins, the largest and most complex group of IF proteins, including at least 30 proteins that are characteristic of epithelial cells (Table 2). Type III IF Proteins. Type III IF proteins include vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin (Table 2). Vimentin, the most widely expressed type III IF, is produced by mesenchymal cell types and by a variety of transformed cell lines and tumors. Vimentin is often expressed in 217

Table 1. Main Features of Cytoskeleton Fibers Cytoskeleton fibers

Diameter

Main component

Function

Microfilaments

4--7 nm

Actin (42 kd)

Contraction of muscle fibers Cell movement and movements within cells Stress fibers

Microtubules

-23 nm

Tubulin (50 kd)

Support and shape cells Movement within cells Flagella and cilia Mitotic spindle

Intermediate filaments

7--11 nm

See Table 2

Structural reinforcement inside cells Hold organelles in place Shape cells

cultivated cells and at certain stages in developing cells. Desmin is displayed in smooth, skeletal and cardiac muscle cells, GFAP in filaments of glial cells, and peripherin in neurons and ganglia of the peripheral nervous system.

Type V IF Proteins. This group of IF proteins comprise the nuclear lamins that constitute the filaments beneath the nuclear envelope. As expressed in higher eukaryotes, lamins seem to provide a framework for the nucleus.

Type IV IF Proteins. Type IV IF proteins group neurofilament proteins and ~-internexin. These proteins together with microtubules constitute the main structural element of neural axons, dendrites and perikarya.

Methods of Purification Procedures for the purification of muscle actin (Spudich and Watt, 1971) and brain tubulin (Shelanski et al., 1973; Mead et al., 1980) are available.

Table 2. Main Features of IF Proteins Type

III

IV

Generic name

Specific name

Molecular weight

Location

Acidic keratins (pKi 4--6)

K9--K20 (epithelial keratins) Hal-Ha4 (hair keratins)

ranging between 40--67 kd

epithelial cells

Basic keratins (pKi 6-8)

K1--K8 (epithelial keratins) Hbl--Hb4 (hair keratins)

ranging between 40--67 kd

epithelial cells

Vimentin

Vimentin

57 kd

Desmin Glial fibrillary acidic protein Peripherin

Desmin GFAP

53--54 kd 50 kd

Peripherin

57 kd

mesenchymal cells (transforming cell lines and tumors) muscle cells astrocytes and some Schwann cells neurons

Neurofilaments

NF-L NF-M NF-H

62 kd 102 kd 110 kd 66-70 kd

a-internexin Nuclear lamins

218

type A type B type C

70 kd 63--68 kd 60 kd

neurons neurons nucleus

Intermediate filament proteins can be purified from cells by treatment with a nonionic detergent that leaves insoluble cytoskeleton and actin filaments. Microfilaments can be removed by exposing the cells to high-salt buffers. IF proteins can then be obtained by the presence of a strong ionic detergent (Lazarides, 1982). Keratins can be extracted at low pH or by denaturing solvents (for example, 8M urea); a reducing agent is also required for the efficient solubilization of keratins from the terminally differentiated cells (Lazarides, 1980). Methods to obtain vimentin from pig lens and desmin from chicken gizzard are also available (Geisler and Weber, 1980; 1981).

Commercial Sources At present, most cytoskeleton proteins can be obtained commercially (Sigma; Boehringer Mannheim).

AUTOANTIBODIES

Terminology Autoantibodies to cytoskeleton proteins found in sera from most healthy humans and animals are considered natural autoantibodies (Avrameas, 1991). Antiactin, antitubulin and anti-IF reactivities are commonly associated with smooth muscle antibodies (SMA) (Mead et al., 1980). Although SMA and antibodies against cytoskeletal proteins correlate significantly, some SMA-positive sera contain antibodies that are either not directed against known cytoskeleton proteins or might be reactive with cytoskeleton-associated proteins or epitopes which are related to the tertiary structure of polymerized cytoskeletal proteins (Dighiero et al., 1990). The specificities of antibodies against cytokeratins and the so-called antikeratin antibodies differ; the latter react with the stratum corneum of esophagus epithelium and constitute a specific serological criterion for the diagnosis of rheumatoid arthritis. The antigenic proteins recognized by the antikeratin antibodies were recently characterized biochemically as three noncytokeratin late-differentiation proteins referred to as A, B and C (Sebbag et al., 1995).

Pathogenetic Role It is doubtful that the anticytoskeletal antibodies found in a proportion of healthy individuals and considered

natural autoantibodies (Avrameas 199 l; Senecal et al., 1993) have a role in pathogenesis.

Human Disease. Present in a wide range of diseases, anticytoskeletal antibodies are attributed, in most cases, to nonspecific stimulation of B cells, i.e., bacterial, viral, parasitic or other factors activate B cells to produce polyclonally natural autoantibodies to cytoskeleton proteins. This hypothesis is supported by an increase in anticytoskeletal antibodies produced after the induction of infection in animals (MortazaviMilani et al., 1984a) and by experiments which show that most EBV-immortalized human B-cell lines produce autoantibodies reactive with cytoskeleton proteins (Seigneurin et al., 1988). Moreover, some anticytoskeletal antibodies are polyreactive, i.e., they react with more than one antigen, including some unrelated to cytoskeleton proteins (Dellagi et al., 1984; Blaschek et al., 1988; Nikkari et al., 1993). Animal Models. Antibodies to actin and tubulin are found in mice with an SLE-like syndrome (Hentati et al., 1991). Anti-IF antibodies occur spontaneously in rabbits (Osborn et al., 1977). Rats with adjuvant arthritis have high activities of antivimentin and anticytokeratin antibodies in advanced phases of the disease (Franch et al., 1994). Antiactin, antivimentin and anticytokeratin antibodies are also detected in a model of progressive systemic sclerosis (Muryoi et al., 1992). In some diseases that involve tissue destruction, anticytoskeletal antibodies might result from an antigen-driven response; for example, the antiactin, antitubulin, antivimentin and antidesmin antibodies found in chronic liver diseases might be the result of hepatocyte destruction. Likewise, because vimentin is an IF protein that is prominent in proliferating synoviocytes (Osung et al., 1982) and because there is damage to cytokeratin-containing cells of the synovial endothelium (Borg et al., 1993), antivimentin and anticytokeratin antibodies in rheumatoid arthritis patients might follow destruction of synovia. Studies of adjuvant arthritis are consistent with the appearance of anti-IF antibodies in response to tissue destruction in that antivimentin and anticytokeratin antibodies appear in the chronic phase of adjuvant-induced arthritis in rats, when the inflammatory response in the hind paw is well established but not before, even though the adjuvant arthritic model is induced by mycobacterial adjuvant which is a polyclonal activator (Franch et al., 1994). Likewise, the anticytokeratin 219

antibodies in Crohn's disease and in psoriatic arthropathy and psoriasis might result from epidermal cell destruction (Borg et al., 1994). In all these cases, however, autoantibodies to cytoskeleton proteins reflect pathology (a consequence of the disease) rather than pathogenesis, i.e., able to cause certain disease manifestations. Nevertheless, when anticytoskeletal antibodies are produced by an increase in natural autoantibodies, the accumulation of these antibodies in the sera and their participation in the formation of circulating immune complexes might be important in pathogenesis (Louzir et al., 1992).

Factors in Pathogenicity The study of isotypes of anticytoskeletal antibodies does not provide conclusive results. IgM, IgG and even IgA can be the predominant class of autoantibodies against actin (Mayet et al., 1990; Louzir et al., 1992), against tubulin (Louzir et al., 1992) and against IF-proteins (Nikkari et al., 1993; Borg et al., 1993).

Methods of Detection Indirect immunofluorescence, the first method employed for detection of anticytoskeletal antibodies, is still useful for the identification of these antibodies, even though more specific methods such as immunoblotting and ELISA are now available. Cryostat sections of several tissues, including stomach, liver or kidney, can be used to study the fluorescence pattern obtained after sequential incubation of sera and anti-Ig conjugated to a fluorochrome. Anti-IF activities are now commonly detected with

monolayers of cultured cells, including fibroblasts and HEp-2 cells. By immunofluorescence on fibroblasts, antiactin reaCtivity presents a cable pattern (stress fibers) that cross the cell surface longitudinally (Figure 1A). If these cells are pretreated with cytochalasin B, actin depolymerizes and the pattern changes to small clusters of fine-punctate fluorescence (Figure 1B). Antibodies to tubulin and antibodies to IF proteins incubated on cultured cells produce a similar pattern, consisting of a fibrous meshwork extending throughout the cytoplasm and particularly abundant around the nucleus (Figure 2A,C,E). Differentiation among these reactivities is permitted by drug treatment of cells; colchicine produces the destruction of microtubules and redistributes vimentin filaments into a characteristic perinuclear coil (Figure 2B); whereas, cytokeratin filaments are only partially affected (Figure 2D). Vinblastine sulfate leads to the organization of microtubules into crystal-like structures. Besides treating with colchicine to differentiate between antibodies to vimentin and to cytokeratin, cells of different embryonal origin can be used. Thus, the IF found in mesenchymal cells are of the vimentin type; whereas, cytokeratin filaments are present in epithelial cells or cells of epithelial origin. However, it must be taken into account that cells growing in culture often develop vimentin filaments in addition to their tissue-specific IF. Use of immunoblotting to improve the characterization of anticytoskeletal antibodies yields conflicting data. Indirect immunofluorescence results do not always agree with immunoblots, and sera negative by immunofluorescence can be immunoblot positive,

Figure 1. Fluorescent pattern characteristic of actin microfilaments. Human skin fibroblasts fixed with 4% paraformaldehyde, permeabilized with methanol, stained by FITC-conjugated phalloidin (Sigma). A: Nonpretreated fibroblasts. B: Fibroblasts pretreated with 1 ~aMcytochalasin (Sigma) for 30 min at 37~ (courtesy of S. Vilaro from Unit of Cell Biology, University of Barcelona). 220

Figure 2. Fluorescent pattern characteristic of vimentin, cytokeratin and tubulin filaments. HEp-2 cells fixed with absolute acetone. A: Cells stained with antimouse vimentin monoclonal antibody (Boehringer Mannheim) and F(ab') 2 antimouse IgG-FITC (Sigma). B: Cells pretreated with 0.25 jag/mL colchicine (12 h at 370C) (Gibco) and stained with the same monoclonal antibodies. C: Cells stained with antimouse cytokeratin monoclonal antibody (ICN ImmunoBiologicals) and F(ab') 2 antimouse IgG-FITC. D: Cells pretreated with 0.25 lag/mL colchicine (12 h at 37~ and stained with the same monoclonal antibodies. E: Cells stained with antimouse tubulin monoclonal antibody (Boehringer Mannheim) and F(ab') 2 antimouse IgG-FITC. F: Cells pretreated with 0.25 ~g/mL colchicine (12 h at 37~ and stained with the same monoclonal antibodies.

221

Table

3. Disease Association of Actin and Tubulin Autoantibodies Frequency of Positive Results (%)

Antibody/Disease

Patients

Work dilution

Normal

Work dilution

44.5% 80% 51.9% 39% IgA 88.7% IgG 71%; IgM 58%

1/200 1/200 1/200 1/100 1/100

4.8% 10% **

1/200 1/200 1/200 1/100 1/100

38% 26% 9% 56% 57% 14%

1/100 1/100 1/50-- 1/200 1/100 1/100 1/50-1/400

10% 10% 0% 10% 10% 0%

1/100 1/100 <1/20 1/100 1/100 < 1/20

IgM 48.3% IgG 36.7%; IgA 26.7%

1/500

**

IgG 68.4%

1/100-1/900

Chronic liver disease -- Primary biliary cirrhosis -- Autoimmune chronic active hepatitis -- Hepatitis B virus-related chronic liver disease

55.5% 35.2% IgG 51.6% IgM 32%; IgA 27.4%

Infectious diseases Active visceral leishmaniasis - - Infectious mononucleosis

86%-IGG IgM 86%; IgG 34%

Methodology

Reference

ELISA ELISA ELISA

Dighiero et al., 1990 Cunningham et al., 1985 Dighiero et al., 1990 Girard and Senecal, 1995 Louzir et al., 1992

Antiaetin

Chronic liver disease -- Primary biliary cirrhosis -- Alcoholic cirrhosis - - Autoimmune chronic active hepatitis -- Hepatitis B virus-related chronic liver disease Connective tissue diseases Rheumatoid arthritis -- SLE --

Polymyositis/dermatomyositis -- Systemic sclerosis --

Chronic inflammatory bowel diseases Crohn's disease

--

Infectious diseases Active visceral leishmaniasis

--

4.8% *

IB

ELISA

IIF (PTK2)

Girard and Senecal, Girard and Senecal, Senecal et al., 1985 Girard and Senecal, Girard and Senecal, Senecal et al., 1995

1/500

ELISA

Mayet et al., 1990

6.6%

1/100--1/900

ELISA

Louzir et al., 1994

1/200 1/200 1/100

4.8% 4.8% **

1/200 1/200 1/100

ELISA ELISA ELISA

Dighiero et al., 1990 Dighiero et al., 1990 Louzir et al., 1992

1/100-- 1/900 1/1000

10% *

1/100-1/900 1/1000

ELISA RIA

Louzir et al., 1994 Mead et al., 1980

IB IB

IIF (PTK2) IB IB

Antitubulin

--

*Significant difference between patient levels and normal levels (p < 0.001); **positive values higher than normal mean + 2 SD.

1995 1995 1995 1995

Table 4. Disease Association of Intermediate Filament Autoantibodies Frequency of Positive Results (%) Disease

-

Ab Specificity

-

-

-

-

-

Chronic liver disease Primary biliary cirrhosis -

-- Autoimmune chronic active hepatitis

-

Chronic inflammatory bowel diseases Crohn's disease -

-

-

-

-

-

a c c

1/50--1/400 1/50--1/800 1/80 1/80

42.5% 42.5% 1.7% 1.7%

1/10--1/20 1/10--1/20 1/80 1/80

IIF (PTK2) IIF (PTK2) ELISA ELISA

a a d d

35.3% 41.1% 27.6% 43.4%

1/200 1/200 1/200 1/200

0% 0% 0% 0%

1/200 1/200 1/200 1/200

ELISA ELISA ELISA ELISA

e e e e

IgG IgM IgG IgM IgG IgM

20%; IgA 13.3%; 6.7% 3.3%; IgA 10%; 16.7% 6.7%; IgA 5%; 15.1%

1/500

**

1/500

ELISA

f

1/500

**

1/500

ELISA

f

1/500

**

1/500

ELISA

f

IgG 5.4%; IgA 3.6% IgM 12.5%; IgG 3.6% IgG 23.2%; IgM 54%

1/500 1/500 1/500

** ** **

1/500 1/500 1/500

ELISA ELISA ELISA

g g g

48%--IGM 82%--IGM

1/16--1/512 1/20--1/320

-8%

1/10--1/20

IIF (fibroblasts) IIF (fibroblasts, HEp2)

h i

97%--IGM

1/8--1/512

IgM 23.5%

1/8--1/16

IIF (fibroblasts)

j

75%

1/64--1/512

0%

< 1/30

IIF (fibroblasts)/IB

k

vimentin vimentin

IgM 25% IgM 38%; IgG 16% IgM 36%; IgG 64%

1/50--1/400 1/10 1/500

cytokeratin 18 cytokeratin 18

IgM IgM IgA IgA

vimentin desmin vimentin desmin cytokeratin 18

cytokeratin 18 vimentin desmin

-

-

IIF (PTK2) IIF (fibroblasts) ELISA

17.5% 1.4%

-

Mycoplasma infections

Lymphoproliferative diseases Angioimmunoblastic lymphadenopathy

1/10--1/20 1/10

1/50-1/400 1/80

Infectious diseases Acute viral hepatitis Acute malaria -

42.5% IgM 17%; IgG 6% **

IgM 54.5% IgA 39.6%

desmin

Vascular disease Coronary artery disease

a b

cytokeratin 18

vimentin

-

IIF (PTK2) ELISA

Work dilution

-- SLE

-

1/50-1/100 1/80

Normal

-

Polymyositis/dermatomyositis -- Systemic sclerosis Psoriatic arthropathy Psoriasis

Ref.

Work dilution

Connective tissue diseases Rheumatoid arthritis

-

Methodology Patients

vimentin

71.4% 42.8% 58% 75%

Note: a = Senecal et al., 1985; b = Borg et al., 1993; c = Blaschek et al., 1988; d = Borg et al., 1994; e = Dighiero et al., 1990; f = Mayet et al., 1990; g = Nikkari et al., 1993; h = Pedersen et al., 1981; i = Mortazavi-Milani et al., 1984b; j = Bretherton et al., 1981; k = Dellagi et al., 1984.

1",3 t,,3

because this technique has greater sensitivity; or vice versa, due to denaturing electrophoretic conditions that cause loss of cytoskeleton protein antigenicity (Franch et al., 1994). To quantify anticytoskeletal antibodies, ELISA techniques have been applied. Purified proteins such as actin, tubulin, vimentin, certain cytokeratins and desmin, are coated to a solid-phase to quantify IgG, IgM and IgA anticytoskeletal antibodies (Louzir et al., 1992; Nikkari et al., 1993). In all these methods, commercially available polyclonal or monoclonal anticytoskeletal antibodies can be used as standards.

CLINICAL UTILITY Many diverse diseases are associated with high activity and significant prevalence of anticytoskeletal antibodies. Therefore, no diagnostic meaning can be ascribed to these autoantibodies, and they are not useful in clinical practice. Perhaps in the future, autoantibodies to a specific cytoskeleton protein or epitopes thereof might play an important role in the diagnosis and prognosis of a disease that causes cell destruction. Disease Association

Among the diseases associated with anticytoskeletal antibodies, the most relevant and recent are summarized (below and Tables 3 and 4).

disease (Mayet et al., 1990), and parasite infections (Louzir et al., 1994) can also show antiactin activity. Tubulin Autoantibodies (Antitubulin). Antitubulin are often described in patients who have antiactin activity, including chronic liver diseases (Dighiero et al., 1990; Louzir et al., 1992) and visceral leishmaniasis (Louzir et al., 1994). Antitubulin are also found in patients with infectious mononucleosis (Mead et al., 1980). Autoantibodies to IF Proteins. Serum autoantibodies to vimentin, cytokeratins or desmin are reported in many diseases (Table 4) including several connective tissue diseases, such as rheumatoid arthritis, SLE, polymyositis/dermatomyositis, systemic sclerosis and some spondyloarthropathies (Senecal et al., 1985; Blaschek et al., 1988; Borg et al., 1993; 1994). Patients with Crohn's disease can develop antibodies to vimentin, cytokeratin 18 and desmin (Mayet et al., 1990); whereas, patients with coronary artery disease mainly produce antidesmin antibodies (Nikkari et al., 1993). Anti-IF antibodies are reported in varying frequencies in viral (Pedersen et al., 1981), parasitic (Mortazavi-Milani et al., 1984b) and mycoplasmal infections (Bretherton et al., 1981), as well as in lymphoproliferative diseases such as angioimmunoblastic lymphadenopathy (Dellagi et al., 1984) (Table 4).

CONCLUSION Actin Autoantibodies (Antiactin). Following their description in 1965 in patients with liver diseases (Johnson et al., 1965), the presence of antiactin was confirmed in chronic liver diseases, including cirrhosis (Cunningham et al., 1985; Dighiero et al., 1990) and chronic hepatitis (Dighiero et al., 1990; Louzir et al., 1992; Girard and Senecal, 1995). Antiactin are sometimes found in certain rheumatic diseases, including rheumatoid arthritis, systemic lupus erythematosus (SLE), polymyositis/dermatomyositis and systemic sclerosis (Senecal et al., 1985; Girard and Senecal., 1995). Chronic inflammatory diseases, such as Crohn's

Anticytoskeletal antibodies constitute a complex family of autoantibodies that has multiple specificities and is weakly associated with many different diseases. At present, a pathogenetic role for these autoantibodies is unlikely, but a pathologic role of antibodies to a specific component of the cytoskeleton is possible in some diseases. Further analysis in order to identify the epitopes recognized by natural and by pathologic anticytoskeletal antibodies and the time course of their appearance in diseases, might reveal an important role for anticytoskeletal antibodies. See also ACTIN AUTOANTIBODIES and FILAGGRIN (KERATIN) AUTOANTIBODIES.

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Avrameas S. Natural autoantibodies: from "horror autotoxicus" to "gnothi seauton". Immunol Today 1991;12:154--159. 224

Blaschek MA, Boehme M, Jouquan J, Simitzis AM, Fifas S, Le Goff P, Youinou P. Relation of antivimentin antibodies to anticardiolipin antibodies in systemic lupus erythemat~ Ann Rheum Dis 1988;47:708--716.

Borg AA, Dawes PT, Mattey DL. Increased levels of IgA antibodies to cytokeratin and epidermal keratin in rheumatoid arthritis. Arthritis Rheum 1993;36: 229-233. Borg AA, Nixon NB, Dawes PT, Mattey DL. Increased IgA antibodies to cytokeratins in the spondyloarthropathies. Ann Rheum Dis 1994;53:391-395. Bretherton L, Toh BH, Jack I. IgM autoantibody to intermediate filaments in Mycoplasma pneumoniae infections. Clin Immunol Immunopathol 1981 ;18: 425-430. Cunningham AL, Mackay IR, Frazer IH, Brown C, Pedersen JS, Toh BH, Tait BD, Clarke FM. Antibody to G-actin in different categories of alcoholic liver disease: quantification by an ELISA and significance for alcoholic cirrhosis. Clin Immunol Immunopathol 1985 ;34:158-- 164. Dellagi K, Brouet JC, Seligmann M. Antivimentin autoantibodies in angioimmunoblastic lymphadenopathy. N Eng J Med 1984;310:215-218. Dighiero G, Lymberi P, Monot C, Abuaf N. Sera with high levels of antismooth muscle and antimitochondrial antibodies frequently bind to cytoskeleton proteins. Clin Exp Immunol 1990;82:52--56. Franch A, Castellote C, Vila JL, Vilaro S, Castell M. Anticytoskeletal autoantibody development in adjuvant arthritis. J Rheumatol 1994;21:489-497. Fuchs E, Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem 1994;63:345--382. Gabbiani G, Ryan GB, Lamelin JP, Vassali P, Majno G, Bouvier CA, Cruchaud A, Luscher EF. Human smooth muscle autoantibody. Its identification as antiactin antibody and a study of its binding to "nonmuscular" cells. Am J Pathol 1973;72:473-- 488. Geisler N, Weber K. Purification of smooth-muscle desmin and a protein-chemical comparison of desmins from chicken gizzard and hog stomach. Eur J Biochem 1980;111:425-433. Geisler N, Weber K. Isolation of polymerization-competent vimentin from porcine eye lens tissue. FEBS Lett 1981;125: 253--256. Girard D, Senecal J-L. Antimicrofilament IgG antibodies in normal adults and in patients with autoimmune diseases: immunofluorescence and immunoblotting analysis of 201 subjects reveals polyreactivity with microfilament-associated proteins. Clin Immunol Immunopathol 1995;74: 193--201. Hentati B, Ternynck T, Avrameas S, Payelle-Brogard B. Comparison of natural antibodies to autoantibodies arising during lupus in (NZB • NZW)F1 mice. J Autoimmun 1991;4:341--356. Johnson GD, Holborow EJ, Glynn LE. Antibody to smooth muscle in patients with liver disease. Lancet 1965;2:878--879. Kurki P, Linder E, Virtanen I, Stenman S. Human smooth muscle autoantibodies reacting with intermediate (100 A) filaments. Nature 1977;268:240-- 241. Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature (London) 1980;283:249--256. Lazarides E. Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu Rev Biochem 1982;51:219--250. Louzir H, Ternynck T, Gorgi Y, Tahar S, Ayed K, Avrameas S. Autoantibodies and circulating immune complexes in sera

from patients with hepatitis B virus-related chronic liver disease. Clin Immunol Immunopathol 1992;62:160-- 167. Louzir H, Belal-Kacemi L, Sassi A, Laouini D, Ben Ismail R, Dellagi K, the Leishmania study group. Natural autoantibodies, IgG antibodies to tetanus toxoid and CD5+ B cells in patients with Mediterranean visceral leishmaniasis. Clin Exp Immunol 1994;95:479--484. Mayet WJ, Press AG, Hermann E, Moll R, Manns M, Ewe K, Meyer zum Buschenfelde KH. Antibodies to cytoskeletal proteins in patients with Crohn's disease. Eur J Clin Invest 1990;20:516-524. Mead GM, Cowin P, Whitehouse JM. Antitubulin antibody in healthy adults and patients with infectious mononucleosis and its relationship to smooth muscle antibody (SMA). Clin Exp Immunol 1980;39:328-336. Mortazavi-Milani SM, Facer CA, Holborow EJ. Induction of anti-intermediate filament antibody in rabbits experimentally infected with Trypanosoma brucei. Immunology 1984a;52: 423-426. Mortazavi-Milani SM, Badakere SS, Holborow EJ. Antibody to intermediate filaments of the cytoskeleton in the sera of patients with acute malaria. Clin Exp Immunol 1984b;55: 177-182. Muryoi T, Andre-Schwartz J, Saitoh Y, Daian C, Hall B, Dimitriu-Bona A, Schwartz RS, Bona CA, Kasturi KN. Selfreactive repertoire of tight skin (TSK/+) mouse: immunochemical and molecular characterization of anticellular autoantibodies. Cell Immunol 1992;144:43--54. Nikkari ST, Solakivi T, Sisto T, Jaakkola O. Antibodies to cytoskeletal proteins in sera of patients with angiographically assessed coronary artery disease. Atherosclerosis 1993;98: 11-16. Osborn M, Franke WW, Weber K. Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy. Proc Natl Acad Sci USA 1977;74:2490-2494. Osung OA, Chandra M, Holborow EJ. Intermediate filaments in synovial lining cells in rheumatoid arthritis and other arthritides are of vimentin type. Ann Rheum Dis 1982;41: 74--77. Pedersen JS, Toh BH, Locarnini SA, Gust ID, Shyamala GN. Autoantibody to intermediate filaments in viral hepatitis. Clin Immunol Immunopathol 1981 ;21:154-- 161. Sebbag M, Simon M, Vincent C, Masson-Bessiere C, Girbal E, Durieux JJ, Serre G. The antiperinuclear factor and the socalled antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies. J Clin Invest 1995;95:26722679. Seigneurin JM, Guilbert B, Bourgeat MJ, Avrameas S. Polyspecific natural antibodies and autoantibodies secreted by human lymphocytes immortalized with Epstein-Barr virus. Blood 1988:71: 581--585. Senecal JL, Oliver JM, Rothfield N. Anticytoskeletal autoantibodies in the connective tissue diseases. Arthritis Rheum 1985;28:889-898. Senecal JL, Ichiki S, Girard D, Raymond Y. Autoantibodies to nuclear lamins and to intermediate filament proteins: natural, pathologic or pathogenic? J Rheumatol 1993 ;20:211--219.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

dsDNA AUTOANTIBODIES Ruud J.T. Smeenk, Ph.D. a, Jo H.M. Berden, M.D., Ph.D. b and Antonius J.G. Swaak, M.D., Ph.D. c

aDepartment of Autoimmune Diseases, C.L.B., NL-1066 CX Amsterdam; bDivision of Nephrology, Academic Hospital St. Radboud, NL-6525 GA Amsterdam; and CDepartment of Rheumatology; Dr. Daniel den Hoed Clinic; NL-3085 EA Rotterdam, The Netherlands

HISTORICAL NOTES Following the first description of serum components reactive with DNA in patients with systemic lupus erythematosus (SLE) (Ceppelini et al., 1957), several techniques were developed for the detection, characterization and quantitation of anti-DNA antibodies (anti-DNA) (Feltkamp, 1975; Maini and Holborow, 1977). The strong association of anti-DNA in serum with SLE emphasized the diagnostic value of antiDNA detection (Stollar, 1975). Later, with more sensitive methods anti-DNA was also reported in other clinical syndromes (Grennan et al., 1977). The specificity for SLE of anti-DNA was regained when reaction conditions were more carefully controlled and purely double-stranded DNA (dsDNA) was used as the antigen (Aarden et al., 1975).

THE AUTOANTIGEN Definition Deoxyribonucleic acid (DNA) as antigen may be either double-stranded (dsDNA) or single-stranded (ssDNA). In vivo, DNA will almost always occur in the form of nucleosomes, i.e., closely associated with histones. Because the epitopes situated on DNA partially reflect the repetitive negative charge of the molecule, synthetic polynucleotides are often also recognized by anti-DNA antibodies. How much DNA from various species differ in antigenicity is not known in great detail, but differences clearly exist. Anti-DNA in human serum reacts with DNA of all species tested, though not to the

same extent (Stollar et al., 1962), as do monoclonal anti-DNA (Wu et al., 1990).

Methods of Purification/Commercial Sources For use in anti-DNA assays, DNA can be purified (through standard DNA purification protocols) from tissue (e.g., calf thymus), from eukaryotic cells, from bacteria or from bacteriophages. The DNA from bacteriophage PM2 (which can easily be grown in its host the bacterium Pseudomonas B AL31) is very useful, because it can be radiolabeled in vivo and easily isolated in a purely double-stranded form (Aarden and Smeenk, 1981). Plasmid DNA (e.g., from the vector pUC9) is a suitable alternative that is easily iodinated after isolation (Smeenk, 1993). Commercially obtainable DNA includes calf thymus DNA which is often used in anti-DNA assays. Care should be taken to avoid protein contamination of the employed preparation. In experienced hands, PM2 DNA as well as plasmid (pUC9) DNA both provide excellent double-stranded DNA antigens, especially suited for the detection of high avidity anti-dsDNA in radioimmunoassays. Because of its giant mitochondrion (the kinetoplast) which is composed of pure DNA, the hemoflagellate Crithidia luciliae can be used for the measurement of anti-DNA by indirect immunofluorescence (IIF). Although there have been occasional reports on the putative presence of histones in kinetoplasts, it has not been we have failed to show that histones indeed are present in these organelles.

Sequence Information Backbone determinants of DNA as well as specific

227

nucleotide sequences can be the targets of anti-DNA recognition. Backbone determinants on either singlestranded or double-stranded DNA are short regions of DNA helix. Here the interaction between B-cell paratope and dsDNA epitope seems based on electrostatic interactions, because this binding is extremely sensitive to salt concentration and pH (Smeenk et al., 1983). Yet, especially in the case of high avidity antiDNA, secondary hydrogen bonding also plays a role (van Oss et al., 1985). Most likely, such dsDNA epitopes are constituted by the sugar-phosphate backbone of the DNA because of the repetitive negative charges of the phosphate moieties. Specificity of autoantibodies for such epitopes might also explain the known extent of anti-DNA cross-reactivity (Shoenfeld et al., 1983). The SLE serum antibodies that react with DNA of animal, bacterial, viral and plant origin probably recognize the backbone epitopes that occur on dsDNA from any species (Stollar, 1991). Apart from backbone recognition there also is selective recognition of DNA sites, variably expressed on different DNAs (Wu et al., 1990). Such a binding seems more pronounced in the case of single-stranded DNA and is presumably based on recognition of defined nucleotide sequences (Pisetsky and Reich, 1994). Although anti-DNA specific for ssDNA may exist as a separate entity, most of what is generally called anti-ssDNA reactivity actually is anti-dsDNA of a low avidity (Aarden and Smeenk, 1982). When dsDNA is denatured, the strands of DNA become more flexible. Upon cooling, internal duplex formation over short stretches of DNA occurs. The reactivity of antidsDNA with ssDNA is mainly due to this kind of internal duplex formation (Stollar and Papalian, 1980). Epitopes formed by these internal duplexes are completely different than in dsDNA. The difference lies in the flexibility of the DNA backbone, which is of extreme importance in terms of allowing multipoint attachment (and thus high avidity binding) of antibodies to DNA. Therefore, the greater flexibility of ssDNA could lead to higher avidity binding (Stollar and Papalian, 1980; Aarden and Smeenk, 1982). The actual combining site of an anti-DNA autoantibody encompasses only about six nucleotides (Stollar et al., 1986), but most anti-DNA antibodies require DNA fragments from 40 to several hundred basepairs in length for stable interaction. The size dependency, however, differs very much among antibodies (Ali et al., 1985). These findings suggest that both F(abs) of an anti-DNA antibody need to be

228

bound for a stable interaction via (monogamous) bivalent interactions with antigenic sites distributed along the DNA molecule.

AUTOANTIBODIES Pathogenetic Role Human Disease. Antibodies to DNA have long been thought to play an important role in the pathogenesis of SLE, which traditionally reflects anti-DNA binding to DNA with resultant deposition of immune complexes in tissues (Koffler et al., 1971). This binding of DNA by antibodies may occur in the circulation or in situ (Izui et al., 1977). At the site of deposition, subsequent complement activation then leads to inflammation and the characteristic disease features of SLE. Challenges to this concept of the pathophysiology of SLE (Eilat, 1986) catalyzed a modified hypothesis based on studies showing that anti-DNA can interact with tissue structures such as heparan sulfate, the major glycosaminoglycan side chain of the glomerular basement membrane (GBM) (Faaber et al., 1986; Brinkman et al., 1990a). Although the binding of anti-DNA to heparan sulfate was originally thought to reflect cross-reactivity, this binding is in fact mediated by nucleosomes (Termaat et al., 1992) which are present in plasma of SLE patients (Rumore and Steinman, 19901) perhaps as a result of increased or defective apoptosis (Emlen et al., 1994; CasciolaRosen et al., 1994).

Animal Models. Various strains of mice (e.g., NZB/W F1 and MRL/lpr) spontaneously develop an autoimmune disorder, which resembles SLE (Theofilopoulos and Dixon, 1985), including production of autoantibodies to DNA as well as to other antigens (histones, Sm, nRNP, rRNP). Another animal model claimed to be comparable with human SLE is obtained by inducing chronic graft-versus-host disease (GVHD) in mice through the injection of parental spleen and/or lymph node cells in F1 mice (Gleichmann et al., 1984). Monoclonal antibodies to DNA, derived from MRL/lpr, NZB/W F1, and GVHD mice behave quite comparably in four different anti-DNA assays (Brinkman et al., 1990b). An induced model for SLE is based on perturbation of the idiotypic network (Mendlovic et al., 1988; Mozes et al., 1989); these mice, too, develop anti-DNA antibodies.

Genetics Susceptibility to SLE is associated with certain MHCencoded genes. HLA-DR2, DQw 1 and the rare allele DQ[~I.AZH confer high relative risk (RR - 14) for lupus nephritis (Fronek et al., 1990). DR4 is significantly decreased in patients with lupus nephritis. Of the patients with lupus nephritis, 50% have either the DQ~I.1, the DQ[~I.AZH or the DQ~I.9 allele. These alleles, therefore, seem to have a direct role in the predisposition to lupus nephritis (Fronek et al., 1990). Although SLE is generally considered not to be an inherited disease, estimates of twin concordance in monozygotic twins vary between 24 and 69% (Gregersen, 1993). So there is at least an inherited factor predisposing to susceptibility for SLE. Another finding pointing in this direction is the observation that family members of SLE patients often show increased incidences of antinuclear and anti-DNA antibodies (Le Page et al., 1989). Studies of V H and V L gene usage show that both V chains are necessary for DNA reactivity of an antiDNA antibody (Mahmoudi et al., 1995), and that no unique V, D or J gene segments are used to construct the antibody (Tsao et al., 1990). Genetic studies suggest that anti-DNA is produced by a process of somatic mutation and clonal expansion favoring sequences with accumulated positively charged amino acids in the complementary-determining regions (Rahman and Isenberg, 1994). The antigens that trigger this process are not known, but nucleosomes might be implicated (Mohan et al., 1993). Indeed, in MRL/lpr mice, the autoantibody response is initially directed to nucleosomes, with anti-DNA appearing later in the disease (epitope spreading) (Burlingame et al., 1993). Anti-DNA are thought to play a pivotal role in the development of SLE disease features. Flares of SLE are generally preceded by a rise in anti-DNA levels, followed by a steep drop during the exacerbation (Swaak et al., 1979). An example is given in Figure 1. From these studies, it was considered necessary to follow patients on a regular basis: anti-DNA must be measured preferably every 6 weeks. Although Swaak defined an increase in anti-DNA in terms of at least doubling in 6 weeks occurring for at least two periods in succession, more recently a less stringent definition defined a rise in anti-DNA as an increase of 25% of the level in the previous sample (ter Borg et al., 1990; Bootsma et al., 1995). In 89% of clinical relapses in patients positive for anti-DNA, a pronounced rise in

Figure 1. Longitudinal study of anti-dsDNA level in relation to a renal exacerbation.

anti-DNA could be detected 10 weeks before the relapse occurred. These studies made use of the Farr assay to quantitate anti-DNA levels. Other anti-DNA assays (e.g., the PEG assay) proved less useful in this respect (Nossent et al., 1989).

Factors in Pathogenicity Lupus nephritis is especially correlated with anti-DNA of high avidity (Leon et al., 1977); central nervous system involvement correlates with low avidity antiDNA. Studies in murine models of SLE show that an initial IgM anti-DNA response is followed in time with an IgG response and affinity maturation of the antibodies (Steward and Hay, 1976). Nephritis occurs in these mice only after the development of IgG antiDNA. In general, IgG antibodies are of greater relevance to the disease than IgM antibodies, because the latter are more common in non-SLE patients (Hylkema et al., 1994). Expression of IgG autoantibodies to chromatin and its substructures in NZB/W F1 mice increases significantly in the period from 14 to 2 weeks before the onset of nephritis. IgG subclass analysis showed that antichromatin reactivity consisted mainly of IgG2b and IgG3 antibodies; antihistone and

229

anti-DNA reactivity was restricted to the IgG2b subclass. Kidney eluates of mice with nephritis contained IgG3 and IgG2b antibodies (Hylkema et al., 1995). Certain common anti-DNA idiotypes might also play a role in the pathogenesis of SLE. Immunization of mice with a human monoclonal anti-DNA antibody carrying the 16/6 idiotype, with a 16/6 carrying monoclonal antibody without anti-DNA reactivity, or even with a monoclonal anti- 16/6-idiotype antibody all induce anti-DNA production and SLE-like disease (Shoenfeld and Mozes, 1990).

Methods of Detection Among the multitude of assays for antibodies to DNA the most commonly used are IIF on Crithidia luciliae, radiobinding assays (RBAs) (Farr assay and PEG assay) and enzyme-linked immunosorbent assays (ELISAs) (Smeenk, 1993). These methods can either be obtained in kit form or be employed as in-house assays. The IIF on C. luciliae is a method that couples a good sensitivity to a high disease specificity and is therefore one of the preferred methods for screening for the presence of anti-DNA. The method is specific for antibodies to dsDNA (of intermediate to high avidity) and, in able hands, is very reproducible (Smeenk, 1993). For good reproducibility, it is of utmost importance never to let the slides dry out during the IIF procedure since local increases in salt concentration will lead to dissociation of low avidity DNA-antibody interactions (Smeenk et al., 1982). In RBAs, the choice of antigen again is of great importance. The DNA employed has to be bigger than 105 but smaller than 107 d. Furthermore, the DNA must

be double-stranded and, to allow quantitation of antibody reactivity, monodisperse in size. This indicates that circular double-stranded bacteriophage DNA (such as from PM2) or plasmids (such as pUC9) are to be preferred. In ELISA systems, DNA has to be coated to plastic, ssDNA can easily be coated directly, but dsDNA is mostly coated via intermediates such as poly-L-lysine, protamine or methylated BSA. Such precoats introduce problems related to the binding of immune complexes and/or immunoglobulins not directed against DNA to the plates (via the intermediate molecule) (Brinkman et al., 1991). An alternative is to make use of biotinylated DNA and coat this via streptavidin to the plates (Emlen et al., 1990; Hylkema et al., 1994). Different assay systems are not always comparable, for the following reasons: 1) the source of antigen differs DNA may be eukaryotic or prokaryotic in origin, be double-stranded or single-stranded and be polydisperse in size or homogenous; 2) presentation of the antigen to the antibody differs in RBAs it is generally in solution, in EL1SAs it is coated to plastic; in the Crithidia test DNA are mostly presented intact in cells; 3) reaction conditions are d i f f e r e n t - e.g., due to the ammonium sulfate precipitation step used in the Farr assay, anti-dsDNA antibodies of low avidity are missed with this method; in second antibody techniques such as IIF and ELISA the choice of conjugated antibody is of importance. Often, only IgG antiDNA antibodies are measured with these techniques. General comparison of the four mentioned assays using sera of patients with defined SLE leads to high levels of correlation among the various assays (Table 1). However, upon routine screening of sera (not necessarily of SLE patients) large discrepancies

Table 1. Correlations Among Four Anti-DNA Assays. Anti-DNA levels of 75 sera of defined SLE patients were determined by Farr assay, PEG assay, Crithidia test and ELISA. All levels were expressed in Units/mL. Correlations were calculated using linear regression analysis and Kendall's rank correlation test Anti-DNA assay

PEG assay

Crithidia test

ELISA

Farr assay

r = 0.67* (p < 0.001) t - 0.63** (p < 0.0001)

r = 0.85 (p < 0.001) t = 0.69 (p < 0.0001)

r = 0.67 (p < 0.001) t = 0.53 (p < 0.0001)

PEG assay

--

r = 0.83 (p < 0.001) t = 0.69 (p < 0.0001)

r = 0.62 (p < 0.001) t = 0.63 (p < 0.0001)

Crithidia test

*Coefficient of correlation by linear regression analysis; **t: tau; Kendall's rank correlation coefficient.

230

r = 0.70 (p < 0.001) t = 0.66 (p < 0.0001)

Table 2. Comparison of Results Obtained by Four Anti-DNA Assays on 289 Sera Referred to Our Institute for Routine anti-DNA Determination PEG assay

ELISA

Crithidia test

Farr assay

+

+

+

+

22

+

+

-

22

+

+

+

7

+

Sera (n)

15

+

+

+

-

+

-

2

+

+

-

3

+

-

+

1

-

-

-

33

+

-

-

23

+

3 158 94

93

59

30

289

+ indicates a positive result; - indicates a negative result with the indicated assay.

between the different assays are also seen: out of 16 possible reactivity combinations, 11 actually occurred (Table 2). Sensitivity differences can only partly explain the discrepancies observed between the assays. An important cause of discrepant results is to be found in the avidity of the antibodies (Figure 2). Furthermore, histones or n u c l e o s o m e s c o m p l e x e d to antinucleosome antibodies may also cause a positive reaction in the Farr assay (Kramers et al., 1994). Finally, it is advisable to quantitate anti-DNA levels and express these in International Units, to allow comparison a m o n g different studies. A W H O standard serum for this purpose is available from the W H O and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB) (Feltkamp et al., 1988).

practice, SLE specificity is inversely related to the sensitivity of the assay, or, more correctly put, to the avidity spectrum detected. There is a discrepancy between results obtained on sera from patients with defined diseases and on sera sent for routine antiD N A measurement. Testing defined sera, it would seem that Farr assay and Crithidia test have the highest (and comparable) specificity for SLE (Table 3). However, when routine sera are tested, the Farr assay

LOW 4

CLINICAL UTILITY

Application There are two important but different ways in which anti-DNA assays may be used: either as an aid to the diagnosis of SLE or as a tool to monitor the clinical course of a defined SLE patient. For the first purpose the assay should have a high specificity for SLE. In

anti-DNA avidity

HIGH

ELISA PEG-assay IFT Crithidia q

P

Farr-assay

Figure 2. Anti-DNA avidity and assay behavior.

231

Table 3. Comparison of the Disease Specificity of Four Anti-DNA Assays Percentage of sera positive in Disease

Farr assay

Crithidia

PEG assay

ELISA

98

96

96

100

Rheumatoid arthritis

1

0

5

3

Sj/3gren's syndrome

0

0

7

20

Scleroderma

0

0

0

30

Autoimmune hepatitis

0

0

20

15

Myasthenia gravis

0

0

32

20

Autoimmune thyroiditis

0

0

7

13

Autoimmune gastritis

0

0

0

18

Addison' s disease

0

0

0

0

Autoimmune hemolytic anemia

0

0

10

8

Normal donors

0

0

0

0

Active SLE

has a considerably higher SLE specificity (Table 4). Of the Farr-positive sera, 94% had SLE, for the Crithidia test, this figure amounted to 74% and for the PEG assay 73%. Of the five sera that contained antiDNA detectable by Crithidia test only, just one was found to originate from an SLE patient. So if screening for the presence of anti-DNA is done by an assay that is not selective for high avidity anti-DNA, a positive assay result does not always indicate that the patient has SLE because anti-DNA of lower avidity occurs in diseases other than SLE as well. Therefore, screening by either ELISA or Crithidia test should preferably be followed by quantitation using a Farr assay. In case one only wants to perform one (non-

RBA) assay, the Crithidia test is preferred above an ELISA. The specificity of the Farr assay for SLE was evaluated in a group of 441 non-SLE patients with Farr-assay-detectable anti-DNA. More than 85% of these patients developed SLE within 5 years after the first Farr-positive assay result (Swaak and Smeenk, 1985a). Fluctuations in the anti-DNA amounts as well as of the relative avidity of the anti-DNA are valuable in patient monitoring. Repeated serum sampling of individual patients (preferably every 6 weeks) can be very informative about the clinical course of the disease, because a clear-cut relationship exists between anti-DNA and disease activity (in particular, nephritis; see Figure 1).

Table 4. Anti-DNA Determination in 94 Sera Sent to Our Institute for Routine Anti-DNA Screening in Relation to the Diagnosis of the Donors Anti-DNA assay Farr assay

Number of patients with SLE

Crithidia

PEG assay

+

+

32

30

+

+

43

28

+

-

5

1

+

14

7

+ indicates a positive result; - indicates a negative result with the indicated assay.

232

Number of sera

However, as stated before, this correlation is extremely dependent on the anti-DNA assay employed. It would seem that only high avidity anti-DNA (as is measured with the Farr assay and expressed in IU/ mL) follows such a clear-cut disease correlation. Treatment of SLE patients when anti-DNA starts to rise will prevent upcoming exacerbations in a significant number of patients (Bootsma et al., 1995). In this important study, 156 patients with SLE were studied and anti-DNA was measured by Farr assay monthly. Following a 25% rise in anti-DNA level (detected in 46 patients), patients were randomly assigned either conventional treatment or 30 mg. Prednisone added to the current daily dose. The relapse rate was significantly higher in the conventional group than in the prednisolone group (20 versus 2, p < 0.001). Longitudinal studies of SLE patients show that anti-DNA avidity remains more or less constant over time, including patients who, at the beginning of the study, have only low avidity anti-DNA (i.e., Farr-assaynegative), as well as patients with higher avidity antiDNA. Exceptions are mostly found among patients who develop nephritis during the course of the disease. SLE patients initially found to have only low avidity anti-DNA generally have a milder form of SLE with less frequent episodes of nephritis. Furthermore, anti-DNA remained of low avidity during this prospective study (Nossent et al., 1989). A relative index of anti-DNA avidity is obtained by dividing results obtained in the Farr assay by those of the PEG assay. A Farr/PEG ratio indicated a preponderance of high avidity anti-DNA, and vice versa. With this approach, we showed that the Farr/PEG ratio of SLE patients with nephritis was significantly higher than that of patients with central nervous system involvements, which underlines the relevance of high avidity anti-DNA to nephritis (Swaak and Smeenk, 1985b). On the other hand, this approach can not be used to predict for an individual patient whether the patient will develop nephritis in the future or not. Although a causal relation between SLE nephritis and complement-fixing ability of anti-DNA was proposed, complement-fixing titers are more likely to be a direct reflection of anti-DNA titers, because patients with nephritis generally have higher titers of anti-DNA than patients without nephritis. Therefore, more complement-fixing anti-DNA is found in sera of patients with nephritis (Ezparza et al., 1985). Because of this correlation, measurement of complement-fixing anti-DNA is of little additional value above anti-DNA testing and such should not be used regularly.

Effect of Therapy Different treatment of patients with SLE have varying influences on anti-dsDNA levels. Immunosuppressive therapy suppresses production of anti-DNA, and, because anti-DNA are implicated in the development of lupus nephritis, such therapy can reduce kidney injury (unpublished data). Although plasmapheresis dramatically reduces anti-dsDNA initially, the final outcome is unchanged (Lewis et al., 1992). Treatment of NZB/W F1 mice with anti-anti-DNA (anti-idiotypic antibodies) can reduce anti-DNA antibodies, frequency of proteinuria and mortality, but the effect is transient, because in time anti-DNA antibodies lacking the cross-reactive idiotype appear, and the mice develop nephritis (Hahn and Ebling, 1984). Therapy with cytokine antagonists are promising; anti-IL-10 treatment of NZB/W F1 mice reduces anti-DNA and frequency of nephritis, and improves survival (Ishida et al., 1994). Anti-DNA can pass the placenta and can be associated with neonatal lupus syndromes. However, disease features which include rashes and cytopenia are transient; with the disappearance from the circulation of anti-DNA, disease features subside (Buyon, 1994). The only permanent injury is congenital heart block, which is associated with anti-Ro/SS-A and antiLa/SS-B antibodies but not with anti-DNA (Buyon, 1994).

CONCLUSION Detection of anti-dsDNA autoantibodies by an assay selective for high avidity anti-DNA is diagnostic of SLE. In this respect, the Farr assay offers the highest disease specificity. Because low avidity anti-DNA also occur in rheumatic diseases other than SLE, detection of such antibodies is of less diagnostic value. Therefore, for an optimal screening procedure an initial "broad-spectrum" screening employing ELISA or Crithidia test should be followed by confirmation using the Farr assay. Longitudinal studies employing the Farr assay show that exacerbations tend to be preceded by an increase in anti-DNA antibodies in the serum followed by a sharp drop. This makes frequent measurement of anti-DNA (every 4--6 weeks) with the use of a truly quantitative assay selective for high avidity anti-DNA very valuable in the monitoring of individual SLE patients.

233

Just as disease features of SLE vary from patient to patient, so too does anti-DNA avidity. Studies done so far show a relation between high avidity anti-DNA and nephritis, and low avidity D N A and central nervous system involvement. Additional studies are needed to elaborate these findings and to elucidate the m e c h a n i s m behind these associations. With respect to

the pathogenesis of SLE, anti-DNA are not an epip h e n o m e n o n , but are vital to the induction of disease manifestations. For instance, anti-DNA most probably induce nephritis by binding heparan sulphate in the G B M , a binding that is mediated through n u c l e o s o m e s c o m p l e x e d to antibodies.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ENDOMYSIAL AUTOANTIBODIES Helge Scott, M.D. and Per Brandtzaeg, Ph.D.

Laboratory for Immunohistochemistry and Immunopathology, Institute of Pathology, University of Oslo, The National Hospital, Rikshospitalet, N-0027 Oslo, Norway

H I S T O R I C A L NOTES As first observed by indirect immunofluorescence staining (IIF), sera from patients with dermatitis herpetiformis (DH) or celiac disease (CD) often contain IgA antibodies reactive with the lining of smooth muscle bundles (i.e., the endomysium) of monkey esophagus (Chorzelski et al., 1983) (Figure 1). Monkey esophagus was previously used as the substrate of choice for detection of pemphigus and pemphigoid IgG antibodies directed against desmosomal antigens and basement membrane antigens of the epithelium, respectively. Like pemphigus and pemphigoid, DH is a bullous dermatosis and can resemble bullous pemphigoid. IgA endomysial antibodies (IgA-EMA) have a higher specificity for DH and CD than the gluten (or gliadin) antibodies and reticulin antibodies used previously for the laboratory evaluation of the gluten-sensitive enteropathy which is characteristic of DH and CD (Scott et al., 1992).

THE A U T O A N T I G E N

Definition/Origin The amount of endomysial antigen present in monkey esophagus is greater in the lower than in the uppermost part where such reactivity can only be detected around blood vessels (Kumar et al., 1984). The antigen is localized intercellularly and is associated with collagen fibers but is not collagen itself (Karpati et al., 1991). Although not yet purified from monkey esophagus, the endomysial antigen(s) is closely related to collagen-associated antigens present in the esophagus of other species as well as in human and monkey

Figure 1. This micrograph illustrates the IgA-EMA staining pattern in monkey esophagus demonstrated by indirect immunofluorescence. A cryostat section was first incubated with serum (dilution 1:50) from a patient with active CD, washed in phosphate-buffered saline and then reincubated with a fluorescein-labelled rabbit IgG conjugate specific for human IgA. E = epithelium. SM = layer of smooth muscle fibers. Original magnification: x 400.

237

jejunum and in rat kidney, liver and stomach (Valeski et al., 1990). Some but not all IgA-EMA-positive sera stain calf, sheep and goat esophagus (Chorzelski et al., 1984). IgA antibodies in sera of children with DH bind to vascular smooth muscle, the endomysium of the intestinal muscularis mucosae and smooth muscle fibers in human jejunal lamina propria (Karpati et al., 1986); a similar antibody reaction pattern is found in monkey jejunum (Volta et al., 1994). Antibodies in sera of adult patients with CD and DH react with the reticulin components of rat kidney, liver and stomach (Seah et al., 1971). Of the several types of reticulin antibodies, only the R1 variety is associated with gluten-sensitive enteropathy (Rizzetto and Doniach, 1973). Several lines of evidence suggest that the various collagen-associated antigens are similar or even identical and that only the use of different biological substrates makes them seem different. 1. Silver impregnation methods and immunoelectronmicroscopy show that endomysial antibodies, reticulin antibodies and jejunal antibodies all react with extracellular reticulin matrix components. (For a Table comparing reticulin, endomysial and jejunal autoantibodies, see chapter: Reticulin Autoantibodies) 2. The relevant antibodies are primarily of the IgA class. 3. All three categories of antibodies are highly specific for gluten-sensitive enteropathies. 4. The circulating levels of IgA antibodies fluctuate with dietary gluten intake regardless of the test substrate used. Therefore the antibodies may, in fact, be cross-reactive, with only slight differences in antigenic epitopes causing variability in antibody avidity. Nevertheless, there seems to be some species restriction for the endomysial antigen(s) and a broader reactivity with reticulin antigens, although an overlap may occur in some species such as sheep (Valeski et al., 1990). Rat or mouse kidney antigens absorb the reticulin antibodies without affecting the endomysial antibodies (as revealed with monkey esophagus); whereas, absorption with monkey stomach eliminate not only the endomysial antibodies but also the reticulin antibodies (Valeski et al., 1990). Perhaps the relevant antigen is highly conserved and carries several epitopes of which at least one may be restricted to rat and mouse tissue ("reticulin antigen") and another to primate tissue ("endomysial antigen"). The agyrophilia of reticulin as well as the strong 238

periodic acid-Schiff (PAS) reactivity are thought to be due to the high carbohydrate content of the fibers or the proteoglycan content of the interfibrillar matrix (Velician and Velician, 1968; Snodgrass, 1977). "Reticulin" may correspond to a group of different fibrillar and amorphous components such as collagen I fibers, collagen III fibers, fibronectin and proteoglycans. Antibodies to collagen-associated antigens (endomysial, reticulin or jejunal antibodies) may react with unique conformational epitopes of these components, or with distinct antigenic determinants present on one or more of them (K~irp~iti et al., 1991). An antiserum raised against a noncollagenous connective tissue component from human and pig kidney (Pras and Glynn, 1973) produces an immunofluorescence staining pattern similar to that of human reticulin antibodies. Blocking experiments show that the isolated component does not bind CD-specific reticulin antibodies. A similar noncollagenous 90 kd glycoprotein reticulin-associated component isolated from skin does not react with gluten enteropathyspecific reticulin antibodies (Maury and Teppo, 1984). Six human noncollagenous protein molecules from human fetal lung react with serum IgA from patients with CD (M~iki et al., 1991a). The molecules had isoelectric points of 5.0, 5.2, 7.0, 7.5, 7.6 and 7.8 and molecular weights of 18--37 kd measured by gel filtration. These antigens absorbed antibodies from serum samples of CD patients reactive with both reticulin and endomysium. It was further shown that fibroblasts from fetal lung synthesize and secrete in vitro a large molecular weight protein aggregate with similar antigenicity, but without reactivity for gluten antibodies (Marttinen and M~iki, 1993). When this protein complex was treated with 4 M guanidium and 0.1% SDS, several polypeptides were detected by PAGE; four of these (molecular weight, 17--36 kd; isoelectric point, 5.0--7.0) reacted with IgA antibodies from sera of CD children. It was recently shown that endomysial antibodies apparently react with both perimuscular sheaths in human esophagus (Uibo et al., 1995) and with similar structures in human umbilical cord vein (Ladinser et al., 1994). Antibodies to endomysium, jejunum and reticulin are probably directed against various antigenic determinants on noncollagenous proteins produced by fibroblasts; particularly the components detected by endomysial antibodies may be regarded as an autoantigen.

AUTOANTIBODIES

Methods of Detection

Pathogenetic Role

The usual method to detect IgA-EMA is IIF with commercial cryostat sections of monkey esophagus as substrate. Cryostat sections of commercially obtained, fresh-frozen monkey tissue should be documented to contain the appropriate antigenic substrate by microscopic examination of H&E-staining of at least the first and last section in a series. Commercial slides are apparently not always made in this critical manner. Because the monkey is an endangered species, alternative substrates for IgA-EMA determinations are being sought. Human esophagus is quite satisfactory; indeed, IgA-EMA are detected at higher serum dilutions than with monkey substrate (Uibo et al., 1995), but human esophagus is difficult to use for ethical reasons, especially when it comes to the distribution of commercial substrate. Human umbilical cord (Ladinser et al., 1994) is probably a better choice and was also recommended by a task force group organized jointly by the European Medical Research Council (EMRC) and the European Society for Paediatric Gastroenterology and Nutrition (ESPGAN) for serological screening for CD (unpublished observations, Maikammer, Germany, November 1994). Positive staining is detected with cryostat sections of human umbilical cord vein for most IgA-EMA positive sera, but the staining intensity is considerably weaker than with freshly prepared monkey esophagus sections (unpublished observations) (Table 1). Thus, some sera weakly positive for IgA-EMA are deemed to be negative with the umbilical cord substrate. A higher sensitivity for CD with human umbilical cord than with monkey esophagus as substrate was reported (Ladinser et al., 1994); whether this observation depended on an unsatisfactory quality of the commercial monkey cryostat sections is unknown. In screening for CD and DH, sera should be tested at two dilutions (1:5 and 1:50 recommended). Testing also at the higher dilution is strongly recommended to avoid the "masking effect" of coexisting antibodies to

Gluten ingestion in patients with a genetic predisposition for CD or DH provokes IgA-EMA production (M~iki et al., 1991b). However, gluten does not bind to endomysium (Chorzelski et al., 1983) and how gluten elicits the production of these antibodies and how they might contribute to gluten-sensitive enteropathy are unknown. In the suggested autoimmune reaction in CD and DH, fibroblast-derived molecules are considered the putative autoantigen (M~iki et al., 1991a; Marttinen and M~iki, 1993). Jejunal antibodies might be the true autoantibodies in CD (K~irp~iti et al., 1990), a theory consistent with subepithelial deposition of activated complement observed in most untreated CD patients (Halstensen et al., 1992), and the possibility that IgGor IgM-mediated complement activation with resultant damage of the surface epithelium might contribute to the pathogenesis of gluten-sensitive enteropathy. However, the dissimilar deposition of IgA-EMA makes it unlikely that these antibodies are responsible for the observed subepithelial complement activation. Moreover, human IgA does not activate complement by the classical or alternative pathway (Russel and Mansa, 1989). Nevertheless, fibroblasts produce molecules with paracrine and autocrine functions (Rubin et al., 1991; Montesano et al., 1991) that may be important for epithelial cell proliferation and maintenance of the lamina propria structure. Indeed, some fibroblast-derived proteins with regulatory functions might be targets for IgA-EMA; by blocking these regulatory factors, the IgA autoantibodies might have an essential role in the pathogenesis of CD (Marttinen and M~iki, 1993). In rare patients there is an association between lung disease (pulmonary hemosiderosis) and CD; this complication might in theory be caused by antibodies to EMA, perhaps of the IgG class (Perelman et al., 1992).

Table 1. Umbilical Staining Reactions for IgA Endomysium Antibodies in 130 Patients when Monkey Esophagus or Human Umbilical Cord Were Used as Substrate

Substrate Monkey esophagus Human umbilical cord

Staining Reaction +++

++

+

Neg.

21

10

6

93

9

15

11

95

239

smooth muscle (Uibo et al., 1995) and the negative prozone that occasionally occurs at the low dilution. The IgA-EMA test is in practice rather laborious and cannot be applied for large-scale screening because one technician can perform no more than a few dozen tests per day. An enzyme-linked immunosorbent assay based on fibroblast-derived CD autoantigens (crossreacting with IgA-EMA) has been developed (Marttinen and M~iki, 1993), but the antigens are not commercially available.

untreated CD children, the sensitivity is 85--100% (Table 2), being notably reduced in children below 2 years of age (Btirgin-Wolff et al., 1991). The sensitivity for DH is 7 0 - 8 0 % (Table 2) but increases to 100% when the associated gluten-sensitive enteropathy shows severe villus atrophy (Table 3). The specificity of IgA-EMA for active glutensensitive enteropathy is as high as 99.7--100% (Table 1). Similar antibodies are reported in a few patients with food intolerance unrelated to gluten (Chan et al., 1994) but in no skin disease other than DH (Ktihn et al., 1987). Most false-positive results have low IgAE M A titers. Serum dilutions 1:5 or higher are recommended to avoid false-positive staining reactions. In patients with Crohn's disease of the small intestine, IgA-EMA are negative, but titers of gluten antibodies are comparable to those of untreated CD patients. Due to this high specificity, the positive predictive value of IgA-EMA for CD or DH is quite impressive (Table 2). Because the expected frequency of CD is rather

CLINICAL UTILITY

Disease Association Endomysial antibodies of the IgA class (IgA-EMA) occur rather selectively in patients with active CD or DH. The reported sensitivity for IgA-EMA in adult patients with untreated CD is 68--100% (Table 1). In

Table 2. Studies of IgA Endomysium Antibodies in Serum of Untreated Celiac Patients Compared with Controls CD

Controls

Authors

Sensitivity No.

No. Pos.

No.

No. Pos.

Specificity

Positive Predictive Value

Chorzelski et al. (1984)

28*

19

107

0

68

100

100

Kapuschinska et al. (1987)

33c

33

140

0

100

100

100

Rossi et al. (1988)

46c

46

160

0

100

100

100

Kumar et al. (1989)

38c

38

278

0

100

100

100

H~illstr6m (1989)

14c 32a

14 29

24 45

0 0

100 9O

100 100

100 100

340c

306

211

4

90

98

99

McMillan et al. (1991)

28a

25

68

0

89

100

100

Ferreira et al. (1992)

21

21

160

0

100

100

100

Sategna-Guidetti et al. (1993)

91a

85

438

0

93

100

100

Carroccio et al. (1993)

70c

70

60

2

100

97

97

Lerner et al. (1994)

34c

33

41

1

97

98

97

Uibo et al. (1995)

26c 13a

26 13

16 87

0 0

100 100

100 100

100 100

100a

100

109

0

100

100

100

49a

49

53

0

100

100

100

Btirgin-Wolff et al. (1991)

Sategna-Guidetti et al. (1995) Vogelsang et al. (1995) *Not specified aAdults cChildren

240

a

Table 3. Studies of IgA Endomysium Antibodies in Serum of Patients with Dermatitis Herpetiformis on a Gluten-containing Diet DH with Severe Villous Atrophy

DH Authors

No.

No. Pos.

% Pos.

No.

No. Pos.

% Pos.

Chorzelski et al. (1983)

38

26

68

Reunala et al. (1987)

29

22

76

14

14

100

179

127

71

18

18

100

H~illstr6m et al. (1989)

23

19

83

14

14

100

Kumar et al. (1987)

45

29

65

Kumar et al. (1982)

14

20

72

Ktihn et al. (1987)

dissimilar in various populations and because CD and DH patients on a gluten-free diet are negative for IgA-EMA, the exact negative predictive value of IgAEMA cannot be determined. However, it should be close to 100% in most clinical settings. IgA-EMA are strongly related to active glutensensitive enteropathy and therefore correlate strongly with the degree of villus atrophy (Rossi et al., 1988; Sategna-Guidetti et al., 1993). IgA-EMA disappear 1--12 months after introduction of a gluten-free diet (Figure 2) and are usually negative somewhat before the normalization of villus architecture is completed (Sategna-Guidetti et al., 1993). The antibodies re-

appear 1--12 months after the reintroduction of gluten (Figure 2), the titers are dependent on the degree of villus atrophy caused by this challenge. However, IgA-EMA can also be found in low amounts in patients with a histologically normal intestine who later develop CD (M~iki et al., 1990). The antibodies may therefore precede the development of active CD. EMA of the IgG class and IgG antireticulin antibodies are found in IgA-deficient patients with CD (Beutner et al., 1989; Collin et al., 1992). In fact, the frequency of CD is considerably increased in subjects with selective IgA deficiency (Thomas and Jewell, 1979); screening for IgG-EMA may, therefore, be of

Figure 2. Change in percentage of IgA-EMA-positive CD or DH patients during treatment with a gluten-free diet (1-12 months) or during gluten challenge (1-12 months) as compared with original values reported by various authors.

241

value even though this antibody class shows little specificity and can be found in various other diseases (Beutner et al., 1989). CD is associated with juvenile diabetes mellitus, joint diseases and liver diseases (Collin et al., 1990). Screening for CD by IgA-EMA is important especially in young diabetics with abdominal symptoms (Rossi et al., 1993). Likewise, CD and IgA nephropathy are associated (Katz et al., 1979), and increased titers of gliadin antibodies, but not IgA-EMA, are found in some patients with IgA nephropathy (Nagy et al., 1988; Sategna-Guidetti et al., 1992). Moreover, although schizophrenia and CD are said to be associated, an increased frequency of IgA-EMA is not found in schizophrenia (Rybakowski et al., 1990).

CONCLUSION Antibodies to endomysium are most likely directed against noncollagenous proteins produced by fibroblasts and apparently contain different species-dependent epitopes. As originally described, these antibodies react with antigenic determinants present in monkey esophagus, but similar antigens are found in human esophagus, small intestine and umbilical cord vein and in monkey small intestine. EMA of the IgA class are found in patients with active gluten-sensitive enteropathy (either CD or DH) but in virtually no other condition. Silent forms of CD exist (Ferguson et al.,

1993) and the disease may cause vague symptoms. For population screening and selecting patients for intestinal biopsy, IgA-EMA are, therefore, crucial. IgA-EMA have the highest positive and negative predictive value for CD among the available serological tests for this disease (Scott et al., 1992). However, the final diagnosis of gluten-sensitive enteropathy still depends on histological evaluation of intestinal biopsies (Schmitz, 1992), and IgA-EMA cannot replace this invasive procedure. The need for a postchallenge biopsy in children, however, is questionable (Guandalini et al., 1989). IgA-EMA can probably replace such a second biopsy in children who respond to challenge with positive IgA-EMA. Because the sensitivity for IgA-EMA in CD children less than two years of age is reduced, follow-up with measurements of both gluten antibodies and IgA-EMA are recommended (BUrgin-Wolff et al., 1991). Although IgA antibodies to gluten are reported to increase more rapidly after gluten-challenge than IgAEMA (BUrgin-Wolff et al., 1991), serum samples from gluten-challenged CD children, taken a few weeks after the initiation of the challenge, are often positive for IgA-EMA but show normal gluten antibody levels (unpublished observations). Such differences may depend on methodological aspects. It is intriguing, but not unusual for autoantibodies to be employed for clinically useful purposes without knowing how they are elicited and whether they play a pathogenic role. See also RETICULIN AUTOANTIBODIES and SKIN DISEASES AUTOANTIBODIES.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ENDOTHELIAL CELL AUTOANTIBODIES Pier Luigi Meroni, M.D. a and Pierre Youinou, M.D., Ph.D. b

alstituto di Medicina Interna, Malattie Infettive & Immunopatologia, Universitgt degli Studi di Milano, 20122 Milan, Italy; and bLaboratoire d'Immunologie, Centre Hospitalier R6gional et Universitaire, Brest, Cedex France

HISTORICAL NOTES

Antibodies reacting with endothelial cell (EC) structures were first reported independently by two groups in the early seventies (Lindqvist and Osterland, 1971; Tan and Pearson, 1972). Autoantibodies were identified in sera from patients with systemic autoimmune diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) by using an indirect immunofluorescent technique with mouse kidney sections as a substrate. More recently, culture of human EC in vitro permitted development of sensitive radioimmunoassays or enzyme-linked assays to detect anti-EC antibodies (AECA) in sera from patients with a variety of immune-mediated vascular damage. Recent studies include intensive efforts to standardize the assay, define associations with disease or clinical presentation and characterize antigenic and biological properties of these antibodies (Del Papa et al., 1994a; Meroni et al., 1995; Youinou et al., 1995). THE AUTOANTIGENS Definition

Cell Types. Sera positive for AECA react with: (1) human EC obtained from large arterial (aorta) as well as venous vessels (umbilical cord vein) or small vessels, such as omental vessels and renal microvasculature (Koenig et al., 1993; Del Papa et al., 1994a; Meroni et al., 1995), (2) cells from a permanent cell line (Eayh926) obtained by hybridizing human EC from human umbilical vein EC (HUVEC) with epithelioma cell line cells (Edgell et al., 1983), (3) bovine EC from aorta (Carreras and Vermylen, 1982) and (4) murine endothelioma cell line cells (H5V)

(Del Papa and Meroni, unpublished data). AECA are not EC-specific due to unequivocal cross-reactivity with human fibroblasts (Del Papa et al., 1994b). In addition, antiendothelial activity can be partially inhibited by absorption with peripheral blood mononuclear cells (van der Zee et al., 1991a). Characterization

As first manifest by striking differences in the slopes of titration curves with individual antiendothelial cytoplasmic antibody-positive sera (Rosenbaum et al., 1988), AECA are a heterogeneous family of antibodies reacting with different structures on EC. This heterogeneity was confirmed by immunoprecipitation studies using AECA-positive sera, which precipitate different proteins ranging from 25 to 200 kd (van der Zee et al., 1991a; 1991b; Del Papa et al., 1994b; Adler et al., 1994). Although the majority of antigens recognized by AECA appear to be constitutive EC membrane proteins, the demonstration that the radiolabeled preparations extensively washed with high molar buffers lost part of their reactivity with SLE sera suggests that these antibodies are also able to bind unrelated proteins (Del Papa et al., 1994b). For example, both monoclonal and polyclonal anti-DNA antibodies do bind to EC in vitro by reacting with DNA or DNA/histone molecules attached to the cell membranes through electric charges (Chan et al., 1992). In addition, however, SLE sera frequently bind to EC via antibodies reacting with cell-surface-bound bovine serum proteins contributed by the fetal calf serum added to the tissue culture medium (Meroni 1995, unpublished results). One of these proteins is I]2-glycoprotein I (132-GPI), the plasma cofactor for the

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antiphospholipid (aPL) antibodies. Sera from patients with the antiphospholipid antibody syndrome (APS) contain antiphospholipid or anti-[32-GPI antibodies which can bind to endothelial monolayers through ~2GPI on the EC surface (Dueymes et al., 1995; Del Papa et al., 1995; Le Tonqueze et al., 1995a; 1995b). Although AECA recognize antigens present on the cell surface of resting EC, increased anti-endothelial binding (or cytotoxic activity) to cytokine-activated EC is also present in sera from patients with Kawasaki's disease and in SLE sera (Leung, 1990; van der Zee et al., 1994). In Kawasaki's disease, AECA display a complement-dependent cytotoxicity on interferon t~ (IFNct), interleukin 1~ (IL- 1ct) or IL- 1 and tumor necrosis factor (TNF) t~-activated EC but not on resting cells. The target antigen(s) induced by IFNt~ is quite different from those expressed following treatment of the cells with IL-1 ~ or TNFt~. In contrast, the EC cytolytic activity found in hemolyticuremic syndrome (HUS) and in thrombotic thrombocytopenic purpura (TTP) is lower when tested on IFNy-activated EC than on resting cells (Leung et al., 1988); IFNy apparently induces a specific loss or an alteration in structure or accessibility of the ECsurface molecules to autoantibodies in HUS and TTP. Resting EC normally carry both HLA class I and ABO blood group antigens, and the expression of HLA class II molecules can be induced by IFNy. These molecules are not, however, usually involved in the binding, inasmuch as AECA react in a similar manner with cells from different HLA and ABO unrelated donors (Ferraro et al., 1990; Savage et al., 1991). Furthermore, binding is not influenced by IFNy-activation of EC in diseases other than Kawasaki's disease, HUS or TTP (Savage et al., 1991; Del Papa et al., 1992b). Immunoblots of crude HUVEC preparations reveal 12 different bands ranging from 16 to 68 kd reactive with sera from patients with RA complicated by vasculitis (van der Zee et al., 199 l b). With SLE sera, nineteen bands ranging from 15 to 200 kd can be identified (van der Zee et al., 1991a). To obviate contamination by cytoplasmic or nuclear components, immunoprecipitation studies with radiolabeled HUVEC cell surface preparations were undertaken with Wegener's granulomatosis (WG) and SLE sera. The proteins precipitated with WG and SLE sera as analyzed by SDS/PAGE include endothelial antigens ranging in size from 25 to 200 kd. The majority of WG sera precipitate five proteins (25, 68, 125, 155 and180); whereas, SLE sera react with a more hetero-

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geneous group of endothelial proteins. Some antigens can be immunoprecipitated only by WG sera (125 kd) or by SLE sera (200 kd), suggesting that EC reactivity differs from one disease to another (Del Papa et al., 1994b). By immunoblot analysis of cellular proteins derived from human microvascular renal EC (HRMEC), antibodies to a 43 kd, cytosolic and nuclear HRMEC protein can be identified in sera from patients with TTP and HUS. A similar reactivity is detectable occasionally in sera from patients with SLE, antiglomerular basement nephropathy and heparin-associated immune thrombocytopenic purpura (Koenig et al., 1993). Table 1 shows the characteristics of AECA detected in various pathological conditions. Antibodies reacting with extracellular endothelial matrix proteins (quite distinct from those directed against EC) are also present in sera from patients with primary autoimmune vasculitis (Direskeneli et al., 1994).

AUTOANTIBODIES Terminology Antiendothelial reactivity is antibody-mediated, because AECA activity is retained by IgG or IgG F(ab')2 fragments. Likewise, AECA reactivity is not simply due to a nonspecific link of aggregated IgG to the Fc receptors on EC, because immune complex removal does not affect the binding (Del Papa et al., 1994a). Although AECA of IgG, IgA and IgM isotypes are recognized, IgG is the most common. The absence of AECA in diseases with vascular damage clearly mediated by other immune effectors is consistent with the antibodies representing a primary event rather than a mere immune response against determinants exposed in the course of vascular inflammatory processes (Del Papa et al., 1992a). Serum immunoglobulin concentrations do not influence the presence or titer of AECA in primary and secondary vasculitis (Ferraro et al., 1990; D'Cruz et al., 1991), and AECA are not found in sera from patients with signs of polyclonal B-cell activation (Del Papa et al., 1992a).

Pathogenetic Role Human Disease. The above-mentioned disease associations and the fact that AECA react with surface EC antigens strongly support a potential pathogenetic

Table 1. Characteristics of Antiendothelial Cell Autoantibodies in Systemic Autoimmune Vasculitis Correlation with Disease A c t i v i t y

Endothelial Cell ( E C ) Characteristics

Cross-Reactivity

Kawasaki's Disease

Nonimmunoprecipitable IL-1, TNF~, IFNy-inducible

C' cytotoxicity

HUS

Nonimmunoprecipitable downregulated by IFNy

Direct cytotoxicity C' fixation

TTP

Only in part comparable with HUS

C' fixation Cytotoxicity not always detectable

Heparin-assoc. thrombocytopenia

Heparin or heparin-like molecules on EC

Cell-bound heparin (platelets)

Platelet aggregation tissue factor expression

WG/MPA

Constitutive EC surface proteins 120 kd molecule specifically recognized

Fibroblasts and partially with mononuclear cells

C' fixation ADCC EC activation

APS

In part directed against 132-GPI bound to EC

SLE

Directed against a group of constitutive and adhered proteins (15 to 200 kd) or nucleosomal components

RA vasculitis

Directed against a group of molecules (16 to 68 kd)

Pathogenic Mechanism

PGI metabolism E-selectin expression Fibroblasts and partially with mononuclear cells

C' fixation ADCC

EC activation

PSS Higher Reactivity against microvascular EC

Behcet' s disease PM/DM Abbreviations:

APS: C': DM: HUS: MPA: PM:

Antiphospholipid syndrome Complement Dermatomyositis Hemolyticuremic syndrome Micropolyarteritis Polymyositis

role for these autoantibodies. Although AECA are reported to fix complement in vitro (Brasile et al., 1989), most studies do not show direct or complement-mediated cytotoxic activity on EC monolayers by AECA-positive sera from primary or secondary autoimmune vasculitis (Penning et al., 1985; Ferraro et al., 1990). However, some but not all AECApositive sera do mediate antibody-dependent cellular cytotoxicity (ADCC). The lysis, which is mediated by IgG fractions and by NK cells, requires high effector/target ratios and is not enhanced by cytokine activation of EC monolayers (Savage et al., 1991; Del Papa et al., 1992a). An as-yet-unconfirmed exception is acute Kawasaki's disease in which complement-

PSS: RA: SLE: TTP: WG:

Progressive systemic sclerosis Rheumatoid arthritis Systemic lupus erythematosus Thrombotic thrombocytopenic purpura Wegener's granulomatosis

mediated cytotoxicity is reported on cytokine-activated but not on resting EC (Leung, 1990). Besides cytotoxic activity, AECA can affect endothelium by modifying some of its complex functional activities. For example, IgG fractions from patients with APS-associated thrombosis reportedly affect prostacycline production by EC in culture; it is thus possible that the thrombophilic diathesis might be due to the IgG anti-EC activity which results in imbalance between the prostacycline production by EC and that of thromboxane by platelets (Carreras and Vermylen, 1982). Recent data (Del Papa et al., 1995; Le Tonqueze et al., 1995a; 1995b) point to the role of the aPL plasma cofactor, namely 132-GPI, in mediating

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the binding of aPL to endothelial surfaces. 132-GPI, a cationic protein able to bind to negatively charged human EC surfaces, offers suitable epitopes to both [32-GPI-dependent aPL and anti-132-GPI antibodies themselves. Furthermore, binding of affinity-purified anti-~z-GPI antibodies to the EC monolayers causes cell activation, as indicated by the d e n o v o expression of adhesion molecule E-selectin (Del Papa et al., 1995). The relationship between EC activation and the thrombotic diathesis in APS could reflect a procoagulant state of the activated endothelium accompanied by the adherence of mononuclear cells mediated by increased expression of adhesion molecules. Endothelial adhesion p e r se can induce leukocyte activation (Beekhuizen and van Furth, 1993), and activated monocytes display some procoagulant activity (Korneberg et al., 1994). Deposition of AECA on EC surfaces might induce membrane perturbation with resultant cell activation; experiments using IgG from scleroderma and from WG patients support this concept (reviewed in Meroni et al., 1995). WG AECA-IgG, in particular, can induce a dramatic upregulation of ELAM-1, ICAM-1 and VCAM-1 as well as increased secretion of proinflammatory (IL-13, IL-6) and chemoattractant (IL-8, MCP-1) cytokines. Taken together, these findings strongly support a pivotal role for AECA in the vessel wall damage by attracting leukocytes to the inflammatory site, facilitating not only their adhesion to the inflamed vessel walls, but also the extravascular migration of leukocytes and granuloma formation.

Animal Models. AECA can be detected in sera from mice with spontaneous (MRL lpr/lpr) (Le Tonqueze et al., 1995b) or with lupus-like disease experimentally induced by idiotypic manipulation (Blank et al., 1995). Interestingly, murine AECA can also be detected in naive mice three months after injection with human AECA, suggesting that these antibodies are under idiotypic control (Damjanovic et al., 1995).

Radioimmunoassay (RIA). After seeding onto gelatin-coated microtiter plates, confluent monolayers of EC fixed with glutaraldehyde or paraformaldehyde or unfixed are used in solid-phase assay in the usual manner. After incubation with radiolabeled antihuman immunoglobulin and washes, bound material is collected by cell lysis and its radioactivity is counted. The AECA activity of a test serum is usually expressed as a percentage of a positive control serum (Ferraro et al., 1990). Enzyme-linked Assay. Two distinct techniques include: 1) ELISA with whole cells as a substrate and 2) ELISA using EC membranes. In the first type, the assay is performed the same way as RIA, with the only difference in the final step which does not require cell lysis and consists of the regular colorimetric reaction with peroxidase or alkaline phosphatase. AECA activity of the sample is calculated as in the RIA (Del Papa et al., 1992b). The second type employs enriched EC membrane preparations which are coated onto the microtiter plates. The ELISA is then performed as usual (Heurkens et al., 1991).

Cytotoxicity. Complement-fixation ability, direct cytotoxicity, complement-dependent cytotoxicity, ADCC and inhibition of cellular growth (Burns and Zucker-Franklin, 1982; Cohen et al., 1983; Drenk et al., 1985) can be utilized, but there is no general agreement. Therefore, additional studies are necessary to determine optimal conditions to demonstrate cytotoxic effects. However, AECA in primary and secondary systemic vasculitis were detected by a standard complement-dependent microcytotoxicity assay (Brasile et al., 1989). Cytofluorimetry. Recently AECA were detected by immunoglobulin binding to a suspension of EC in a standard cytofluorimetry assay (Westphal et al., 1994).

Immunoblotting (IB). By IB, crude EC preparations Methods of Detection Indirect Immunofluorescence. The use of fluorescein-conjugated antisera to human immunoglobulins permitted the original description of AECA in rodent tissues and then on EC maintained in culture. Other immunohistological techniques (immunoperoxidase) allowed more refined histological observations on kidney biopsies fixed and incubated with patients' sera (Baguley et al., 1987).

248

as well as membrane-enriched preparations can confirm the presence of the antibodies to antigens of defined size (Koenig et al., 1993; van der Zee et al., 1991a; 1991b).

Immunoprecipitation (IP). Selectively radiolabeled EC surface proteins can be immunoprecipitated by AECA-positive sera. The molecular weight of the immunoprecipitated bands is determined by SDSPAGE (McCrae et al., 1991; Del Papa et al., 1994b).

Solid-phase assay (mainly ELISA) with intact cells is the method most widely employed. Different sources of cells, for example the endothelioma Eahy926 cells instead of HUVEC, are still being evaluated in an international standardization program (Meroni et al., 1995; Youinou et al., 1995). The ELISA with EC membrane-coated plates is easy to perform, but the correlation with the whole cell assay is not complete, and false-positive results are possible, probably due to the contamination of the membrane preparations by cytoplasmic or nuclear components. Cytofluorimetric tests are not yet evaluated, because of the requirement for a large number of EC in suspension. EC display different antigenic distributions on their surfaces depending on whether in contact with the solid support (subendothelial matrix) or dispersed in the fluid phase. The cell detachment could induce a redistribution of the cell surface molecules, which in turn could alter the density of the antigens carrying the AECA-specific epitopes. This fact could affect the sensitivity of the cytofluorimetric assay. IB and IP assays are rather complicated and too time consuming to be considered on a routine basis. Large studies comparing results obtained by processing the same sera using different assays are needed. Table 2 summarizes the characteristics of all the AECA assays used so far.

ably autoimmune) vasculitis, both primary and secondary to connective tissue diseases. In fact, the presence of AECA is well documented in primary vasculitis, such as WG, MPA, Kawasaki's disease and idiopathic retinal vasculitis (Edelstein et al., 1992). Moreover, IgG-AECA occur in SLE and also APS, RA-associated vasculitis, mixed connective tissue disease and progressive systemic sclerosis. AECA titers correlate with disease activity in primary autoimmune systemic vasculitis, such as WG, MPA and Kawasaki's disease (Ferraro et al., 1990; Frampton et al., 1990; Chan et al., 1993; Leung 1990). In addition, production of AECA is associated with renal involvement in SLE (D' Cruz et al., 1991), vasculitic manifestations in RA (van der Zee et al., 199 l b), lung complications in dermato-polymyositis (Cervera et al., 1991) and peripheral vascular occlusions in severe progressive systemic sclerosis (reviewed in Meroni et al., 1995). AECA directed against non-HLA molecules on the endothelium of transplanted organs are also detected during accelerated or chronic graft rejection (reviewed in Del Papa et al., 1994a). Although detectable in sera from patients with systemic autoimmune vasculitis, AECA do not display any disease specificity. The highest prevalences are reported in WG, MPA, acute Kawasaki's disease, SLE with renal involvement and APS (Table 3) (reviewed in Del Papa et al., 1994a; Meroni et al., 1995).

CLINICAL UTILITY Correlation with Disease Activity Disease Associations AECA can be detected in clinically distinct, apparently unrelated immunologically mediated diseases (Table 3). The largest group includes patients with (presum-

In autoimmune vasculitis, AECA titers correlate with disease activity. AECA in Kawasaki's disease, although not associated with any particular clinical manifestation (even coronary aneurysms), are detect-

Table 2. Methods for Detection of Antiendothelial Cell Autoantibodies Assay

Specificity

Sensitivity

IIF

high

lowest

Microtoxicity

high

low

Cytofluorimetry

high

high

HUVEC ELISA/RIA

high

high

Eah 926 ELISA

medium

medium

Membrane ELISA

not satisfactory

highest

Immunoblotting (HUVEC membrane)

medium

high

Immunoprecipitation (radiolabeled HUVEC)

high

high

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Table 3. Pathological Conditions in Which Antiendothelial Cell Antibodies Are Reported Disease

Prevalence

Systemic lupus erythematosus

0--88%

Rheumatoid arthritis with vasculitis

80%

Mixed connective tissue disease

45% (20/44)

Progressive systemic sclerosis

20-30%

Polymyositis/dermatomyositis

44% (8/18)

Wegener's granulomatosis

50-80%

Antiphospholipid syndrome

60-63%

Behcet's disease

18--80% (depending on the cell type as substrate)

Idiopathic retinal vasculitis

35%

Kawasaki's disease

65%

Hemolytis uremic syndrome

93% (13/14)

Thrombotic thrombocytopenic purpura

100% (3/3)

Heparin-associated thrombocytopenia

!00% (27/27)

HLA-matched graft rejection Inflammatory bowel diseases

25--43%

Insulin-dependent diabetes mellitus

up to 34%

Multiple sclerosis

23% (7/32)

Immune-mediated hypoparathyroidism

100% (6/6)

Episodic angioedema/hypereosinophilia

100% (3/3)

Acute preeclampsia

50% (9/18)

Berger's disease

32% (26/72)

Viral infections

up to 18%

Rocky mountain spotted fever

50% (7/14)

able only during the active disease but not during remission. In WG and MPA, AECA titers decrease during therapy-induced remission and correlate with other clinical or laboratory parameters of disease activity as well as endothelial damage. AECA are predictive of relapses in systemic vasculitis (Chan et al., 1993), but such a correlation is not clear in SLEassociated vasculitis. Although their presence is reported in active disease (Cines et al., 1984), a higher prevalence of AECA is reported only in SLE patients with (but not without) nephritis (D'Cruz et al., 1991), but others (Van der Zee et al., 1991a) find only a correlation of AECA with skin or joint involvement. IB analysis of the same sera, however, reveals that

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antibodies against endothelial proteins of 38, 41 and 150 kd are associated with renal involvement. Finally, there is a positive correlation between the presence of the antibodies and the severity of vascular lesions in systemic sclerosis (quoted in Meroni et al., 1995). Interestingly, IgA-AECA are reported in sera from patients with IgA nephropathy (Yap et al., 1988).

Associations with Other Autoantibodies AECA are not related to other circulating autoantibodies such as rheumatoid factor or antibodies to nuclear constituents including extractable nuclear antigens (van der Zee et al., 1991a; 1991b). An

exception is the linkage of EC antibodies to DNA antibodies targeting DNA or DNA/histone complexes (Chan et al., 1992). AECA and ANCA are frequently detectable in the same pathological conditions, namely primary autoimmune systemic vasculitis (Gross et al., 1993). Both epidemiological and cross-inhibition studies clearly demonstrate that A N C A and AECA, although associated in many sera, are two distinct antibody populations (Ferraro et al., 1990; Frampton et al., 1990).

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CONCLUSION Several methodological approaches have demonstrated the existence and the potential pathogenic role of antiendothelial antibodies, especially in autoimmune vasculitis. In addition, a huge number of reports in literature support the importance of AECA identification both from a diagnostic and a prognostic point of view. However, further standardization of the procedures for their detection does represent a requisite for a wider use of these autoantibodies as a clinical marker.

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endothelium. Clin Exp Immunol 1971;9:753-760. McCrae KR, DeMichele A, Samuels P, Roth D, Kuo A, Meng QH, Rauch J Cines DB. Detection of endothelial cell-reactive immunoglobulin in patients with antiphospholipid antibodies. Br J Haematol 1991;79:595--605. Meroni PL, Khamashta MA, Youinou P, Shoenfeld Y. Mosaic of antiendothelial antibodies. Review of the first international workshop on antiendothelial antibodies: clinical and pathological significance. Lupus 1995;4:95-99. Penning CA, French MAH, Rowell NR, Hughes P. Antibodydependent cellular cytotoxicity of human vascular endothelium in systemic lupus erythematosus. J Clin Lab Immunol 1985;17:125-130. Rosenbaum J, Pottinger BE, Woo P, Black CM, Louzou S, Byron MA, Pearson JD. Measurement and characterization of circulating antiendothelial cell IgG in connective tissue diseases. Clin Exp Immunol 1988;72:450--456. Savage COS, Pottinger BE, Gaskin G, Lockwood CM, Pusey CD, Pearson JD. Vascular damage in Wegener's granulomatosis and microscopic polyarteritis: presence of antiendothelial cell antibodies and their relation to antineutrophil cytoplasm antibodies. Clin Exp Immunol 1991;85:14--19. Tan EM, Pearson CM. Rheumatic disease sera reactive with capillaries in the mouse kidney. Arthritis Rheum 1972;15: 23-28. van der Zee JM, Heurkens AH, van de Voort EA, Daha MR, Breedveld FC. Characterization of antiendothelial antibodies in patients with rheumatoid arthritis complicated by vasculitis. Clin Exp Rheumatol 1991a;9:589--595. van der Zee JM, Siegert CEH, de Vreede TA, Daha MR, Breedveld FC. Characterization of antiendothelial cell antibodies in systemic lupus erythematosus. Clin Exp Immunol 1991b;84;238--244. van der Zee JM, Miltenburg AMM, Siegert CEH, Daha MR, Breedveld FC. Antiendothelial cell antibodies in systemic lupus erythematosus (SLE): enhanced antibody binding to interleukin-1 stimulated endothelium. Int Arch Allergy Appl Immunol 1994;104:131-136. Westphal JR, Boerbooms AMTh, Schalkwijk CJM, Kwast H, de Weijert M, Jacobs C, Vierwinden G, Ruiter DH, Van de Putte LBA, De Waal RMW. Anti-EC antibodies in sera of patients with autoimmune diseases: comparison between ELISA and FACS analysis. Clin Exp Immunol 1994;96: 444--449. Yap HK, Sakai S, Bahn L, Rappaport V, Woo KT, Ananthurman V, Lim CH, Chiang GS, Jordan SC. Antivascular EC antibodies in patients with IgA nephropathy: frequency and clinical significance. Clin Immunol Immunopathol 1988;49: 450--462. Youinou P, Meroni PL, Khamashta MA, Shoenfeld Y. Standardization program of the antiendothelial cell antibody test. Immunol Today 1995;16:363-364.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

FIBRILLARIN AUTOANTIBODIES Per Hultman, M.D., Ph.D. a and K. Michael Pollard, Ph.D. b

aDepartment of Pathology I, Link6ping University, S-581 85 Link6ping, Sweden; and bW.M. Keck Autoimmune Disease Center, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA

H I S T O R I C A L NOTES Antinucleolar antibodies (ANoA) were first identified in 1961 in the serum of patients with rheumatic diseases (Beck, 1961) and were subsequently found to be especially frequent in patients with scleroderma (Miyawaki and Ritchie, 1974). Of the different nucleolar staining patterns identified by indirect immunofluorescence in sera from patients with scleroderma (Tan et al., 1980), that described as "clumpy" (Bernstein et al., 1982) was later shown to be due to antibodies to a 34 kd nucleolar protein (Reimer et al., 1987) named fibrillarin because of its localization to the dense fibrillar and fibrillar center regions of the nucleolus (Ochs et al., 1985). In 1983, some scleroderma sera were found to precipitate particles containing U3 RNA (Reddy et al., 1983). Fibrillarin is the major antigenic constituent of a family of uridine-rich small nucleolar RNAs (snoRNAs) associated with proteins, localized in the nucleolus and called the small nucleolar ribonucleoprotein particles, or snoRNP particles (Baserga and Steitz, 1993).

THE AUTOANTIGEN

isoelectric form (pI 9.5) has also been described (Celi8 et al., 1992). Fibrillarin shows a high degree of evolutionary conservation, a protein analogous to fibrillarin was detected in 1977 in the slime mold Physarum polycephalum and named B-36 (Christensen et al., 1977).

Native versus Recombinant Antigen Performance There has been no direct comparison of the antigenicity of fibrillarin found in eukaryotic cells with that produced by bacteria using recombinant DNA technology.

Origin, Sources, Organs, Tissue, Cells Fibrillarin is present in all nucleated cell types. A convenient source for immunoblotting are nuclei and nucleoli prepared from rat liver (Lischwe et al., 1985; Chan and Pollard, 1992).

Methods of Purification Fibrillarin can be extracted from purified nucleoli using LiC1 and urea, followed by phosphocellulose column chromatography (Lischwe et al., 1985). There are no commercial sources of purified fibrillarin.

Definition Sequence Information Mammalian fibrillarin (pronounced fi-brill-a-rin) was detected using a serum from a patient with scleroderma which contained autoantibodies monospecific for a protein with an apparent molecular weight of 34 kd and a pI of 8.5 (Ochs et al., 1985). An additional

Cloned and fully sequenced human fibrillarin is 81% identical to the amphibian fibrillarin sequence and 67% identical to yeast fibrillarin sequence (Aris and Blobel, 1991). The mouse sequence is 94% identical

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to the human sequence and 74% identical to yeast fibrillarin sequence (Turley et al., 1993). That a high degree of evolutionary conservation also applies at the level of protein configuration is reflected in the reaction of autoantibodies from scleroderma patients (Reimer et al., 1987; Takeuchi et al., 1995) as well as from mercury-treated mice (Takeuchi et al., 1995) with fibrillarin from a wide variety of species. Fibrillarin contains three major domains. All known sequences of eukaryotic fibrillarin share an Nterminal domain containing 75--85 amino acids sequence which exhibits repetitive stretches rich in glycine and arginine (glycine-arginine rich [GAR] domain) as a result of posttranslational methylation and this motif makes fibrillarin the most heavily methylated protein (4 mol % dimethylarginine) so far identified (Lischwe et al., 1985). This motif as well as a central 80--90 amino acid stretch resembles RNAbinding domains (Aris and Blobel, 1991; Najbauer et al., 1993). The characterized fibrillarin sequences also contain an 8 amino acids long conserved sequence which shows similarities with the RNP consensus motif (Aris and Blobel, 1991). However, as yet there is no evidence supporting direct interaction of fibrillarin with snoRNAs (Turley et al., 1993). Scleroderma sera react with an evolutionary conserved domain on fibrillarin which is also recognized by ANoA-positive sera from mercury-treated, genetically susceptible mouse strains. The epitope common to both human and murine autoantibodies is discontinuous, conformational, nonlinear and is found in the C-terminal end (between amino acid 257--312) together with a region within the N-terminal (the first 156 amino acids) (Takeuchi et al., 1995). In addition, some (3/10) ANoA-positive sera from scleroderma patients appear to react specifically with an epitope within the Cterminal region encompassing amino acid residues 156-327 (Takeuchi et al., 1995). Antifibrillarin antibodies (AFA) arising from immunization follow the general principles for actively elicited antibodies, i.e., they do not react with conserved, nondenaturated antigen in the same way spontaneous antibodies do (Rubin and Tan, 1992). In contrast, a hybridoma (72B9) from the lupus-like murine strain (NZB x NZW)F1 shows evolutionary conservation, reacting with nucleoli in animal as well as plant cells (Reimer et al., 1987), and the same conformational epitope encompasses N- and C-terminal regions of yeast fibrillarin as human and mercury-induced murine autoantibodies (Takeuchi et al., 1995).

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AUTOANTIBODIES

Name Antifibrillarin antibodies (AFA) are sometimes called anti-U3-RNP (Reimer et al., 1988) or anti-(U3) snRNP (Okano et al., 1992) antibodies. Although autoantibodies that immunoprecipitate the U3 snoRNA and associated proteins do react with fibrillarin, it is clear that fibrillarin is also associated with other snoRNAs and, therefore, use of a single snoRNA or snoRNP, such as U3, is now incorrect. The abbreviation AFA, denoting the specific protein antigen, is, therefore, favored (Hultman et al., 1992).

Pathogenetic Role Human Disease. Like other autoantibodies reacting with intracellular antigens, AFA are not known to interact directly with intracellular fibrillarin in vivo, and the effects of AFA on the cellular or molecular function in vitro are unknown. Animal Models. AFA can be present in murine graftversus-host disease (Gelpi et al., 1988). Out of 20 (BALB/c • A/J)F1 mice injected with A/J lymphoid cells, four developed antibodies which precipitated the U3 snoRNA and, in addition, reacted with a 34 kd nucleolar protein in immunoblotting. However, these sera also contained autoantibodies to other U snRNPs, especially the A and 70 kd polypeptides of the UlsnRNP, and the titer of AFA substantially declined during the 20 weeks following the cell injection. In 1989, after injection with mercuric chloride, certain mouse strains were reported to develop ANoA which reacted with a 34--36 kd nucleolar protein identical with fibrillarin (Hultman et al., 1989a) and also precipitated U3snoRNA (Reuter et al., 1989). These AFA recognize the same N- and C-region epitope as AFA of scleroderma patients (Ltibben et al., 1994; Takeuchi et al., 1995) and the antifibrillarin monoclonal antibody 72B9 (Takeuchi et al., 1995). The AFA develop within 2--3 weeks after injection of the metal, are dominated by the IgG class (Hultman et al., 1989a) and often reach high titers of the same magnitude as scleroderma sera (Mirtcheva et al., 1989). The development of AFA in susceptible mice is accompanied by deposition of immune-complexes systemically in vessel walls and in the glomerular mesangium (Hultman et al., 1989b). ANoA/AFA are found in eluates from such kidneys (Hultman and

Enestr6m, 1988) which show a mild mesangial glomerulonephritis (Hultman and Enestr6m, 1987). AFA can also be induced in genetically susceptible mouse strains by mercury vapor (Warfvinge et al., 1995) and the organic mercury compound merthiolate (Hultman and Pollard, unpublished observations). The dose-response relationships for both oral mercuric chloride and mercury vapor exposure are known (Hultman and Enestr6m, 1992; Warfvinge et al., 1995). Silver can also induce AFA (Hultman et al., 1994) with a genetic susceptibility similar to that for mercury (Hultman et al., 1995a).

Genetics In mice, the susceptibility to develop AFA is under the control of MHC (H-2) class II genes, H-2s representing the prototype susceptibility haplotype, but it is evident that also background, non-H-2 genes play an important role in regulating the autoantibody response (Mirtcheva et al., 1989; Hultman et al., 1992). A series of IgG1 and IgG2a monoclonal ANoA from mercury-treated A.SW mice showed little restriction in their V H and V L gene usage, but VI4 J558 and VH3609, and V~: genes from the V~19/38 family are frequently paired (Monestier et al., 1994). Little is known about the genetic requirements for development of AFA in humans. Preliminary studies suggest that AFA appear more frequently in Blacks with scleroderma than Caucasian scleroderma patients. In addition, patients with AFA show significantly greater frequency of the HLA-DQ6 alleles DQB 1"0602 and/or *0604 (carried on HLA-DR2 and DR13 haplotypes) (Arnett et al., 1994).

1989a); treatment with anti-IL-4 MAb achieves only a partial reduction of AFA (Ochel et al., 1991), suggesting that the role of Th2 is limited. Mercury induces IL-1 in vivo and in vitro in mice (Zdolsek et al., 1994), but there are no further published data on the cytokine-profile in mercury-treated mice which develop AFA. Recent findings indicate that T-cell clones from genetically susceptible mercury-treated mice react either with mercury-treated nuclear material or nuclear material alone (Kubicka-Muranyi et al., 1995), and mercury-induced AFA may persist for at least 1 year after treatment with mercury has been stopped (Hultman et al., 1996). Taken together, the data obtained in mercury-treated mice strongly favor an autoantigen-specific mechanism as responsible for the development and persistence of AFA.

Methods of Detection #

The most convenient screening method for detection of AFA is the indirect IF test (IIF) using cultured cells as the substrate; with organ sections, the nucleolar pattern is easily overlooked (Tan et al., 1980). Of the various ANoA patterns described, that described as "clumpy" with bright granules decorating the nucleoli corresponds to AFA (Reimer et al., 1988). AFA yield a distinct staining of the condensed chromosomes in metaphase cells (Figure 1) but do not stain the nucleoplasm of interphase cells; this helps differentiate AFA staining from that caused by PM/ Scl and RNA-polymerase I which give other patterns in metaphase cells and a weak staining of the nucleo-

Factors Involved in Pathogenicity and Etiology The mechanism underlying development of AFA has not been elucidated in humans or in mice. In scleroderma, there is an increased CD4+ helper cell activity (Postlethwaite, 1990) and an increased production of several interleukines (Needleman et al., 1992). The induction of AFA by mercury in mice requires the participation of CD4+ T cells (Hultman, 1995b) which show an increased expression of IL-4 mRNA (van Vliet et al., 1993). A preferential activation of Th2 cells has, therefore, been suggested (Goldman et al., 1991). However, the isotypes of the serum AFA in these mice include not only the Th2/IL-4-dependent IgG1 isotype, but also the Thl/y-interferon-dependent IgG2a isotype of a similar titer (Hultman et al.,

Figure 1. Indirect immunofluorescence (IIF) pattern of antifibrillarin autoantibodies on HEp-2 cells. Nucleolar fluorescence reveals a "clumpy" pattern in interphase cells (short arrow). In late anaphase, fibrillarin is shown decorating the periphery of the replicated chromosomes (large arrows). 255

plasm in interphase cells (Reimer et al., 1988). Recognition of the different nucleolar patterns requires experience and is facilitated by viewing the nucleolar staining in various focal planes and serum dilutions. The clumpy nucleolar staining is often combined with two-to-six brightly staining dots in the nucleoplasm identified as coiled bodies which contain fibrillarin (Raska et al., 1991). Using monospecific antiserum as a standard, AFA can be readily identified by immunoblotting (IB) using purified nuclei or nucleoli as an antigenic source (Chan and Pollard, 1992) (Figure 2). Immunoprecipitation (IP) of radiolabeled extracts from cell culture lines (Steitz, 1989) shows that fibrillarin is an antigenic component of macromolecular complexes containing protein and RNA (Parker and Steitz, 1987). Radiolabelling of protein with [35S]-methionine reveals the presence of at least six proteins of 12.5, 13, 30, 36 (fibrillarin), 59 and 74 kd; the latter two being phosphoproteins. Use of 32po 4 to label nucleic acids showed the association of fibrillarin with a number of small nucleolar RNAs the most predominant of which is the 217 nucleotide long U3 snoRNA (Baserga and Steitz, 1993). The above techniques (IIF, IB and IP (of radiolabeled cell extracts)) do not unequivocally identify antifibrillarin autoantibody because a positive reaction could be due to interaction with an snoRNP component other than fibrillarin, or a nuclear component with the same localization or molecular weight as fibrillarin. Molecular cloning of cDNA encoding fibrillarin from a variety of species permits unambiguous detection of antifibrillarin autoantibodies. Two methods can be used. Radiolabeled protein produced by in vitro transcription, with translation using rabbit reticulocyte lysate (Takeuchi et al., 1995) closely resembling the protein produced in vivo. Synthesis of protein using such a cell-free eukaryotic system can include post translational modifications and di-sulphide bond formation, thereby aiding in protein folding and attainment of correct three-dimensional structure. Such material can then be used in immunoprecipitation (Figure 3) under fluid-phase conditions to maintain structural integrity. Fibrillarin expressed from cDNA in bacteria as a fusion protein can be used to detect antifibrillarin antibodies (Ltibben et al., 1994) but must be purified to reduce nonspecific reaction with bacterial antigens. Use in immunoblot means denaturation of the protein and possible loss of reactivity with autoantibodies directed against conformational determinants. This is of importance in 256

Figure 2. Immunoblotting of antinucleolar (ANoA) sera on rat liver nuclei. The immunoblotting reactivity of five human sera (1-5) with antinucleolar IIF patterns are shown, together with positive control sera for antifibrillarin and anti-PM-Scl sera. Test sera 2, 3 and 4 show reactivity with a 34 kd protein consistent with the presence of antifibrillarin antibodies.

detecting antifibrillarin autoantibodies as a common reactivity appears to be against a highly conserved conformational epitope (Takeuchi et al., 1995).

Figure 3. Immunoprecipitation of [35S]-labeled fibrillarin produced by in vitro transcription and translation of murine fibrillarin cDNA. Twelve sera with antinucleolar autoantibodies by IIF (A-L) were tested for direct interaction with fibrillarin. Positive sera are identified by an *. TnT mFIB: radiolabeled product produced by transcription and translation of mouse fibrillarin; cDNA POS CONT: positive control antifibrillarin; NEG CONT: negative control serum. Detection of antifibrillarin autoantibody should include immunofluorescence detection of the "clumpy" nucleolar staining pattern and, if possible, colocalization of staining to nuclear coiled bodies. Confirmation requires immunoprecipitation with radiolabeled protein from cDNA, or immunoblotting using purified nuclei or nucleoli and standardized positive control.

C L I N I C A L UTILITY

Disease Association When adequate test methods are used, such as a combination of IIF on cultured cells to ascertain reactivity with native autoantigen (clumpy nucleolar staining), and IB or IP to ascertain specificity for fibrillarin, AFA seem to occur very rarely in conditions other than scleroderma (Imai et al., 1993), and AFA are, therefore, included among the marker antibodies for scleroderma (Reimer and Tan, 1993). Interestingly, autoantibodies to other nucleolar antigens such as anticentromere and anti-RNA polymerase I, II or III, which occur rather frequently in scleroderma, only rarely coexist with AFA (Okano et al., 1992). Of patients with scleroderma, 90--95% have autoantibodies to intracellular antigens (Kuwana et al., 1994). Although the majority of the patients have

autoantibodies which target DNA topoisomerase I (Scl-70, a nucleolar and nucleoplasmic antigen), a substantial number have autoantibodies which, on screening using cultured cells, predominantly target the nucleolus (Pollard et al., 1989). However, the fraction of patients with ANoA varies from 7--71% in different studies (Picillo et al., 1993), perhaps reflecting different criteria for scleroderma, differences between ethnic groups or different techniques for detection of ANoA (Reveille et al., 1992). Clinical studies of scleroderma patients employing U3 snoRNP precipitation (Okano et al., 1992; Kuwana et al., 1994) or immunoblotting of fibrillarin (Reimer et al., 1988) showed a similar frequency of AFA (3--6%). In one study, anti-U3 snoRNP antibodies were significantly more frequent in Blacks; the patients with AFA were significantly younger, had more digital scarring and/or ulcers, pigmentation disorders, calcinosis, myositis, small bowel radiographic involvement and primary pulmonary arterial hypertension than scleroderma patients without anti-U3 snoRNP antibodies (Okano et al., 1992). In another study, AFA were significantly more frequent in men, and the patients with AFA were again significantly younger and had fewer joint manifestations than those without ANoA (Reimer et al., 1988). The lower frequency of joint manifestations in scleroderma patients with anti-U3 snoRNP antibodies was confirmed in another study, which also showed a significantly lower frequency of pulmonary interstitial fibrosis in U3 snoRNP-positive patients (Kuwana et al., 1994). There have also been case reports suggesting that AFA are seen more frequently in scleroderma patients with telangiectasias and diffuse cutaneous involvement (Kurzhals et al., 1990). There are no reports on treatment-induced variation in AFA, and there are also no reports on transplacental transfer or neonatal disease variants. Therefore, at present, the main clinical utility of AFA is as a diagnostic marker of scleroderma.

CONCLUSION Fibrillarin is an evolutionary, highly conserved 34 kd nucleolar protein with exceptional, heavy methylation. Fibrillarin is the main antigenic constituent in a rapidly growing family of small nucleolar RNAs associated with protein the small nucleolar ribonucleoprotein, snoRNP, particles. The association of fibrillarin with U3 snoRNA suggests involvement in rRNA processing, and possibly other steps in ribo-

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some biogenesis. Autoantibodies to fibrillarin, reacting with an evolutionary conserved conformational epitope usually encompassing both the N- and C-terminal domains, are found in 3 - 6 % of patients with scleroderma but very rarely in patients with other diseases or in healthy controls. Antifibrillarin antibodies are, therefore, marker antibodies for scleroderma, being significantly more frequent in Blacks and younger patients and associated with a lower frequency of joint manifestations. The role, if any, for A F A in the pathogenesis of scleroderma is unknown. However,

genetically susceptible mouse strains consistently develop high-titered A F A of the IgG class and systemic immune-complex deposits in response to treatment with heavy metals such as mercury and silver. Because the B-cell epitope on fibrillarin is very similar in the mouse model and in scleroderma patients, further elucidation of the pathogenetic mechanisms in the murine model may be anticipated to contribute to the understanding of the pathogenesis of scleroderma. See also AUTOANTIBODIES THAT PENETRATE INTO LIVING CELLS.

REFERENCES

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9 Elsevier Science B.V. All fights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

FIBRONECTIN AUTOANTIBODIES Mustafa S. Atta, M.B., Ch.B., M.Sc., Ph.D., Richard J. Powell, D.M. and Ian Todd, Ph.D.

Division of Molecular and Clinical Immunology, Department of Clinical Laboratory Sciences, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, UK

HISTORICAL NOTES In 1986, antifibronectin autoantibodies (anti-Fn) were detected in serum of 78% of patients with systemic lupus erythematosus (SLE), 40% of those with rheumatoid arthritis (RA) and in association with bacterial and viral infections (Henane et al., 1986). In subsequent studies, anti-Fn were detected in SLE and RA but at lower frequency: 29--34% of SLE patients and 14% of RA patients (Atta et al., 1994a; 1995). AntiFn were also reported in a case of morphea (Stefanato et al., 1992) and a case of undifferentiated connective tissue disease (Girard and Senecal, 1993a; 1993b).

THE AUTOANTIGEN

polypeptide subunit. The two type II repeats are restricted to the part of the Fn molecule that binds to collagen, while type III repeats account for about 60% of the protein sequence and are present in the middle of the molecule. Fifteen of the type III repeats are present in all Fn subunits and numbered from III-1 to III-15. Two extra domains of type III repeats are subject to alternative splicing and are called ED-A (or EIIIA) and ED-B (or EIIIB) (Petersen et al., 1989). Between III-14 and III-15 lies the variable (V), or the connecting segment (CS) sequence. The CS region can be spliced partially; whereas, the ED-A and ED-B are spliced completely. The presence of ED-A or EDB distinguishes the cellular from the plasma Fn as both ED-A and ED-B are excluded in the latter (Petersen et al., 1989). Figure 1 depicts the structure of the Fn molecule.

Definition Origin, Sources, Tissues, Cells Fibronectin (Fn), a high-molecular weight (440 kd), extracellular matrix protein (ECM) present in body fluids, on cell surfaces and associated with basement membranes, is a dimer of two polypeptide chains (each of about 220 kd)which are similar except for splice variations. The two polypeptide chains of the Fn molecule are linked together in an antiparallel arrangement by a pair of disulfide bonds at the carboxyl-terminal end. The complete amino acid sequence for human Fn is established from cloned cDNA and for bovine Fn by protein sequencing (Kornblihtt et al., 1985; Skorstengaard et al., 1986). The Fn molecule is comprised of three types of amino acid repeats (types I, II and III). In each Fn molecule there are 12 type I, two type II and 15--17 type III repeats. Type I repeats are clustered at the amino and the carboxyl-terminal regions of the

260

The Fn molecule binds to a number of macromolecules including fibrin, fibrinogen, heparin, collagen thrombospondin, plasminogen and plasminogen activator, C-reactive protein, DNA, immune complexes, rheumatoid factor, complement components Clq and C3, Staphylococcus aureus proteins, immunoglobulins and cells. The best characterized binding sites are those containing the RGD and LDV amino acid sequences. These sites on the Fn molecule bind specific cell receptors called the integrins (~3~1, ~4~1 and o{5131). The binding of Fn to cells plays an important role in a variety of cell functions such as cell-to-cell, and cell-to-ECM attachment, cell motility and polarity. Fn may also have a role in lymphocyte and macrophage activation (Shimizu and Shaw, 1991). Fn has multiple biological functions which are

Figure 1. Schematic diagram of the structure of the fibronectin dimer. The two subunits are shown with the amino termini (NH2) to the right and the carboxyl termini (COOH) to the left. Regions of alternative splicing (CS, ED-A and ED-B) and the binding domains are shown on one subunit. mediated by the interaction of Fn with cells and macromolecules. Examples of these functions are wound healing and self-defense against infection by opsonizing invading microorganisms. Furthermore, plasma may play a role in clearing immune complexes and tissue debris (Ruoslahti, 1988). Fibronectin is present in soluble and insoluble forms. Produced mainly by hepatocytes, the soluble form has a half-life of 24--72 hours and is widely distributed in plasma, cerebrospinal fluid, synovial fluid, amniotic fluid, seminal fluid, saliva and inflammatory exudates. Although its fate is not proved, circulating Fn may be incorporated into the ECM. It is also reported that soluble Fn is secreted onto and coats mucosal surfaces of the mouth and the vagina (Proctor, 1987). The insoluble form of Fn is widely distributed on cell surfaces and ECMs, where it is cross-linked into

multimeric fibers. A variety of cell types produce this form of Fn, including fibroblasts, vascular endothelial cells, macrophages and synoviocytes. Although also a constituent of thyroid, breast and gut, Fn is not generally found in epithelium except as an artifact of wound repair (Colvin, 1989). Its location in glomerular basement membranes is disputed, but Fn may be a component of the mesangial matrix, and the endotheliomesangial interface and the lamina r a r a of the peripheral glomerular basement membrane (Courtoy et al., 1982). Plasma Fn concentrations vary with gender: in women averaging 400 ~tg/mL and in men 300 ~g/mL. Its production is increased in inflammatory conditions as part of an acute-phase response. Reduced plasma levels are found in serious liver dysfunction and disseminated intravascular coagulopathy (Proctor, 1987). Naturally occurring deficiencies or abnormal-

261

ities of soluble or insoluble Fn are extremely rare. However, partial deficiency is also reported as a familial condition (Shirakami et al., 1986).

THE AUTOANTIBODY Pathogenetic Role Animal Models. Animal studies provide evidence for pathogenic significance of anti-Fn. Rabbits immunized with denatured human Fn monomers developed antiFn and glomerular injuries (Murphy-Ullrich et al., 1984). Mice immunized with native dimeric or denatured multimeric or monomeric serum Fn, in the absence of adjuvant, produce autoantibodies to mouse Fn. However, antibodies induced by one form of Fn do not completely cross-react with antibodies induced by the other forms of Fn. A significant increase in the level of serum immune complexes associated with electron-dense deposits in the renal mesangium suggests an immune complex mechanism of renal damage (Murphy-Ullrich et al., 1986). Rats injected with antibodies to rat plasma Fn developed mesangial immune deposits which were related to an in situ binding of the antibody to the mesangial Fn (Zanetti and Takami, 1984).

Methods of Detection Anti-Fn can be detected by ELISA, indirect immunofluorescence and immunoblotting. Direct ELISA can be employed when using pure (e.g., bovine) Fn (Atta et al., 1994a); however, the detection of impurities in the antigen preparation, necessitates the use of a capture ELISA (Atta et al., 1995) in which a monoclonal Fn autoantibody is coated on wells of microtiter plates. With monkey fibroblasts as substrate for indirect immunofluorescence (Stefanato et al., 1992), the human serum produces an immunofluorescence staining pattern similar to that of the sheep anti-Fn antiserum, but is distinct from the patterns produced by anticollagen and antilaminin. In an inhibition assay, binding of human IgG to the substrate is inhibited by anti-Fn but not other antisera (Stefanato et al., 1992). Anti-Fn were detected by immunoblot in one patient with undifferentiated connective tissue disease but not in patients with rheumatic or other autoimmune diseases (Girard and Senecal, 1993a; 1993b). The low-level detection of anti-Fn by immunoblotting could be related to the denaturation of the Fn molecule during this procedure and the antibodies might recognize native rather than denatured Fn.

CLINICAL UTILITY Human Disease. The pathological significance of anti-Fn is also supported by their interference with Fn functions in vitro. Anti-Fn in SLE sera bind to the collagen binding domain (CBD) of the Fn molecule, block Fn-collagen binding and diminish deposition of Fn and collagen on cultured human fibroblasts (Atta et al., 1994a). The same sera inhibited cell attachment on Fn-coated surfaces. That these effects were due to anti-Fn in the sera was shown when sera depleted of Fn autoantibodies did not reduce the deposition of collagen or Fn on cultured human fibroblasts nor did they inhibit cell attachment (Atta et al., 1994b). Both Fn and collagen are intimately involved in the structure and function of many organs and a reduced amount in tissues could prove to be a significant factor in functional weakness, reduced tensile strength and loss of tissue integrity. Anti-Fn may also prevent Fn from serving as a protein scaffold to direct tissue repair following inflammation. The importance of Fn in tissue repair is manifest by depressed cellular and plasma Fn concentrations associated with impaired healing response to lung injury in infants with cystic. lung disease (Watts and Bruce 1992).

262

Disease Associations The reported frequency of anti-Fn in patients with SLE varies in different studies ranging from 29--78% (Henane et al., 1986; Atta et al., 1994a). This could be related to the variation in the ELISA procedure (direct vs. capture), the source and purity of Fn (bovine vs. human) used in the assay and patient selection. Anti-Fn are also reported in 14--40% of RA patients (Henane et al., 1986, Atta et al., 1995) as well as in 6.6% of patients with Behcet disease (Atta et al., 1995). Anti-Fn are also reported in a number of bacterial and viral infections such as Mycoplasma pneumoniae (70%), Legionella pneumophila (100%), bacterial endocarditis (80%), primary syphilis (5%) and leprosy (20%) (Henane et al., 1986). Anti-Fn cannot be regarded as a specific serological marker for SLE or other autoimmune diseases. Anti-Fn were recently measured by capture ELISA in 65 patients with well-characterized SLE, whose disease activity was assessed by British Islets Lupus Activity Group (BILAG) index and by laboratory

markers of disease activity; namely, the ESR, sIL-2R, anti-dsDNA, C3, C4 and the complement degradation product C3dg levels in blood and neopterin excretion in urine (Atta et al., 1995). Patients with active disease have significantly higher concentrations of anti-Fn than those with inactive disease, and the antibody concentration is correlated with the BILAG disease activity index. Furthermore, the concentration of anti-Fn correlates with ESR, sIL-2R and urine neopterin results. These findings suggest that although anti-Fn are not diagnostic for SLE or other autoimmune diseases, they could be useful markers of disease severity. Analysis matching the presence of anti-Fn with clinical manifestations revealed that, with the exception of higher prevalence in those individuals with musculoskeletal involvement, there was a broadly similar pattern of organ involvement in patients with or without anti-Fn.

IgA-Fibronectin Aggregates High concentrations of IgA-Fn aggregates are found in sera from patients with IgA nephropathy (IgAN) (Cederholm et al., 1988), the most common form of primary glomerulonephritis worldwide (Julian et al., 1988). IgAN disease is characterized by renal injury with increased serum IgA and deposition of IgA in renal tissues. The presence of Fn in these aggregates could mediate the binding of IgA to renal mesangium, but there is no proof (Cederholm et al., 1988). IgA-Fn

complex formation could be a normal binding process, since polymeric IgA has the ability to bind directly to Fn (Davin et al., 1991). But because of its substantial stability, the binding of IgA to Fn in aggregates is unlikely to reflect Fc binding and might be due to an antigen-antibody interaction (Cederholm et al., 1988). However, it is not known yet whether the IgA-Fn aggregates are antigen-antibody complexes or represent a nonspecific binding of Fn to IgA. Because of the strong association (p < 0.0001) of IgA-Fn aggregates and IgA nephropathy (93%) compared with the frequencies in normal healthy controls (6.7%) and in patients with other types of glomerular diseases (11.7%) (Jennette et al., 1991), and because IgA-Fn aggregates are not correlated with serum IgA concentrations or IgA rheumatoid factor (Peter et al., 1990), IgA-Fn aggregates are a useful serological marker for IgA nephropathy (Jennette et al., 1991, Peter et al., 1990). Further studies of the role of IgA-Fn aggregates as a serological marker of IgA nephropathy (Baldree et al., 1993) detected IgAFn aggregates in only 48% of patients with IgA nephropathy. However, the concentrations of IgA-Fn aggregates in patients with IgA nephropathy were significantly higher than in those with other forms of glomerulonephritis and were significantly correlated with IgA and C3 activation. Furthermore, there was a small increase in the frequency of IgA-Fn aggregates in patients who had or subsequently developed chronic renal insufficiency. Thus, IgA-Fn aggregates might be

Table 1. Summary of Characteristics of Fibronectin Autoantibodies Discovery date

1986

Autoantigen

Human fibronectin is an extracellular matrix glycoprotein (440 kd) present in body fluids, on cell surfaces and associated with basement membranes.

Functions of fibronectin

Mediate cell-cell, cell-extracellular matrix attachment, cell motility and polarity, lymphocyte and macrophage activation, wound healing, opsonization of invading microorganisms and clearing immune complexes and tissue debris.

Autoantibody target

The collagen-binding domain of the fibronectin molecule.

Origin of the autoantibody

Unknown.

Detection

Direct ELISA when using pure fibronectin (e.g., bovine fibronectin), and capture ELISA when impurities were detected in the preparation (e.g., human fibronectin). Immunofluorescence staining and immunoblots have also been used.

Clinical association

Fibronectin autoantibodies are found in systemic lupus erythematosus (SLE, 29-34%), rheumatoid arthritis (14%) and a number of bacterial and viral infections. In SLE, higher concentrations of anti-Fn autoantibodies are found in patients with active compared to those with inactive disease. The presence of these autoantibodies is consistently associated with musculoskeletal involvement.

263

a useful marker for differentiating IgA nephropathy from non-IgA nephropathy (Baldree et al., 1993). The mean binding capacity of plasma IgA for Fn is significantly higher in patients with IgA nephropathy compared with healthy controls (Davin et al., 1991). This enhancement might be attributed to the increase in the concentrations of the circulating macromolecular IgA, which correlate significantly with plasma concentrations of IgA-Fn complexes. Because an increased Fn binding by IgA is also observed in patients with alcoholic liver cirrhosis without renal impairment, and there is no apparent correlation between concentrations of IgA, IgA-Fn complexes and the various biological variables in primary IgA glomerulonephritis, IgA-Fn complexes are thought to be formed from a normal binding process which is enhanced in IgA nephropathy, and these complexes are not responsible for renal injury (Davin et al., 1991). At this point, the role of IgA-Fn aggregates in the pathology of IgA nephropathy is not entirely clear (Cederholm et al., 1988).

REFERENCES Atta MS, Powell RJ, Hopkinson ND, Todd I. Human antifibronectin antibodies in systemic lupus erythematosus: occurrence and antigenic specificity. Clin Exp Immunol 1994a;96:20--25. Atta MS, Powell RJ, Todd I. The influence of antifibronectin antibodies on interactions involving extracellular matrix components and cells. Clin Exp Immunol 1994b;96:28--30. Atta MS, Lim KL, Ala'Aldeen DA, Powell RJ, Todd I. Investigation of the prevalence and clinical associations of antibodies to human fibronectin in systemic lupus erythematosus. Ann Rheum Dis 1995;54:117--124. Baldree LA, Wyatt RJ, Julian BA, Falk RJ, Jennette C. Immunoglobulin A-fibronectin aggregate levels in children and adults with immunoglobulin A nephropathy. Am J Kidney Dis 1993;22:1--4. Cederholm B, Wieslander J, Bygren P, Heinegard D. Circulating complexes containing IgA and fibronectin in patients with primary IgA nephropathy. Proc Natl Acad Sci USA 1988;85:4865-4868. Colvin RP. Fibronectin in wound healing. In: Mosher DF, editor. Fibronectin. San Diego: Academic Press, 1989:213255. Courtoy PJ, Timpl R, Farquhar MG. Comparative distribution of laminin, type IV collagen and fibronectin in the rat glomerulus. J Histochem Cytochem 1982;30:874--886. Davin JC, Vecchi ML, Nagy J, Foidart JM, Foidart JB, Barba264

CONCLUSION Anti-Fn antibodies are detectable in patients with rheumatic diseases such as SLE (29--34%), RA (14%) and morphea (Table 1). Evidence in support of these antibodies having pathological significance is provided by animal studies in which immunization with Fn causes the development of anti-Fn and renal damage. Furthermore, in vitro studies show the ability of antiFn to inhibit cell attachment and reduce collagen and Fn deposition on cell surfaces. Clinical studies in SLE patients suggest that anti-Fn are associated with active disease and correlate with markers of disease activity such as ESR and s-IL-2R levels. A consistent association is observed between the presence of anti-Fn and the presence of active musculoskeletal involvement in SLE patients. Further studies are required to confirm the clinical value of anti-Fn in predicting disease activity and active musculoskeletal involvement in SLE patients. IgA-Fn aggregates are found frequently (93%) in serum of patients with IgA nephropathy. However, whether these aggregates provide a useful serological marker to differentiate IgA nephropathy from other nephropathies is controversial.

gallo Sangiorgi G, Malaise M, Machieu P. Evidence that the interaction between circulating IgA and fibronectin is a normal process enhanced in primary IgA nephropathy. J Clin Immunol 1991;11:78--94. Girard D, Senecal JL. A novel human IgG3 ~: autoantibody reactive with the cell-binding domain of fibronectin inhibits cell-adhesion in vitro. Arthritis Rheum 1993a;36:S150. Girard D, Senecal JL. Characterization of a human IgG3 ~antifibronectin autoantibody. Clin Res 1993b;41:A370. Henane T, Rigal D, Monier JC. Antifibronectin autoantibodies in patients with systemic lupus erythematosus, rheumatoid polyarthritis and bacterial or viral infections. Pathol Biol (Paris) 1986;34:165-171. Jennette JC, Wieslander J, Tuttle R, Falk RJ. Serum IgAfibronectin aggregates in patients with IgA nephropathy and Henoch Schonlein purpura diagnostic value and pathogenic implications. Am J Kidney Dis 1991;18:466--471. Julian BA, Waldo FB, Rifai A, Mestecky J. IgA nephropathy, the most common glomerulonephritis worldwide. A neglected disease in the United States? Am J Med 1988;84:129--132. Kornblihtt AR, Umezawa K, Vibe-Pedersen K, Baralle FE. Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J 1985;4:1755-1759. Murphy-Ullrich JE, Oberley TD, Mosher DF. Detection of autoantibodies and glomerular injury in rabbits immunized with denatured human fibronectin monomer. Am J Pathol 1984;117:1--11.

Murphy-Ullrich JE, Oberley TD, Mosher DF. Serologic and pathologic studies of mice immunized with homologous fibronectin. Am J Pathol 1986;125:182-190. Peter JB, Hollingsworth PN, Dawkins RL, Delaney C, Thomas M, Jennette JC. Serologic diagnosis of IgA nephropathy: clinical utility of assay for IgA fibronectin aggregates. J Am Soc Nephrol 1990;1:565. Petersen TE, Skorstensgaard K, Vibe-Pedersen K. Primary structure of fibronectin. In: Mosher DF, ed. Fibronectin. San Diego: Academic Press, 1989:1--24. Proctor RA. Fibronectin: a brief overview of its structure, function and physiology. Rev Infect Dis 1987;9:317--321. Ruoslahti E. Fibronectin and its receptors. Annu Rev Biochem 1988;57:375--413. Shimizu Y, Shaw S. Lymphocyte interactions with the extracellular matrix. FASEB J 1991;5:2292--2299.

Shirakami A, Shigekiyo T, Hirai Y, Takeichi T, Kawauchi S, Saito Miyoshi K. Plasma fibronectin deficiency in eight members of one family. Lancet 1986;1:473-474. Skorstengaard K, Jensen MS, Sahl P, Petersen TE, Magnusson S. Complete primary structure of bovine plasma fibronectin. Eur J Biochem 1986;161:441-453. Stefanato CM, Gorkiewicz-Petkow A, Jarzabek-Chorzelska M, Jablonska S, Chorzelski T. Morphea with high titer of fibronectin antibodies. Int J Dermatol 1992;31:190-- 192. Watts CL, Bruce MC. Effect of dexamethasone therapy on fibronectin and albumin levels in lung secretions of infants with bronchopulmonary dysplasia. J Pediatr 1992;121:597607. Zanetti M, Takami T. Mesangial immune deposits induced in rats by antibodies to fibronectin. Clin Immunol Immunopathol 1984;31:353--363.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

56-kd NUCLEAR PROTEIN AUTOANTIBODIES Ruth Sperling, Ph.D. a and Joseph Sperling, Ph.D. b

aDepartment of Genetics, The Hebrew University of Jerusalem, Jerusalem 91904; and bDepartment of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

HISTORICAL NOTES

THE AUTOANTIGEN

Myositis, an idiopathic inflammatory myopathy, is characterized by an intense mononuclear cellular infiltration leading to muscle degeneration (Dalakas, 1991). Difficulties in diagnosis of myositis frequently arise due to the overlap of symptoms with other muscle diseases. Accurate diagnosis is important for treatment. Accumulated evidence supports the notion that myositis is a disorder of immune function (Bohan and Peter, 1975; Morrow and Isenberg, 1987); attempts to link myositis with a single dominant autoantibody have met with mixed success. Autoantibodies directed to certain proteins (including t-RNA synthetases) are commonly identified in myositis patients. Specific antibodies are present in only a small proportion (<30%), and sometimes only in a subgroup of myositis patients (Arad-Dann et al., 1989; Bakimer and Sperling, 1994; Miller, 1993). First observed in a survey of sera from patients with myositis and patients from the overlap groups (Arad-Dann et al., 1987a; 1987b), anti-56 kd autoantibodies detect specific antigenic polypeptide on immunoblots of mammalian nuclear ribonucleoprotein (RNP) particles (Sperling and Sperling, 1990). The anti-56 kd autoantibodies, which recognize a nuclear RNP protein of 56 kd, are present in 85% of patients with myositis. Furthermore, they are specific to the disease as they are not found in healthy individuals, nor in patients with other muscle diseases (Arad-Dann et al., 1987a; 1987b; 1989; Sperling, 1988; Cambridge et al., 1994).

Definition The 56-kd antigen is a component of a large complex of RNA and protein (Arad-Dann et al., 1987b), designated as large nuclear ribonucleoprotein (lnRNP) particles, which constitute the nuclear pre-mRNA processing machinery in mammalian cells (Sperling and Sperling, 1990). Important components of these particles are several U small nuclear RNP (U snRNP) particles, which are a group of conserved and abundant particles composed each of a small uridine-rich RNA molecule complexed with a set of proteins, among which the Sm antigen is abundant. The lnRNP particles contain the U1, U2, U4/U6 and U5 snRNPs, which are all required for pre-mRNA splicing, as well as heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and non-snRNP splicing factors (Miriami et al., 1995). Autoantibodies directed against some of these antigens are found in sera of patients with autoimmune rheumatic diseases (Ast et al., 1994; Sperling, 1992). As a component of the nuclear premRNA processing machinery, the anti-56 kd is expected to be a conserved protein, present in every tissue or cell type active in transcription and processing. Indeed, human anti-56 kd autoantibodies detect the antigen in Syrian hamster and mouse cell nuclei and in rat myogenic cell line as well as in HeLa cell nuclei (Arad-Dann et al., 1987b; and unpublished).

Origin/Sources With nuclear supernatants enriched for lnRNP particles as the source of protein (Arad-Dann et al., 1989;

266

1987b; Cambridge et al., 1994), immunoblots of sera from myositis patients identified the 56-kd protein. Some of the myositis patients' sera reacted only with the 56-kd protein by immunoblotting. When tested by indirect immunofluorescence on HEp2 cells, these specific sera exhibited a speckled nuclear pattern which is typical of antibodies to components of U snRNP (e.g., anti-Sm or anti-RNP antibodies) (Ast et al., 1994).

Methods of Purification The 56-kd antigen is isolated by immunoaffinity chromatography of lnRNP particle-enriched nuclear supernatants prepared from pure nuclei of Syrian hamster cells, HeLa cells or myogenic cell lines (Miriami et al., 1994; 1995). Typically, cells swollen in hypotonic buffer are lysed in a Dounce homogenizer. From the pure nuclei collected by sedimentation through a glycerol cushion, nuclear supernatants are released by mild sonication and enriched for lnRNP particles by precipitating the chromatin in the presence of excess tRNA. The 56-kd protein is further purified by immunoaffinity chromatography on a column of immobilized anti-56 kd autoantibodies.

detected by immunoblotting the total protein content of nuclear RNP extracts prepared from pure nuclei of Syrian hamster cells or from other mammalian cells after separation on polyacrylamide/SDS gels (AradDann et al., 1987b; 1989; Cambridge et al., 1994) (Figure 1). Samples containing 40 ~g of total proteins are precipitated with 10% trichloroacetic acid for 30 min on ice, washed twice with 5% trichloroacetic acid, redissolved in SDS gel sample buffer and heated for 3 min at 100~ After separation on 12% polyacrylamide/SDS gels, proteins are transferred to a nitrocellulose membrane by electrophoresis for 3 hrs at 160 mA and 4~ in a buffer containing 15 mM Tris-base and 115 mM glycine, pH 8.3. After incubation for 1 hr in a blocking solution consisting of 10% long-life low-fat milk, 0.02% Triton X- 100 and 0.01% NaN3 in phosphate-buffered saline (PBS), the blots are exposed to sera diluted 1:20 in PBS (containing 0.01% Triton X-100 and 0.01% NAN3), followed by incubation for 1--2 hr in heat-sealed plastic bags with gentle shaking. After washing three times for 10 min with blocking solu-

Affinity Purification of the 56-kd Antigen. The immunoglobulin fraction of anti-56 kd autoantibodies was prepared by ammonium sulfate precipitation and DE 52 fractionation. An immunoaffinity matrix was prepared by binding this anti-56 kd immunoglobulin fraction to cyanogen bromide-activated Sepharose 4B CL using a standard coupling protocol. Nuclear supernatant was diluted 16-fold with 10 mM Tris HC1, pH 7.9, 0.5 M NaC1, 2 mM PMSF and passed four times through a column containing an equal volume (packed beads) of the affinity matrix. The column was extensively washed with the above dilution buffer. Elution of the 56-kd protein was performed with 0.2 M acetic acid. Each fraction was neutralized with 1 M K2HPO 4 and the pure protein was dialyzed against PBS and concentrated in a Centricon microconcentrator. The identification of the eluted protein as the 56-kd protein was confirmed by immunoblotting.

AUTOANTIBODIES

Methods of Detection Anti-56 kd antibodies in patients' sera are routinely

Figure 1. Immunoblots of proteins from nuclear RNP extracts probed with sera from myositis patients. The antigenic source, gel fractionation and immunoblotting were as described by Arad-Dann et al., 1989. Lanes: 1) normal human serum; 2) JDM patient; 3 and 4) DM patients; 5 and 6) PM patients. 267

tion, the filters are probed with 1251 protein A (106 cpm/lane) in PBS containing 0.01% Triton X- 100 and 0.01% NaN 3 for 1 hr and then washed three times for 10 min with blocking solution, dried and autoradiographed. All the steps are carried out at room temperature. To determine the relative abundance of anti-56 kd antibodies at various stages of the disease, several bleeds of the same patient taken during the course of the disease are analyzed on the same gel. Quantitation of the anti-56 kd antibodies is performed by densitometry (Arad-Dann et al., 1989; Cambridge et al., 1994).

CLINICAL UTILITY Disease Associations

Sera analyzed from patients with polymyositis (PM) and dermatomyositis (DM), all of whom met the criteria for inflammatory muscle disease, were classified into five subgroups (Bohan and Peter 1975): Group I, primary idiopathic polymyositis; Group II, primary idiopathic dermatomyositis; Group III, DM (or PM) associated with neoplasia; Group IV, juvenile dermatomyositis (JDM) associated with vasculitides; Group V, PM or DM associated with other autoimmune rheumatic diseases (overlap group). The utility of the anti-56 kd antibodies as a diagnostic marker for myositis is now substantiated by study of a large number of patients from all five subgroups of the disease and from controls (Tables 1 and 2) (Arad-Dann et al., 1987a; 1987b; Cambridge et al., 1994; Sperling et al., unpublished). Anti-56 kd antibodies were present in 36 out of 43 patients with idiopathic PM, and in 11 out of 13 patients with idiopathic DM. Eleven out of the 13 patients with another rheumatic disorder (overlap group) and three out of four patients with myositis associated with an

underlying neoplasia expressed the anti-56 kd antibodies. Finally, in juvenile dermatomyositis, 24 out of 26 patients had antibodies to the 56-kd antigen (AradDann et al., 1987b; 1989; Sperling, 1988; Cambridge et al., 1994; Sperling, unpublished data). The overall sensitivity of the anti-56 kd antibodies is thus 85%. The specificity of anti-56 kd antibodies for myositis was tested by screening the following control groups. Patients with other myopathies: multiple sclerosis with proximal muscle wasting (two patients), myasthenia gravis (four patients), muscular dystrophy (three patients), steroid myopathy, glycogen storage disease and idiopathic scoliosis with proximal muscle weakness (one patient each). Out of these 12 patients, none had the anti-56 kd antibodies. Of patients with other autoimmune rheumatic diseases, only two out of 30 patients with SLE without myositis had anti-56 kd antibodies; only one out of 12 patients with rheumatoid arthritis and two out of six patients with scleroderma responded weakly to the 56-kd antigen. Finally, none of 50 healthy individuals (five pools each of 10 sera) had anti-56 kd antibodies (no anti-56 kd antibodies were also observed with individual sera of healthy people). The occurrence of the anti-56 kd antibodies in females with PM, DM or PM with cancer, corresponds to the gender distribution of these diseases (female:male = 3:1). However, the percentage of women carrying the antibodies among patients with PM and another autoimmune rheumatic diseases is higher (female:male = 9:1). In JDM, there appears to be no discrimination between females and males (Table 3) (Arad-Dann et al., 1987a; 1987b; Cambridge et al., 1994; Sperling et al., unpublished). Correlation with Disease Activity

All adults with myositis and anti-56 kd antibodies had creatine kinase (CK) values greater than three times

Table 1. Sensitivity of Anti-56 kd Autoantibodies Diagnosis

Number Tested

Number (%) Anti-56 kd Autoantibodies

Idiopathic polymyositis

43

36 (84)

Idiopathic dermatomyositis

13

11 (85)

4

3 (75)

Myositis and other autoimmune diseases

13

11 (85)

Juvenile dermatomyositis

26

24 (92)

Total

99

85 (86)

Myositis in patients with cancer

268

Table 2. Specificity of Anti-56 kd Autoantibodies

Diagnosis

Number Tested

Number (%) with Anti-56 kd Autoantibodies

Other myopathies

12

0 (0)

SLE without myositis

30

2 (6.6)

6

2 (33)*

Rheumatoid arthritis

12

1 (83)*

Healthy individuals

50

0

Scleroderma

Total

110

5 (95)

*Weak positives.

normal, suggesting that the concentrations of anti-56 kd antibodies correlate with disease activity (AradDann et al., 1989). In serial sera obtained from two patients during the course of their disease, the anti-56 kd activity reflected the clinical activity of the myositis (Arad-Dann et al., 1989). Because fluctuating levels of anti-56 kd antibodies in adult myositis correlate with disease activity, it is important to test for the antibodies at a few time points during suspected myositis, in order to confirm the diagnosis (Arad-Dann et al., 1989). Disease Associations in C h i l d r e n

The presence of anti-56 kd antibodies is especially notable in childhood DM which is typically quite distinct from adult-onset disease due to fixed contraction deformities and calcinosis, absence of underlying neoplasms and histologic presence of distinctive vascular lesions (Cambridge, 1984). In addition, although approximately 30% of patients with adult onset-polymyositis have Jo-1 antibodies, these are very rarely found in children with the disease (Miller et al., 1993). Furthermore, studies correlating anti-56 kd anti-

bodies in childhood with DM, with disease activity and with the presence of circulating antinuclear antibodies (ANA) by indirect immunofluorescence, led to the classification of JDM into two categories (Cambridge et al., 1994). Group 1 comprises those patients with both ANA and anti-56 kd antibodies in whom there is a significant correlation between amount of anti-56 kd antibodies and disease activity. This was true for individual patients and also in study of serial sera from several patients. Group 2 comprises those patients with anti-56 kd antibodies whose sera are ANA-negative and in whom there is no observed correlation between disease activity and antibody titer. In most of the childhood cases, the 56-kd antigen was the only nuclear protein recognized by immunoblotting. However, only antibodies from sera of Group 1 displayed positive ANA by immunofluorescence; whereas, Group 2 was negative. If the ANA signal is due to binding to the 56-kd protein, it is possible that the anti-56 kd antibodies in the two groups recognize different epitopes of the protein. Follow-up studies of severity and prognosis of the disease in the two groups are needed.

Table 3. Occurrence of Anti-56 kd Autoantibodies in Female and Male Patients

Category with Anti-56 kd Antibodies

Females (%)

Males (%)

Polymyositis

81

19

Dermatomyositis

75

25

Myositis + Cancer

66

34

Myositis + Other Diseases

90

10

Juvenile Dermatomyositis

50

50

269

CONCLUSION Antibodies which recognize a specific 56-kd nuclear RNP protein are associated with myositis. Patients with every type of myositis are capable of expressing anti-56 antibodies. Anti-Jo-1 antibodies which are present in 30% of patients with adult onset of PM are generally considered the most prevalent of the disease-specific autoantibodies in myositis (Miller et al., 1993). Anti-56 kd antibodies, however, are found in patients with myositis much more frequently than any of the previously described disease-specific autoanti-

REFERENCES Arad-Dann H, Isenberg DA, Often D, Sperling J, Sperling R, Shoenfeld Y. Anti-56 kDa nuclear RNP antibody in myositis. Arthritis Rheum 1987a;30:549. Arad-Dann H, Isenberg DA, Shoenfeld Y, Often D, Sperling J, Sperling R. Autoantibodies against a specific nuclear protein in sera of patients with autoimmune rheumatic diseases associated with myositis. J Immunol 1987b;138:2463-2468. Arad-Dann H, Isenberg D, Ovadia E, Shoenfeld Y, Sperling J, Sperling R. Autoantibodies against a nuclear 56 kDa protein: a marker for inflammatory muscle disease. J Autoimmun 1989;2:877--888. Ast G, Goldblatt D, Waisman A, Sperling R, Mozes E, Sperling J. An autoantibody derived from mice with experimental systemic lupus erythematosus is directed against the essential splicing factor SF53/4: a possible role for large nuclear ribonucleoprotein particles in autoimmune disorders. Int Immunol 1994;6:1097-1105. Bakimer R, Sperling R. Myositis diagnosis- the importance of serology. Isr J Med Sci 1994;30:917--919. Bohan A, Peter JB. Polymyositis and dermatomyositis. N Engl J Med 1975;292:344--347. Cambridge G. Aetiology and pathogenesis of inflammatory muscle disease. Clin Exp Rheumatol 1984;2:263--269. Cambridge G, Ovadia E, Isenberg DA, Dubowitz V, Sperling J, Sperling R. Juvenile dermatomyositis: serial studies of

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bodies. On average, they are present in 85% of all five subtypes of adult myositis patients and in 92% of JDM cases. Furthermore, the specificity with which nonmyositis sera recognize the 56-kd antigen is 95%. In adults and in Group 1 of JDM, the amount of anti56 kd autoantibodies correlates with the severity of the disease. These autoantibodies can therefore be used as a reliable marker for the differential diagnosis of myositis. See also AMINOACYL-TRNA HISTYDL (JO-1) SYNTHETASE AUTOANTIBODIES, PM-SCL AUTOANTIBODIES and SIGNAL RECOGNITION PARTICLE AUTOANTIBODIES.

circulating autoantibodies to a 56-kd nuclear protein. Clin Exp Rheumatol 1994;12:451--457. Dalakas MC. Polymyositis, dermatomyositis, and inclusionbody myositis. N Engl J Med 1991;325:1487--1498. Miller FW. Myositis-specific autoantibodies. Touchstones for understanding the inflammatory myopathies. JAMA 1993; 270:1846--1849. Miriami E, Sperling J, Sperling R. Heat shock affects 5' splice site selection, cleavage and ligation of CAD pre-mRNA in hamster cells, but not its packaging in InRNP particles. Nucleic Acids Res 1994;22:3084--3091. Miriami E, Angenitzki M, Sperling R, Sperling J. Magnesium cations are required for the association of U snRNPs and SRP proteins with pre-mRNA in 200S large nuclear ribonucleoprotein (lnRNP) particles. J Mol Biol 1995;246:254-263. Morrow WJ, Isenberg DA, eds. Autoimmune Rheumatic Diseases. London: Blackwell Scientific Publication, 1987. Sperling R. Autoantibodies against nuclear ribonucleoprotein (RNP) complexes. Isr J Med Sci 1988;24:358-360. Sperling R, Sperling J. Large nuclear RNP particles of specific polymerase II transcripts. In: Strauss PR, Wilson SH, eds. The Eukaryotic Nucleus, Molecular Biochemistry and Macromolecular Assemblies, Volume 2. New Jersey: Telford Press, 1990:453--476. Sperling R. Autoantibodies against protein factors of the nuclear RNA processing apparatus. Isr J Med Sci 1992;28:120--123.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

FILAGGRIN (KERATIN) AUTOANTIBODIES Guy Serre, M.D., Ph.D. and Christian Vincent, M.D.

Department of Biology and Pathology of the Cell Purpan Medical School University of Toulouse, 31059 Toulouse Cedex, France

HISTORICAL NOTES Since the description of serum autoantibodiesto the superficial layers of the cornified epithelium of rat esophagus (antikeratin antibodies, AKA) in rheumatoid arthritis (RA) in 1979 (Young et al., 1979) the diagnostic value has been repeatedly confirmed. These autoantibodies, which are also present in about onethird of rheumatoid factor (RF)-negative RA, are linked with the biological and clinical parameters that classically define the more active and severe forms of the disease (Scott et al., 1981; Johnson et al., 1981; Miossec et al., 1982; Quismorio et al., 1983; Mallya et al., 1983; Kirstein and Mathiesen 1987; Vincent et al, 1989; Hoet and van Venrooij, 1992; Youinou and Serre, 1995). These antibodies can precede the clinical onset of RA (Kurki et al., 1992; Paimela et al., 1992).

THE AUTOANTIGEN(S)

Definition/Characteristics Because keratins (or cytokeratins) are the major protein components of the stratum corneum (Galvin et al., 1989), the RA-specific antibodies reactive with rat esophagus were named "antikeratin" despite the absence of any immunochemical evidence for such a specificity. At least four features indicate that human epidermal cytokeratins are not a target of AKA. (1) Presaturation of rat esophagus cryosections with a rabbit antiserum to human epidermal cytokeratins does not remove or decrease the reactivity of RA sera to the stratum corneum. (2) Preabsorption of RA sera on human epidermal cytokeratins does not modify their subsequent reactivity to the stratum corneum of rat

esophagus (Quismorio et al., 1983). (3) The titers of natural IgG autoantibodies to human epidermal cytokeratins assayed by a specific ELISA and the titers of AKA'determined on a large series of RA sera are entirely independent (Vincent et al., 1991); in addition, cytokeratins extracted from rat esophagus epithelium are not (Hoet et al., 1991). Finally, (4) immunoblotting reactivity of RA sera with cytokeratins extracted either from the stratum corneum or the rat esophagus epithelium is generally weak or absent and always unrelated to the AKA activity of the sera (Serre et al., unpublished; Hoet et al., 1991; Girbal et al., 1993).

Human Epidermis Filaggrin. The protein filaggrin is now known to be the human epidermal target of AKA (Simon et al., 1993). A 40-kd protein, extracted from human epidermis and specifically immunodetected by AKA-positive RA sera, was purified and identified as a neutral/acidic isoform of basic filaggrin by peptide mapping studies and by the following evidence: 1) monoclonal antibodies specific for filaggrin react with the 40-kd protein; 2) the autoantibodies, affinity-purified from RA sera on the 40kd protein, immunodetect purified filaggrin; 3) the reactivity of RA sera to the 40-kd protein is abolished after immunoadsorption with purified filaggrin; 4) the 40-kd protein and filaggrin have similar amino acid compositions; 5) AKA and autoantibodies against the 40-kd protein are largely the same by immunoadsorption experiments. Epidermal filaggrin, a 37,000 apparent molecular weight cationic protein, is synthesized in the granular layer of the epidermis as profilaggrin, a large, highly phosphorylated polyprotein precursor which is stored in keratohyalin granules and consists of tandemly arranged repeats of filaggrin units separated by linker

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peptides. During later stages of differentiation, profilaggrin is dephosphorylated and proteolytically converted to functional basic filaggrin molecules. Both filaggrin and profilaggrin are rich in basic amino acids and serine, but are devoid of methionine and sulfur; they were originally called "histidine-rich proteins" because they contain 11--12% histidine (LonsdaleEccles et al., 1984; Dale et al., 1990). After its function in cytokeratin-filament aggregation is fulfilled, basic arginine residues of filaggrin are partly modified by a peptidylarginine deiminase to neutral acidic residues resulting in a lower affinity of the more acidic molecule to cytokeratins (Harding and Scott, 1983). Epidermal filaggrin shows two sequential functions: in lower cornified cells, basic filaggrin interacts with and aligns cytokeratin filaments into macrofibrils, promoting the formation of disulfide bonds between cytokeratins. These densely packed filaments and filaggrins form the dense cellular matrix of the corneocytes (Steinert et al., 1981; Manabe et al., 1991). In addition to this structural role as an intermediate filament aggregating protein in upper cornified cells, filaggrin is completely proteolyzed into free amino acids and their derivatives such as urocanic and pyrrolidone carboxylic acids. The high concentration of these components in the intracellular space of the superficial cornified layers plays a key role in maintaining a high osmotic pressure and absorbing ultraviolet light (Scott et al., 1988). Finally, filaggrin may have a third function as a precursor of cornified envelopes (Richards et a1.,1988; personal data).

Commercial Sources At present, filaggrin, its neutral/acidic isoform and its precursor profilaggrin are not commercially available.

Differentiation Molecules of Rat Esophagus Epithelium Related to (Pro)filaggrin. By immunoblotting with RA sera on different extracts of rat esophagus epithelium separated by various one- and twodimensional electrophoreses, three low-salt-soluble antigens sensitive to proteinase K and, therefore, of protein nature are the target of AKA (Girbal and Sebbag et al., 1993). Two proteins, with apparent molecular masses of 210 and 120--90 kd, share isoelectric points ranging from 5.8 to 8.5; the third protein exhibits isoelectric points from 4.5 to 7.2 and its molecular mass ranges from 130 to 60 kd. Immunoadsorption of RA serum samples onto cytokera272

tins extracted from the stratum corneum of rat esophagus epithelium does not change their immunoreactivity with the three antigenic proteins. Widely used deglycosylation and dephosphorylation methods fail to modify either the electrophoretic migration of the proteins or their immunoreactivity with RA serum samples. These proteins differ from rat epidermal (pro)filaggrin and may correspond to a tissue-specific variant of this molecule. The cellular function of these proteins is unknown. If their relationship to (pro)filaggrin is confirmed, their cellular function will probably resemble that of epidermal (pro)filaggrin.

Sequence Information Human Antigen. The isolation and characterization of profilaggrin cDNAs and the profilaggrin gene (Presland et al., 1992; Markova et al., 1993) reveal a polyprotein precursor formed by a large number of tandemly arranged filaggrin units 324 amino acid long (972 bp). The profilaggrin mRNA, 13 kb long, is transcribed in the granular layer from three exons, the first of which is untranslated. Profilaggrin gene is located at chromosome region l q21, is highly polymorphic and contains 10, 11 or 12 repeats in humans; neighboring repeats display as much as 20% difference in amino acid sequences, with resultant heterogeneity of the filaggrin isoelectric point (7.2 to 9.4). The deduced amino- and carboxy-terminal sequences of profilaggrin show that the filaggrin repeats are flanked by truncated units fused to non-filaggrin proteins. The amino-terminal peptide shows sequence similarity to calcium-binding proteins carrying EFhand domains, e.g., calmodulin; profilaggrin is, indeed, a functional calcium-binding protein. Finally, the 5' region of the gene reveals many putative regulatory sequences such as the retinoic acid response element.

AUTOANTIBODIES

Names: synonyms, preferred terminology* Name Abbreviation Antikeratin antibodies AKA Antibodies to cornified layer of rat esophagus Antistratum corneum antibodies ASCA Antirat esophagus antibodies AREA Antifilaggrin autoantibodies* AFA

Pathogenetic Role To date, no direct evidence for a pathogenetic role of AKA in RA has been obtained. Although the presence of AKA is not strictly related to the HLA-DR4 phenotype (Hoet and van Venrooij, 1992), AKA titer is significantly higher in RA patients with DR4/DR4, DR4/DR1 or DR1/DR1 genotype than in patients with none of these risk alleles (Serre et al., unpublished).

Factors in Pathogenicity Classes, Subclasses, Epitopes. Although some human sera contain IgM, which react with the stratum corneum of rat esophagus, only the corresponding IgG are specific for RA (Vincent et al., 1989). Although the IgG subclass profile of AKA shows interindividual heterogeneity, AKA reactivity is IgG1 subclass in 87% and IgG4 subclass in 35% of AKA-positive RA sera. The profiles IgG1 (alone) or IgG (1+4) are largely predominant (Vincent et al., 1990). The sequence and timeframes for appearance of the isotypes remain to be studied. The epitopes recognized by AKA on the various epithelial protein antigens are unknown. Methods of Detection Indirect Immunofluorescence (IIF)-Rat Esophagus Epithelium (Figure 1). Indirect immunofluorescence was the first (Young et al., 1979) and, until recently, the only method available to detect AKA (Hoet and van Venrooij, 1992). Cryosections (4 to 6 ~M) preferably from the middle-third of rat esophagus are used

Figure 1. Indirect immunofluorescence on rat esophagus: typical linear-laminated labeling pattern produced by a rheumatoid serum with a high AKA titer.

without any previous chemical fixation. Sera tested at 1/10 dilution are considered positive or negative for AKA based on a subjective threshold of fluorescence intensity. More generally used is the classical dilutiontitration method which allows the titer to be determined at the point of extinction of fluorescence. A semiquantitative evaluation of fluorescence intensity at 1/10 dilution allows a titer-like value to be determined. Although it requires some training, the latter method saves time and reagent and permits use of an objective threshold of fluorescence intensity which is related to predetermined diagnostic indices (Serre et a1.,1986; Vincent et a1.,1989). The typical labeling pattern of AKA corresponds to a linear-laminated labeling restricted to the upper cornified layer, the stratum corneum, of the epithelium. All the other histological patterns of labeling, including those which involve both the stratum corneum and the other epithelial layers, have no specificity for RA. Because only IgG AKA are specific for RA (Vincent et al., 1989), the use of a secondary antibody highly specific for IgG is recommended to avoid a decrease in diagnostic specificity. I I F - H u m a n Epidermis. Human epidermis is a cornified, stratified stratum squamous epitheliumsimilar to rat esophagus epithelium. In small series of typical high-titered RA sera (Scott et al., 1981; Quismorio et al., 1983) and later with large series of RA and non-RA sera (Serre et al., 1986; Simon et al., 1995), AKA were shown to label the stratum corneum of human epidermis. Nevertheless, this tissue substrate also detects the natural IgG autoantibodies to epidermal cytokeratins which produce a simultaneous labeling of the stratum corneum and the suprabasal layers of epidermis (Serre et al., 1987). Because these natural autoantibodies are present with large interindividual variations of titer in all human sera, normal (Serre et al., 1987) or not (Serre et al., 1985), the labeling pattern produced on epidermis by an AKApositive RA serum always corresponds to a superimposition of the two families of antibodies. Therefore, human skin is not suitable for the detection of AKA. l I F - O t h e r Animal Epithelia. Various mucous membranes with cornified squamous epithelium such as rat lip, rabbit and monkey esophagus, were tested with small series of RA sera and found suitable for detection of AKA. However, because all results in the international literature were obtained with rat esophagus, that tissue remains the reference substrate.

273

Immunoblotting Detection. Detection of AKA by immunoblotting with rat esophagus protein antigens is possible (Gombs-Daudrix et al., 1994). Separation of the antigens by polyacrylamide gel electrophoresis in nondenaturing conditions clearly isolates the three molecules (Girbal and Sebbag et al., 1993). In the same way, after separation by one-dimensional sodium dodecylsulphate-polyacrylamide gel electrophoresis, either the neutral/acidic water-soluble isoform (Figure 2) or the basic urea-soluble human epidermal filaggrin can be used for the detection of filaggrin autoantibodies (Simon et al., 1993). Indirect immunofluorescent detection of autoantibodies to the rat esophagus protein antigens and detection of autoantibodies to filaggrin are highly significantly correlated witheach other. Nevertheless, overlapping of the results of the three assays is not absolute (Serre et al., unpublished). Other Techniques. To date, no other immunochemical techniques to detect AKA, such as ELISA or immunoprecipitation, have been published.

CLINICAL UTILITY

Disease Association The diagnostic value of AKA detection by indirect immunofluorescence has been assessed by 12 research groups in 4,080 patients including 1,694 with RA. Overall, the diagnostic sensitivity varies from 55 to 40% when the diagnostic specificity varies from 95 to 99%. The relatively low diagnostic sensitivity of AKA detection implies that a negative result does not exclude the diagnosis of RA, regardless of the preva-

lence of the disease in the population studied. By contrast, the high diagnostic specificitysuggests a high positive predictive value. Moreover, as reported by the large majority of investigators and in contrast to rheumatoid factor, AKA are found as rarely in patients with non-RA inflammatory rheumatic diseases as in patients with noninflammatory rheumatic diseases (Hoet and van Venrooij, 1992). Therefore, in a population of patients with inflammatory rheumatic diseases where the prevalence of RA is 0.30 or more, the posttest probability of RA could reach 0.98. The presence or the titer of AKA are not related to the age or gender of patients. Similarly, the presence of AKA is independent of disease duration (Vincent et al., 1989). Moreover, the presence of AKA can precede the clinical onset of the disease, sometimes by several years (Kurki et al., 1992; Paimela et al., 1992). Although AKA titers can vary in the course of the disease evolution (Serre et al., unpublished), the possible effect of various therapies on the presence or the titer of AKA is unknown. The presence of a significant titer of AKA, even if found only once in the disease course, is sufficient to define the patient as "AKA-positive", because the diagnostic value of the test seems to be independent of the long-term persistence of the antibodies in the serum. AKA are also potential prognostic markers. Indeed, the presence and the titer of AKA are related to several serological indices of disease activity and/or severity such as erythrocyte sedimentation rate, increased levels of C-reactive protein RF, or soluble immune complexes. Their association with functional indexes and subcutaneous nodules also suggests that the presence of AKA probably defines more severe forms of RA (Hoet and van Venrooij, 1992). More-

Figure 2. A series of rheumatoid sera with increasing AKA titers, detected by immunoblotting on the neutral/acidic isoform of human epidermal filaggrin, following one-dimensional SDS-PAGE separation. C: control; MW: molecular weight. 274

over, although AKA and RF are statistically correlated with each other; the autoantibodies do not define the same RA subpopulation since AKA are present, sometimes with high titers, in one-third of the RF-negative RA patients. The large majority of the studies on the diagnostic value of A K A detection was performed by European research groups and, thus, mainly on Caucasian populations. Whether the prevalence of AKA varies in genetically different populations is unknown.

CONCLUSION Recently, AKA and antiperinuclear factor (APF), another diagnostic marker of RA, were shown to be the same autoantibodies that recognize human epidermal filaggrin and (pro)filaggrin-related proteins of

REFERENCES Dale BA, Resing KA, Haydock PV. Filaggrins. In: Goldman RD, Steinert PM, eds. Cellular and Molecular Biology of Intermediate Filaments. New York: Plenum Press, 1990: 393--412. Galvin S, Loomis C, Manabe M, Dhouailly D, Sun TT. The major pathways of keratinocyte differentiation as defined by keratin expression: an overview. Adv Dermatol 1989;4:277299. Girbal E, Sebbag M, Gom~s-Daudrix V, Simon M, Vincent C, Serre G. Characterisation of the rat oesophagus epithelium antigens defined by the so-called "antikeratin antibodies," specific for rheumatoid arthritis. Ann Rheum Dis 1993;52: 749--757. Gom~s-Daudrix V, Sebbag M, Girbal E, Vincent C, Simon M, Rakotoarivony J, Abbal M, Fourni6 B, Serre G. Immunoblotting detection of so-called "antikeratin antibodies": a new assay for the diagnosis of rheumatoid arthritis. Ann Rheum Dis 1994;53:735--742. Harding CR, Scott IR. Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation. J Mol Biol 1983;170:651--673. Hoet RM, Boerbooms AM, Arends M, Ruiter DJ, van Venrooij WJ. Antiperinuclear factor, a marker autoantibody for rheumatoid arthritis: colocalization of the perinuclear factor and profilaggrin. Ann Rheum Dis 1991;50:611--618. Hoet RM, Van Venrooij WJ. The antiperinuclear factor (APF) and antikeratin antibodies (AKA) in Rheumatoid Arthritis. In: Smolen JS, Kalden JR, Maini RN, eds. Rheumatoid Arthritis. Berlin, Heidelberg: Springer Verlag Publishers, 1992:299318. Johnson GD, Carvalho A, Holborow EJ, Goddard DH, Russell G. Antiperinuclear factor and keratin antibodies in rheumatoid arthritis. Ann Rheum Dis 1981;40:263--266. Kirstein H, and Mathiesen FK. Antikeratin antibodies in

buccal epithelial cells (Sebbag et al., 1995). The nature of the antigen that drives the antifilaggrin response is not yet known. It could be involved in the onset and/or the pathophysiology of RA, because AKA and APF appear before its clinical manifestations. Although filaggrin might be this antigen, it is not considered to be expressed by synoviocytes or chondrocytes. Alternatively, the antifilaggrin-autoantibodies-inducing antigen may be a cross-reactive molecule expressed by these cells. Characterization of the epitopes defined by antifilaggrin autoantibodies on filaggrin and on a hypothetical articular autoantigen might help to elucidate RA etiology and open the way to preventive and/or specific immunosuppressive therapeutics. See also PERINUCLEAR FACTOR (PROFILAGGRIN) AUTOANTIBODIES.

rheumatoid arthritis. Methods and clinical significance. Scand J Rheumatol 1987;16:331--338. Kurki P, Aho K, Palosuo T, Heliovaara M. Immunopathology of rheumatoid arthritis. Antikeratin antibodies precede the clinical disease. Arthritis Rheum 1992;35:914-917. Lonsdale-Eccles JD, Resing KA, Meek RL, Dale BA. Highmolecular-weight precursors of epidermal filaggrin and hypothesis for its tandem repeating structure. Biochemistry 1984;23:1239-1245. Mallya RK, Young BJJ, Pepys MB, Hamblin TJ, Mace BE, Hamilton EB. Antikeratin antibodies in rheumatoid arthritis: frequency and correlation with other features of the disease. Clin Exp Immunol 1983;51:17-20. Manabe M, Sanchez M, Sun TT, Dale BA. Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and ichthyosis vulgaris. Differentiation 1991;48:43--50. Markova N, Marekov LN, Chipev CC, Gan SQ, Idler WW, Steinert PM. Profilaggrin is a major epidermal calciumbinding protein. Mol Cell Biol 1993;13:613-625. Miossec P, Youinou P, Le Goff P, Moineau MP. Clinical relevance of antikeratin antibodies in rheumatoid arthritis. Clin Rheumatol 1982;1:185-- 189. Paimela L, Gripenberg M, Kurki P, Leirisalo-Repo M. Antikeratin antibodies: diagnostic and prognostic markers for early rheumatoid arthritis. Ann Rheum Dis 1992;51:743--746. Presland RB, Haydock PV, Fleckman P, Nirunsukrisi W, Dale BA. Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100like calcium binding domain at the amino terminus. J Biol Chem 1992;267:23772-23781. Quismorio FP, Kaufman RL, Beardmore T, Mongan ES. Reactivity of serum antibodies to the keratin layer of rat esophagus in patients with rheumatoid arthritis. Arthritis Rheum 1983;26:494-499. Richards S, Scott IR, Harding CR, Liddell JE, Powell GM, 275

Curtis CG. Evidence for filaggrin as a component of the cell envelope of the newborn rat. Biochem J 1988;253:153-160. Scott DL, Delamere JP, Jones LJ, Walton KW. Significance of laminar antikeratin antibodies to rat oesophagus in rheumatoid arthritis. Ann Rheum Dis 1981 ;40:267-271. Scott IR, Richards S, Harding C, Liddell JE, Curtis CG. Does catabolism of stratum corneum proteins yield functionally active molecules? Ann NY Acad Sci 1988;548:125--136. Sebbag M, Simon M, Vincent C, Masson-Bessiere C, Girbal E, Durieux JJ, Serre G. The antiperinuclear factor and the socalled "antikeratin antibodies" are the same rheumatoid arthritis-specific autoantibodies. J Clin Invest 1995;95:26722679. Serre G, Vincent C, Viraben R, Soleilhavoup JP. Autoantibodies to keratins in normal human sera, systemic l u p u s erythematosus, rheumatoid arthritis and psoriasis. Eur J Clin Invest 1985;115:$36. Serre G, Vincent C, Fourni6 B, Lapeyre F, Soleilhavoup JP, Fourni6 A. Antistratum corneum antibody in the rat esophagus, antiepidermal keratin and antiepidermis autoantibodies in rheumatoid polyarthritis and other rheumatic diseases. Diagnostic value and basic aspects. Rev Rhum Mal Osteoartic 1986;53:607--614. Serre G, Vincent C, Viraben R, Soleilhavoup JP. Natural IgM and IgG autoantibodies to epidermal keratins in normal human sera. I: ELISA titration, immunofluorescence study. J Invest Dermatol 1987;88:21--27. Simon M, Girbal E, Sebbag M, Gom~s-Daudrix V, Vincent C, Salama G, Serre G. The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis. J Clin Invest 1993;92:1387-1393.

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Simon M, Vincent C, Haftek M, Girbal E, Sebbag M, GombsDaudrix V, Serre G. The rheumatoid arthritis-associated autoantibodies to filaggrin label the fibrous matrix of the cornified cells but not the profilaggrin-containing keratohyalin granules in human epidermis. Clin Exp Immunol 1995; 100:90-98. Steinert PM, Cantieri JS, Teller DC, Lonsdale-Eccles JD, Dale B A. Characterization of a class of cationic proteins that specifically interact with intermediate filaments. Proc Natl Acad Sci USA 1981;78:4097-4101. Vincent C, Serre G, Lapeyre F, Fourni6 B, Ayrolles C, Fourni6 A, Soleilhavoup JP. High diagnostic value in rheumatoid arthritis of antibodies to the stratum corneum of rat oesophagus epithelium, so-called 'antikeratin antibodies'. Ann Rheum Dis 1989;48:712--722. Vincent C, Serre G, Basile JP, Lestra HC, Girbal E, Sebbag M, Soleihavoup JP. Subclass distribution of IgG antibodies to the rat oesophagus stratum corneum (so-called antikeratin antibodies) in rheumatoid arthritis. Clin Exp Immunol 1990;81:83--89. Vincent C, Serre G, Fourni6 B, Fourni6 A, Soleilhavoup JP. Natural IgG to epidermal cytokeratins vs IgG to the stratum corneum of the rat oesophagus epithelium, so-called 'antikeratin antibodies', in rheumatoid arthritis and other rheumatic diseases. J Autoimmun 1991;4:493-505. Youinou P, Serre G. The antiperinuclear factor and antikeratin antibody systems. Int Arch Allergy Immunol 1995;107:508-518. Young BJ, Mallya RK, Leslie RD, Clark CJ, Hamblin TJ. Antikeratin antibodies in rheumatoid arthritis. Br Med J 1979; 2:97--99.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

GANGLIOSIDE AUTOANTIBODIES Hugh J. Willison, Ph.D., M.B.B.S.

Department of Neurology, University of Glasgow, Southern General Hospital, Glasgow G51 4TF, UK

HISTORICAL NOTES Over the last 15 years, antibodies reactive with a wide range of gangliosides were recognized in sera of patients with peripheral nerve disorders. IgM paraproteins reactive with myelin-associated glycoprotein (anti-MAG) were found in patients with IgM paraproteinemic neuropathy; these also react with a novel sulphated glucuronic acid-containing glycosphingolipid termed "sulphated glucuronyl paragloboside (SGPG)" (Latov, 1990). Some patients with IgM paraproteinemic neuropathy are known to be anti-SGPGnegative but are positive for antibodies reactive with various other glycolipid and ganglioside antigens (Thomas and Willison, 1994). After anti-GM 1ganglioside IgM were recognized in a high proportion of patients with a syndrome termed "multifocal motor neuropathy (MMN)", research on the association between anti-GM 1 antibodies and other motor nerve syndromes intensified (Pestronk, 1991). At the same time, antiganglioside antibodies were identified in Guillain-Barr6 syndrome (GBS) (Ilyas et al., 1988); this area of research remains highly topical (Willison and Kennedy, 1993; Hartung et al., 1995).

THE AUTOANTIGEN(S) Nomenclature

Gangliosides are sialic acid containing glycosphingolipids composed of a long-chain aliphatic amine (ceramide) attached to between one and five hexoses, at least one of which must be sialylated (Table 1). The presence of sialic acid molecule(s) attached to galactose residue(s) in the hexose core defines a glycosphingolipid as a ganglioside. In the human

nervous system, the sialic acid is N-acetylneuraminic acid, often abbreviated to NeuNAc, NeuAc or NANA. In ganglioside nomenclature (IUPAC-IUB Commission, 1977), the prefix G refers to ganglio; M, D, T and Q refer to the number of sialic acid molecules (mono, di, tri and quad); and the Arabic numerals and lower case letters refer to the order of migration of the gangliosides on thin layer chromatograms (TLC). In general, higher molecular weight gangliosides, having a longer oligosaccharide core and more sialic acid residues, migrate more slowly than the smaller gangliosides which will run closer to the solvent front on TLC. Methods of Purification

Although total synthesis of some gangliosides, such as GD 3, is possible (Ishida et al., 1993), most clinical investigators either extract and purify gangliosides from bovine brain or purchase the purified gangliosides from commercial sources. Different tissues vary widely in their ganglioside composition; the nervous system is highly enriched and is, therefore, the usual source for extraction. Gangliosides purified from bovine brain by chloroform/methanol extraction and DEAE-Sephadex chromatography (Ledeen and Yu, 1982) are characterized by chemical and enzymatic derivatization, thin layer chromatography and more sophisticated techniques such as fast atom bombardment mass spectrometry and high performance liquid chromatography. The major gangliosides in brain are GM 1, GDla, GDlb and GTlb but there are also many minor gangliosides in brain, peripheral nerves and other tissues. Ganglioside patterns vary widely across different species at different developmental stages with considerable functional diversity (Tettamanti and Riboni, 1993).

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Table 1. Ganglioside Structures Name

Carbohydrate Sequence

GM 1

Gal (131-3) GalNAc (131-4) Gal ([31-4) Glc (131-1) Ceramide (~2-3) NeuNAc

Asialo-GM 1 (GA1)

Gal (131-3) GalNAc (131-4) Gal (131-4) Glc (131-1) Ceramide

GM 2

GalNAc (131-4) Gal (131-4) Glc (131-1) Ceramide (~2-3) NeuNAc

GD1a

Gal (131-3) GalNAc (131-4) Gal (131-4) Glc (131-1)Ceramide (~2-3) (~2-3) NeuNAc NeuNAc

GD1b

Gal (131-3) GalNAc (131-4) Gal (131-4) Glc (131-1)Ceramide (~2-3) NeuNAc (~2-8) NeuNAc

GD 2

GalNAc (131-4) Gal (131-4) Glc (131-1) Ceramide (c~2-3) NeuNAc (~2-8) NeuNAc

GD 3

Gal (131-4) Glc (131-1) Ceramide (c~2-3) NeuNAc (c~2-8) NeuNAc

GTlb

Gal (131-3) GalNAc (131-4) Gal (131-4) Glc (131-1) Ceramide (o~2-3) (~2-3) NeuNAc NeuNAc (~2-8) NeuNAc

GTla

Gal (131-3) GalNAc (131-4) Gal (131-4) Glc (131-1) Ceramide (~2-3) (~2-3) NeuNAc NeuNAc (c~2-8) NeuNAc

GQlb

Gal (131-3) GalNAc (131-4) Gal (131-4) Glc (131-1) Ceramide (~2-3) (o~2-3) NeuNAc NeuNAc (c~2-8) (~2-8) NeuNAc NeuNAc

LM 1 (= SPG)

Gal (131-3) GalNAc (131-4) Gal (131-4) Glc (131-1) Ceramide (~2-3) NeuNAc

Commercial Sources Major gangliosides can be purchased from a variety of

278

.companies; rare gangliosides should be sought by personal requests to relevant investigators. Many preparations are only partially pure; for example

commercial preparations of GTla and GTlb are often contaminated by significant amounts of GQ1 b and vice versa. Care should be taken to control for this in nondiscriminatory immunoassays such as ELISAs or dot blots by also using TLC overlay.

AUTOANTIBODIES Terminology Antiganglioside autoantibodies are referred to by their specificity, either in terms of individual gangliosides (e.g., anti-GM 2 antibodies) or in terms of the reactive carbohydrate epitope (e.g., anti-Gal(131-3)GalNAc antibodies). When generalizing, the term "antiglycolipid antibodies" is more appropriate than "antiganglioside antibodies," because many neuropathy-associated autoantibodies react with glycolipids such as SGPG, sulphatide, asialo-GM 1 and galactocerebroside, which are not gangliosides. Antibodies to these glycolipids are discussed elsewhere (Thomas and Willison, 1994; Hartung et al., 1995).

Pathogenetic Role The evidence that antiglycolipid and antiganglioside antibodies have a pathogenetic role in neuropathy is controversial for several reasons (Lange and Trojaborg, 1994; Parry, 1994). Firstly, clinically identical syndromes may or may not be associated with a particular antibody marker. Secondly, antiganglioside antibodies are often present in disease-free individuals. Thirdly, positive evidence from animal models produced across different laboratories is still lacking. Despite these caveats, there is strong circumstantial evidence from clinical data in support of a central role for antiganglioside antibodies in pathogenesis. Many of the animal studies have focussed on anti-GM a antibodies. Following intraneural injection or perfusion of anti-GMl-positive sera in rodent nerve and nerve muscle preparations, nerve conduction block is observed in some (Uncini et al., 1993; Arasaki et al., 1993; Roberts et al., 1995) but not all studies (Harvey et al., 1995). Anti-GQlb antibody-positive sera from Miller-Fisher syndrome patients (MFS, a variant of GBS) blocks nerve-evoked transmission in the mouse phrenic nerve hemidiaphragm preparation (Roberts et al., 1994). The mechanism(s) by which this takes place is unknown. In addition to activation of inflammatory pathways, antiganglioside antibodies might act

pharmacologically by perturbing membrane ion channel function directly (Takigawa et al., 1995).

Genetics V gene sequencing studies of human anti-GM 1 and asialo-GM 1 IgM antibodies cloned from peripheral blood of affected patients (Weng et al., 1992; Willison et al., 1994a) show no particular restriction in V gene usage apart from a possible preferential usage of the VH3 gene Hv3005, but only small numbers of antibodies were studied (Weng et al., 1992; Paterson et al., 1995). The high degree of somatic mutation of all the sequenced antibodies suggests antigen-driven mechanisms. Because antibody responses to pure carbohydrate structures are not known to be processed through the MHC class II pathway and therefore cannot be presented to T cells, there are no data on TCR usage and very little interpreted data on HLA associations in antiganglioside antibody-associated peripheral neuropathy.

Factors in Pathogenicity/Etiology In the chronic peripheral neuropathy syndromes, the antiganglioside antibodies are invariably IgM, either occurring as paraproteins (so-called monoclonal gammopathies of undetermined significance, MGUS) or as polyclonal antibodies (Thomas and Willison, 1994). For example, isoelectric focusing of affinitypurified multifocal motor .neuropathy sera has shown that the anti-GM 1 IgM antibodies are generally present as polyclonal IgM with or without a proportion of the anti-GM 1 being resolvable as monoclonal peaks of IgM (Willison et al., 1994a). These data suggest that monoclonal peaks of IgM may arise out of a background of polyclonal IgM, possibly because the antigen that stimulates anti-GM 1 antibody production drives vulnerable B cells to a state of autonomous proliferation. Very rarely, chronically elevated IgG antiganglioside antibodies are associated with chronic syndromes (Garcia Guijo et al., 1992; Kornberg et al., 1994). In GBS which is an acute onset, spontaneously remitting neuropathy, the clinical illness often starts 10-14 days following an infectious illness in a temporal pattern consistent with a primary immune response to that infection (McFarlin, 1990). In GBS, the antiganglioside antibodies (which are present in up to 50% of cases) are often at highest titer in the IgG class, with lower and more rapidly falling titers in the IgM and IgA classes. Although the IgG antibodies

279

disappear over ensuing months concomitant with clinical recovery, occasionally persistent elevations after recovery raise doubts about relevance to the disease. The IgG subclasses of both anti-GM 1 antibodies in GBS and anti-GQ1 b antibodies in MFS are restricted to IgG1 and IgG3, subclasses traditionally associated with T-cell dependent responses to protein antigens (Willison and Veitch, 1994). Lower amounts of IgG2 antibodies may be seen. Molecular mimicry between gangliosides and microbial carbohydrate antigens, particularly bacterial lipopolysaccharides (LPS) might be clinically relevant. For example, GBS is often preceded by Campylobacter jejuni enteritis (Kaldor and Speed, 1984), and LPS from C. jejuni isolates belonging to the Penner 19 serotype derived from an anti-GM 1 antibody-positive GBS patient bear the GM 1 ganglioside-like tetrasaccharide structure, Gal~ 1-3GalNAcl31-4(NeuAca2-3)Gal~ 1 (Yuki et al., 1993). A GDla-like epitope is also present on a Penner 4 C. jejuni isolate (Yuki et al., 1994a). Sera from patients with anti-GM 1 antibodypositive GBS and MMN bind certain C. jejuni LPS (Wirguin et al., 1994). Likewise, LPS in C. jejuni isolates from MFS cases are recognized by anti-GQ1 b antibodies (Yuki et al., 1994b; Jacobs et al., 1995). Antiganglioside antibodies are now thought to arise fortuitously as part of an immune response directed against bacterial LPS and possibly other, as yet unidentified, microbial carbohydrate antigens.

will therefore be removed at least in part from the polystyrene plate). Two percent bovine serum albumin (BSA) is a suitable blocking agent, although human serum albumin can be substituted in situations where test sera contain anti-BSA antibodies. Assay results are usually reported as titers calculated by end point analysis. A normal range must be established for each laboratory, because in common with many other autoantibodies, antiganglioside and antiglycolipid antibodies are part of the normal autoantibody repertoire and are found at low titer in both normal and disease control sera (Willison and Kennedy, 1993). A positive control sample producing a known OD should be used with each test plate or a quality control measure. These factors are discussed at length in many papers on the subject (Zielasek et al., 1994). In addition to ELISA, the thin layer chromatography overlay (TLC overlay) technique is described in detail and widely used (Willison et al., 1993). Although not readily amenable to quantitative titration, TLC overlay has the advantage of allowing unambiguous identity of a particular glycolipid and is thus considered the gold standard method. Most centers use a combination of enzyme-linked immunosorbent assay (ELISA) with thin layer chromatography overlay (TLC-overlay) as a confirmatory test.

Methods of Detection

Application

Although detectable by a variety of methods, ELISA is the principle screening method for antiganglioside antibodies. Despite some attempts to recommend standard methodology, most laboratories employ inhouse protocols. In two multicenter comparative studies, there was good (>90%) agreement on clearly positive or negative cases but variable results with borderline samples (Marcus et al., 1989; Zielasek et al., 1994). Important factors in setting up the ELISA include: 1) the choice of ELISA plates, including batch variations and storage conditions (screen plates from different manufacturers for optimal signal-tonoise ratio and lowest coefficients of variance across the plate using a standard positive control serum); 2) the temperature at which the assay is performed, 4~ being most commonly recommended; 3) the duration of serum incubation which should be at least 4 hours; and 4) the presence or absence of Tween 20 as a detergent (gangliosides are soluble in detergent and

Detection of antiganglioside antibodies is not absolutely essential for diagnosis of a particular subtype of peripheral neuropathy, because independent investigation, principally electrodiagnostic tests in the context of an appropriate clinical picture and exclusion of other causes is usually adequate. However, detection of antiganglioside antibodies can be useful to: 1) confirm a diagnosis, such as multifocal motor neuropathy or Miller-Fisher syndrome (MFS); 2) to subclassify an existing diagnosis such as IgM paraproteinemic neuropathy into those with anti-NeuAc(a2-8)NeuAc activity or anti-SGPG activity, etc.; and 3) to exclude or differentiate among several possible diagnoses such as botulism, brain stem demyelinating disease and MFS.

280

CLINICAL UTILITY

Disease Associations Well-defined antiglycolipid antibody specificities are

associated with several clinical syndromes (Table 2). An increasingly recognized paraproteinemic neuropathy syndrome is a chronic large fiber sensory neuropathy with prominent ataxia (Ilyas et al., 1985; Willison et al., 1993). The IgM paraprotein reacts with gangliosides beating NeuNAc(c~2-8)NeuNAc-linked disialosyl groups, including but not limited to GD 3, GDlb, GTla, GTlb and GQ1 b. The patients may also have cold agglutinin disease by virtue of the presence of sialylated glycoprotein epitopes on human red blood cells with which the IgM paraproteins can cross-react. Ophthalmoplegia is also variably present (Herron et al., 1994), reminiscent of MFS. In MFS, an acute selflimiting variant of GBS comprising ataxia, areflexia and ophthalmoplegia, anti-GQ1 b (Chiba et al., 1992) and anti-GTla IgG antibodies are present in over 90% of cases; the antibodies also react with GDlb and/or GD 3 in about half the cases (Willison et al., 1994b). In a pattern suggestive of a primary immune response, the antibodies are at their highest titer on presentation and tend to disappear in parallel with clinical recovery. The clinical syndrome is mainly a sensory neuropathy with ophthalmoplegia; transient IgG antibodies are associated with the acute remitting form and a persistently elevated IgM paraprotein with the chronic form of MFS. In multifocal motor neuropathy (MMN) with demyelinating conduction block, antibodies to GM 1 ganglioside are present in 50% (Kornberg and Pestronk, 1994). Early reports suggesting an important association between anti-GM 1 antibodies and motor neuron disease/amyotrophic lateral sclerosis appear to be incorrect. Patients with MMN and anti-GM 1 antibodies are mostly male and have a clinical picture

comprising a chronic asymmetric motor syndrome, usually with distal onset in an upper limb. In eight patients recently reported, the mean age of onset was 33 years and the mean duration was 15 years at which time all patients were still ambulant and physically independent (Roberts et al., 1995). The electrophysiological examination classically shows focal motor conduction block over at least one segment of peripheral nerve (Lewis et al., 1982); although strict diagnostic criteria were applied to this in the past, some flexibility is warranted in that some patients have more diffuse demyelinating conduction block or apparently pure axonal degeneration or a combination of these characteristics. All these clinical subtypes can be associated with anti-GM 1 antibodies. In chronic inflammatory demyelinating polyneuropathy (CIDP), anti-GM 1 antibodies may be found in up to 20% of cases (Simone et al., 1993). In GBS, a wide variety of antiganglioside and antiglycolipid antibodies are reported in up to 50% of cases in over 30 published series (Hartung et al., 1995; O'Leary and Willison, 1995); no unifying patterns have yet emerged. The significance of antiGM 1 antibodies in GBS remains uncertain. In some studies, anti-GM 1 antibodies mark a particularly severe form of the illness with prominent motor axonal involvement and poor recovery (Yuki et al., 1990; Nobile-Orazio et al., 1992), but this association is not detected by others (Vreisendorp et al., 1993; Enders et al., 1993).

Effect of Therapy Treatment

of

antiganglioside

antibody-mediated

Table 2. Peripheral Neuropathy Syndromes Associated with Antiganglioside Antibodies Clinical Syndrome

Antibody Isotype

Antibody Specificity

Antibody Frequency

Multifocal motor neuropathy

Monoclonal or polyclonal IgM

GM 1 and related Gal ([51-3) GalNAc bearing gangliosides (GDlb and GA1)

50%

Chronic large fiber sensory neuropathy with ataxia

Monoclonal IgM

NeuNAc (~2-8) NeuNAc epitopes (on GDlb,

100% (i.e., syndrome defined by antibody)

"MAG-negative" paraproteinemic neuropathy*

Monoclonal IgM

GM2, LM 1, GalNAc-GDlb, GalNAc-GDla and unknown unidentified glycolipids/gangliosides

Miller-Fisher syndrome

Polyclonal IgG, IgM and IgA

GQ1b and GTla

Guillain-Barr6 syndrome

Polyclonal IgG, IgM and IgA

GM l, GDla, LM l, GTlb, GDlb, GalNAc-GDla -50%

GTlb, GQlb, GD3)

>90%

*MAG-negative refers to the 50% of cases of IgM paraproteinemic neuropathy who are anti-MAG/anti-SGPG antibody-negative.

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neuropathy is generally determined by the clinical pattern of illness and degree of disability rather than the serological features. Acute GBS is treated with either plasma exchange or intravenous immunoglobulin (IVIg) according to protocols established in multicenter clinical trials (GBS Study Group, 1985; French Cooperative Group, 1987). Patients with MMN respond well, albeit temporarily, to IVIg (Chaudhry et al., 1993; Thornton and Griggs, 1994) which can be regularly repeated. Cyclophosphamide can also be effective (Feldman et al., 1991; Pestronk et al., 1994). In paraproteinemic neuropathy, treatment is more difficult to assess in view of the chronic nature of the diseases; again IVIg is increasingly used and other therapies include cytotoxic drugs such as chlorambucil and plasma exchange (Thornton and Griggs, 1994; Glass and Cornblatt, 1994). Steroid therapy has no known place in the management of either the acute or chronic syndromes associated with antiganglioside antibodies.

CONCLUSION In common with many antibody-associated autoimmune diseases, the precise relationship between autoantibodies and peripheral neuropathy is obscure. Some syndromes, such as MFS are very tightly associated with a particular antibody which appears likely to play a primary role in pathogenesis. In other diseases, such as MMN, the clinico-serological association (although less well defined) is nevertheless sufficiently strong to aid diagnosis and for research into pathogenesis. A large body of confusing evidence, particularly in Guillain-Barr6 syndrome suggests involvement of antiganglioside antibodies which as yet are not of value for aiding diagnosis or researching the cause.

REFERENCES Arasaki K, Kusonoki S, Kudo N, Kanazawa I. Acute conduction block in vitro following exposure to antiganglioside sera. Muscle Nerve 1993;16:587-593. Chaudhry V, Corse AM, Cornblath DR, Kuncl RW, Drachman DB, Freimer ML, Miller RG, Griffin JW. Multifocal motor neuropathy: response to human immune globulin. Ann Neurol 1993;33:237-242. Chiba A, Kusonoki S, Shimizu T, Kanazawa I. Serum IgG antibody to ganglioside GQ l b is a possible marker of Miller Fisher syndrome. Ann Neurol 1992;31:677-679. Enders U, Karch H, Toyka KV, Michels M, Zeilasek J, Pette M, Heesemann J, Hartung H-P. The spectrum of immune 282

Methodology for antibody detection presents a major difficulty, largely because the physical properties of gangliosides do not lend themselves well to development of uniform assays. ELISAs must be set up with great care and results confirmed with an independent method, ideally thin layer chromatography overlay; specimen protocols for these methods are widely available in the literature. A current overview that assumes a mechanistic role for antiganglioside antibodies is as follows. Low affinity, possibly polyreactive antibodies with specificity for carbohydrate antigens exist in the natural autoantibody repertoire with the primary function of acting as defense against lipopolysaccharide-bearing microbes; LPS molecules often bear structurally similar carbohydrate epitopes to gangliosides. During the course of bacterial infections, antigen-driven affinity maturation processes aimed at increasing antibody affinity for LPS may inadvertently drive these antibodies toward ganglioside reactivity. This may occur either as an acute process with recruitment of noncognate T-cell help, thereby generating a high affinity IgG response as seen in GBS, or as a chronic process, generating a lower affinity T-cell-independent IgM response, as seen in MMN. Antibodies thus derived would react with ganglioside antigens in nerve at sites to which they have access, possibly enhanced by T-cell- or cytokine-mediated disruption of the blood nerve barrier. In nerve, antibodies exert autopathogenic effects through either activation of proinflammatory pathways or pharmacological blockade of ganglioside-mediated physiological processes. This model is currently being investigated at many different levels. See also GLYCOLIPID (EXCLUDING GANGLIOSIDE) AUTOANTIBODIES and MYELIN-ASSOCIATED GLYCOPROTEIN AUTOANTIBODIES.

responses to Campylobacter jejuni and glycoconjugates in Guillain Barr6 syndrome and other neuroimmunological disorders. Ann Neurol 1993;34:136-- 144. Feldman EL, Bromberg MB, Albers JW, Pestonk A. Immunosuppressive treatment in mUltifocal motor neuropathy. Ann Neurol 1991;30:397-401. French Cooperative Group of Plasma Exchange in GuillainBarr6 Syndrome. Efficiency of plasma exchange in GuillainBarr6 syndrome: role of replacement fluids. Ann Neurol 1987:22;753-761. Garcia Guijo C, Garcia-Merino A, Rubio G, Guerrero A, Cruz Martinez A, Arpa J. IgG antiganglioside antibodies and their subclass distribution in two patients with acute and chronic motor neuropathy. J Neuroimmunol 1992;37:141-148.

Glass JD, Cornblath DR. Chronic inflammatory demyelinating polyneuropathy and paraproteinaemic neuropathy. Curr Opin Neurol 1994;7:393--397. Guillain-Barr6 Syndrome Study Group. Plasmapheresis and acute Guillain-Barr6 syndrome. Neurology 1985;35:1096-1104. Hartung H-P, Pollard JD, Harvey GK, Toyka KV. Immunopathogenesis and treatment of Guillain-Barr6 s y n d r o m e Parts 1 and 2. Muscle Nerve 1995;18:137--164. Harvey GK, Toyka KV, Zielasek J, Keifer R, Simonis C, Hartung H-P. Failure of anti-GM1 IgG or IgM to induce conduction block following intraneural transfer. Muscle Nerve 1995;18:388--394. Herron B, Willison HJ, Veitch J, Roelcke D, Illis LS, Boulton FE. Monoclonal IgM cold agglutinins with anti-Prl d specificity in a patient with peripheral neuropathy. Vox Sang 1994;67:58--64. Ilyas AA, Quarles RH, Dalakas MC, Fishman PH, Brady RO. Monoclonal IgM in a patient with paraproteineamic polyneuropathy binds to gangliosides containing disialosyl groups. Ann Neurol 1985;18:655--659. Ilyas AA, Willison HJ, Quarles RH, Jungalwala FB, Cornblath DR, Trapp BD, Griffin DE. Serum antibodies to gangliosides in Guillain-Barr6 syndrome. Ann Neurol 1988;23:440-447. Ishida H, Ohta Y, Tsukada Y, Kiso M, Hasegawa A. A synthetic approach to polysialogangliosides containing alphasialyl-(2-8)-sialic acid: total synthesis of ganglioside GD3. Carbohydr Res 1993;246:75-88. IUPAC-IUB Commission on Biochemical Nomenclature (CBN). The nomenclature of lipids. Eur J Biochem 1977;79:11-21. Jacobs BC, Endtz H, Van Der Meche FG, Hazenberg MP, Achtereekte HA, Van Doom PA. Serum anti-GQlb antibodies recognize surface epitopes on Campylobacterjejuni from patients with Miller Fisher syndrome. Ann Neurol 1995;37:260-264. Kaldor J, Speed BR. GBS and Campylobacterjejuni: a serological study. Br Med J 1984;288:1867--1870. Kornberg AJ, Pestronk A, Bieser K, Ho TW, McKhann GM, Wu HS, Ziang Z. The clinical correlates of high titre IgG anti-GM1 antibodies. Ann Neurol 1994;35:234-237. Kornberg AJ, Pestronk A. The clinical and diagnostic role of anti-GM 1 testing. Muscle Nerve 1994; 17:100-104. Lange DJ, Trojaborg W. Do anti-GM1 antibodies induce demyelination? Muscle Nerve 1994; 17:105-- 107. Latov N. Antibodies to glycoconjugates in neurological disease. Clinical Aspects of Autoimmunity 1990;4:18--29. Ledeen RW, Yu RK. Gangliosides: structure, isolation and analysis. In: Ginsburg V, ed. Methods In Enzymology. New York: Academic Press, 1982;83:139-191. Lewis RA, Sumner AJ, Brown MJ, Asbury AK. Multifocal demyelinating neuropathy with persistent conduction block. Neurology 1982;32:958--964. Marcus DM, Latov N, Hsi BP, Gillard BK. Measurement and significance of antibodies against GM1 ganglioside. J Neuroimmunol 1989;25:255--259. McFarlin DE. Immunological parameters in Guillain Barr6 syndrome. Ann Neurol 1990;29:$25-$29. Nobile-Orazio E, Carpo M, Meucci N, Grassi MP, Capitani E,

Sciacco M, Mangioni A, Scarlato G. Guillain Barr6 syndrome associated with high titers of anti-GM1 antibodies. J Neurol Sci 1992;109:200-206. O'Leary C, Willison HJ. Immunological Investigation. Curr Opin Neurol 1995;in press. Parry GJG. Antiganglioside antibodies do not necessarily play a role in multifocal motor neuropathy. Muscle Nerve 1994; 17:97--99. Paterson G, Wilson G, Kennedy PGE, Willison HJ. Analysis of anti-GM1 ganglioside IgM antibodies cloned from motor neuropathy patients demonstrates diverse variable region gene usage with extensive somatic mutation. J Immunol 1995;in press. Pestronk A. Invited review: motor neuropathies, motor neuron disorders and antiglycolipid antibodies. Muscle Nerve 1991 ;14:927--936. Pestronk A, Lopate G, Kornberg AJ, Elliott JL, Blume G, Yee W-C, Goodnough LT. Distal lower motor neuron syndrome with high titre serum IgM anti-GM 1 antibodies: improvement following immunotherapy with monthly plasma exchange and intravenous cyclophosphamide. Neurology 1994;44:20272031. Roberts M, Willison HJ, Vincent A, Newsom-Davis J. Serum factor in the Miller Fisher variant of Guillain Barr6 syndrome blocks neurotransmitter release. Lancet 1994;343:454--455. Roberts M, Willison HJ, Vincent A, Newsom-Davis J. Multifocal motor neuropathy human sera block distal motor nerve conduction in mice. Ann Neurol 1995;38:569--576. Simone IL, Annunziata P, Maimone D, Liguori M, Leante R, Livrea P. Serum and CSF anti-GM1 antibodies in patients with Guillain Barr6 syndrome and chronic inflammatory demyelinating polyneuropathy. J Neurol Sci 1993;114:49-55. Takigawa T, Yasuda H, Kikkawa R, Shigeta Y, Saida T, Kitasata H. Antibodies against GM1 affect K+ and Na + currents in isolated rat myelinated nerve fibres. Ann Neurol 1995;37:436-442. Tettemanti G, Riboni L. Gangliosides and modulation of function of neural cells. Adv Lipids Res 1993;25:235--267. Thomas PK, Willison HJ. Paraproteineamic neuropathies. In: McLeod EJ, ed. Inflammatory Neuropathies, Balliere's Clinical Neurology Series. London: Balliere Tindall, 1994. Thornton CA, Griggs RC. Plasma e~change and intravenous immunoglobulin treatment of neuromuscular disease. Ann Neurol 1994;35:260-268. Uncini A, Santoro M, Corbo M, Lugaresi A, Latov N. Conduction abnormalities induced by sera of patients with multifocal motor neuropathy and anti-GM1 antibodies. Muscle Nerve 1993;16:610-615. Vreisendorp FJ, Mishu B, Blaser MJ, Koski CL. Serum antibodies to GM1, GDlb, peripheral nerve myelin and Campylobacter jejuni in patients with Guillain Barr6 syndrome and controls: correlation and prognosis. Ann Neurol 1993;34:130-135. Weng N-P, Yu-Lee L-Y, Sanz I, Patten BM, Marcus DM. Structure and specificity of antiganglioside autoantibodies associated with motor neuropathies. J Immunol 1992;149: 2518. Willison HJ, Kennedy PGE. Gangliosides and bacterial toxins

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in Guillain Barr6 syndrome. J Neuroimmunol 1993;46:105-112. Willison HJ, Paterson G, Veitch J, Inglis G , Barnett sc. Peripheral neuropathy associated with anti-Pr2 cold agglutinins: biochemical and immunocytochemical studies. J Neurol Neurosurg Psychiatr 1993 ;56:1178-- 1184. Willison HJ, Veitch J. Immunoglobulin subclass distribution and binding characteristics of anti-GQ 1b antibodies in Miller Fisher syndrome. J Neuroimmunol 1994;50:159-165. Willison HJ, Paterson G, Kennedy PGE, Veitch J. Cloning of human anti-GM 1 antibodies from motor neuropathy patients. Ann Neurol 1994a;35:471--478. Willison HJ, Almemar A, Veitch J, Thrush D. Acute ataxic neuropathy with cross-reactive antibodies to GD 1b and GD3 gangliosides. Neurology 1994b;44:2395-2397. Wirguin I, Sunurkova-Milsevic L, Della-Latta P, Fisher T, Brown RHJ, Latov N. Monoclonal IgM antibodies to GM1 and asialo-GM1 in chronic neuropathies cross-react with Campylobacter jejuni lipopolysaccharides. Ann Neurol 1994;35:698--703. Yuki N, Yoshino H, Sato S, Miyatake T. Acute axonal poly-

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neuropathy associated with anti-GM1 antibodies following Campylobacter enteritis. Neurology 1990;40:1900-1902. Yuki N, Taki T, Inagaka F, Kasama T, Takahashi M, Saito K, Handa S, Miyatake T. A bacterium lipopolysaccharide that elicits Guillain-Barr6 syndrome has a GM1 ganglioside-like structure. J Exp Med 1993;178:1771--1775. Yuki N, Taki T, Takahashi M, Saito K, Tai T, Miyataki T, Handa S. Penner's serotype 4 of Campylobacterjejuni has a lipopolysaccharide that bears a GM1 epitope as well as one that bears a GD 1a epitope. Infect Immun 1994a;62:2101-2103. Yuki N, Taki T, Takahashi M, Saito K, Yoshino H, Tai T, Handa S, Miyataki T. Molecular mimicry between GQlb ganglioside and liposaccharides of Campylobacter jejuni isolated from patients with Fisher's syndrome. Ann Neurol 1994b;36:791-793. Zielasek J, Ritter G, Magi S, Hartung HP, Toyka KV, Participating Laboratories. A comparative trial of antiglycoconjugate antibody assays: IgM antibodies to GMI. J Neurol 1994 ;241: 475-480.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editor~.

GLIADIN ANTIBODIES Carlo Catassi, M.D.

Department of Pediatrics, University of Ancona, 60123 Ancona, Italy

HISTORICAL NOTES

Sequence Similarity

The presence of agglutinating antibodies against wheat gluten in the sera of patients with celiac disease (CD) was first reported in the late 1950s (Berger, 1958). The development of sensitive methods for measuring antibodies to gliadin (AGA), the ethanol-soluble fraction of gluten, led to the recognition of the validity of the AGA test as a screening tool for CD in the early 1980s (Unsworth et al., 1981). The widespread use of AGA and other serological tests, such as the antiendomysial antibody (AEA) test in recent years, shows that CD is not only more common than previously thought, but also that this disorder is characterized by a high degree of clinical variability.

The relationship between the antigenicity and toxicity of gluten components remains an open question. Several studies show that AGAs are directed against any of several gliadin fractions rather than a specific "celiac-reactive" gliadin fraction. The difference in AGA response between CD patients and controls is quantitative rather qualitative, i.e., low AGA titers with the same spectrum of reactivity can be found in normal sera (Levenson et al., 1985). On the other hand, ~-gliadin celiac nontoxic cereals share regions of sequence similarity. Indeed, monoclonal antibodies raised against a 54-amino acid peptide of ~-gliadin, which is thought to exacerbate CD, cross-react with celiac nontoxic cereal prolamins in rice, maize, millet and sorghum (Ellis et al., 1993). Monoclonal antibodies also cross-react with the 206--217 sequence of A-gliadin and the 54 kd Elb protein of adenovirus 12 (Ellis et al., 1992).

THE AUTOANTIGEN(S)

Definition/Origin Gliadin (molecular weight 16--40 kd) is a mixture of about 50 components. On the basis of electrophoretic mobility, gliadins can be divided into four major fractions: ~z-gliadins, [3-gliadins, y-gliadins, and cogliadins. In vivo and in vitro studies indicate that all these fractions, as well as the prolamins contained in oats, barley and rye, have toxic effects on CD patients (Ciclitira et al., 1984). A-gliadin, a component of ~gliadin of known primary amino acid sequence, contains 32 glutamines and 15 prolines per 100 amino acid residues (Kasarda et al., 1984). The PSQQ and QQQP sequences are thought to be present in all celiac-active peptides.

THE ANTIBODIES Pathogenetic Role Although increased serum titers of specific IgE, IgD and IgM against gliadin are found in untreated celiacs, only the serum IgG and IgA ACA response has been extensively investigated. Because the IgG-AGA belong mainly to the IgG1 and IgG3 subclasses, these antibodies are capable of complement fixation and cellular activation resulting in damage to the gut mucosa (Husby et al., 1986). However, animal studies show no pathological changes in the intestine suggestive of possible local antigen-antibody complex

285

formation or deposition (Smart et al., 1992). The high AGA titer in the serum of untreated CD patients might be a secondary event reflecting altered intestinal permeability. Serum IgA-AGA are more frequently monomeric and mainly belong to the IgA1 subclass (Volta et al., 1990). Concentrations of IgA and IgM are also increased in the jejunal fluid of CD subjects. Jejunal IgA-AGA are in the polymeric form and belong to IgA1 and IgA2 in equal proportion. They might be synthesized in loco by the plasma cells of the lamina propria (Volta et al., 1990). Significant amounts of IgM-AGA persist in the intestinal secretion of treated CD patients who show a normal jejunal histology (O'Mahony et al., 1991). The celiac-like intestinal pattern of IgA- and IgM-AGA might represent a marker of latent gluten-sensitive enteropathy (Arranz and Ferguson, 1993). Increased IgA-AGA levels are also found in the saliva of patients with active CD. Measurement of salivary IgA-AGA is reported to discriminate between children with CD and controls (Hakeem et al., 1992); this finding merits confirmation. Atypical and Silent Cases of CD. Serum AGA determination is also a reliable screening test for atypical or silent cases of CD, i.e., those cases of gluten-sensitive enteropathy with mild complaints, nonintestinal manifestations in apparently healthy subjects. Perhaps the most striking finding is that AGA screening can detect a large number of cases of CD in the general population. In a group of 3,351 Italian healthy students aged 11--15 years, 11 cases of CD were found by the determination of both IgGAGA and IgA-AGA on capillary blood samples as the first diagnostic step (Figure 1). The prevalence of subclinical celiac disease in that study group was 3.3 per 1,000, and the numbers detected by the AGA screening were five or six times greater than those who had previously received a clinical diagnosis. For a biopsy-proven CD diagnosis, the positive predictive value of a positive AGA test for screening was 15.5% (Catassi et al., 1994). Methods of Detection Methods used for AGA determination include indirect immunofluorescence (IIF), mixed reverse (solid phase) passive antiglobulin hemadsorption, several ELISA tests (enzyme immunoassay, fluorescence immunoassay, diffusion-in-gel ELISA) and a solid-phase radio-

286

immunoassay. In a direct comparison, IIF and ELISA give comparable results in screening celiac patients (Volta et al., 1985). Both methods are available as commercial kits. The AGA assay can be performed on a single drop of whole blood with a rapid, noninvasive strip test. In this test, purified a-gliadin absorbed as a spot onto nitrocellulose sheets that are immobilized on plastic strips (Not et al., 1993). The results of the AGA test can be expressed in different ways, such as the highest dilution giving a certain optical density (OD) in ELISA or in arbitrary units calculated as the percentage of the OD of a standard positive serum or a pool of highly positive sera (Troncone and Ferguson, 1991). An ELISA assay for measuring AGA in absolute units (microgram protein/ mL) is described (Perticarari et al., 1992). The use of a quantitative method could overcome problems of quality control in the preparation of kits and facilitate the comparison of the results of different studies. CD Diagnosis: Which Test Works Better? Although the comparison of CD diagnostic tests has conflicting results (McMillan et al., 1991; Lerner et al., 1994), the following points seem to be generally accepted: (1) the AGA assay is better than the 1-hr blood xylose test for CD screening due to its increased specificity and sensitivity for diagnosis of atypical cases (Lifschitz et al., 1989). Unlike the AGA and other immunological assays, the small intestine function tests (such as the blood xylose and the intestinal permeability tests) can give false-negative results because they reflect the extent of the mucosal damage, and this could be limited in atypical cases of CD; (2) no significant difference in sensitivity seems to exist between the combined determination of IgG and IgAAGA and the IgA antiendomysial antibody (AEA) assay as both tests approach a 95--100% value (Btirgin-Wolff et al., 1991). The AGA test could be preferable in the diagnostic work-up of children less than two years of age, since it becomes positive earlier than the AEA test (Btirgin-Wolff et al., 1991; Troncone and Ferguson, 1991). Other advantages of AGA determination are the low cost and the positivity of IgG-AGA in CD patients with selective IgA deficiency who lack the AEA, which is a class A antibody; (3) the AGA test has lower specificity compared to the AEA test, especially in disease controls (e.g., subjects with nonceliac enteropathies or with Down's syndrome). For this reason, many experts consider the AEA test as the best available serological marker of CD.

Figure 1. Diagnostic algorithm of CD screening in the general population.

CLINICAL UTILITY Disease Associations CD, also known as gluten-sensitive enteropathy (GSE), is characterized by permanent gluten intolerance leading to severe villous atrophy of the duodenum and jejunum (Report of Working Group, European Society of Paediatric Gastroenterology and Nutrition, 1990). In typical cases CD becomes clinically manifest during the first years of life with signs of malabsorption (e.g., failure to thrive, chronic diarrhea, vomiting and abdominal distension). Although the diagnosis of CD relies on the intestinal biopsy, the role of serum AGA determination in the diagnosis and follow-up of this condition is well

established (Guandalini et al., 1989). The reported sensitivity of the AGA assay for clinically suspected cases of CD is very high, ranging mostly between 95 and 100%. In two large European multicenter studies, 100% of children with active CD (untreated; always characterized by a flat mucosa at the intestinal biopsy) had serum IgG-AGA, while IgAAGA were detected in 89--90.5% of cases. Overestimation of the AGA sensitivity is possible, because a negative result in the AGA test was often taken as a criterion for avoiding the intestinal biopsy. Therefore, some AGA-false-negative CD patients probably went unnoticed. IgG-AGA were also detectable in --21% of subjects with other gastroenterological disorders (disease controls); whereas, IgA-AGA were only found in --3% of them (Btirgin-Wolff, 1989; Guan-

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dalini et al., 1989). Because IgG-AGA are more sensitive but less specific, and IgA-AGA are more specific but less sensitive; determination of both IgGAGA and IgA-AGA is usually recommended (BtirginWolff et al., 1989). CD is 10 times more common in subjects with selective IgA deficiency (Collin et a1.,1992), because patients with both selective IgA deficiency and C D lack IgA-AGA they can only be detected by screening with an IgG class antibody such as IgG-AGA.

celiacs are indicative of poor compliance to the glutenfree diet (GFD) remains to be proven. A common problem in treated CD patients is the ingestion of traces of "hidden" gluten in commercial food, such as ice cream or sausages. Recent data suggest that the AGA assay is not very sensitive indetecting minimal dietary transgressions, as the ingestion of 100 mg of gliadin per day for 4 weeks could not elicit a significant rise of AGA in most CD children (Catassi, 1993).

Genetics Effect of Therapy When gluten is withdrawn from the diet of the CD patient, the IgA-AGA titer decreases rapidly to normal values while the IgG-AGA decreases slowly and may persist at low titer for months or years. During a diagnostic gluten challenge, both IgG and IgA-AGA usually reach pathological values after some weeks or months of gluten ingestion (Bottaro et al., 1988). The assumption that persistent IgA-AGA in treated

Family Studies. The determination of serum AGA in first-degree relatives of CD patients has given conflicting results. Among 328 relatives, 21 were AGApositive and 13 of the 21 had celiac disease by intestinal biopsy (Corazza et al., 1992). In contrast to this evidence were results of intestinal biopsy of 122 relatives showing 13 had gluten-sensitive enteropathy, but the AGA test was positive in only half of these cases (Maki et al., 1991).

Table 1. Clinical Conditions Requiring AGA Determination. Intestinal disturbances

Extraintestinal disorders

Chronic/recurrent diarrhea

Iron-deficient anemia

Failure to thrive/malnutrition

Dermatitis herpetiformis

Vomiting

Insulin-dependent diabetes mellitus

Abdominal distension

Selective IgA deficiency

Recurrent abdominal pain

Short stature

Dental enamel hypoplasia

Pubertal delay

Recurrent apthous stomatitis

Epilepsy with cerebral calcifications

Anorexia

Mood disturbances

Elevated serum transaminases

Infertility

Constipation

Obstetrical problems (recurrent abortions, intrauterine growth retardation) Autoimmune thyroid disease Osteoporosis Down's syndrome Sj6gren's syndrome Sarcoidosis Dementia Psoriasis Malignancies (non-Hodgkin lymphoma, small intestine adenocarcinoma, pharynx and esophagus cancer)

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CD-Associated Diseases. A G A screening of large groups of patients with insulin-dependent diabetes mellitus (IDDM) shows that up to 4.6% of these subjects are also affected with a (usually) silent form of CD (Sigurs et al., 1993). However, many patients show only a transitory, nonspecific increase in AGA at the onset of IDDM (Catassi, 1987). Dermatitis herpetiformis is regarded as a glutensensitive disorder frequently associated with a typical celiac enteropathy. Increased A G A values are more commonly found in patients with dermatitis herpetiformis whose intestinal biopsies show villous atrophy (Kilander et al., 1985). The prevalence of CD is increased by up t o 5% in subjects with Down's syndrome. However, the specificity of the IgA-AGA in these patients is rather poor, since 26% of 155 children with D o w n ' s syndrome show increased levels of serum IgA-AGA (Castro et al., 1993). The reliability of the serological CD markers in patients affected with CD-associated malignancy is still unclear. Not one of 16 patients with enteropathy-associated T-cell lymphoma had raised levels of A G A (O'Farrelly et al., 1986).

Increased serum AGA, not always associated with a biopsy proven CD, are also reported in some patients with pemphigoid, psoriasis, atopic eczema, IgA mesangial nephropathy, rheumatoid arthritis, Sj6gren's syndrome, autoimmune thyroid disorders, HIV infection, cystic fibrosis, chronic liver diseases and sarcoidosis, but the clinical significance of these associations is dubious. A list of the clinical conditions where the A G A assay should be routinely included in the diagnostic work-up is shown in Table 1.

REFERENCES

ent effects of protracted ingestion of small amounts of gliadin in coeliac disease children: a clinical and jejunal morphometric study. Gut 1993;34:1515-1519. Catassi C, R~itsch IM, Fabiani E, Rossini M, Bordicchia F, Candela F, Coppa GV, Giorgi PL. Coeliac disease in the year 2000: exploring the iceberg. Lancet 1994;343:200--203. Ciclitira PJ, Evans DJ, Fagg NL, Lennox ES, Dowling RH. Clinical testing of gliadin fractions in coeliac patients. Clin Sci 1984;66:357-364. Collin P, M~iki M, Keyril~iinen O, H~illstrOm O, Reunala T, Pasternack A. Selective IgA deficiency and celiac disease. Scand J Gastroenterol 1992;27:367--371. Corazza G, Valentini RA, Frisoni M, Volta U, Corrao G, Bianchi FB, Gasbarrini G. Gliadin immune reactivity is associated with overt and latent enteropathy in relatives of celiac patients. Gastroenterology 1992;103:1517-- 1522. Ellis HJ, Doyle AP; Sturgess RP, Ciclitira PJ. Coeliac disease: characterisation of monoclonal antibodies raised against a synthetic peptide corresponding to amino acid residues 206--217 of antigliadin. Gut 1992;33:1504-1507. Ellis HJ, Doyle AP, Wieser H, Sturgess RP, Ciclitira PJ. Specificities of monoclonal antibodies to domain I of alphagliadins. Scand J Gastroenterol 1993;28:212-216. Guandalini S, Ventura A, Ansaldi N, Giunta AM, Greco L, Lazzari R, Mastella G, Rubino A. Diagnosis of coeliac disease: time for a change? Arch Dis Child 1989;64:13201325. Hakeem V, Fifield R, al Bayaty HF, Aldred MJ, Walker DM,

Arranz E, Ferguson A. Intestinal antibody pattern of celiac disease: occurrence in patients with normal jejunal biopsy histology. Gastroenterology 1993;104:1263--1272. Berger E. Zur allergischen pathogenese der Z61iakie. Bibliotheca Paediatrica 1958:67:1-55. Bottaro G, Sciacca A, Failla P, Cagnina M, Di Pietro MC, Ricca O, Iudica ML, Castiglione N, Patane R. Antigliadin antibodies in the various stages of celiac disease in children. Pediatr Med Chir 1988;10:409--413. Btirgin-Wolff A, Berger R, Gaze H, Huber H, Lentze MJ, Nussle D. IgG, IgA and IgE gliadin antibody determinations as screening test for untreated coeliac disease in children, a multicentre study. Eur J Pediatr 1989;148:496-502. Btirgin-Wolff A, Gaze H, Hadziselimovic F, et al. Antigliadin and antiendomysium antibody determination for celiac disease. Arch Dis Child 1991;66:941--947. Castro M, Crino A, Papadatou B, Purpura M, Giannotti A, Ferretti F, Colistro F, Mottola L, Digilio MC, Lucidi V, et al. Down's syndrome and celiac disease: the prevalence of high IgA-antigliadin antibodies and HLA-DR and DQ antigens i n trisomy 21. J Pediatr Gastroenterol Nutr 1993;16:265-268. Catassi C, Guerrieri A, Bartolotta E, Coppa GV, Giorgi PL. Antigliadin antibodies at onset of diabetes in children. Lancet 1987;2:158. Catassi C, Rossini M, R~itsch IM, Bearzi I, Santinelli A, Castagnani R, Pisani E, Coppa GV, Giorgi PL. Dose depend-

CONCLUSION The A G A assay is a valuable screening test for CD. The combined determination of IgG and IgA-AGA is recommended to maximize both sensitivity and specificity. A two-step procedure for CD screening seems advisable at present, with the A G A determination as the first-level test and the AEA test performed on AGA-positive cases. Suspected cases should receive an intestinal biopsy for the definitive diagnosis of CD. See also ENDOMYSIAL AUTOANTIBODIES and RETICULIN AUTOANTIBODIES.

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Williams J, Jenkins HR. Salivary IgA antigliadin antibody as a marker for coeliac disease. Arch Dis Child 1992;67:724727. Husby S, Foged N, Oxelius VA, Svehag SE. Serum IgG subclass antibodies to gliadin and other dietary antigens in children with coeliac disease. Clin Exp Immunol 1986;64: 526-535. Kasarda DD, Okita TW, Bernardin JE, Baecker PA, Nimmo CC, Lew EJ, Diertler MD, Greene FC. Nucleic acid (cDNA) and amino acid sequences of (x-type gliadins from wheat (Triticum aestivum L.). Proc Natl Acad Sci USA 1984;81: 4712-4716. Kilander AF, Gillberg RE, Kastrup W, Mobaken H, Nilsson LA. Serum antibodies to gliadin and small intestinal morphology in dermatitis herpetiformis. A controlled clinical study of the effect of treatment with a gluten-free diet. Scand J Gastroenterol 1985;20:951-958. Lerner A, Kumar V, Iancu TC. Immunological diagnosis of childhood coeliac disease: comparison between antigliadin, antireticulin and antiendomysial antibodies. Clin Exp Immunol 1994;95:78--82. Levenson SD, Austin RK, Dietler MD, Kasarda DD, Kagnoff MF. Specificity of antigliadin antibody in celiac disease. Gastroenterology 1985;89:1-5. Lifschitz CH, Polanco I, Lobb K. The urinary excretion of polyethlene glycol as a test for mucosal integrity in children with celiac disease: comparison with other noninvasive tests. J Pediatr Gastroenterol Nutr 1989;9:49-57. Maki M, Holm K, Lipsanen V, Hallstrom O, Viander M, Collin P, Savilahti E, Koskimies S. Serological markers and HLA genes among healthy first-degree relatives of patients with coeliac dlsease. Lancet 1991;338:1350-1353. McMillan SA, Haughton DJ, Biggart JD, Edgar JD, Porter KG, McNeill TA. Predictive value for coeliac disease of antibodies to gliadin, endomysium, and jejunum in patients attending the jejunal biopsy. Br Med J 1991;303:1163--1165.

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Not T, Ventura A, Perticarari S, Basile S, Torre G, Dragovic D. A new, rapid, non invasive screening test for celiac disease. J Pediatr 1993;123:425--427. O'Farrelly C, Feighery C, O'Brian DS, Stevens F, Connoly CE, McCarthy C, Weir DG. Humoral response to wheat protein in patients with coeliac disease and enteropathy associated T cell lymphoma. Br Med J 1986;293:908--910. O'Mahony S, Arranz E, Barton JR, Ferguson A. Dissociation between systemic and mucosal humoral immune responses in coeliac disease. Gut 1991;32:29-35. Perticarari S, Not T, Cauci S, Luchesi A, Presani G. ELISA method for quantitative measurement of IgA and IgG specific antigliadin antibodies. J Pediatr Gastroenterol Nutr 1992;15: 302--309. Report of Working Group, European Society of Paediatric Gastroenterology and Nutrition. Revised criteria for diagnosis of celiac disease. Arch Dis Child 1990;65:909-911. Sigurs N, Johansson C, Elfstrand PO, Viander M, Lanner A. Prevalence of coeliac disease in diabetic children and adolescents in Sweden. Acta Paediatr 1993;82:748--751. Smart CJ, Trejdosiewicz LK, Howdle PD. Specific circulating antigliadin IgG-class antibody does not mediate intestinal enteropathy in gliadin-fed mice. Int Arch Allergy Immunol 1992;97:160-166. Troncone R, Ferguson A. Antigliadin antibodies. J Pediatr Gastroenterol Nutr 1991; 12:150-158. Unsworth DJ, Manuel PD, Walker-Smith JA, Campbell CA, Johnson GD, Holborow EJ. New immunofluorescent blood test for gluten sensitivity. Arch Dis Child 1981 ;56:864-868. Volta U, Lenzi M, Lazzari R. Antibodies to gliadin detected by immunofluorescence and a micro-ELISA method: markers of active childhood and adult coeliac disease. Gut 1985;26: 667--671. Volta U, Molinaro N, Fratangelo D, Bianchi FB. IgA subclass antibodies to gliadin in serum and intestinal juice of patients with celiac disease. Clin Exp Immunol 1990;80:192--195.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

G L O M E R U L A R BASEMENT MEMBRANE AUTOANTIBODIES Thomas Hellmark, M.Sc. a, M~trten Segelmark, M.D., Ph.D. a, Per Bygren, M.D. a and J6rgen Wieslander, Ph.D. b

aDepartment of Nephrology, Lund University Hospital, S-221 85 Lunc# and bWieslab AB, S-233 70 Lund, Sweden

HISTORICAL NOTES In 1919 an American pathologist (Goodpasture, 1919) drew attention to the association of massive pulmonary hemorrhage with associated glomerulonephritis (GN). The name Goodpasture's syndrome was later used (Stanton and Tange, 1958) to describe a subset of patients presenting with acute or subacute renopulmonary syndromes of unknown etiology in recognition of Goodpasture's first report. In these patients, continuous linear deposition of immunoglobulins along their glomerular basement membrane (GBM) is demonstrable by direct immunofluorescence (Sheer and Grossman, 1964). The pathogenic role of these antibodies was later demonstrated in primates by transfer of the disease with serum or kidney-eluted antibodies (Lerner et al., 1967). The disease was then called Goodpasture's syndrome (GP) and characterized by lung hemorrhage, renal failure and anti-GBM antibodies. Less than one-third of the patients with reno-pulmonary syndromes have antibodies against GBM; the majority have either PR3-ANCA or MPOANCA.

THE AUTOANTIGEN(S) Definition Basement membranes are thin, sheet-like extracellular structures that form an anatomical barrier wherever cells meet connective tissue. They provide a substrate for organs and cells and relay important signals for the development of organs and for the differentiation and maintenance of the tissue. An additional function of GBM is ultrafiltration of blood. Composed of several specific molecules such as type IV collagen,

laminin, proteoglycans and entactin/nidogen, basement membranes are produced mainly by the endothelial cell layer. The GBM, which is thicker than other basement membranes, may be a fused membrane of endothelial and epithelial cells. Type IV collagen has self-aggregating properties and forms a matrix in which the other basement membrane molecules are integrated. Four type IV collagen molecules are connected in their N-terminal ends and two molecules interact in their C-terminal end forming a chicken wire-like network (Figure l a). Each type IV collagen molecule is furthermore composed of three subunits called ~ chains that are intertwined in a rope-like fashion (Figure l b) except for the C-terminal end where it is folded into a globular domain called NC1. Six ~(IV) chains are known and different basement membranes have a different composition of their ~(IV) chains (Hudson et al., 1993a). The c~I(IV) and cz2(IV) chains are found in most basement membranes; whereas, the c~3(IV) and ~4(IV) chains are found in some specialized membranes, for example the GBM. The (z5(IV) and c~6(IV) chains also have a limited tissue distribution. A network comprised of ~3(IV) and (z4(IV) found in the GBM (Johansson et al., 1992) provides the GBM with the strength and flexibility needed in this specialized basement membrane. The best known autoantigen in anti-GBM nephritis is the Goodpasture antigen (reviewed by Hudson et al., 1989; 1993b). In 1984, the antigen was isolated' and shown to be a 29 kd collagenase-resistant molecule of the GBM derived from the C-terminal end of type IV collagen (Wieslander et al., 1984a; 1984b), and was later shown to be localized to the NC1 domain of the (z3(IV) chain (Butkowski et al., 1987). The GP antigen is found in GBM, lung, lens, cochlea,

291

Figure 1. a: The type IV collagen network in which the other basement membrane components are integrated, b: An enlargement of one type IV collagen molecule. It is composed of three c~(IV) chains that are intertwined in a triple helix except in the C-terminal end, where each chain forms a globular domain (NC 1). e: A model of the NC 1 domain of the c~3(IV) chain. An epitope suggested by Kalluri et al., 1991 is indicated with shaded balls.

brain and testis (Kleppel et al., 1989). The majority of the antibodies are directed to ~3(IV) and most patients have a response to the other ~ (IV) chains, even though it is weaker than the response to the ~3(IV) chain. Many patients with elevated levels of anti~I(IV) also have anti-~4(IV), while anti-~2(IV) antibodies seem to be rare (Segelmark et al., 1990, Hellmark et al., 1994). In a typical GP patient, about 1% of the total IgG and almost 90% of the autoantibodies are directed to a specific epitope on the ~3(IV) chain. Ten percent of the autoantibodies are directed to cross-reactive epitopes on the other chains. Antibodies eluted from the kidneys of GP patients show the same specificity as circulating ones (Saxena et al., 1989b). Origin and Sources

The Goodpasture epitope is a cryptope, that is, the antibodies prefer a denatured structure to the native antigen. If the NC1 domain is isolated as a hexamer form, the reactivity of the antibodies is limited. Once the hexamer is dissociated into monomers and dimers, the epitope is exposed. However, if the disulfide bonds are reduced, all reactivity is lost. The autoantibodies do recognize the recombinant protein produced in E. coli (Neilson et al., 1993), but the reactivity is 25% compared to the antigen purified from human tissue. When expressed in a baculovirus system (Turner et al., 1994), the ~3(IV) chain yield is low but well recognized by Goodpasture antibodies. All six chains from type IV collagen are cloned

292

and sequenced, and the genes encoding for the ~ 1(IV) to the ~6(IV) chains are called COL4A1 to COL4A6, respectively (Hudson et al., 1993a). The gene for the GP antigen, i.e., the NC 1 domain of the ~3(IV) chain, is localized to chromosome 2 segment q36. There is one dominant epitope, and it is conformational. Nevertheless, a 36 amino acid-long synthetic peptide is proposed to contain the epitope (Figure lc) (Kalluri et al., 1991). Methods of Purification

GBM are isolated from kidney cortex by differential sieving followed by sonication to remove cell material. The resulting basement membrane is further purified by extraction with detergents and/or denaturing solutions, such as 6M guanadinium HC1 (gu HC1). After solubilization of the NC1 domains from type IV collagen from the GBM by bacterial collagenase digestion, further purification is done under nondenaturing conditions by ion exchange chromatography and gel filtration (Freytag et al., 1976, Hellmark et al., 1994). Collagenase treatment of the basement membrane releases the NC1 in a hexamer form, that is, the NC1 domains from two collagen molecules connected via their C-terminal ends. Solubilized NC1 hexamer treated with denaturing agents like 6M gu HC1 dissociates into monomers (the NC1 domain from one single ~[IV] chain) and dimers (two cross-linked c~[IV] chains from two different molecules). These can easily be separated from each other by gel filtration under denaturing conditions. The monomers or

dimers can be further separated and purified by reversed-phase HPLC in acetonitrile gradients (Butkowski et al., 1985). The 150 kd protein entactin/nidogen can be prepared from the crude 6M gu HC1 extract of GBM with chromatographic methods as described before (Saxena et al., 1990).

AUTOANTIBODIES Definitions Antibodies to glomerular basement membrane (antiGBM), are sometimes termed Goodpasture-antibodies (GP antibodies) (Turner et al., 1993). Non-GP anti-GBM are antibodies to other a chains of type (IV) collagen than o~3(IV) and antibodies to entactin or laminin, etc.

Pathogenetic Role The ability of anti-GBM to cause disease was demonstrated and Koch's postulate fulfilled with a now classic transfer experiment (Lerner et al., 1967). Primates developed GN after injection of autoantibodies eluted from the kidneys of a nephrectomized patient suffering from anti-GBM disease. Indirect proof of the pathogenic potential of the antibodies was given by the reappearance of disease in a renal transplant given to a patient with persistent high levels of circulating anti-GBM. Temporal relationships between relapse and recurrence of autoantibodies are also documented. The titer of circulating anti-GBM, as measured by ELISA, has prognostic importance (Herody et al., 1993). No spontaneous anti-GBM disease is known to occur in laboratory animals and many animals do not develop GN when challenged with basement membrane. In the passive nephrotoxic serum nephritis model, rabbits are immunized with renal cortex (Masugi, 1934). The rabbits are unaffected, but when their serum is injected into rats, a disease with two distinct phases can be observed. In the first heterologous phase, complement-dependent neutrophil invasion is seen. The second phase occurs as a consequence of the immune reaction to the rabbit IgG fixed to GBM. This phase is macrophage dependent and can be accelerated by preimmunization of recipients with rabbit IgG. In the active immunization model first described in

sheep using heterologous GBM and Freund's complete adjuvant (Steblay, 1962), the sheep recognize the same epitope(s) as patients with human disease. More recently, an active model was established in certain strains of rats using bovine GBM. When matrix from the basement membrane-producing cell line EHS (which is very low in the o~3(IV)) was used as antigen, antibodies to type IV collagen were produced, but no disease was detected (Bolton et al., 1995). A self-limiting disease with anti-GBM and glomerulonephritis can be seen in Brown Norway rats injected with mercuric chloride. However, these animals also exhibit autoantibodies with other specificities including ANA and MPO-ANCA.

Genetics Genetic studies reveal a strong link between antiGBM disease and HLA-DR2. RFLP (Restricted Fragment Length Polymorphism) technique studies indicate an association with the haplotypes DRwl5(DR2) DQw6(DQwl) and DR4(DQw7) (Bums et al., 1995). HLA-B7 also seems to be over-represented and patients expressing this antigen were shown to have a more severe renal disease. Anti-GBM disease is reported in pairs of identical twins, siblings and first cousins. Although anti-GBM are generally of IgG isotype/ IgG1 subclass, IgA anti-GBM are recognized. Some patients also have a substantial IgG4 titer (Segelmark et al., 1990). In contrast to IgG1, the reoccurrence of a high IgG4 anti-GBM-titer is not associated with a relapse, suggesting that the subclass composition of the autoantibodies may have importance. Although many reports link the onset of Goodpasture's syndrome with infections, nothing is known concerning specific organisms. Anti-GBM are normally polyclonal. A patient with a monoclonal lambda IgG3 directed to the NC1 portion of the c~l(IV) did not have a progressive GN despite high antibody titers in contrast to patients with polyclonal reactivity to the ~3(IV) chain (Johansson et all, 1993). The evidence concerning T-cell participation in the immune response in anti-GBM disease is only circumstantial. The autoantibody lgG subclass distribution is compatible with a T-cell-mediated reaction toward a protein antigen. Factors that block the interaction between T cells and antigen-presenting cells attenuate antibody response and disease expression in experimental animals (Nishikawa et al., 1994). A similar effect is seen after the administration of the

293

T-cell inhibitor cyclosporin A. A mononuclear interstitial cell infiltrate is invariably seen in human antiGBM disease, consisting mainly of CD4 + cells. In an experimental T-cell dependent model, bursectomized chicks do not produce antibodies but develop GN on immunization with GBM (Bolton et al., 1988). The disease could also be transferred using T cells to unchallenged syngeneic birds.

Methods of Detection The traditional way to demonstrate the presence of anti-GBM is to visualize bound antibodies along renal basement membranes by direct immunofluorescence (IF) of renal biopsy specimens (Figure 2). This

method can give false-positive results in cases of diabetes and in biopsies from renal transplants (Querin et al., 1986). Circulating anti-GBM can be detected by indirect IF with serum overlaid on a normal kidney. A good substrate and a good pathologist are needed because nonspecific staining can be difficult to distinguish from the true linear staining pattern. Low levels of circulating autoantibodies can usually not be detected with this method. In 1974, a radioimmunoassay based on a collagenase digest of crude GBM was developed for detection of anti-GBM (Wilson et al., 1974). In 1981, the first ELISA based on a collagenase digest was published (Wieslander et al., 1981). Assays using crude extracts were the only alterative until 1984

Figure 2. Typical smooth linear pattern of IgG antibody deposition in" classical anti-GBM nephritis, visualized by direct immunofluorescence staining of a renal biopsy specimen. 294

when specific assays were developed using the Cterminal end of type IV collagen (Wieslander et al., 1984a; 1984b). Sensitive and specific assays were subsequently developed (Saxena et al., 1989a). The performance of these assays depends on the purity of the antigen preparation. The assays may, for instance, give positive results for antibodies to entactin (antientactin), if entactin is present in the preparation. Antientactin can be found in certain patients with chronic GN and in many patients with SLE, but usually not in anti-GBM disease (Saxena et al., 1990). Although the sensitivity and specificity to detect anti-GBM by ELISA is high, no actual figures from large studies are available; the best estimate is 98-99%. The positive and negative predictive values are of course very high since the presence of the antibody is a diagnostic criterion. False-positive reactions occur mainly in SLE and other diseases with polyclonal activation. Normally, around 1% of samples sent to a laboratory contain nonspecific reactivities. Checking for background reactivities in each sample can be used to control for this.

CLINICAL UTILITY

Disease Association The usual clinical presentation of anti-GBM disease which alerts clinicians is rapidly progressive glomerulonephritis with or without lung hemorrhage. Far less dramatic clinical symptoms may dominate such as recurrent hemoptysis, unexplained pulmonary infiltrates, red urine, anemia with breathlessness, and, particularly in older people, a silent progression to uremia. Many other much more frequent disorders may have similar clinical features, necessitating a high degree of clinical suspicion and ultimately a serological confirmation (Kelly and Haponik, 1994). A negative result by ELISA almost certainly excludes a diagnosis of active classical anti-GBM disease, provided that specific and pure antigen is used as a target in the assay. On the other hand, relatively high antibody titers may persist in patients in clear clinical remission and decline only slowly over a year or so. Recurrences of the disease after a year are very uncommon (Turner et al., 1993). In general, renal transplantation should be postponed until antibody titration is negative to avoid a recurrence of the disease in the transplant recipient (Glassock et al., 1989).

Antibody Frequencies A wider serological evaluation is important in all patients presenting with pulmonary-renal syndromes or with rapidly progressive GN, because only about 15% have anti-GBM. The majority have ANCA, leaving a minority with other disorders (Saxena et al., 1995). The antibody found is also of importance for the prognosis. Indeed, no other clinical or laboratory parameter has a similar impact on prognosis (Saxena et al., 1995). Patients with anti-GBM have the poorest prognosis, followed b y those with PR3-ANCA-associated systemic vasculitis, i.e., Wegener's granulomatosis (WG) and MPO-ANCA-associated systemic microscopic polyangiitis. The remaining have milder, secondary forms of rapidly progressive GN such as poststreptococcal GN, Henoch Sch6nlein purpura, systemic lupus erythematosus and mixed cryoglobulinemia. There are two peaks of age-dependent incidence, in the third and in the seventh decades. It is uncommon before puberty. The male to female ratio is about equal, but lung hemorrhage is at least twice as common in male patients (Glassock et al., 1989). In published series from New Zealand, Australia, the British Isles, the US and Scandinavia, estimated frequencies vary from 0.5-1 case per million inhabitants per year. The incidence in other parts of the world is unknown. Patients with classical disease comprise a medical emergency in nephrological referral centers because mortality rates exceed 75% without treatment. Quick recognition before tissue damage has advanced too far, using the reliable autoantibody assays as markers, is therefore crucial. Serial analysis of autoantibody is also useful in monitoring the effect of therapy. Modem treatment is based on fast removal of toxic antibodies by plasma exchange or preferably by more effective extracorporeal immune adsorption methods (Figure 3). Supplementation with pharmacological suppression of inflammatory and immune cell responses is necessary. A reduction of mortality rates to <15% can be expected. Oliguric patients almost never regain useful renal function in spite of therapy. The classical antibody should be differentiated from non-Goodpasture anti-GBM since antibody reactivities against other GBM proteins are rather common in human GN (Bygren et al., 1989). About 40% of the patients in one biopsy series of patients with GN had such antibodies. The fine specificity of the latter are only partially defined. One category of

295

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patients has IgA nephropathy by clinicopathological definition and antibodies to fibronectin, probably occurring as IgA-fibronectin complexes (Cederholm et al., 1988). Another relatively distinct subset of patients with GN form antibodies against the basement m e m b r a n e protein entactin/nidogen (Saxena et al., 1990). The influence of these antibodies on the disease process is not known.

CONCLUSION Goodpasture's syndrome is a classical example of autoimmune disease (Table 1). The disease can be transferred with the anti-GBM. The antigen is well known as the C-terminal end of the cz3 chain of type IV collagen. The cryptic epitope is localized in a cross-linking domain of the type IV collagen network.

Table 1. Features of Anti-GBM in Goodpasture's Syndrome Main Feature

Comments

Antigen

Type IV collagen

Other GBM proteins (e.g., entactin, fibronectin) frequently recognized in other GN forms, but of uncertain significance.

Epitope

Cryptic conformational epitope on NC1 domain of alpha 3 chain

Other epitopes (e.g., linear epitopes, alpha 1[IV]) sometimes recognized, but of uncertain significance.

Heavy chain

IgG 1

Other heavy chains (e.g., IgG4, IgA) sometimes present, but of uncertain significance.

Incidence

0.5-1.0 per million

Data from USA and Europe

HLA linkage

HLA-DR2 and B7 overrepresented

Significance

Diagnostic tool in nephritic syndrome

Positive test heralds urgent need for treatment, unfavorable prognosis and high risk for lung hemorrhage. Negative test rules out active anti-GBM disease.

Detection

ELISA with purified antigen

Lower sensitivity and specificity with immunofluorescence

296

Reliable diagnosis is based on clinical signs of lung hemorrhage and rapidly progressive renal failure combined with the presence of serum anti-GBM. Clinical expression of the disease can vary, but antiG B M disease in man has a poor prognosis. Rapidly progressive GN combined with lung purpura and Goodpasture's syndrome often results in death of the

patient if not treated. Results obtained with combined immunosuppression and plasma exchange in GP yield a much improved prognosis, but it is still important to initiate therapy before renal damage has advanced too far. Early recognition is therefore mandatory and can be confirmed with the use of sensitive assays.

REFERENCES

structure, gene organisation, and role in human disease. J Biol Chem 1993a;268:26033-26036. Hudson BG, Kalluri R, Gunwar S, Noelken ME, Mariyama M, Reeders ST. Molecular characteristics of the Goodpasture autoantigen. Kidney Int 1993b;43:135-139. Johansson C, Butkowski R. The structural organization of type IV collagen. Identification of three NC1 populations in the glomerular basement membrane. J Biol Chem 1992;267: 24533-24537. Johansson C, Butkowski R, Swedenborg P, Aim P, Wieslander J. Characterization of a non-Goodpasture autoantibody to type IV collagen. Nephrol Dial Transplant 1993;8:1205-1210. Kalluri R, Gunvar S, Reeders ST, Morrison KC, Mariyama M, Ebner KE, Noelken ME, Hudson BG. Goodpasture Syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha3(IV) chain of basement membrane collagen. J Biol Chem 1991;266: 24018-24024. Kelly D, Haponik E. Goodpasture syndrome: molecular and clinical advances. Medicine (Baltimore) 1994;73:171-- 185. Kleppel MM, Santi PA, Cameron JD, Wieslander J, Michael AF. Human tissue distribution of novel basement membrane collagen. Am J Pathol 1989;134:813--825. Lerner R, Glassock RJ, Dixon FJ. The role of antiglomerular basement membrane antibodies in the pathogenesis of human glomerulonephritis. J Exp Med 1967;26:989-1004. Masugi M. Uber die experimentelle Glomerulonephritis durch das spezifischen Antinierenserum. Beitr Pathol Anat Alg Pathol 1934;92:429--466. Neilson EG, Kalluri R, Sun MJ, Gunwar S, Danoff T, Mariyama M, Myers JC, Reeders ST, Hudson BG. Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type IV collagen. J Biol Chem 1993 ;268:8402- 8405. Nishikawa K, Linsley PS. Collins AB, Stamenkovitch I, McCluskey RT, Andres G. Effect of CTLA-4 chimeric protein on rat autoimmune antiglomerular basement membrane glomerulonephritis. Eur J Immunol 1994;24:1249-- 1254. Querin S, Noel LH, Grunfeld JP, Droz D, Mahieu P, Berger J, Kreis H. Linear glomerular IgG fixation in renal allografts: incidence and significance in Alport's syndrome. Clin Nephrol 1986;25:134--140. Saxena R, Isaksson B, Bygren P, Wieslander J.. A rapid assay for circulating glomerular basement membrane antibodies in Goodpasture syndrome. J Immunol Methods 1989a;118: 73-78.

Bolton WK, Chandra M, Tyson TM, Kirkpatrick PR, Sadovnic MJ, Sturgill BC. Transfer of experimental glomerulonephritis in chickens by mononuclear cells. Kidney Int 1988;34:598610. Bolton WK, Luo AM, Fox PL, May WJ, Sturgill BC. Study of EHS type IV collagen lacking Goodpasture's epitope in glomerulonephritis in rats. Kidney Int 1995;47:404--410. Burns AP, Fisher M, Li P, Pusey CD, Rees AJ. Molecular analysis of HLA class II genes in Goodpasture's disease. QJM 1995;88:93-100. Butkowski R, Wieslander J, Wisdom BJ, Barr JF, Noelken M, Hudson BG. Properties of the globular domain of type IV collagen and its relationship to the Goodpasture antigen. J Biol Chem 1985;260:3739-3747. Butkowski R, Langeveld J, Wieslander J, Hamilton J, Hudson BG. Localization of the Goodpasture epitope to a novel chain of basement membrane collagen. J. Biol Chem 1987;262: 7874-7877. Bygren P, Cederholm B, Heinegard D, Wieslander J. NonGoodpasture anti-GBM antibodies in patients with glomerulonephritis. Nephrol Dial Transplant 1989;4:254--261. Cederholm B, Wieslander J, Bygren P, Heinegard D. Circulating complexes containing IgA and fibronectin in patients with primary IgA nephropathy. Proc Natl Acad Sci USA

1988;85:4865--4868. Freytag JW, Ohno M, Hudson G. Bovine renal glomerular basement membrane. Assessment of proteolysis during isolation. Biochem Biophys Res Commun 1976;72:796-802. Glassock RJ, Adler SG, Ward HJ, Cohen AH. Secondary glomerular disease. In: Brenner BM, Rector Jr. FC, eds: The Kidney. 4th edition. Philadelphia: W.B. Saunders, 1989: 1301-1305. Goodpasture, EW. The significance of certain pulmonary lesions in relation to the etiology of influenza. Am J Med Sci 1919;158:864--870. Hellmark T, Johansson C, Wieslander J. Characterization of anti-GBM antibodies involved in Goodpasture's syndrome. Kidney Int 1994;46:823--829. Herody M, Bobrie G, Gourin G, Grunfeld JP, Noel LH. AntiGBM disease: predictive value of clinical, histological and serological data. Clin Nephrol 1993;40:249--255. Hudson BG, Wieslander J, Wisdom B, Noelken ME. Goodpasture syndrome. Molecular architecture and function of the basement membrane antigen. Lab Invest 1989;61:256-269. Hudson BG, Reeders ST, Tryggvason K. Type IV collagen:

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Saxena R, Bygren P, Butkowski R, Wieslander J. Specificity of kidney-bound antibodies in Goodpasture's syndrome. Clin Exp Immunol 1989b;78:31--36. Saxena R, Bygren p, Butkowski R, Wieslander J. Entactin: a possible autoantigen in the pathogenesis of non-Goodpasture anti GBM nephritis. Kidney Int 1990;38:263--272. Saxena R, Bygren P, Arvastson B, Wieslander J. Circulating autoantibodies as serological markers in the differential diagnosis of pulmonary renal syndrome. J Intern Med 1995;237"143--152. Segelmark M, Butkowski R, Wieslander J. Antigen restriction and IgG subclasses among anti-GBM autoantibodies. Nephrol Dial Transplant 1990;5:991-996. Sheer R L, Grossman MA. Immune aspects of glomerulonephritis associated with pulmonary hemorrhage. Ann Intern Med ' 1964 ;60:1009-- 1021. Steblay RW. Glomerulonephritis induced in sheep by injections of heterologous glomerular basement membrane and Freund' s complete adjuvant. J Exp Med 1962;116:253-281. Stanton MC, Tange JD. The Goodpasture's syndrome (pulmonary haemorrhage associated with glomerulonephritis). Australas Ann Med 1958;7"132--144.

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Turner N, Lockwood CM, Rees AJ. Antiglomerular basement membrane antibody-mediated nephritis. In: Schrier RW, Gottschalk CW, eds. Diseases of the Kidney. Boston: Little, Brown and Company, 1993:1865. Turner N, Forstova J, Rees A, Pusey CD, Mason PJ. Production and characterization of recombinant Goodpasture antigen in insect cells. J Biol Chem 1994;269:17141-17145. Wieslander J, Bygren P, Heinegard D. Antibasement membrane antibody: immunoenzymatic assay and specificity of antibodies. Scand J Clin Lab Invest 1981;41:763--772. Wieslander J, Bygren P, Heinegard D. Isolation of the specific glomerular basement membrane antigen involved in Goodpasture syndrome. Proc Natl Acad Sci USA 1984a;81:15441548. Wieslander J, Barr JF, Butkowski RJ, Edwards SJ, Bygren P, Heinegard D, Hudson BG. Goodpasture antigen of the glomerular basement membrane: localization to noncollagenous regions of type IV collagen. Proc Natl Acad Sci USA 1984b;81:3838-3842. Wilson CB. Marquardt H. Dixon FJ. Radioimmunoassay (RIA) for circulating antiglomerular membrane(GBM) antibodies. Kidney Int 1974;6:114.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

GLUTAMIC ACID DECARBOXYLASE AUTOANTIBODIES IN DIABETES MELLITUS Robert S. Schmidli, M.B., Ch.B. and Leonard C. Harrison M.D., D.Sc.

Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia

HISTORICAL NOTES

THE AUTOANTIGENS

Circulating autoantibodies to pancreatic islets of Langerhans in insulin-dependent diabetes mellitus (IDDM), termed islet cell antibodies (ICA), were first demonstrated in 1974 (Bottazzo et al., 1974). Although this stimulated considerable effort to identify target antigens, not until 1982 were sera from IDDM patients shown to immunoprecipitate a specific 64 kd islet protein from lysates of 35S-labeled rat islets (Baekkeskov et al., 1990). This 64 kd protein was further characterized (DeAizpurua et al., 1992b) and 64 kd antibodies were shown to be present prior to the onset of IDDM (Bottazzo, 1993). In 1988, glutamic acid decarboxylase (GAD) was shown to be an autoantigen in the rare neurological condition, stiffman syndrome (SMS) (Solimena and DeCamilli, 1991). The frequent association of SMS with organspecific autoimmune disease including IDDM (Blum and Jankovic, 1991) and the similarity of the molecular weights of GAD and the 64 kd antigen were the clues that led in 1990 to tlae identification of GAD as the 64 kd autoantigen in IDDM (Baekkeskov et al., 1990). Since its discovery in the central nervous system (CNS) in the 1950s, GAD has been studied extensively as the enzyme that catalyses the synthesis of the inhibitory neurotransmitter gamma amino butyric acid (GABA). GAD was first cloned from feline brain (Kaufman et al., 1986) and .was subsequently cloned from human brain and pancreas (Faulkner-Jones et al., 1995).

The two isoforms of GAD of predicted molecular weight 65 kd and 67 kd are known as GAD65 and GAD67. These are encoded by separate genes and share 65% identity and 80% similarity at the amino acid level; most of the differences are within the Nterminal 110 amino acids (Erlander et al., 1991) (Figure 1). Enzymatic activity requires binding of the cofactor pyridoxal 5'-phosphate (P-5-P) to a fourresidue motif situated near the C-terminus. GAD65 exists as an apoenzyme; whereas, GAD67 exists as a holoenzyme bound to P-5-P.

Tissue Expression The highest levels of GAD are in the brain (FaulknerJones et al., 1995) and significant GAD is expressed in the islets of Langerhans, pituitary, gonad, kidney, adrenal and liver in the rat. Because of its role as an autoantigen in IDDM, GAD has now been extensively studied in the pancreatic islets where different patterns of expression are found in the human, rat and mouse. At the protein level, human islets contain only GAD65; rat islets contain both forms and mouse islets predominantly GAD67, expressed at a lower level than in rat islets. In situ hybridization reveals that GAD65 and GAD67 gene expression is restricted to [3 cells in the rat and probably the mouse, but the lack of clear demarcation of [3 from o~ and other islet cells precludes such exact localization in human mouse (Faulkner-Jones et al., 1995). At the mRNA level, human islets contain GAD65 and GAD67 at a ratio of 200 to 1 (Cram et al., 1995); rat islets contain both

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Figure 1. Amino acid (aa) sequence comparison, Coxsackie P2-C protein similarity and pyridoxal-5'-phosphate P-5-P binding site of GAD65 and GAD67.

forms, and GAD67 predominates in the mouse. GAD protein is detectable in several immortalized mouse ~cell lines, including the Simian virus 40 (SV40) transformed ~-cell lines 13-TC3 and [3HC. The [3TC3 cell line expresses both GAD mRNAs, GAD65 being more abundant. GAD67 mRNA is present in the SV40 transformed nonobese diabetic (NOD) mouse ~-cell line NIT-l, but not in the rat insulinoma line RINm5F nor the SV40 transformed hamster islet cell line HIT (Faulkner-Jones et al., 1995).

Recombinant GAD Recombinant GAD expressed in E. coli expression systems often lacks enzymatic activity, presumably due to improper folding. Production in eukaryotic hosts (such as Sf9 insect cells from baculovirus-based vectors) allows synthesis of large amounts of enzymatically active, immunoreactive GAD (Mauch et al., 1993). GAD can also be synthesized in small quantities by in vitro transcription and translation (Ujihara et al., 1994). Although IDDM sera react with native and recombinant GAD at similar frequencies (Schmidli et al., 1995), differences in antibody binding are documented. For example, the GAD65-specific monoclonal antibody GAD6 immunoprecipitates both GAD65 and GAD67 from brain (Butler et al., 1993), but only precipitates recombinant GAD65. The formation of heterodimers in tissue extracts is thought to give rise to coprecipitation of both GAD forms.

Methods of Purification Native GAD-purified by a combination of gel filtration, calcium phosphate gel and DEAE-Sephadex

300

chromatography was used to raise monoclonal antiGAD antibodies (Gottlieb et al., 1986), which are now used to purify GAD in a one-step immunoaffinity purification. Recombinant GAD, expressed in the baculovirus-Sf9 cell system, is purified by anion exchange chromatography or immunoaffinity chromatography (Moody et al., 1995). Recombinant GAD produced as a fusion protein with polyhistidines on the C-terminus is conveniently purified by nickelchelation affinity chromatography (Mauch et al., 1993). Purified, enzymatically active, baculovirusexpressed human GAD65 and GAD67 are available through the authors. E. coli or C. perfringens-derived GAD reported to be enzymatically active is available as a crude acetone powder or a partially purified powder from Sigma (St. Louis, Missouri, USA).

GAD Epitopes The patterns of antibody recognition of GAD in SMS and IDDM appear to differ. Generally, antibodies in sera from patients with SMS react with denatured GAD in immunoblots, whereas, those in IDDM sera only recognize GAD in its native form by immunoprecipitation (Baekkeskov et al., 1990). Additionally, GAD antibodies in SMS patients precipitate both GAD65 and GAD67; whereas, <20% of IDDM sera immunoprecipitate GAD67 (Butler et al., 1993). Furthermore, the titer of antibodies in SMS is generally higher than that in IDDM (Kim et al., 1994). Reported GAD65 antibody epitopes in SMS and IDDM are summarized in Figure 2. The study of GAD antibody epitopes is complicated by the presence of conformational epitopes and complex determinants spanning different regions of the molecule.

Figure 2. Summary of GAD65 epitopes in SMS and IDDM. Black rectangles denote conformational epitopes, grey rectangles denote linear epitopes.

AUTOANTIBODIES Pathogenetic Role Available evidence indicates that GAD antibodies do not have a pathogenic role in IDDM. In fact, GAD antibodies may signify relative protection from progressive ~-cell destruction. Animal Models. Transfer of peripheral blood mononuclear cells from patients with IDDM to mice with severe combined immunodeficiency disease (SCID) led to formation of GAD antibodies in some mice, but impairment of ~-cell function or evidence of islet cell damage did not occur (Petersen et al., 1993). Additionally, GAD antibodies are found in ICA-positive subjects without IDDM or impaired pancreatic ~-cell insulin release (Wagner et al., 1994). Further evidence of a "protective" effect of GAD antibodies is provided by a study in the NOD mouse. In this model of

spontaneous autoimmune IDDM, GAD antibodies occur at higher frequency and concentration and are detected at an earlier age in female mice with a lower incidence of diabetes (DeAizpurua et al., 1994). There are now a number of reports, however, which directly demonstrate a pathogenic role of GAD-reactive T cells in the NOD mouse. Intrathymic, intravenous or intraperitoneal injection of GAD65, and intraperitoneal injection of GAD67 delayed or prevented the appearance of insulitis and diabetes (Faulkner-Jones et al., 1995). This was accompanied by a reduction of Tcell proliferative responses to GAD in two of the studies (Tisch et al., 1993; Kaufman et al., 1993). Similar experiments were not undertaken in humans to determine whether GAD is a pathogenic autoantigen, because the presence of GAD in the central nervous system and the association of CNS disease with GAD autoimmunity raise serious concerns about tolerizing humans against GAD.

301

Human Disease. Despite the lack of direct evidence for a pathogenic role of GAD in human IDDM, partial evidence is provided by the existence of T-cell responses to GAD65 in newly diagnosed patients (Atkinson et al., 1992), to GAD67 in "at-risk" and newly diagnosed patients (Honeyman et al., 1993) and to a peptide sequence of the C-terminal of GAD65, aa473-555 (Lohman et al., 1994). Additionally, GADspecific cytotoxic T cells can be identified in IDDM patients (Panina-Bordignon et al., 1995). By contrast with SMS, all but one study shows that in IDDM, antibodies react only with conformational epitopes. IDDM sera immunoprecipitate fulllength GAD65 and a large fragment containing aa188--585, but do not precipitate smaller fragments (Ujihara et al., 1994). Using deletion mutants of GAD65 and five monoclonal antibodies derived from a patient with newly diagnosed IDDM, the presence of a midregion and C-terminal epitope was demonstrated in GAD65 (Richter et al., 1993). Antibody reactivity occurred against aa244--585, but further deletion of amino acids at the N-terminus did not affect antibody binding. The last 44 amino acids at the C-terminus were necessary for binding of three of the antibodies. Four of the five antibodies did not recognize the mutant missing aa363--422, which contains the P-5-P binding site. Binding by sera was abolished by deletion of aal--295, and deletion of 41 amino acids at the C-terminus prevents binding by four sera. Similar epitope patterns were found in a study in which chimeras of GAD65 and GAD67 were used to examine antibody binding (Daw and Powers, 1995); two distinct antibody specificities to GAD were found, one located in aa240-435 and the other in aa451--5 70. Mapping of overlapping fragments (-~100aa) encompassing human GAD67 by ELISA revealed a lower frequency of reactivity compared to GAD65 with 20% of newly diagnosed IDDM or "at risk" sera reacting with at least one GAD67 fragment. Most epitopes were in the mid- and C-terminal thirds of the protein, a region that shares a high degree of homology with GAD65. However, in another study (DeAizpurua et al., 1992b) using three fragments spanning mouse GAD67, reactivity by ELISA occurred with all fragments. The presence of a short region of similarity between GAD and the P2-C protein of coxsackievirus B 4 (Kaufman et al., 1992) (Figure 1) led to the suggestion that molecular mimicry might play a role in the genesis of GAD autoimmunity. Coxsackie virus B 4 can infect [3 cells and is epidemiologically linked

302

to IDDM in humans. The molecular mimicry theory is supported by the demonstration of T-cell proliferative responses to GAD65 peptides containing the shared sequence KXXPEVKEK, in "at-risk" and newly diagnosed IDDM subjects (Atkinson et al., 1994). Three out of three GAD peptide-reactive IDDM patients in this study also responded to the homologous coxsackie viral peptide; whereas, none of 13 controls did. In another study, immunization of mice with 16-mer coxsackie P2-C and GAD65 peptides containing the conserved sequence KXXPEVKEK generated T-cell responses only in NOD mice and not in nine other mouse strains (Tian et al., 1994). This was also the only strain in which cross-reactive T-cell recognition occurred. Thus, T-cell cross-reactivity in mice was restricted to the diabetes-associated NOD MHC class II allele, I-A g7. Direct proof of functional mimicry requires demonstration that pathogenic T-cell clones recognize the shared sequence. At the antibody level, molecular mimicry based on similarities between linear sequences is open to argument. Antibodies raised against either P2-C or GAD65 peptides cross-react with the other peptide in one report (Hou et al., 1994). Sera from coxsackie virus-infected mice contain antibodies to GAD65 protein and P2-C peptide, and some react to GAD65 peptide. Additionally, several IDDM sera react with GAD65 protein in addition to P2-C and GAD65 peptide. However, in another study with six humanderived, anti-GAD monoclonal antibodies which require the P2-C homology sequence of GAD65 for reactivity, no binding was found to a range of viral antigens derived from coxsackie B l--B6 (Richter et al., 1994). Furthermore, none of 15 coxsackie B 4positive human sera immunoprecipitated GAD65. Other infectious agents with sequence similarities to GAD include mycobacterial and human heat shock protein 60 (hsp60), Kunjin virus, Japanese encephalitis virus, Western Nile virus and Murray Valley encephalitis virus (Honeyman et al., 1993), but their significance is unknown.

Factors in Pathogenicity Subclasses. IgG subclasses may reflect the nature of T-cell responses and, therefore, the nature of immunemediated pathology. There are no published studies examining the IgG subclasses that react with GAD. Newly diagnosed IDDM patients have predominantly IgG1 _+ IgG3 antibodies to native brain GAD; on the other hand, ICA-positive relatives who do not pro-

gress to IDDM have predominantly IgG2 _+ IgG4 antibodies to GAD (Couper and Harrison, unpublished). Thus, the measurement of IgG subclass antibodies to GAD may be a practical way of refining the prediction of risk for progression to clinical IDDM. Genetics

Inheritance plays a major role in the susceptibility to IDDM, as indicated by twin studies in which pairwise concordance for IDDM ranges from 13--34% in monozygotic twins, compared to 2.5--5% in dizygotic twins. GAD antibodies may also be influenced by genetic factors. In a study of identical twins, 15% of siblings who were long-term discordant for IDDM were GAD Ab-positive (Bottazzo, 1993). Class II HLA genes are the major single genetic determinant of risk for IDDM, the highest risk haplotype in caucasoids being HLA DR3,4; DQ2,8, with "protection" being afforded by DR2 and DQ6 (Tait and Harrison, 1991). In one study, GAD antibodies were more prevalent (85%) in DR3, DR4 IDDM patients than in DR 3,4,X (48%) patients (Serjeantson et al., 1992). In a larger study, DQ2 was significantly more common in patients with GAD antibodies, and GAD antibodieswere detected in 64% of DQ2,8; 55% of DQ2,2 and 41% of patients with other HLA-DQB 1 alleles. The HLA-DQ2,8 association was more marked in those with onset of IDDM after the age of 14 years (Serjeantson et al., 1993). Methods of Detection

Among several methods to detect GAD antibodies, the most widely used are ELISA, radiobinding assay (RBA) and enzymatic immunoprecipitation (EIP). Indirect immunofluorescence (Genovese et al., 1992) and immunoprecipitation followed by immunoblotting (Baekkeskov et al., 1990) are used less frequently. In the ELISAs (DeAizpurua et al., 1992a; 1992b), purified recombinant or native GAD is bound to a microtiter plate, serum is incubated in the wells, and bound antibody is detected with an appropriate labeled secondary antibody. RBAs utilize radiolabeled recombinant (Hagopian et al., 1993a; Petersen et al., 1994; Grubin et al., 1994) or native (Rowley et al., 1992) GAD, which is precipitated with serum antibodies and detected by gamma or beta counting. 125I-labeled, immunoaffinity-purified pig brain GAD is often used as a source of native GAD for this type of assay.

Most assays that use recombinant GAD rely on biosynthetic labelling with 35S-methionine, but 125I can also be used successfully. There is little difference in assay sensitivity or specificity when recombinant and native GAD are used, or when 125I or 35S are used as the radiolabel (Schmidli et al., 1995). In the EIP assays (Baekkeskov et al., 1990; Martino et al., 1991; DeAizpurua et al., 1992b; Schmidli et al., 1994a), GAD is quantified after immunoprecipitation by enzymatic activity, usually measured as conversion of 14C-labeled glutamate to GABA and 14C02, the latter detected by adsorption to hyamine hydroxidesoaked filter paper disks. Assay performance was compared in two international GAD antibody workshops. Despite a wide range of methods, a surprisingly high degree of concordance was noted between laboratories. After standardization of assay sensitivity to give a specificity of 100%, the mean sensitivity for IDDM of RBAs was 66.7%, ELISAs 24.7% and EIPs 40.6%. Eight RBAs and one ELISA had a sensitivity >80% (Schmidli et al., 1995). RBA is, therefore, currently the preferred method, with 35S-labeled in vitro translated recombinant GAD being the most widely used source of antigen (Petersen et al., 1994; Grubin et al., 1994). Despite their obvious advantage of convenience, most currentlyavailable ELISAs are not sufficiently sensitive for applications such as preclinical screening for IDDM. There is no international standard measure of GAD antibodies at present, but dilutions of a standard serum and the monoclonal GAD65 antibody MICA6 generate a standard curve from which results can be interpolated, thus allowing standardization between laboratories.

CLINICAL UTILITY Disease Associations

GAD antibodies are specific for SMS and IDDM and are not detected in other disorders, except those, such as polyendocrine autoimmunity, which are associated with IDDM. GAD antibodies corroborate the clinical diagnosis of SMS, with a diagnostic sensitivity reported at 100% in well-characterized patients (Kim et al., 1994). As this condition coexists with IDDM, false-positive results may occur where IDDM is present and the diagnosis of SMS is in doubt. GAD antibodies may be useful for identifying adult-onset diabetic patients who have a slow onset of IDDM

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typical of adults, variously referred to as latent autoimmune diabetes of adults, or "type 1.5" diabetes. Among newly diagnosed adult diabetic subjects, 65% of the GAD antibody-positive patients require a change from an oral agent to insulin within 18 months (Hagopian et al., 1993a). In another study, 76% of adult patients presenting with diabetes and impaired insulin response to glucagon had GAD antibodies, compared to 12% with a normal insulin response (Tuomi et al., 1993a). Antibodies to GAD are found in SMS and IDDM. The frequency of GAD antibodies in SMS was initially reported to be about 60% (Solimena and DeCamilli, 1991), but in a recent study (Kim et al., 1994), 100% of patients had GAD antibodies. The frequencies of GAD antibodies in IDDM range from 25% (Martino et al., 1991) to 79% (Hagopian et al., 1993a). Although this difference may reflect the genetic, racial (Serjeantson et al., 1992), age and gender (Schmidli et al., 1994b) composition of study groups, the lower figures are more likely due to less sensitive assays and the true positivity is probably 60--80%. In a study of 23 patients with polyendocrine autoimmunity, 21 who were ICA-positive were also positive for GAD antibodies (Wagner et al., 1994). Of these 21, six developed impaired first-phase insulin release in response to intravenous glucose, three developed IDDM, and the remaining 12 did not develop IDDM in 22--82 months follow-up. These results suggest that polyendocrine autoimmunity is associated with a high prevalence of GAD Ab, although these patients do not progress rapidly to IDDM. In our hands, however, only 2 out of 27 unselected patients with thyroid disease had elevated GAD antibody concentrations. No definite associations between GAD antibodies and diabetes complications are found. Despite an early report which suggested that diabetic neuropathy in a small number of patients was associated with high levels of GAD antibodies (Kaufman et al., 1992), subsequent studies have not shown this association (Tuomi et al., 1993b). ICA and GAD antibodies positivity are strongly correlated in preclinical and clinical IDDM subjects; in several studies, GAD antibodies were almost exclusively found in the presence of ICA (Martino et al., 1991; Schmidli et al., 1994b). Experimental evidence indicates that GAD represents a component of the ICA antigen. Incubation of ICA-positive sera with rat brain homogenate (Genovese et al., 1992), E. coli lysates containing recombinant human GAD65 (Atkinson et al., 1993) or purified recombinant human

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islet GAD (Marshall et al., 1994) reduced or abolished ICA staining. In two of these studies, these "GADabsorbable" ICA associated with a low rate of progression to IDDM (Genovese et al., 1992; Atkinson et al., 1993). "GAD-adsorbable" ICA are associated with very high levels of GAD antibodies (Yu et al., 1994). Six anti-GAD monoclonal antibodies derived from an ICA-positive, newly diagnosed IDDM patient were designated monoclonal islet cell antibodies (MICA) 1--6 (Richter et al., 1993). The prevalence of GAD antibodies is also influenced by age, gender and perhaps race. In ICApositive first-degree relatives and newly diagnosed IDDM subjects, GAD antibodies are higher in postpubertal females than in males and prepubertal females (Schmidli et al., 1994b). The frequency of GAD antibodies in Asian IDDM patients is controversial; a lower prevalence of GAD antibodies was found in a small number of Hong Kong Cantonese, Korean and Japanese patients compared to Caucasians (Serjeantson et al., 1992), but in another study GAD antibodies were detected in over 80% of Japanese patients (Kawasaki et al., 1994). There is some evidence that high levels of GAD antibodies correlate with a lower risk of IDDM and a slower rate of decline in [~-cell function in ICApositive first-degree relatives (a group at-risk for IDDM). [3-cell destruction is considered to be mediated by cellular immunity, and in one study IDDM developed in ICA-positive relatives with evidence of cellular rather than humoral immunity (Harrison et al., 1993). In another study of ICA-positive relatives, GAD antibodies were inversely related to the loss of glucose-stimulated insulin release and the presence of insulin autoantibodies, a marker of high risk for IDDM (Yu et al., 1994). A number of potential preventive therapies for IDDM are being evaluated, including nicotinamide and induction of immune tolerance to insulin. Intervention therapy prior to diagnosis necessitates screening tests of high sensitivity and specificity. Currently, ICA is the most commonly used screening test for "preclinical" IDDM, but it is relatively labor-intensive, poorly reproducible and only semiquantitative. With the availability of recombinant GAD, large scale screening based on GAD antibodies is now possible. However, as the incidence of IDDM is relatively low in the general population, the presence of even a small number of false-positive cases lowers the positive predictive value of a screening test. This was the case when ICA were used to predict IDDM in the general

population. For this reason, screening studies continue to concentrate on first-degree relatives of patients with IDDM, who have an approximately 15-fold increase in risk for IDDM. No large-scale prospective study of the predictive value of GAD antibodies has yet been published in first-degree relatives or in an unselected population. In a retrospective study of pregnant women sampled during the antenatal period, GAD antibodies were detected in 82% of subjects who later developed IDDM and 36% who developed NIDDM, compared to 0% of 100 nonmatched controls. Several studies examined GAD antibodies in ICA or IAApositive first-degree relatives of patients with IDDM, a group in which 30-50% eventually develop IDDM. In two of these studies, the presence of GAD antibodies did not influence IDDM-free survival in firstdegree relatives with either ICA or IAA (Schmidli et al., 1994b) or ICA (Bingley et al., 1994). GAD antibodies in the absence of ICA are uncommon, reflecting the strong relationship between the two; only one GAD antibody-positive, ICA-negative subject was found in several separate studies of firstdegree relatives who were tested prior to onset of IDDM (Schmidli et al., 1994a; Thivolet et al., 1992).

GADAb

(nU/ml) 5000-

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u u u 1 6 5 1 6 5 1u6u5 1 6 5 u1 16 65 5

2500-

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2b

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Figure 3. Levels of GAD antibodies in a patient with SMS, treated with plasmapheresis followed by cyclophosphamide. 12 months. Although the effect of insulin therapy on GAD antibodies has not been formally examined, similar frequencies of GAD antibodies are found in recent-onset and established IDDM (Rowley et al., 1992; Schmidli et al., 1994a), suggesting that insulin therapy does not alter GAD antibody positivity.

CONCLUSION

Effect of Therapy There are few studies examining the effect of therapy on GAD antibodies. In one patient with SMS who was treated with plasmapheresis and cyclophosphamide (Figure 3), an immediate almost two-fold reduction in GAD antibody levels was followed by a further gradual decline to about a third of initial amount levels. However, GAD antibodies returned to pretreatment values when plasmapheresis was stopped, despite treatment with cyclophosphamide. This patient had muscular symptoms which did not alter when GAD antibodies were reduced by plasmapheresis (Schmidli and Harrison, unpublished). The effect of immunotherapy on GAD antibody levels in newly diagnosed IDDM has been examined in a placebocontrolled study of 132 patients; therapy with cyclosporin did not affect GAD antibodies over a period of

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GAD antibodies are present in the majority of patients with SMS and IDDM. Although results from several studies indicate a pathogenic role for GAD in driving T-cell-mediated ~-cell destruction in the NOD mouse model, there is no direct evidence as yet that GAD is a pathogenic autoantigen in human IDDM. Nevertheless, GAD antibodies are a sensitive and specific marker of islet autoimmunity and may be used to identify humans "at-risk" for IDDM, including asymptomatic relatives of IDDM patients and a subgroup of people with adult-onset diabetes. The availability of technically simple GAD antibody assays could allow GAD antibodies to displace ICA as the "gold standard" immune marker of IDDM and become the initial screening test for preclinical IDDM. See also GAD IN STIFF-MAN SYNDROME, INSULIN AUTOANTIBODIES and ISLET CELL AUTOANTIBODIES.

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1986;83:8808-8812. Grubin CE, Daniels T, Toivola B, Landin-Olsson M, Hagopian WA, Li L, Karlsen AE, Boel E, Michelsen B, Lernmark A. A novel radioligand binding assay to determine diagnostic accuracy of isoform-specific glutamic acid decarboxylase

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antibodies in childhood IDDM. Diabetologia 1994;37:344-350. Hagopian WA, Karlsen AE, Gottsater A, Landin-Olsson M, Grubin CE, Sundkvist G, Petersen JS, Boel E, Dyrberg T, Lernmark A. Quantitative assay using recombinant human islet glutamic acid decarboxylase (GAD65) shows that 64K autoantibody positivity at onset predicts diabetes type. J Clin Invest 1993a;91:368--374. Hagopian WA, Michelsen B, Karlsen AE, Larsen F, Moody A, Grubin CE, Rowe R, Petersen J, McEvoy R, Lernmark A. Autoantibodies in IDDM primarily recognize the 65,000-M(r) rather than the 67,000-M(r) isoform of glutamic acid decarboxylase. Diabetes 1993b;42:631-636. Harrison LC, Honeyman MC, DeAizpurua HJ, Schmidli RS, Colman PG, Tait BD, Cram DS. Inverse relation between humoral and cellular immunity to glutamic acid decarboxylase in subjects at risk of insulin-dependent diabetes. Lancet 1993 ;341:1365-- 1369. Honeyman MC, Cram DS, Harrison LC. Glutamic acid decarboxylase 67-reactive T cells: a marker of insulin-dependent diabetes. J Exp Med 1993;177:535--540. Hou J, Said C, Franchi D, Dockstader P, Chatterjee NK. Antibodies to glutamic acid decarboxylase and P2-C peptides in sera from coxsackie virus B4-infected mice and IDDM patients. Diabetes 1994;43:1260-1266. Kaufman DL, McGinnis JF, Krieger NR, Tobin AJ. Brain glutamate decarboxylase cloned in lambda gt-ll: fusion protein produces gamma-aminobutyric acid. Science 1986; 232:1138-1140. Kaufman DL, Erlander MG, Clare-Salzler M, Atkinson MA, Maclaren NK, Tobin AJ. Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus. J Clin Invest 1992;89:283-292. Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GS, Robinson P, Atkinson MA, Sercarz EE, Tobin AJ, Lehmann PV. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993;366:69-72. Kawasaki E, Takino H, Yano M, Uotani S, Matsumoto K, Takao Y, Yamaguchi Y, Akazawa S, Nagataki S. Autoantibodies to glutamic acid decarboxylase in patients with IDDM and autoimmune thyroid disease. Diabetes 1994;43:80-86. Kim J, Namchuk M, Bugawan T, Fu Q, Jaffe M, Shi Y, Aanstoot HJ, Turck CW, Erlich H, Lennon V, Baekkeskov S. Higher autoantibody levels and recognition of a linear NH2-terminal epitope in the autoantigen GAD65, distinguish stiff-man syndrome from insulin-dependent diabetes mellitus. J Exp Med 1994;180:595--606. Li L, Hagopian WA, Brashear HR, Daniels T, Lernmark A. Identification of autoantibody epitopes of glutamic acid decarboxylase in stiff-man syndrome patients. J Immunol 1994;152:930-934. Lohmann T, Leslie RD, Hawa M, Geysen M, Rodda S, Londei M. Immunodominant epitopes of glutamic acid decarboxylase 65 and 67 in insulin-dependent diabetes mellitus. Lancet 1994 ;343:1607-1608. Marshall MO, Hoyer PE, Petersen JS, Hejnaes KR, Genovese S, Dyrberg T, Bottazzo GF. Contribution of glutamate

decarboxylase antibodies to the reactivity of islet cell cytoplasmic antibodies. J Autoimmun 1994;7:497-508. Martino GV, Tappaz ML, Braghi S, Dozio N, Canal N, Pozza G, Bottazzo GF, Grimaldi LM, Bosi E. Autoantibodies to glutamic acid decarboxylase (GAD) detected by an immunotrapping enzyme activity assay: relation to insulin-dependent diabetes mellitus and islet cell antibodies. J Autoimmun 1991;4:915--923. Mauch L, Seissler J, Haubruck H, Cook NJ, Abney CC, Berthold H, Wirbelauer C, Liedvogel B, Scherbaum WA, Northemann W. Baculovirus-mediated expression of human 65 kDa and 67 kDa glutamic acid decarboxylases in Sf9 insect cells and their relevance in diagnosis of insulindependent diabetes mellitus. J Biochem 1993;113:699--704. Moody AJ, Hejnaes KR, Marshall MO, Larsen FS, Boel E, Svendsen I, Mortensen E, Dyrberg T. Isolation by anionexchange of immunologically and enzymatically active human islet glutamic acid decarboxylase 65 overexpressed in Sf9 insect cells. Diabetologia 1995;38:14--23. Panina-Bordignon P, Lang R, van Endert PM, Benazzi E, Felix AM, Pastore RM, Spinas GA, Sinigaglia F. Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes. J Exp Med 1995; 181:1923-- 1927. Petersen JS, Marshall MO, Baekkeskov S, Hejnaes KR, HoierMadsen M, Dyrberg T. Transfer of type 1 (insulin-dependent) diabetes mellitus associated autoimmunity to mice with severe combined immunodeficiency (SCID). Diabetologia 1993;36:510--515. Petersen JS, Hejnaes KR, Moody A, Karlsen AE, Marshall MO, Hoier-Madsen M, Boel E, Michelsen BK, Dyrberg T. Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay. Diabetes 1994;43:459--467. Richter W, Shi Y, Baekkeskov S. Autoreactive epitopes defined by diabetes-associated human monoclonal antibodies are localized in the middle and C-terminal domains of the smaller form of glutamate decarboxylase. Proc Natl Acad Sci USA 1993;90:2832-2836. Richter W, Mertens T, Schoel B, Muir P, Ritzkowsky A, Scherbaum WA, Boehm BO. Sequence homology of the diabetes-associated autoantigen glutamate decarboxylase with coxsackie B4-2C protein and heat shock protein 60 mediates no molecular mimicry of autoantibodies. J Exp Med 1994; 180:721--726. Rowley MJ, Mackay IR, Chen QY, Knowles WJ, Zimmet PZ. Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus. Diabetes 1992;41:548--551. Schmidli RS, Colman PG, Harrison LC. Do glutamic acid decarboxylase antibodies improve the prediction of IDD in first-degree relatives at risk for IDDM? J Autoimmun 1994a;7:873--879. Schmidli RS, DeAizpurua HJ, Harrison LC, Colman PG. Antibodies to glutamic acid decarboxylase in at-risk and clinical insulin-dependent diabetic subjects: relationship to age, sex and islet cell antibody status, and temporal profile. J Autoimmun 1994b;7:55-66.

Schmidli RS, Colman PG, Bonifacio E. Disease sensitivity and specificity of 52 assays for glutamic acid decarboxylase antibodies. The second international glutamic acid decarboxylase antibody workshop. Diabetes 1995;44:636--640. Serjeantson SW, Kohonen-Corish MR, Rowley MJ, Mackay IR, Knowles W, Zimmet P. Antibodies to glutamic acid decarboxylase are associated with HLA-DR genotypes in both Australians and Asians with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1992;35:996-1001. Serjeantson SW, Court J, Mackay IR, Matheson B, Rowley MJ, Tuomi T, Wilson JD, Zimmet P. HLA-DQ genotypes are associated with autoimmunity to glutamic acid decarboxylase in insulin-dependent diabetes mellitus patients. Hum Immunol 1993;38:97--104. Solimena M, DeCamilli P. Autoimmunity to glutamic acid decarboxylase (GAD) in stiff-man syndrome and insulindependent diabetes mellitus. TINS 1991;14:452-457. Tait BD, Harrison LC. Overview: the major histocompatibility complex and insulin dependent diabetes mellitus. Baillieres Clin Endocrinol Metab 1991;5:211--228. Thivolet CH, Tappaz M, Durand A, Petersen J, Stefanutti A, Chatelain P, Vialettes B, Scherbaum W, Orgiazzi J. Glutamic acid decarboxylase (GAD) autoantibodies are additional predictive markers of type 1 (insulin-dependent) diabetes mellitus in high risk individuals. Diabetologia 1992;35:570-576. Tian J, Lehman PV, Kaufman DL. T cell cross-reactivity between Coxsackievirus and glutamate decarboxylase is associated with a murine diabetes susceptibility allelle. J Exp Med 1994;180:1979--1984. Tisch R, Yang XD, Singer SM, Liblau RS, Fugger L, McDevitt HO. Immune response to glutamic acid decarboxylase correlates with insulitis in nonobese diabetic mice. Nature 1993;366:72--75. Tuomi T, Groop LC, Zimmet PZ, Rowley MJ, Knowles W, Mackay IR. Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with a noninsulin-dependent onset of disease. Diabetes 1993a;42:359-362. Tuomi T, Zimmet PZ, Rowley MJ, Serjeantson SW, Mackay IR. Persisting antibodies to glutamic acid decarboxylase in type 1 (insulin-dependent) diabetes mellitus are not associated with neuropathy. Diabetologia 1993b;36:685. Ujihara N, Daw K, Gianani R, Boel E, Yu L, Powers AC. Identification of glutamic acid decarboxylase autoantibody heterogeneity and epitope regions in type I diabetes. Diabetes 1994;43:968--975. Wagner R, Genovese S, Bosi E, Becker F, Bingley PJ, Bonifacio E, Miles KA, Christie MR, Bottazzo GF, Gale EA. Slow metabolic deterioration towards diabetes in islet cell antibody positive patients with autoimmune polyendocrine disease. Diabetologia 1994;37:365-371. Yu L, Gianani R, Eisenbarth GS. Quantitation of glutamic acid decarboxylase autoantibody levels in prospectively evaluated relatives of patients with type I diabetes. Diabetes 1994:43: 1229--1233.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

GLUTAMIC ACID DECARBOXYLASE AUTOANTIBODIES IN STIFF-MAN SYNDROME Daniel L. Kaufman, Ph.D.

Department of Molecular and Medical Pharmacology, Brain Research and Molecular Biology Institute, University of California, Los Angeles, CA 90095-1735, USA

HISTORICAL NOTES Glutamic acid decarboxylase (GAD) catalyzes the conversion of glutamate to gamma amino butyric acid (GABA), a widely utilized neurotransmitter throughout the central and peripheral nervous systems (reviewed in Kaufman and Tobin, 1993; Cooper et al., 1986; Erlander and Tobin, 1991; Erdo and Wolff, 1990). There has been intense interest in GAD due to the crucial role GABA plays in brain development and function, the possible involvement of alterations in GAD and GAB A in neurological diseases and the association of GAD autoimmunity with stiff-man syndrome (SMS) and insulin dependent diabetes mellitus (IDDM). Described over four decades ago in studies of neurotransmission in invertebrates (Roberts and Frankel, 1950; Awapara et al., 1950), brain GAD has been widely studied by biochemists, neurobiologists, molecular biologists and immunologists. This review will focus on GAD antibodies, with special emphasis on the antigen itself and on the clinical relevance of GAD autoantibodies in SMS.

THE AUTOANTIGEN Definition/Structure For many years, studies of GAD were hampered by the difficulty of purifying GAD from brain. Preparation of the first GAD antiserum used 9,000 mouse brains and conventional protein separation techniques to achieve a 700-fold purification of the antigen. The purified GAD had a native molecular weight of 85 kd

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by sedimentation equilibrium analysis; whereas, SDSPAGE analysis revealed components of 60 and 44 kd. To reconcile the different molecular weights of native and denatured GAD, a GAD hexamer of identical 15 kd subunits was proposed (Wu et al., 1973). This purified GAD provided the first antigen for generating polyclonal antisera to GAD and visualized GAD expression in the brain by immunohistochemistry (Matsuda et al., 1973; Saito et al., 1974; McLaughlin et al., 1974). Further experiments with this antisera also showed the association of GAD with synaptic vesicles in inhibitory neurons and provided detailed maps of GAB Aergic neurons in the brain (reviewed in Erlander and Tobin, 1991). In the ensuing years, brain GAD was purified by different laboratories and used to generate additional GAD antisera. However, the reported molecular weights of the purified GADs varied considerably, raising concerns about the molecular specificity of these antisera (reviewed in Erlander and Tobin, 1991). These antisera recognized generally similar patterns of neurons in immunohistochemical studies. The subtle differences in staining patterns between the antisera can now be attributed to their different specificity for the two forms of GAD and the different intracellular distribution of these two forms (Kaufman et al., 1991; Esclapez et al., 1994). Further resolution of the molecular weight of GAD and the specificity of the GAD antibodies awaited the isolation and molecular characterization of the GAD genes. The first gene for GAD, now known as GAD67, was isolated in 1986 when a GAD antiserum (Oertel et al., 1981) was used to screen a brain cDNA expression library in recombinant E. coli for the expression of an immunoreactive protein (Kaufman et al., 1986).

The identity of the putative GAD clone was confirmed when the recombinant bacteria were shown to synthesize a protein that actively converted glutamate to GABA. Immunoblot analysis of the antisera raised against GADs of different reported molecular weights revealed that they, in fact, recognized the same two major proteins in brain homogenates (now known to be GAD67 and GAD65), and all recognized the protein encoded by the recombinant cDNA (Kaufman et al., 1986). This GAD cDNA was used to express and subsequently purify GAD from recombinant E. coli to generate a polyclonal antibody highly specific for GAD67 (Kaufman et al., 1991). Experiments using this GAD67-reactive antibody together with a monoclonal antibody specific for the smaller form of GAD (now known to be GAD65) (Chang and Gottlieb, 1988) demonstrated that the two forms of GAD had different intracellular distributions and interactions with their cofactor (Kaufman et al., 1991). Although GAD67 is widely distributed throughout the neuron, GAD65 is transported to axon terminals and associates with membranes. And, while most of GAD67 is in an active holoenzyme form (saturated with its cofactor), less than half of GAD65 exists as holoenzyme. Extending previous observations, GAB A production was suggested to be regulated by modulation of GAD65 apo/holoenzyme levels in nerve terminals (Kaufman et al., 1991). Recent evidence suggests that the association of GAD with its cofactor may be regulated through phosphorylation of GAD (Bao et al., 1995).

specific for each form of GAD were raised from gene-specific synthetic peptides (Li et al., 1995). The amino terminus of GAD65, which is the most divergent region from GAD67, has been shown to target GAD65 to nerve terminals (Solemina et al., 1994a; Dirkx et al., 1995). Posttranslational modifications to GAD65 anchor the molecule to the cytosolic side of neuron synaptic vesicles and the small microvesicles of ~ cells (Christgau et al., 1991; 1992). There is, however, no evidence that either form of GAD is present on the cell surface as native protein for immunorecognition by autoantibodies. However, like all intracellular proteins, GAD is broken into peptides and displayed on the cell surface by the major histocompatibility complex (MHC) molecules for surveillance by T cells. If T cells recognize a GAD peptide presented by MHC on the cell surface, they may initiate or augment autoreactive T-cell responses (Kaufman et al., 1993; Tisch et al., 1993; Atkinson et al., 1992; 1994; Tian et al., 1994), as well as provide help for B cells, thus generating the GAD autoantibodies that are associated with SMS and IDDM. However, while I] cells are generally thought to express class I MHC molecules and can be made to induce class II MHC molecules, neurons are not thought to express either molecule (Pujol-Borrell et al., 1986; Joly and Oldstone, 1992).

Sequence/Information

There is scant evidence that humoral immunity to GAD is involved in the etiology or pathogenesis of SMS. Forty percent of SMS patients do not have autoantibodies. There do not appear to be differences in the symptoms of SMS patients with or without GAD antibodies. A few of the SMS patients who lack detectable GAD antibodies have been shown to have autoantibodies to another synaptic vesicle-associated neuronal antigen, amphiphysin. All of these patients were women affected by breast cancer, suggesting that the condition may have an autoimmune paraneoplastic basis (De Camilli et al., 1993, Folli et al., 1993). Autopsy examination of a few patients have shown some inflammation of the brain stem and spinal cord, but most reports have found no evidence of immunoreactivity to CNS components (Mitsumoto et al., 1991; Meinch et al., 1994). Moreover, of the 60% of SMS patients who have GAD antibodies, approxi-

In 1990, a second GAD gene (GAD65) was isolated using PCR techniques (Erlander et al., 1991). The two GAD genes have about 65% sequence similarity, are found on different chromosomes and encode polypeptides of 67 and 65 kd (Bu et al., 1992). There are no reported sequence differences between the GADs expressed in the brain and those expressed in peripheral tissues. Sequence analysis of another (third) putative GAD cDNA (Huang et al., 1990) suggests it is a homolog of myosin. Following the association of GAD autoimmunity with both SMS and IDDM (Solemina et al., 1988; Baekkeskov et al., 1990), biochemical characterization of GAD has proceeded at a rapid pace. Using the GAD65 and GAD67 predicted amino acid sequences, additional polyclonal and monoclonal antibodies

AUTOANTIBODIES Pathogenetic Role

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mately two-thirds have other autoimmune disorders, such as IDDM (primarily), Hashimoto's thyroiditis and Graves' disease; whereas, only a few percent of the GAD antibody-negative SMS patients have associated autoimmune diseases, indicating that a broad dysfunction of the immune system has occurred in SMS patients with GAD autoantibodies (reviewed in Gorin et al., 1990; Solemina and De Camilli, 1991; Ellis and Atkinson, 1996; Solemina et al., 1994b). Genetics

HLA typing of SMS patients revealed that nearly three-fourths of the patients carry the DQB*0201 allele, which is also a susceptibility allele for IDDM (Pugliese et al., 1993). SMS patients carry the DQB 1 *0602 allele and the related DQB 1"06 alleles at the same frequency as control individuals. These alleles are rarely found in IDDM patients and are considered to confer protection from IDDM. Among a limited number of SMS patients, those lacking DQB 1"06 have a greater tendency to have IDDM compared to those with this allele, suggesting that the presence of DQB 1"0602 and other DQB 1"06 alleles may protect SMS patients from developing IDDM.

CLINICAL UTILITY Disease Associations

Stiff-man syndrome (SMS) is a rare neurological disease that is characterized by muscle rigidity and painful spasms (Moersch and Woltman, 1957; Solemina et al., 1988; 1990; 1994b; Baekkeskov et al., 1990; Solemina and De Camilli, 1991; Ellis and Atkinson, 1996). Only a few hundred cases of SMS have been diagnosed since its first description. SMS primarily affects women, generally arises late in adulthood and often follows a slowly progressing course. In contrast, IDDM affects almost one in 300 individuals, usually occurs in childhood and affects both sexes equally. CNS dysfunction in SMS is suggested by the findings that agonist and antagonist muscle groups are often simultaneously contracted and the finding that GAB A agonists such as benzodiazepines sometimes ameliorate disease symptoms (reviewed in Solemina and De Camilli, 1991; Solemina et al., 1994b). Interestingly, about 10% of SMS patients also have epilepsy (Solemina et al., 1990). The discovery that SMS patients have autoantibodies

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which recognize a GAB Aergic pattern of neurons by immunohistochemistry, lead to the discovery that a major component of this antineuronal response is directed against GAD (Solemina et al., 1988). About 60% of SMS patients have GAD autoantibodies in both their serum and CSF (Solemina et al., 1990; 1994b). About 30% of SMS patients also have IDDM which led to the identification of GAD as the 64 kd autoantigen in IDDM (Baekkeskov et al., 1990). GAD antibodies in SMS differ from GAD autoantibodies in IDDM patients in a number of significant ways (Solimena et al., 1990; 1994b; Baekkeskov et al., 1990; Solimena and De Camilli 1991; Ellis and Atkinson, 1996; Butler et al., 1993). First, unlike the GAD antibodies of IDDM patients, the titers of GAD antibodies of SMS patients are very high. In fact, the sera of SMS patients can be used in immunohistochemistry and immunoblots at dilutions similar to those of experimentally raised GAD antisera. Second, GAD antibodies in SMS patients, recognize both conformational and linear GAD epitopes (Baekkeskov et al., 1990; Solemina and De Camilli, 1991; Solemina et al., 1994b; Butler et al., 1993). In contrast, about 97% of IDDM patients with GAD autoantibodies have detectable immunoreactivity only against native GAD (i.e., conformational epitopes) and not against denatured GAD (Baekkeskov et al., 1990). Indeed, the ability of SMS-associated GAD autoantibodies to recognize denatured GAD in immunohistochemical experiments and on immunoblots was key to the identification of GAD as the target autoantigen in SMS (Baekkeskov et al., 1990). Third, GAD antibodies in SMS recognize both GAD65 (predominantly) and GAD67. The immunoreactivity against GAD67 is only detectable through assays which preserve a conformationally dependent epitope (Butler et al., 1993). In contrast, only about 30% of IDDM sera recognize GAD67, although this could be due to cross-reactivity of GAD65 antibodies with similar epitopes on GAD67 Solemina and De Camilli, 1991; Kaufman et al., 1992; Ellis and Atkinson, 1996; Solemina et al., 1994b). Because GAD65 and GAD67 are most divergent near their amino terminus, one would predict that the specificity of GAD antibodies from SMS patients for GAD65 would be due to a dominant epitope(s) in the amino-terminal portion of GAD65. Indeed, one study found that most SMS sera recognize a GAD epitope formed by the first eight amino acids of GAD65 (Kim et al., 1994). Another study also localized a major epitope in the aminoterminal region; however, this study found that the

predominant epitope recognized by SMS sera was near the carboxy-terminus of GAD65 (Butler et al., 1993). While the carboxy-terminal is highly conserved between GAD65 and GAD67, this epitope is only present in GAD65. Deletion of a few amino acids on either side of this region causes loss of immunoreactivity, indicating that the epitope is conformation dependent. A third study localized the major GAD epitopes to the middle of GAD, overlapping with the co-factor binding site (Li et al., 1994). If a unique pattern of immunoreactivity to GAD65 epitopes can be identified in SMS patients, it might provide a diagnostic marker for SMS. Differences in the properties of GAD autoantibodies in SMS and IDDM patients might arise from differences in how GAD is presented to the immune system by [~ cells vs. neurons. [3 cells may be rapidly lysed, releasing native islet-cell GAD leading to GAD antibodies that react almost exclusively with conformational epitopes. Alternatively, differences in antigen presentation (due to differential antigen processing by the different cell types), the lack of MHC class I and accessory molecule expression on neurons, as well as the microenvironment of the CNS vs. the islet, might lead to priming of cells that recognize different GAD epitopes. Differences in the microenvironment in which GAD is presented to the immune system could also bias the type of T-cell response. For example, if factors in the brain favored a Th2 response to GAD (a T-cell subset that provides B-cell help), it might account for the large amounts of GAD65 autoantibodies observed in SMS patients. Autoantibodies to GAD are present in the CSF of all SMS patients who have GAD autoantibodies in their sera, but any possible relationship of GAD antibody-mediated impairment of GAD-containing neurons to disease manifestations is unclear (Solemina and De Camilli, 1991; Ellis and Atkinson, 1996). As an intracellular protein, GAD is not considered accessible to autoantibodies. In addition, incubation of SMS sera containing GAD autoantibodies with brain homogenates typically does not interfere with GAD enzymatic activity (Baekkeskov et al., 1990; Kaufman, unpublished observations). However, there is one report of antibodies in SMS sera interfering with GAD enzymatic activity (Bjork et al., 1994). Furthermore, impairment of GAB Aergic function appears to be limited to neurons involved in motor activity. In

contrast to SMS, GAD autoantibodies are not found in neurodegenerative diseases which involve GABAergic neurons, such as Huntington's chorea and cerebellar ataxia. If GAD autoimmunity is not an initiating or pathogenetically relevant factor, the high degree of association of GAD autoimmunity with SMS, but not other neurological diseases, suggests that GAD autoimmunity is a common secondary consequence of a disease process that is unique to SMS. On the other hand, a role for GAD autoimmunity in SMS pathogenesis is supported by the observations that plasmapheresis is beneficial to some SMS patients and that administration of GABA agonists (e.g., benzodiazepines) ameliorates the muscle rigidity. Immune system involvement in SMS pathogenesis is also suggested by the association of an HLA allele with SMS and the observation that the administration of steroids and immunoglobulins are beneficial to some patients. However, none of these treatments substantially affect the concentration of GAD autoantibodies in the serum or CSF (Solimena et al., 1994b; Ellis and Atkinson; 1996).

CONCLUSION The isolation and characterization of GAD begun by neuroscientists four decades ago has now led to new diagnostic tests for SMS and IDDM, based on the detection of GAD autoantibodies. Furthermore, analysis of GAD-reactive T cells has lead to a better understanding of autoimmune mechanisms and potential immunotherapeutics (Kaufman et al., 1993; Tisch et al., 1993; Atkinson et al., 1992; 1994; Lohmann et al., 1994). While the role of GAD autoimmunity in SMS is unclear, it is clear that autoimmunity to GAD is a major contributing factor to the pathogenesis of murine IDDM, as the early induction of tolerance to GAD prevents disease (Kaufman et al., 1993; Tisch et al., 1993). Several companies are now developing GAD-based diagnostic tests which are likely to be available in the near future. Thus, a long string of basic science discoveries over several decades and in several different scientific fields has led to a new generation of prediagnostics and potential therapeutics. See also AUTOANTIBODIES THAT PENETRATE INTO LIVING CELLS AND GAD AUTOANTIBODIES IN DIABETES MELLITUS.

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the 65-kda isoform of glutamic acid decarboxylase. J Biol Chem 1995;270:2241-2246. Ellis TM, Atkinson MA. Glutamic acid decarboxylase autoimmunity in stiff-man syndrome and insulin-dependent diabetes. Nature-Medicine 1996;(in press). Erdo SL, Wolff JR. 7-Aminobutyric acid outside the mammalian brain. J Neurochem 1990;54:363--372. Erlander MG, Tobin AJ. The structural and functional heterogeneity of glutamic acid decarboxylase: a review. Neurochem Res 1991;16:215--226. Erlander MG, Tillakaratne NJK, Feldblum S, Patel N, Tobin AJ. Two genes encode distinct glutamate decarboxylases with different responses to pyridoxal phosphate. Neuron 1991;7:91-100. Esclapez M, Kaufman DL, Tobin AJ, Houser CR. Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in the rat brain supports the concept of functional differences between the forms. J Neurosci 1994; 14:1834--1855. Folli F, Solimena M, Cofiell R, Austoni M, Tallini G, Fussetta G, Bates D, Cartlidge N, Bottazzo GF, Piccolo G, De Camilli P. Autoantibodies to a 128-kd synaptic protein in three women with the stiff-man syndrome and breast cancer. N Engl J Med 1993;328:546--551. Gorin F, Baldwin B, Tait R, Pathak R, Seyal M, Mugnaini E. Stiff-man syndrome: a GABAergic autoimmune disorder with autoantigenic heterogeneity. Ann Neurol 1990;28:71--75. Huang WM, Reed-Fourquet L, Wu E, Wu JY. Molecular cloning and amino acid sequence of brain L-glutamate decarboxylase. Proc Nat Acad Sci USA 1990;87:8491-8495. Joly E, Oldstone MB. Neuronal cells are deficient in loading peptides onto MHC class I molecules. Neuron 1992;8:1185--1192. Kaufman DL, McGinnis JF, Krieger NR, Tobin AJ. Brain glutamate decarboxylase cloned in )~gt-ll: Fusion protein produces gamma-aminobutyric acid. Science 1986:232:1138-1140. Kaufman DL, Houser CR, Tobin AJ. Two forms of the yaminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. J Neurochem 1991 ;56:720-723. Kaufman DL, Erlander MG, Clare-Salzler M, Atkinson M, Maclaren NK, Tobin AJ. Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus. J Clin Invest 1992;89:283--292. Kaufman DL, Tobin AJ. Glutamate decarboxylases and autoimmunity in insulin-dependent diabetes. Trends Pharmacol Sci 1993;14:107--109. Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GS, Robinson P, Atkinson MA, Sercarz EE, Tobin AJ, Lehmann PV. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993;366:69-72. Kim J, Namchuk M, Bugawan T, Fu Q, Jaffe M, Shi Y, Aanstoot JH, Turck CW, Erlich H, Lennon V, Baekkeskov S. Higher autoantibody levels and recognition of a linear NH2 terminal epitope in the auto antigen GAD65, distinguish stiff-man syndrome from insulin dependent diabetes mellitus.

J Exp Med 1994; 180:595-606. Li L, Hagopian WA, Brashear HR, Daniels T, Lernmark A. Identification of auto antibody epitopes of glutamic acid decarboxylase in stiff-man syndrome patients. J Immunol 1994;152:930-934. Li L, Jiang J, Hagopian WA, Karlsen AE, Skelly M, Baskin DG, Lernmark ~. Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequencespecific pep tide antibodies. J Histochem Cytochem 1995;43:53--59. Lohmann T, Leslie RDG, Hawa M, Geysen M, Rodda S, Londei M. Immunodominant epitopes of glutamic acid decarboxylase 65 and 67 in insulin-dependent diabetes mellitus. Lancet 1994;343:1606-1608. Matsuda T, Wu J-Y, Roberts E. Immunochemical studies on glutamic acid decarboxylase (EC4.1.1.15) from mouse brain. J Neurochem 1973;21:159--166. McLaughlin B, Wood J, Saito K, Barber R, Vaughn J, Roberts E, Wu J-Y. The fine structural localization of glutamate decarboxylase in synaptic terminals of rodent cerebellum. Brain Res 1974;76:377--391. Meinch H-M, Ricker K, Htilser P-J, Schmid E, Peiffer J, Solimena M. Stiff-man syndrome: clinical and laboratory findings in eight patients. J Neurol 1994;241:157-166. Mitsumoto H, Schwartzman MJ, Estes ML, Chou SM, La Franchise EF, De Camilli P, Solimena M. Sudden death and paroxysmal autonomic dysfunction in stiff-man syndrome. J Neurol 1991 ;238:91-96. Moersch FP, Woltman HW. Progressive fluctuating muscular rigidity and spasm ("stiff-man" syndrome): report of a case and some observations in 13 other cases. Mayo Clin Proc 1957;31:421-427. Oertel WH, Schmechel DE, Tappaz ML, Kopin IJ. Production of a specific antiserum to rat brain glutamic acid decarboxylase by injection of an antigen-antibody complex. Neuroscience 1981:6:2689--2700. Pugliese A, Solemina M, Awdeh ZL, Alper CA, Bugawan T, Erlich HA, De Camilli P, Eisenbarth G. Association of HLADQBI*0201 with stiff-man syndrome. J Clin Endocrinol Metab 1993;77:1550-1553.

Pujol-Borrell R, Todd I, Doshi M, Gray D, Feldmann M, Bottazzo GF. Differential expression and regulation of MHC products in the endocrine and exocrine cells of the human pancreas. Clin Exp Immunol 1986;65:128-139. Roberts E, Frankel S. Gamma-aminobutyric acid in brain: its formation from glutamic acid. J Biol Chem 1950;187:55-63. Saito K, Barber R, Wu J-Y, Matsuda T, Roberts E, Vaughn J. Immunohistochemical localization of glutamate decarboxylase in the rat cerebellum. Proc Natl Acad Sci USA 1974;71:269-273. Solemina M, Folli F, Denis-Donini S, Comi GC, Pozza G, De Camilli P, Vicari AM. Autoantibodies to glutamic acid decarboxylase in a patient with stiff-man syndrome, epilepsy, and type 1 diabetes. N Engl J Med 1988;318:1012--1020. Solemina M, Folli F, Aparisi R, Pozza G, De Camilli P. Autoantibodies to GABA-ergic neurons and pancreatic beta cells in stiff-man syndrome. N Engl J Med 1990;322:1555-1560. Solimena M, De Camilli P. Autoimmunity to glutamic acid decarboxylase (GAD) in Stiff-Man syndrome and insulindependent diabetes mellitus. Trends Neurosci 1991; 14:452--457. Solimena M, Dirkx R Jr, Radzynski M, Mundigl O, De Camilli P. A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region. J Cell Biol 1994a;126:331--341. Solemina M, Butler MH, De Camilla P. GAD, diabetes and stiff-man syndrome: some progress and more questions. J Endocrinol Invest 1994b;17:509-520. Tian J, Lehmann PV, Kaufman DL. T cell cross-reactivity between Coxsackie virus and glutamate decarboxylase is associated with a diabetes susceptibility allele. J Exp Med 1994; 180:1970-- 1984. Tisch R, Yang X-D, Singer SM, Liblau RS, Fugger L, McDevitt HO. Immune response to glutamic acid decarboxylase correlates with insulitis in nonobese diabetic mice. Nature 1993;366:72--75. Wu J-Y, Matsuda T, Roberts E. Purification and characterization of glutamate decarboxylase from mouse brain. J Biol Chem 1973;248:3029-3034.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

GLYCOLIPID (EXCLUDING GANGLIOSIDE) AUTOANTIBODIES Martin A. Winer, Ph.D. and Jeff W. Terryberry, B.S.

Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES

THE AUTOANTIGENS

to a lesser extent, glucuronic acid, uronic acid and mannose; the pentose saccharide inositol is also present on glycophospholipids. GSLs containing sialic acid (either N-acetyl or N-glycosyl neuraminic acids) form a special subclass, the gangliosides, which are discussed elsewhere in this volume (also see Tables 1 and 2). Variations in the composition of the carbohydrate moieties include alternate core structures, degrees of branching and various anomeric linkages (i.e., spatial variations in the attachment to an asymmetric carbon atom). The oligosaccharides can be modified further by sulfation, phosphorylation, amidation, lactonization and deacylation (Hakomori, 1983). In addition to classification according to their biochemical modifications (sulfolipids, phospholipids, neutral and acidic GSLs), glycolipids can be further subclassifled by the biosynthetic pathway (Table 3), i.e., the ganglio-, globo-, isoglobo-, lacto- and neolacto-series of GSLs (IUPAC-IUB Commission on Biochemical Nomenclature, 1977). Glycolipids are also categorized according to their cellular or functional distributions, such as blood group antigen, myelin, or tumor-associated glycolipids.

Definition and Nomenclature

Function

Glycolipids can be divided broadly into two classes based on the lipid moiety: glycoglycerolipids (generally considered a special class of phospholipids) and glycosphingolipids (GSLs). GSLs contain a long chain basic amine, typically sphingosine (C 18:0), and a fatty acid of 16 to 24 carbons, which together comprise ceramide. The ceramide portion shows microheterogeneity both in terms of chain length and degree of saturation. The lipids are variably glycosylated by one or more saccharides, including N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, fucose, and

As integral membrane components, glycolipids regulate cell membrane structural integrity (including myelination), cell adhesion and signal transduction. In neural tissue, glycolipids bind to adhesion molecule glycoproteins in myelin compaction (Needham and Schnaar, 1993). This heterophilic molecular interaction is mediated by noncovalent carbohydrate-carbohydrate binding. The HNK-1 epitope (sulfoglucuronylneolacto-series carbohydrates) is important in this cellcell adhesion phenomenon, and is found on glycolipids such as sulfoglucuronylparagloboside (SGPG)

Glycolipids were established as the major lipid component of brain tissue as early as 1874, but much of the characterization, including the initial investigations on hematologic (Yamakawa and Suzuki, 1952) and transformation-associated glycolipids (Hakomori and Murakami, 1968), was not accomplished until the 1950s and 60s (Hakomori, 1983; Ishizuka and Yamakawa, 1985). Early studies on blood group antigens (ABH, Lewis, Forssman, P group) established the antigenic determinants as the carbohydrate moieties of glycolipids. Transformation-associated glycolipid changes seen in cancer were identified soon after (Hakomori and Young, 1983). Antineuronal glycolipid autoreactivities found in central and peripheral nervous system inflammatory demyelinating diseases such as multiple sclerosis, motor neuropathy disease and Guillain-Barr6 syndrome are also well established (Quarles et al., 1986).

314

and s u l f o g l u c u r o n y l l a c t o p a r a g l o b o s i d e ( S G L P G ) , and on i m m u n o g l 0 b u l i n superfamily adhesion m o l e c u l e s such as m y e l i n - a s s o c i a t e d glycoprotein, N - C A M and P0 in central and peripheral nervous s y s t e m myelin, and L2 and J 1 on l y m p h o c y t e s and natural killer cells. G l y c o l i p i d s bind or affect important signal m o l e c u l e s such as selectins (Suzuki et al., 1993), and glycosphingolipid b r e a k d o w n products and metabolites act as second m e s s e n g e r s in signal transduction pathways.

G l y c o l i p i d s also regulate the activities of proinflamm a t o r y m o l e c u l e s such as leukotrienes and histamine (Lichey et al., 1994) as well as platelet adhesion ( C u n n i n g h a m et al., 1993). C h a n g e s in oligosaccharide and c e r a m i d e microh e t e r o g e n e i t y are d o c u m e n t e d with respect to n o r m a l d e v e l o p m e n t , t u m o r i g e n e s i s and neoplasia (Hakomori, 1981), cell activation (Kannagi et al., 1983), viral infections ( M a t s u d a et al., 1993) and neurological

Table 1. Glycans of Disease-Associated Glycolipid (Including Ganglioside) Antigens Glycolipid

Structure

digalactosyldiacylglyceride

Galp~l ---~6Galp[31---)3DAG

cerebroside (GalCer)

Gal~ 1~ 1Cer

sulfatide

SO4---~3Ga1131--~1Cer (Glc)

SGPG

SO4--~3GlcUA]31~ 3Gal]31--~4GlcNAc]31~ 3Gall31---~4Glc[31~ 1Cer

galactosylgloboside

Gall31~ 3GalNAc ]31---)3Galc~1---)4Gall31---~4Glc[31--~1Cer

globotriaosylceramide

Galc~l---) 4Gall]l---~4Glc~l~ 1Cer

sulfoglobotriaosylceramide

SO4---)3Gala 1---~4Gal131---~4Glc~ 1--~1Cer

globotetraosylceramide (globoside)

GalNAc ~ 1~ 3Gal~ 1---)4Gal~ 1---~4Glc~ 1---)1Cer

GalNAc-globotetraosylceramide

GalNac ]31~ 3GalNAc ~ 1--~3Gal~ 1---~4Gal131---~4Glc~ 1---)1Cer

neolactotetraosylceramide

Gal~ 1---)4GlcNAc[31---~3Gal~ 1---~4Glc[31~ 1Cer

Blood Group Antigens ABH blood antigens Aa

GalNAc o~1--~3Gal~ 1---)4GlcNAc~ 1~ 3Gal~ 1---~4Glc]31~ 1Cer 2 1" Fuc~l

Ab

GalNAc~ 1~ 3Gal]31---~4GlcNAc~ 1--~3Gall31--~4GlcNAc[31~ 3Gall31---~4Glc~ 1--~1Cer 2 1" Fuc~l

Ac

Fuc~l + 2 GalNAc ~ 1--~3Gall] 1--~4GlcNAc~ 1-~ 3Gall31--~4GlcNAc~ 1--~3Gal~ 1--~4Glc~ 1--~1Cer 6 q" GalNAc~ 1--~3Gall31--~4GlcNAc]31 2 1" Fuc~l

(continued) 315

Table 1. Continued Glycolipid

Structure

Ad

Fuc~l

$

2 GalNAc~ 1-~ 3 Gall31 --~4GlcNAc 131--~6Gall31 --~4GlcNAc 131--~3 Gall31 --~4Glc 131--~ 1Cer 3 GalNAc ~ 1-+ 3 Ga1131--~4GlcNAc 131--~3 Gall31 --~4GlcNAc 131 2

1, Fuc~l BI

Galc~ 1---~3 Gall31 ---~4GlcNAc 131---)3Gall31 --+4Glc [31--~ 1Cer 2

q,

Fucc~l B II

Gal~l ---)3Gall31 ---~4GlcNAc ~ 1---)3Gal l] 1---~4GlcNAc ~ 1~ 3Gal~ 1---~4Glc131--~ 1Cer 2 Fucal

HI

Fuc~l ---~2Gall31---~4GlcNAc 131~ 3Gal~ 1---~4Glc~ 1---)1Cer

H2

Fuc~l ---~2Gal ~ 1---~4GlcNAc ~ 1---~3Gal ~ 1---~4GlcNAc ~ 1---)3Gal~ 1---~4Glc~ 1~ 1Cer

H3

F u c a l ---)2Gal~ 1---~4GlcNAc ~ 1---)3 Gall] 1---~4GlcNAc 131~ 3Gall31 ---~4Glc131--~ 1Cer 6

q,

Fuc~ 1--~2Gal~ 1---~4GlcNAc ~ 1 Forssman antigen

GalNAc c~1~ 3 GalNAc ~ 1~ 3Gal~ 1---~4Gall] 1---~4Glcl] 1~ 1Cer

I antigen

Gal~ 1---~4GlcNAc 131---~6Gall31 --~4GlcNAc ~ 1~ 3Gall31 --)4Glc ~ 1---)1Cer 3

q,

Gal~ 1---~4GlcNAcl] 1 i antigen

Gall31 ---~4GlcNAc ~ 1--~3Gall31 ---~4GlcNAc ~ 1~ 3Gall31 --->4Glc~ 1~ 1Cer

Lewis a antigen (Le a)

Gall31 ---)3GlcNAc 131---)3 G al ~ 1---~4Glc~ 1---)1Cer 4

q,

Fuc~l Lewis x antigen (Le x)

Gall31 ---~4GlcNAc ~ 1---~3 G all31 ---~4Glc~ 1---)1Cer 3

?

Fucal Pr2 antigen

NeuAc c~2---~3Gal ~ 1---~4GlcNAc ~ 1---~3Gall31 ---~4GlcNAc [31~ 3 Gall31 ---)4Glc 131--~ 1Cer

Gangliosides

GA1

Gal~ 1--~3 GalNAc [31---)4Gal ~ 1---~4Glc[31~ 1Cer

(continued)

316

Table 1. Continued Glycolipid

GDIa

Structure NeuAc~2 ~ 3Gal~ 1~ 3 GalNAc [31-+4Gal ~ 1-+4Glc ~ 1---)1Cer 3

?

NeuAcc~2 GDlb

Gall31 --~3GalNAc[31 ---~4Gal[31---~4Glc~ 1--~ 1Cer 3

?

NeuAca2---~8NeuAc~2 GD 2

GalNAc ~ 1---~4Gal~ 1-+4Glc ~ 1-+ 1Cer 3

1" NeuAca2-+8NeuAcc~2 GD 3

NeuAc a2 --~8NeuAc c~2~ 3 Gall31 -+4Glc ~ 1~ 1Cer

GM l

Gall31 -+ 3 GalNAc 131---~4Gal131---~4Glc131-+ 1Cer 3

t"

NeuAco~2 GM 2

GalNAc 1]1-+4Gal~ 1-+4Glc l] 1~ 1Cer 3

?

NeuAcc~2 GM 3

NeuAc a2 -+ 3 Gall] 1--->4Glc131-+ 1Cer

GM4

NeuAc ~2 ~ 3 Gall31 ---)1Cer

GQlb

NeuAc c~2-+ 8NeuAca2 ~ 3Gal~ 1~ 3GalNAcGal ~ 1---~4Glc1]1-+ 1Cer 3

1" NeuAcc~2--+8NeuAc~2

GTIb

NeuAca2 ~ 3Gal~ 1~ 3 GalNAc ~ 1-+4Gall31 -+4Glc [31--> 1Cer 3

1" NeuAc~2-+8NeuAca2 GT 3

NeuAc-->NeuAc-->NeuAc2--> 3Gall] 1---~4Glc~ 1--~ 1Cer

hematoside (Hanganutziu-Deicher (H-D) antigen)

NeuGc~2 ~ 3 Gall] 1-+4Glcl] 1~ 1Cer

Sialyl Le a (SLe a)

NeuAc~2 ~ 3Gal~ 1--93 GlcNAc ~ 1-+3 Gal ~ 1-+4Glc ~ 1---)1Cer 4

1, Fucc~l Sialyl Le x (SLe x)

NeuAco~2--~3 Gall31 --+4GlcNAc 131-+ 3 Gall31 ---~4Glc131-+ 1Cer 3

1" Fucocl sialosylparagloboside (SPG, LM1)

3 NeuAc~2 --~6Gall] 1---~4GlcNA~ 1---)3Gal~ 1--~4Glc ~ 1~ 1Cer

317

Table 2. Observed Antiglycolipid (Including Ganglioside) Autoantibody Reactivities in Specific Disease States Disease State Hematologic antiphospholipid syndrome Gaucher's disease hemolytic anemia idiopathic thrombocytopenic purpura Wiskott-Aldrich syndrome Infectious chagasic cardiomyopathy chronic fatigue syndrome neuroborreliosis leprosy Inflammatory Graves' disease Hashimoto' s thyroiditis hepatitis Heymann's nephritis insulin-dependent diabetes mellitus kidney transplant mixed connective tissue disease rheumatoid and osteoarthritis systemic lupus erythematosus Neurological amyotrophic lateral sclerosis chronic inflammatory demyelinating polyradiculoneuropathy diabetic neuropathy Guillain-Barr6 syndrome Lambert-Eaton myasthenic syndrome lower motor neuron disease Miller Fisher syndrome motor neuropathy multiple sclerosis myelopathy neuropsychiatric systemic lupus erythematosus paraneoplastic sensory neuropathy paraproteinemic neuropathy primary polyneuropathy + monoclonal gammopathy sensory polyneuropathy + monoclonal gammopathy

Autoantigen sphingomyelin, p/Tja blood group antigen sulfatide Pr2, Me blood group antigen neolactotetraosylceramide, SLea, SLex I/i blood group antigen

sulfatide, GM~, GM 2, GM 3 sphingomyelin, GA~ GM 2, GM 3 sulfatide

Forssman antigen, GM~ Forssman antigen

neolactotetraosylceramide, GQlb, H-D antigen sulfoglobotriaosylceramide GT3 ABH blood group antigen GalNAc-globotetraosylceramide, neolactotetraosylceramide GalNAc-globotetraosylceramide globotriaosylceramide, globoside, neolactotetraosylceramide

GA1, GDla, GM1, GM 2 sulfatide, SGPG, GA~, GD1b, GM l, GM3, LM~

sulfatide, GM1 sulfatide, GalCer, SGPG, Forssman antigen, GA~, GD1a, GDlb, GD2, GD 3, GM~, GM 2, GM3, GQlb, GTlb, LM1 GDla, GTlb, LM1 GA1, GDlb, GM1 GDlb, GQlb, GTlb sphingomyelin, Forssman antigen, GA 1, GD1a, GD1b, GM~, GM 2, GM4, LM 1 sulfatide, GalCer, sphingomyelin, digalactosyldiacylglyceride, GDlb, GM1, GM 2, GM 3 GalCer sulfatide, GalCer, GA 1, GM~, GM 3 GM1 sulfatide, SGPG, Pr2 antigen sulfatide, GDlb, GM 1 GD1a, GDlb, GD 3, GM 3, GQlb, GT1b, LM1

(continued)

318

Table 2. Continued Disease State schizophrenia sudden deafness transverse myelitis Tumor-related adenocarcinoma breast cancer cervical cancer colon cancer glioma leukemia/lymphoma lung cancer hepatocarcinoma melanoma renal carcinoma

Autoantigen sphingomyelin, GA~, GM 1 sulfatide sulfatide

Le a antigen

sulfatide GA1 LeXantigen, H-D antigen GD2 globoside, GalNAc-globotetraosylceramide, neolactotetraosylceramide, GA 1 sulfatide, Forssman antigen, galactosylgloboside, ABH blood group antigens GM~ GD2, GD3, GM2, GM3 G D 2, G M 2

pathologies such as multiple sclerosis (Miyatani et al., 1990) and alcohol or drug abuse. Abnormal sphingolipid accumulation is seen in congenital lysosomal storage diseases and gangliosidoses, such as TaySachs, Nieman-Pick and Krabbe's diseases (Kaye et al., 1992). Glycolipids thus play a complex and important role in normal cellular function, as well as in neuroimmunology, immuno-oncology and autoimmune diseases (Table 2). The initial events of lymphocyte homing via selectin binding are regulated by the cell surface density of sulfolipids and sialylated Lewis x glycolipids. Metastatic cells mimic this mechanism of adhesion and extravasation (Takada et al., 1991); tumors can evade the host's antitumor immune response by shifting to secretion and expression of inhibitory glycolipids with subsequent deactivation of CD8 + T killer cells and activation of CD4 + T suppressor cells (Ladisch et al., 1994). Pathogens also utilize molecular mimicry to evade

the host's immune surveillance mechanism and maintain chronic infections. This is exemplified by the molecular mimicry between Mycobacterium and sulfatide (Wheeler et al., 1994) and spirochetes and GSLs (Garci~i-Monc6 et al., 1993). In addition, toxigenic bacteria produce glycolipid-binding toxins and viruses can bind GSLs directly. Infections can also polyclonally activate the proliferation of autoantibody-secreting B cells, and thus can upregulate natural antiglycolipid antibody responses that can further deregulate the glycolipid network and its influence on cellular immunity. Purification and Detection Glycolipids are usually isolated by chromatography on ion exchange resins that separate the lipids based on carbohydrate electrostatic and hydrogen-bonding interactions with the resin. Separations based on reverse-phase hydrophobic interactions are also

Table 3. Classification of Glycosphingolipids by Carbohydrate Core Structure Series

Core Structure

ganglio -

GalNAc 131-->4Gall31-+3 GalNAc l] 1-+4Gall] 1-->4Glc

globo -

Gall] 1--~3GalNAc l] 1~ 3GaRz1-+4Gall31-+4Glc

isoglobo -

GalNAc 131-->3Galo~1~ 3Gal~ 1--~4Glc

lacto -

(Gall]l 3GlcNAc)n~I-+3GalI31-->4Glc

neolacto -

(Gall31---~4GlcNAc)n131--~3Gall] 1-+4Glc

319

Table 4. Estimated Clinical Sensitivities of Antiglycolipid Autoantibodies Associated with Specific Pathologies Glycolipid

Pathology

Sensitivity*

Sulfatide

chagasic cardiomyopathy diabetic neuropathy leprosy neuropsychiatric systemic lupus erythematosus paraproteinemic neuropathy sudden deafness Guillain-Barr6 syndrome primary biliary cirrhosis + neuropathy chronic inflammatory demyelinating polyneuropathy transverse myelitis

100 88 86 50 50 50 43 25 20 20

Neolactotetraosylceramide

hepatitis leukemia/lymphoma idiopathic thrombocytopenic purpura mixed connective tissue disease systemic lupus erythematosus

57 33 31 17 17

Galactocerebroside

neuropsychiatric-systemic lupus erythematosus multiple sclerosis Guillain-Barr6 syndrome

25 9

GalNAc-globotetraosylceramide

rheumatoid and osteoarthritis leukemia/lymphoma mixed connective tissue disease

55 42 17

Sphingomyelin

chronic fatigue syndrome multiple sclerosis schizophrenia

23 20 18

Sulfoglucuronylparagloboside (SGPG)

paraproteinemic neuropathy Guillain-Barr6 syndrome chronic inflammatory demyelinating polyneuropathy

50 15 10

Forssman

Guillain-Barr6 syndrome

Galactosylgloboside

lung cancer

Phenolic glycolipid I-III

leprosy

81

5 17 100

*Estimated clinical sensitivities; diagnostic cut-off = mean + 2SD of normal controls.

utilized. To ensure a high level of purity for a particular glycolipid, multiple chromatographic steps are employed. Preparative thin-layer chromatography (TLC) also can be used to isolate glycolipids. TLC is useful for one or two-dimensional mapping of components in a complex mixture and for confirming the identity and purity of glycolipids. TLC requires the proper selection of solvent mixtures, as well as an efficient detection method. Resorcinol- and orcinolbased colorimetric stains are widely employed for detecting GSLs, while azure A can detect sulfolipids.

320

THE AUTOANTIBODIES

Natural antiglycolipid allo- and autoantibodies exist in the normal immune network (Kaise et al., 1985) and might function in clearing cellular debris, as a firstline antimicrobial host defense mechanism (phagocytosis and opsonization), in the regulation of hematopoiesis, and in further refining the effects of glycolipids on cell signaling (Hakomori, 1981). Both the lipid and carbohydrate domains can serve as antigenic determinants; antibodies against carbohydrate epitopes often cross-react with similar carbohydrate domains

occurring in glycoproteins, particularly in the case of blood group and myelin antigens.

Pathogenetic Role In idiopathic demyelinating polyneuropathies, elevated levels of autoantibodies are derived from either polyclonal activation due to antecedent or multiple infections, or from paraproteinemic gammopathies. Such increased concentrations promote tissue deposition and subsequent proinflammatory complement activation, cytokine and eicosanoid alterations and cytotoxic cellular immunity; this can result in destruction of Schwann cells, oligodendrocytes, dorsal root neurons or other target cells (Hughes, 1994). With continued inflammation, autospecific affinity maturation can ensue and lead to selective secretion of highaffinity pathogenic IgG autoantibodies. Neuropathy-associated paraproteins (usually IgM) often possess increased pathogenic concentrations and/or affinities. Acute neuropathies such as the autoimmune inflammatory demyelinating polyradiculoneuropathy and acute motor axonal neuropathy forms of Guillain-Barr6 syndrome show a rapid increase in autoantibody concentration, which then declines due to transient autoantibody production, reinstated T suppressor function and compensatory anti-idiotype antibodies (Sun, 1993). However, with continued antigen-driven responses due to recurrent infections, relapses can evolve to a chronic progressive course, as demonstrated in chronic inflammatory demyelinating polyradiculoneuropathy. Direct evidence of a pathologic role of antiglycolipid antibodies in neurologic disease is exemplified by cerebroside (GalCer)-induced experimental allergic neuritis and trypanosomiasis as well as by the administration of anti-GalCer which causes demyelination (Hughes, 1994). While it is increasingly clear that antiglycolipid antibodies mediate target-cell cytotoxicity (i.e., antibody-dependent complementmediated cytotoxicity (ADCC)-induced demyelination and axonal degeneration) and cellular dysimmunity, the exact requirements for glycolipid immunogenicity are obscure (Ishizuka and Yamakawa, 1985).

Molecular Mimicry/Cross-Reactivity. In chronic neuropathies, neurotropic viruses (herpes-, entero-, influenza, corona- and paramyxoviruses) that develop latent or abortive infections in nervous or neuroendocrine tissue can lead to continued sensitization to a self antigen via molecular mimicry and/or antigen

shedding. In multiple sclerosis, virus particles incorporate myelin-derived GalCer into their envelopes during budding and thus can promote the development of demyelinating lesions via an antigen-driven response (Pathak et al., 1990). Anti-GalCer autoantibodies are found in multiple sclerosis cerebrospinal fluid (Kasai et al., 1986) as well as in neuropsychiatric systemic lupus erythematosus (Costallat et al., 1990). Antibodies to digalactosyl-diacylglycerol are also found in multiple sclerosis (Ishizuka and Yamakawa, 1985). Initial polyclonal humoral activation can result in highly cross- and/or polyreactive antiglycolipid antibodies, wherein the primary cognate antibody paratope involves mostly electrostatic binding, as exemplified by antibodies directed against the mycobacterial phenolic glycolipids (PGL-I through III) that cross-react with cardiolipin, sulfatide, DNA and other polyanions (Shoenfeld et al., 1986). Other pathogens (i.e., T. cruzi) can induce antisulfatide antibodies cross-reactive with neural tissue and skeletal and cardiac muscle, resulting in cardiomyopathy and Chagas' disease (Avila et al., 1993). The specificity of antiglycolipid antibodies is thus highly variable. For example, antibodies to digalactosyl-diacylglycerol do not cross-react with GalCer, while anti-galactosyldiacylglycerol antibodies obtained from rabbits immunized with Treponema galactosyl-diacylglycerol cross-react with spinal cord galactosyl-diacylglycerol, galactosyl-alkylacylglycerol and GalCer (Ishizuka and Yamakawa, 1985). Monoclonal antiglycolipid antibodies, including Mproteins, also show patterns of cross-reactivity that are useful in subclassifying neuropathies. Similarities exist in the isotype and specificity of human monoclonal antisulfoglycolipid antibodies produced in vitro from Epstein-Barr virus-transformed lymphoblastoid cells derived from multiple sclerosis patients and those obtained from proteinemic neuropathies (Kirschning et al., 1995). Epstein-Barr virus is implicated in the in vivo development of several lymphoproliferative disorders; it can also trigger Guillain-Barr6 syndrome and myelitis. Other human antisulfolipid monoclonal antibodies have been derived from cancer patients (Miyake et al., 1992).

Methods of Detection Antiglycolipid antibodies are most often detected by enzyme-linked immunosorbent assays (ELISA). However, highly purified antigen preparations are necessary to pinpoint antibody specificity in ELISA. 321

Crude or concocted glycolipid mixtures can be used in ELISA and in TLC with immunofixation for screening for antiglycolipid reactivity. High performance TLC is the method of choice to confirm ELISA autoreactivity results. Various improvements and modifications have generated highly sensitive ELISA techniques that can detect natural antiglycolipid allo- and autoantibodies, as well as antibodies (murine, human or chimeric) used in cancer immunotherapy (Harada et al., 1992). Establishment of standardized cut-off values in terms of titer or arbitrary units per liter (AU/L) is crucial to the definition of pathologic antiglycolipid autoantibodies. Alternatively, glycolipid antibodies can be detected by liposome immune lysis assays (LILA), a method that employs fluorescent dye-loaded liposomes incorporating the presumptive glycolipid antigen. Upon addition of the biological fluid to be tested (i.e., serum, CSF, synovial fluid), complement-mediated lysis of the liposomes and subsequent release of the fluorescent dye indicates the presence of antiglycolipid antibodies (Yasuda et al., 1981). Antiglycolipid antibodies can be purified using glycolipid-coated or neoglycoconjugatederivatized affinity matrices.

CLINICAL UTILITY

Disease Association Detection of specific antiglycolipid antibodies in biological tissues and fluids is useful for diagnosis of a variety of neurological, oncogenic and autoimmune diseases (Table 4), including lower motor neuron syndromes, sensory and/or motor peripheral neuropathies (Pestronk et al., 1991), tumors (Kaise et al., 1985), liver diseases, systemic lupus erythematosus with and without neuropsychiatric manifestations (Kaise et al., 1985; Costallat et al., 1990), multiple sclerosis (Kasai et al., 1986), leprosy (Wheeler et al., 1994) and chagasic cardiomyopathy (Avila et al., 1993). Other antiglycolipid-mediated autoimmune diseases include idiopathic thrombocytopenic purpura and hemolytic anemia involving pathogenic antiblood group glycolipid antibodies (Murakami et al., 1991), antiphospholipid syndrome where thrombosis, infarcts and recurrent fetal loss are mediated in part by antiphospholipid and antiblood group glycolipid autoantibodies (Lindstrom et al., 1992), and experimental Heymann's nephritis, where kidney tubule glycolipids are targeted (Susani et al., 1994). In 322

addition to direct assessment for the presence of autoantibodies, antiglycolipid antibodies provide sensitive probes for detection of tumors (Miyake et al., 1992) as well as bacterial (Garcifi-Monc6 et al., 1993) and viral infections (Pathak et al., 1990). Autoreactivity to glycolipids is seen in up to 50% of idiopathic neuropathies and in systemic autoimmunity with or without neurologic overlap and neuroinfections; these autoantibodies are usually of higher titer than those found in normal sera. Elevated antisulfatide IgG is found in several peripheral mixed sensorimotor, sensory and autonomic neuropathies, including Guillain-Barr6 syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, diabetic neuropathy, neuropsychiatric systemic lupus erythematosus, nerve-mediated deafness and primary biliary cirrhosis and neuropathy as well as transverse myelitis and multiple sclerosis (Terryberry et al., 1995). Whether antisulfatide antibodies predispose systemic autoimmunity patients to develop neurologic involvement is unclear. Antisulfatide IgM can be found in sensory polyneuropathy with monoclonal gammopathy of undetermined significance (MGUS), where a paraprotein directed to the sulfate moiety cross-reacts in some cases with myelin-associated glycoprotein, P0, SGPG, SGLPG and sulfatide (Pestronk et al., 1991). However, differences in fine specificities exist between autoantibodies directed against epitopes of myelin-associated glycoprotein, SGPG and sulfatides (Brouet et al., 1992). Sulfolipids are also the targets for some antitumor antibodies and natural auto/alloantibodies directed against blood group glycolipids (Miyake et al., 1992). Cross-reactivity of blood group glycolipids to nerve glycolipids has been shown in Prld- and Pr2-specific paraproteinemic neuropathy (Willison, 1993). Responses to lacto-, neolacto-, and globo-series neutral GSLs (including Lewis a and x, and Forssman glycolipids) occur in the natural antibody repertoire, and can be expanded in some instances of Graves' disease, cancer and neuropathy. Certain phosphoglycolipids such as sphingomyelin show antigenicity in systemic lupus erythematosus, schizophrenia and chronic fatigue and immune dysfunction syndrome (Terryberry et al., 1995).

Therapeutic Utility Antibody-based immunotherapy of cancers indicates that antitumor glycolipid antibodies are effective in causing tumor regression (Minasian et al., 1994); this

may necessitate laboratory monitoring of antibody levels. Immunomodulating therapies for neuropathies such as intravenous immunoglobulins, plasmapheresis and corticosteroid treatment also require monitoring for decreasing antiglycolipid antibody levels as a putative marker of recovery.

CONCLUSION The clinical utility of antiglycolipid antibodies is expanding; applications are being found in chronic infections, systemic autoimmunity and neuropathy. The detection of natural and pathogenic allo- and autoantibodies in healthy individuals as well as in cancer and autoimmunity patients is also receiving increasing coverage; appropriate investigations into what constitutes a pathogenic antiglycolipid response are currently being pursued. It has been established that antisulfatide, anti-GalCer and anti-SGPG are important for differentiating chronic and acute sensori-

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Pestronk A, Li F, Griffin J, Feldman EL, Cornblath D, Trotter J, Zhu S, Yee WC, Phillips D, Peeples DM, Winslow B. Polyneuropathy syndromes associated with serum antibodies to sulfatide and myelin-associated glycoprotein. Neurology 1991;41:357-362. Quarles RH, Ilyas AA, Willison HJ. Antibodies to glycolipids in demyelinating diseases of the human peripheral nervous system. Chem Phys Lipids 1986;42:235--248. Shoenfeld Y, Vilner Y, Coates ARM, Rauch J, Lavie G, Shaul D, Pinkhas J. Monoclonal antituberculosis antibodies react with DNA, and monoclonal anti-DNA autoantibodies react with Mycobacterium tuberculosis. Clin Exp Immunol 1986;66:255-261. Sun JB. Autoreactive T and B cells in nervous system diseases. Acta Neurol Scand 1993;142:S1-$56. Susani M, Schulze M, Exner M, Kerjaschki D. Antibodies to glycolipids activate complement and promote proteinuria in passive Heymann nephritis. Am J Pathol 1994;144:807-819. Suzuki Y, Toda Y, Tamatani T, Watanabe T, Suzuki T, Nakao T, Murase K, Kiso M, Hasegawa A, Tadano-Aritomi K, Ishizuka I, Miyasaka M. Sulfated glycolipids are ligands for a lymphocyte homing receptor, L-selectin (LECAM-1), binding epitope in sulfated sugar chain. Biochem Biophys Res Commun 1993;190:426-434. Takada A, Ohmori K, Takahashi N, Tsuyuoka K, Yago A, Zenita K, Hasegawa A, Kannagi R. Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen sialyl Lewis A 1. Biochem Biophys Res Commun 1991;179:713--719. Terryberry J, Sutjita M, Shoenfeld Y, Gilburd B, Tanne D, Lorber M, Alosachie I, Barka N, Lin H-C, Youinou P, Peter JB. Myelin- and microbe-specific antibodies in Guillain-Barr6 syndrome. J Clin Lab Anal 1995;9:308--319. Wheeler PR, Raynes JG, McAdam KP. Autoantibodies to cerebroside sulphate (sulphatide) in leprosy. Clin Exp Immunol 1994;98:145--150. Willison HJ, Paterson G, Veitch J, Inglis G, Barnett SC. Peripheral neuropathy associated with monoclonal IgM anti Pr 2 cold agglutinins. J Neurol Neurosurg Psychiatry 1993;56: 1178--1183. Yamakawa T, Suzuki S. The chemistry of the lipids of posthemolytic residue or stroma of erythrocytes. III. Globoside, the sugar-containing lipid of human blood stroma. J Biochem 1952;39:393--402. Yasuda T, Naito Y, Tsumita T, Tadakuma T. A simple method to measure antiglycolipid antibody by using complementmediated immune lysis of fluorescent dye-trapped liposomes. J Immunol Methods 1981;44:153-158.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. ' J.B. Peter and Y. Shoenfeld, editors.

GOLGI APPARATUS AUTOANTIBODIES Gilles Renier, M.D. a, Marvin J. Fritzler, M.D. b and Alain Chevailler, M.D. a

aLaboratoire d'Immuno-Pathologie, Centre Hospitalier Universitaire d'Angers, Cedex 01, France; and bDepartment of Medicine, The University of Calgary, Calgary, Alberta T2N 4N1, Canada

H I S T O R I C A L NOTES First described in 1982 in a patient with Sj6gren's syndrome and lymphoma (Rodriguez et al., 1982), autoantibodies directed against the Golgi apparatus (AGAA) were subsequently found during routine examination of patients' sera and as a result of a few systematic surveys (Renier et al., 1994a). Some of the autoantigens are now characterized (Fritzler et al., 1993; Kooy et al., 1992; 1994; Rios et al., 1994; Seelig et al., 1994a; Sohda et al., 1994). In a murine model, AGAA were induced by an isolate of lactate dehydrogenase-elevating ;virus (LDV) (Weiland et al., 1987).

THE AUTOANTIGEN(S)

Origin/Sources The Golgi apparatus is a complex cytoplasmic organelle that has a prominent function in the processing, transporting and sorting of intracellular proteins (Gonatas, 1994; Mollenhauer et al., 1994; Nilsson and Warren, 1994). Structurally, the Golgi apparatus is localized in the perinuclear region of most mammalian cells and is characterized by stacks of membranebound cisternae as well as a functionally distinct trans-Golgi network (Mollenhauer and Morre, 1994). The intracellular transport of newly synthesized and recycled proteins requires directed movement of intracellular vesicles between the endoplasmic reticulum and the cis-, medial- and trans-compartments of the Golgi complex and the plasma membrane (Nilsson and Warren, 1994). The signals and molecular characteristics of the proteins that control this intracellular

traffic are poorly understood, but intracellular microtubules are known to be important structural and functional components (Nilsson and Warren, 1994; Kreis, 1990). Other components of the Golgi apparatus believed to play a role in these processes include families of proteins such as the adaptins, the 'coatomer' proteins, GTP-binding proteins, including ADP ribosylation factors, and resident enzymes (Nilsson and Warren, 1994).

Methods of Purification Among several approaches to the purification of the Golgi complex (Seelig et al., 1994a; Marks et al., 1994), most rely on rodent liver as the source of Golgi proteins, but the autoantigens are not tissue specific and are evolutionarily conserved (Renier et al., 1994b). These antigens have not, however, been systematically studied in various Golgi complex preparations. Immunoelectron microscopy studies suggest that the autoantigens reside in the membrane of cisternal and vesicular Golgi structures (Hong et al., 1992; Kooy et al., 1992; Renier et al., 1994b; Rios et al., 1994).

Sequence/Information Although recognized as a target of autoantibodies for almost a decade, only in the last three years were any of the Golgi apparatus autoantigens characterized and sequenced (Table 1). Golgins 95 and 160, the first Golgi complex autoantigens to be cloned (Fritzler et al., 1993), were followed by reports identifying giantin as another Golgi autoantigen (Seelig et al., 1994b; Linstedt and Hauri, 1993). Macrogolgin was reported to be a new Golgi autoantigen (Seelig et al.,

325

Table 1. Cloned Golgi Complex Autoantigens Name

MW native protein (kd)

Disease screening Ab*

Features

Accession Number

Reference

golgin 95

95

SLE/cerebellar ataxia

coiled-coil

L06147

Fritzler et al., 1993

golgin 160

160

SLE/cerebellar ataxia

coiled-coil

L06148

Fritzler et al., 1993

golgin 97

97

glomerulonephritis

coiled-coil

n/s

Fritzler et al., 1994

golgin 180

180

SjOgren's syndrome

coiled-coil

n/s

Fritzler et al., 1994

macrogolgin**

376

Sj6gren's syndrome

coiled-coil

X75304

Seelig et al., 1994

GCP372**

372

rheumatoid arthritis

coiled-coil

D25542

Sohda et al., 1994

Abbreviations: SLE: systemic lupus erythematosus n/s: not submitted * diagnosis of patient whose antibody was used for screening cDNA library ** macrogolgin, giantin and GCP372 are likely identical.

1994a) but was subsequently found to be identical to giantin (Seelig et al., 1994b). A 372 kd Golgi complex autoantigen identified as GCP372 (Sohda et al., 1994) was found to have >95% identity to giantin when sequences were aligned. A 261 kd autoantigen is also being characterized (Seelig et al., personal communication) and various bands detectable by immunoblotting are also reported (Hong et al., 1992; Kooy et al., 1994; Renier et al., 1994b; Rios et al., 1994; Rossie et al., 1992).

THE AUTOANTIBODIES Terminology Because AGAA tend not to be tissue specific, a wide range of tissue substrates (e.g., kidney, liver) produce a Golgi staining pattern (Fritzler et al., 1984; Renier et al., 1994b; Rios et al., 1994; Rodriguez et al., 1982). The isotype is predominantly IgG, especially when associated with viral infections. The terminology AGAA should be preferentially used because antibodies reacting with Golgi cells on brain sections are also reported (Greenlee et al., 1988). Because the Golgi apparatus plays a major role in the processing of synthesized proteins, the appellation "antibody" should be particularly restricted to the cases with proved antibody activity. The IgG F(ab') 2 fragments prepared in two cases retained the full antibody reactivity (Rodriguez et a1.,1982; Renier et al., 1994b), ruling out nonspecific binding such as carbohydrate interactions, e.g., several murine ascites monoclonal

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antibody preparations contain contaminants that bind to a blood group A-related epitope localized in the Golgi complex (Spicer et al., 1994).

Pathogenetic Role Animal Model. AGAA can be detected in STU mice as early as seven days after lactic dehydrogenase virus injection and a week before antiviral antibodies. After a peak of reactivity at day 16 post infection, the antibody activity decreases but remains detectable on certain target cells throughout the whole observation period (Grossmann et al., 1989). Detected only after infection with live LDV but not after immunization with inactivated virus, AGAA do not show any crossreactivity with antiviral antibodies (Grossmann and Weiland, 1991). All of the six mouse strains studied developed AGAA after LDV injection but with different autoantibody titers (Weiland et al., 1987). There is no evidence for a direct pathogenic role of the AGAA in human diseases or in animal models. Although LDV causes lifelong viremia in infected mice and alters a variety of immune functions, no overt autoimmune disease is reported in STU mice (Grossmann et al., 1989). Moreover, these autoantibodies appear to be part of the normal B-cell repertoire (Underwood et al., 1985). H u m a n Model. It is worth noting that all the viruses found in association with AGAA (Table 2) acquire an envelope in the Golgi apparatus (Grief et al., 1991; Griffiths and Rottier, 1992). On the other hand, the Golgi apparatus is also involved in antigen processing,

Table 2. Diseases with High Frequencies of AGAA in Population-Based Studies Diagnosis

Positive patients (%)

Normal controls

10

Sj6gren's syndrome

40.5

Virus Infections Cytomegalovirus Epstein-Barr virus (infectious mononucleosis) HIV-1 Rubeola

35.5 33 36 19.5

(Blaschek et al., 1988; Gentric et al., 1991; Huibtichel et al., 1991)

and macrophage Golgi complexes are particularly reactive with human autoantibodies (Fritzler et al., 1984). Virus-induced AGAA may be clinically relevant to the study of this organelle as a target of autoantibodies. Methods of Detection

By indirect immunofluorescent (IIF) techniques, AGAA display a characteristic fluorescent staining located in a limited region of the cytoplasm just outside the nuclear membrane (Figure 1). On a routine basis, commercially available HEp-2 cells perform very well. Similar results can be obtained on other

Figure 1. Characteristic staining pattern of anti-Golgi apparatus autoantibodies as found by immunofluorescence on HEp-2 cells (original magnification x 400).

tissue culture cells but not with rat organ sections. In a study of over 100 sera with AGAA detected on HEp-2 substrates, <75% of the sera produce a detectable anti-Golgi staining pattern on cryopreserved sections of mouse liver, kidney or stomach (Fritzler et al., unpublished results). In fact, adherent cells such as HEp-2, HeLa or MRC5 cells are larger and have a large spreading cytoplasm, and most AGAA are devoid of specificity for a particular substrate. A notable exception are antibodies directed to the Purkinje cell (Yo) antigen where cryopreserved sections of cerebellum are a more sensitive and specific substrate. The Yo autoantigen may be a glycoprotein processed through the Golgi complex rather than a true Golgi apparatus component (Rodriguez et al., 1988). Because the gerbil fibrosarcoma cell line IMR-33 used in some population-based surveys has higher sensitivity (Blaschek et al., 1988), a panel of various cell lines should be considered for population-based studies. The use of F(ab') 2 fragments of antihuman immunoglobulins as the detector reagent should be useful for increasing sensitivity by minimizing background staining so that low titer antibodies are easier to observe. Unfortunately, the cost of F(ab') 2 detector antibodies hampers their routine use. The precise location of the AGAA binding (membrane or intraluminal, in the cis-, medial- or transsubcompartments of the Golgi complex) can be further determined by immunoelectron microscopy. The molecular weight of their target can be estimated by immunoblotting (Hong et al., 1992; Rossie et al., 1992; Kooy et al., 1994; Renier et al., 1994b; Rios et al., 1994). Although easily prepared whole cell extracts can be used, they will hamper the interpretation when other autoantibodies (especially antinuclear) are associated with AGAA. In this case, the use of

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Golgi complex purified fractions is recommended (Seelig et al., 1994a; Marks et al., 1994). Immunodiffusion, counterimmunoelectrophoresis and ELISA techniques have not been appropriately developed to detect anti-Golgi antibodies. Future studies are likely to determine if there are clinical and technical advantages to the use of recombinant proteins or purified Golgi complex proteins.

CLINICAL UTILITY

Disease Association AGAA, although only rarely found in routine examination of sera (overall frequency between 1/500 and 1/800), are usually at high levels and in many cases associated with other antibodies, especially antinuclear antibodies of various specificities. One group of AGAA-positive patients (Table 3) have systemic diseases; Sj6gren's syndrome, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are the most often reported diagnoses, with overlap syndromes in many cases (Fritzler et al., 1984; 1993; Hong et al., 1992; Kooy et al., 1992; Renier et al., 1994a; Rios et al., 1994; Rodriguez et al., 1982; Rossie et al., 1992; Seelig et al., 1994a; Sohda et al., 1994). No correlation with disease activity is reported.

In one case the AGAA were first discovered during recrudescence of the patient' s rheumatoid arthritis, but no variation in the titer was observed in a second sample obtained two years later (Renier et al., 1994b). A second group of patients present with cerebellar disorders (Fritzler et al., 1993; Gaspar et al., 1988) or malignancies (Rodriguez et a1.,1982; Mohan et al., 1991; Renier et al., 1994b). Paraneoplastic cerebellar degeneration is the typical example of these cases. Indeed, Golgi labeling by a subset of the Purkinje cell antibodies is recognized (Rodriguez et al., 1988; Tanaka et al., 1992). Of interest, patients with Sj6gren's syndrome can develop central nervous system disease including cerebellar degeneration (Terao et al., 1994). Finally, AGAA are also reported in some miscellaneous conditions such as hepatitis or glomerulonephritis (Fritzler et al., 1984; Mohan et al., 1991). AGAA at low titer are also found in populationbased surveys (Blaschek et al., 1988; Gentric et al., 1991; Huibuchel et al., 1991). Because the methods used in these experiments detected antibodies in low concentrations, AGAA were found in up to 10% of normal controls. With similarly sensitive assay conditions, AGAA are found especially in Sj6gren's syndrome and in various infections due to enveloped viruses (Table 2). With the renal cancer SK-RC11 cell line, AGAA are also reported in Wegener's granulomatosis (Mayet et al., 1991).

Table 3. AGAA in the 30 Individual Cases Reported Disease Systemic Diseases Sji3gren' s syndrome

systemic lupus erythematosus rheumatoid arthritis scleroderma polymyositis bullous pemphigoid

References Fritzler et al., 1984; 1994; Kooy et al., 1994; 1992; Renier et al., 1994; Rios et al., 1994; Rodriguez et al., 1982; Rossie et al., 1992; Seelig et al., 1994. Fritzler et al., 1984; 1993; Hong et al., 1992; Rossie et al., 1992. Fritzler et al., 1984; Hong et al., 1992; Renier et al., 1994; Seelig et al., 1994; Sohda et al., 1994 Seelig et al., 1994a Rossie et all., 1992 Kooy et al., 1992

Cerebellar dysfunction and/or malignancies cerebellar ataxia paraneoplastic cerebellar degeneration lymphoma adenocarcinoma

Fritzler et al., 1993; Gaspar et al., 1988 Rodriguez et al., 1988; Tanaka et al., 1992 Mohan et al., 1991; Rodriguez et al., 1982 Renier et al., 1994

Miscellaneous glomerulonephritis hepatitis others

Fritzler et al., 1994; Mohan et al., 1991 Fritzler et al., 1984; Mohan et al., 1991 Mohan et al., 1991; Renier et al., 1994

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CONCLUSION The structural and functional complexity of the Golgi apparatus is mirrored by the diversity of the findings about AGAA. More data are needed before the relationships among diseases, autoantigens and autoantibodies will be fully understood. The presence of A G A A cannot actually confirm or exclude any diagnosis. Although reported mainly in patients with systemic diseases such as Sj6gren's syndrome, infections due to enveloped viruses and cerebellar ataxia, A G A A are also found in some patients with overlap

REFERENCES Blaschek MA, Pennec YL, Simitzis AM, Le Goff P, Lamour A, Kerdraon G, Jouquan J, Youinou P. Anti-Golgi complex autoantibodies in patients with primary Sj6gren's syndrome. Scand J Rheumatol 1988;17:291-296. Fritzler MJ, Etherington J, Sokoluk C, Kinsella TD, Valencia DW. Antibodies from patients with autoimmune disease react with a cytoplasmic antigen in the Golgi apparatus. J Immunol 1984;132:2904-2908. Fritzler MJ, Hamel J, Ochs RL, Chan EK. Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex. J Exp Med 1993;178:49--62. Gaspar ML, Marcos MA, Gutierrez C, Martin MJ, Bonifacino JS, Sandoval IV. Presence of an autoantibody against a Golgi cisternal membrane protein in the serum and cerebrospinal fluid from a patient with idiopathic late onset cerebellar ataxia. J Neuroimmunol 1988;17:287--299. Gentric A, Blaschek MA, Julien Ch, Jouquan J, Pennec Y, Berthelot J-M, Mottier D, Casburn-Budd D, Youinou P. Nonorgan-specific autoantibodies in individuals infected with type 1 human immunodeficiency virus. Clin Immunol Immunopathol 1991 ;59:487-494. Gonatas NK. Rouse-Whipple Award Lecture. Contributions to the physiology and pathology of the Golgi apparatus. Am J Pathol 1994;145:751--761. Greenlee JE, Brashear HR, Herndon RM. Immunoperoxidase labelling of rat brain sections with sera from patients with paraneoplastic cerebellar degeneration and systemic neoplasia. J Neuropathol Exp Neurol 1988;47:561--571. Grief C, Farrar GH, Kent KA, Berger EG. The assembly of HIV within the Golgi apparatus and Golgi-derived vesicles of JM cell syncytia. AIDS 1991;5:1433--1439. Griffiths G, Rottier P. Cell biology of viruses that assemble along the biosynthetic pathway. Semin Cell Biology 1992;3: 367-381. Grossmann A, Weiland E. Immunization with inactivated lactate dehydrogenase-elevating virus followed by virus infection favors the selection of hybridomas synthesizing antibodies cross-reacting with intermediate filaments and the LDV envelope protein VP3. Autoimmunity 1991;8:329-233.

syndromes (Fritzler et al., 1984; Hong et al., 1992; Rossie et a1.,1992; Fritzler et al., 1993; Kooy et al., 1992). Rather than a random finding, the association of particular features such as cerebellar involvement or liver dysfunction may be clues to the clinical importance of AGAA, e.g., particular subsegments of patients (Fritzler et al., 1984; Hong et al., 1992; Mohan et al., 1991). Some reports suggest that A G A A are a hallmark of the relationships between viral infections and autoimmunity (Blaschek et al., 1988). See also PURKINJE CELL ANTIBODIES, TYPE 1 (YO).

Grossmann A, Weiland F, Weiland E. Autoimmunity induced by lactate dehydrogenase-elevating virus: monoclonal autoantibodies against Golgi antigens and other subcellular elements. Autoimmunity 1989;2:201-211. Hong HS, Morshed SA, Tanaka S, Fujiwara T, Ikehara Y, Nishioka M. Anti-Golgi antibody in rheumatoid arthritis patients recognizes a novel antigen of 79 kDa (doublet) by westem blot. Scand J Immunol 1992;36:785-792. Huidbuchel E, Blaschek M, Seigneurin JM, Lamour A, Berthelot JM, Youinou P. Antiorganelle and anticytoskeletal autoantibodies in the serum of Epstein-Barr virus,infected patients. Ann Med Interne (Paris) 1991;142:343--346. Kooy J, Toh BH, Gleeson PA. Heterogeneity of human antiGolgi autoantibodies: reactivity with components from 35 to 260 kDa. Immunol Cell Biol 1994;7:123-127. Kooy J, Toh BH, Pettitt JM, Erlich R, Gleeson PA. Human autoantibodies as reagents to conserved Golgi components. Characterization of a peripheral, 230-kDa compartmentspecific Golgi protein. J Biol Chem 1992;267:20255--20263. Kreis TE. Role of microtubules in the organization of the Golgi apparatus. Cell Motil Cytoskeleton 1990;15:67-70. Linstedt AD, Hauri HP. Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa. Mol Biol Cell 1993;4:679-693. Marks DL, Larkin JM, McNiven MA. Association of kinesin with the Golgi apparatus in rat hepatocytes. J Cell Sci 1994;107:2417--2426. Mayet WJ, Hermann E, Csernk E, Knuth, Poralla T, Gross WL, Meyer zum Btischenfelde KH. A human renal cancer line as a new antigen source for the detection of antibodies to cytoplasmic and nuclear antigens in sera of patients with Wegener' s granulomatosis. J Immunol Methods 1991;143: 57--68. Mohan TC, Jalil HA, Nadarajah M, Sng EH. Four patients in Singapore with anti-Golgi antibodies. Singapore Med J 1991;32:332-334. Mollenhauer HH, Morre DJ. Structure of Golgi apparatus. Protoplasma 1994; 180:14028. Nilsson T, Warren G. Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus. Curr Opin Cell Biol 1994;6:517-521. Renier G, Carrere F, Chevailler A. Les autoanticorps diriges

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contre l'appareil de Golgi. Rev Med Interne 1994a;15:174181. Renier G, Rousselet MC, Carrere F, Croue A, Andre C, Oksman F, Chevailler A, Hurez D. Golgi autoantibodies and autoantigens. J Autoimmunity 1994b;7:133-143. Rios RM, Tassin AM, Celati C, Antony C, Boissier MC, Homberg JC, Bornens A. A peripheral protein associated with the cis-Golgi network redistributes in the intermediate compartment upon Brefeldin A treatment. J Cell Biol 1994; 125:997-- 1013. Rodriguez JL, Gelpi C, Thomson M, Real FJ, Fernandez J. Anti-Golgi complex autoantibodies in a patient with Sj~3gren's syndrome and lymphoma. Clin Exp Immunol 1982;49: 579-586. Rodriguez M, Truh LI, O'Neill BP, Lennon DA. Autoimmune paraneoplastic cerebellar degeneration: ultrastructural localization of antibody-binding sites in Purkinje cells. Neurology 1988;38:1380-1386. Rossie KM, Piesco NP, Charley MR, Oddis CV, Steen VD, Fratto J, Deng JS. A monoclonal antibody recognizing Golgi apparatus produced using affinity purified material from a patient with connective tissue disease. Scand J Rheumatol 1992;21:109-115. Seelig HP, Schranz P, Schroter H, Wiemann C, Renz M. Macrogolgin- a new 376 kD Golgi complex outer membrane protein as target of antibodies in patients with rheumatic diseases and HIV infections. J Autoimmun 1994a;7:67--91.

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Seelig HP, Schranz P, Schroter H, Wiemann C, Griffiths G, Renz M. Molecular genetic analyses of a 376-kilodalton Golgi complex membrane protein (giantin). Mol Cell Biol 1994b; 14:2564--2576. Sohda M, Misumi Y, Fujiwara T, Nishioka M, Ikehara Y. Molecular cloning and sequence analysis of a human 372kDa protein localized in the Golgi complex. Biochem Biophys Res Commun 1994;205:1399-1408. Spicer SS, Spivey MA, Ito M, Schulte BA. Some ascites monoclonal antibody preparations contain contaminants that bind to selected Golgi zones or mast cells. J Histochem Cytochem 1994;42:213-221. Tanaka K, Igarashi S, Yamazaki M, Nakajima T. Paraneoplastic cerebellar degeneration: successful early detection and treatment of cancer through characterization of the antiPurkinje cell antibody. Intern Med 1992;31:1339-1342. Terao Y, Sakai K, Kato S, Tanabe H, Ishida K, Tsukamoto T. Antineuronal antibody in Sj~3gren's syndrome masquerading as paraneoplastic cerebellar degeneration. Lancet 1994;343: 790. Underwood JR, Pedersen JS, Chalmers PJ, Toh BH. Hybrids from normal, germ free, nude and neonatal mice produce monoclonal autoantibodies to eight different intracellular structures. Clin Exp Immunol 1985;60:417-426. Weiland E, Weiland F, Grossmann A. Lactate dehydrogenaseelevating virus induces anti-Golgi apparatus antibodies. J Gen Virol 1987; 68:1983-1991.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

GRANULOCYTE-SPECIFIC ANTINUCLEAR ANTIBODIES Allan Wiik, M.D., D.Sc.

Department of Autoimmunology, Statens Seruminstitut, DC-2300 Copenhagen 5, Denmark

HISTORICAL NOTES

THE AUTOANTIGENS

Human blood smears were already used in the 1950s for demonstration of antinuclear antibodies (ANA) by indirect immunofluorescence technique (IIF). However, a clear distinction between these and antibodies reacting selectively with the nuclear/perinuclear region of neutrophils and monocytes, but not with lymphocytes or nuclei of conventional ANA substrates, was not made until 1964. Leucocyte-specific antibodies were then described in a patient with Felty's syndrome (FS) and subsequently in rheumatoid arthritis (RA) and in some patients with systemic lupus erythematosus (SLE) (Faber et al., 1964; Faber and Elling, 1966). When similar studies were repeated using washed, isolated human buffy coat smears and human thyroid sections as ANA control substrate, the existence of such leucocyte-specific antibodies was strongly supported, and the name granulocyte-specific ANA (GS-ANA) was introduced (Wiik, 1980). The neutrophil/monocyte-reactive autoantibodies associated with RA, and FS are termed GS-ANA throughout. No clear data prove whether the autoantigens targeted in these particular diseases are nuclear or cytoplasmic in origin, and thus, GS-ANA might equally well be termed perinuclear antineutrophil cytoplasmic antibodies (P-ANCA), an expression which is commonly used in recent literature on the subject (Mulder et al., 1993; Cambridge et al., 1994). GS-ANA in RA and FS are mainly IgG antibodies with a tendency to engage in circulating and synovial fluid immune complexes and most likely are produced locally in the rheumatoid synovium indicating a probable relationship to pathogenesis (Wiik, 1980).

Origin/Structure The nature and origin of the autoantigens recognized by GS-ANA surprisingly evaded identification for three decades. Early studies indicated that the antigens were easily extracted from ethanol-fixed neutrophils even after short-term washing of smears with isotonic phosphate-buffered saline (PBS). Although always destroyed by trypsin and sometimes rendered nonreactive by DNAse treatment, GS-ANA are not affected by RNAse or periodate treatment (Wiik, 1980). This indicates that some of the neutrophil autoantigens are saline-soluble, nonglycosylated proteins, and some are associated or can become associated with DNA under in vitro conditions. The conditions that are important for demonstration of GSANA by IIF are those recommended as a standard technique for detection of ANCA (Wiik, 1989). The use of ethanol fixation/permeabilization makes the intracellular autoantigens accessible to antibodies and solubilizes cellular membranes so that hydrophilic, soluble antigens can move freely around in the partially delipidated cell. This leads to redistribution of such antigens to other cell compartments or outside the cell ("extraction"). The many cationic constituents of the azurophilic and specific granules commonly migrate to oppositely charged constituents of the nucleus, giving rise to an artefactual antigen localization on the nucleus in the final preparation, so that autoantibodies to these antigens are visualized much like ANA (Falk and Jennette, 1988). The use of a protein cross-linking agent like paraformaldehyde in combination with a permeabilizing agent is one way used to preserve an autoantigen in its original cellular

331

compartment, but many GS-ANA positive reactions of RA patient sera turn negative or borderline positive after such cross-linking procedures, so conclusions cannot be drawn about the site of origin of the antigens recognized. It is, however, the experience of several groups of investigators that some sera will still react with nuclear material and others with cytoplasmic material after paraformaldehyde/acetone treatment, suggesting that both nuclear and cytoplasmic autoantigens may be targeted by GS-ANA in RA (Gross, Kallenberg, Wiik, personal communication). Many different nuclear/perinuclear staining patterns can be discerned in different patients with RA, but serial specimens from an individual patient produce identical staining patterns (Wiik, 1980). Attempts at characterizing neutrophil autoantigens through inhibition of IIF have all been fruitless. As shown by immunoblotting, RA sera typically react with the neutrophil granule protein lactoferrin (LF) or with two sets of unidentified neutrophil antigens with an apparent molecular weight of 67/66 kd and 63/54 kd, respectively (Mulder et al., 1993), as shown by immunoblotting technique. The neutrophil extraction procedure used in this study to produce the antigen source did not permit conclusions regarding the derivation of these latter antigens. Another study which primarily looked at RA patients with vasculitis similarly found ANCA directed to lactoferrin in a substantial proportion of the patients (43%), but LFANCA are not common in RA p e r se (Coremans et al., 1992). Antibodies to myeloperoxidase (MPO) (140 kd) occur in 22% of rheumatoid vasculitis patients and in 12% of RA patients (Coremans et al., 1992; Cambridge et al., 1994).

Lactoferrin Granules of neutrophils containing lactoferrin may be the main recognizable cytoplasmic target of GSANA/P-ANCA which are found in 20% of RA patients (Mulder et al., 1993). LF, a single chain cationic glycoprotein with a molecular weight of 80 kd and two iron-binding domains (Birgens, 1991), is produced in mammary glands, kidneys, pancreas, salivary, lachrymal and bronchial exocrine glands as well as in bone marrow myelocytes. LF is stored in specific granules of mature neutrophils from which it is exocytosed upon activation. In addition to its antimicrobial and antiinflammatory properties, LF might also cause increased extravasation of neutrophils from the cir-

332

culation to the tissues and might regulate myelopoiesis negatively (Peen, 1995). LF possesses a DNA-binding sequence and has a role in gene transcription (He and Furmanski, 1995). Nothing is yet known about autoantigenic epitopes on LF. Purified human milk LF can easily be obtained from commercial sources, and as yet no differences have been found between milk- and neutrophil-derived LF as autoantigen for in vitro studies.

AUTOANTIBODIES Terminology The early designation of GS-ANA was "leucocytespecific antinuclear factors" (Faber and Elling, 1966); whereas, more recently the term "P-ANCA" is typically used (Mulder et al., 1993; Cambridge et al., 1994). Commonly, buffer-soluble antigens not delimited by lipid membranes are artifactually redistributed to the perinuclear area making standardized procedures indispensable for reliably distinguishing GS-ANA from non-organ-specific ANA in a routine serology setting (Wiik et al., 1974). Thus, GS-ANA were first defined as antibodies reacting with nuclei of human neutrophils and monocytes, but not with other mature cells in the blood or tissues; at that time GS-ANA were studied with human thyroid tissue as control. By definition, demonstration of GS-ANA in sera containing both GS-ANA and ANA requires that the titers on neutrophils exceed the titers on thyroid nuclei by at least two dilution steps. With very sensitive ANA substrates such as HEp-2 cells as controls (Humbel, 1993), this distinction between GS-ANA and ANA cannot readily be used, unless ANA are negative or morphologically distinct from GS-ANA (e.g., antinucleolar, anticentromere patterns), because HEp-2 cells have much higher sensitivity for ANA detection (Bang La Cour et al., unpublished). GS-ANA are found in about 75% of patients with active RA and 90% of patients with active FS (Wiik et al., 1974; Wiik and Munthe, 1974); whereas, they are found in lower frequency (--50%) in random RA patients (Wiik, 1980). Because no large studies of non-RA inflammatory arthritides (e.g., Reiter's syndrome and other long-standing arthritides) are available, exact figures for diagnostic specificity as well as positive and negative predictive value cannot be calculated. Experience derived from a large routine serology laboratory indicates that GS-ANA of the IgG

class are very infrequent in non-RA inflammatory arthritides and in osteoarthritis. In normal healthy donors between the age of 1 and 60 years, only 2% harbor IgG GS-ANA (Wiik, 1976), but at ages above 60 years IgG GS-ANA are more common (--15-20%) both in healthy controls and in osteoarthritis patients (Wiik et al., 1974). IgG GS-ANA are similarly common in polyarthritic juvenile RA (JRA), but not in Still's disease or in oligoarticular JRA (Permin et al., 1978). Though represented in all five main Ig classes, GSANA are predominantly IgG and IgM in RA and FS; GS-ANA titers are higher in FS (Wiik, 1980); the IgG antibodies are found in all four subclasses; IgG1 and IgG3 predominate (Wiik and Munthe, 1972). Nothing is yet known about idiotypes on GS-ANA, but mixing of strongly positive GS-ANA sera with normal donor sera commonly eliminates reactivity of the original serum; this might indicate presence of anti-idiotypic activity to GS-ANA in some normal sera (Wiik, unpublished). Pathogenetic Role

Although there is no direct evidence, a role for GSANA in pathogenesis is suggested by: (1) the higher prevalence and titers found in active disease, (2) the presence of GS-ANA in local and circulating immune complexes (Wiik, 1975a; 1975b), (3) the ability of GS-ANA to activate complement in neutropenic cases of RA (Wiik and Munthe, 1974) with leakage of the antibodies into the urine in nonnephritic patients (Wiik et al., 1975a), probably as a subclinical sign of immune complex-mediated renal damage. Direct immunofluorescence studies on neutrophils from the circulation of FS patients indicate that immune complexes may be ingested by neutrophils in vivo (Gupta et al., 1976). When normal neutrophils are incubated at 37~ in vitro with sera from FS patients, immune complexes are quickly phagocytosed by neutrophils (Hurd et al., 1970; Permin et al., 1984). Thus, particular types of immune complexes found in FS sera show an unusual propensity to interact with neutrophils and possibly with some of their bone marrow precursors to cause neutropenia (Permin et al., 1984). Whether this propensity can be attributed to the rich representation of GS-ANA in the circulating immune complexes in FS (Wiik, 1975b) and in cryoglobulins isolated from such sera (Weisman and Zvaifler, 1976) is not known, but seems unlikely, because F(ab') 2 fragments of IgG isolated from FS

sera show no reactivity with surface molecules on mature neutrophils in suspension, indicating that GSANA p e r se have no influence on mature neutrophils in the circulation (Petersen and Wiik, 1983). However, immune complex phagocytosis as evidenced by the number of large inclusion-containing neutrophils is inversely correlated to the number of circulating neutrophils in FS patients; this argues in favor of a direct pathophysiologic relationship between the immune complexes and neutropenia (Pertain et al., 1984). These large inclusions in neutrophils in FS contain IgG, IgA and C3 consistent with the surface receptors on neutrophils for these constituents. The activation caused by immune complex ingestion might cause margination in the circulatory bed or elimination of neutrophils by the mononuclear phagocytic system. IgE constitutes part of the circulating immune complexes in FS (Meretey et al., 1984), and IgE GSANA are found in these complexes (Permin et al., 1984). Whether IgE-containing immune complexes contribute to the induction of neutropenia is not known. Nor is it known whether immune complexes composed of [32-microglobulin and autoantibodies to ~2-microglobulin contribute to the pathogenesis of neutropenia in FS (Falus et al., 1983). Methods of Detection

The main limitation of GS-ANA detection is that the antibodies are readily demonstrable only by IIF (Wiik, 1980). All attempts to demonstrate the antibodies by double immunodiffusion are unsuccessful, and ELISA assays with purified and crude neutrophil antigen preparations give inconclusive results or infrequent antigen reactivity. In addition, ELISA results are not comparable from one laboratory to another because of the lack of standard reagents and methodology; hence, even ELISA data on LF-ANCA are not comparable. Immunoblots show reactivity with only a few antigens (Mulder et al., 1993, Cambridge et al., 1994), and no data are available to evaluate whether the IIF results are in fact attributable to reactivity with the antigens recognized by ELISA or immunoblotting. The tendency of some sera to show false reactivity with neutrophils adds to the problem. For example, normal sera heated to inactivate complement yield a finely speckled, P-ANCA-like immunofluorescence pattern, which is probably caused by Fc-receptor interaction with small IgG aggregates (Rasmussen and Wiik, 1989).

333

CONCLUSION Because G S - A N A detected by IIF are c o m m o n l y found in certain subpopulations of RA patients, including those with spontaneous neutropenia and those who go on to develop extensive erosive arthritis, the determination of these autoantibodies in RA.is of promising clinical value. Recognition of the main

REFERENCES B irgens HS. The interaction of lactoferrin with human monocytes. Dan Med Bull 1991;38:244--252. Cambridge G, Williams M, Leaker B, Corbett M, Smith CR. Antimyeloperoxidase antibodies in patients with rheumatoid arthritis: prevalence, clinical correlates, and IgG subclass. Ann Rheum Dis 1994;53:24--29. Coremans IE, Hagen EC, Daha MR, van der Voort EA, Kleijburg-van der Keur C, Breedveld FC. Antilactoferrin antibodies in patients with rheumatoid arthritis are associated with vasculitis. Arthritis Rheum 1992;35:1466-1475. Faber V, Elling P, Norup G, Mansa B, Nissen NI. An antinuclear factor specific for leucocytes. Lancet 1964;2:344-345. Faber V, Elling P. Leucocyte-specific antinuclear factors in patients with Felty' s syndrome, rheumatoid arthritis, systemic lupus erythematosus and other diseases. Acta Med Scand 1966;179:257--267. Falk RJ, Jennette JC. Antineutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 1988;318:1651--1657. Falus A, Wiik A, Permin H, Brandslund I, Svehag SE. High serum beta-2-microglobulin levels and circulating immune complexes containing beta-2-microglobulin and anti-beta-2microglobulin antibodies in Felty's syndrome. Arthritis Rheum 1983;26:721--727. Gupta RC, Laforce FM, Mills DM. Polymorphonuclear leukocyte inclusions and impaired bacterial killing in patients with Felty's syndrome. J Lab Clin Med 1976;88:183--193. He J, Furmanski P. Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA. Nature 1995;373:721--724. Humbel RL. Detection of antinuclear antibodies by immunofluorescence. In: van Venrooij WJ, Maini RN, eds. Manual of Biological Markers of Disease. Dordrecht: Kluwer Academic Publishers, 1993:A1--16. Hurd ER, LoSpolluto J, Ziff M. Formation of leukocyte inclusions in normal polymorphonuclear cells incubated with synovial fluid. Arthritis Rheum 1970;13:724--733. Meretey K, Falus A, Bohm U, Permin H, Wiik A. IgE class immune complexes in Felty's syndrome: characterization of antibody reactivity in isolated complexes. Ann Rheum Dis 1984;43:246-250. Mulder AH, Horst G, van Leeuwen MA, Limburg PC, Kallen-

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autoantigens involved should lead to reliable, specific quantification which will allow critical evaluation of G S - A N A in clinical diagnostics and patient management. See also A N C A IN INFLAMMATORY BOWEL DISEASES, A N C A WITH SPECIFICITY FOR MYELOPEROXIDASE, A N C A WITH SPECIFICITY FOR PROTEINASE 3 and A N C A WITH SPECIFICITY OTHER THAN PR 3 AND M P O (X-ANCA).

berg CG. Antineutrophil cytoplasmic antibodies in rheumatoid arthritis. Arthritis Rheum 1993;36:1054-- 1060. Peen E. Lactoferrin and antilactoferrin antibodies. A clinical and experimental study [Dissertation]. Halsouniversitetet: Linkoping, 1995. Permin H, Horbov S, Wiik A, Knudsen JV. Antinuclear antibodies in juvenile chronic arthritis. Acta Paediatr Scand 1978;67:181-185. Permin H, Wiik A, Djurup R. Phagocytosis by normal polymorphonuclear leucocytes of immune complexes from sera of patients with Felty's syndrome and rheumatoid arthritis with special reference to IgE immune complexes. Acta Pathol Microbiol Immunol Scand [C] 1984;92:37--42. Petersen J, Wiik A. Lack of evidence for granulocyte specific membrane-directed autoantibodies in neutropenic cases of rheumatoid arthritis and in autoimmune neutropenia. Acta Pathol Microbiol Immunol Scand [C] 1983;91:15--22. Rasmussen N, Wiik A. Indirect immunofluorescence examination for IgG-ANCA in sera. APMIS 1989;96:16-20. Weisman M, Zvaifler NJ. Cryoglobulinemia in Felty's syndrome. Arthritis Rheum 1976;19:103--110. Wiik A. Circulating immune complexes involving granulocytespecific antinuclear factors in Felty's syndrome and rheumatoid arthritis. Acta Pathol Microbiol Scand [C] 1975a;83: 354-364. Wiik A. Joint fluid immune complexes involving granulocytespecific antinuclear factors in rheumatoid arthritis. Acta Pathol Microbiol Scand [C] 1975b;83:365--369. Wiik A. Antinuclear factors in sera from healthy blood donors. Acta Pathol Microbiol Scand [C] 1976;84:215--220. Wiik A. Granulocyte-specific antinuclear antibodies. Possible significance for the pathogenesis, clinical features and diagnosis of rheumatoid arthritis. Allergy 1980;35:263--289. Wiik A. Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. APMIS 1989;6: 12--13. Wiik A, Munthe E. Restrictions among heavy and light chain determinants of granulocyte-specific antinuclear factors. Immunology 1972;23:53-60. Wiik A, Munthe E. Complement-fixing granulocyte-specific antinuclear factors in neutropenic cases of rheumatoid arthritis. Immunology 1974;26:1127--1134. Wiik A, Jensen E, Friis J. Granulocyte-specific antinuclear factors in synovial fluids and sera from patients with rheumatoid arthritis. Ann Rheum Dis 1974;33:515--522. Wiik A, Henriksen K, Faber V. Urinary excretion of granulo-

cyte-specific antinuclear factors in rheumatoid arthritis. Predominance in neutropenic cases showing high titers of complement fixation. Acta Pathol Microbiol Scand [C] 1975a;83:273-279.

Wiik A, Jensen E, Friis J, Bach-Andersen R. Participation of granulocyte-specific antinuclear factors in rheumatoid joint fluid cryoprecipitates. Acta Pathol Microbiol Scand [C] 1975b;83:265-272.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

HEAT S H O C K PROTEIN A U T O A N T I B O D I E S Merrill J. Rowley, Ph.D. and Christopher Karopoulos, B.Sc. (hons)

Centre for Molecular Biology and Medicine, Monash University, Clayton, Victoria 3168, Australia

HISTORICAL NOTES

T H E AUTOANTIGENS

The first suggestion that an immune response to heat shock proteins might be implicated in autoimmunity came from adjuvant arthritis induced in susceptible rats by injection with Freund's complete adjuvant containing mycobacterial antigens (van Eden et al., 1985). This arthritis was passively transferred by rat T-cell lines specific for the mycobacterial 65 kd heat shock protein (hsp 65), and one T-cell clone (A2b) cross-reacted with hsp 65 and a component of cartilage proteoglycan (van Eden et al., 1985). Epitope mapping for the arthritogenic T-cell clone showed specificity for amino acids 180--188 of mycobacterial hsp 65. The cross-reactivity was presumed due to sequence similarity with proteolgycan link protein, albeit limited to 4 of the 9 amino acids (van Eden, 1991). Subsequently, both T- and B-cell reactivity in humans to mycobacterial hsp 65 were described in rheumatoid arthritis (Bahr et al. 1988; Tsoulfa et al., 1989). Also, in the nonobese diabetic (NOD) mouse, antibodies to a [3-islet cell target antigen were reported to cross-react with mycobacterial hsp 65 (Elias et al., 1990). Although no disease-specific increase in autoantibodies to heat shock proteins is present in human insulin-dependent diabetes mellitus (Tun et al., 1994), these findings, coupled with the conserved nature and strong immunogenicity of heat shock proteins suggested that molecular mimicry might cause breakdown of tolerance if an immune response against a heat shock protein of an infective organism elicited a cross-reactive autoimmune response to another self protein.

Heat shock proteins (or stress proteins) are induced by different stressors, including heat, nutrient deprivation, attack by reactive oxygen metabolites and metabolic disruption induced by anoxia, low pH or exposure to toxins. The major heat shock proteins are among the most highly conserved and abundant proteins found in nature and are dominant antigens of many pathogens (Young and Elliott, 1989). Concentrations vary from low to moderate in nonstressed cells and very high in stressed cells. In nonstressed cells, heat shock proteins act as chaperones in the stabilization of protein structure, the facilitation of protein folding and the transport of proteins across membranes (Hartl et al., 1994). Heat shock proteins can be grouped according to their molecular size, amino acid sequence similarities and the cellular localization and function of the major groupings, including hsp 90, hsp 70, hsp 60 and small heat shock proteins (sHsp) with molecular mass of 90, 70 or 60 and <40 kd (Table 1).

336

Hsp 90. The hsp 90 family plays a critical role in cell functions under both stress and nonstress conditions; hsp 90, the most abundant cytosolic protein in eukaryotic cells, is an ATP-independent molecular chaperone reactive with or regulating specific proteins, including calmodulin, actin, tubulin, kinases and steroid receptors (Jakob and Buchner, 1994). Hsp 70. The hsp 70 family are the mammalian counterparts of E. colb and mycobacterial proteins. As molecular chaperones in the cytosol, in the endoplasmic reticulum and the mitochondria, they maintain newly synthesized proteins in their unfolded or

Table 1. The Major Mammalian Heat Shock Proteins Family Hsp 90

Hsp 70

Most abundant constitutive hsp. Critical role in cell function. ATP-independent chaperones.

Maintains proteins in unfolded and extended conformations. ATP-dependent chaperones, ATPase activity

Members

Alternate Names

Function/Localization

hsp 90a hsp 90b

hsp83

Cytoplasmic; maintains steroid receptors and kinases inactive until appropriate

GRP94

endoplasmin

Golgi, plasma membranes, similar function to hsp90; may interact with MHC class II

hsp 70

72K, hsx70, hsp71, sp71, Cytosol (nucleus after stress); major heathsp68, hsp70 inducible hsp70; some basal expression; higher expression in dividing cells

hsp 72

hsp70B, hsp70

hsp 73

p72, 73K hscp73, scp73, Cytosol (nucleus after stress); high basal hsc70, hsc71, hsp70, expression; slightly heat-inducible hsp73 Endoplasmic reticulum; high basal expression BiP, p78, grp80 in secretory cells; protein import, glycosylation, assembly within organelles; binds unassembled subunits of multisubunit ER proteins

GRP78

Cytosol (nucleus after stress); no basal expression; heat-inducible

GRP75

Hsp 60 Small Hsp

"Chaperonin family"; mediates folding of nonnative proteins to the native state; binds ATP hsp 28

Mt-hsp70, p71

Mitochondria; protein import, folding and assembly

P1, Hsp58

Mitochondria; promotes folding and assembly of imported proteins Large oligomer in perinuclear cytoplasm and Golgi

Ubiquitin Cytoplasm/nucleus; association with histone H 2 A - possible role in gene regulation ~-crystallin

ta~

extended conformation. Hsp 90 has ATPase activity and energy derived from ATP hydrolysis is required for dissociation of bound polypeptides (Hartl et al., 1994). Member(s) of the hsp 70 family play a role in antigen processing and presentation (Pierce, 1994) and have adjuvant activity (Del Guidice, 1994).

Hsp 60. The only mammalian member of this family (Jindal et al., 1989), is constitutively expressed in mitochondria and is involved in the ATP-dependent import of proteins into mitochondria and their subsequent refolding (Hartl et al., 1994).

sHsp. This disparate group of uncertain functions interacts with other heat shock proteins; several act as chaperones in protein folding and unfolding (Jakob and Buchner, 1994). An abundant structural protein of eye lens, o~-crystallin, can be classified as a member of the sHsp family, based on similarity in domain structure over a conserved sequence of 90-100 amino acid residues in the carboxy-terminus of the molecule (Jakob and Buchner, 1994; Groenen et al., 1994). It has been shown to have Hsp function in vitro (Jakob and Buchner, 1994). The amount of a-crystallin is raised in myelin isolated from multiple sclerosisaffected brain, and it has been proposed as a potential autoantigen in multiple sclerosis (van Noort et al., 1995).

Characteristics Despite their phylogenetic conservation, heat shock proteins are the most commonly documented immunogenic proteins of infectious agents, perhaps because they are produced in large quantities by pathogens after exposure to various host defense mechanisms. An immune response to heat shock proteins occurs after a wide variety of infections (Young and Elliott, 1989), and after injection with vaccines. For example, IgG antibodies to hsp 65 and hsp 70 can be detected in 90% of infants after injection with triple vaccine against diphtheria, tetanus and pertussis. The anti-hsp 65 antibodies induced by the whole cell pertussis vaccine cross-react with E. coli hsp but not with its human hsp 60 counterpart (Del Guidice et al., 1993). Because they are so highly conserved, frequent exposure to heat shock proteins from organisms of low virulence could lead to constant boosting of the immune response, while the peptide-binding capacity of heat shock proteins may favor their entrance into antigen-processing pathways

338

(Kaufman, 1991). Indeed, at least in an experimental system, peptides of heat shock proteins have been shown to function as T-cell carrier epitopes to enhance the response to a poorly immunogenic polysaccharide antigen, the Vi antigen of Salmonella typhi (Konen-Waisman et al., 1995). Given that bacterial and human heat shock proteins have about 60% sequence similarity (Young and Elliott, 1989), a response to such highly conserved proteins could represent a risk for autoimmunity. Indeed, there is considerable speculation about hsps as critical autoantigens in rheumatoid arthritis, multiple sclerosis or systemic lupus erythematosus (SLE) (Gaston, 1991). The observation that T cells and antibodies against the mycobacterial hsp 65 crossreact with the human heat shock proteins is compatible with a role for microbial heat shock proteins, especially mycobacterial hsp 65, in the induction of autoimmunity (van Eden et al., 1991).

AUTOANTIBODIES Heat shock proteins can be increased in target tissues in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis (Karlsson-Para et al., 1990; Dhillon et al., 1994; van Noort et al., 1995). Although T- and B-cell reactivities to various heat shock proteins are described in autoimmune diseases, particularly rheumatoid arthritis and SLE, such reactivity is seen also in healthy subjects (Gaston, 1991; van Eden, 1991). In addition, autoantibodies to recombinant mycobacterial hsp 65 cannot be taken as direct evidence for autoimmune reactivity with either autologous or even mammalian-specific heat shock proteins. Following the initial studies on cell-mediated immunity to mycobacterial hsp 65 in animals with adjuvant arthritis, the first studies in humans involved patients with rheumatoid arthritis. Almost all studies used recombinant hsp 65 prepared from M. tuberculosis, although hsp 65 from M. Leprae can also be used as antigen. Serum IgG antibodies to mycobacterial hsp 65 were reportedly increased in patients with rheumatoid arthritis (Bahr et al., 1988; Tsoulfa et al., 1988; 1989; McLean et al., 1990; Tun et al., 1994), but other workers detected no difference from amounts in healthy subjects (Karopoulos et al., 1995). IgG anti-hsp65 antibodies vary widely in twins with rheumatoid arthritis and in control groups, with no significant differences between disease-discordant twin

pairs (Worthington et al., 1993). Moreover, immunoblotting using polypeptide fragments of mycobacterial hsp 65 revealed no differences between epitopes recognized by disease sera or normal sera (Karopoulos et al., 1995). Thus, antibodies to hsp65 are not specific for RA; indeed, such antibodies are found in a number of diverse diseases including atherosclerosis (Xu et al., 1993), multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease (Gao et al., 1994) and onchocerciasis or "river blindness" (Meilof et al., 1993). Methods of Detection

Methods to detect autoantibodies to mammalian hsps are more varied than those used for antibodies to mycobacterial hsp 65. Immunoblotting using cell lysates after one- or two-dimensional SDS-polyacrylamide gel electrophoresis is most widely used; ELISAs are being used more often as recombinant proteins become available. Immunoblotting has the advantage that antibodies to heat shock proteins from several families can be detected in the same preparation, although the exact identification of a given reactive antigen in a reactive band based solely on molecular weight is problematic.

CLINICAL UTILITY Disease Associations

Although not specific for any particular autoimmune disease, autoantibodies to mammalian heat shock proteins can be present in some disease at higher frequencies and greater amounts than in health. By immunoblotting with cell lysates, or ELISA with recombinant human hsp 60, autoantibodies to mammalian hsp 60 are detected in conditions as varied as rheumatoid arthritis (Girouard et al., 1993) (Table 2), atherosclerosis (Wick et al., 1995), dilated cardiomyopathy and myocardial infarction (Latif et al., 1993) and bacterial infections including Lyme disease, (Girouard et al., 1993). Antibodies to hsp 70 are also described in SLE (Minota et al., 1988a), after thermal burns (Qureishi et al., 1995) and in progressive sensorineural hearing loss (Billings et al., 1995), and antibodies to hsp 90 are described in SLE (Minota et al., 1988b; al-Dughayam et al., 1994; Conroy et al., 1994) and in patients with various infections (alDughayam et al., 1994).

In one study, sera from 268 patients with various rheumatic, inflammatory bowel, and autoimmune skin diseases were tested for autoantibodies to hsp 60, hsp 70 and hsp 90 by immunoblotting against a lysate of the human cell line E6-1 (Jarjour et al., 1991). Blotting conditions optimized for detection of antibodies to heat shock proteins discriminated serum antibody reactivity in disease from that in health. There was little evidence that a humoral autoimmune reaction to heat shock proteins contributed to the pathogenesis of autoimmune diseases and no indication of diagnostic utility, because IgG and IgM autoantibodies against heat shock proteins werepresent in <15% of patients with SLE, rheumatoid arthritis, Sj6gren's syndrome, ankylosing spondylitis, Reiter's syndrome and systemic sclerosis. Antibodies to hsp 60 and hsp 70 occurred more frequently in certain other disorders, but titers were still low, generally <1:50. The diseases with the most frequent occurrence of antibodies to hsp 60, 20-30%, were mixed connective tissue disease, polymyositis or dermatomyositis, psoriatic arthritis, ulcerative colitis, Crohn's disease, epidermolysis bullosa acquisita and bullous pemphigoid; for antibodies to hsp 70, the highest frequencies were Lyme disease, 33% and ulcerative colitis, 23%. Antibodies to hsp 90 were rare in all groups, being present in <2% overall. These studies indicate that autoantibodies to heat shock proteins can occur in a range of diseases, but are present also in healthy individuals (Jarjour et al., 1991; Kindas-Mugge et al., 1993; Konen-Waisman et al., 1995). There is no evidence that patients with autoimmune disease develop hightiter autoantbodies to heat shock proteins like other autoantibodies in autoimmune diseases. Instead, autoantibodies to heat shock proteins may be considered to be a part of the repertoire of natural antibodies. Although there is little evidence for specific disease-associated autoimmune responses to heat shock proteins, the occurrence of autoantibodies to heat shock proteins under conditions of tissue stress, as after thermal burns (Qureishi et al., 1995) or myocardial infarction (Latif et al., 1993), might indicate a response to an increase in amounts of those heat shock proteins. For example, mucosal expression of human hsp 60 is reported in B7-positive antigenpresenting mononuclear cells in the ileum and colon of patients with Crohn's disease and ulcerative colitis (Peetermans et al., 1995), and autoantibodies to hsp 60 occur in 20--30% of patients with the same diseases (Jarjour et al., 1991). Similarly hsp 90 is

339

Table 2. Autoantibodies to Mammalian Heat Shock Proteins in Various Diseases 1

Disease

Autoantibodies to

Source

Hsp 90

Hsp 70

Hsp 60

No Yes Yes Yes

No Yes No

No --

Mixed connective tissue disease

No

No

Yes

Jarjour et al., 1991

Polymyositis/dermatomyositis

No

No

Yes

Jarjour et al., 1991

Rheumatoid arthritis

No

No

No Yes

Jarjour et al., 1991 Girouard et al., 1993

Psoriatic arthritis

No

No

Yes

Jarjour et al., 1991

Ulcerative colitis

No

Yes

Yes

Jarjour et al., 1991

Crohn's disease

No

No

Yes

Jarjour et al., 1991

Epideromolysis bullosa acquisita

No

No

Yes

Jarjour et al., 1991

Bullous pemphigoid

No

No

Yes

Jarjour et al., 1991

Lyme disease

No

Yes

No Yes

Jarjour et al., 1991 Girouard et al., 1993

Nonspirochete infections

Yes

m

al-Dughayam et al., 1994

SLE

Jarjour et al., 1991 Minota et al., 1988a; 1988b Conroy et al., 1994 al-Dughayam et al., 1994

Sensorineural hearing loss

Yes

--

Billings et al., 1995

Thermal burns

Yes

m

Qureishi et al., 1995

Yes

Wick et al., 1995

Atherosclerosis

~

Ischemic heart disease

No

No

Yes

Latif et al., 1993

Dilated cardiomyopathy

No

No

Yes

Latif et al., 1993

1Disease in which antibodies were not detected include systemic sclerosis, polymyalgia rheumatica, Reiter's syndrome, ankylosing spondylitis and Sj6gren's syndrome (Jarjour et al., 1991).

reported to be overexpressed in peripheral blood mononuclear cells from patients with SLE (Dhillon et al., 1994), and as noted above, antibodies to hsp 90 are found in SLE (Minota et al., 1988b; al-Dughayam et al., 1994; Conroy et al., 1994). Overexpression of hsp 90 also occurs in the spleen of the lupus-prone MRL/lpr-lpr mouse prior to the onset of disease (Faulds et al., 1994). Although hsp 90 is primarily an intracytoplasmic protein, cell surface expression is described in patients with SLE during active disease (Erkeller-Yuksel et al., 1992), and elevation of hsp 90 expression may be particularly associated with active neuropsychiatric and cardiorespiratory diseases and the antiphospholipid syndrome (Dhillon et al., 1994). The epitopes on hsp 90 recognized by sera from patients with SLE differ from those recognized by patients with infections (al-Dughayam et al., 1994).

340

Because of the wide degree of overlap in reactivity among normal individuals and disease subjects, measurement of autoantibodies to heat shock proteins has no clinical utility for the diagnosis.

CONCLUSION From the practical standpoint of diagnostic assistance from the clinical laboratory, there is no present utility in measuring levels of antibody or even T-lymphocyte responsiveness to any of the readily available preparations of heat shock protein. From the basic standpoint, studies on cellular and humoral immune responses to such important and ubiquitous reactants as heat shock proteins should provide insights into unresolved issues of generation and maintenance of self-tolerance and

induction of autoimmunity. In addition, heat shock proteins themselves may play a role in the generation of an i m m u n e response quite apart from that as an autoantigen. Heat shock proteins as markers of cell stress might indicate the likely presence of d a m a g e d proteins that could directly enter antigen-processing pathways within the cell. Alternatively, autoantigens c o m p l e x e d to heat shock

proteins and released from dying cells during stress could expose cryptic epitopes on the unfolded (or incompletely folded) protein. T- and B-cell responses to heat shock proteins that occur as part of the normal repertoire might be amplified as autoimmune responses to c o m p l e x e d autoantigens. See also NATURAL AUTOANTIBODIES.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

HEPARIN-ASSOCIATED AUTOANTIBODIES Gowthami Arepally, M.D. a and Douglas B. Cines, M.D. b

aUniversity of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104; and bDepartment of Pathology~Laboratory Medicine, Hospital of the University of Pennsylvania, Pennsylvania, PA 19104, USA

HISTORICAL NOTES

THE AUTOANTIGENS

Heparin was first introduced into clinical medicine in the mid-1930s and has since become the drug used most commonly in the initial treatment of thromboembolism (Hirsh et al., 1992). It was immediately evident that heparin has a narrow therapeutic window, as bleeding occurs commonly at dosages that provide clinical benefit. Appreciation of other adverse effects developed more slowly. Although acute, transient and asymptomatic thrombocytopenia beginning within minutes of infusion of heparin Was soon discovered to occur in dogs, the data in man were less conclusive and were attributed to impurities (Fratantoni et al., 1975). Recurrent arterial thrombi in human subjects treated with heparin was first described in the late 1950s and early 1960s (Roberts et al., 1964). Although an immunologic basis was suspected, thrombocytopenia was not documented and theinvolvement of platelets was not considered in detail. The concurrent development of thrombosis and thrombocytopenia was first described in the late 1960s and heparin-dependent antiplatelet antibodies in affected patients were first reported shortly thereafter (Fratantoni et al., 1975). Prospective studies (Bell et al., 1976) helped to establish the frequency and elucidate the typical clinical presentation of heparin-induced thrombocytopenia (HIT), which is now widely recognized as a common and severe complication of heparin that requires surveillance of platelet counts in all patients who receive this drug. The immunologic basis of the disease is now clearly established and provides a model to study the development of pathogenic autoantibodies.

Definition Recent evidence suggests that the pathogenic autoantibodies in HIT most often recognize a complex between the heterologous mucopolysaccharide, heparin and an endogenous protein, platelet factor 4 (PF4) (Amiral et al., 1992). Although a good deal is known about the individual components of the heparin/PF4 complex and its structure, the epitope that elicits the immune response is unknown, and the reason why autoantibodies appear in only a subset of exposed individuals remains unresolved.

Heparin. Heparin is a naturally occurring glycosaminoglycan (GAG) generated by posttranslational modification of the polysaccharide chains of heparin proteoglycan (Rosenberg and Bauer, 1994). The nonsulfated precursor, containing alternating D-glucuronic acid and N-acetyl-D-glucosamine residues, undergoes N-deacetylation, N-sulfation, epimerization (D- to L-uronic acid) and O-sulfation to yield the active polymer (Rosenberg and Bauer, 1994). These modifications are variably completed, yielding a mixture of differing sulfated disaccharide units within each polysaccharide chain (Jacobsson et al., 1985; Rosenberg and Bauer, 1994). The anticoagulant activity of heparin requires a specific pentasaccharide sequence which binds to critical basic amino acid residues of the serpin, antithrombin III (AT-III) (Blajchman et al., 1992; Hirsh et al., 1992). AT-III then undergoes a reversible conformation change which lowers its Km for serine proteases such as thrombin, factor IXa and Xa (Blajchman et al., 1992;

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Rosenberg and Bauer, 1994), which, in turn, leads to the formation of inactive complexes that are cleared by serpin receptors in the liver (Mast et al., 1991). Heparins of sufficient size also enhance the activity of AT-III by forming ternary complexes in which the inhibitor and protease are bound to the same polysaccharide chain (Hirsh et al., 1992; Rosenberg and Bauer, 1994). Proteoglycans containing heparan sulfate and other less highly sulfated GAGs are synthesized by endothelial cells and deposit in vascular matrices where they modulate cell growth, differentiation, and migration in addition to their anticoagulant activity (Rosenberg and Bauer, 1994; Linhardt et al., 1992). Commercial heparins are prepared from bovine or porcine lung or intestine by enzymatic digestion, salt precipitation and gel chromatography (Rosenberg and Bauer, 1994). The resulting product is a heterogenous mixture of polysaccharide chains with molecular weights ranging from 5 to 30 kd with a mean molecular weight of 15 kd and a mean chain length of 50 monosaccharide residues (Hirsh et al., 1992). Because of its negative charge, heparin forms stable salts with various proteins, organic bases and basic dyes (Coon and Willis, 1966). However, only approximately onethird of the heparin molecules in commercial preparations bind AT-III with sufficient affinity to express cofactor activity in vivo (Hirsh et al., 1992). Indeed, for a "heparin" to meet USP requirements, it must possess only a minimal potency based on the recalcification of sheep plasma and it must be nonpyogenic (Walton et al., 1966). This means that its other chemical and biological properties may vary (Walton et al., 1966). Low molecular weight heparin (LMWH) fractions, ranging in molecular weight from 1 to 10 kd (mean molecular weight 4--5 kd) are generated by enzymatic or chemical depolymerization and are now commercially available. LMWH fragments retain antiXa activity but do not inhibit thrombin directly (Hirsh et al., 1992). LMWH and unfractionated heparin may differ in their capacity to initiate or to propagate HIT (see below). Differences between the structure of human and heterologous heparin, especially bovine heparin, have been identified (Linhardt et al., 1992), but whether these differences are involved in the development of HIT remains to be determined. Platelet Factor 4 (PF4). PF4 was initially discovered by its capacity to neutralize the activity of heparin (Conley et al., 1948). PF4 is a 70 amino acid (MW 7800) peptide synthesized by megakaryocytes and 344

stored in the a-granules of platelets (Zucker and Katz, 1991) and released on platelet activation. In addition to antagonizing plasma heparin activity, PF4 is a chemotactant for neutrophils, monocytes and fibroblasts and in vitro is an inhibitor of angiogenesis and megakaryopoeisis (Zucker and Katz, 1991). mRNA transcripts for PF4 have been found to date only in megakaryocytes and platelets, although the protein is also identified in mast cell granules (Zucker and Katz, 1991). The basis of the lineage-specific expression of PF4 is not established. PF4 can be purified from biologic fluids through its high affinity for heparin (Handin and Cohen, 1976). Recombinant PF4 expressed in E. coli retains the biologic and antigenic properties of the native protein (Park et al., 1990; Amiral et al., 1995). PF4 polymerizes to form noncovalently linked tetramers at physiologic pH and ionic strength (Zucker and Katz, 1991). The heparin binding region of PF4 is localized to the C-terminus which contains an (zhelix with two closely spaced Lys-Lys residues (Zucker and Katz, 1991). PF4 binds other anionic sulfated glycosaminoglycans with lower affinity relative to heparin (heparin > heparan sulfate > dermatan sulfate > chondroitin 6-sulfate > chondroitin 4-sulfate) (Handin and Cohen, 1976). Binding of heparin to PF4 does not involve the pentasaccharide sequence which binds to AT-III (Zucker and Katz, 1991), but binding requires a minimum chain length of six monosaccharides (Maccarana and Lindahl, 11993). As PF4 and heparin interactions are mostly charge dependent, the PF4 binding efficiency for heparin increases with chain length of the oligosaccharide (Denton et al., 1983; Zucker and Katz, 1991). The stoichiometry of complex formation also depends on the length of the available oligosaccharide chains. Heparin molecules >9 kd bind to two or more PF4 tetramers; whereas, the binding ratio is inverted when PF4 combines with smaller heparin chains (Denton et al., 1983). Many heparin or heparin-like molecules with little anticoagulant activity may form complexes with PF4 in vivo. PF4 tetramers are incorporated into the developing platelet a-granules complexed with two molecules of chondroitin sulfate (Zucker and Katz, 1991). PF4 remains within these granules during platelet formation and senescence unless the platelets become activated. The basal plasma concentrations of PF4 are exceedingly low (1.8 ng/mL) with optimal collection technique compared with platelet content of PF4 (18 + 4 pg/109 plts) (Files et al., 1981; Zucker and Katz,

1991). Plasma levels may exceed 600 ng/mL when platelets are activated (Files et al., 1981). Secreted PF4 rebinds to an unknown site on the surface of activated platelets (Capitanio et al., 1985) as well as to the endothelium, presumably to heparan sulfate proteoglycans (Zucker and Katz, 1991). It is presumed that heparin administered to patients displaces at least a portion of endogenous heparan sulfate from PF4 at both sites due to its higher affinity for PF4 (Zucker and Katz, 1991).

The Heparin/PF4 Complex. The antibodies that are presumed to cause HIT bind to heparin/PF4 with far greater avidity than to either component alone (Amiral et al., 1992). This finding implies the antigen is formed through a conformational change in one or both reactants or that the epitope forms at the interface between the two molecules. Therefore, knowledge concerning the structure of the antigen awaits a more complete description of the structure of the heparin/PF4 multimer itself. The crystal structure of recombinant human PF4 has been resolved (Zhang et al., 1994). Each monomer consists of an extended N-terminal loop, an intervening [3-sheet composed of three antiparallel strands and an c~-helical C-terminus (Zhang et al., 1994). Dimers form through association of 13-sheets in antiparallel orientation. Tetramers, composed of two dimers, have the antiparallel [3-sheets sandwiched by the o~-helices which are facing outward on the protein

surface (Figure 1). The positively charged Lys residues lie in two clusters in cis-orientation across the external surface of these (x-helices permitting solvent interactions. The surface distribution of positive charges on the (x-helices and [3-sheets, are approximated to be at a distance of 10 * from each other, comparable to the average distance between the negative charges on heparin (Zhang et al., 1994). Using computer-based three-dimensional structures of PF4 and heparin, two modes of interaction between heparin and PF4 have been described where heparin lies parallel or perpendicular to the (x-helices of the dimer (Stuckey et al., 1992). Based on charge and energy constraints, heparin is predicted to lie across the surface of PF4 at right angles to the (x-helices (Stuckey et al., 1992; Zhang et al., 1994). Recent sitedirected mutagenesis studies of the arginine residues in the [3-sheets suggest that it is the spatial proximity of the positive residues on both the (x- and [3-chains of PF4 rather than the clustering of lysines on the ~helices that are critical to heparin binding. The requirement for the carbohydrate portion of the epitope appears to be less strict. Dextran sulfate, pentostan sulfate, LMWH and certain synthetic GAGs can substitute for heparin in at least some cases (Greinacher et al., 1992; Wolf et al., 1983). However, N-desulfated heparin and heparinoids which are less highly sulfated show less cross-reactivity (Greinacher et al., 1992). Critical features of the carbohydrate determinants involved in HIT antibody binding have been recently described and include degree of sulfation, branching of the carbohydrate backbone, as well as size and concentration of the heparin species (Greinacher et al., 1995). The structural basis for these observations as well as the somewhat higher incidence of HIT in patients receiving bovine versus porcine heparin are not elucidated (Chong, 1995). Whether binding of heparin to PF4 alters the structure of the tetramer is not established.

AUTOANTIBODIES Terminology Figure 1. PF4 Tetramer. Dimers, AB and CD, are formed by the association between the ~-sheets of each monomer (A & B or C & D) in antiparallel fashion. Surface interactions between the dimer [3-sheets leads to tetramer formation with the Cterminal c~-helices oriented on the surface of the molecule exposed to solvent. Binding of heparin of PF4 is thought to occur through a "ring" of positive charges across the A-D and B-C interfaces (see text; Zhang et al., 1994).

The antibodies are variously named based on their appearance in patients who develop thrombocytopenia after being treated with heparin, and the requirement for heparin as well as plasma to activate platelets in vitro (Chong, 1995). All of the described patients in the literature have had the same clinical presentation

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and it is likely that all descriptions refer to antibodies now recognized to bind to heparin/PF4. Pathogenetic Role An immunologic basis for HIT is strongly suggested by the fact that thrombocytopenia and thrombosis, when present, typically develop seven days after treatment with heparin has been initiated in naive individuals, but develop sooner in patients with prior drug exposure (Laster et al., 1989). Heparin-dependent, platelet-reactive antibodies are found in plasma from at least 85% of affected patients during the acute phase of the disease; the titer falls days to weeks after heparin is discontinued (Cines et al., 1980; Fratantoni et al., 1975; Laster et al., 1989; Kelton et al., 1988; Chong, 1995). Antibody binding to platelets requires heparin concentrations that are often several orders of magnitude below those achieved clinically (Visentin et al., 1994a; Cines et al., 1980), while the capacity of heparin to aggregate platelets directly is only observed at much higher concentrations than are usually attained. A syndrome resembling disseminated intravascular coagulation has been described in a few patients with otherwise typical clinical features of HIT in whom drug-dependent platelet antibodies were not sought (Klein and Bell, 1974). There is as of yet no animal model of the disease. Platelet Activation. HIT is distinct from other antibody-mediated platelet disorders in that symptomatic patients may suffer from thrombosis rather than bleeding (Chong et al., 1981). Therefore, the effect of antibody must differ in a fundamental way from other platelet antibodies (Deckmyn and De Reys, 1995). In the presence of heparin, HIT antibodies cause platelets to aggregate and secrete the contents of their storage granules, including thromboxane A 2 (Fratantoni et al., 1975; Chong et al., 1981). Some HIT antibodies fix sufficient complement to lyse platelets (Cines et al., 1980). However, auto- and alloantibodies from patients with diverse thrombocytopenic disorders characterized by bleeding cause identical changes in vitro. Of potential interest, the ability of HIT-IgG to activate platelets requires that it bind not only to cell-associated heparin/PF4 via the Fab end of the molecule, but that Fc~IIA receptors on adjacent platelets become cross-linked through the Fc end of the molecule (Kelton et al., 1988; Chong, 1995). Further, platelets from individuals homozygous for a specific allele of this receptor (Arg TM) are not activated by HIT anti-

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bodies in vitro; whereas, those with a single or double copy of the allelic counterpart (His TM) respond normally (Denomme et al., 1994). Whether only patients with the permissive genotype are at risk for thrombosis and whether cross-linking platelet Fc~ receptors is a critical intermediate step in the pathogenesis of thrombosis are unknown. Endothelial Cell Activation. In addition to activation of platelets, HIT antibodies might also cause thrombosis by initiating procoagulant reactions on endothelial cells which secrete heparan sulfate and bind both heparin and PF4 (Cines et al., 1987; Rosenberg and Bauer, 1994; Zucker and Katz, 1991). PF4 released from activated platelets may bind to the endothelium in vivo forming complexes recognized by HIT antibodies (Visentin et al., 1994b). These antibodies stimulate tissue factor expression by endothelial cells in vitro (Cines et al., 1987) and undoubtedly modulate other coagulant reactions. Patients with perturbed endothelial cell function, such as those with cardiovascular disease, may be more susceptible to immune vascular injury and thrombosis (Boshkov et al., 1993). Genetics A genetic susceptibility to develop HIT has not been identified. There is one case report describing the occurrence of HIT in family members (Kosfeld et al., 1985). As noted above, only platelets with Fc~,IIA containing at least one copy of His TMcan be activated in vitro. There are no published data on preferential V H and V L gene usage by B cells producing HIT antibodies. Factors in Pathogenicity IgG and/or IgM antibodies are found in >85% of patients at presentation (Kelton et al., 1988; Visentin et al., 1994a; Amiral et al., 1995); IgA antibodies are occasionally reported (Amiral et al., 1995). IgG antibodies predominate in some studies (Amiral et al., 1995), but not in others (Visentin et al., 1994b) and are presumably responsible for platelet activation in vitro. Thrombosis is reported in a few patients in whom only IgM antibodies were detected (Amiral et al., 1995). There is no evidence that these antibodies cross-react with other anionic compounds, such as cardiolipin (Arepally et al., 1995) or that other disturbances in the immune system occur in susceptible individuals. Autoantibodies that bind to heparan

sulfate and other GAGs in the absence of PF4 can be identified in patients with systemic lupus, progressive systemic sclerosis and poststreptococcal glomerulonephritis; a role for these antibodies in renal vascular injury is posited (Aotsuka et al., 1988) but neither thrombocytopenia nor thrombi at other sites were described.

Methods of Detection HIT antibodies cause normal platelets to aggregate and secrete serotonin in vitro (Fratantoni et al., 1975; Sheridan et al., 1986). The serotonin release assay (SRA) provides a more objective endpoint (Sheridan et al., 1986) than platelet aggregation studies which have variable sensitivity and specificity (Chong, 1995). The SRA is performed by incubating plateletrich plasma from normal donors and loading the platelet granules with 14C-serotonin. Plasma from suspected patients or controls is added along with various amounts of heparin, and the secreted radioactivity is measured. Plasmas must be shown to be devoid of heparin or depleted of residual heparin by cation exchange. Heparin is then reintroduced to establish the drug-dependence of the reaction. The test should be considered positive only when aggregation or secretion requires the addition of heparin at concentrations at or below those attained clinically (0.2-0.5 U/mL) (Sheridan et al., 1986; Cines et al., 1980). The specificity of the test may be improved by demonstrating that high concentrations of heparin (100 U/mL) suppress platelet activation (Sheridan et al., 1986). Identification of normal donors is an important variable, because platelets from some healthy individuals are resistant to HIT antibodies in vitro (Chong, 1995), at least in part as a result of their Fc~IIA receptor phenotype. Platelets from some affected patients are mor~ sensitive than those from normal donors (Chong, 1995). The SRA is reported to have an analytical sensitivity of 94% and a specificity approaching 100% under optimal conditions (Chong, 1995). The results of an enzyme-linked immunosorbent assay (ELISA) using wells precoated with heparin/PF4 complexes are in accord with platelet activation assays in--80% of cases (Arepally et al., 1995; Amiral et al., 1995). A positive ELISA combined with a negative SRA is seen in --10% of cases, presumably due to the greater sensitivity of the former assay. Low titers of antibodies can be detected by ELISA in -20% of patients receiving heparin who are not thrombocyto-

penic. A positive SRA combined with a negative ELISA occurs in --5--10% of cases, possibly due to antigens consisting of heparin-binding proteins other than PF4. The ELISA does not depend on donor platelets, is technically simpler to perform and does not involve the use of radioactive materials. However, the ELISA, being more sensitive than the SRA, detects low titer antibodies in a substantial proportion of patients treated with heparin who are not thrombocytopenic, while potentially missing antibodies directed to complexes between heparin and other proteins (Arepally et al., 1995; Greinacher et al., 1994c). No ELISA kit is presently available in the United States.

CLINICAL UTILITY Disease Association Heparin-Induced Thrombocytopenia. HIT, the most common drug-induced thrombocytopenia, occurs in approximately 1% of patients who receive intravenous unfractionated heparin for at least 1 week (Schmitt and Adelman, 1993; Warkentin and Kelton, 1.989). Although thrombocytopenia is typically moderate (platelet count 20--100,000/~L), HIT should be considered in any patient who is at risk and whose platelet count falls by >50% without explanation (Chong, 1995). HIT occurs sooner in previously exposed individuals (Laster et al., 1989). Approximately 10% of patients with HIT develop thrombi (Warkentin and Kelton, 1989). Arterial thrombi occur most often in the peripheral vessels, especially those which have been entered surgically, but any vessel can be affected (Boshkov et al., 1993). Venous thrombi and pulmonary emboli also occur frequently (Boshkov et al., 1993). A falling platelet count accompanying thrombosis is an important clue in distinguishing HIT from thrombosis caused by inadequate anticoagulation. Thrombi may be recurrent unless the disease is recognized and all exposure to the drug is stopped. Antibody Frequencies in Disease Although HIT occurs rarely in patients who have received only low doses of heparin (e.g., subcutaneous prophylaxis, IV flushes or indwelling heparin-bonded catheters), exposure to less than 10 units of heparin/day is sufficient to sustain the disease and precipitate

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thrombosis in sensitized individuals (Laster et al., 1989; Warkentin et al., 1995; Chong, 1995). HIT occurs somewhat more commonly in patients receiving bovine than porcine heparin and unfractionated compared with LMWH (Chong, 1995; Warkentin et al., 1995). HIT is also reported in some patients treated exclusively with sulfated heparinoids (Wolf et al., 1983). HIT is rare in children while the frequency is highest in patients undergoing vascular surgery, either because of recurrent drug exposure, concomitant platelet activation from vascular trauma or predisposition to thrombosis due to underlying cardiovascular diseases (Boshkov et al., 1993).

Application Although HIT is fundamentally a clinical diagnosis (Chong, 1995), detection of heparin-dependent antiplatelet antibodies in plasma from a patient suspected of having HIT provides strong supportive evidence, since false-positive tests are extraordinarily rare (Sheridan et al., 1986; Chong, 1995) even among unaffected individuals who have received heparin for comparable periods of time (Cines et al., 1980). On the other hand, a negative SRA occurs in -~10--15% of patients with a typical clinical presentation (Chong, 1995), a few of whom have evidence of a DIC-like syndrome manifest by hypofibrinogenemia and fibrin split products (Bell et al., 1976). In addition, occasional patients have a strongly positive ELISA, with a negative SRA, presumably because the former is more sensitive, especially for detecting IgM antibodies. Also, a substantial fraction of patients receiving heparin have a weakly positive ELISA and the diagnosis of HIT may require confirmation by SRA in cases where the index of suspicion is low. Whether IgG, IgM and IgA antiheparin/PF4 antibodies detected by ELISA have the same clinical significance is unknown. Finally, a small minority of patients in whom a strong clinical suspicion of HIT exists have a positive SRA with a negative ELISA, presumably because of the involvement of other heparin-binding proteins (Greinacher et al., 1994a). Neither the SRA nor the ELISA can distinguish patients with HIT from those considered to have asymptomatic transient thrombocytopenia ascribed to heparin (Greinacher et al., 1994b).

Effect of Therapy Heparin is contraindicated in any patient in whom

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HIT is suspected. Since the morbidity and mortality associated with HIT is due to thrombosis rather than bleeding, treatment is directed at diagnosis and the institution of alternative forms of anticoagulation rather than therapy typical of other autoimmune platelet disorders designed to prevent bleeding (Chong, 1995). HIT is self-limited if exposure to even small amounts of heparin is avoided. Further, the risk of recurrent thrombosis falls rapidly within the first 36 hours after heparin is discontinued; the platelet count returns towards normal in 3 - 5 days in the typical patient (Chong, 1995). Disappearance of antibody as detected by SRA parallels the resolution of the clinical disease in most patients, but residual antibody can be detected for several weeks on occasion (Laster et al., 1989; Cines et al., 1980). Since HIT is self-limited if properly managed, there is no compelling reason to interfere with antibody production or platelet clearance. Indeed, measures designed to raise the platelet count, such as platelet transfusion, may be contraindicated while the risk of recurrent thrombosis persists. Commercial intravenous IgG contains antiidiotype antiheparin/PF4 antibodies, but effects on the resolution of thrombosis or thrombocytopenia are not documented (Greinacher et al., 1994b).

CONCLUSION HIT provides an interesting model of autoimmunity in which a heterologous mucopolysaccharide combines with a normal endogenous protein released in specific clinical settings to generate autoantibodies in susceptible, but otherwise immunologically "normal" individuals to cause thrombocytopenia and thrombosis. It is unsettled whether the clinical consequences of autoantibody formation, specifically the risk of thrombosis, are modulated by a separate set of genetic factors, i.e., a polymorphism in the platelet Fc~,IIA receptor which renders platelets susceptible to aggregation. Whether these antibodies recognize neoepitopes within the protein, the carbohydrate or the complex of the two is also unclear. In addition to potentially shedding light on how exogenous molecules alter the antigenicity of host proteins, elucidating the role of the carbohydrate moiety may be important in the design of alternative, nonantigenic anticoagulant heparin-like molecules which may reduce the frequency of this potentially devastating disease.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

HETEROPHILE ANTIBODIES Roger L. Dawkins, M.D., D.Sc., Stephen C. Pummer, B.Sc., Romano G. Krueger, B.Sc. and Peter N. Hollingsworth, D. Phil.

Department of Clinical Immunology, Royal Perth Hospital, Sir Charles Gairdner Hospital, The Centre for Molecular Immunology and Instrumentation, University of Western Australia, Perth 6001, Western Australia, Australia

HISTORICAL NOTES For the present purposes, "heterophile" is used to describe those human antibodies which react with animal rather than human tissues. By contrast, autoantibodies are thought to be tissue rather than species specific; however, these definitions are overly simplistic. In this chapter, the focus is on those heterophile antibodies which mimic immunofluorescent patterns found when autoantibodies are tested on animal sections. The purpose is to illustrate these patterns so that confusion can be minimized. There is no doubt that the failure to distinguish between heterophile antibodies and autoantibodies has created substantial difficulty in the past. In the decade from 1967 there was considerable interest in antibodies induced by alloimmunization. Some of these react with antigens carried by nonhuman tissues. No doubt, some of these antibodies react with antigens that are highly conserved but polymorphic and, therefore, alloimmunogenic in some but not all humans. For example, humans of blood groups A and O can be induced to produce antibodies resembling anti-B isohemagglutinins but reactive with antigens found in most strains of rat (McDonald et al., 1977). In 1971, such antibodies were shown to react with gastric parietal cells in the rat and were recognized as easily confused with autoantibodies reactive with parietal cells in humans and other species (Muller et al., 1971). Fortunately, the heterophile antibodies produce characteristic patterns on other tissues which permit their recognition and classification (Hawkins et al., 1977). It became apparent that many patterns previously

attributed to autoantibodies were in fact due to heterophile antibodies. For example, the patterns on heart tissue have been misinterpreted in the past (Nicholson et al., 1977). Some of these patterns are illustrated (Figures 1--15).

IMMUNOFLUORESCENT PATTERNS HETEROPHILE ANTIBODIES

OF

Heterophile antibodies can be readily mistaken for other common autoantibodies (Table 1). For example, the immunofluorescence patterns of heterophile and antiparietal cell antibodies can be confused on rat stomach alone (Figures 1, 2) but not if both mouse and rat stomachs are used. The gastric parietal cells of the mouse stomach are negative with heterophile (Figure 3) but positive with antiparietal cell antibody (Figure 4). Similarly, by using both mouse and rat stomach, anti-smooth muscle (Figures 5, 6) and heterophile (Figures 1, 3) antibodies can be distinguished. The absence of smooth muscle fiber staining and the presence of gastric parietal cell staining in the rat stomach confirm the presence of heterophile antibodies (Figure 1). Antimitochondrial antibodies bind gastric parietal cells in both rat and mouse stomachs; whereas, heterophile antibodies bind rat but not mouse gastric parietal cells (Figures 1, 3). Antimitochondrial antibodies bind to heart muscle fibers, producing a streaky immunofluorescent pattern (Figure 7); whereas, heterophile binds only to the endomysium (Figure 8). Antimitochondrial antibodies bind to the cytoplasm

351

L/I I,,3

Table 1. Heterophile Antibodies and Some Autoantibodies with Which They Are Confused

Rat Stomach

Mouse Stomach

Rat Kidney

Parietal Cells

Parietal Muscularis Between Cells Mucosae & Gastric Glands Externa

Tubules

Heterophile

+ (Fig. 1)

Parietal Cell

+ (Fig. 2)

Mitochondrial

+

Muscularis Between Mucosae & Gastric Glands Externa

+ (Fig. 3)

+ (Fig. 6)

vertical smooth muscle fibers (Fig. 6)

+ (Fig. 5)

vertical smooth muscle fibers (Fig. 5)

Reticulin (R1) -

peripheral connective tissue

intergastric connective tissue

peripheral connective tissue

intergastric connective tissue

peripheral connective tissue

intergastric connective tissue

-

Glomeruli

Rat Heart

Blood Vessels

brush border _+ peritubular

_+ Kupffer cells endomysium + capillaries (Fig. 8) (Fig. 15)

cytoplasmic (Fig. 9)

_+ cytoplasmic

Guinea Pig Skeletal Muscle

endomysium (Fig. 11)

+ (Fig. 4)

Smooth muscle-

Endomysial

Rat Liver

-

-I-

+

blood vessels

peritubular

periglomerular

+

portal vein portal duct bile duct _+ sinusoids

peritubular

periglomerular

+

cytoplasmic streaky intermyofibrillar (Fig. 7)

endomysium

endomysium (negative with IgG)

endomysium

endomysium

Heart

diffuse of peripheral (Fig. 13)

Striational

Striations (Fig. 14)

Striations (Fig. 12)

Figure 1. Heterophile antibodies, x 400. Staining of gastric parietal cells of rat stomach. The muscularis is at the right of the photograph. Note the differential staining intensity of the parietal cells and their dark nuclei.

Figure 2. Antiparietal cell antibodies, x 400. Staining of gastric parietal cells of rat stomach. The muscularis is at the right of the photograph. The pattern of staining is the same as heterophile (Figure 1).

Figure 3. Heterophile antibodies, x 400. Staining between the parietal cells of mouse stomach, but note that the antibody does not bind to the parietal cells. The muscularis is at the bottom of the photograph.

Figure 4. Antiparietal cell antibodies, x 400. Staining of the gastric parietal cells of mouse stomach but not between the cells. The muscularis is at the bottom of the photograph.

Figure 5. Anti-smooth-muscle antibodies, x 400. Staining of the smooth muscle fibers oriented horizontally between the gastric glands, in the muscularis mucosae and in the muscularis externa (at the right of the photograph) of mouse stomach.

Figure 6. Anti-smooth-muscle antibodies, x 400. Staining of the smooth muscle fibers oriented horizontally between the gastric glands in the muscularis mucosae, in a blood vessel and in the muscularis externa (at the right of the photograph) of rat stomach.

353

Figure 7. Antimitochondrial antibodies, x 400. Cytoplasmic streaky intermyofibrillar pattern in rat heart.

Figure 8. Heterophile antibodies, x 600. Staining of the endomysium of rat heart.

Figure 9. Antimitochondrial antibodies, x 400. Cytoplasmic staining of rat kidney tubule cells. Note the absence of staining of the brush border.

Figure 10. Heterophile antibodies, x 400. Staining of the brush border of rat kidney tubule cells. The cytoplasm of the tubular cells does not bind heterophile antibodies.

Figure 11. Heterophile antibodies, x 600. Staining of the endomysium of guinea pig skeletal muscle.

354

Figure 12. a,b: Antistriational antibodies, (x 1200). Striational staining pattern in guinea pig skeletal muscle.

Figure 13. a,b: Antiheart antibodies. (a: x 600, b: x 1200). Diffuse pattern on rat heart. A peripheral pattern may also be seen with these antibodies.

Figure 14. a,b: Anti-striational antibodies. (a: x 600, b: x 1200). Cross striations can clearly be seen in the rat heart.

355

tinguished from anti-heart antibodies which have a peripheral or diffuse pattern (Nicholson et al., 1977) (Figure 13) without striations (as seen with antistriational antibodies on rat heart [Figure 14]). Another pattern seen with many, but not all, heterophile antibodies is staining associated with Kupffer cells and capillaries (Hawkins et al., 1977) (Figure 15).

CONCLUSION

Figure 15. Heterophile antibodies, x 400. Staining pattern in rat liver Kuppfer cells and capillaries.

of rat kidney tubule cells (Figure 9); whereas, heterophile has a characteristic brush border pattern (Figure 10). The characteristic antistriational antibodies pattern can be distinguished from the heterophile pattern of each on guinea pig skeletal muscle. Heterophile stains only the endomysium (Figure 11) and lacks the striational pattern seen with antistriational antibodies (Figure 12). The endomysial pattern due to heterophile antibodies should not be confused with the antiendomysial antibody associated with celiac disease which, unlike heterophile, is an IgA antibody. On rat heart tissue, heterophile antibodies, which bind to the endomysium only (Figure 8), may be dis-

REFERENCES

Degli-Esposti MA, Dallas PB, Dawkins RL. Neuromuscular function and polymorphism of the acetylcholine receptor gamma gene. Muscle Nerve 1992;15:543--549. Garlepp MJ, Kay PH, Dawkins RL, Bucknall R, Kemp A. Cross-reactivity of antiacety!choline receptor autoantibodies. Muscle Nerve 1981;4:282--288. Hawkins BR, McDonald BL, Dawkins RL. Characterization of immunofluorescent heterophile antibodies which may be confused with autoantibodies. J Clin Pathol 1977;30:299-307. Hawkins BR, Saueracker GC, Dawkins RL, Davey MG, O'Con-

356

In addition to the importance of recognizing such patterns and avoiding misdiagnosis, it may be important to emphasize a more general conclusion. An individual is capable of producing antibodies which react with antigens which are not present in that individual. Such antigens may be classified as allo- or xenoantigens as distinct from autoantigens. The distinction, however, may not always be obvious. Any antigen which is polymorphic (varies within the species) is a potential source of confusion when evaluating autoantibodies irrespective of the detection system used. Further potential complexity is relevant because at least some autoantigens are polymorphic to some degree (Garlepp et al., 1981; Degli-Esposti et al., 1992). Thus, it can be expected that some reaction patterns will be the consequence of a mixture of antibodies of different specificities and some of these may be attributed to autoantibodies incorrectly (McDonald et al., 1977; Hawkins et al, 1980; Garlepp et al., 1981; Degli-Esposti et al., 1992).

ner KJ. Population study of heterophile antibodies. Vox Sang 1980;39:339--342. McDonald BL, Hawkins BR, Dawkins RL, Davey MG. Characterization of human heterophile antibodies apparently induced by alloimmunization. Vox Sang 1977;33:143--149. Muller HK, McGiven AR, Nairn RC. Immunofluorescent staining of rat gastric parietal cells by human antibody unrelated to pernicious anaemia. J Clin Pathol 1971;24:1314. Nicholson GC, Dawkins RL, McDonald BL, Wetherall JD. A classification of anti-heart antibodies: differentiation between heart-specific and heterophile antibodies. Clin Immunol Immunopathol 1977;7:349--363.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

HIDDEN A U T O A N T I B O D I E S Margalit Lorber, M.D. a, Jacob George, M.D. b and Yehuda Shoenfeld, M.D. b

alnstitute of Clinical Immunology and Allergy, Rambam Medical Center, The B. Rappaport Faculty of Medicine, Technion, Haifa; and bDepartment of Medicine "B", Research Unit of Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel

H I S T O R I C A L NOTES Over the last decade, a vast amount of data has been accumulated in order to create a better understanding of the function and role of naturally occurring antibodies that have the capacity to bind to self-antigens. These antibodies, called "natural" autoantibodies (NAA), are present in the sera of healthy individuals and rodents (Avrameas et al., 1988) and can be generated in vitro by activating normal human B lymphocytes. AUTOANTIBODIES Terminology

Hidden autoantibodies are preformed antibodies directed against self-constituents. They are produced by the normal immune system, but they cannot be detected by the conventional methods used to screen sera of animals and humans; hence, the term "hidden". Hidden autoantibodies are natural, polyreactive autoantibodies produced by human CD5-positive B cells (Ly 1+ B in mice) as opposed to antigen-driven, monoreactive antibodies which are produced by CD5negative B cells (Casali and Notkins, 1989). Like NAA, hidden autoantibodies constitute a physiologic repertoire that may play a role in immune regulation. Methods of Detection ,

The conventional assays used to detect antibodies in normal human sera will not detect hidden autoantibodies. Yet, in all comparative studies in which autoreactivity was examined, 1--5% of "normal" controls

carry autoantibodies; the frequency depends on the statistical cut-off used (p<0.05-5% and p<0.01--1%). Among different methods employed to "reveal" hidden autoantibodies (Table 1), most are based on positive selection of immunoglobulins (Ig) from the serum, i.e., the hidden autoantibodies are revealed in Ig fractions obtained by elution from anti-Ig or protein A columns. The Ig eluted from the various affinity chromatography columns often yields a reaction with autoantigen(s); whereas, the original, intact, unfractionated serum shows no reactivity with the same autoantigen(s) in serum containing hidden autoantibodies. When the corresponding whole serum or the flow-through from the column is added to the Ig fraction, the binding of the autoantibodies to its antigen will be inhibited. These findings suggest the presence of certain "natural inhibitors" which are probably a normal part of the immune system in healthy individuals (Kra-Oz et al., 1993). Other methods of detection include acid gel filtration on Sephadex G-200 to reveal hidden IgM rheumatoid factor (RF) (Moore et al., 1978; 1980; Dorner et al., 1983) and heat inactivation of normal human sera to disclose the presence of antibodies to cardiolipin (aCL) (Cheng, 1991).

CLINICAL UTILITY Hidden Antiphospholipid Antibodies. After the passage of normal human sera through Sepharose antihuman IgG or protein-A columns, the bound fractions (purified IgG) can be eluted with either MgC12 or low pH. Antiphospholipid antibodies are detected by ELISA (Figure 1). Normal human sera do

357

k~

Table 1. Methods Employed to Reveal Hidden Autoantibodies Antigen

Separation of Antibodies

Elution

Assay of Antibody Activity

Reference

CL, PS, PI, DNA, TG,

Protein-A chromatography antihuman IgG chromatography

MgC12, pH3

ELISA

Kra-Oz, 1993; Lorber, 1995

PDH

ELISA, Immunoblot

CL

Heat inactivation

Histones

Protein-G agarose

DNA

Q A E - Sephadex A-50

TG, DNA, Laminin, Transferin, Myoglobin, Factor VIII IF

ELISA

Cheng, 1991

pH3

ELISA

Bustos, 1994

0.5M Na CL

ELISA

Saenko, 1992

Protein-A chromatography

ELISA, RIA

Hurez, 1993a; 1993b

Tubulin, Albumin, Actin, TG, Myoglobulin, Fetuin, Transferin, Albumin, Collagen

Immunoadsorption columns of unsolubilized antigens

ELISA

Guilbert, 1982

Antiribosomal P

Affinity chromatography

ELISA

Stafford, 1995

IgM Rheumatoid Factor

Acid-gel filtration on Sephadex G-200 QAE Sephadex A-50

ELISA, srbc-hemolytic assay

Allen, 1966; Magsaam, 1987; Moore, 1978

CL: cardiolipin; PS: phosphatidylserine; PI: phosphatidylinositol; TG: thyroglobulin; PDH: pyruvate dehydrogenase; IF: intrinsic factor.

Figure 1. Binding of IgG and sera in anticardiolipin-ELISA. Binding of IgG fractions (open bars, 50 ~tl/mL) and serum (black bars, 1:200) from eight healthy individuals (NHS) (116,148,149,252,253,254,255,264) and four patients with antiphospholipid syndrome (APLS) (159,257,494,484) to cardiolipin (CL) in anti-CL-ELISA assay. The IgGs were purified on a Sepharose anti human IgG column and eluted by 4.9 MgC12.

not show anti-CL activity; whereas, all the IgG fractions react with CL and give high levels of binding, similar to the IgG from patients with antiphospholipid syndrome (APS). Competition assays confirm the specific binding of the IgG fractions (Figure 2). Preincubation of the IgG fractions with increasing concentrations of CL yields nearly total inhibition of the anti-CL binding. To examine the possibility that the detection of natural anticardiolipin antibodies (aCL) in normal human sera is blocked by certain inhibitory factors present in the normal human sera, the purified IgG fractions are mixed with increasing concentrations of normal human sera. Normal human sera inhibit the binding of CL-aCL in a dosedependent manner (Figure 2). The inhibitory effect is found not only in normal human sera but also in bovine, chicken, horse and mouse sera (Figure 3). Binding of the purified IgG from normal human sera is inhibited in a dose-dependent manner by the 132glycoprotein-I (~2-GPI); whereas, the binding of the IgG fractions from patients with APS is either enhanced or is not greatly influenced by the addition of this cofactor (Figure 4). In addition to aCL, normal human sera react with phosphatidylserine, phosphati-

Figure 2. Specificity of IgG fraction binding by anti-CL competition assay. IgG fractions (25 ~tg/mL) from six healthy individuals (NHS) were preincubated with increasing concentrations of cardiolipin (5--100 ~g/mL). The mixture (50 ~tL) was then added to aCL-ELISA assay. Each point represent the mean _+ SD of the inhibition percentage of six different IgGs. Each sample was tested three times.

dylinositol and phosphatidylcholine (Figure 5). However, the inhibitory effect of the ~2-GPI occurs only with negatively charged phospholipids (data not shown).

Figure 3. Inhibition of the binding of IgG fraction from healthy individuals to CL by various animal sera. IgG fractions of six human sera (25 ~tg/mL) were preincubated with dilutions of 1:50-- 1:3200 of sera from various animals. The residual activity was calculated as the percentage of activity of the same amount of the IgG preincubated with buffer only (100%). Each point represents the mean _+SD of eight IgG fractions. O, bovine; O, chicken; FI, horse; II, mouse; A, rabbit.

359

Figure 4. The effect of I]2-GPI on aCL antibodies. IgG fractions (50 ~tg/mL) from healthy individuals (NHS) (A) and from serum of APLS patients (B) were preincubated with 132-GP1 (10 ~g/mL, 30 ~g/mL) for 30 minutes. Each mixture (50 laL) was then added to aCL-ELISA. The calculation of the residual binding was the same as in Figure 3. Each line-connected character represents serum of one individual.

Figure 5. Binding of IgG fractions and their corresponding sera from 11 healthy individuals to various phospholipids. The assay was done by ELISA as described previously. CL = cardiolipin, PC = phosphatidylcholine, PS = phosphatidylserine, PI = phosphatidylinositol, PEA - phosphoethanolamine. 360

These data support the notion of the existence of both hidden antiphospholipid antibodies and natural inhibitors (in this case, [32-GPI) in sera of healthy individuals (Kra-Oz et al., 1993, Lorber et al., 1995).

Hidden Anti-Pyruvate Dehydrogenase Antibodies. Hidden autoantibodies to pyruvate dehydrogenase (PDH) can be detected in primary biliary cirrhosis, an organ-specific autoimmune disease. As with autoantibodies to certain phospholipids, anti-PDH antibodies are not detected in whole sera from healthy individuals, but all the corresponding IgG fractions bind in the anti-PDH assays, including ELISA and immunoblot (Figure 6) (Lorber et al., 1995). Hidden Antibodies in Pregnancy. Preparations of normal human IgG from pregnant women as well as healthy individuals contain a wide autoreactive repertoire, including autoantibodies to antigens such as intrinsic factor, transferrin, dsDNA, myoglobin and laminin as well as hidden antibodies to gliadin, tetanus toxoid and infectious agents (Hurez et al., 1993b). Although autoantibody activity is very low in whole sera, it can be easily detected in all purified IgG fractions. IgG purified from sera of pregnant

women shows higher binding activity than IgG from infants or adults (Hurez et al., 1993b).

Hidden Anti-dsDNA Antibodies. The presence of hidden, high-avidity dsDNA antibodies can also be demonstrated in normal human immunoglobulin preparations. Normal human sera significantly inhibits the DNA-binding activity of normal IgG (Saenko et al., 1992). Hidden Antihistone Antibodies. Purified IgG fractions from healthy donors react with histones in an antihistone ELISA; the activity is higher in the IgG fraction than in the corresponding serum. Histonebinding components are found in Ig fractions from normal donors and patients with SLE. Serum protein components inhibitory to the histone-anti-histone binding may play a protective role against both highaffinity anti-histone antibodies found in SLE sera and natural low-avidity antihistone antibodies in healthy donors (Bustos et al., 1994). Hidden Rheumatoid Factors. Rheumatoid factors (RF) are autoantibodies reactive with the Fc of the IgG molecule. Although not highly specific, RF

Figure 6. Immunoblotting of antipyruvate dehydrogenase (PDH) antibodies. Immunoblotting was done on polyacrylamide gel 10%. The first five lanes (1--5) are sera from healthy individuals diluted 1:100. Lanes 6--10 are their corresponding IgG fractions 10 ~al/mL. Lane 11 shows serum from a patient with primary biliary cirrhosis, positive for anti PDH. 361

commonly accompany rheumatoid arthritis (RA) and can be detected in about 70% of adult RA. In juvenile RA, (JRA) most of the sera do not contain conventional RF and are therefore called "sero-negative", as defined by standard tests for serum of RF. Hidden RF were detected in the IgM fractions of serum in the absence of other serum proteins as early as 1966 (Allen and Kunkel, 1966). 19S hidden IgM-RF were subsequently detected in 68% of patients with JRA using acid gel filtration of sera (Moore et al., 1978; 1980). Similar results occurred using an ion exchange procedure with QAE Sephadex A-50 or acid gel filtration and ELISA; RF were detected in 57% (Magsaam et al., 1987) and 100% of JRA sera, respectively (Roizenblatt et al., 1993).

Natural Serum Inhibitors. Hidden autoantibodies in the normal human sera are apparently bound by some serum factors in a way which inhibits their activity. The inhibitory factors can be a variety of serum components. Following the observation that heating of sera reveals the presence of aCL (Cheng, 1991), the possibility of complement components as inhibitors was investigated. However, addition of anti-Clq and anti-C4 monoclonal antibodies to the inhibitory effluent does not abolish the inhibitory effect on CL-aCL activity (unpublished data). In this case, anti-idiotypic activity is also excluded, because the inhibitory factor is in the Ig-free fraction; therefore, it is probably not an immunoglobulin (unpublished data). Hidden Antiribosomal P Antibodies Sera from healthy individuals were applied to affinity columns coated with ribosomes. Antiribosomal P anti

REFERENCES Allen JC, Kunke HG. Hidden rheumatoid factors with specificity for native gamma globulins. Arthritis Rheum 1966;9: 758--768. Avrameas S, Guilbert B, Mahana W, Matsiota P, Ternynck T. Recognition of self- and non-self-constituents by polyspecific autoreceptors. Int Rev Immunol 1988;3:1-15. Bustos A, Boimorto R, Subiza JL, Pereira LF, Marco M, Figueredo MA, de la Concha EG. Inhibition of histone/antihistone reactivity by histone-binding serum components; differential effect on anti-H 1 versus anti-H2B antibodies. Clin Exp Immunol 1994;95:408--414. Casali P, Notkins AL. Probing the human B-cell repertoire with EBV: polyreactive antibodies and CD5+ B lymphocytes. Annu Rev Immunol 1989;7:513--535.

362

bodies were detected in serum only after affinity chromatography, were predominantly IgG and demonstrated specificity for all three ribosomal P phosphoproteins. Autologous serum contained an IgG inhibitor of anti-P antibodies.

CONCLUSION The presence of hidden N A A in the sera of healthy individuals is now established. The fact that there are so many different hidden autoantibodies and that they are directed both against self- and non-self-constituents (Hurez et al., 1993a; 1993b) merits explanatory hypotheses. N A A are a normal part of the immune system; a great variety of N A A can be detected in normal human sera. How the N A A found in healthy individuals differ from those in pathologic conditions is not fully established. How N A A become pathogenic might depend on differences in natural inhibitors in serum. The autoantibodies formed in normal individuals are the same antibodies as those formed in pathologic conditions (such as autoimmune diseases) in terms of antigen specificity and avidity. Because they are inhibited by normal constituents of serum, N A A are typically not pathogenic. Since serum inhibitors of N A A are normal constituents of the immune system, once a perturbation in their production or some other alteration occurs, the N A A are no longer "covered" or "hidden." Their binding sites are exposed to bind to their respective antigen and they then become pathogenic antibodies, perhaps with a crucial role in the evolution of autoimmune conditions. See also NATURAL AUTOANTIBODIES.

Cheng HM. Antiphospholipid antibodies are masked in normal human serum. Immunol Today 1991;12:96. Dorner RW, Moore TL, Alexander RL Jr. Rapid determination of hidden rheumatoid factor. J Immunol Methods 1983;57: 221--226. Guilbert B, Dighiero G, Avrameas S. Naturally occurring antibodies against nine common antigens in human sera. I. Detection, isolation and characterization. J Immunol 1982; 128:2779-2787. Hurez V, Dietrich G, Kaveri SV, Kazatchkine MD. Polyreactivity is a property of natural and disease-associated human autoantibodies. Scand J Immunol 1993a;38:190--196. Hurez V, Kaveri SV, Kazatchkine MD. Expression and control of the natural autoreactive IgG repertoire in normal human serum. Eur J Immunol 1993b;23:783-789. Kra-Oz Z, Lorber M, Shoenfeld Y, Scharff Y. Inhibitor(s) of

natural anticardiolipin antibodies. Clin Exp Immunol 1993; 93:265--268. Lorber M, Kra-Oz Z, Guilbrud B, Shoenfeld Y. Natural (Antiphospholipid-PDH,-DNA) autoantibodies and their physiologic serum inhibitors. Isr J Med Sci 1995;31:31-35. Magsaam J, Ferjencik P, Tempels M, Dippell J. A new method for the detection of hidden IgM rheumatoid factor in patients with juvenile rheumatoid arthritis. J Rheumatol 1987; 14:964--967. Moore TL, Zuckner J, Baldassare AR, Weiss TD, Dorner RW. Complement-fixing hidden rheumatoid factor in juvenile rheumatoid arthritis. Arthritis Rheum 1978;21:935-941. Moore TL, Dorner RW, Weiss TD, Baldassare AR, Zuckner J.

Hidden 19S IgM rheumatoid factor in juvenile rheumatoid arthritis. Pediatr Res 1980;14:1135--1138. Roizenblatt S. Goldenberg J, Gabriel A Jr. Hilario MO, Atra E. 7S IgG rheumatoid factor and hidden 19S IgM rheumatoid factor in juvenile chronic arthritis. Allergol Immunopathol (Madr) 1993 ;21:197--200. Saenko V, Kabakov AE, Poverenny AM. Hidden high-avidity anti-DNA antibodies occur in normal human gamma globulin preparations. Immunol Lett 1992;34:1--5. Stafford HA, Anderson CJ, Reichlin M. Unmasking of antiribosomal P autoantibodies in healthy individuals. J Immunol 1995;155:2754-2761.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

HISTONE (H2A-H2B)-DNA AUTOANTIBODIES Robert L. Rubin, Ph.D.

W.M. Keck Autoimmune Disease Center, Department of Molecular & Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA

HISTORICAL NOTES Although antibodies to the (H2A-H2B)-DNA complex (anti-[(H2A-H2B)-DNA]) were demonstrated only relatively recently, these antibodies were almost certainly involved in the 1948 description of the lupus erythematosus cell (LE cell) phenomenon, which is generally considered to be the seminal discovery linking autoantibodies to rheumatologic diseases (Hargraves et al., 1948). Anti-[(H2A-H2B)-DNA] can also be implicated retrospectively in much of the early work using a variety of immunoassays to "nucleoprotein" and other histone-DNA complexes that led to the discovery of anti-DNA antibodies in systemic lupus erythematosus (SLE). However, it was not until knowledge about the structure of chromatin and methods for isolating histone-histone and histoneDNA complexes was combined with solid-phase immunoassay methodology that the unique antigenicity of the H2A-H2B complex was first detected (Rubin et al., 1982). By definition, these antibodies do not react with (or at least bind significantly less well to) the individual component histones H2A or H2B. This discovery was soon followed by detection of antibodies to the H2A-H2B complex in patients with procainamide-induced lupus (Totoritis et al., 1988); this activity was subsequently shown to reflect a partial epitope requiring both the H2A-H2B dimer and DNA for full antigenicity (Burlingame and Rubin, 1991). Isolation of mouse monoclonal antibodies with similar specificities provided further support for the existence and importance of this antibody (Kotzin et al., 1984; Losman et al., 1992). More recent work demonstrated that the presence of anti-[(H2A-H2B)-DNA] is one of the earliest signs of lupus in BXSB and MRL/lpr mice (Burlingame et

364

al., 1993; Amoura et al., 1994) These antibodies are found in the majority of patients with idiopathic SLE (Burlingame et al., 1994b) and in lupus induced by a variety of drugs (Rubin et al., 1992b).

THE AUTOANTIGEN Terminology, Structure and Features The five major histone classes (HI, H2A, H2B, H3 and H4) constitute a set of interacting proteins that organize and constrain the topology of DNA in most eukaryotic cells into a particle called the "nucleosome", the fundamental repeat unit of chromatin structure. The kernel of the nucleosome is composed of two molecules of both H3 and H4 in the form of a tetramer. Flanking this tetramer are two dimers of histones H2A and H2B (Figure 1). The (H3-H4) 2 tetramer and the H2A-H2B dimer are stable in solution, and, therefore, will assemble when the individual histones are mixed. The (H2A-H2B-H3-H4) 2 octamer is stabilized by a nearly double wrap of approximately 146 base pairs of DNA to form the core particle of the nucleosome. This structure is linked to the adjacent core particle by 20 to 60 base pairs of DNA with one molecule of histone H1, producing the mononucleosome (McGhee and Felsenfeld, 1980). Controlled digestion of chromatin with micrococcal nuclease produces oligonucleosomes and core particles due to the susceptibility of the linker DNA to hydrolytic cleavage. After extensive digestion of chromatin with micrococcal nuclease and treatment with 3 M urea, subnucleosome particles are released, consisting of H1 plus 60--70 base pairs of DNA, H2A-H2B with 50--60 base pairs of DNA, and (H3-H4) 2 with 70--80

Figure 1. Exploded diagrammatic structure of the core particle of the nucleosome (right) and its relationship to other nucleosomes in the solenoid-like polynucleosome fiber (center), which undergoes additional supercoiling into chromatin fibers of increasing diameter (left). See text for additional details. base pairs of DNA (Nelson et al., 1982). A complex between H2A-H2B and DNA (and H3-H4 and DNA) can also be formed in vitro by annealing DNA with H2A-H2B as described (Rubin and Lahita, 1992). When properly prepared, these histone-DNA complexes are soluble and stable in physiologic solution.

Sources The H2A-H2B dimer or the dimer-DNA is not currently available commercially. A number of vendors (Boehringer-Mannheim, U.S. Biochemical Corp., Sigma) offer the individual histones from which the H2A-H2B and the (H2A-H2B)-DNA complex can be produced as described (Burlingame and Rubin, 1990; Rubin and Lahita, 1992). However, validation of the claimed identity requires chemical credential information from the supplier or in-house laboratory confirmation (Figure 2). In addition, in vitro assembly of the H2A-H2B complex is incomplete, leaving monomeric H2A and H2B which can bind antibodies to these individual histones and produce false-positive results in immunoassays. Therefore, the preferred source of H2A-H2B is the preformed complex isolated from native chromatin. Since the primary and higher ordered structure of the (H2A-H2B)-DNA complex is highly conserved among all eukaryotes (including plants), almost any tissue (except sperm) can be used. Quiescent lymphocytes are an especially good source of chromatin because their sparse cytoplasm simplifies the extraction. Calf thymus, the tissue of choice because of its large size and low cost, should be obtained either fresh from a slaughterhouse or by special order from a commercial source such as

Pel-Freeze Biologicals, (Rogers, Arkansas) to insure that it is frozen within a few hours after harvesting.

Methods of Purification Purification of the H2A-H2B dimer from calf thymus (Godfrey et al., 1990; Burlingame and Rubin, 1990) can be accomplished in a few days using the standard equipment of a biochemistry laboratory including an ultracentrifuge and column chromatography. In addition, polyacrylamide gel electrophoretic analysis is required to establish quality (Figure 2). (H2AH2B)-DNA is easily produced by mixing the dimer with DNA in 2.0 M NaC1 followed by removal of the salt by dialysis (Rubin and Lahita, 1992). The size and species origin of the DNA is apparently unimportant to the antigenicity of the (H2A-H2B)-DNA complex. However, the DNA must be freed of singlestranded regions by S 1-nuclease digestion in order to avoid false-positive signals due to antibodies to denatured (single-stranded) DNA. Antinative (doublestranded) DNA antibodies will unavoidably produce false-positives with (H2A-H2B)-DNA.

Sequence Information and Epitopes The amino acid sequence of the major variant of bovine and human H2A and of bovine and human H2B are identical (Figure 3), so calf thymus is an appropriate source for isolating these histones for screening human autoantibodies. The region within the H2A-H2B dimer that contributes to the conformational epitope(s) recognized by anti-[(H2A-H2B)-DNA] is unknown. However, based on the X-ray crystal-

365

nakis, 1993) and presumably also contributes to the epitope(s) recognized by most idiopathic and druginduced lupus sera. Linear epitopes on histones H2A and H2B (and H3, H4 and H1) are recognized by separate populations of antihistone antibodies commonly found in SLE, drug-induced lupus, drug-induced autoimmunity (without accompanying symptoms), juvenilerheumatoid arthritis and some other syndromes, as summarized elsewhere (Rubin and Lahita, 1992; Burlingame et al., 1994b). Most (although not all) studies place these epitopes at the amino and carboxy-terminal trypsin-sensitive tails of histones H2A and the amino terminus of H2B, distinctly different from the region of the H2A-H2B dimer where anti-[(H2A-H2B)-DNA] bind and to which access is actually enhanced after trypsin digestion (Burlingame and Rubin, 1991).

AUTOANTIBODIES

Terminology

Figure 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of histones in various chromatin-derived preparations. Lanes from left to right are total histones acid-extracted from chromatin, the (H2A-H2B-H3-H4)2 octamerof thecore particle, the (H3-H4)2 tetramer and the (H2A-H2B) dimer.

lographic solution of the three-dimensional structure of the octamer (Arents et al., 1991), the solventexposed surface likely to be accessible for antibody binding is quite limited. These discontinuous peptides are underlined in Figure 3. DNA is closely juxtaposed to this region in the nucleosome (Arents and Moudria-

366

Anti-[(H2A-H2B)-DNA] can be shortened to anti(dimer-DNA) antibodies for simplicity within a relevant context. These antibodies react with an epitope created by the quaternary interaction between H2A, H2B and DNA. Anti-[(H2A-H2B)-DNA] frequently bind to the H2A-H2B complex in the absence of DNA albeit usually with lower avidity. However, H2A-H2B is a less specific antigen because of the existence of antibodies to the component histones that often react with the H2A-H2B dimer; the latter appear to be largely inhibited when the dimer is bound to DNA (Burlingame and Rubin, 1991; Rubin et al., 1992a). Antibodies to total or individual histones are definitely not synonymous with anti-[(H2AH2B)-DNA]. These "antihistone antibodies" react with denatured histones in ELISA or immunoblot, are detected in a number of rheumatic diseases and generally do not show clinical associations. Anti[(H2A-H2B)-DNA] will react strongly with native chromatin; antichromatin and anti-[ (H2A-H2B)-DNA] are usually identical. However, the DNA in chromatin and dimer-DNA can also react with anti-DNA antibodies, so the presence of anti-DNA antibodies should be ruled out by appropriate immunoassay.

Pathogenetic Role As with most autoantibodies, anti-[(H2A-H2B)-DNA]

H2A

1 21 41 61 81 101 121

ser gly arg gly lys gin gly gly lys ala arg ala lys ala lys thr arg ser ser arg ala gly leu gin phe pro pal gly arg val his arg leu leu arg lys gly asn tyr ala glu arg val gly ala gly ala pro val tyr leu ala ala val leu glu tyr leu thr ala glu ile leu glu leu ala gly asn ala ala arg asp asn lys lys thr arg ile ile pro arg his leu gin leu ala ile arg asn asp glu glu leu asn lys leu leu gly lys val thr ile ala gin gly gly val leu pro asn ile gin ala val leu leu pro lys lys thr glu ser his his lys ala lys gly lys H2B

1 21 41 61 81 101

pro glu pro ala lys ser ala pro ala pro lys lys gly ser lys lys ala val thr lys ala gln lys lys asp gly lys lys arg lys arg ser arg lys glu ser tyr ser ile tyr val tyr lys val leu lys gln val his pro asp thr gly ile ser ser lys ala met gly ile met asn ser phe val asn asp ile phe glu arg ile ala gly glu ala ser arg leu ala his tyr asn lys arg ser thr ile thr ser arg glu ile gln thr ala val arg leu leu leu pro gly glu leu ala lys his ala val ser glu gly thr lys ala val thr lys

Figure 3. Amino acid sequences of the predominant forms of bovine and human H2B (Ohe et al., 1979) and H2A (Hayashi et al., 1980). The underlined regions are exposed in the core particle of the nucleosome and are likely to constitute part of the (H2A-H2B)DNA epitope. 40% of human spleen H2A has Lys at position 99 (H2A l) and 30% has Arg (H2A2). H2A 3 (15%) has Ser instead of Thr at position 16. H2A4 has a Ser at position 16 and the His at position 124 is deleted. Bovine H2A 1 is identical to human H2A 1, and bovine H2A2 has a Ser at position 16 and a Met instead of Leu at position 51 (Wu et al., 1986).

are not proved to be pathogenetic. Nevertheless, clinical correlations of SLE and drug-induced lupus sera enriched in these antibodies suggest pathogenic potential. In SLE and drug-induced lupus, anti-[(H2AH2B)-DNA] are predominantly IgG1 and IgG3, potent complement-fixing immunoglobulins (Rubin et al., 1986) with capacity to mediate the deposition of C3, C4 and properdin on nuclei of HEp-2 cells (Kanayama et al., 1986). The LE cell phenomenon is accompanied by complement fixation (Robbins et al., 1957) and phagocytosis. Depressed complement levels are reported in patients with lupus induced by procainamide (Utsinger et al., 1976) and hydralazine (Weinsteill, 1978), and elevated C4d/C4 ratios can be observed in procainamide-induced lupus (Rubin et al., 1989). Because anti-[(H2A-H2B)-DNA] are the predominant autoantibody in procainamide-induced lupus (Burlingame and Rubin, 1991) and in some SLE patients (Burlingame et al., 1994b), and because nucleohistone has been detected in SLE and normal serum (Rumore and Steinman, 1990), immune complex formation and classical pathway activation presumably involves this antigen-antibody system. Deposition or in situ formation of immune complexes on the glomerular basement membrane (Schmiedeke et al., 1989; Kramers et al., 1994) may be mediated by nucleohistone binding to heparan sulfate (Termaat

et al., 1990) or type IV collagen (Bernstein et al., 1995) due to affinity between these macromolecules in glomeruli.

Animal Models. Anti-[(H2A-H2B)-DNA] spontaneously arise in the mouse strains BSXB and MRL/lpr (Burlingame et al., 1993; Amoura et al., 1994) and NZB x N Z W F1. Anti-[(H2A-H2B)-DNA] are the earliest autoantibodies detectable in BSXB and MRL/lpr mice and account for the bulk of the early antichromatin activity in these SLE-prone mice. These animals have a serology similar to that of 14 out of 40 newly diagnosed SLE patients, and the mouse and human sera efficiently co-blocked, indicating a similar target epitope (Burlingame et al., 1994b). Chromatinspecific T-cell clones derived from the lupus prone S W R x NZB F1 mice display proliferative and cytokine responses to nucleosomes but not the component macromolecules; these clones also provide helper activity to primary B cells for secretion of IgG anti"histone-DNA" in vitro (Mohan et al., 1993). T cells with similar properties can be isolated from peripheral blood of SLE patients (Desai-Mehta et al., 1995). Immunization of normal mouse strains with various sources of histone and chromatin fails to produce autoantibodies reactive with native forms of chromatin (Rubin et al., 1990). However, (H2A-H2B)-

367

DNA-specific antibodies can be induced in C57BL/6 x DBA/2 F1 mice by in vivo transfer of DBA/2 T cells accompanying the graft-versus-host reaction (Rubin et al., 1990). Monoclonal antibodies reactive with the H2A-H2B dimer and/or the dimer-DNA complex derived from MRL/+, MRL/lpr and NZB x NZW F1 mice (Kotzin et al., 1984; Losman et al., 1993) use similar V n gene segments consistent with a dominant role for the heavy chain gene in antibody binding. The third complementarity-determining region in these heavy chains has an unusually high frequency of D-D genetic element fusions and many arginine residues, similar to anti-DNA antibodies. Some of these antibodies appear to be clonally related, apparently differing only through somatic mutations, suggesting that these clones are positively selected by engagement of chromatin in vivo (Losman et al., 1992; 1993). Methods of Detection Enzyme-linked immunosorbent assay (ELISA) is the only reliable method for detection of anti-[(H2AH2B)-DNA] (Burlingame and Rubin, 1990). It is important to use an IgG-specific, rather than a polyvalent detecting reagent because IgM anti-[(H2A-H2B)DNA] are commonly found in patients who remain asymptomatic (Burlingame and Rubin, 1991; Rubin et al., 1995). Use of H2A-H2B dimer in the absence of DNA is less satisfactory because of false-positive results with antibodies to the individual histones as previously discussed. In addition, if SLE is part of the differential diagnosis, antibodies to native DNA must be ruled out when employing the (H2A-H2B)-DNA complex. Ideally, detection of antinative DNA should be done in the same immunoassay format as antibodies to dimer-DNA as described (Rubin and Lahita, 1992). Antibodies to denatured (single-stranded) DNA could also produce a false-positive signal if the DNA in the preparation is denatured and should be monitored by including a prototype anti-(single-stranded) DNA antibody as a negative control in all immunoassays. Chromatin can also be used in ELISA to screen for anti-[(H2A-H2B)-DNA] because the dimerDNA is the dominant B-cell epitope in chromatin (Burlingame et al., 1994a; Burlingame and Rubin, 1991). Immunofluorescence on standard cell substrates generally produces a homogeneous, chromosomepositive staining pattern of the nucleus (Rubin et al., 1982). However, because this pattern is not specific

368

for anti-[(H2A-H2B)-DNA] and can be difficult to distinguish from a fine-speckled nuclear fluorescence pattern, this method is not considered definitive. Use of a three substrate system for detecting antihistone antibodies (normal, acid-extracted and histone-reconstituted cells) (Tan et al., 1976) is remarkably specific for anti-[(H2A-H2B)-DNA] (Burlingame et al., 1994a), but is labor-intensive, nonquantitative and largely of historical interest.

CLINICAL UTILITY Application and Disease Association A positive IgG anti-[(H2A-H2B)-DNA] is strong confirmation of a diagnosis of drug-induced lupus in a patient taking a known lupus-inducing drug. Since this antibody activity can be detected prior to development of symptoms of drug-induced lupus in some patients, a positive reaction may also be considered prognostic for development of symptomatic disease. However, IgG anti-[H2A-H2B]-DNA is not specific for drug-induced lupus, occurs commonly in patients with idiopathic SLE (Burlingame et al., 1994b) and is reported in a subset of patients with scleroderma-like disease (Wallace et al., 1994). Therefore, the clinical setting is important, but a diagnosis of drug-induced lupus is almost certain if a patient without a history of rheumatological disease develops arthralgias, myalgias, serositis and/or constitutional symptoms after having taken a known lupus-inducing drug for a few months or more and produces a positive test result for IgG anti-[H2A-H2B]-DNA. Since drug-induced anti[H2A-H2B]-DNA displays an effective serum half-life of approximately 3 months (Rubin et al., 1995), confidence in diagnosis is assured if assay results are decreased a few months after discontinuation of therapy. A negative result for IgG anti-[H2A-H2B]-DNA does not exclude a diagnosis of drug-induced lupus or SLE. Although only about 10% of patients with lupus induced by procainamide are negative for IgG anti[H2A-H2B]-DNA, approximately 50% of quinidineinduced lupus and two-thirds of hydralazine-induced lupus patients are negative on this antigen (Burlingame and Rubin, 1991; Rubin et al., 1992b). On the other hand, limited numbers of reports of lupus induced by penicillamine, isoniazid, acebutalol, methyldopa, timolol and sulfasalazine showed that these patients invariably had high levels of IgG anti[(H2A-H2B)-DNA] at symptom presentation.

Subclassifying The amount of IgG anti-[(H2A-H2B)-DNA] activity in symptomatic drug-induced lupus or SLE does not correlate with any measure of disease activity except proteinuria in SLE (Burlingame et al., 1994b). However, the absence of IgG anti-(dimer-DNA) in patients undergoing long-term therapy with procainamide is generally inconsistent with a diagnosis of drug-induced lupus. Asymptomatic procainamide-treated patients commonly produce IgM and/or IgA antibodies to the (H2A-H2B)-DNA complex and exhibit antinuclear antibody activity by immunofluorescence, but there is no evidence that these patients have an increased risk for converting to IgG anti-(dimer-DNA) and symptomatic drug-induced lupus. Approximately 40% of patients taking procainamide for 1 year or more have the IgM and/or IgA antibodies which appear to be retained indefinitely without IgG seroconversion and development of disease (Rubin et al., 1995). SLE and drug-induced lupus can usually be differentiated serologically by testing for antibodies to native DNA, SS-A/Ro, Sm and/or nRNP. Other than a few reports claiming antinative DNA antibodies in drug-induced lupus, these antibodies are generally not associated with drug-induced lupus but are often found in patients with SLE. It is important to be sure that the antinative DNA assay employed does not detect antibodies to denatured (single-stranded DNA) because antidenatured DNA antibodies are commonly present in the rheumatic diseases including druginduced lupus, so this false-positive finding will confound the ability to differentiate between SLE and drug-induced lupus.

Antibody Frequencies and Disease Associations Other than one report on this activity in sclerodermarelated disorders, all studies of anti-[(H2A-H2B)DNA] in rheumatic diseases have related to druginduced or idiopathic SLE (Table 1). The substantially higher prevalence (70 vs. 31%) of IgG anti-([H2AH2B]-DNA) in SLE (reported by Burlingame et al., 1994b compared to Wallace et al., 1994) may be due to the fact that most of the Oriental patients in the former study were newly diagnosed and largely untreated. Anti-([H2A-H2B]-DNA) in an American SLE patient population had significantly lower frequency (59%) and lower average activities than in the untreated oriental SLE patients (70%) (Burlingame et

al., 1994b). Immunosuppressive anti-inflammatory agents including corticosteroids are likely to reduce the anti-(dimer-DNA) activity. Although there are relatively few reports evaluating anti-[(H2A-H2B)DNA] in lupus induced by the "rare" lupus-inducing drugs, the prevalence of positive signals in those tested was very high.

Sensitivity and Specificity The sensitivity of IgG anti-[(H2A-H2B)-DNA] was calculated only for procainamide-induced lupus. Sensitivity was reported to be 84% of patients at time of diagnosis and low but significantly elevated reactivity was detected in 70% of patients one year before recognition of symptoms (Rubin et al., 1995). Sensitivity for SLE can be as high as 70% (Burlingame et al., 1994b), but is substantially lower after treatment with immunosuppressive or anti-inflammatory therapy. Specificity of IgG anti-[(H2A-H2B)-DNA] for druginduced lupus or for either drug-induced or idiopathic SLE has not been formally determined. Use of a nonIgG-specific detecting reagent in ELISA will preclude the ability to discriminate symptomatic from asymptomatic drug-treated patients.

CONCLUSION Anti-[(H2A-H2B)-DNA] is remarkably common in drug-induced and idiopathic SLE, explaining why this activity probably accounted for the first reports of autoantibodies in the rheumatic diseases. IgG but not other classes of anti-[(H2A-H2B)-DNA] provides a sensitive diagnostic test for lupus induced by a wide variety of drugs and has substantial prognostic value in the forewarning of clinical disease as a side-effect of procainamide therapy. This antibody specificity is also present in the majority of untreated SLE patients and is the earliest autoantibody in murine lupus. The complete epitope is present in the solvent-accessible region of polynucleosomes and chromatin; it is produced by a higher ordered structure in the stable subnucleosome particle (H2A-H2B)-DNA and probably requires the intermolecular interface between DNA and histones H2A and H2B. In patients with ambiguous clinical histories, further testing on other common autoantigens can help to distinguish various rheumatic diseases from druginduced lupus which usually has a more restricted serology. In addition, false-positive reactions can be

369

Table 1. Prevalence of IgG Anti-[(H2A-H2B)DNA] in Various Diseases

Disease

Patients Evaluated (N)

Patients with Elevated Anti- [(2A-H2B)DNA] (%)

Average Antibody Level (O.D.* _+ S.D.)

Reference

Systemic lupus erythematosus, Oriental untreated

40

28 (70%)**

4.1 + 6.0

Burlingame et al., 1994b

Systemic lupus erythematosus, American, treated

37

22 (59%)

0.8 _+ 1.2

Burlingame et al., 1994b

Systemic lupus erythematosus

100

31 (31%)

N.R.**

Wallace et al., 1994

Scleroderma-related disorders

26

16 (62%)

N.R.**

Wallace et al., 1994

Procainamide-induced lupus

24

23 (96%)

8.5 + 4.9

Burlingame and Rubin, 1991; Rubin et al., 1992

Quinidine-induced lupus

17

9 (53%)

1.8 + 1.7

Burlingame and Rubin, 1991; Rubin et al., 1992b

Hydralazine-induced lupus

14

6 (43%)

7.0 _+7.4

Burlingame and Rubin, 1991

1 (100%)

4.1

Enzenauer et al., 1990; Rubin et al., 1992b

1 (100%)

1.3

Salazar-Paramo et al., 1992; Rubin et al., 1992b

Acebutalol-induced lupus

1 (100%)

2.3

Rubin et al., 1992b

Methyldopa-induced lupus

2 (100%)

1.4 _+0.7

Nordstrom et al., 1989; Rubin et al., 1992b

Timolol-induced lupus

1 (100%)

4.8

Zamber et al., 1992

Sulfasalazine-induced lupus

2 (100%)

2.3 _+ 1.5

Bray et al., 1994

Penicillamine-induced lupus Isoniazid-induced lupus

1

*Normal serum binding <0.05 O.D.; **DNA binding activity absent or removed by absorption; *** N.R. = not reported.

produced by antibodies to native and denatured D N A as well as to individual histones. Use of high-quality preparations of the ( H 2 A - H 2 B ) - D N A complex can largely eliminate the latter two reactions, but subsequent testing for antinative D N A may be required to rule out non-drug-induced (idiopathic) SLE. Use of H 2 A - H 2 B complex (without D N A ) avoids the potential problems associated with anti-DNA antibodies, but this antigen is less specific due to binding of antihistone antibodies and less sensitive because it corn-

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prises a partial epitope. The H 2 A - H 2 B complex isolated from chromatin is a superior preparation to that produced by mixing H 2 A with H2B, but its production requires biochemistry laboratory procedures; native chromatin is easier to prepare and may be a suitable alternative antigen for screening anti-[(H2AH2B]-DNA]. See also HISTONE AUTOANTIBODIES OTHER THAN ( H 2 A - H 2 B ) - D N A AUTOANTIBODIES and D s D N A AUTOANTIBODIES.

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Arents G, Moudrianakis EN. Topography of the histone octamer surface: repeating structural motifs utilized in the docking of nucleosomal DNA. Proc Natl Acad Sci USA 1993 ;90:10489-10493. Bernstein KA, Di Valerio R, Lefkowith JB. Glomerular binding activity in MRL lpr serum consists of antibodies that bind to a DNA/histone/type IV collagen complex. J Immunol 1995; 154:2424--2433. Burlingame RW, Rubin RL. Subnucleosome structures as substrates in enzyme-linked immunosorbent assays. J Immunol Methods 1990; 134:187-- 199. Burlingame RW, Rubin RL. Drug-induced antihistone autoantibodies display two patterns of reactivity with substructures of chromatin. J Clin Invest 1991;88:680-690. Burlingame RW, Rubin RL, Balderas RS, Theofilopoulos AN. Genesis and evolution of antichromatin autoantibodies in murine lupus implicates T-dependent immunization with self antigen. J Clin Invest 1993;91:1687-1696. Burlingame RW, Boey ML, Starkebaum G, Rubin RL. The central role of chromatin in autoimmune responses to histones and DNA in systemic lupus erythematosus. J Clin Invest 1994a:94:184-192. Burlingame RW, Rubin RL, van Venrooij WJ, Maini RN, eds. Manual of Biological Markers of Disease. Section B: The Autoantigens. Dordrecht: Kluwer Academic Publishers, 1994b:B2.2/1--B2.2/18. Desai-Mehta A, Mao C, Rajagopalan S, Robinson T, Datta SK. Structure and specificity of T cell receptors expressed by potentially pathogenic anti-DNA autoantibody-inducing T cells in human lupus. J Clin Invest 1995;95:531--541. Enzenauer RJ, West SG, Rubin RL. D-penicillamine-induced systemic lupus erythematosus. Arthritis Rheum 1990:33: 1582--1585. Godfrey JE, Baxevanis AD, Moudrianakis EN. Spectropolarimetric analysis of the core histone octamer and its subunits. Biochemistry 1990;29:965--972. Hargraves MM, Richmond H, Morton R. Presentation of two bone marrow elements: the "Tart" cell and the "LE" cell. Proceedings of the Staff Meeting in Mayo Clinic 1948;23:25. Kanayama Y, Peebles C, Tan EM, Curd JG. Complement activating abilities of defined antinuclear antibodies. Arthritis Rheum 1986;29:748--754. Kotzin BL, Lafferty JA, Portanova JP, Rubin RL, Tan EM. Monoclonal antihistone autoantibodies derived from murine models of lupus. J Immunol 1984;133:2554. Kramers C, Hylkema MN, van Bruggen MCJ, van de Lagemaat R, Dijkman HBP, Assmann KJM, Smeenk RJT, Berden JHM. Antinucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo. J Clin Invest 1994;94:568-577. Losman MJ, Fasy TM, Novick KE, Monestier M. Monoclonal autoantibodies to subnucleosomes from a MRL/Mp-+/+ mouse. J Immunol 1992;148:1561--1569. Losman MJ, Fasy TM, Novick KE, Monestier M. Relationship among antinuclear antibodies from autoimmune MRL mice reacting with histone H2A-H2B dimers and DNA. Int Immunol 1993;5:513. McGhee JD, Felsenfeld G. Nucleosome structure. Annu Rev

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

HISTONE AUTOANTIBODIES OTHER THAN (H2A-H2B)-DNA AUTOANTIBODIES Christine Stemmer, B.Sc. and Sylviane Muller, Ph.D.

Institut de Biologic Mol6culaire et Cellulaire CNRS, UPR 9021 Immunochimie des Peptides et Virus, 67000 Strasbourg, France

HISTORICAL NOTES Early in the 1970s, observations of specific oligomeric histone complexes and a tandemly repeated beaded morphology for chromatin led to the description in 1974 of the nucleosome entity. Subject to intense research efforts over the two last decades, the nucleosome was shown to consist of a histone octamer (two H2A-H2B dimers and a histone H3-H4 tetramer), 146 base pairs (bp) of DNA wrapped 1.75 times around the octamer (forming the so-called core particle), and linker DNA (20 to 60 bp) joining adjacent core particles to form the nucleosomal array. Histone H1 binds outside the core particle to the linker DNA region. Whereas, initially considered simply constitutive proteins with the capacity to compact DNA within the chromosomes, histones are now known to play very specific roles in regulatory events, e.g., in the transcription of individual genes. Autoantibodies to histones were first detected in the serum of autoimmune patients by a complement fixation technique (Kunkel et al., 1960; Stollar, 1971). Subsequent studies showed that histones themselves tend to be anticomplementary and other immunoassays were then developed (Hekman and Sluyser, 1973).

THE AUTOANTIGENS

Definition The several nomenclatures for histone fractions extant prior to 1974 were standardized (and subsequently universally accepted) during the CIBA Foundation Symposium on the structure and function of chroma-

tin. The standard nomenclature is based on chromatographic fractionation behavior. The four core histones are H2A, H2B, H3 and H4. Linker histone species (also called lysine-rich histones) are H1, H5 and H1 o. In fact, each histone fraction is composed of isoprotein species or variants, designated, for example, as H3.1, H2A.X, H lb. Because they occur in specific tissues (see below), H1 o, H5 or the germ cell-specific H1, H lt are considered special variants. Some characteristics of calf thymus histones are shown (Table 1).

Native vs. Recombinant Antigen Performance cDNA of the major somatic histones (including human histones) as well as of a number of variants is available. However, probably because histones are found in abundance in all eukaryotes and are particularly well conserved during evolution (at least for core histones), recombinant histones are rarely (if ever) used for the study of human and mouse antibodies.

Origin, Sources, Organs, Tissue, Cells Histones are present in all eukaryotic nuclei (animal, plant, fungi and protista kingdoms) with a few exceptions, e.g., yeast does not contain H1. Each mammalian diploid nucleus contains about 40% (w/w) DNA (5 x 109 bp of DNA), 40% (w/w) histones, and 20% (w/w) of other compounds, more particularly nonhistone proteins and RNA. Found in inactive nucleated erythrocytes from birds and some fishes, H5 replaces a part of H1 during the process of genetic inactivation. H1 o accumulates in nondividing cells or in cells that have been chemically induced to differen-

373

Table 1. Principal Characteristics of Calf Thymus Histones

Fraction Mra

Number of residues

N-terminal o~-helix residue content (%)

[3-pleated 1 mg/mL Cationic sheet (%) E 275 nm charge densityb

Molar staining coefficients Coomassie blue Amido black

H1

26,500

220

Ac-Ser

55

5

0.079

37.7

1.00

1.00

H2A

14,000

125

Ac-Ser

35

20

0.300

21.4

0.44

0.49

H2B

13,770

129

Pro

40

20

0.510

21.4

0.22

0.59

H3

15,340

135

Ala

39

15

0.300

22.2

0.65

0.78

H4

11,280

102

Ac-Ser

28

31

0.500

23.9

1.67

0.47

aMr values have been taken from Dayhoff (Dayhoff, 1978) except for H1 calculated from the sequence. bCationic charge density was calculated from the amino acid composition, that is, the sum of the mol % Lys, His, Arg and the Nterminus divided by the sum of Mr values of the constituent amino acids (i.e., charge per 10,000 Da). In most proteins this figure is close to the mol % basic amino acids present (adapted from von Holt et al., 1989). tiate. In tissues with very little or no replication activity, H5 and H 1~ represent 7--50% of the total H 1. Germ cell-specific histones are either absent from oocytes or present in much lower concentrations than in comparable stages of male germ cells. Methods of Purification

In general, histones are prepared from calf thymus, rat liver or chicken erythrocytes (Von Holt et al., 1989). The tertiary structure of histone molecules appears to be affected by the purification procedure since the antigenic properties of histones isolated by the salt and acid extraction procedures are not the same. In the so-called saline method, which is recommended, extreme pH conditions are avoided; protamine is used to displace histones from DNA and a series of runs on Sephadex G-100 columns, equilibrated in 50 mM sodium acetate successively at pH 5 and 4, are used to separate the five histone fractions. Because the different histones are very similar in molecular weight, charge and secondary structure and have a high tendency to copolymerize, particular care in the preparation of pure fractions free of mutual contamination is necessary to avoid mistakes in the characterization of human or mouse antibodies tested (Muller et al., 1985b). Purified histones can be kept in lyophilized form for years under dry conditions at room temperature. However, if samples are stored cold and opened when cold, moisture will condense in the protein powder, and histones will slowly deteriorate. Furthermore, when resuspended in solution, histones should not be stored longer than 1 or 2 weeks at 4~ since they form homoaggregates, especially at high ionic strength and concentration,

374

leading to changes in their antigenic properties. Proteolytic and chemical methods for producing natural histone fragments are useful for locating histone regions which are recognized by autoantibodies (Gohill et al., 1990; Monestier et al., 1989). The purity of histone fractions and fragments can be evaluated by electrophoresis in different gel systems including SDS-PAGE and acetic acid-urea gels with or without triton (Von Holt et al., 1989). These procedures, however, are not very sensitive and it is not uncommon to find that fractions which appear to be pure by biochemical criteria are in fact contaminated by as much as 1--2% of other histone classes. In some cases, and depending on the conditions used for immunoassays, this degree of contamination can be sufficient to influence the results (Muller et al., 1985a). Commercial Sources

Histone mixtures and individual histones are available from different sources including Boehringer-Mannheim (Germany), Sigma (St. Louis, MO, USA) and Worthington (Freehold, NJ, USA). Ambion (Austin, Texas, USA) offers highly purified calf histone H1, originally provided for molecular biology. Commercial histones are generally prepared from calf thymus by the acid method; they should be checked for purity and net protein content. Considerable variability of quality and content can be observed between different batches provided by the same company. When evaluating the purity of histone fractions, it should be kept in mind that each histone type presents different affinities for the usual protein stains, such as Coomassie blue or amido black (Table 1).

H3 and H4 are much more conserved than H2A and H2B, probably because they form a tetramer with a critical role in nucleosome formation. There are, for example, only two conservative amino acid substitu-

Sequence Information Histone Sequences. Histones display a remarkable phylogenetic conservation in their primary structure.

H2A

H2B

SGRGKQGGKARAKAKTRSS 20 R A G L Q F P V G R V H R L L R K G N 39 Y A E R V G A G A P V Y L A A V L E Y L 59 T A E I L E L A G N A A R D N K K T R I I 80 P R H L Q L A I R N D E E L N K L L G K LW V T I A Q G G V L P N I Q A V L L P K K 120 T E S H H K A K G K

PEPAKSAPAPKKGSKKAVTK 22 A Q K K D G K K R K R S R K E S Y S V 41 Y V Y K V L K Q V H P D T G I S S K A M so G I M N S F V N D I F E R I A G E A S R L 81 A H Y N K R S T I T S R E I Q T A V R L L 102 L P G E L A K H A V S E G T K A V T K 121 Y T S S K

H3

H4

ARTKQTARKSTGGKAPRKQL ATKAARKSAPATGGVKKPH 40 R Y R P G T V A L R E I R R Y Q K S T E 60 L L I R K L P F Q R L V R E I A Q D F K T 81 D L R F Q S S A V M A L Q E A C E A Y too L V G L F E D T N L C A I H A K R V T 119 I M P K D I Q L A R R I R G E R A

SGRGKGGKGLGKGGAKRHR KVLRDNIQGITKPAIRRLARR 41 G G V K R I S G L I Y E E T R G V L K V 61 F L E N V I R D A V T Y T E H A K R K T 81 V T A M D V V Y A L K R Q G R T L Y G loo F G G

I

i

i

i

20

21

Hlb 1 S e T A E A A P A A P A P A E K T P ~ S A G ~ G P P V S E L I T K A V A A S K52 E I 1111 II I :I I I I I I I I: I I H.5 1 TESLV.LSPAPmICRVK.... A S m m T Y S E M I A A A I R A E K S 41

.... .RIKLGLKSLVSKGTLVQTICGTGAS 101

53 RSGVS-GYDVEXNNS

I I I : I : I I I I : : I: I I Ill1 1 1 1 4 2 RGGSSRQSIQ~IKSE.YKVG~LQIKLSIRRLLAAGnKQTKGVGAS90

..

..

102 GSFKLNPCKAASGEAK. P KAKXAG AbKARRPAGAAUXPKKATGAATP 14 6 III:I I I II I 1 111 II 11 :[:I1 1 91 GSFR L A K . . . SDRAKRSPGKKKKAVRRSTSPICK...AA. RPRKARSPA.. 131 1 4 7 K K S A K K T P K K A K W A A A A G ~ P ~ K K A P K S P ~ V K 196 PK

II I 1 :11:1 1 1111 II I II :11 I Ill 11 132 K K . P K A T m . S R A S P m K T V K . A K S R K A . . S . . K A K K V K R .

173

197 AAKP.Kl'RKPKMKPKKAAAKKK 218 11: 11 I :I 111 174 .SICPR.AKSGA...RK.SPKKK 189

Figure 1. Amino acid sequences of the four core histones H2A, H2B, H3 and H4 from calf thymus, human H l b and chicken erythrocyte H5 (Von Holt et al., 1989). H l b and H5 sequences have been aligned using the Bestfit program.

tions between pea and calf H4. This makes histone H4 the most highly conserved protein known after ubiquitin. A much higher heterogeneity among species is observed for H1. The amino acid sequences of the four core histones from calf thymus, human H lb and chicken H5 are shown in Figure 1. In fact a large number of histone variants exists. By using histone sequences available from the D N A database GenBank and the protein database PIR (a total of 265 nonredundant complete sequences corresponding to 71 H2As, 58 H2Bs, 73 H3s, and 63 H4s), a complete phylogenetic analysis of the core histones was recently carried out (Thatcher and Gorovsky, 1994). A great number of variants exist also due to the presence of posttranslational modifications affecting histones. Thus, acetyl, phosphoryl, methyl and poly (ADP-ribosyl) groups as well as ubiquitin can be added and removed at specific amino acid sites by specific enzymes at different stages of the cell cycle and development and at different nuclear microenvironments in a highly specific manner. About 5 - 1 0 % of H2A and 1% of H2B in chromatin is in the form of ubiquitinated H2A

HISTONE

and H2B. Postsynthetic modifications of histones are found to affect chromatin structure thereby regulating its functions. The location of the main sites of histone modifications is shown (Figure 2). Antigenic S t r u c t u r e of Histones. Approaches to the identification of histone epitopes include, (1) testing natural or synthetic fragments in direct binding or inhibition assays (microcomplement fixation, ELISA, RIA) with polyclonal or monoclonal antihistone antibodies; (2) testing antipeptide antibodies for their ability to react with histone molecules; and (3) measuring the interaction of histone antibodies with histone variants presenting one or more residues substitutions or postsynthetic changes (e.g., acetylated lysine residues in H4, phosphorylated serine residues in H3 and HI). To identify histone epitopes exposed in chromatin, either antibodies raised against nucleosomes are characterized with synthetic peptides (Muller et al., 1989) or antipeptide antibodies are used as probes with core particles, oligonucleosomes or chromatin. All these methods lead to the identification

DOMAINS

Figure 2. Structure of individual histones. Different structural and functional domains have been identified in histones by means of nuclear magnetic resonance, circular dichroism, and protease digestion studies. All four of the core histones have randomly coiled unstructured N-terminal tails and globular, structured central domains. In addition, H2A, H2B, and H3 have short C-terminal tails. In contrast, HI/H5 have a three-domain structure consisting of an amino terminal unfolded tail of about 20 to 40 residues, a central globular domain of approximately 80 residues, and a 100 amino acid-long lysine-rich carboxyl-terminal tail which contains an inducible m-helical region. The histone domains that are accessible within polynucleosomal chains are of particular interest since they are the potential sites for postsynthetic modifications and for interactions with nonhistone proteins. In the core histone octamer structure recently elucidated to a resolution of 3.1 ,~, the folding and packaging of individual histones are significantly different: each core histone contains three helices and short-strand motifs between helices which are presumably located at the surface of the histone octamer (Arents et al., 1991). 376

Figure 3. Linear B-cell epitopes on individual histones identified with rabbit antibodies raised against whole histones (complexed with RNA or uncomplexed, FI), total histone mixture complexed with RNA (n) or nucleosomes (1771).Antigenic regions were identified using 6 to 29 residue long synthetic peptides directly adsorbed to ELISA microtiter plates. Globular domains of individual histones are shown (~1). (Adapted from Muller and Van Regenmortel, 1993 and Stemmer et al., 1994.)

of so-called continuous epitopes (Muller and van Regenmortel, 1993). A number of continuous epitopes have been localized in the six histones H1, H5, H2A, H2B, H3 and H4 by means of histone peptides and

antibodies raised against histones and nucleosomes (Figure 3). Conformational epitopes are obviously present on histone-histone and histone-DNA complexes and on nucleosomes.

377

AUTOANTIBODIES

interact locally with histones (or nucleosomes) planted on the glomerular basement membrane (GBM) via highly negatively charged GBM constituents (e.g., heparan sulfate) (Vogt et al., 1993). Planted histones (or nucleosome structures) accessible for circulating antibodies might serve as the starting point of in situ immune complex formation. Another possibility is that immune deposits leading to lupus nephritis form from antigen-antibody complexes. These complexes may involve DNA and histones circulating in lupus patients as oligonucleosome-like structures and anti-DNA, histone or nucleosome antibodies (Kramers et al., 1994).

Names Antihistone antibodies (AHA).

Pathogenetic Role Human Disease. AHA are found in a number of systemic diseases such as systemic lupus erythematosus (SLE) (Table 2), drug-induced lupus (DIL), rheumatoid arthritis (RA), juvenile chronic arthritis (JCA), Felty's syndrome, localized scleroderma, systemic sclerosis, certain autoimmune disorders of the liver such as primary biliary cirrhosis and certain neurological diseases such as subacute sensory neuropathy, Alzheimer's disease and vascular dementia. Although some investigators report that the presence of circulating AHA has no relation to a given clinical state of the patients, others find an association (Leak and Woo, 1991; Sato et al., 1994; Stemmer et al., 1995). As with the majority of autoantibodies, AHA fluctuate greatly during the course of lupus. To explain this observation, one could argue that if an antibody is nephritogenic and the epitopes of the target antigen are not saturated, its level in the circulation of patients with glomerulonephritis will be rather low (Vogt et al., 1993). This has been shown in the case of anti-DNA antibodies and in one study in the case of AHA (Muller et al., 1990). AHA may

Animal Models. Several mouse strains such as (NZB/NZW)F1 (females) and MRL/Mp-lpr/Ipr mice spontaneously develop a lupus-like syndrome, which can also be induced in normal hybrid F1 mice by injection of allogenic T cells from a parent strain. The recipient mice develop a chronic graft-versus-host (GVH) reaction with production of antinuclear antibodies (including AHA) and renal disease. The canine lupus model is also particularly interesting because of its clinical similarity with human SLE. Lupus mice of different genetic backgrounds are extremely useful for studying the production and specificity of AHA during the course of the disease and for analyzing their possible role in pathogenesis. AHA time of appearance, titers and specificity differ among the autoimmune strains. IgG AHA production

Table 2. A Summary of the Prevalence of Histone Autoantibodies in Several Studies (SLE patients) Reference

1

2

3

4

5

6

7

8

Method

FIA a

ELISA a

ELISA a

IB b

ELISA b

ELISA b

ELISA b

ELISA b

N u m b e r o f sera

82

39

151

127

32

46

12

40

Histones:

%

%

%

%

%

%

%

%

H1

62

95

52

98

6

59

67

60

H2A

43

54

23

50

34

nd

42

55

H2B

68

79

21

98

63

72

25

55

H3

57

44

47

63

56

59

33

35

H4

54

nd

21

53

22

nd

42

38

ELISA, enzyme-linked immunosorbent assay; FIA, fluorimetric immunoassay; IB, Immunoblotting; nd, not determined; a, test of IgG + IgM autoantibodies; b, test of IgG antibodies only. References:

1. B e r n s t e i n et al., 1985

2. Gohill et al., 1985 3. Muller et al., 1989 4. Costa and Monier, 1986

378

5. Shoenfeld et al., 1987 6. Kohda et al., 1989 7. Muller et al., 1990 8. Burlingame et al., 1994

frequently correlates with or appears slightly after the production of IgG anti-DNA antibodies (Monestier and Kotzin, 1992; Amoura et al., 1994; Elouaai et al., 1994; M6zibre et al., 1994). GVH mice develop high titers of IgG autoantibodies directed to histones, and more particularly to the regions 204--218 of H1, 1--25 of H2B and 1--29 of H4. The level of these antibodies decreases significantly before the appearance of proteinuria, thus suggesting their involvement in glomerular injury (M6zi~re et al., 1994). The presence of histones (or nucleosomes?) in kidneys from diseased lupus mice (Schmiedeke et al., 1992) as well as the identification of AHA in glomerular eluates from diseased MRL-lpr/lpr, NZB/NZW mice (Elouaai et al., 1994) support this idea.

Genetics Very little is known about the genetics of AHA production in autoimmune patients. A significant association between HLA-A2 and the presence of AHA in children with pauciarticular onset JCA, may simply reflect the known association between HLAA2 and this form of the disease (Malleson et al., 1992). Although AHA have been found in asymptomatic relatives of patients with RA (Youinou et al., 1989) and SLE (Shoenfeld et al., 1987), there are no specific genetic studies in such families or in AHApositive twins. The sequences of V regions of a number of monoclonal antibodies (mAbs) from autoimmune mice are known, as well as the nucleotide sequence of the V H and V L of two human IgM~, mAbs specific for H1 (Tuaillon et al., 1994). Sequencing studies of mAbs allow the following conclusions: (1) the V regions of mAbs to histones, nucleosomes and DNA bear striking similarities suggesting that common pathways lead to the expansion of B-cell clones reactive with several chromatin components; (2) AHA are often somatically mutated, in agreement with the fact that histones (in nucleosome structures) presumably act as a direct antigenic stimulus; (3) the complementary determining regions (CDRs) of the H chains of these mAbs contain negatively charged amino acid residues that may play a role in the binding to cationic histones; (4) there are striking similarities between segments of murine and human CDRs of AHA.

Factors Involved in Pathogenicity and Etiology The possible pathogenic role of AHA remains a

matter of controversy. Little attempt has been made to study AHA in patients bled serially in well-defined disease subgroups (Rubin, 1989); information is thus extremely fragmentary and concerns only procainamide-induced lupus (PIL). Avidity of AHA has not been evaluated. In contrast, the fine specificity of AHA is the subject of intense research. A number of epitopes recognized by human and murine autoantibodies have been identified (Table 3) (Figure 4). Although the results can vary appreciably with the experimental conditions used for epitope mapping, a remarkable agreement between independent studies is evident (Burlingame and Rubin, 1994; Muller and van Regenmortel, 1993). Major linear epitopes are found in the N-terminal end of H2A, H2B, H3 and H4 and in the C-terminal end of H2A, H3 and H1 (Table 3) (Figure 4). In general, the same regions are recognized by antibodies raised in rabbits against whole histones in the presence or absence of Freund's adjuvant (Figure 3). It should be noted that the terminal regions in the histones which are recognized by autoantibodies correspond also to the major sites of postsynthetic modifications (Figure 3) and are particularly exposed in chromatin. Additional linear epitopes have been characterized with short synthetic peptides which are in the internal primary structure of histones (Table 3) (Figure 4). Epitopes recognized by antibodies from patients with PIL, RA and JCA as well as by rheumatoid factors reacting with histones are generally identical to those recognized by antibodies from SLE patients (Burlingame and Rubin, 1994; Stemmer et al., 1994; Tuaillon et al., 1992). By contrast, in hydralazine-induced lupus (HIL), antibodies react preferentially with epitopes located in the internal regions of the primary sequence of H3 and H4 (Burlingame and Rubin, 1994). The peptide regions recognized by antibodies from lupus patients are generally the same as those recognized by antibodies raised in New Zealand white rabbits against nucleosomes and the total histone mixture complexed with RNA (Muller et al., 1989; Atanassov et al., 1991). In contrast New Zealand rabbits immunized with total histone mixture in the absence of RNA produced antibodies that reacted with very few histone peptides. Together these results are consistent with the idea that chromatin is the antigenic stimulus that gives rise to AHA in SLE and PIL, but not in HIL. Whether antibody subsets reacting with particular domains of histones are pathogenic is unknown. However, histone deposits present in the kidney of lupus patients and

379

Table 3. Some Human and Murine Monoclonal Antibodies Specific for Histones and Characterized with Histone Synthetic Peptides in ELISA mAb

Subclass

Specificity

Origin

Reference

BWA3

IgG1

1-20 H2A, 1-29 H4

NZB/W

Monestier et al., 1993

KM2

IgG2a

1-20 H2A, 1-29 H4

MRL- lpr/lpr

Kramers et al., submitted

D1 1

IgG

116-129 H2A

NZB/W

Eilat and Muller (n.p.)

73C3

IgM

1-11 H2B

MRL - lpr/lpr

Muller et al., 1985b

LG2-2

IgG2a

1-13 H2B

MRL - lpr/lpr

Monestier et al., 1993

LG 11-1

IgG2a

1-25 H2B

MRL - lpr/lpr

Monestier and Muller (n.p.)

LG11-2

IgG2a

1-25 H2B

MRL- lpr/lpr

Monestier and Muller (n.p.)

PL2-2

IgG2b

1-25 H2B

MRL- lpr/lpr

Kramers et al., submitted

41A5

IgM

1-25 H2B

MRL- lpr/lpr

Muller et al., 1985b

69 B4

IgG2a

26-40 H2B

MRL- lpr/lpr

Muller et al., 1985b

34

IgG2a

18-32 H3

NZB/W

Kramers et al., submitted

LG2-1

IgG2a

30-45 H3

MRL- lpr/lpr

Monestier et al., 1993

2

IgG2b

83-100 H3

GVH

Kramers et al., submitted

42

IgG2a

83-100 H3

NZB/W

Kramers et al., submitted

53

IgG2b

83-100 H3

NZB/W

Kramers et al., submitted

56

IgG2a

83-100 H3

NZB/W

Kramers et al., submitted

BEN 27

IgM

204-218 human Hlb

human

Tuaillon et al., 1994

WRI 170

IgM

1-16, 204-218 human Hlb

human

Tuaillon et al., 1994

n.p., not published. lupus mice could only be identified with the use of antibodies against N-terminal regions of histones and not with antisera against other regions of histones nor with antisera against whole histones (Schmiedeke et al., 1992; St6ckl et al., 1994). This means that the Nterminal regions are the only parts of histones which are accessible in these deposits and, thus, that autoantibodies directed against epitopes on the N-terminal part of histones (alone or within antigen-antibody complexes) may play a role in the development of the glomerular inflammation in SLE.

Pathogenetic Mechanism Molecular mimicry between host antigens and unrelated exogenous proteins (usually from infectious agents) is generally proposed as a primary event to explain how antibodies to self-components may arise, break tolerance and lead to autoimmune disease. Sequence comparison reveals regions of homology in foreign antigens and histones. However, in the case of

380

histones very few examples of molecular mimicry supported by immune cross-reaction studies are known (Eriksen et al., 1995; Jarjour et al., 1992; Stemmer et al., 1995). Peripheral blood mononuclear leukocytes from normal individuals rarely produce AHA spontaneously; however, the presence of histone-specific B cells can be detected in normal peripheral blood after pokeweed mitogen stimulation (O'Dell et al., 1988). Normal mice of different haplotypes injected with Freund's adjuvant do not develop antibody response against histones (Ravirajan et al., 1995). T-cell epitopes for histones have not yet been identified. However, two T-helper lines generated from lupus patients and responding to nucleosomal histones were recently described (Desai-Mehta et al., 1995). Both are DR-restricted and one responds to histone H4. Histone-derived peptides from nucleosomes probably have been claimed to form the Tcell stimulus for the production of anti-DNA antibodies.

Figure 4. Linear B-cell epitopes on individual histones recognized by antibodies from SLE patients and lupus mice (see Burlingame and Rubin, 1994 and text for histone epitopes recognized by antibodies from patients with HIL, PIL and JCA). Histone epitopes were identified by ELISA using peptides of 40 residues or less. Lupus patients: (Ul), Muller and Van Regenmortel, 1993; (n), Pauls et al., 1993; ([]), Gohill et al., 1985; ([]), Stemmer et al., 1994. Lupus mice: (rm), M6zi~re et al., 1994; (:~:~), Monestier and Kotzin, 1992.

Methods of Detection Some clinical laboratories still use histone reconstitution immunofluorescence assay for the detection of AHA. This laborious technique is specific but insen-

sitive to AHA reactive with H3 and H4 and cannot be used with sera in which anti-DNA antibodies coexist (Burlingame and Rubin, 1994). Counter-immunoelectrophoresis, immunodiffusion and immunoprecipitation are unsuitable methods for detecting AHA. Immuno-

381

blotting, dot-immunoassays and ELISA provide efficient and sensitive methods to detect AHA with whole histone proteins and with histone peptides (Brinet et al., 1988; Burlingame and Rubin, 1994; Gompertz et al., 1990; M6zi~re et al., 1994; Stemmer et al., 1994). ELISA was found to be slightly less sensitive than immunoblotting (Brinet et al., 1988). Furthermore, although both ELISA and immunoblotting essentially reveal reactivities to partially denatured histones, important discrepancies have been found between these two techniques. For example, in one study (Tuaillon et al., 1990), it was shown that anti-H3 antibodies were mostly detected in immunoblot; whereas, anti-H 1 antibodies preferentially reacted with Hi-coated ELISA plates. Detection of AHA directed against histone-histone and histone-DNA complexes is described elsewhere (Rubin, 1989). Autoantibodies reacting specifically with ubiquitinated H2A (and not with ubiquitin or H2A) can be studied using a branched octapeptide containing residues of both ubiquitin and the histone or by immunoblotting (Plau6 et al., 1989). As discussed above, specificity of ELISA and dot immunoassays is dependent on the purity of histones (not always achieved with commercial histones). Furthermore, due to the basic and sticky nature of histones, tests with high sensitivity are needed to decrease the amount of histones used as antigen and to increase the dilution of test sera (1:500-1:1000 if possible). Both in ELISA and immunoblotting, absence of reaction with normal sera has to be paid particular attention. Finally, circulating DNA as well as histone-binding serum proteins such as ~2-macroglobulin, C-reactive protein, nucleolin, actin and myosine can affect the detection of circulating AHA.

CLINICAL UTILITY

Application AHA are found in a number of systemic and organspecific autoimmune diseases as well as in neurological diseases and certain infections including infectious mononucleosis. AHA are also present in some asymptomatic relatives of patients with SLE. Whether circulating AHA reflect the disease activity of SLE or other autoimmune conditions is controversial. Thus, although AHA may be involved in the pathogenesis of SLE, in terms of clinical utility these antibodies are

382

not useful to classify, to confirm or to exclude a diagnosis.

Disease Associations Not only are circulating AHA detected in patients with a number of different diseases, but there is also substantial patient-to-patient variability in regard to the most reactive histones in any particular disease. Thus, it has not been possible to establish a clear association between a particular disease and AHA subsets. Although major histone linear epitopes recognized by circulating AHA are defined at the level of short peptide sequences, this knowledge does not assist diagnosis (Stemmer et al., 1994; 1995). In general, no major difference is observed in the levels of circulating AHA with respect to the overall activity of lupus or RA. Contradictory observations have been published, however, showing either a positive (Kohda et al., 1989), negative (Muller et al., 1990) or no correlation between levels of AHA and the severity of lupus nephritis.

Effect of Various Therapies A number of drugs including procainamide, sulfasalazinc, penicillamine, quinidine, acebutolol and isoniazid elicit antibodies reacting with free histones or histoneDNA complexes (Rubin, 1989). In asymptomatic patients, AHA display broad IgG and especially IgM reactivity with all five histone classes. Patients with PIL have AHA reactivity mostly with H2A-H2B histone dimers.

CONCLUSION Critical appraisal of studies on circulating AHA does not support any clinical utility of these autoantibodies except in the assessment of lupus nephritis. Relevant experiments suggest that DNA and histones circulate in some SLE patients as oligonucleosome-like complexes which are potentially available for forming complexes with anti-DNA antibodies and AHA. Since AHA in SLE are directed almost exclusively against epitopes available on nucleosome particles, these components rather than free histone molecules may represent the important immunogen. Oligonucleosomes (free or associated with antibodies) may also be nephritogenic, acting as planted antigens in the negatively charged GBM. Further studies are required

to determine the exact role of AHA. For example, while circulating A H A are present in DIL or in RA, patients with these diseases do not develop characteristic glomerulonephritis. Identification of A H A present in tissue deposits rather than circulating might provide more insight into the identification of pathogenic antibodies. See also HISTONE ( H 2 A - H 2 B ) - D N A AUTOANTIBODIES and NUCLEOSOME-SPECIFIC AUTOANTIBODIES.

REFERENCES Amoura Z, Chabre H, Koutouzov S Lotton C, Cabrespines A, Bach J-F, Jacob L. Nucleosome-restricted antibodies are detected before anti-dsDNA and/or antihistone antibodies in serum of MRL-Mp lpr/lpr and +/+ mice, and are present in kidney eluates of lupus mice with proteinuria. Arthritis Rheum 1994;37:1684--1688. Atanassov C, Briand J-P, Bonnier D, Van Regenmortel MHV, Muller S. New Zealand white rabbits immunized with RNAcomplexed total histones develop an autoimmune-like response. Clin Exp Immunol 1991;86:124--133. Bernstein RM, Hobbs RN, Lea DJ, Ward DJ, Hughes GR. Patterns of antihistone antibody specificity in systemic rheumatic disease. I. Systemic lupus erythematosus, mixed connective tissue disease, primary sicca syndrome, and rheumatoid arthritis with vasculitis. Arthritis Rheum 1985; 28:285--293. Brinet A, Fournel C, Faure JR, Venet C, Monier JC. Antihistone antibodies (ELISA and immunoblot) in canine lupus erythematosus. Clin Exp Immunol 1988;74:105--109. Burlingame RW, Rubin RL. Histones. Manual Bio Markers Dis 1994;B2.2:1--28. Costa O, Monier JC. Antihistone antibodies detected by ELISA and immunoblotting in systemic lupus erythematosus and rheumatoid arthritis. J Rheumatol 1986;13:722--725. Desai-Mehta A, Mao C, Rajagopalan S, Robinson T, Datta SK. Structure and specificity of T-cell receptors expressed by potentially pathogenic anti-DNA autoantibody-inducing T cells in human lupus. J Clin Invest 1995;95:531-541. Elouaai F, Lule J, Benoist H, AppolinaireP, Pilipenko S, Atanassov C, Muller S, Fournie GJ. Autoimmunity to histones, ubiquitin and ubiquitinated histone H2A in NZB x NZW and MRL-lpr/lpr mice. Antihistone antibodies are concentrated in glomerular eluates of lupus mice. Nephrol Dial Transplant 1994;9:362--366. Eriksen N, Kumar SB, Fukuchi K-I, Martin GM, Benditt EP. Molecular mimicry: histone H3 and mycobacterial protein epitopes. Proc Natl Acad Sci USA 1995;92:2150-2153. Gohill J, Pauls JD, Fritzler MJ. Purification of histone H1 polypeptides by high-performance cation-exchange chromatography. Chromatography 1990;502:47--57. Gohill J, Cary PD, Couppez M, Fritzler MJ. Antibodies from patients with drug-induced and idiopathic lupus erythematosus react with epitopes restricted to the amino and carboxyl

A CKNOWLEDGEMENTS The authors wish to thank Marc Monestier (Philadelphia) and Steve Batsford (Freiburg, G e r m a n y ) for stimulating discussions and critical reading of the manuscript.

termini of histone. J Immunol 1985;135:3116--3121. Gompertz NR, Isenberg DA, Turner BM. Correlation between clinical features of systemic lupus erythematosus and levels of antihistone antibodies of the IgG, IgA and IgM isotypes. Ann Rheum Dis 1990;49:524--527. Hekman A, Sluyser M. Antigenic determinants on lysine-rich histones. Biochim Biophys Acta 1973;295:613--620. Jarjour WN, Minota S, Roubey RA, Mimura T, Winfield JB. Autoantibodies to nucleolin cross-react with histone H1 in systemic lupus erythematosus. Mol Biol Rep 1992;16:263-266. Kohda S, Kanayama Y, Okamura M, Amatsu K, Negoro N, Takeda T, Onoue T. Clinical significance of antibodies of antibodies to histones in systemic lupus erythematosus. J Rheumatol 1989; 16:24-28. Kramers C, Hylkema MN, van Bruggen MCJ, Dijkman HB, Assman KJM, Smeenk RJ, Berden JH. Antinucleosome antibodies complexed to nucleosomal antigens show antiDNA reactivity and bind to rat glomerular basement membrane in vivo. J Clin Invest 1994;94:568--577. Kunkel HG, Holman HR, Deicher HRG. Multiple "autoantibodies" to cell constituents in systemic lupus erythematosus. Ciba Found Symp 1960;8:429--437. Leak AM, Woo P. Juvenile chronic arthritis, chronic iridocyclitis, and reactivity to histones. Ann Rheum Dis 1991 ;50:653-657. Malleson PN, Fung MY, Petty RE, Mackinnon MJ, Schroeder ML. Autoantibodies in chronic arthritis of childhood: relations with each other and with histocompatibility antigens. Ann Rheum Dis 1992;51:1301--1306. M6zi6re C, Stockl F, Batsford S, Vogt A, Muller S. Antibodies to DNA, chromatin core particles and histones in mice with graft-versus-host disease and their involvement in glomerular injury. Clin Exp Immunol 1994;98:287--294. Monestier M, Fasy TM, Losman MJ, Novick KE, Muller S. Structure and binding properties of monoclonal antibodies to core histones from autoimmune mice. Mol Immunol 1993; 30:1069-- 1075. Monestier M, Fasy TM, Bohm L. Monoclonal antihistone H1 autoantibodies from MRL lpr/lpr mice. Mol Immunol 1989;26:749--758. Monestier M, Kotzin BL. Antibodies to histones in systemic lupus erythematosus and drug-induced lupus syndromes. Rheum Dis Clin North Am 1992;18:415-436. Muller S, Barakat S, Watts R, Joubaud P, Isenberg D. Lon-

383

gitudinal analysis of antibodies to histones, Sm-D peptides and ubiquitin in the serum of patients with systemic lupus erythematosus, rheumatoid arthritis and tuberculosis. Clin Exp Rheum 1990;8:445--453. Muller S, Bonnier D, Thiry M, Van Regenmortel MH. Reactivity of autoantibodies in systemic lupus erythematosus with synthetic core histone peptides. Int Arch Allergy Appl Immunol 1989;89:288--296. Muller S, Couppez M, Briand JP, Gordon J, Sautiere P, van Regenmortel MH. Antigenic structure of histone H2B. Biochim Biophys Acta 1985a;827:235--246. Muller S, Jockers-Wretou E, Sekeris CE, van Regenmortel MH, Bautz FA. Characterization of a monoclonal antibody reacting with histone H3. FEBS Lett 1985b;182:459-464. Muller S, van Regenmortel MH. Histones. In: van Regenmortel MH, ed. Structure of Antigens. Boca Raton: CRC Press, 1993;2:149-178. O'Dell JR, Bizar-Schneebaum A, Kotzin BL. In vitro antihistone antibody production by peripheral blood cells from patients with systemic lupus erythematosus. Clin Immunol Immunopathol 1988;47:343--353. Pauls JD, Edworthy SM, Fritzler MJ. Epitope mapping of histone 5 (H5) with systemic lupus erythematosus, procainamide-induced lupus and hydralazine-induced lupus sera. Mol Immunol 1993 ;30:709--719. Plau6 S, Muller S, van Regenmortel MH. A branched synthetic octapeptide of ubiquitinated histone H2A as target of autoantibodies. J Exp Med 1989; 169:1607-- 1617. Ravirajan CT, Muller S, Katz DR, Isenberg DA. Effect of histone and histone-RNA complexes on the disease process of murine systemic lupus erythematosus. Autoimmunity 1995; in press. Rubin RL. Autoimmune reactions induced by procainamide and hydralazine. In: Kammuller ME, Bloksma N, Seinen W, eds. Autoimmunity and Toxicology. Amsterdam: Elsevier Science Publishers B.V., 1989:119--150. Sato S, Ihn H, Kikuchi K, Takehara K. Antihistone antibodies in systemic sclerosis. Arthritis Rheum 1994;37:391--394. Schmiedeke T, Stoeckl F, Muller S Sugisaki Y, Batsford S, Woitas R, Vogt A. Glomerular immune deposits in murine lupus models may contain histones. Clin Exp Immunol 1992;90:453--458. Shoenfeld Y, Segol G, Segol O, Neary B, Klajman A, Stollar BD, Isenberg DA. Detection of antibodies to total histones and their subfractions in systemic lupus erythematosus

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patients and their asymptomatic relatives. Arthritis Rheum 1987:30:169--175. Stemmer C, Briand JP, Muller S. Mapping of linear epitopes of human histone H1 recognized by rabbit anti-Hi/H5 antisera and antibodies from autoimmune patients. Mol Immunol 1994 ;31:1037-- 1046. Stemmer C, Tuaillon N, Prieur AM, Muller S. Mapping of Bcell epitopes recognized by antibodies to histones in subsets of juvenile chronic arthritis. Clin Immunol Immunopathol 1995;76:82--89. St6kl F, Muller S, Batsford S, Schmiedeke T, Waldherr R, Andrassy K, Sugisaki Y, Nakabayashi K, Nagasawa T, Rodriguez-Iturbe B, et al. A role for histones and ubiquitin in lupus nephritis? Clin Nephrol 1994;41:10--17. Stoller BD. Reactions of systemic lupus erythematosus sera with histone fractions and histone-DNA complexes. Arthritis Rheum 1971;14:485-492. Thatcher TH, Gorovsky MA. Phylogenetic analysis of the core histones H2A, H2B, H3 and H4. Nucleic Acids Res 1994; 22:174--179. Tuaillon N, Martin T, Knapp A-M, Pasquali J-L, Muller S. Double reactivity of monoclonal and polyclonal rheumatoid factors for IgG and histones: mapping of binding sites by means of histone synthetic peptides and anti-Id antibodies. J Autoimmun 1992;5:1-14. Tuaillon N, Muller S, Pasquali JL, Bordigoni P, Youinou P, van Regenmortel MH. Antibodies from patients with rheumatoid arthritis and juvenile chronic arthritis analyzed with core histone synthetic peptides. Int Arch Allergy Appl Immunol 1990;91:297-305. Tuaillon N, Watts RA, Isenberg DA, Muller S. Sequence analysis and fine specificity of two human monoclonal antibodies to histone H1. Mol Immunol 1994;31:269--277. Vogt A, Batsford S, Morioka T, Woitas R. Changing concepts in the pathogenesis of lupus nephritis. In: Andreucci VE, Fine LG, eds. International Yearbook of Nephrology. Berlin: Springer-Verlag, 1993:25-44. Von Holt C, Brandt WF, Greyling HJ, Linsey GG, Retief JD, Rodrigues JD, Schwager S, Sewel BT. Isolation and characterization of histones. Methods Enzymol 1989; 170:431--523. Youinou P, Williams W, Le Goff P, Tuaillon N, Jouquan J, Muller S, Isenberg DA. Serological abnormalities, including common idiotype PR4, in families with rheumatoid arthritis. Ann Rheum Dis 1989;48:898--904.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

HORMONE NONPEPTIDE AUTOANTIBODIES: THYROID Douglas C. Aziz, M.D., Ph.D.

Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES

In 1956, thyroxine (T4) binding to human serum gamma globulin was described in a patient treated with 131I for papillary carcinoma of the thyroid (Robbins et al., 1956). Subsequently, thyroid hormone autoantibodies (THAA) were detected in six patients with Hashimoto's lymphocytic thyroiditis (Premachandra and Blumenthal, 1967). Since then, autoantibodies to T4 and 3-5-3'-triiodothyronine (T3) were described in over 200 patients with thyroid disease, nonthyroidal disorders and in patients without underlying illness.

THE AUTOANTIGEN(S)

The thyroid produces an iodinated glycoprotein, thyroglobulin (Tg), which is hydrolyzed at the peptide bonds between the iodinated thyronyl residues to yield T3 and T4 which are released into the circulation. Tg is the most likely candidate autoantigen for the formation of THAA (Byfield et al., 1982; 1984; 1985; Premachandra et al., 1963; Chopra et al., 1971a). Both nondenatured and denatured (heat, acid or alkaline) Tg are potent immunogens. Iodine in the phenolic ring of the thyronine is a requisite for antigenic activity; 3'-monoiodothyronine is the minimum structure (Margherita and Premachandra, 1969; Chopra et al., 1971a). The first radioimmunoassay for T4 used antithyroxine antibodies raised by injecting human Tg into rabbits (Chopra et al., 197 lb). Over 99.9% of serum thyroxine (T4) is proteinbound, including thyroxine-binding globulin (TBG) (70%), and prealbumin and albumin (20%), and lipoproteins (3-6%); whereas, T3 is bound (99.6%) to TBG, albumin and lipoproteins (Ekins, 1990). At least 80% of the T3 and essentially all of the inactive

reverse T3 (rT3) result from the deiodination of T4 by the liver or other peripheral tissues. Although the total T3 and total T4 assays are analytically precise, the free T3 and free T4 dictate the physiological effect and are a better indication of thyroid function, particularly when there is wide physiological variation in the concentration of thyroid-binding serum proteins, e.g., in pregnancy (Larsen and Ingbar, 1993). Circulating autoantibodies directed against thyroid hormones interfere with radioimmunoassays (RIA) for serum total and free thyroid hormone concentrations and cause a discrepancy between the laboratory result and the clinical features. Binding of such antibodies can be markedly decreased by previous absorption of the serum with human thyroglobulin (Ochi et al., 1972). Because THAA can be present in euthyroidism, hypothyroidism and hyperthyroidism, their presence must be excluded whenever the clinical and thyroid function data are in disagreement. Effect of THAA on Thyroid Hormone Determinations

In a patient with THAA, results for serum T3 and T4 will be falsely elevated or depressed in the presence of THAA depending on the analytical method employed. Total T3 and total T4 are measured by RIA, using radiolabeled thyroid hormone, after separation of endogenous hormone with barbitol buffer and 8anilino-l-naphthalalene sulfonic acid (ANS). Free T3 and free T4 can be measured directly by RIA after separation by equilibrium dialysis, ultrafiltration or gel chromatography, but these techniques are cumbersome for the routine clinical laboratory. Most laboratories previously calculate the free T3 and free T4 indices by multiplying the respective total T3 and total T4 by

385

the T3 resin uptake, an estimation of the unoccupied binding sites available (Teitz, 1987). Most laboratories use immunoassays that are designed so that the equilibrium between free and bound T3 and T4 are not disturbed (Teitz, 1987). When a single-antibody precipitation technique is used, such as polyethylene glycol (PEG) precipitation of gamma globulin or adsorption of free thyroid hormone with dextran-coated charcoal, the reported concentration of thyroid hormone is lower in the presence of THAA, because PEG precipitates both THAA and exogenous antibody to thyroid hormone (Figure 1). In contrast, when using double-antibody techniques, THAA causes falsely high reported values because the secondary antibodies only precipitate exogeneous antibody, not THAA which competes for the thyroid hormone. Falsely high values are also present in solid-phase immunoassays because THAA competes with solid-phase antibody which specifically captures thyroid hormone (Sakata et al., 1985; Benvenga et al., 1987). In liquid-phase RIA for the detection of thyroid hormones, labeled hormone is added to the serum

LIQUID PHASE RIA OF THYROID HORMONE A. Normal Plasma xxxxxx

i

OOOOOO I

OOOOOO I

PEG o r 2rid Ab-

XXX OOO

OOOO XOXOXOOC

X= 0= y = LIe=

Labeled TH Endogenous TH Exogenous Ab to TH THAA

B. Plasma with THAA

•

OOOOOO OOOOOO

PEG

~oo l 100

f ~

"-

m

C. Plasma with THAA I XXXXXX J

~

[ 2nd

IA 0L

!I

[TH]

!

A. Normal Plasma XXXXXX J

XXX 000

WAS.= 2_~176 ~ <;

;>

X= O= y = LIe=

Labeled TH Analog Endogenous TH Ab to Analog THAA

B. Plasma with THAA XXXX)~ O000C O000C

_
XX OOOO

WASH

V~~%=-

-~--~o

o~

o 1 0 0 ~

50

o

l FREE TH ]

Figure 2. Interference of THAA in the AMERLEX Free T 4 o r Free T 3 kits.

with an antithyroid hormone antibody and endogenous thyroid hormone competes with the labeled hormone for binding to the exogenous antibody. Using nonspecific precipitants, such as PEG, hormone bound to both endogenous THAA and exogenous antithyroid hormone antibody (the RIA procedure reagent) will precipitate, causing higher bound to total ratio (B/Be) and lower hormone concentration calculated from the standard curve. In contrast, when a secondary antibody is used to precipitate the hormone bound to the exogenous primary antibody, only exogenous primary antibody is recognized by the secondary antibody, not THAA and hormone that is bound to the THAA is not available, resulting in lower bound to total ratio (B/Be) and higher calculated hormone levels (Sakata et al., 1985; Benvenga et al., 1987). In solid-phase RIA, overestimation also occurs, because exogenous antibody is covalently bound to the tube wall, THAA competes for radiolabeled tracer hormone, and less radioactivity is bound to the tube, simulating increased endogenous hormone (Figure 2).

Xoo Ab

YYYY l

Figure 1. Interference of THAA in the liquid phase RIA for thyroid hormones. 386

SOLID PHASE RIA OF FREE THYROID HORMONE

AUTOANTIBODIES Methods of Detection THAA are measured by several different methods, but all involve incubating THAA with calibrated radiolabeled thyroid hormone (or an analogue), separation

and counting of bound radioactivity. Prior removal of endogenous thyroid hormone improves the analytical sensitivity and precision of the assay as does inhibition of binding of thyroid hormone to serum proteins such as TBG. Dextran-coated charcoal effectively removes endogenous thyroid hormone, including bound hormone after acid-liberation from TBG and other thyroid-hormone-binding serum proteins, thus allowing measurement of most THAA that may otherwise be obscured by the endogenous thyroid hormone (Nakamura et al., 1986). THAA-bound thyroid hormone, including radiolabeled tracer hormone (or an analogue), can be separated from inbound hormone and tracer by PEG precipitation of gamma globulins, or absorption of free hormone and radiolabeled tracer to dextran-coated charcoal. Paper or agarose electrophoresis, immunoelectrophoresis and column chromatography offer no advantage over RIA, which is most often used because of its simplicity and high precision (Sakata et al., 1985; Savastano et al., 1995). With RIA, an appropriate radiolabeled thyroid hormone analogue is advantageous over radiolabeled thyroid hormone p e r s e because the analogue does not bind to TBG (Midgley and Wilkins, 1982; Wilkins et al., 1985; Allan et al., 1982).

Pathogenetic Role Thyroid hormones are not immunogenetic in the free form, but as haptens can initiate an antibody response when conjugated to serum proteins such as albumin. Tg can elicit the production of antithyroid hormone antibodies in animals, especially when injected with Freund's complete adjuvant; Tg, the precursor of thyroid hormones, is the most plentiful iodinated thyroid macromolecule, is not normally present in the blood and is recognized as a foreign substance when it leaks into the blood following damage to the thyroid. Titers of THAA in the serum demonstrably decrease after absorption with human Tg (Ochi et al., 1972). THAA, like thyroglobulin antibodies, frequently recognize Tg, and there is a direct association between titers of anti-Tg antibodies and THAA (Benvenga et al., 1987).

Animal Models. Thyroid hormone antibodies can be elicited in guinea pigs, rabbits, mice, rats, baboons and monkeys by isoimmunization with thyroid extracts or heteroimmunization with human Tg or other Tg (Sakata et al., 1985; Benvenga et al., 1987). Spon-

taneously occurring thyroid hormone antibodies occur in obese strain (OS) and Cornell strain of chickens. The OS have an overall prevalence of anti-thyroid hormones of 16%, but higher frequencies occur in chickens with high titers of anti-Tg antibodies (36%) versus those without (4%) and in those with spontaneous autoimmune thyroiditis (22%) versus 9% in those without (Nilsson et al., 1971). Immunization with human Tg of various strains of mice with different H-2 genes shows that the genes which control the immune response to Tg are different than the genes associated with the presence of antithyroid hormone antibodies; this might explain why THAA are not found in all patients with anti-Tg antibodies (Sakata et al., 1983).

CLINICAL UTILITY

Disease Associations The frequency of THAA in the normal healthy population is 0.0 to 0.2% (Vyas and Wilkin, 1994). Anti-Tg antibodies are detected in 65% of patients with THAA; 76% are women and 24% men. THAA bind most commonly to T3 (53%), to T4 (8%), T3 and T4 (14%), T4 and rT3 (14%), T4, T3 and rT3 (11%) and rarely rT3 (0%) (Merlin et al., 1984). The isotype is nearly always IgG, with only 4 reported cases of IgM, IgA or IgE alone or in addition to IgG (Benvenga et al., 1987). THAA are generally associated with euthyroidism (44%), but both hypothyroidism (40%) and hyperthyroidism (16%) are also observed. THAA bind to circulating thyroid hormone. However, because the physiological effect of thyroid hormone is related to the concentration of free thyroid hormone, the increases in the concentration of THAA result in more thyroid hormone bound to THAA (like that bound to TBG and other serum proteins), but this has very little effect on free thyroid hormone concentration because only a small proportion of thyroid hormone is free (Ekins, 1990). Rare cases of very high concentrations of THAA (10 times what is normally observed) can be associated with hypothyroidism, which presumably reflects THAA binding of all available T3 (Karlsson et al., 1977). Among patients with THAA, Hashimoto's lymphocytic thyroiditis is most frequently associated (54%). Presumably, the lymphocytic destruction of the thyroid causes a release of Tg which serves as an immunogen for the production of

387

THAA. Another 25% of all THAA are found in Graves' disease and 7% in nodular and diffuse goiter. With extremely sensitive assays for THAA, nonthyroid autoimmune disorders frequencies of about 7.5% (Vyas and Wilkin, 1994). Other nonthyroid illnesses sometimes associated with THAA include Waldenstr6m' s macroglobulinemia (1 case), hepatocellular carcinoma (1 case) and laryngeal cancer (1 case). In the case of laryngeal cancer, the patient was given external radiation to the neck, the damage to the thyroid conceivably could have elicited the abnormal immune response (Doi et al., 1983). Although THAA are thought to reflect damage to the thyroid, such as in autoimmune thyroiditis, THAA p e r se do not cause clinical disease, except for the rare case of hypothyroidism without thyroiditis secondary to such extensive binding of the thyroid hormone that the free concentration is greatly reduced (Karlsson et al., 1977). The most important clinical problem associated with THAA is the discrepancy between the laboratory result and the clinical signs because the THAA interfere with the assay, either positively or negatively depending on the type of assay. Depending on the separation procedure used, T3 or T4 bound to THAA will be associated with the free or the bound fraction, thus causing a spuriously increased or decreased thyroid hormone concentration (Table 1). The presence of THAA should be suspected and evaluated, if the laboratory results and clinical

disorder are inconsistent. If the clinician knows what methodology is used to detect T3 and T4, the bias (positive or negative) can be deduced (Sakata et al., 1993; Desai et al., 1988; John et al., 1990). Patients with primary hypothyroidism can easily be misdiagnosed if THAA binding produces a spurious increase in free T3 or T4 to within the normal reference interval. Finally, if the free T3 and T4 is reanalyzed in serum stripped of THAA, the laboratory result will confirm the clinical suspicion. Removal of THAA from the sera of THAA-positive patients lowers measured values of free T4 analyzed by RIA and PEG precipitation, and the effect correlates with THAA titers (Vyas and Wilkin, 1994).

CONCLUSION Although, THAA do not usually cause thyroid disease treatment of the associated thyroid disorder such as autoimmune thyroiditis is the cornerstone of therapy and can decrease the THAA titers. The association of THAA with anti-Tg antibodies reflects the development of THAA following thyroid damage. THAA are important because they interfere with assays used to measure thyroid hormones and can cause confusion if the results are not correlated with the patient's condition.

p e r se,

Table 1.

Analyte

Methodology

Kit (Manufacturer)

Effect of THAA

Free T4

125I-labeled analogue of T4

Amerlex-M (Amersham) Coat-a-Count (Diagnostic Products)

1"

Free T4

125I-labeled analogue of T4 and PEG pretreatment

Amerlex-M (Amersham)

N

Free T4

*HRP-labeled T4 (chemiluminescence)

Amerlite (Amersham)

1"

Total T4

Labeled-antibody direct, one step

Amerlex-MAB (Amersham)

N

Total T3

Two-step direct immunoassay

Amerlex (Amersham)

N

Free T3

125I-labeled analogue of T3

Amerlex-M (Amersham) Seria (Sereno)

1"

Free T3

Adsorption chromatography and Free T3 RIA in eluate

Lisophase (Metachem Diagnostics)

N

*HRP = horseradish peroxidase.

388

REFERENCES Allan DJ, Murphy F, Needham CA, Barron N, Wilkins TA, Midgley JEM. Sensitive test for thyroid hormone autoantibodies in serum [Letter]. Lancet 1982;2:824. Benvenga S, Trimarchi F, Robbins J. Circulating thyroid hormone autoantibodies. J Endocrinol Invest 1987;10:605-619. Byfield PG, Bond A, Copping S, Himsworth RL. Restricted antigenicity of thyroxyls in human thyroglobulin. Biochem J 1982;207:471--478. Byfield PG, Clingan D, Himsworth RL. Exposure of thyroxine residues in human thyroglobulin. Two-site binding studies. Biochem J 1984;219:405-410. Byfield PG, Clingan d, Himsworth RL. Structural basis for the reaction of 3, 5, 3'-tri-iodothyronine-specific antibodies with thyroxine-containing thyroglobulin. Biochem J 1985;228: 155--160. Chopra IJ, Nelson JC, Solomon DH, Beall GN. Production of antibodies specifically binding triiodothyronine and thyroxine. J Clin Endocrinol 1971a;32:299-308. Chopra IJ, Solomon DH, Ho RS. A radioimmunoassay of thyroxine. J Clin Endocrinol Metab 1971b;33:865-868. Desai RK, Brdenkamp B, Jialal I, Omar MAK, Rajput MC, Joubert SM. Autoantibodies to thyroxin and triiodothyronine. Clin Chem 1988;34:944-946. Doi K, Takeuchi Y, Maekawa M, Nakabashi N, Hasegawa K, Okishio T. A case of T3 autoantibody found in blood. Clin Endocrinol (Tokyo) 1983;31:45-49. Ekins R. Measurement of free hormones in blood. Endocr Rev 1990; 11:5--46. John R, Henley R, Shankland D. Concentrations of free thyroxin and free triiodothyronine in serum of patients with thyroxin- and triiodothyronine-binding autoantibodies. Clin Chem 1990;36:470--473. Karlsson FA, Wibell L, Wide L. Hypothyroidism due to thyroid-hormone-binding antibodies. N Engl J Med 1977; 296; 1146-- 1148. Larsen PR, Ingbar SH. The thyroid gland. In: Wilson JD, Foster DW, eds. Textbook of Endocrinology. 8th edition. Philadelphia: W.B. Saunders, Co., 1993:372. Margherita SS, Premachandra BN. Studies on thyroglobulin immunity. VI. Thyroid hormones and thyroglobulin specificity. J Immunol 1969;102:1511--1522. Merlin P, Sapelli S, Testori O, Mongardi L, De Filippis V. Auto anticorpi antiormoni tiroidei (AbOT) nelle diverse malattie della tiroide [Abstract]. Seconde Giornate Toscane della Tiroide. Padova, October 1984:57.

Midgley JEM, Wilkins TA. A defense of the Amerlex free thyroxin kit [Letter]. Clin Chem 1982;28:2183-2184. Nakamura S, Sakata S, Komaki T, Kamikubo K, Yasuda K, Miura K. An improved and simplified method for the detection of thyroid hormone autoantibodies (THAA) in serum. Endocrinol Jpn 1986;33:415-422. Nilsson LA, Rose NR, Witebsky E. Spontaneous thyroiditis in the obese strain chickens. VI. Thyroxine-binding antibodies. J Immunol 1971; 107:997-1003. Ochi Y, Shiomi K, Hachiya T, Yoshimura M, Miyazaki T. Immunological analysis of abnormal binding of thyroid hormone in the gamma globulin. J Clin Endocrinol Metab 1972;35:743--752. Premachandra BN, Ray AK, Hirata Y, Blumenthal HT. Electrophoretic studies of thyroxine and triiodothyronine binding by the sera of guinea pigs immunized against thyroglobulin. Endocrinology 1963;73:135-- 144. Premachandra BN, Blumenthal HT. Abnormal binding of thyroid hormone in sera from patients with Hashimoto's disease. J Clin Endocrinol Metab 1967;27:931--936. Robbins J, Rail JE, Rawson RW. An unusual instance of thyroxine-binding by human serum gamma globulin. J Clin Endocrinol Metab 1956; 16:573--579. Sakata S, Nakamura S, Kojima N, Okuyama M, Miura K, Tarutani O, Aihara Y, Okuda K. Investigations on the mechanism(s) of the production of antithyroid hormone antibodies. 1. Antithyroid hormone antibodies in rabbits and mice immunized with human thyroglobulin. Nippon Naibunpi Gakkai Zasshi 1983;59:64-71. Sakata S, Nakamura S, Miura K. Autoantibodies against thyroid hormones or iodothyronine. Ann Intern Med 1985;103:579-589. Sakata S, Komaki T, Ogawa T, Takuno H, Matsui I, Sarui H, Kojima N, Takamatsu J, Miura K. Evaluation of thyroid function in patients with thyroid hormone autoantibodies. Clin Chem 1993;219:23--34. Savastano S, Tommaselli AP, Valentino R, Carlino M, Selleri A, Randazzo G, Benvenga S, Lombardi G. A quick method to detect circulating antithyroid hormone autoantibodies. J Endocrinol Invest 1995;18:9--16. Teitz NW, ed. Fundamentals of Clinical Chemistry, 3rd edition. Philadelphia: W.B. Saunders, 1987. Vyas SK, Wilkin TJ. Thyroid hormone autoantibodies and their implications for free thyroid hormone measurement. J Endocrinol Invest 1994;17:15--21. Wilkins TA, Midgley JE, Barron N. Comprehensive study of a thyroxin-analog-based assay for free thyroxin (Amerlex FT4). Clin Chem 1985;31:1644--1653.

389

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

HORMONE PEPTIDE AUTOANTIBODIES Diana S. Trundle, Ph.D.

Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

Hormone Peptide Autoantibodies Autoantigen

Autoantibody

Clinical Manifestation

Methods

ACTH

Not characterized

Hypocortisolism (Addison's disease)

Serum extraction followed by indirect RIA method

FSH/LH

Not characterized

Premature menopause; suboptimal response to exogenous gonadotropins in female.

ELISA and immunohistochemistry, indirect immunofluorescence

hGH

IgG K IgM

None

False elevation in radiobinding and ELISA assays

PTH

IgG )~ IgM

Hypocalcemia Hyperphosphatemia

R I A - False elevation of PTH

Prolactin

IgG K

Minimal galactorrhea

1. RIA or RRA with Sephadex isolation 2. PEG precipitation

390

ADRENOCORTICOTROPIC HORMONE AUTOANTIBODIES

H I S T O R I C A L NOTES Isolated ACTH deficiency is recognized (Steinberg et al., 1954) but its diverse clinical characteristics are attributed to heterogeneous causes and to its frequent association with other autoimmune endocrinopathies (Notsu et al., 1994). Lymphocytic hypophysitis and selective absence of corticotrophs are thought to be due to an autoimmune process (Sugiara et al., 1986; 1987).

THE AUTOANTIGEN

ACTH is a peptide of 39 amino acids with biological activity residing in the first 18. Smaller than other anterior pituitary hormones, ACTH has no structural similarity to prolactin, FSH, LH, TSH or GH. Growth hormone and prolactin have approximately 200 amino acids and share 16% structural similarity. Of similar size, TSH, LH and FSH dimeric glycoproteins, share a common alpha subunit, but have a variable beta subunit which confers unique biological activity. The structural dissimilarity between ACTH and the other pituitary hormones might explain the production of ACTH-specific antibodies rather than antibodies which cross-react because of similar sequence and common epitopes. A case of isolated ACTH deficiency is reported with an autoantibody to a corticotroph antigen which is not ACTH (Sauter et al., 1990). Immunohistochemical tests indicate the unidentified corticotroph-specific antigen is granular. This negative finding is supported by later reports (Mau et al., 1993).

(Pranzatelli et al., 1993). The presence of antibodybound ACTH did not interfere with cortisol secretion. ACTH autoantibodies are not sensitive predictors of patients with pituitary ACTH deficiency, i.e., secondary adrenal failure. A group of 11 patients with pituitary diseases was evaluated to determine whether pituitary hormone antibodies correlate with hormone deficiencies. Six controls were negative for pituitary disease and antibodies. In the five patients with ACTH antibodies, only one had hypocortisolism. A second patient meeting the criteria for secondary adrenal insufficiency did not demonstrate ACTH antibodies (Mau et al., 1993) (Table 1). Thus, the presence of ACTH antibodies had a sensitivity of 50% and a specificity of 60% for diagnosis of secondary adrenal deficiency in this small study. The majority of cases of primary adrenal cortical insufficiency are of autoimmune etiology with parenchymal atrophy and lymphocytic infiltrates similar to Hashimoto's thyroiditis. There is a frequent association of ACTH deficiency with insulin-dependent diabetes mellitus, thyroiditis and polyglandular deficiency (Sauter et al., 1990). ACTH autoantibodies are characteristically absent in primary adrenal cortical insufficiency (Orth et al., 1992). The most common clinical explanation for a high ACTH concentration in the presence of persistent hypocortisolemia is primary adrenal failure (Addison' s disease). If serum ACTH concentrations are normal or low in patients with clinical Addison's disease, the discrepancy might reflect the presence of ACTH antibodies which bind to the circulating ACTH. A reliable assay for measuring ACTH in these patients should include measurement of both free and total ACTH after extraction to dissociate antibody-bound ACTH.

AUTOANTIBODIES

The autoantibodies associated with Addison's disease include both cytoplasmic and cell surface antiadrenal autoantibodies (direct immunofluorescence using adrenal gland), surface autoantibodies to (indirect immunofluorescence using rat pituitary) (Notsu et al., 1994), and steroidal cell autoantibodies. Antibodies to ACTH detected by RIA were present in serum of a child with opsoclonus-myoclonus for 24 weeks after discontinuing chronic ACTH therapy

CLINICAL UTILITY One case of isolated ACTH deficiency associated with antipituitary antibodies, pituitary cyst, sphenoidal cyst and pineal tumor is reported in a 68-year-old male (Notsu et al., 1994). Isolated ACTH deficiency can be associated, albeit rarely, with antipituitary antibodies (Notsu et al., 1994). There is a frequent association of ACTH 391

Table 1. Pituitary Hormone Autoantibody (APHA) Levels Using Scanning Densitometry* Subjects

Anti-hGH

Anti-ACTH

Anti-TSH

Anti-FSH/LH

APHAs Detected

Adrenal Function

Empty sella syndrome 1.BK

0<)5*

0-05

0<)5

0"05/0.05

2.RC

0<)5

0"05

0<)5

0"05/0"05

3.WB

1.08

0.72

0<)5

0.05/0.05

+ Anti-hGH, Anti-ACTH

WNL

4.MH

1.20

1"05

0<)5

0.05/0.05

+ Anti-hGH, Anti-ACTH

WNL

5.JD

0<)5

0"05

0<)5

0"05/0.05

6.SP

0<)5

0"05

0<)5

0"05/0.05

0<)5

0"86

1.17

0"05/0"05

+ Anti-ACTH, Anti-TSH

Low

2.ReH

0<)5

0-05

0<)5

0"05/0-05

3.HS

0<)5

0"05

0<)5

0"05/0.05

4.CS

0<)5

1.35

1.03

0.05/0.05

+ Anti-ACTH, Anti-TSH

WNL

5.RH

0<)5

1.18

0<)5

0.05/0.05

+ Anti-ACTH

WNL

0<)5

0"05

0<)5

0"05/0"05

Pituitary tumor 1. RO

Controls 1- 6

* Reported in absorbance units (AU). * Baseline levels minus background. WNL Within normal limits. Modified from Mau et al., 1993.

deficiency with insulin-dependent diabetes mellitus, thyroiditis and polyglandular deficiency (Sauter et al., 1990). But autoantibodies to A C T H are found in only in some cases of pituitary disease (Mau et al., 1993).

CONCLUSION Patients with unexplained depression of serum cortisol with elevated concentrations of circulating immuno-

REFERENCES Mau M, Phillips TM, Ratner RE. Presence of antipituitary hormone antibodies in patients with empty sella syndrome and pituitary tumours. Clin Endocrinol (Oxf) 1993;38:495--500. Notsu K, Oka N, Sohmiya M, Sato T, Ando S, Moritake K, Inada K, Osamura Y, Kato Y. Isolated adrenocorticotrophin deficiency associated with antipituitary antibodies, pituitary cyst, sphenoidal cyst and pineal tumor. Endocr J 1994;41: 631--637.

392

reactive A C T H should be evaluated for A C T H autoantibodies. Subclinical adrenocortical insufficiency presents with nonspecific symptoms and hormonal abnormalities only recognized by careful endocrine evaluation and periodic testing of cortisol reserve. This is of particular importance in organ-specific autoimmune diseases in which the subclinical period is marked only by the presence of specific autoantibodies. See also STEROID CELL AUTOANTIBODIES.

Orth DN, Kovacs WJ, DeBold CR. The adrenal cortex. In: Wilson JD, Foster DW, eds. Textbook of Endocrinology. Philadelphia: W.B. Saunders & Co., 1992:525. Pranzatelli MR, Kao PC, Tate ED, Chaves E, Chez M, Dobyns WB, Kang H, Rothner DA. Antibodies to ACTH in opsoclonus-myoclonus. Neuropediatrics 1993;24:131--133. Sauter NP, Toni R, McLaughlin CD, Dyess EM, Kritzman J, Lechan RM. Isolated adrenocorticotropin deficiency associated with an autoantibody to a corticotroph antigen that is not adrenocorticotropin or other proopiomelanocortin-

derived peptides. J Clin Endocrinol Metab 1990;70:1391-1397. Steinberg A, Schechter FR, Segal HI. The pituitary Addison's disease- a pituitary unitropic deficiency. J Clin Endocrinol Metab 1954;14:1519. Sugiura M, Hashimoto A, Shizawa M, Tsukada M, Maruyama S, Ishido T, Kasahara T, Hirata Y. Heterogeneity of anterior pituitary cell antibodies detected in insulin-dependent

diabetes mellitus and adrenocorticotropic hormone deficiency. Diabetes Res 1986;3:111--114. Sugiura M, Hashimoto A, Shizawa M, Tsukada M, Saito T, Hayami H, Maruyama S, Ishido T. Detection of antibodies to anterior pituitary cell surface membrane with insulin dependent diabetes mellitus and adrenocorticotropic hormone deficiency. Diabetes Res 1987;4:63--66.

393

FOLLICLE STIMULATING HORMONE AND LUTEINIZING HORMONE AUTOANTIBODIES

HISTORICAL NOTES The development of gonadotropin antibodies in humans has been reported in children, hypogonadotropic men and women treated with pituitary extracts and urinary preparations of gonadotropin and human chorionic gonadotropin (hCG) (Meyer et al., 1990; Cameron et al., 1988). Investigation of gonadotropin (LH and FSH) antibodies in women was prompted by suboptimal response to human menopausal gonadotropins (hMG) (Meyer et al., 1990). Interspecies gonadotropin antibody formation has also been demonstrated in rhesus monkeys receiving hMG injections (Platia et al., 1984). Interfering antibody formation was verified after clinical response declined despite rising serum hormone concentrations.

tile women with tubal or male factor infertility and a normal response to hMG, showed no detectable autoantibodies to gonadotropins or ovarian tissue.

CLINICAL UTILITY Disease Associations

Although not fully characterized, the autoantigens are FSH (human FSH: NIA DOK-hFSH 1-3 AFP 4822B), LH (human LH: NIADDK-hLH 1-2 AFP 8207B), human ovarian tissue components (Meyer et al., 1990; Pellicer et al., 1994) and monkey ovarian tissue containing both theca and granulosa cells (Cameron et al., 1988; Ebbiary et al., 1994)

Women under 40 years old with normal estradiol concentrations and elevated FSH show a higher prevalence of FSH autoantibodies when their sera are tested for 20 antibodies by indirect immunofluorescence using monkey or rat tissue (Ebbiary et al., 1994). In an age-matched control group of 48 women with normal FSH: 27% had one antibody, 8% had two and 2% had three. However, in the group of women with elevated FSH, the distribution was: 58% one antibody, 27% two and 8% three antibodies (Table 2). The presence of gonadotropin autoantibodies in occult ovarian failure supports an immunologic cause for low ovarian stimulation by exogenous gonadotropin and an explanation for the failure of in vitro fertilization efforts. This is probably an early stage of premature ovarian failure and the autoantibodies identify a group at risk to develop polyglandular autoimmune disease.

AUTOANTIBODIES

CONCLUSION

The most common clinical explanation for a high serum FSH concentration is gonadal failure. However, there is a report of gonadotropin autoantibodies in women with suboptimal response to exogenous gonadotropin stimulation (Meyer et al., 1990). In a group of 26 normally menstruating women demonstrating suboptimal response to hMG, 92% had FSH antibodies, 65% had LH antibodies and 77% had ovarian antibodies. These autoantibodies were measured by an enzyme-linked immunosorbent assay (ELISA) and an immunohistochemical method using human ovarian tissue. The control group of 25 infer-

An attempt to demonstrate autoantibodies blocking FSH action in male infertility has failed (Simoni et al., 1993). However, in women the evidence for this etiology is more convincing. Ovarian follicle exhaustion is responsible for reproductive aging in women, a characteristic finding during the menopause transition. Ovarian failure is the most likely cause of elevated FSH although in some patients this may be explained by antibodies to FSH. Thus, a connection to autoimmune premature ovarian failure is suggested by the presence of circulating antibodies.

THE AUTOANTIGENS

394

Table 2. Type and Frequency (Number of Patients) of Autoantibodies Detected in the Study Groups

Normal FSH Organ-specific Antibodies Antiovarian Antiadrenal Antithyroid Antigastric parietal Antipituitary Antisalivary duct Non-organ-specific antibodies Anti-DNA Antinuclear Antismooth muscle Antireticulin Antimitochondrial Antispermatozoid Rheumatoid factor

m

5 1

High FS H

3 2 11 2 1 1 14 7 2 2 1 1 2

Modified from Ebbiary et al., 1994.

REFERENCES

Cameron I, O'Shea F, Rolland J, Hughes E, de Kretsher D, Healy D. Occult ovarian failure: a syndrome of infertility, regular menses and elevated FSH concentrations. J Clin Endocrinol Metab 1988;67:1190-- 1194. Ebbiary NA, Lenton EA, Salt C, Ward AM, Cooke ID. The significance of elevated basal follicle stimulating hormone in regularly menstruating infertile women. Hum Reprod 1994; 9:245--252. Meyer W, Lavy G, Decherney A, Visintin I, Economy K, Luborsky J. Evidence of gonadal and gonadotropin antibodies in women with a suboptimal ovarian response to exogenous gonadotropin. Obstet Gynecol 1990;75:795-799.

Pellicer A, Ballester M, Serrano M, Amparo M, Serra-Serra V, Remohi J, Bonilla-Musoles M. Aetiological factors involved in the low response to gonadotrophins in infertile women with normal basal serum follicle stimulating hormone levels. Hum Reprod 1994;9:806--811. Platia M, Bloomquist G, Williams R, Hodgen G. Refractoriness to gonadotropin therapy: how to distinguish ovarian failure versus pseudo ovarian resistance caused by neutralizing antibodies. Fertil Steril 1984;42:779-784. Simoni M, Paschke R, Nieschlog E. A search for circulating immunoglobulins blocking follicle-stimulating hormone action in male idiopathic infertility. Int J Andrology 1993; 16:129-135.

395

GROWTH HORMONE AUTOANTIBODIES

HISTORICAL NOTES

AUTOANTIBODIES

Genetic studies of children with deletions in the growth hormone (GH) gene show that these children have impaired GH synthesis, attenuated growth and spontaneous human GH (hGH) autoantibodies. However, treatment with exogenous GH remains effective (Llera et al., 1993). Expectedly, children treated with pituitary or recombinant human growth hormone (rhGH) develop GH antibodies (Tyllstrom et al., 1985; Takano and Shizume, 1986). In one study, GH antibodies were found in 48% of children who received either methionyl-GH or natural sequence GH (DiSilvio et al., 1987). Some variability in the capacity of these antibodies to interfere with the laboratory estimation of total and free growth hormone is attributed to differences in antibody affinity (Pringle et al., 1989). Immunocytochemical studies of sera from patients with multiple sclerosis (MS) that autoantibodies are directed toward antigenic determinants on the surface of pituitary and brain cells (Moller et al., 1985), but no conclusive evidence regarding specificity of this immunoreactive component is available.

A group of 35 Caucasian children with a clinical diagnosis of idiopathic hypopituitarism with GH deficiency were tested for autoantibodies prior to treatment (Llera et al., 1993). Some of these patients demonstrated a gene deletion with impaired GH synthesis; others had hypothalamic/pituitary masses. They were selected on the basis of documented growth failure and serum GH concentration <5 ng/mL in two provocative tests. IgG autoantibodies measured by competitive radiobinding assay and ELISA were found in 6/35 patients and in 1/85 control children. IgM antibodies were found in only 3/35 patients and 0/85 controls. Of the children with hGH autoantibodies, 80% also had other pituitary deficiencies, particularly thyroid stimulating hormone.

THE AUTOANTIGEN Exogenous hGH induces hGH antibodies and other antibodies which although unreactive with hGH react with bovine and equine GH (Perez et al., 1985). Biochemically purified hGH exhibits different antigenic epitopes in different patients when used therapeutically (Poskus et al., 1982). Similar epitope diversity has not been reported for therapy with rhGH. Long-term follow-up studies of GH-deficient children treated with methionyl recombinant hGH (met-rhGH) and rhGH showed that met-rhGH has high immunogenicity and induces antibodies detectable throughout the treatment period (Massa et al., 1993). The antibodies do not influence the therapeutic effect of met-rhGH and disappear when met-rhGH is discontinued or changed to rhGH.

396

CLINICAL UTILITY A spontaneous GH autoantibody (IgG ~:) was documented in 1/38 patients with myasthenia gravis (MG) (Okada et al., 1990). A serum antibody to GH was also reported in a patient with MG with very high serum GH concentrations and no symptoms of gigantism or acromegaly. This 44-year-old male was admitted for thymectomy and the total GH immunoreactivity measured in his serum by RIA was very high (225 mcg/L), but the free GH measured after precipitation of antibodies with 25% PEG was markedly reduced. This concentration did not change after provocative endocrine stimuli, e.g., insulin and bromocriptine. This result was confirmed by acid dissociation of the antibody complex. In the presence of GH autoantibodies, total GH is markedly increased relative to free GH concentrations (Pringle et al., 1989). However, GH autoantibodies found in children treated for one year with rhGH did not affect growth, IGF-I concentration or fasting insulin concentration.

CONCLUSION There are documented reports of spontaneous GH autoantibodies in one patient with MG and in six patients with idiopathic hypopituitarism. Both were IgG isotype. The presence of the spontaneous autoantibodies does not interfere with the therapeutic replacement of GH, but they do induce variable aberrations and false elevation in measured concentration

REFERENCES DiSilvio L, Hindmarsh P, Pringle P, Brook C, Kurtz A. A radioimmunoprecipitation assay for antibodies to growth hormone [Abstract]. J Endocrinol 1987;112 (Suppl):239. Llera A, Cardoso AI, Stumpo RR, Martinez AS, Heinrich JJ, Poskus E. Detection of autoantibodies against hGH in sera of idiopathic hypopituitary children. Clin Immunol Immunopathol 1993;66:114-119. Massa G, Vanderschueren-Lodeweyckx M, Bouillon R. Five year follow-up of growth hormone antibodies in growth hormone-deficient children treated with recombinant human growth hormone. Clin Endocrinol 1993;38:137-142. Moller A, Hansen BL, Hansen GN, Hagen C. Autoantibodies in sera from patients with multiple sclerosis directed against antigenic determinants in pituitary growth hormone-producing cells and in structures containing vasopressin/oxytocin. J Neuroimmunol 1985;8:177--184. Okada S, Saito E, Oshima H, Kinoshita M. Autoantibody to growth hormone in a patient with myasthenia gravis. J Endocrinol 1990;127:533--538.

of serum GH. Little is known about the effect of induced GH antibodies on the bioavailability of exogenously administered GH, but some evidence suggests that GH antibodies enhance biological activity. It is unclear whether the spontaneous hGH autoantibodies are isolated markers of autoimmunity or components of a major autoimmune process involving somatotropic cells of the pituitary.

Perez AR, Pena C, Poskus E, Paladini AC, Domene HM, Martinez AS, Heinrich JJ. Antibodies against animal growth hormones appearing in patients treated with human growth hormone: their specificities and influence on growth velocity. Acta Endocrinol (Copenh) 1985;110:24-31. Poskus E, Pena C, Perez AR, Vita N, Heinrich JJ, Paladinin AC. Human, bovine and equine growth hormone antibodies in patients treated with human growth hormone. J Clin Endocrinol Metab 1982;55:13--17. Pringle PJ, Hindmarsh PC, Di Silvio L, Teale JD, Kurtz AB, Brook CG. The measurement and effect of growth hormone in the presence of growth hormone-binding antibodies. J Endocrinol 1989;121:193-- 199. Takano K, Shizume K. Clinical experience with Somatrem in Japan. Acta Paediatr Scand Suppl 1986;325:19-25. Tyllstrom J, Karlen B, Guilbaud O. Somatonorm (Somatrem): Immunological aspects. In: Flodh H, Milner RDG, eds. Immunological Aspects of Human Growth Hormone. Proceedings of a Workshop. Oxford: Oxford Medical Education Services, 1985:19-32.

397

PARATHYROID HORMONE AUTOANTIBODIES

HISTORICAL NOTES Among three reports of spontaneous parathyroid hormone (PTH) antibodies (anti-PTH) resulting in high concentrations of PTH, two were associated with hypocalcemia (McElduff et al., 1992; Kasono et al., 1991), and one with normal to borderline elevated serum calcium (Wilkinson et al., 1990). Each of these three patients had a preceding episode of thyrotoxicosis. One case is explained by an anti-idiotypic PTH autoantibody (McElduff et al., 1992), one by IgG )~ (Kasono et al., 1991) and the third involved a circulating IgM (Wilkinson et al., 1990). The spuriously elevated immunoreactive PTH (iPTH) caused by the IgG )~ showed cross-reactivity with antiserum specific for the C-terminal region of PTH (65--69); however, only 70% bound to the anti-IgG )~ suggesting that this was not monoclonal. A fourth patient developed an anti-PTH after repeated subcutaneous injections of PTH-(1--34) (Audran et al., 1987).

THE AUTOANTIGEN

Definition Parathyroid hormone is secreted by the chief cells of the parathyroid glands after synthesis as a precursor molecule, preproparathyroid hormone. The principal form stored and released is a single amino acid chain with a molecular weight of 9425. The first 27 amino acids of the N-terminal are required for biological activity, receptor binding and activation of adenylate cyclase.

Origin/Sources The multiple forms of PTH in plasma and their heterogeneity are explained by secretion of both intact hormone and inactive fragments. Cleared rapidly by the liver and the kidneys, intact PTH has a half-life less than 10 minutes. Native PTH has several antigenic sites accessible to endogenous autoantibody or reagent antibody production. Two-site diagnostic assays detect intact PTH

398

with one antibody to the N-terminal and one antibody directed toward epitopes in the mid-region or Cterminal. Those assays with antibodies directed toward epitopes in the mid-region show the greatest sensitivity and closest correlation with clinical status.

AUTOANTIBODIES

Pathogenetic Role The most common clinical explanation for a high PTH concentration in the presence of persistent hypocalcemia is secondary hyperparathyroidism caused by chronic renal failure, malabsorption or magnesium deficiency. However, in these three case reports, the related PTH results were artifactual due to interference by spontaneous anti-PTH (Wilkinson et al., 1990, Kasono et al., 1991; McElduff et al., 1992). 1. Chromatography on a C18 solid-phase extraction column showing 90% flow through exclusion compared with 0-12% in sera from normal controls and <2% of native PTH. PTH immunoreactivity retained in the patient serum immunoglobulin fraction by separation of small hydrophilic molecules on octodecyl bonded to silica. PTH is retained by the column; IgG is excluded. Analysis of the recovered patient serum peak by SE-HPLC and radioimmunoassay showed only trace amounts of normal PTH. 2. Precipitation with 15% PEG, indicating molecular size in excess of 10 kd. Only 7% of normal serum PTH precipitates. 3. Molecular mass chromatography revealing molecular size closer to human immunoglobulins (158 kd) than PTH (10 kd). 4. Affinity for Protein A and antibodies to human IgG. Further evaluation of this patient serum showed iPTH demonstrable with six antisera from 5 different species; dilutions of serum did not dilute in parallel with standard PTH, and binding studies with labeled hPTH failed to reveal a circulating PTH binder in the patient' s serum.

CLINICAL UTILITY

Disease Associations Patients with unexplained clinical hypoparathyroidism associated with normal or elevated concentrations of circulating immunoreactive PTH should be evaluated for PTH autoantibodies. The following three cases have been documented. A 69-year-old man is reported with hypocalcemia in the presence of immunoreactive PTH (McElduff et a1.,1992). The patient came to medical attention because of hematuria and acute urinary retention caused by a Grawitz' tumor and was treated with a right total nephrectomy. At the initial presentation, he was normocalcemic. One year later, hypocalcemia and hyperthyroidism led him back for medical care. His TBII test was negative for TSH receptor antibodies, and the hyperthyroidism responded to propylthiouracil and the calcium depletion to oral calcium supplement. Three years later, he returned to his physician with worsening hypocalcemia and demonstrable parasthesias. Full clinical evaluation ruled out malabsorption, chronic renal failure and magnesium deficiency as causes of his condition. Provocative tests with human PTH (i.e., isolated from human gland) infusions were normal, thus excluding PTH resistance. So, after common causes of hypoparathyroidism and PTH resistance were excluded, the patient was diagnosed with acquired hypocalcemia due to hypoparathyroidism in the presence of normal PTH sensitivity.

REFERENCES Audran M, Basie M-F, DeFontain A, Jallet P, Bidet M-T, Ermias A, Tanguy G, Pouplard A, Reeve J, Zanelli J, Renier J-C. Transient hypoparathyroidism induced by synthetic human parathyroid hormone-(1--34) treatment. J Clin Endocrinol Metab 1987;64:937--943. Kasono K, Sato K, Suzuki T, Ohmura E, Demura R, Shizume K, Tsushima T, Demura H. Falsely elevated serum parathyroid hormone levels due to immunoglobulin G in a patient with idiopathic hypoparathyroidism. J Clin Endocrinol Metab

A 76-year-old woman presented with painful red nodules on her shins (Wilkinson et al., 1990). Four months prior to admission she had a mastectomy for infiltrating ductal carcinoma of the breast and also was treated with tamoxifen. At admission, she had a diagnosis of pancreatitis and elevated PTH with a paradoxical normal to borderline hypercalcemia. The circulating binding component in her serum was identified as IgM. Serum from a 73-year-old patient with idiopathic hypoparathyroidism was negative for PTH-binding protein, heterophilic antibodies and antibody to goat IgG (Kasono et al., 1991). Gel filtration identified the iPTH-like substance as IgG )~. Thus, the false elevation of PTH was due primarily to IgG cross reactivity with the PTH antiserum used in the diagnostic test.

CONCLUSION Patients with unexplained hypocalcemia and hypoparathyroidism associated with normal levels of circulating immunoreactive PTH should be evaluated for PTH autoantibodies. Although these antibodies are a rare occurrence, treatment with supplemental vitamin D and oral calcium relieves symptoms and avoids misdiagnosis indicating surgical treatment. Interestingly, all of the patients studied to date had a preceding episode of hyperthyroidism. Not only would this have aggravated calcium loss, it may have triggered an autoimmune cascade resulting in pathogenic changes and ultimate disease.

1991;72:217--222. McElduff A, Lackmann M, Wilkinson M. Anti-idiotypic PTH antibodies as a cause of elevated immunoreactive parathyroid hormone in idiopathic hypoparathyroidism a second case: another manifestation of autoimmune endocrine disease? Calcif Tissue Int 1992;51:121--126. Wilkinson M, McElduff A, Wilson J, Haber P, Freeman A, Robertson M, Mathews P. Spontaneously occurring antibodies to parathyroid hormone. J Clin Endocrinol Metab 1990;70:1744--1749.

399

PROLACTIN AUTOANTIBODIES

HISTORICAL NOTES Autoimmune hypophysitis, long suspected on histological grounds, was finally associated with an autoantibody which reacted with anterior pituitary tissue in 1975 (Bottazzo et al., 1975). Among 287 patients with various autoimmune endocrine disorders, 19 were identified with pituitary antibodies in sera. Immunofluorescent staining of these sera revealed the antigen to be cytoplasmic organelles of the prolactinsecreting cells. More recently, the presence of prolactin autoantibodies was demonstrated in five patients with idiopathic hyperprolactinemia (Hattori et al., 1992b). The total prolactin concentrations were markedly elevated (685 +/- 386 mcg/L). However, the clinical features did not substantiate the hyperprolactinemia (regular menses and no galactorrhea). Scatchard analysis revealed a low-affinity, high-capacity antibody; immunoprecipitation showed it to be a kappa type immunoglobulin G. This was the first report of an autoantibody-related hyperprolactinemia. Prior to that time, prolactin levels greater than 200 ~ag/L in nonpregnant women were pathognomonic of pituitary microadenomas and always associated with signs and symptoms of the disease. Now, in asymptomatic patients with markedly elevated prolactin levels and no radiologic evidence of a pituitary adenoma, the possibility of prolactin antibodies should be evaluated (Hattori et al., 1992a; 1994b).

THE AUTOANTIGEN Prolactin is synthesized primarily by the pituitary, although other tissues can form prolactin including lymphatics, endometrium and ectopic production is reported in some tumors. Prolactin, a single-chain polypeptide with 199 amino acids, three disulfide bonds and 16% shared sequence with growth hormone, circulates predominantly in 23 kd form but also exists as a dimer ("big" prolactin, 50 kd) and "big, big" prolactin 150--170 kd (Lindsteadt, 1994; Leite et al., 1992; Fraser et al., 1989). The 150--170 kd form has reduced receptor-binding activity and biological activity; and was thought to be an oligomer, but now appears to be a complex of 400

prolactin with IgG (Hattori et al., 1992a, 1992b, ). Native prolactin has several antigenic sites and may be accessible to both endogenous autoantibody and reagent antibody (i.e., those employed in capture assays). Human prolactin (NIDDK-hPRLI-7 and NIDDK-hPRL-RP-1) is available from the National Hormone and Pituitary Program in Baltimore, MD.

AUTOANTIBODIES Prolactin autoantibodies are IgG kappa (Hattori et al., 1992b). Combination with prolactin autoantibodies prevents bound prolactin from reaching the glomerular filtrate, thus prolonging its biological half life. The elevation prolactin concentrations in patients with the autoantibodies are not directly related to the severity of the clinical symptoms nor do the antibodies indicate an underlying systemic autoimmune disease. The mechanism of prolactin autoantibody production is unknown (Buskila et al., 1991). Macroprolactinemia (i.e., presence of sustained large molecular weight prolactin) is similar to the hyperprolactinemia due to prolactin autoantibodies; neither condition evidence significant clinical symptoms of hyperprolactinemia (Hattori et al., 1992a; Lindstedt, 1994). Methods of Detection

A solid-phase radioimmunosorbent assay is available for detection of circulating antibodies to polypeptide hormones, including prolactin (Ericsson et al., 1984). Prolactin antibodies bind radiolabeled prolactin; the complexes are then separated on a Sepharose column coupled to an immunosorbent. Prolactin autoantibodies can also be assayed by nonextraction methods (e.g., Sephadex chromatography followed by radioimmunoassay (RIA) or radioreceptor assay (RRA) (Farkouh et al., 1979); the results are similar or lower than those of competitive RIA. Prolactin autoantibodies cause underestimation of serum prolactin when a double-antibody RIA method is used; however, results by immunoradiometric assay (IRMA) are unaffected (Hattori et al., 1994a). Serum prolactin autoantibodies can also be detected

by PEG precipitation after displacement of labeled prolactin by unlabeled prolactin which binds to protein G. Because this method requires partial dissociation of the endogenous prolactin-antibody complex, high affinity antibodies will not be detected by this method (Fraser et al., 1989). In this method, 100 ~aL serum is incubated with iodine 125I-labeled prolactin with and without variable amounts of unlabeled prolactin (0.08--5 ~tg) for an hour at ambient temperature (Hattori et al., 1992a, 1994b); 200 ~L of 25% PEG is added, mixed and centrifuged. Prolactin antibodies are assumed to be present when these three criteria are met: the ratio of radioactivity in the precipitate is greater than 9%, there is demonstrable dose-dependent displacement by unlabeled prolactin, and immunoglobulin structure is confirmed by capture of prolactin IgG complexes on a protein G column. Scatchard analysis of the autoantibodies precipitated by 12.5% PEG show low affinity, high capacity, Ka 3.1+/- 3.6 x 106 L/mol, B max 1543 +/- 1427 ~tg/L (Hattori et al., 1992a). Problems with this assay include partial disassociation of the endogenous prolactin-antibody complex and the possibility that high affinity antibodies will escape detection (Fraser et al., 1989).

CLINICAL UTILITY Normal basal prolactin values are <20 ~g/L in the adult female and <10 ~g/L in the male. Hyperprolactinemia is associated with menstrual disturbances, infertility and galactorrhea in women and loss of libido and impotence in men. Similar to thyroid stimulating hormone, there is a diurnal variance in the circulating hormone with a peak at the end of the night and a nadir in midmorning. In the past, serum prolactin concentrations greater than 200 were considered pathognomonic of prolactinoma. This assumption is no longer reliable because the range of values in idiopathic hyperprolactinemia extends from 30--935 ~g/L. Individuals with hyperprolactinemia must be checked for a pituitary adenoma. When clinical and associated symptoms are absent and radiologic examinations are negative; autoantibody-associated hyperprolactinemia is, more likely than prolactinoma. Laboratories offering prolactin testing should make available the determination of prolactin autoantibodies when elevated results of prolactin assays do not support the clinical impression. Some authorities suggest that continuing medical treatment is not indicated for patients with marked

hyperprolactinemia associated with autoantibodies to prolactin because clinical symptoms rarely occur and pregnancy has a normal course (Hattori et al., 1992a; 1994b; Whittaker et al., 1981; Buskila et al., 1991). However, because this has not been the experience of all clinicians, it may be unwise to exclude these patients from further investigation because a few may progress to demonstrate clinical symptoms. For example: Five patients with idiopathic hyperprolactinemia and markedly elevated prolactin concentrations (685 +/386 ~g/L), demonstrated prolactin autoantibodies and mild clinical signs and symptoms, e.g., minimal galactorrhea and transient hypermenorrhea (Hattori et al., 1992b). In a patient with normal ovulation and transient galactorrhea secondary to treatment with an antidopaminergic drug, hyperprolactinemia persisting for a year was attributed to demonstrated prolactin autoantibodies delaying prolactin clearance and/or altering central regulation (Hattori et al., 1992a). A very small subgroup (11 female) of patients with hyperprolactinemia and measurable antibodies exhibited the characteristic signs and symptoms: galactorrhea, oligomenorrhea and amenorrhea (Leite et al., 1992). Complexes with low stability (i.e., with lower affinity for prolactin than for receptors) are thought to dissociate and to exacerbate the hyperprolactinemia syndrome (Lindsteadt et al., 1994). Therefore, patients with gross elevations of prolactin should be evaluated for prolactin autoantibodies. In a study of 208 patients with elevated serum prolactin, autoantibodies were present in 16% of those with idiopathic hyperprolactinemia, 4% of those with drug-induced hyperprolactinemia, 3% of those with prolactinoma and 3 % of those with other causes. Only 1.3% of the control group (228 individuals with normal serum prolactin concentrations) had detectable autoantibodies (Hattori et al., 1994b). The frequency of idiopathic hyperprolactinemia is greater than hyperprolactinemia with a proven cause; there is a significant positive correlation between titers of autoantibodies and serum prolactin levels (r =0.74, p <0.01). The sulpiride test for prolactin responsiveness is reduced or absent in prolactinoma and normal or augmented in individuals with prolactin autoantibodies. Therefore, when prolactin autoantibodies are present, the prolactin response to sulpiride is normal,

401

and the prolactin response to bromocriptine or dopamine is blunted (Bjcro et al., 1993; Fraser et al., 1989).

Methods of Detection Using the PEG precipitation method for demonstrating the presence of prolactin autoantibodies, sensitivity is 98.4% and specificity is 92.9%. PPV is 91.8% and N P V 98.6% (Hattori et al., 1994b).

REFERENCES BjCro T, Johansen E, Frey H, Turter A, Torjesen P. Different responses in little and big big prolactin to metoclopramide in subjects with hyperprolactinemia due to 150--170 kd (big big) prolactin. Acta Endocrinologica 1993;128:308--312. Bottazzo G, Pouplard A, Florin-Christensen A, Doniach D. Autoantibodies to prolactin-secreting cells of human pituitary. Lancet 1975;2:97--101. Buskila D, Sukenik S, Shoenfeld Y. The possible role of prolactin in autoimmunity. Am J Reprod Immunol 1991;26: 118--123. Ericsson U, Larsson I, Murne A., Thorell J. A new sensitive immunosorbent radioassay for the detection of circulating antibodies to polypeptide hormones and proteins. Scand J Clin Lab Invest 1984;44:487-493. Farkouh N, Packer M, Frantz A. Large molecular size prolactin with reduced receptor activity in human serum: high proportion in basal state and reduction after thyrotropin-releasing hormone. J Clin Endocrinol Metab 1979;48:1026-1032. Fraser I, Lun Z, Zhou J, Herington A, McCarron G, Caterson I, Tan K, Markham R. Detailed assessment of big big prolactin in women with hyperprolactinemia and normal ovarian function. J Clin Endocrinol Metab 1989;69:585--592. Hattori N, Ishihara T, Ikekubo K, Moridera K, Hino M, Kura-

402

CONCLUSION All patients with hyperprolactinemia should be subclassified according to the presence of prolactin autoantibodies. Most patients with a predominance of large ( 1 5 0 - 1 7 0 kd) prolactin-antibody complexes lack the clinical manifestations of the syndrome as shown by normal menses and maintained fertility. Medical intervention would be unnecessary for this type of hyperprolactinemia.

hachi H. Autoantibody to human prolactin in patients with idiopathic hyperprolactinemia. J Clin Endocrinol Metab 1992a;75:1226-1229. Hattori N, Ikekubo K, Ishihara T, Moridera K, Hino M, Kurahachi H. A normal ovulatory woman with hyperprolactinemia: presence of antiprolactin autoantibody and the regulation of prolactin secretion. Acta Endocrinol (Copenh) 1992b; 126:497--500. Hattori N, Ikekubo K, Ishihara T, Moridera K, Hino M, Kurahachi H. Effects of antiprolactin autoantibodies on serum prolactin measurements. Eur J Endocrinol 1994a;130:434-437. Hattori N, Ikekubo K, Ishihara T, Moridera K, Hino M, Kurahachi H. Correlation of the antibody titers with serum prolactin levels and their clinical course in patients with antiprolactin autoantibody. Eur J Endocrinol 1994b;130:438--445. Leite V, Cosby H, Sobrinho G, Fresnoza A, Santos M, Friesen H. Characterization of big, big prolactin in patients with hyperprolactinaemia. Clin Endocrinology 1992;37:365-372. Lindstedt G. Endogenous antibodies against prolactin- a "new" cause of hyperprolactinemia. Eur J Endocrinol 1994;130: 429--432. Whittaker P, Wilcox T, Lind T. Maintained fertility in a patient with hyperprolactinemia due to big, big prolactin. J Clin Endocrinol Metab 1981;53:863--866.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

H U M A N A N T I M O U S E ANTIBODIES Joachim R. Kalden, M.D., Ph.D.

Department of Medicine III, and Institute for Clinical Immunology and Rheumatology, University ErlangenNiirenberg, 91054 Erlangen, Germany

HISTORICAL NOTES Monoclonal antibody treatment and monoclonal antibody-based diagnostic procedures are increasingly used in cancer patients (Schroff et al., 1985; Courtenay-Luck et al., 1986), organ transplantation (Thistlethwaite et al., 1988; Ortho Multicenter Transplant Study Group, 1985) and in autoimmune diseases, especially rheumatoid arthritis (Horneff et al., 1995; Herzog et al., 1987; Hailer and Weiner, 1988). Development of human antimouse antibodies (HAMA) is important first with regard to repeated diagnostic or therapeutic applications, and second with regard to the interference with specific in vitro immunoassays. The interference of HAMA in vitro with immunoassays using murine monoclonal antibodies for the detection of hormones, oncofetal and tumor-associated antigens in serum (Horneff et al., 1991a; Zweig et al., 1987; Primus et al., 1988; Baum et al., 1994) can possibly be overcome by addition of normal mouse serum to absorb the HAMA. The strong immune response against mouse reagents is not surprising. Because HAMA generally prohibit further in vivo applications of mouse monoclonal antibodies in diagnostic and treatment situations, attempts to circumvent the induction of HAMA have included the chimerization (or humanization) of the mouse monoclonal antibodies or the concomitant application of immunosuppressive agents.

THE ANTIGENS Patients receiving mouse monoclonal antibodies for diagnostic or treatment purposes develop HAMA

between 7 and 15 days after the beginning of antibody application. In all the studies applying monoclonal antibodies for diagnostic or treatment purposes, IgM as well as IgG HAMA are reported (Horneff et al., 199 l b). HAMA can bind specifically to the Fc portion or can have anti-idiotype or anti-isotype specificity. Anti-idiotype HAMA will be formed even in situations where a humanized mouse monoclonal antibody was employed. Indeed, even in situations where a bispecific F(ab')2 monoclonal antibody was given to patients with an ovarian carcinoma, in addition to HAMA, a significant proportion of the HAMA response was directed against the idiotype of the bispecific monoclonal antibody (Lammers et al., 1995). In RA patients treated with a mouse monoclonal antiCD4 antibody, anti-idiotype-specific antibodies were detected in 25% of the treated patients (Horneff et al., 1991b). The frequency and intensity of development of HAMA when humanized mouse monoclonal antibodies are used, as well as how commonly such HAMA interfere with the humanized antibody reactivity are unknown. In this respect, two reports are of interest: (1) myeloablative therapy strongly suppresses the HAMA and the anti-idiotypic response to murine monoclonal antibodies in patients with stage IV neuroblastoma (Cheung et al., 1994); (2) application of the mouse monoclonal antibody 17-1A was followed by a HAMA response of the IgG and IgM class in all the patients, and 41 out of 43 patients also developed anti-idiotypic antibodies and 20 developed anti-anti-idiotypic antibodies (Frodin et al., 1992). Whether development of such anti-anti-idiotypic antibodies might be beneficial in terms of suppressing tumor development and supporting a longer survival is unknown. The development of high titers of HAMA

403

did not cause significant clinical problems due to repeated infusions of the monoclonal antibody 17-1A (Frodin et al., 1992).

THE ANTIBODIES Methods of Detection

Detection of a newly formed human antibody response against xenogeneic antibodies is influenced by pre-existing anti-immunoglobulin antibodies, especially of the rheumatoid factor (RF) type, RFs are human anti-IgG antibodies primarily detectable in the IgM isotype fraction (Horneff et al., 1991b). Antimurine immunoglobulin antibodies can be detected by an ELISA using plates coated either with the whole therapeutic antibody or its F(ab')2 fragment. With the whole antibody molecule, there is a significant correlation between the IgM responses to the mouse monoclonal antibody and the titers of rheumatoid factors in the Waaler-Rose test. In contrast, there are no significant correlations between titers of rheumatoid factors and the IgM response against the F(ab')2 fragment or the IgG responses against both the whole antibody or the F(ab')2 fragment. These data (Figure 1) indicate that in a given ELISA system, the whole antibody molecules can be used as antigen for demonstrating antibodies to the monoclonal antibody; whereas, the F(ab')2 fragment should be used when IgM antibody responses to the monoclonal are being evaluated.

The demonstration of pre-existing HAMA suggests that HAMA might be attributable either to multireactive antibodies (Casali and Notkins, 1989) or to low levels of IgG rheumatoid factors (Courtenay-Luck et al., 1986). An alternative explanation for the presence of pre-existing HAMA-like activities might be contact with pet rodents which are known to elicit IgE-mediated allergic reactions. Data on IgE or IgG4 HAMA isotypes are not yet available, possibly due to the lower sensitivity of the assay systems. Pre-existing human antimurine antibodies, either as part of the Bcell repertoire or due to a previous contact with mouse proteins, interfere with different technologies including immunoscintography. For example, three patients previously exposed to radiolabeled monoclonal antibodies showed an abnormal body distribution of the antibody and an increased hepatic uptake during subsequent immunoscintography; antimurine antibodies and immune complexes were demonstrated in all the patients (Torres et al., 1993). Problems in the detection of antimouse antibodies include rheumatoid factor-like activities. In one study of RA patients undergoing mouse antihuman, antiCD4 monoclonal antibody treatment, the demonstrated HAMA activity was primarily of the IgG isotype, indicating a secondary immune response (Horneff et al., 1991b). However, increased concentrations of specific HAMA activity of the IgM isotype against the F(ab')2 fragment were the most pronounced finding after a second treatment cycle. The demonstration of human antimouse immunoglobulin antibodies is a

IgG

IgM 1000

400

800 300 600 O

200

E o~ 400 r

100

200 0 16H5 F(ab)2 16H5 F(ab)2 Normal Donors

RA Patients

16H5 F(ab)2

Normal Donors

16H5 F(ab)2 RA Patients

Figure 1.

Human antimouse response measured in the sera of 9 normal donors and 18 untreated RA patients (IgG in mg/liter, IgM in OD). A considerable HAMA activity of the IgM class may be due to the presence of rheumatoid factors as indicated by the marked reduction of HAMA activity against the F(ab') 2 fragment as compared to the whole antibody. There was a significant difference of HAMA responses between the normal donors and RA patients, both against the whole antibody and the F(ab') 2 fragment (p < 0.01, asterisks). Pre-existing HAMA activity of the IgG class did not correlate to rheumatoid factors and was found in normal donors as well as in RA patients without a significant difference (p > 0.10).

404

particular problem in individuals with rheumatoid factors present in their sera, including not only patients with autoimmune diseases but also patients with infectious diseases or cancer. One way to circumvent the problem of pre-existing HAMA activity and monitoring patients undergoing therapy with monoclonal murine antibodies is the utilization of F(ab') 2 fragments in the ELISA system. Even though the sensitivity of detection decreases, since the Fc part represents an important immunogenic region, the specificity appears to be greatly enhanced, especially in patient groups with high rheumatoid factor activities. From one study (Horneff et al., 1991b), it appears that use of F(ab') 2 fragments of the therapeutic antibody will be a great advantage in monitoring specific immune responses to the antibody used. In addition to ELISA systems, flow cytometry for the quantitation of HAMA to approximately 1 ng/mL is also useful; results obtained parallel those with ELISA (Labus and Petersen, 1992). Radioimmunoassays can also be used for detection of specific and nonspecific HAMA. In one report, the nonspecific HAMA assay identified both pre-existent and monoclonal-induced HAMA; whereas, the specific HAMA assays identified specific immune responses occurring after the respective monoclonal antibody injection (Massuger et al., 1992). Such radioimmunoassays for HAMA are of importance prior to further administration of monoclonal antibodies for diagnosis or treatment. The difficulties encountered in measuring human antimouse antibody activity in body fluids has been recently and critically summarized (HAMA Survey Group, 1993). For example, nonspecific immunoglobulin interaction with the F(ab')2 region and Fc piece of human IgG can lead to false-positive results in patients being monitored for naturally occurring or monoclonal antibody-induced human antimouse responses (Papoian, 1992). Specificity of HAMA reactions can be distinguished from false-positive reactions by competitive inhibition with mouse Ig.

CLINICAL UTILITY One of the major problems associated with repeated application of mouse monoclonal antibodies is the induction of HAMA, including some with neutralizing activity. Attempts to circumvent the induction of HAMA by chimerizing or humanizing mouse mono-

clonal antibodies are only partly successful. Human antichimeric antibody responses including anti-isotype and anti-idiotype responses were demonstrated after an anti-TNF-a monoclonal antibody treatment in RA patients (Elliott et al., 1994); repeated injection of the chimerized anti-TNF-~ resulted in a decrease of the clinical response intervals. Problems might also arise from the significantly longer half-life of chimerized or humanized monoclonal antibodies with resultant accumulation.

Effect of Therapies To circumvent the induction of antimouse antibodies, different approaches have been tested. In therapeutic trials, antibodies with different immunosuppressive potentials were used (Benjamin et al., 1988; Goronzy et al., 1986; Goldstein et al., 1986; Jaffers et al., 1986); the application of anti-CD4 in association with antigens resulted in a persistent tolerance to the respective antigen in animal models (Goronzy et al., 1986). Anti-CD4 monoclonal antibodies p e r s e seem to be more effective in suppressing HAMA responses, as compared to the anti-CD3 monoclonal reagent which is directed against all major T cells and is used for treatment of organ rejection episodes. Anti-CD3 monoclonal antibodies elicit considerable antibody reactivity (Goldstein et al., 1986; Jaffers et al., 1986). These observations led to further experiments using anti-CD4 monoclonal antibodies to induce T-cell tolerance for subsequently administered proteins including monoclonal antibody preparations; at least in animal models, this is a useful practice (Qin et al., 1993). Other methods to suppress human antimouse antibody reactivity include the application of cyclosporin A prior to antibody treatment (Weiden et al., 1994) and also the protein engineering of antibody preparations (Sandhu, 1992). Furthermore, single chain antibodies and fusion proteins with antibody specificity joined to nonimmunoglobulin sequences will in the future provide a source of antibody-like molecules with novel properties. Finally, combinatorial libraries produced in bacteriophage present an alternative possibility besides the hybridoma technology for the production of antibodies with the desired antigen-binding specificity and less antigenicity. A further problem for the application of monoclonal antibodies for diagnostic or treatment purposes is the complex formation with the respective antigen, e.g., with CEA in patients with colorectal carcinomas.

405

In such situations, the application of a high-affinity anti-CEA monoclonal antibody is superior in immunoscintography studies as compared to a low-affinity anti-CEA monoclonal antibody (Sharkey et al., 1993).

CONCLUSION Investigations of the development of human antimouse antibodies in patients with cancer, in patients undergoing diagnostic investigations and in patients with autoimmune diseases led to the following principal observations: 1. Pre-existing antibodies of the IgM class with an antimouse antibody activity show a strong correlation to the presence of IgM rheumatoid factors. The possibility of pre-existing antibodies of the IgM class produced by B cells of the multireactive B-cell component cannot be excluded. In addition, a presensitization with mouse proteins has to be considered. 2. In contrast to HAMA of the IgM isotype, IgG

REFERENCES Baum RP, Niesen A, Hertel A, Nancy A, Hess H, Donnerstag B, Sykes TR, Sykes CJ, Suresh MR, Noujaim AA, H6r G. Activating anti-idiotypic human antimouse antibodies for immunotherapy of ovarian carcinoma. Cancer 1994;73: S1121-S1125. Benjamin RJ, Qin SX, Wise MP, Cobbold SP, Waldmann H. Mechanisms of monoclonal antibody-facilitated tolerance induction: a possible role for the CD4 (L3T4) and CDlla (LFA-1) molecules in self-nonself-discrimination. Eur J Immunol 1988;18:1079--1088. Casali P, Notkins AL. Probing the human B-cell receptor with EBV: polyreactive antibodies and CD5+ B lymphocytes. Annu Rev Immunol 1989;7:513--535. Cheung NK, Cheung IY, Canete A, Yeh SJ, Kushner B, Bonilla MA, Heller G, Larson SM. Antibody response to murine anti-GD2 monoclonal antibodies: correlation with patient survival. Cancer Res 1994;54:2228--2233. Courtenay-Luck NS, Epentos AA, Moore R, Larche M, Pectasides D, Dhokia B, Ritter MA. Development of primary and secondary immune responses to mouse monoclonal antibodies used in the diagnosis and therapy of malignant neoplasms. Cancer Res 1986;46:6489--6493. Elliott MJ, Maini RN, Feldmann M, Long-Fox A, Charles P, Bijl H, Woody JN. Repeated therapy with monoclonal antibody to tumour necrosis factor (cA2) in patients with rheumatoid arthritis. Lancet 1994;344:1125-1127. Frodin JE, Lefvert AK, Mellstedt H. The clinical significance of HAMA in patients treated with mouse monoclonal antibodies. Cell Biophys 1992;21:153--165.

406

antibodies with HAMA activity do not show correlation to rheumatoid factors, especially in RA. 3. There is a difference in the development of HAMA depending on the specificity of the mouse monoclonal antibody used. Thus, treatment with an anti-CD4 monoclonal antibody resulted in low amounts of HAMA, even after a second treatment course, which is in contrast to human antimouse antibody response in situations where other monoclonal antibodies were applied. 4. Approximately 25% of the human antimouse monoclonal antibody responses are of an idiotypic or isotypic specificity; this suggests that use of chimerized or humanized mouse monoclonal antibody for diagnostic or treatment purposes might also be limited by the host antibody response. Hopefully, further developments of monoclonal antibody molecules and their use will help minimize human antimonoclonal antibody responses in situations in which antibodies are administered for diagnostic or treatment purposes.

Goldstein G, Fuccello AJ, Norman DJ, Shield CF 3rd, Colvin RB, Cosimi AB. OKT3 monoclonal antibody plasma levels during therapy and the subsequent development of host antibodies to OKT3. Transplantation 1986;42:507--510. Goronzy J, Weyand CM, Fathman CG. Long-term humoral unresponsiveness in vivo, induced by treatment with monoclonal antibody against L3T4. J Exp Med 1986;164:911--925. Hailer DA, Weiner HL. Immunosuppression with monoclonal antibodies in multiple sclerosis. Neurology 1988;38:$42-$47. HAMA Survey Group. Survey of methods for measuring human antimouse antibodies. Clin Chim Acta 1993;215:153--163. Herzog C, Walker C, Pichler W, Aeschlimann A, Wassmer P, Stockinger H, Knapp W, Rieber P, Muller W. Monoclonal anti-CD4 in arthritis. Lancet 1987;2:1461--1462. Horneff G, Becker W, Wolf F, Kalden JR, Burmester GR. Human antimurine immunoglobulin antibodies as disturbing factors in TSH determination.Klin Wochenschr 1991a;69: 220-223. Horneff G, Winkler T, Kalden JR, Emmrich F, Burmester G. Human antimouse antibody response induced by anti-CD4 monoclonal antibody therapy in patients with rheumatoid arthritis. Clin Immunol Immunopathol 1991b;59:89-103. Horneff G, Burmester GR, Emmrich F, Kalden JR. Treatment of rheumatoid arthritis with an anti-CD4 monoclonal antibody. Arthritis Rheum 1995:In press. Jaffers GJ, Fuller TC, Cosimi AB, Russell PS, Winn HJ, Colvin RB. Monoclonal antibody therapy. Anti-idiotypic and nonanti-idiotypic antibodies to OKT3 arising despite intense immunosuppression. Transplantation 1986;41:572--582. Labus JM, Petersen BH. Quantitation of human antimouse

antibody in serum by flow cytometry. Cytometry 1992;13: 275--281. Lammers CH, Gratma JW, Wamaar SO, Stoter G, Bolhuis RL. Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human antimouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T lymphocytes. Int J Cancer 1995;60:450-457. Massuger LF, Thomas CM, Segers MF, Corstens FH Verheijen RH, Kenemans P, Poels LG. Specific and nonspecifc immunoassays to detect HAMA after administration of indium-11 l-labeled OV-TL 3 F(ab') 2 monoclonal antibody to patients with ovarian cancer. J Nucl Med 1992;33:1958-1963. Ortho Multicenter Transplant Study Group. A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplant. N Eng J Med 1985;313: 337--342. Papoian R. Nonspecific immunoglobulin interactions may lead to false-positive results in assays for human antimouse monoclonal antibodies (HAMA). J Immunoassay 1992;13: 289-296. Primus FJ, Kelley EA, Hansen HJ, Goldenberg DM. "Sandwich"-type immunoassay of carcinoembryonic antigen in patients receiving murine monoclonal antibodies for diagnosis and therapy. Clin Chem 1988;34:261-264. Qin S, Cobbold SP, Pope H, Elliott J, Kioussis D, Davies J, Waldmann H. Infectious transplantation tolerance. Science 1993;259:974--976.

Sandhu JS. Protein engineering of antibodies. Crit Rev Biotechnol 1992;12:437--462. Schroff RW, Foon KA, Beatty SM, Oldham RK, Morgan AC Jr. Human antimurine immunoglobulin responses in patients receiving monoclonal antibody therapy. Cancer Res 1985;45: 879--885. Sharkey RM, Goldenberg DM, Murthy S, Pinsky H, Vagg R, Pawlyk D, Siegel JA, Wong GY, Gascon P, Izon DO, Vezza M, Burger K, Swayne LC, Pinsky CM, Hansen HJ. Clinical evaluation of tumor targeting with a high-affinity, anticarcinoembryonic-antigen-specific, murine monoclonal antibody, MN-14. Cancer 1993;71:2082--2096. Tistlethwaite JR Jr, Stuart JK, Mayes JT, Gaber AO, Woodle S, Buckingham MR, Stuart FP. Complications and monitoring of OKT3 therapy. Am J Kidney Dis 1988;11:112-119. Torres G, Bema L, Estorch M, Juarez C, Martinez-Duncker D, Carrio I. Pre-existing human antimurine antibodies and the effect of immune complexes on the outcome of immunoscintography. Clin Nucl Med 1993;18:477-481. Weiden PL, Wolf SB, Breitz HB, Appelbaum JW, Seiler CA, Mallett R, Bjorn MJ, Su FM, Fer MF, Salk D. Human antimouse antibody suppression with cyclosporin A. Cancer 1994 ;73 :S 1093-S 1097. Zweig MH, Csako G, Benson CC, Weintraub BD, Kahn BB. Interference by anti-immunoglobulin G antibodies in immunoradiometric assays of thyrotropin involving mouse monoclonal antibodies. Clin Chem 1987;33:840-844.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

IDIOTYPES AND ANTI-IDIOTYPIC ANTIBODIES Mahmoud Abu-Shakra, M.D. a, Dan Buskila, M.D. a and Yehuda Shoenfeld, M.D. b

aRheumatic Diseases Unit, Department of Medicine, Ben-Gurion University, Soroka Medical Centre, Beer-Sheva; and bDepartment of Medicine "B" Research Unit of Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel

H I S T O R I C A L NOTES Over the past 25 years, there has been a significant transformation in our understanding of the immunogenetics and biochemical structure of the immunoglobulin molecules. Table 1 shows the landmark events in the study of humoral immunity and idiotypes (Grabar, 1984; Nisonoff, 1991). Forty years ago, immunoglobulin molecules were known to be proteins and thought to be antigenic (Abbas et al., 1991). In 1963, immunization of animals with immunoglobulins was shown to result in the production of anti-immunoglobulin antibodies (Oudin and Michel, 1963). This activity might be directed against: (1) immunoglobulin determinants located in the conserved region of the antibodies as a result of differences in amino acid sequences; (2) determinants known as idiotopes located in the light and heavy chains of the variable region of the immunoglobulin molecules. Idiotopes are defined serologically by the reaction of anti-idiotopic antibodies with the antibody bearing the idiotope. The collection of idiotopes on an individual antibody constitute its idiotype (Nisonoff, 1991). Idiotypes are antigenic determinants of immunoglobulins that have the ability to bind to antiidiotypic antibodies as well as to induce an immune response. The terms "idiotypes" and "anti-idiotypic antibodies" also fit the definition of "epitopes" and "paratopes," respectively. Epitopes are defined as those antigenic determinants of a protein that are specifically recognized by the binding site of certain immunoglobulins, i.e., idiotypes are epitopes of immunoglobulins located in the variable region of the molecule. The concept paratope refers to the antigen binding site of the immunoglobulins which binds

408

specifically to certain antigens. Therefore, epitopes require complementary paratopes for their operational recognition. The concept of idiotypes emerged in 1955 when human myeloma proteins were shown to contain antigenic determinants not found on other immunoglobulins from patients with myeloma or from normal donors (Slater et al., 1955). Only in 1963 was the presence of these antigenic sites confirmed with heterologous antisera, and they were called "individual antigenic determinants for isolated antibodies" (Kunkel et al., 1963). In the same year, the antigenic determinants of anti-Salmonella antibodies were identified with isologous antisera and were named "idiotypes" (Oudin and Michel, 1963). Subsequently, the term "individual determinants" was abandoned and the term "idiotype" adopted. Idiotypic markers of immunoglobulins can be used to follow the appearance and persistence of antibodies (Pan et al., 1995). The potential regulatory role of idiotypic/anti-idiotypic interactions in the immune system became the center of many studies starting in 1974 with the proposal that the immune response might be regulated via the idiotypes (Jerne, 1974). This hypothesis predicts that the idiotypic determinants of each antibody molecule are recognized by those of another antibody, thus creating an "idiotypic network" through which immunoglobulin expression might be controlled. Idiotypic dysregulation may have a significant role in the pathogenesis of autoimmune diseases (Shoenfeld, 1994). Various experimental models of autoimmune diseases including systemic lupus erythematosus (SLE)(Mendlovic et al., 1988), antiphospholipid syndrome (APS) (Bakimer et al., 1992) and Wegen-

Table 1. Landmark Events in the Study of Immunoglobulins

Year

Researcher(s)

Event

1886

Foder

The first to observe a direct action of sera on microbes

1894

Pfeiffer

Described Pfeiffer phenomenon: Cholera vibrios injected into the peritoneum of immunized guinea pigs lost mobility

1894

Bordet, Ehrlich

The terms antigen and antibody were introduced

1902

Metchnikoff

Antileukocyte antisera were produced

1938

Tiselius, Kabat

Antibodies were found to be globulins

1940

Felton

Obtained purified preparation of antibodies

1959

Nisonoff

Structure and formation of immunoglobulins

1963

Oudin, Michel, Kunkel

The term idiotype was introduced

1974

Jerne

The idiotypic network was proposed

1975

Kohler, Milstein

Described hybridoma technology

er's granulomatosis (WG) (Shoenfeld, 1994; Tomer et al., 1995) were induced in naive mice following idiotypic immunization. Intensive research directed to downregulating pathogenic idiotypes includes: (1) the prevention of autoimmune diseases by immunization with antiidiotypic antibodies (Blank et al., 1994), (2)controlling malignant lymphoproliferative diseases by using anti-idiotypic antibodies directed against idiotypes located on surface immunoglobulins of malignant B cells (Levy and Miller, 1990), and (3) the use of intravenous gamma globulins (IVIg) to treat various autoimmune diseases. It has been suggested that IVIg preparations contain natural anti-idiotypic antibodies that might suppress the pathogenic idiotypes (Ronda et al., 1993).

THE AUTOANTIGEN(S) Definition and Classifications

Idiotypes, are the antigenic determinants of immunoglobulin molecules that are located in the variable region of the antibodies (Pan et al., 1995). Idiotypes are subdivided into those that reside at the antigenbinding site, the "paratope", of the antibody molecule and those on the areas adjacent to this site, the "framework" determinants. Idiotypes on myeloma proteins are specific for each myeloma antibody (Slater et al., 1955). Because such idiotypes are markers for individual myeloma

proteins, the term "private" idiotype was coined (Slater et al., 1955). Likewise, because antibodies from different individuals share some idiotypes, the terms "recurrent," "public" and "cross-reactive" (CRI) idiotypes were used for those antigenic determinants (Nisonoff, 1991). Anti-idiotypic antibodies are antibodies directed against the idiotypic determinants. They are classified into (Figure 1): 1. "Ab2 alpha" if they are directed against idiotypes which are distinct from the antigen-binding site (paratope) on Abl. The Ab2 alpha anti-idiotypic antibodies recognize Abl framework region antigens. Those anti-idiotypes are also referred to as antigen-noninhibitable since the idiotype/antiidiotype interaction cannot be inhibited by a hapten that binds specifically to the antigen-binding site (paratope). 2. "Ab2 beta" if they fit the antigen-binding site of the antibody molecule. Those idiotypes are antigen-inhibitable. As proposed in 1974, the term "internal image" indicates that anti-idiotype antibodies interact with the binding site of an antibody through structures that resemble the relevant epitope of the antigen; this suggests that external antigens are potentially represented within the immune system as idiotypic determinants on antiidiotype antibodies (Jerne, 1974). The concept of the internal image does not, however, mean that the Ab2 molecule carries a structure resembling the entire antigenic site. Rather, the internal image represents an image of a specific epitope within

409

reovirus hemagglutinin (Bruck et al., 1986). 3. "Ab2 gamma" interfere with antigen binding (antigen-inhibitable) and are directed against idiotopes close to, rather than within the antigenbinding site. Their antigen-inhibitable effect is because of steric hindrance with the antigenbinding site. The Ab2 gamma recognizes combining site-associated idiotopes, but they do not carry the internal image of the antigen. 4. "Ab3", are anti-anti-idiotypic antibodies which are induced by the presence of Ab2 and may have binding characteristics similar to Abl.

AUTOANTIBODIES

PathogeneticRole ldiotypic Network.

According to the theory of the idiotypic network presented in 1974 (Jerne, 1974), all individuals possess thousands of idiotypes reflecting the infinite possibilities of foreign antigen structure. Any antigenic stimulation leads to the production of idiotypes (Abl) and anti-idiotypes (Ab2 and Ab3) as a network of interacting antibodies; the idiotypic determinants of each antibody molecule are complemented by those of another (Figure 2). The idiotypic network is thought to play a crucial physiologic role

Figure 1. The two aspects of Jerne's original theory of idiotypic immunization that lead to generation of a network. A) Antigen (Ag) antibody 1 (Abl) which is anti-Ag. The unique structure of Abl is recognized by the immune system which generates Ab2, which is anti-anti-Ag. The antigen binding characteristics of Ab2 resemble the structure of the antigen. Ab3, induced by the presence of Ab2, may have binding capabilities similar to those of Abl. B) An alternative pathway is shown where Ab2 is anti-idiotypic to a structure residing out of the binding site (framework).

THE PATHOGENICASPECTOF THE JERNE'S IDIOTYPIC NETWORK Ab3

]~

] ~ Abl +ADJUVANT 2-3 WEEK Abe'

the antigen-binding site and not the whole binding site. Anti-idiotypic antibodies with internal image activity include polyclonal rabbit anti-idiotypic antibodies which bind the cellular receptors for insulin (Sege and Peterson, 1978) and retinolbinding protein (Shechter et al., 1982). The peptide sequence of a monoclonal anti-idiotypic antibody directed against an antibody specific for the virusneutralizing epitope on a reovirus hemagglutinin shows an amino acid sequence similarity to the

410

Figure2. Immunization with an antibody (autoantibody or antimicrobial antibody) that carries a pathogenic idiotype to the generation of Ab2. After a period of incubation, Ab3 is produced, which may have binding characteristics similar to the original pathogenic idiotype.

in regulating the immune response to nonself-antigens and preventing the development of pathogenic autoantibodies (Jerne, 1974). Indeed, manipulation of the network may lead to the development of autoimmune diseases (Shoenfeld, 1994). Under normal physiological conditions, the idiotypic network is thought to play a major role in the regulation of immune responses to external antigens (Jerne, 1974). The antigens stimulate the generation of Ab 1 and then the serologically unique structure of its antigen-binding site stimulates the immune system to produce Ab2 which recognize the antigen-binding site of Ab 1. This idiotypic/anti-idiotypic interaction has a regulatory role in the immune response to the eliciting antigen. Therefore, similar to the manner in which an antibody removes an antigen circulating in the blood stream, an anti-idiotype may be triggered and terminate the production of another idiotype. As manifest by natural autoantibodies (NAA), autoimmune activity is ubiquitous in healthy people. At least 20% of all immunoglobulins correspond to polyreactive NAA. Natural autoantibodies, which are normal components of the immune system have important physiologic roles (Avrameas, 1992), including binding of damaged or degraded self-tissue to facilitate opsonization and phagocytosis. NAA clearly have a role in the phagocytosis of defective erythrocytes (Heegard, 1990) and might have a role in selftolerance by preventing autoreactive clones from reacting vigorously with self-antigens by binding to those antigens and masking their antigenic determinants from autoreactive clones; NAA might also block the receptors on autoreactive CD5-positive cells and thereby downregulate their own synthesis (Avrameas, 1992). Injection of NAA with anti-idiotypic activity into mice reduces the titer of the corresponding idiotype (Vakil and Kearney, 1986). Natural monoclonal antibodies of neonates interact extensively among themselves through their idiotopes and this crossreactivity among idiotypes normally persists throughout life and fluctuates in complex dynamic patterns (Lundkvist et al., 1989). Intravenous injection of a pair of complementary idiotypes suppresses the fluctuation in the serum concentration of both idiotypes. No similar phenomenon is observed after immunization with nonrelated idiotypes (Lundkvist et al., 1989). Immunization of newborn mice with immunoglobulins derived from spleens of perinatal mice reduces the concentrations of the corresponding idiotypes of

the injected antibodies. Immunization at different stages of the life of the mice is associated with upregulation or downregulation of the immune responses of the mice (Vakil and Kearney, 1986). The anti-idiotypic activity of NAA plays a major role in immunoregulation and might prevent expansion of autoreactive cells.

Structure of Idiotypes. The light and heavy chains of immunoglobulin molecules contain series of repeats, each about 110 amino acid residues in length, defined as immunoglobulin domains. The amino-terminal domains constitute the variable region which includes the three hypervariable regions, also called complementarity-determining regions (CDR), and four more conserved framework regions (FR1--4). The three CDRs are each about 10 amino acids long (Abbas et al., 1991). Idiotypes are associated partially or entirely with CDRs and framework regions. Usually, the full expression of idiotypes requires CDRs from light and heavy chains (Pan et al., 1995). The conformation of binding sites is determined by the amino acid sequence of the CDRs. The antigenbinding site of the Fab fragment from a monoclonal antibody to lysozyme is a rather flat surface (Amit et al., 1986). As expected, anti-idiotypic antibodies react with their idiotypic targets through their CDRs, of which several can be involved on each antibody of the pair (Bently et al., 1990). Data show that an idiotype can consist of 13 amino acids from 5 CDRs and one framework region (Bently et al., 1990). In other cases, the heavy chain can dominate binding. Antibody 3 (anti-anti-idiotypic antibody) reactive with human angiotensin has an affinity similar to that of antibody 1 for angiotensin and binds angiotensin via six CDRs (Garcia et al., 1992). Taken together, the data indicate that antigen sequence can be preserved through the idiotope and can reappear in the structure of Ab2 (internal image). Genetics The immunoglobulin heavy and light V region genes are composed of multiple gene families. In each gene family, there is more than 80% similarity in the nucleotide sequences of the individual genes. All cells, with the exception of B cells, contain germline immunoglobulin genes. In B cells, there is a process of somatic rearrangement of the germline genes to enable the genes to produce functional 411

proteins. This process occurs in the absence of antigenic stimulation. Following exposure to an antigen the immunoglobulin genes undergo somatic mutation in the V region genes to allow affinity maturation of antibodies. Cross-reactive idiotypes (CRI), i.e., public idiotypes, are encoded by germline genes; whereas, the genes of private idiotypes undergo somatic mutation (Pan et al., 1995). The idiotypes associated with natural autoantibodies (NAA) are the prototype of germline geneencoded idiotypes. They are primarily polyreactive IgM autoantibodies with low affinity to their autoantigens; these features are characteristic of a B-cell response prior to antigenic stimulation. Furthermore, some autoantibodies from patients with SLE and other autoimmune diseases are encoded by germline genes (Chen et al, 1988). However, genomic studies of the high affinity IgG anti-dsDNA antibodies and their idiotypes revealed that these immunoglobulins are produced by a process of somatic mutation which is clustered mainly in the CDRs of the variable regions (Demaison et al., 1994). Factors in Pathogenicity Idiotypes of Autoantibodies. The majority of 30 antiDNA idiotypes can be detected on human monoclonal IgM anti-DNA antibodies derived from patients with SLE or leprosy. However, anti-DNA idiotypes from healthy people are also described (Buskila and Shoenfeld, 1994). One of the most studied anti-DNA idiotypes, the 16/6 idiotype, binds single-stranded DNA, other nucleic acids, nucleoproteins, cell membrane antigens and phospholipids. Detected in the sera of 50% of patients with active SLE and in 40% of immunoglobulin deposits in the skin and kidneys of patients with SLE, the 16/6 idiotype is also present on human autoantibodies directed against RNP, Sm, SS-A, and on the SA-1 antibody, an autoantibody derived from a patient with polymyositis (Buskila and Shoenfeld, 1994). The major cross-reactive idiotypes of RF include the Wa, Po and Bla idiotypes (Posnett et al., 1986). The Wa idiotype was identified initially on IgM RF from a patient with Waldenstr6m's macroglobulinemia; whereas, Bla idiotype is present on a unique subset of RF that cross-react with DNA-histones (Barnes et al, 1990). Seven polyclonal antiribonucleoprotein idiotypes raised following immunization with three anti-La 412

autoantibodies bound only to the immunizing antibody (Horsfall et al., 1986). Y2, a cross-reactive idiotype found on a mouse monoclonal anti-Sm antibody from lpr/lpr mouse, is present in the sera of 41% of SLE patients, 27% of their first-degree relatives and 6% of healthy controls (Pisetsky et al., 1984).

CLINICAL UTILITY Disease Association High titers of pathogenic idiotypes detected in the sera of patients with autoimmune diseases include the pathogenic anti-DNA idiotype 16/6 found in the sera of 50% of patients with active SLE (Buskila and Shoenfeld, 1994). Similarly, antiacetylcholine receptor idiotypes and their anti-idiotypic antibodies can be identified in the sera of patients with myasthenia gravis (Cleveland et al., 1983). Immunization of naive mice with an autoantibody to a weak immunogenic antigen can lead to the generation of Ab2 (anti-idiotype). After a long followup period of 3--8 months, Ab3 is produced which has binding characteristics similar to the original pathogenic autoantibodies (Bakimer et al., 1992). Thus, naive (i.e., never exposed to the antigen per se) mice can secrete autoantibodies (Figure 2). Immunization of naive and other strains of mice, including BALB/c (H-2a), C3h (H-2b), AKR (H-2k) and SJL (H-25), with monoclonal/polyclonal human/ mice anti-DNA antibodies carrying the pathogenic DNA idiotype designated 16/6 Id is associated with the development of SLE-like disease. The disease is characterized by the production of anti-DNA antibodies and other autoantibodies, thrombocytopenia, leukopenia and clinical features of SLE including nephritis (Mendolovic et al., 1988; Shoenfeld et al., 1994). The F(ab') 2 fragments of the anti-DNA antibody which carry the 16/6 idiotype retain the specificity and pathogenic activity of the whole antibody (Ruiz et al., 1994). Furthermore, immunization of mice with a synthetic peptide based on the CDRs sequence of the heavy chain of a murine monoclonal anti-DNA antibody, is also associated with the development of SLE (Waisman et al., 1995). The autoimmune disease is thus triggered by the pathogenic idiotype and production of pathogenic anti-idiotypes. Similarly, models of the antiphospholipid syndrome and Wegener's granulomatosis develop after

immunization of the mice with anticardiolipin antibodies (aCL) and antineutrophil cytoplasmic antibodies (ANCA), respectively. Immunization of various strains of mice with aCLs was followed after 3--4 months by the generation of anticardiolipin antibodies, development of thrombocytopenia and prolonged activated thromboplastin time as well as low fecundity and an increased rate of fetal resorption in immunized females (Bakimer et al., 1992) (Figure 3). In a third model, immunization of B ALB/c mice with ANCA led either to the death of the mice from multiple nonbacterial lung abscesses or to the appearance of perivascular mononuclear infiltration and immunoglobulin deposition (Tomer, et al., 1995). In humans, the development of autoimmune diseases might be related to exposure to an external antigen mimicking pathogenic or regulatory idiotypes. Support for this hypothesis comes from the presence of high levels of the 16/6 idiotype following infection with mycobacteria, Klebsiella and other microbial agents (Abu-Shakra and Shoenfeld, 1991). Exposure to a microbe might trigger autoimmune phenomena, including antimicrobial antibodies carrying the pathogenic idiotype of an autoantibody perhaps via adjuvant effects in people with the appropriate genetic and hormonal background (Figure 4). The model of immunological homunculus suggests that antimicrobial antibodies may carry a limited number of pathogenic idiotypes according to their representation in the naive immune system (Cohen, 1992).

I D I O T Y P I C I N D U C T I O N OF A U T O I M M U N E DISEASES

INFECTING AGENT ( e.g. TB, Klebsiella

AB1

~

~'

(anti-BACTERIALAB) MAY CARRYA PATHOGENICId (e.g. 16/6 Id)

"~

=!= |HELPER

AB2

(anti-ld e.g. anti 16/6)

HEALTHY SUBJECT

>MALE HLA-DR6 N-IgA N-C'

ADJUVANT BACTERIALWALL SUPERANTIGEN

INFECTINGAGENT ~ e.g. TB, Klebsiella AB1 ~ (anti-BACTERIALAB) MAYCARRYA PATHOGENICId (e.g. 16/6Id)

w-. |HELPER

3W

(anti-ld e . g . anti 16/6)

AB3 ' anti-anti-ld /

=autoantibody

HEALTHYSUBJECT AUTOIMMUNE

B

> MALE HLA-DR6 N-IgA N-C'

>FEMALE HLA DR2,3,4 C2,C4def.

Figure 4. Idiotypic induction of autoimmune disease. A) Infection may trigger autoimmune diseases by inducing antibacterial antibodies carrying the pathogenic idiotypes of autoantibodies (Abl). B) In the presence of the adjuvant effect (or super antigen) attributed to the bacterial agents, Abl leads to the generation of Ab3 in patients with the "proper" genetic and hormonal background.

Therapeutic Implications Immunization of animals with the relevant anti-

Figure 3. Fetal resorption (the equivalent to human fetal loss) is demonstrated in mice with experimental antiphospholipid syndrome. The lower part serves as a normal uterus with fetuses from mouse immunized with normal serum immunoglobulins.

idiotypic antibodies can produce antibody response against several infectious agents, including hepatitis B antigen (Kennedy et al., 1986) and HIV envelope proteins (Zaghouani et al., 1991). Immunization of chimpanzees with anti-idiotypic antibodies of hepatitis B surface antigen causes the production of antihepatitis antibodies and prevents the development of hepatitis (Kennedy et al., 1986). An alternative application of the idiotypic network involves administration of anti-idiotypic antibodies to animal models of autoimmune diseases, as well as to humans with established autoimmune disease. Longterm idiotypic suppression can be achieved by treatment with anti-idiotypic antibodies, e.g., long-term suppression of CRI in mice that receive rabbit antiidiotypic antibodies. Suppression of pathogenic antibodies to DNA in NZB/NZW female mice follows repeated inoculation of the mice with monoclonal antiidiotypic antibodies (Hahn and Ebling, 1984).

413

Idiotypic manipulation can also prevent or suppress experimental models of autoimmune diseases, e.g., inhibition of the development of experimental autoimmune thyroiditis by the generation of anti-idiotypic antibodies (Ab2 beta) that recognize the paratope of an antithyroglobulin monoclonal antibody specific for a pathogenic epitope of the thyroglobulin molecule (Roubaty et al., 1990). Development of SLE in naive mice induced by immunization with anti-DNA idiotypes can be suppressed by treatment with specific anti-idiotypic antibodies conjugated to immunotoxin with resultant decreases in titers of autoantibodies and mild clinical features (Blank et al., 1994). IVIg is sometimes effective in selected human autoimmune disorders, including autoimmune thrombocytopenia, polymyositis, SLE and Kawasaki disease (Ronda et al., 1993). Two major hypotheses for the mechanism of action of IVIg are proposed: (1) Fc receptor blockade and, (2) natural anti-idiotypic antibodies directed against the pathogenic autoantibodies. By electron microscopy, a high proportion of IVIg is in the form of dimers, compatible with an idiotype/anti-idiotype interaction. Anti-idiotypic antibodies might also be an effective biologic therapy for malignant diseases. Anti-idiotypic antibodies directed against idiotypes located at the surface immunoglobulins of B cells were used with some success in the treatment of malignant lymphoma and leukemia (Levy and Miller, 1990). These data, albeit preliminary reveal a potential beneficial role for the induction of anti-idiotypic antibodies.

CONCLUSION Under normal conditions, idiotypes and their antiidiotypic antibodies (idiotypic network) might have a major role in regulating the immune response to selfand foreign antigens. Auto-anti-idiotypic antibodies are components of the normal immune system. Manipulation of the idiotypic network might lead to the development of pathogenic idiotypes and autoimmune diseases. The Koch criteria for classic autoimmune disease include: the presence of autoantigen, autoantibody or autoreactive T cells and induction of the disease by active immunization with the autoantigen or by passive transfer of the autoantibody. Because autoimmune diseases can also be induced by immunization with anti-idiotypic antibodies, another criterion for autoimmune disease might include induction of autoimmune disease by active immunization with the autoantibody or the idiotype (Shoenfeld, 1994). Ongoing research is directed toward manipulation of the idiotypic network in an attempt to downregulate the immune system in autoimmune diseases or to upregulate anti-idiotypic antibodies with activity against tumor antigens or anti-anti-idiotype antibodies against bacterial antigens. Clearly, further research is required to develop effective therapy for autoimmune and malignant diseases. See also NATURAL AUTOANTIBODIES.

Table 2. Summary Table Idiotype

Antigenic determinants located on the variable region of immunoglobulin molecules and defined serologically by the reaction of anti-idiotypic antibodies

Types of idiotypes

Private and cross reactive

Anti-idiotypic antibodies

Antibodies directed against idiotypic determinants

Types of anti-idiotypic antibodies

Ab2 alpha, Ab2 beta, Ab2 gamma and Ab3

Idiotypic network

The immune response might be regulated via the idiotypic determinants of immunoglobulins

Structure of idiotypes

Idiotypes are associated with complementarity determining and framework regions of the immunoglobulin molecules

Genetics of idiotypes

Public idiotypes are encoded by germline genes whereas the genes of private idiotypes undergo somatic mutation

Pathogenicity

Idiotypic manipulation of pathogenic idiotypes results in the development of overt autoimmune disease

Therapeutic implications

Anti-idiotypic antibodies might be used to suppress pathogenic idiotypes

414

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Grabar P. The historical background of immunology. In: Stites DP, Stobo JD, Fudenberg HH, Wells JV, eds. Basic and Clinical Immunology. Los Altos: LANGE, 1984:1--12. Hahn BH, Ebling FM. Suppression of murine lupus nephritis by administration of an anti-idiotypic antibody to anti-DNA. J Immunol 1984;132:187--190. Heegard NH. Immunochemical characterization of interaction between circulating autologous immunoglobulin G and normal human erythrocyte membrane proteins. Biochim Biophys Acta 1990;1023:239--46. Horsfall AC, Venables PJ, Mumford PA, Maini RN. Idiotypes on antibodies to the La antigens are restricted and associated with the antigen binding site. Clin Exp Immunol 1986;6: 292-299. Jeme NK. Towards a network theory of the immune system. Ann Immunol (Paris) 1974;125:373-389. Kennedy RC, Eichberg JW, Lanford RE Dreesman GR. Antiidiotypic antibody vaccine for type B viral hepatitis in chimpanzee. Science 1986;232:220-223. Kunkel HG. Mannik M, Williams RC. Individual antigenic specificity of isolated antibodies. Science 1963; 140:1218-1219. Levy R, Miller A. Therapy of lymphoma directed at idiotypes. Mongr Natl Cancer Inst 1990; 10:61--68. Lundkvist I, Coutinho A, Varela F, Holmberg D. Evidence for a functional idiotypic network among natural antibodies in normal mice. Proc Natl Acad Sci 1989;86:5074-5078. Lymberi P, Dighiero G, Temynck T. A high incidence of crossreactive idiotype among murine natural autoantibodies. Eur J Immunol 1985;15:702--707. Mendlovic S, Brocke S, Shoenfeld Y, Ben-Bassat M, Meshorer A, Bakimer R, Mozes E. Induction of a systemic lupus erythematosus-like disease in mice by a common anti-DNA idiotype. Proc Natl Acad Sci 1988:85:2260-2264. Nisonoff A. Idiotypes: concepts and application. J Immunol 1991 ;147:2429--2438. Oudin J, Michel M. Une nouvelle forme d'allotypie des globulines gamma du serum de lapin, apparemment liee a la fonction et a la specificite anti corps. CR Acad Sci III 1963;257:805-808. Pan Y, Yuhasz SC, Amzel LM. Anti-idiotypic antibodies: biological function and structural studies. FASEB J 1995;9: 43--49. Pisesky DS, Semper KF, Eisenberg DA. Idiotypic analysis of a monoclonal anti-Sm antibody. J Immunol 1984;133:20852092. Posnett DN, Wisniewolski R, Pernis B, Kunkel HG. Dissection of human antigammaglobulin idiotype system with monoclonal antibodies. Scand J Immunol 1986;23:169--176. Ronda N, Kaveri SV, Kazatchkine MD. Treatment of autoimmune diseases with normal immunoglobulins through manipulation of the idiotypic network. Clin Exp Immunol 1993 ;93 :S 14-S 15. Roubaty C, Bedin C, Charreire J. Prevention of experimental autoimmune thyroiditis through the anti-idiotypic network. J Immunol 1990;144:2167-2172. Ruiz PJ, Zinder H, Mozes E. Induction of experimental systemic lupus erythematosus in mice by immunization with

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F(ab') 2 fragment of the human anti DNA monoclonal antibody carrying the 16/6 idiotype. Immunol Lett 1994;41: 79--84. Sege K, Peterson PA. Use of anti-idiotypic antibodies as cell surface receptor probes. Proc Natl Acad Sci 1978;75:2443-2447. Shechter Y, Maron R, Elias D, Cohen IR. Autoantibodies to receptor spontaneously develop anti-idiotypic antibodies in mice immunized with insulin. Science 1982;216:542-545. Shoenfeld Y. Idiotypic induction of autoimmunity: a new aspect of the idiotypic network. FASEB J 1994;8:1296-1301. Slater RJ, Ward SM, Kunkel HG. Immunological relationship among myeloma proteins. J Exp Med 1955;101:85--90. Tomer Y, Gilburd B, Blank M, Lider O, Hershkoviz R, Fishman P, Zigelman R, Meroni PL, Wiik A, Shoenfeld Y. Characterization of biologically active ANCA-induced in

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mice: Pathogenetic role in experimental vasculitis. Arthritis Rheum 1995;38:1375--1381. Vakil M, Kearney F. Functional characterization of monoclonal auto-anti-idiotypic antibodies isolated from the early B cell repertoire of BALB/C mice. Eur J Immunol 1986;16:1151-1158. Waisman A, Ruiz PJ, Mozes E. Induction and modulation of systemic lupus erythematosus by two complementary determining region peptides of a pathogenic anti-DNA monoclonal antibody. Lupus 1995;4:$2:49. Zaghouani HD, Goldstein H, Shah S, Anderson S, Lacroix M, Dionne G, Kennedy R, Bona C. Induction of antibodies to the envelope protein of the human immunodeficiency virus by immunization with monoclonal anti-idiotypes. Proc Natl Acad Sci 1991 ;88:5645--5650.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

IgA AUTOANTIBODIES Charlotte Cunningham-Rundles, M.D., Ph.D.

Departments of Medicine, Pediatrics and Biochemistry, The Mount Sinai Medical Center, New York, NY 100296574, USA

H I S T O R I C A L NOTES After their initial description in IgA-deficient patients with ataxia telangiectasia (Strober et al., 1968), autoantibodies to IgA were detected in selective IgA deficiency (IgA <10 mg/dL) in the study of sera from 250 normal adult volunteers and 250 patients with a variety of disorders (Fudenberg et al., 1968). The original intent of the serological studies was to identify sera containing anti-IgA antibodies for use in investigations of genetically determined antigens on the ~ chain of IgA. However, the medical importance of IgA antibodies in producing severe, nonhemolytic, anaphylactic transfusion reactions soon emerged (Vyas et al., 1968). An increased prevalence of anti-IgA antibodies is now recognized in patients with hypogammaglobulinemia and other immunodeficiencies (including especially selective IgA deficiency), multiply transfused patients, patients with autoimmune diseases and pregnant women. With sensitive methods, anti-IgA antibodies can also be detected in normal adults even in the absence of severe deficiency of IgA.

THE AUTOANTIGENS Definition

The target antigen of immunoassays for anti-IgA antibodies is human IgA obtained either from normal serum, exocrine secretions or, in some cases, IgA myeloma proteins. The autoantigen is restricted to IgA; anti-IgA antibodies do not react with other immunoglobulins and are thus often termed "class

specific". The antigenic target(s) are determinants on, or related to the ~ chain. Some IgA autoantibodies are broadly reactive and bind all IgA immunoglobulins, others are subclass specific and react with either IgA1 or IgA2 (Koistinen et al., 1977; Petty et al., 1985; Sennhauser et al., 1988). Still other anti-IgA antibodies react only with IgA Am(l) or Am(2) molecules (allotype specific) (Vyas and Fudenberg, 1969). In most cases, anti-IgA antibodies in sera bind both IgA subclasses, but pregnant IgA-deficient women who have anti-IgA antibodies, may more commonly have antibodies binding to IgA2 (Petty et al., 1985). The significance of this antibody under these circumstances is unknown. The detection of antibodies of this specificity has generally not been sought, but methods using myeloma IgA1 proteins may not detect anti-IgA antibodies with specificity for IgA2 proteins. Methods of Purification/Commercial Sources

Purification of the target antigen, IgA, varies with the source of the IgA used for testing. For convenience, myeloma IgA is often used as the antigen in test systems, although in some cases, polyclonal serum IgA or secretory IgA is used. To detect anti-IgA antibodies of IgA1 and IgA2 specificity, one could theoretically use a mixture of several myeloma IgA proteins (containing both IgA1 and IgA2). Purification of serum or myeloma IgA can be performed by chromatographic means (Vyas et al., 1969) or by lectin separation using jacalin. Chromatographic means are most commonly employed. Commercial sources are scarce, and contamination with other serum immunoglobulins is common. Contamination of commercial IgA proteins with even small amounts of

417

IgG produces unacceptable results using ELISA techniques; when coated red blood cells serve as the indicator system, small amounts of IgG or IgM are of less concern.

been detected in all sera in which IgE anti-IgA is present. Anti-IgA antibodies of the IgG isotype are mainly of the IgG 1 subclass, but may also be of IgG2, IgG3 and/or IgG4 subclasses (Bj6rkander et al., 1987).

Genetic Associations AUTOANTIBODIES Pathogenetic Role The relationship of transfusion-related anaphylaxis to IgA antibodies was widely confirmed (Vyas et al., 1968; 1969; Schmidt et al., 1969; Miller et al., 1970; Bjerrum and Jersild, 1971; Leikola et al., 1973). Transfusion reactions due to anti-IgA antibodies are often severe. After the infusion of a few milliliters of blood or blood products, patients with anti-IgA antibodies can have a rapid progression of anaphylactic symptoms. While rare, stroke events, myocardial infarction, anuria and death are reported (Pineda and Taswell, 1975). The reasons why these reactions can be so severe is not completely understood, although complement activation certainly occurs (Wahn et al., 1984; Cunningham-Rundles et al., 1986) with profound depletion of complement components C lq, C2 to C6 and increased serum concentrations of C3 split products (Wahn et al., 1984). In addition to the IgGmediated reactions, IgE anti-IgA reactions are also reported (Burks et al., 1986). Although IgE anti-IgA antibodies were not sought in each case and when sought were not commonly found (Bj6rkander et al., 1987), an IgE-mediated pathway could also explain the rapid onset and severity of some of these transfusion and immunoglobulin infusion reactions.

Factors in Pathogenicity Isotype and Subclass. Anti-IgA antibodies are primarily of the IgG class, but IgM anti-IgA antibodies are occasionally found. IgE anti-IgA antibodies can also be detected in the sera of some patients; antibodies of this isotype may produce especially severe infusion reactions (Burks et al., 1986). The incidence of anti-IgA antibodies of the IgE class in IgA deficient serum is unknown. Few studies have sought such antibodies; the methods used in one study did not detect them (Bj6rkander et al., 1987); in another study, of a group of 86 patients with primary immunodeficiency, only one subject had anti-IgA of the IgE isotype as well as anti-IgA of the IgG isotype. It should be noted that IgG anti-IgA antibodies have 418

Attempts to predict which IgA-deficient individuals might be more likely to develop anti-IgA antibodies, on the basis of HLA and DR typing, included systematic screening of 2,782 blood-donor samples, among whom 67 donors with selective IgA deficiency were identified. In addition to a significant association of IgA deficiency with HLA-A1 (x 2 = 10.24, p < 0.005) and B 14 (x2= 14.43, p < 0.0005) antigens in 36 of these 67 donors, the frequency of HLA-A1 and B8 antigens was 54 and 26% in the groups of IgAdeficient donors with and without anti-IgA antibodies, respectively. Although too small to show a clear statistical significance (0.05 < p < 0.1), this study suggests a possible HLA association with anti-IgA antibodies (Strothman et al., 1986). A weak association between IgA deficiency and DR3 was observed for 46 unrelated IgA-deficient donors (RR = 2.07); DR7 seemed to be associated with those who developed IgA antibodies (RR = 2.94); whereas, DR1 was associated with those who did not (RR = 2.42) (Strothman et al., 1989). IgA deficiency is relatively common in the Caucasian population; this incidence is usually cited at 1:500 to 1:1000 for healthy individuals in such populations (Cunningham-Rundles, 1995), however, the prevalence of selective IgA deficiency in Japan is only 0.007%. On the other hand, anti-IgA antibodies were found in three of the 12 Japanese IgA-deficient subjects with IgA levels <5 mg/dL, a prevalence rate comparable to that of donors of European ancestry. Possible HLA or other genetic associations in Japanese are unknown (Kanoh et al., 1986). In the Swedish population, a statistically significant association between the IgG G lm-2 phenotype, and the presence of anti-IgA antibodies was noticed (Hammarstr6m et al., 1985) as well as a nonrandom distribution between Gm phenotypes and HLA-B8/ DR3-positive individuals who had anti-IgA antibodies.

Methods of Detection The standard means of detecting anti-IgA antibodies in test serum is passive hemagglutination using human blood group O, Rh-negative erythrocytes sensitized by

CLINICAL UTILITY

chromic chloride and then coated with target myeloma IgA antigens (Vyas et al., 1969). More sensitive ELISA can detect and quantitate anti-IgA antibodies in sera of various patients and even normal subjects. With a modified sandwich ELISA, the IgA target antigen is immobilized on microtiter wells coated with streptococcal protein B, which binds -to the Fc portion of IgA but does not bind other immunoglobulins (Eckrich et al., 1993). Detection of anti-IgA antibodies using flow cytometry and microbeads coated with highly purified serum IgA instead of myeloma IgA proteins is advantageous, because anti-IgA antibodies sera with restricted specificities would not often be missed using polyclonal IgA as a target as opposed to monoclonal IgAs (Syrjala et al., 1991). Purification of polyclonal IgA from serum is, however, more timeconsuming than purifying monoclonal IgA proteins which admittedly are not representative of total serum IgA. Demonstration that highly purified polyclonal IgA is truly representative of total serum IgA and that it does not contain other immunoglobulins or other possibly relevant impurities is difficult. Patients who have anti-IgA antibodies are skintest-negative to red blood cells, plasma, buffy coat cells from IgA-containing blood (Schmidt et al., 1969) and even IgA (Nadorp et al., 1973).

Detection of anti-IgA antibodies in serum is important for prevention to avoid adverse reactions upon infusion of blood or blood products. The autoantibody is not known to produce any pathology in the person in whose serum it is found. Disease Association

Following their detection in the sera of five IgAdeficient patients with ataxia telangiectasia, (Strober et al., 1968) anti-IgA antibodies were found in some individuals with selective IgA deficiency, with or without deficiencies of other immunoglobulins (Table 1). The wide range for the prevalence of this autoantibody is notable: 53 of 185 (29%) of patients with selective IgA deficiency, 15 of 68 (22%) of patients with hypogammaglobulinemia and 6 of 10 (60%) subjects with combined IgA and IgG2 subclass deficiency had anti-IgA antibodies (Bj6rkander et al., 1987). No anti-IgA antibodies were found in the serum of 25 patients who had a serum IgA of greater than 5 mg/dL, although lower titered anti-IgA antibodies can sometimes be found in such sera (Bj6rkander et al., 1987).

Table 1. Prevalence of Anti-IgA Antibodies

Subjects

Prevalence (%)

Reference

2

Fudenberg et al., 1968

Ataxia telangiectasia

23

Fudenberg et al., 1968

Persons having anaphylactoid urticarial transfusion reactions

86

Vyas et al., 1969

Selective IgA deficiency and ataxia telangiectasia

40

Vyas et al., 1969

Selective IgA deficiency

16

Vyas et al., 1975

Selective IgA deficiency

42

Hammarstr6m et al., 1983

IgA-deficient blood donors

17

Petty et al., 1985

IgA-deficient pregnant women

16

Petty et al., 1985

Selective IgA deficiency

53.9

Bj6rkander et al., 1987

Common variable immunodeficiency

29

Bj6rkander et al., 1987

IgA and IgG2 subclass deficiency

60

Bj6rkander et al., 1987

Selective IgA deficiency

28.5

Ferriera et al., 1988

IgA and IgG2 subclass deficiency

50

Ferriera et al., 1988

IgA-deficient children

63

Sennhauser et al., 1988

Normal donors

419

The prevalence of anti-IgA antibodies is greater in IgA-deficient individuals who have other autoimmune diseases than in those who do not. For example, antiIgA antibodies were found in the sera of 25--30% of healthy or nonaffected IgA-deficient subjects; whereas, anti-IgA antibodies were found in the Ig sera of 50% of those with IgA deficiency and rheumatoid arthritis, 77% of those with IgA-deficiency juvenile rheumatoid arthritis and 100% of those with IgAdeficiency systemic lupus erythematosus (Petty et al., 1979). Anti-IgA antibodies are also more common in IgA-deficient subjects who have pernicious anemia (Bjerrum and Jersild, 1971; Katka et a1.,1988). Although the stimulus to the production of antiIgA antibodies is unclear, their presence is certainly not traceable to prior exposure to blood or blood products in each case (Leikola et al., 1973; Vyas et al., 1975). Prior treatment with intramuscular immunoglobulin can, however, sensitize immunodeficient patients (Cunningham-Rundles et al., 1986; Ferreira et al., 1989). Although anti-IgA antibodies of high titer are most commonly found in the sera of IgA-deficient subjects (especially for those with IgA <5 mg/dL), low titered anti-IgA antibodies of both the IgG and IgM isotype can also be detected in the serum of normal subjects using enzyme-linked immunosorbent assay (ELISA) techniques. These antibodies are directed at the Fab of IgA (Jackson et al., 1987). In addition to anti-IgA antibodies that react with native IgA, human serum also contains antibodies to pepsin-digested IgA, or otherwise enzymatically altered IgA (Wilson et al., 1970; Koistenen and Leikola, 1977). These antibodies are of unknown biological relevance, but could potentially result in false-positive anti-IgA antibody tests if the IgA proteins have become "aged," or partially enzymatically degraded during imperfect storage conditions.

Antibody Frequencies Class-specific anti-IgA antibodies (antibodies reacting with all IgA molecules) or antibodies binding to only IgA1 or IgA2 proteins, or antibodies binding to IgA molecules of selected allotypes, can cause transfusion reactions. For example, of sera from 158 patients with transfusion reactions, four had class-specific anti-IgA antibodies and 10 had antibodies with more limited specificity (Koistinen and Leikola, 1977). How common are IgA-antibody-induced transfusion reactions? The answer to this question is prob-

420

lematic. IgA deficiency is a common immune defect (frequency of 1:500 to 1:1000) and it is even more prevalent in hospital populations (1:300 to 1:500). The estimated frequency of anti-IgA antibodies in IgA deficiency is also quite h i g h - up to 63% (Table 1). Although expected to be reasonably common, the actual prevalence of documented IgA-antibody-induced-transfusion reactions or immunoglobulin infusion reactions is quite rare. For example, during a 6-year period (1977 to 1982), blood samples from 152 Canadian patients were referred to a national laboratory because of severe nonhemolytic blood transfusion reactions. Twenty-one of these infused patients were found to be IgA-deficient, and of these only 12 had strong, class-specific anti-IgA antibodies. In these cases, the IgA antibody was presumed to be responsible for the reactions. However, considering the overall number of transfusions of blood given in this period, the actual incidence of anti-IgA antibodyinduced reactions was only 1.3 reactions per million units of blood or blood products infused (Laschinger et al., 1984). Anti-IgA-IgA-based transfusion reactions are sufficiently rare that routine testing for anti-IgA antibodies is not recommended. More difficult to assess is the relative merit of screening for anti-IgA antibodies in subjects known to be IgA deficient. Although this is a reasonable procedure, it has not entered common usage, usually because (1) lack of readily available laboratory expertise, and (2) the urgent need of blood transfusion or immunoglobulin infusion precludes prior testing for the presence of anti-IgA antibodies. A reasonable recommendation would be to test all IgA-deficient individuals (persons having an IgA <10 mg/dL), at the same time this deficiency is diagnosed, for anti-IgA antibodies. Because some individuals develop anti-IgA antibodies only after exposure to blood or blood products, a second test could be performed several months after transfusion or infusion to determine if this happened (people with anti-IgA antibodies should wear a MedicAlert tag). When a patient known to have anti-IgA antibodies needs a blood transfusion: autologous blood can be collected, an IgA-deficient donor can be used or multiply washed red blood cells can be supplied. Platelets, if needed, can also be washed prior to use. Liver transplantation in three sensitized IgA-deficient subjects was uneventful using these means (Davenport et al., 1992). Patients with hypogammaglobulinemia or symp-

tomatic combined deficiencies of IgA and IgG2 are sometimes given prophylactic intravenous immunoglobulin (IVIg); when these individuals have anti-IgA antibodies, serious infusion reactions can ensue. IVIg depleted of IgA is, however, readily available and safe to use, even for maintenance infusions given monthly for years (Cunningham-Rundles, et al., 1993; Ferriera et al., 1989; Bj6rkander et al., 1987). In such patients, the titer of anti-IgA is usually stable over time, despite monthly infusions of IVIg preparations which are not devoid of IgA. These solutions contain up to 1 pg/mL IgA; thus patients are infused with 100 to 500 pg of IgA on each occasion (Bj6rkander et al., 1987; Cunningham-Rundles et al., 1993).

tions (2 SD below the normal mean), five had mothers with anti-IgA antibodies (Petty et al., 1985). Because sera with high-titered anti-IgA antibodies can selectively inhibit IgA synthesis in vitro, anti-IgA antibodies might influence development of the IgA system (Hammarstr6m, et al., 1983). Anti-IgA antibodies can also be detected in the sera of women who are not IgA-deficient, including 15 percent of mothers of recently delivered infants; in some cases, these antibodies are directed at allotypic determinants (Cassidy et al., 1969). Similarly, infants can produce antibodies to determinants on IgA unique to maternal IgA, suggesting exposure to minute amounts of these proteins (Vyas et al., 1969). The biological role of these antibodies is unknown.

Anti-IgA Antibodies in Pregnancy Because the IgG of the neonate is supplied by the transplacental passage of maternal IgG, the passage of IgG anti-IgA antibodies across the placenta should be a common event in IgA-deficient women with antiIgA antibodies. What would be the biological effects, if any, on the developing IgA system of the neonate? Families with IgA deficiency in a number of generations have a higher frequency of mother-to-child than father-to-child inheritance of IgA deficiency (Petty et al., 1985). This suggests that anti-IgA antibodies not only cross the placenta, but also inhibit IgA development in the fetus. Studying 61 serum samples from IgA-deficient pregnant women, 2% had antibody to IgA1 only, 16% had antibodies to IgA1 and IgA2, and 20% had antibody to IgA2 only. Children of IgAdeficient mothers (but not fathers) had serum IgA concentrations below the normal means (21 of 27); of seven infants with the lowest serum IgA concentra-

REFERENCES Bjerrum OJ, Jersild C. Class-specific anti-IgA associated with severe anaphylactic transfusion reactions in a patient with pernicious anaemia. Vox Sang 1971;21:411-424. Bj6rkander J, Hammarstr/3m L, Smith CL, Buckley RH, Cunningham-Rundles C, Hanson LA. Immunoglobulin prophylaxis in patients with antibody deficiency syndromes and anti-IgA antibodies. J Clin Immunol 1987;7:8-15. Burks AW, Sampson HA, Buckley RH. Anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia. N Engl J Med 1986;314:560-564. Cassidy JJ, Burt A, Petty R, Sullivan D. Selective IgA deficiency in connective tissue diseases. N Engl J Med 1969;280: 275.

CONCLUSION Anti-IgA antibodies are common in the serum of immunodeficient subjects and are an important cause of nonhemolytic transfusion reactions, which while actually quite rare are important because of their severity. Anti-IgA antibody-induced infusion reactions can also occur after infusion of immunoglobulins if anti-IgA antibodies are present. In most cases, antiIgA antibodies of significant titer occur in patients who have IgA concentrations of <5 mg/dL serum. These antibodies can be directed at IgA1, IgA2 or both immunoglobulin molecules. For patients with anti-IgA antibodies, a history of prior sensitization by blood or blood products is not always present. Various means can be used to make transfusion of blood or blood products a safe procedure for patients with antibodies to IgA.

Cunningham-Rundles C, Wong S, Bj6rkander J, Hanson LA. Use of an IgA-depleted intravenous immunoglobulin in a patient with an anti-IgA antibody. Clin Immunol Immunopathol 1986;38:141-149. Cunningham-Rundles C, Zhou Z, Mankarious S, Courter S. Long-term use of IgA-depleted intravenous immunoglobulin in immunodeficient subjects with anti-IgA antibodies. J Clin Immunol 1993;13:272-278. Cunningham-Rundles C. Disorders of the IgA system. In: Steim ER, ed. Disorders in Infants and Children. Fourth Edition. Philadelphia: W.B. Saunders Company, 1995: in press. Davenport RD, Burnie KL, Barr RM. Transfusion management of patients with IgA deficiency and anti-IgA during liver transplantation. Vox Sang 1992;63:247-250. Eckrich RJ, Mallory DM, Sandler SG. Laboratory tests to

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exclude IgA deficiency in the investigation of suspected antiIgA transfusion reactions. Transfusion 1993;33:488--492. Ferreira A, Garcia Rodriguez MC, Lopez-Trascasa M, Pascual Salcedo D, Fontan G. Anti-IgA antibodies in selective IgA deficiency and in primary immunodeficient patients treated with gamma globulin. Clin Immunol Immunopathol 1988;47: 199-207. Ferreira A, Garcia Rodriguez MC, Font AN. Follow-up of antiIgA antibodies in primary immunodeficient patients treated with gamma globulin. Vox Sang 1989;56:4:218-222. Fudenberg HH, Gold ER, Vyas GN, MacKenzie MR. Human antibodies to human IgA globulins. Immunochemistry 1968;5:203-206. Hammarstr6m L, Persson MA, Smith CI. Anti-IgA in selective IgA deficiency. In vitro effects and Ig subclass pattern of human anti-IgA. Scand J Immunol 1983;18:509-513. Hammarstr6m L, Grubb R, Smith CI. Gm allotypes in IgA deficiency. J Immunogenet 1985;12:125-130. Jackson S, Montgomery RI, Mestecky J, Czerkinsky C. Normal human sera contain antibodies directed at Fab of IgA. J Immunol 1987;138:2244--2248. Kanoh T, Mizumoto T, Yasuda N, Koya M, Ohno Y, Uchino H, Yoshimura K, Ohkubo Y, Yamaguchi H. Selective IgA deficiency in Japanese blood donors: frequency and statistical analysis. Vox Sang 1986;50:81-86. Katka K, Eskola J, Granfors K, Koistinen J, Toivanen A. Serum IgA deficiency and anti-IgA antibodies in pernicious anemia. Clin Immunol Immunopathol 1988;46:55-60. Koistinen J, Leikola J. Weak anti-IgA antibodies with limited specificity and nonhemolytic transfusion reactions. Vox Sang 1977;32:77--81. Koistinen J, Cardenas RM, Fudenberg HH. Anti-IgA antibodies of limited specificity in healthy IgA-deficient subjects. J Immunogenet 1977;4:295-300. Laschinger C, Sheppard FA, Naylor DH. Anti-IgA-mediated transfusion reactions in Canada. Can Med Assoc J 1984; 130: 141-144. Leikola J, Koistinen J, Lehtinen M, Virolainen M. IgA-induced anaphylactic transfusion reactions: a report of four cases. Blood 1973;42:111--119. Miller WV, Holland PV, Sugarbaker E, Strober W, Waldmann TA. Anaphylactic reactions to IgA: a difficult transfusion problem. Transfusion 1970;54:618--621. Nadorp JH, Voss M, Buys WC, van Munster PJ, van Tongeren JH, Aalberse RC, van Loghem E. The significance of the presence of anti-IgA antibodies in individuals with an IgA deficiency. Eur J Clin Invest 1973;3:317--323. Petty RE, Palmer NR, Cassidy JT, Tubergen DG, Sullivan DB.

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The association of autoimmune diseases and anti-IgA antibodies in patients with selective IgA deficiency. Clin Exp Immunol 1979;27:83--88. Petty RE, Sherry DD, Johannson J. Anti-IgA antibodies in pregnancy. N Engl J Med 1985;313:1620-- 1625. Pineda AA, Taswell HF. Transfusion reactions associated with anti-IgA antibodies: report of our cases and review of the literature. Transfusion 1975;15:10-- 15. Schmidt AP, Taswell HF, Gleich GJ. Anaphylactic transfusion reactions associated with anti-IgA antibody. N Engl J Med 1969;280:188-- 193. Sennhauser FH, Hosking CS, Jones CL, MacDonald RA, Mermelstein N, Roberton DM. Anti-IgA antibodies in IgAdeficient children. J Clin Immunol 1988;8:356-361. Strober W, Wochner RD, Barlow MH, McFarlin DE, Waldmann TA. Immunoglobulin metabolism in ataxia telangiectasia. J Clin Invest 1968;47:1905-1915. Strothman R, White MB, Testin J, Chen SN, Ball MJ. HLA and IgA deficiency in blood donors. Hum Immunol 1986;16: 289--294. Strothman RA, Sedestrom LM, Ball MJ, Chen SN. HLA association of anti-IgA antibody production. Tissue Antigens 1989;34:141--144. Syrjala MT, Tolo H, Koistinen J, Krusius T. Determination of anti-IgA antibodies with a flow cytometer based microbead immunoassay (MIA). J Immunol Methods 1991; 139:265--270. Vyas GN, Perkins HA, Fudenberg HH. Anaphylactoid tranfusion reactions associated with anti-IgA. Lancet 1968;2: 312--315. Vyas GN, Fudenberg HH. Isoimmune anti-IgA causing anaphylactoid transfusion reactions. N Engl J Med 1969;280:1073-1074. Vyas GN, Holmdahl L, Perkins HA, Fudenberg HH. Serologic specificity of hun anti-IgA and its significance in transfusion. Blood 1969;34:573--581. Vyas GN, Perkins HA, Yang YM, Basantani GK. Healthy blood donors with selective absence of immunoglobulin A: prevention of anaphylactic transfusion reactions caused by antibodies to IgA. J Lab Clin Med 1975;85:838-842. Wahn V, Good RA, Gupta S, Pahwa S, Day NK. Evidence of persistent IgA/lgG circulating immune complexes associated with activation of the complement system in serum of a patient with common variable immune deficiency: anaphylactic reactions to intravenous gamma globulin. Acta Pathol Microbiol Immunol Scand 1984;284:49-58. Wilson ID, Soltis RD, Williams RC Jr. Naturally occurring human antibodies to pepsin-digested IgA. Blood 1970;36: 390-398.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

IgE RECEPTOR AUTOANTIBODIES Michihiro Hide, M.D., Ph.D. a, Robert M. Barr, Ph.D. b, David M. Francis, B.Sc. b and Malcolm W. Greaves, M.D., Ph.D. b

aDepartment of Dermatology, Onomichi General Hospital, Onomichi 722, Japan; and bSt. John's Institute of Dermatology, UMDS, St. Thomas's Hospital, London SE1 7EH, UK

HISTORICAL NOTES

As with other receptors for immunoglobulins, the high affinity IgE receptor (FceRI) is activated by crosslinking of multiple receptors, with resultant histamine release and cytokine production by mast cells and basophils (Marone et al., 1989a; Gordon et al., 1990). The binding of multivalent antigens to IgE on the receptors triggers the activation of FceRI under physiological conditions, but antibodies against IgE or FceRI also cross-link and activate FceRI by di- or multivalent binding in vitro (Metzger, 1991). The possibility of circulating histamine-releasing factors in chronic urticaria was first suggested by the finding of basophilic leukopenia (Rorsman, 1962) and reduced reactivity of peripheral basophils to anti-IgE antibodies (Greaves et al., 1974, Kern and Lichtenstein 1976). Serum histamine-releasing factors induce a wheal-and-flare reaction when autologous serum is injected intradermally in patients with chronic urticaria (Grattan et al., 1986). Autoantibodies against IgE can be identified in some of those patients (Grattan et al., 1991), but other patients with urticaria were subsequently found to have autoantibodies to FceRI (Hide et al., 1993). Of those patients with serum histaminereleasing activity, about half have activity characteristic of autoantibodies primarily to FceRI (Hide et al., 1995). The nature of the serum histamine-releasing activity in the remaining patients is not known (Kermani et al., 1995).

THE AUTOANTIGENS

The DNA sequences of the genes for two types of IgE

receptor are known (Ravetch, 1994; Gould et al., 1991). The high affinity receptor (FceRI) is abundantly expressed on mast cells and basophils. It is also expressed on Langerhans cells (Bieber et al., 1992; Wang et al., 1992), eosinophils (Gounni et al., 1994) and activated monocytes (Maurer et al., 1994). The low-affinity receptor (Fc~RII/CD23) is expressed by eosinophils, monocytes, platelets, Langerhans cells and some lymphocytes (Sutton and Gould, 1993). Mac-2/eBP, an IgE-binding protein on the surface of neutrophils, is also involved in the IgE-dependent activation of the neutrophils (Truong et al., 1993), but to date, only FceRI has been identified as a target of disease-related autoantibodies. Characteristics

FceRI consists of one c~, one ~ and two disulfidebonded 7 subunits, which are noncovalently associated on the plasma membrane (Metzger, 1991) (Figure 1). Both IgE and the autoantibodies bind to the extracellular region of the cz-subunit (FceRI~), which consists of two immunoglobulin-like structures with seven sites for glycosylation. The binding site of IgE was mapped to the second (C-terminal side) domain of FceRI~ (Hulett et al., 1993; Robertson, 1993). IgE binding is not affected by glycosylation (Blank et al., 1991), and the recombinant FceRIcz fragments produced by phage and E. coli systems, which intrinsically lack capacity for glycosylation, have high-affinity binding for IgE (Robertson, 1993). Urticaria is often triggered or aggravated in association with infections, raising the possibility of sequence similarity of epitopes of microbial antigens and of the m-chain of FceRI. Although precise epitopes for the anti-FceRI~ auto-

423

Figure 1. Schematic representation of an IgE molecule bound to FceRI. FceRI consists of non-covalently associated subunits" one IgEbinding ~-subunit, one 4-fold membrane spanning ~-subunit and two disulfide-linked y-subunits. The ~-subunit contains two immunoglobulin-like structures. Both IgE and a monoclonal antibody that inhibits interaction of IgE with FceRI bind to a region in the second (C-terminal side) immunoglobulin-like domain. Sugar moieties, whose total molecular weight is even larger than that of the primary structure of the receptor, covering seven potential sites in the c~-subunit, were not illustrated for clarity. (Adapted from Sutton and Gould, 1993 with modifications.)

antibodies are not known, the presence of multiple epitopes, some of which presumably overlap with the region for IgE binding, is likely (Hide et al., 1994). Inhibition of autoantibody-induced histamine release by three different preparations of the recombinant extracellular fragment of FceRIc~ with substantially different forms of glycosylation suggests that differences in glycosylation have little or no influence on autoantibody binding to FceRI~ (Figure 2). The [3 and y subunits of FceRI have small extracellular regions which consist of 47 and 5 amino acids, respectively (Blank et al., 1989; Ktister et al., 1992), but there are no reports of autoantibodies that functionally interact with these fragments.

424

AUTOANTIBODIES

Pathogenetic Role Biological activity, rather than physiological binding, provided the evidence for autoantibodies in chronic urticaria. Histamine-releasing activity in sera can be readily detected by observing wheal and flare formation at 30 minutes in response to intradermal injection of autologous sera (Grattan et al., 1986). These reactions to autologous sera diminish during clinical remission thereby suggesting a causal relationship of serum histamine-releasing factors to disease activity (Grattan et al., 1986). Electron microscopy shows mast cell degranulation at the site of the skin test

Figure 2. Inhibition of serum-induced histamine release from human basophils by various types of recombinant fragments of FceRI(~), produced by mammalian, yeast and insect cell systems. Sera from four patients (RH, SK, FC, AP) containing autoantibodies against FceRI were preincubated at 37~ for 30 minutes with buffer (pin-striped bar) or 30 ng/mL of various types of extracellular fragment of FcERI~, produced by Chinese hamster ovary (CHO) cells (filled bar), Pichia (strip bar) and Baculovirus-insect cell system (hatched bar) (gifts from Dr. J. Hakimi and Dr. J. Kochan, Hoffmann La Roche, Nutley, NJ). The mixtures were then incubated with leukocytes containing basophils from a healthy volunteer whose serum IgE level was less than 1 IU/mL and the amount of histamine release was determined (Hide et al., 1993). (Grattan et al., 1990). In vitro assays of histamine release from mast cells in human skin and from basophil leukocytes confirm that sera from skin testpositive patients (- 60% of severely affected chronic urticaria patients) contain histamine-releasing factors.

Methods of Detection Approximately half of the sera from skin test-positive patients release histamine from basophils, indicative of the presence of functional autoantibodies against either FceRI~ (Hide et al., 1993) or IgE (Grattan et al., 1991). Sera from all skin test-positive patients release histamine from human mast cells; those that fail to release from basophil leukocytes contain non-IgG histamine-releasing activity specific for mast cells (Kermani et al., 1995). Sera from skin test-negative patients fail to release histamine from either basophil leukocytes or mast cells. The histamine-release assay using basophil leukocytes of healthy volunteers is a

more definitive method than skin testing for detection of histamine-releasing autoantibodies. The assay is very sensitive relative to the minimum detectable concentration of monoclonal antibodies against FceRI (approximately 3 ng/mL) (Hide et al., 1993). It requires no pretreatment of serum samples, but it is affected by the characteristics of basophils, especially by their degree of IgE sensitization. Basophils of most healthy donors are essentially fully sensitized with endogenous IgE. They respond to anti-IgE antibodies and autoantibodies against FceRI that are not competitive with IgE but are not responsive to the autoantibodies against FceRI that are competitive with IgE (Hide et al., 1993). Histamine-releasing activity of both types of autoantibodies against FceRI, therefore, should be tested on basophils with little or no sensitization by IgE. Either basophils obtained from a volunteer with a very low serum IgE concentration or basophils treated with lactic acid to remove IgE can be used for this purpose (Hide et al., 1993). Antibody

425

specificity against FceRI~ is confirmed by neutralization of the activity by the soluble recombinant fragment of the ~-subunit. Lack of histamine release from rat basophilic leukemia (RBL-2H3) cells by sera containing autoantibodies against FceRI suggests the epitopes for the autoantibodies are human specific (Hide et al., unpublished data). Comparative studies with monoclonal antibodies against FceRI~ suggest that epitopes for IgE-competitive type autoantibodies are located in the Cterminal side domain, and those for non-IgE-competitive type in the N-terminal domain (Riske et al., 1991). IgG autoantibodies from patients with chronic urticaria bind to transfected human FceRI~ expressed on Chinese hamster ovary cells (Fiebiger et al., 1995). ELISA using immobilized, recombinant FceRI~ detects binding of IgG which correlates well with basophil histamine-releasing activity when serial serum samples are available from patients studied over an extended period (Hide et al., unpublished results). However, control sera from some healthy subjects also show appreciable binding in the ELISA, in the absence of histamine-releasing activity. Such binding, which is partly dependent on the cellular source of recombinant FceRI~, might reflect antibody interaction with nonhuman types of glycosylation on the recombinant FceRI~ (Borrebaeck et al., 1993).

Factors in Pathogenecity Sera from some healthy individuals might contain biologically inactive anti-FceRI~ antibodies that lack sufficient avidity or are otherwise unable to cross-link receptors and induce degranulation. Concentrations of functional autoantibodies in the sera of patients, estimated by comparison with histamine-release, doseresponse curves of monoclonal anti-FceRIa antibodies, are usually not more than 200 ng/mL which is 5 • 10 4 times lower than the concentration of total IgG in serum. Thus, no immunoassay with reliable specificity for the functional autoantibodies has been established. It has been recently reported that IgEmediated release of mediators and cytokines is inhibited by cross-linking of FceRIa to low-affinity IgG receptors (FcyRII) (DaCron et al., 1995). The binding of some autoantibodies to FceRIc~, even with high avidity, might partially inhibit histamine release from basophils because of simultaneous binding to FcyRII via their Fc; such, however, should not affect human skin mast cells which lack Fc y receptors (Valent and Bettelheim, 1992). 426

To date, only IgG antibodies have been identified as functional autoantibodies against FceRI.

CLINICAL UTILITY

Disease Association Chronic urticaria, a common disorder characterized by recurrent, transitory wheals and flares with itching, is induced by immunological and/or nonimmunological activation of skin mast cells followed by release of mediators such as histamine (Kaplan et al., 1978). Although the sensitivity of the cutaneous vasculature to vasopermeability-enhancing agents, including histamine, is elevated in some patients with urticaria (Smith et al., 1992), the causes of mast cell activation remain obscure in the majority of patients. Indeed, although 60% of the patients with chronic idiopathic urticaria are autologous serum skin test-positive (Table 1), only about 25% have basophil and mast cell histamine-releasing activities in their sera with characteristics of autoantibodies against FceRIc~. The male to female ratio in the patients with anti-FceRI~like histamine-releasing activities is 1:5.3; whereas, that in the whole population of chronic urticaria patients studied is 1:2.7. Approximately one-third of anti-FceRIc~ autoantibodies are competitive with IgE and two thirds are non-, or only partially competitive with IgE (Hide et al., 1995). Serum IgE concentrations were low in four patients reported to have the IgE-competitive type autoantibodies. Pathological activation of mast cells by autoantibodies against FceRI is described only in chronic urticaria; whereas, autoantibodies against IgE, which can also activate mast cells and basophils via crosslinking of FceRI, are reported not only in chronic urticaria (Grattan et al., 1991) but also in allergic diseases such as asthma and atopic dermatitis (Nawata et al., 1984, Marone et al., 1989b). Sera from patients with chronic urticaria containing anti-FceRIc~ autoantibodies induce histamine release in vitro not only from basophils and skin mast cells (Francis et al., 1994), but also from mast cells of lung, heart and small intestine (Barr et al., unpublished observations). It is, therefore, feasible that autoantibodies against FceRI may be involved in the pathogenesis of a variety of diseases. The reason why in patients with chronic urticaria only skin responds clinically to the autoantibodies is obscure, but could reflect increased acces-

Table 1. Autologous Serum Skin Test in Patients with Chronic Urticaria, Symptomatic Dermographism and Apparently Healthy Controls

Chronic urticaria Symptomatic dermographism Apparently healthy controls

Total Tested

Positive (wheal volume >9 mm3)

163

98 (60%)

6

0 (0%)

18

0 (0%)

sibility of the autoantibodies in the cutaneous microcirculation to skin mast cells or hyperreactivity of mast cells in chronic urticaria skin primed by locally generated factors such as cytokines or neuropeptides.

Effect of Various Therapies Substantial numbers of patients have urticaria which is resistant to conventional drug therapies, including antagonists acting on histamine H~-receptors, sometimes in combination with those for Hz-receptors. Among several types of immunoregulatory therapies undertaken with success in some severely affected patients, plasmapheresis induced temporary remission or improvement in six of eight patients with chronic urticaria who gave positive skin test reactions to autologous serum (Grattan et al., 1992). In this study, the serum histamine-releasing activity of a patient followed for nine months varied in parallel with urticarial activity score. It was later confirmed that the histamine-releasing activity in the serum of this patient was due to anti-FceRI autoantibodies (Hide et al., 1993). Intravenous infusion of high dose immunoglobulin (IVIG) induced clinical improvement and reduction of skin test reaction on autologous serum injection in six of eight patients (O'Donnell et al., 1994). Administration of cyclosporin A 6 mg/kg/day dramatically suppressed severe urticaria of three patients but with troublesome side effects (Fradin et al., 1991). When low-dose cyclosporin A (2.5--3.5 mg/kg/day) was tried for 12 severe, unselected cases, nine resolved or improved during the treatment for four weeks without apparent side effects (Barlow et al., 1993). Interestingly, two of them relapsed on withdrawal of cyclosporin A, but seven showed a sustained improvement for at least a month after stopping the therapy. Subsequent experience with lowdose cyclosporin A supports its value in severe

chronic urticaria (Greaves et al., unpublished results), but the relationship of responsiveness to the presence or absence of anti-FceRI autoantibodies has not yet been defined. In spite of severe and nonremitting clinical symptoms of urticaria, apparent concentrations of the histamine-releasing autoantibodies in sera are generally low (<200 ng/mL), and can be neutralized in vitro by relatively low concentrations of the soluble fragment of FceRI~ (Hide et al., 1993). Trials of more specific therapies such as specific immunoadsorption by FceRIc~ or its analogues are expected in the near future. The administration of the soluble FceRI~ fragments (Ra et al., 1993) or FceRI~-human IgG chimeric molecules (Haak-Frendscho et al., 1993) are reported to prevent passive cutaneous anaphylaxis (PCA) reactions in laboratory animals. Full information on the epitopes, including tertiary structures might allow development of new drugs which specifically block FceRI receptor-autoantibody interactions.

CONCLUSION Approximately one-quarter of the patients with chronic idiopathic urticaria have autoantibodies to FceRI, a third of which are competitive with IgE for the binding to FceRI~. The functional significance of anti-FceRI autoantibodies is that they activate rather than impair receptor function, leading to mediator release from mast cells. Several immunotherapies developed for autoimmune disorders including plasmapheresis, high-dose immunoglobulin infusion and administration of low-dose cyclosporin A are effective for some patients with severe, refractory urticaria. Elucidation of precise epitopes for the autoantibodies might facilitate selective immunotherapy for urticaria and possibly other mast cell-mediated diseases.

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REFERENCES Barlow RJ, Black AK, Greaves MW. Treatment of severe chronic urticaria with cyclosporin A. Eur J Dermatol 1993;3: 273-275. Bieber T, de la Salle H, Wollenberg A, Hakimi J, Chizzonite R, Ring J, Hanau D, de la Salle C. Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (FceRI). J Exp Med 1992;175:1285-1290. Blank U, Ra C, Miller L, White K, Metzger H, Kinet JP. Complete structure and expression in transfected cells of high affinity IgE receptor. Nature 1989;337:187--189. Blank U, Ra C, Kinet JP. Characterization of truncated {x-chain products from human, rat and mouse high affinity receptors for immunoglobulin E. J Biol Chem 1991;266:2639-2646. Borrebaeck CA, Malmborg AC, Ohlin M. Does endogenous glycosylation prevent the use of mouse monoclonal antibodies as cancer therapeutics? Immunol Today 1993;14: 477-479. Daeron M, Malbec O, Latour S, Arock M, Fridman WH. Regulation of high-affinity IgE receptor-mediated mast cell activation by murine low-affinity IgG receptors. J Clin Invest 1995;95:577-585. Fiebiger E, Maurer D, Holub H, Reininger B, Hartmann G, Woisetschler M, Kinet JP, Stingl G. Serum IgG autoantibodies directed against the or-chain of FceRI: a selective marker and pathogenetic factor for a distinct subset of chronic urticaria patients? J Clin Invest 1995;in press. Fradin MS, Ellis N, Goldfarb MT, Voorhees JJ. Oral cyclosporine for severe chronic idiopathic urticaria and angioedema. J Am Acad Dermatol 1991;25:1065-1067. Francis DM, Niimi N, Hide M, Kermani F, Barr RM, Black AK, Greaves MW. Histamine release from human skin in vitro induced by sera containing IgG anti-FceRIo~ autoantibodies from patients with chronic urticaria. J Invest Dermatol 1994;103:399. Gordon JR, Burd PR, Galli SJ. Mast cells as a source of multifunctional cytokines. Immunol Today 1990; 11:458--464. Gould H, Sutton B, Edmeades R, Beavil A. CD23/FceRII: Ctype lectin membrane protein with a split personality? In: Gordon J, ed. CD23-a Novel Multifunctional Regulator of the Immune System that Binds IgE. Monographs in Allergy. Basel: Karger, 1991 ;29:29--49. Gounni AS, Lamkhioued B, Ochiai K, Tanaka Y, Delaporte E, Capron A, Kinet JP, Capron M. High-affinity IgE receptor on eosinophils is involved in defense against parasites. Nature 1994 ;367:183-186. Grattan CE, Wallington TB, Warin RP, Kennedy CT, Bradfield JW. A serological mediator in chronic idiopathic urticariaa clinical immunological and histological evaluation. Br J Dermatol 1986;114:583--590. Grattan CE, Boon AP, Eady RA, Winkelmann RK. The pathology of the autologous serum skin test response in chronic urticaria resembles IgE-mediated late-phase reaction. Int Arch Allergy Appl Immunol 1990;93:198-204. Grattan CE, Francis DM, Hide M, Greaves MW. Detection of circulating histamine-releasing autoantibodies with functional properties of anti-IgE in chronic urticaria. Clin Exp Allergy

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1991;21:695-704. Grattan CE, Francis DM, Slater NG, Barlow RJ, Greaves MW. Plasmapheresis for severe, unremitting, chronic urticaria. Lancet 1992;339:1078--1080. Greaves MW, Plummer VM, McLaughlan P, Stanworth DR. Serum and cell bound IgE in chronic urticaria. Clin Allergy 1974;4:265--271. Haak-Frendscho M, Ridgway J, Shields R, Robbins K, Gorman C, Jardieu P. Human IgE receptor {x-chain IgG chimera blocks passive cutaneous anaphylaxis reaction in vivo. J Immunol 1993;151:351-358. Hide M, Francis DM, Grattan CE, Hakimi J, Kochan JP, Greaves MW. Autoantibodies against the high affinity IgE receptors as cause of histamine release in chronic urticaria. N Eng J Med 1993;328:1599--1604. Hide M, Francis DM, Grattan CE, Barr RM, Winkelmann RK, Greaves MW. The pathogenesis of chronic urticaria: new evidence suggest an autoimmune basis and implications for treatment. Clin Exp Allergy 1994;24:624--627. Hide M, Francis DM, Barr RM, Greaves MW. Skin mast cell activation by autoantibodies in urticaria and therapeutic implications. In: Kitamura Y, Yamamoto S, Galli SJ, Greaves MW, eds. Biological and Molecular Aspects of Mast Cell and Basophil Differentiation and Function. New York: Raven Press, 1995:183-192. Hulett MD, McKenzie IF, Hogarth PM. Chimeric Fc receptors identify immunoglobulin-binding regions in human Fc~,RII and FceRI. Eur J Immunol. 1993;23:640-645. Kaplan AP, Horakova Z, Katz SI. Assessment of tissue fluid histamine levels in patients with urticaria. J Allergy Clin Immunol 1978;61:350-354. Kermani F, Niimi N, Francis DM, O'Donnell BF, Black AK, Hafizi S, Yacoub M, Greaves MW, Barr RM. Characterization of a novel mast cell-specific histamine releasing activity in chronic idiopathic urticaria (CIU). J Invest Dermatol 1995;105:452. Kern F, Lichtenstein LM. Defective histamine release in chronic urticaria. J Clin Invest 1976;57:1369-1377. Ktister H, Zhang L, Brini AT, MacGlashan DW, Kinet JP. The gene and cDNA for the human high affinity immunoglobulin E receptor 13 chain and expression of the complete human receptor. J Biol Chem 1992;267:12782--12787. Marone G, Casolaro V, Cirillo R, Stellato C, Genovese A. Pathophysiology of human basophils and mast cells in allergic disorders. Clin Immunol Immunopathol 1989a;50: $24-$40. Marone G, Casolaro V, Paganelli R, Quinti I. IgG anti-IgE from atopic dermatitis induces mediator release from basophils and mast cells. J Invest Dermatol 1989b;93:246-252. Maurer D, Fiebiger E, Reininger B, Wolff-Winiski B, Jouvin MH, Kilgus O, Kinet JP, Stingl G. Expression of functional high affinity immunoglobulin E receptors (FceRI) on monocytes of atopic individuals. J Exp Med 1994;179:745--750. Metzger H. The high affinity receptor for IgE in mast cells. Clin Exp Allergy 1991;21:269--279. Nawata Y, Koike T, Yanagisawa T, Iwamoto I, Itaya T, Yoshida S, Tomioka H. Anti-IgE autoantibody in patients with bronchial asthma. Clin Exp Immunol 1984;58:348--356.

O'Donnell BF, Barlow RJ, Black AK, Greaves MW. Response of severe chronic urticaria to intravenous immunoglobulin (IVIG). Br J Dermatol 1994;131:23. Ra C, Kuromitsu S, Hirose T, Yasuda S, Furuichi K, Okumura K. Soluble human high affinity receptor for IgE abrogates the IgE-mediated allergic reaction. Int Immunol 1993;5:47-54. Ravetch JV. Fc receptors: Rubor redux. Cell 1994;78:553--560. Riske F, Hakimi J, Mallamaci M, Griffin M, Pilson B, Tobkes N, Lin P, Danho W, Kochan J, Chizzonite R. High affinity human IgE receptor (FceRI): Analysis of functional domains of the (x-subunit with monoclonal antibodies. J Biol Chem 1991 ;266:11245-11251. Robertson MW. Phage and escherichia coli expression of the human high affinity immunoglobulin E receptor (x-subunit ectodomain. J Biol Chem 1993;268:12736--12743. Rorsman H. Basophilic leucopenia in different forms of urticaria. Acta Allergol 1962;17:168--184.

Smith CH, Atkinson B, Morris RW, Hayes N, Foreman JC, Lee TH. Cutaneous responses to vasoactive intestinal polypeptide in chronic idiopathic urticaria. Lancet 1992;339:91--93. Sutton BJ, Gould HJ. The human IgE network. Nature 1993; 366:421--428. Truong M-J, Gruart V, Kusnierz JP, Papin JP, Loiseau S, Capron A, Capron M. Human neutrophils express immunoglobulin E (IgE)-binding proteins (Mac-2/eBP) of the S-type lectin family: Role in IgE-dependent activation. J Exp Med 1993;177:243--248. Valent P, Bettelheim P. Cell surface structures on human basophils and mast cells: Biochemical and functional characterization. Adv Immunol 1992;52:333--423. Wang B, Rieger A, Kilgus O, Ochiai K, Maurer D, F6dinger D, Kinet JP, Stingl G. Epidermal Langerhans cells from normal human skin bind monomeric IgE via FceRI. J Exp Med 1992;175:1353--1365.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

INSULIN AUTOANTIBODIES Tamara S. Galloway, B.Sc., Ph.D. and Terence J. Wilkin, M.D.

University of Plymouth, Division of Medicine, Drake Circus, Plymouth, PL4 8AA, UK

HISTORICAL NOTES In 1965, insulin antibodies were considered "prima facie evidence of previous therapy with insulin" because there was no evidence for autoimmunity to endogenous insulin (Berson and Yalow, 1965). In 1967, studies of sera from nondiabetics and from diabetics before and after insulin treatment, using both porcine and human insulin ligands, revealed no insulin-binding antibodies before insulin treatment (Deckert, 1967). Although claims for the presence of insulin antibodies in the sera of insulin-naive patients were made during the early 1960s, the "no autoantibodies" view prevailed for several years. In 1970, a paper in Japanese described insulin autoimmunity in a patient with spontaneous hypoglycemia (Hirata et al., 1970) who had never been treated with insulin. The serum had significant insulinbinding activity in the globulin fraction and a large amount of immunoreactive insulin was extractable. Pancreas from partial pancreatectomy undertaken to exclude insulinoma showed instead diffuse beta-cell hyperplasia and no evidence of insulitis. After six further cases were described from Japan (Hirata et al., 1972), the association of hypoglycemia and insulin antibodies in the absence of insulin treatment became known as the "insulin autoimmune syndrome" (Hirata's disease). Since then, more than 200 cases have been reported world-wide, principally from Japan (Hirata and Uchigata, 1994); occasionally the islet histology shows diffuse hyperplasia and absence of insulitis. Normal or raised human C-peptide concentrations distinguish these patients from cases of factitious hyperinsulinemic hypoglycemia due to the clandestine injection of insulin. In 1983, insulin binding by serum globulin fractions was described in 18 of 112 children at diag-

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nosis of insulin-dependent diabetes mellitus (IDDM) (Palmer et al., 1983). Insulin binding (IgG) antibodies in the sera of patients with non-diabetes-related autoimmunity were reported a few months later (Wilkin and Nicholson, 1984). The discovery of a potential new marker for IDDM, with the promise of wide availability and ease of standardization, generated a great deal of interest in the immunochemistry and clinical implications of insulin autoantibodies (IAA). Insulin research has featured in many important scientific developments, and although a number of autoantibody markers is standardized, none has perhaps generated more information and basic understanding of autoantibody behavior than autoantibodies to insulin.

THE AUTOANTIGEN Definition

The insulin molecule is a 51-amino acid peptide comprising an A chain and B chain linked by two disulfide bonds. Human differs from porcine insulin by a single substitution of the B30 residue, and from bovine by a B30 substitution and two further substitutions at A8 and A10. The insulin molecule has no repeating structures. Origin Insulin is secreted by the [~-cell fraction of the islets of Langerhans from a precursor, proinsulin. Like many secretory products of endocrine glands, insulin is gland specific. None of the other antigens so far described in association with islet autoimmunity is totally tissue

specific. Many can, however, be linked to the insulinsecretory pathway of the 13 cell, including proinsulin, gangliosides of the Golgi apparatus and membrane components of the insulin granule membrane. Others are enzymes such as glutamic acid decarboxylase (GAD) and carboxypeptidase H. In this respect, the immune response in IDDM resembles certain other organ-specific autoimmune conditions such as thyroiditis, adrenalitis, gastritis and primary biliary cirrhosis, where the dominant (though not necessarily causative) antigens are enzymes. Insulin therefore occupies a unique position as both a central focus of the immune response in diabetes, and the only ~-cell-specific antigen so far identified. This adds to its important place in biomedical research as the first peptide hormone to be sequenced, the first structure to be defined by crystallography and the first licensed medication to be engineered genetically.

THE AUTOANTIBODIES Terminology IAA are defined as antibodies which bind insulin in insulin-naive patients, as distinct from the insulin antibodies (IA) found in the majority of patients treated with exogenous insulin. Although endogenous insulin is intrinsically a poor immunogen in humans, recombinant human insulin provokes a strong immune response, both cellular and humoral (de Beaufort et al., 1993). Immunogenicity is boosted by giving insulin in depot preparations containing zinc or protamine which decrease its solubility and allow aggregation or polymerization at the site of subcutaneous injection. The initial description of IAA in the insulin autoimmune syndrome made relatively little impact in the West where the syndrome is rare. In addition to IDDM, IAA are found after administration of thiolcontaining drugs such as methimazole and penicillamine, in patients with polyautoimmunity or with other organ-specific immune conditions.

Pathogenetic Role Human Disease. The pathogenetic role for IAA in the hypoglycemia of the insulin autoimmune syndrome, involves the dissociation of antibody-insulin complexes (Caillat-Zucman et al., 1992). In IDDM, the

major effectors of [3-cell destruction and resultant hyperglycemia are generally agreed to be T lymphocytes (Atkinson and Maclaren, 1994), although the finding of immunoglobulin and complement deposition within diabetic insulitis suggests some involvement of the humoral response (Bottazzo et al., 1985).

Animal Models. Of the two main animal models of IDDM, the nonobese diabetic (NOD) mouse, like humans, expresses IAA before clinical onset of diabetes, but whether IAA correlate with risk of diabetes development is disputed (Ziegler et al., 1989; Reddy et al., 1990). No systematic relationship is found in the bio-breeding (BB) rat, in which the presence of IAA is strain specific rather than insulitis specific (Diaz et al., 1991). Genetic Association Higher levels of IAA are significantly associated with the alleles HLADRB I*0406/DQA 1"0301/DQB 1"0302 in Japanese patients with the insulin autoimmune syndrome (Hirata and Uchigata, 1994). In Caucasians with IDDM, high IAA titers are associated with HLA-DR4 haplotypes, but DR4 does not account for all such cases (Ziegler et al., 1991). In first-degree relatives of patients with IDDM, two different subsets of DQA1 alleles were associated with phenotypic variation in IAA titer (Pugliese et al., 1994). Homozygosity for lineage 4 alleles was associated with low levels of IAA; whereas, lineage 1--3 DQA1 alleles (including DQAl*0301, implicated in the insulin autoimmune syndrome) are associated with high IAA levels. Perhaps the DQA1 sequences shared by the lineage 1--3 alleles facilitate the presentation of insulin to the immune system and this determines higher levels of autoantibodies once tolerance to insulin is broken during the prediabetic period (Pugliese et al., 1994).

Epitope Restriction Autoantibodies reactive only with human insulin were first described in 1984 in sera from patients with polyautoimmune serology (Wilkin and Nicholson, 1984). The restriction of IAA binding to particular variants of insulin is associated with IDDM (Wilkin et al., 1988a). This finding, confirmed and extended by others (Castano et al., 1993) shows that antibodies recognizing epitopes which incorporate threonine B30 (the residue that distinguishes human from porcine

431

insulin) are not associated with IDDM, whereas antibodies which recognize a conformational epitope spanning the A-chain residues A8--A13 and the Bchain residues B l--B3 appear to predict IDDM. Subtly different profiles of cross-reactivity were apparent when a panel of seven artificial human insulin analogues, in which the B30 threonine residue was substituted with residues of increasingly complex side chain (glycine, alanine, serine, threonine, valine, leucine, phenylalanine, tyrosine) in competitive binding experiments with several IAA sera specific for human (B30 threonine) insulin. The different profiles suggest that, although all human insulinspecific IAA recognize an epitope incorporating B30 threonine, they probably do not recognize identical idiotypes (Diaz, 1988). The exact relationship between dominant epitope and IDDM prediction is unknown. However, the heterogeneity of IAA binding, and the variable predictive values of different IAA idiotypes, have their parallel in islet cell antibodies (ICA). As with human insulin-specific IAA, the original observations in ICA serology were made in polyendocrine patients, rather than IDDM patients. Amid the heterogeneity of the immunofluorescence patterns of ICA staining, the "nonselective" staining of ~ and [3 cells of the islets is predictive of IDDM; whereas, "selective" ~-cell staining characteristic of polyendocrine sera is not (Yu et al., 1994). Epitope spreading during the maturation of GAD65 immunity has been described (Kaufman et al., 1993), but has yet to be investigated for insulin. The apparent diversity of IAA sera that cross-react with several insulin variants is difficult to assess. Cross-reaction could equally imply the recognition of a single epitope common to many insulins by a monoclonal serum as it could be binding of unshared epitopes by a polyclonal serum. Individual clones equally cross-reactive with human, porcine and bovine insulin that exist within the immune repertoire do not recognize isolated human B chain, and are presumed to react with an epitope of the A chain, such as that incorporating A4 glutamic acid (Storch et al., 1985; Mirza et al., 1987). Evidence for clonal restriction in cross-reactive IAA sera similar to that of human insulin-specific sera remains controversial, although a case report suggests that an IAA serum cross-reactive with human, porcine and bovine insulin was restricted to an epitope incorporating B3 asparagine (Uchigata et al., 1989). The antibody specificity of an immune response to insulin can be considerably narrower than the cor-

432

responding T-cell response, as demonstrated for the allo-immune response to insulin (Nell et al., 1985). Similarly, T cells were sensitized to a much broader range of insulin epitopes in a patient with apparently monoclonal, human-insulin-restricted insulin autoantibodies (Sklenar et al., 1987).

Molecular Mimicry In NOD mice, endogenous retroviruses including intracisternal type A particles (IAP) are expressed in the cells and IAA in these mice cross-react with retroviral protein p73 (the IAP gag gene product) (Serreze et al., 1988). Studies of cross-reactivity between insulin autoantibodies and insulin antibodies from insulin-naive and treated diabetics show that 65% of sera which bind insulin by ELISA also bind p73. The simultaneous appearance of antibody binding to p73 and to insulin in some individuals suggests that IAA and IA recognize an epitope shared between insulin and p73, consistent with endogenous retrovirus infection as a precipitating event in human IDDM (Hao et al., 1993).

Methods of Detection IAA can be measured either by radiobinding assay or by direct ELISA. The radiobinding assay employs a minimum quantity of high specific activity ligand and is thereby affinity sensitive, in contrast to the antigenexcess ELISA which is capacity sensitive. Many of the discrepancies evident in earlier reports of IAA associations are attributable to these differences in methodology. The theoretical implications of both methods were detailed in the five international workshops designed to standardize IAA measurement (Wilkin et al., 1987; 1988b; Palmer et al., 1990; Kuglin et al., 1990; Greenbaum et al., 1992). The clinically important conclusion was that liquid-phase (affinity-based) assays detect IAA of predictive value for IDDM more efficiently than ELISA. In direct comparisons, both systems detected IAA in nondiabetic sera, such as those from patients with polyautoimmune disease and insulin autoimmune syndrome. With both assays, a competition step is required to express displacement of insulin binding rather than merely raw insulin binding. Binding in liquid phase is maximal at 4~ but, although many centers operate a 24-hour incubation protocol, equilibrium between antibody and ligand at 4~ is not complete until 4--6

days. Radiobinding assays should always be read at equilibrium to optimize precision. The conjecture that distortion of the insulin molecule by monoiodination might increase the affinity of binding to IAA was demonstrated by experiments in which the relative affinities of autoantibodies by radiobinding assay and by ELISA were compared by displacement with native insulin and by 1251 monoA14 human-insulin. For some sera, iodination increases the affinity of the binding reaction by up to 1,000-fold. The artefact, however, does not apply to every serum (Stumpo et al., 1994).

CLINICAL UTILITY IAA are sometimes considered of little value because they are seldom found in more than 40% of new onset diabetics. Importantly, however, the titer and, by implication, frequencies of IAA vary inversely with age (Vardi et al., 1988). Although found at onset of diabetes in only 4% of adults, IAA are almost universally present in children less than four years old at onset. The prevalence of ICA, on the other hand, is much the same in both groups. The explanation may lie in the genetic differences in IDDM of different ages at onset (Caillat-Zucman et al., 1992). Although the titers of IAA may be a marker for time-to-onset of clinical disease in prediabetics (Jackson et al., 1988), it is not clear whether IAA titers predict the duration to diabetes at all ages, or whether IAA titre and duration of the prodrome are independent functions of age. The clinical utility of IAA also reflects their increased predictive values in combination with other autoantibodies. Indeed, prediction of IDDM improves dramatically according to the number of islet-specific autoantibodies present (Bingley et al., 1994). Based on the observation of 101 family members for up to 14 years, the risk for IDDM was 8% if ICA alone was present, rising to 88% if three or more islet-specific autoantibodies, including IAA, were present. The increased risk associated with multiple autoantibodies is independent of age. In a prospective study of 25 nondiabetic, identical twins of patients with IDDM, a combination of cellular and humoral immune abnormalities and their tendency to persist was highly predictive of IDDM (Tun et al., 1994). Many other reports have evaluated combinations of immune, genetic and metabolic markers for their predictive ability, both in high-risk

groups and in the general population (Hagopian et al., 1995; Reijonen et al., 1994); autoantibodies are virtually never associated with diabetes. There is a strong correlation between insulin binding in the cord blood of neonates and binding in the circulation of their insulin-treated diabetic mothers (Ziegler et al., 1991). Of the neonates, 74% were IAA-positive, presumably due to the transplacental passage of alloantibodies (IA). During follow-up, the majority of infants lost their insulin-binding, leaving only 3.3% positive by nine months. However, by two years of age the figure had doubled again to 7.7%, a rise attributed to the de novo synthesis of autoantibodies (IAA) as a possible predictor of future diabetes (Ziegler et al., 1991). The mean insulin binding in samples of children born to nondiabetic mothers was higher at birth than later in childhood. The Early Bird study, which is a community-based investigation of immune response to islet cell antigens in consecutive births, shows that cord blood in births to nondiabetic mothers has two (rather than one) discreet distributions of insulin binding. The first is similar to the background binding seen in other children and in adults (<2%); the second, accounting for some 7% of consecutive cord samples, shows insulin displaceable binding at levels between 2 and 37%. None of the mothers was diabetic, and none was exposed to insulin. The study will monitor the progress of these children and their controls throughout childhood. These IAA are largely IgG, highly insulin specific, poorly displaceable with proinsulin and IgF-1 and not associated with other disease-linked autoantibodies (Galloway et al., 1995).

CONCLUSION Insulin is the only autoimmune antigen unique to the islets, although insulin autoantibodies occur in conditions other than diabetes. IAA are simple to measure, but have gained limited popularity because of their relatively low prevalence overall in IDDM. However, they may have high predictive value in very young onset diabetes. Overall, IAA add more to IDDM prediction as one of a combination of autoantibodies than they do alone. Refinements in IAA measurement, and possibly in their predictive value, might come with further study of epitope restriction and its maturation. See also GLUTAMICACID DECARBOXYLASE AUTOANTIBODIESIN DIABETES MELLITUS and ISLET CELL AUTOANTIBODIES.

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REFERENCES Atkinson MA, Maclaren NK. The pathogenesis of insulindependent diabetes mellitus. N Engl J Med 1994;331:1428-1435. Berson SA, Yalow RS. Some current controversies in diabetes research. Diabetes 1965;14:459-472. Bingley PJ, Christie MR, Bonifacio E, Bonfanti R, Shattock M, Fonte MT, Bottazo GF, Gale EA. Combined analysis of autoantibodies improves prediction of IDDM in islet cell antibody-positive relatives. Diabetes 1994;43:1304-1310. Bottazzo GF, Dean BM, McNally JM, Mackay EH, Swift PGF, Gamble DR. In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis. N Engl J Med 1985;313:353-360. Caillat-Zucman S, Garchan HJ, Timsit J, Assan R, Boitard C, Djilali Saiah I, Bougneies P, Bach J-F. Age-dependent HLA genetic heterogeneity of type I insulin-dependent diabetes mellitus. J Clin Invest 1992;90:2242-2250. Castano L, Ziegler AG, Ziegler R, Shoelson S, Eisenbarth GS. Characterisation of insulin autoantibodies in relatives of patients with type I diabetes. Diabetes 1993;42:1202-1209. de Beaufort CE, Sodoyez JC, Koch M, Bruining GF, SodoyezGoffaux F. Insulin autoantibodies and immune response to human insulin therapy in 24 type I (insulin-dependent) diabetic children: superiority of radiobinding assay over solid phase assay. Diabetes Res Clin Pract 1993;21:19-24. Deckert T. Autoimmunological aspects of diabetes mellitus. Acta Med Scand (Suppl) 1967;476:29-32. Diaz JL. The autoimmune response to insulin [Thesis]. University of Southampton, Southampton, UK, 1988. Diaz JL, Daneman D, Martin JM, Sochett E, Wilkin TJ. The relationship between insulin autoantibodies and islet cell histology in the diabetes prone BB rat. Autoimmunity 1991;11:45-51. Galloway TS, Millirard B A, Crocker D, Noor M, Wilken TJ. Autoimmune insulitis may start before birth; the earlybird project. Diabetologia 1995;38(Suppl)A90. Greenbaum CJ, Wilkin TJ, Palmer JP. Fifth International Serum Exchange Workshop for Insulin Autoantibody (IAA) Standardization. Diabetologia 1992;35:798--800. Hagopian WA, Sanjeevi CB, Kockum I, Landin-Olsson M, Karlsen AE, Sundkvist G, Dahlquist G, Palmer J, Lernmark A. Glutamate decarboxylase-, insulin-, and islet cell-antibodies and HLA typing to detect diabetes in a general population-based study of Swedish children. J Clin Invest 1995 ;95:1505-1511. Hao W, Serreze DV, McCulloch DK, Neifing JL, Palmer JP. Insulin autoantibodies from human IDDM cross-react with retroviral antigen p73. J Autoimmun 1993;6:787-798. Hirata Y, Ishizu H, Ouchi N, et al. Insulin autoimmunity in a case of spontaneous hypoglycaemia. Jpn Diabetes Soc 1970; 13:312-320. Hirata Y, Uchigata Y. Insulin autoimmune syndrome in Japan. Diabetes Res Clin Pract 1994; 24(Suppl):153-157. Hirata Y, Nishima H, Tominaga N, Arimichi T, Kogushi T. On insulin autoimmune syndrome. Jpn Diabetes Soc 1972; 15(Suppl); 179-183.

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Jackson R, Vardi P, Herskowitz R, Soeldner JS, Eisenbarth GS. Dual parameter linear model for prediction of onset of type I diabetes in islet cell antibody positive relatives. Clin Res 1988;36:585A. Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GS, Robinson P, Atkinson MA, Sercarz EE, Tobin AJ, Lehmann PV. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993;366:69-72. Kuglin B, Kolb H, Greenbaum C, MacLaren NK, Lernmark ~, Palmer JP. The Fourth International Workshop on the Standardisation of Insulin Autoantibody Workshop. Diabetologia 1990;33:638--639. Mirza IH, Wilkin TJ, Cantarini M, Moore K. A comparison of spleen and lymphnode cells as fusion partners for the raising of monoclonal antibodies after different routes of immunisation. J Immunol Methods 1987;105:235-243. Nell LJ, Virta VJ, Thomas JW. Recognition of human insulin in vitro by T cells from subjects treated with animal insulins. J Clin Invest 1985;76:2070-2077. Palmer JP, Asplin CM, Clemons P, Lyen K, Tatpati O, Raghu PK, Paquette TL. Insulin antibodies in insulin dependent diabetics before insulin treatment. Science 1983;222:13371339. Palmer JP, Wilkin TJ, Kurtz AB, Bonifacio E. The Third International Workshop on the Standardisation of Insulin Autoantibody Measurement. Diabetologia 1990;33:60-61. Pugliese A, Bugawan T, Moromisato R, Awdeh ZL, Alper CA, Jackson RA, Erlich HA, Eisenbarth GS. Two subsets of HLA-DQA1 alleles mark phenotypic variation in levels of insulin autoantibodies in first degree relatives at risk for insulin-dependent diabetes. J Clin Invest 1994 ;93:2447-2452. Reddy S, Bibby N, Elliot RB. Longitudinal study of islet cell antibodies and insulin autoantibodies and development of diabetes in nonobese diabetic (NOD) mice. Clin Exp Immunol 1990;81:400-405. Reijonen H, Vahasalo P, Karjalainen J, Ilonen J, Akerblom HK, Knip M. HLA-DQB 1 genotypes and islet cell antibodies in the identification of siblings at risk for insulin-dependent diabetes (IDDM) in Finland. Childhood Diabetes in Finland (DiMe) Study Group. J Autoimmun 1994;7:675--686. Serreze DV, Leiter EH, Kuff EL, Jardieu P, Ishizaka K. Molecular mimicry between insulin and retroviral antigen P73. Development of cross-reactive autoantibodies in sera of NOD and C57 BL/KsJ db/db mice. Diabetes 1988;37:351358. Sklenar I, Wilkin TJ, Diaz JL, Erb P, Keller U. Spontaneous hypoglycaemia associated with autoimmunity specific to human insulin. Diabetes Care 1987;10:152-159. Storch MJ, Peterson KG, Licht T, Kerp L. Recognition of human insulin and proinsulin by monoclonal antibodies. Diabetes 1985;34:808-811. Stumpo RR, Ilera AS, Cardosa AL, Poskus E. Solid versus liquid phase assays in detection of insulin antibodies. Influence of iodination site on labelled insulin binding. J Immunol Methods 1994;169:241--249. Tun RY, Peakman M, Alviggi L, Hussain MJ, Lo SS, Shattock M, Pyke DA, Bottazzo GF, Vergani D, Leslie RD. Impor-

tance of persistent cellular and humoral changes before diabetes develops: prospective study of identical twins. Br Med J 1994;308:1063-1068. Uchigata Y, Yao K, Takayama-Hasumi S, Hirata Y. Human monoclonal IgG1 insulin autoantibody from insulin autoimmune syndrome directed at determinant at asparagine site on the insulin B chain. Diabetes 1989;38:663--666. Vardi P, Ziegler AG, Mathews JH, Dib S, keller RJ, Ricker AT, Wolfsdorf JI, Herskowitz RD, Rabizadeh A, Eisenbarth GS, et al. Concentration of insulin autoantibodies at onset of type I diabetes. Inverse log-linear correlation with age. Diabetes Care 1988;11:736--739. Wilkin T, Palmer J, Kurtz A, Bonifacio E, Diaz JL. The Second International Workshop on the Standardisation of Insulin Autoantibody (IAA) Measurement. Diabetologia 1988a;31: 449-450. Wilkin TJ, Mirza I, Armitage M, Casey C, Scott-Morgan L. Insulin autoantibody polymorphisms with greater discrimination for diabetes in humans. Diabetologia 1988b;31:670--674.

Wilkin TJ, Palmer J, Bonifacio E, Diaz JL, Kruse V. First International Workshop on the Standardisation of Insulin Autoantibodies. Diabetologia 1987;30:676--677. Wilkin TJ, Nicholson S. Autoantibodies against human insulin. Br Med J 1984;288:349-352. Yu L, Gianani R, Eisenbarth GS. Quantitation of glutamic acid decarboxylase autoantibody levels in prospectively evaluated relatives of patients with type I diabetes. Diabetes 1994;43: 1229--1233. Ziegler AG, Vardi P, Ricker AT, Hattori M, Soeldner JS, Eisenbarth GS. Radioassay determination of insulin autoantibodies in NOD mice. Correlation with increased risk of progression to overt diabetes. Diabetes 1989;38:358--363. Ziegler R, Alper CA, Ardeh ZL, Castano L, Brink SJ, Soeldner JS, Jackson RA, EisenbarthGS. Specific association of HLADR4 with increased prevalence and level of insulin autoantibodies in first degree relatives of patients with type I diabetes A. Diabetes 1991;40:709--714.

435

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

INTERFERON-INDUCIBLE PROTEIN IFI 16 AUTOANTIBODIES Hans Peter Seelig, M.D. and Manfred Renz, Ph.D.

Institute of Immunology and Molecular Genetics, D-76133 Karlsruhe, Germany

HISTORICAL NOTES

Autoantibodies against interferon (IFN) inducible protein IFI 16 (anti-IFI 16), which constitute a new antibody specificity of antinuclear antibodies (ANA), occur in about one-third of SLE patients (Seelig et al., 1994). The IFI 16 encoding gene Ifi16 (Trapani et al., 1992) exhibits structural similarities with a family of highly homologous IFN-inducible genes first described on the mouse chromosome 1 as Ifi201 - Ifi204 gene cluster (Samanta et al., 1986; Engel et al., 1985; 1988; Kingsmore et al., 1989; Choubey et al., 1989; Opdenakker et al., 1989). Another human homologue of this gene family encodes the myeloid nuclear differentiation antigen (MNDA) (Burrus et al., 1992; Briggs et al., 1992). Ifi16 transcription and concomitant IFI 16 translation can be induced by IFNy in myeloid cells, but lymphoid cells (Trapani et al., 1992) as well as HeLa and HEp-2 cells (Seelig et al., 1994) express the protein constitutively. The autoantigenic nature of IFI 16 was detected by immunoscreening of a HeLa cell complementary DNA expression library with the ANA-positive IgG fraction from a female patient with rheumatic complaints. In these experiments a cDNA corresponding to Ifi16 coding regions was isolated and the nuclear localization of the encoded protein was demonstrated (Seelig et al., 1994).

THE AUTOANTIGEN Definition/Nomenclature

lfi16 is localized on human chromosome lq21-32 (Trapani et al., 1992, Dawson et al., 1995). The genomic organization spans a segment of at least 28 kilobases with 10 exons and nine intervening introns

436

(Trapani et al., 1994) (Figure 1). The protein is composed of 729 amino acids with a calculated molecular weight of (82,114 D). However, native IFI 16 shows an apparent molecular weight within the range of 95--100 kd as demonstrated with SDS-PAGE separated and immunoblotted nuclear proteins probed with monospecific rabbit anti-IFI 16. Identical results were obtained with immunoprecipitated, 35S-methionine-labeled HEp-2 cell proteins (Seelig et al., 1994). Therefore, additional posttranslational modifications of the protein have to be considered. An immunoreactive, full-length, recombinant IFI 16 MS2-polymerase fusion protein was used for antibody production in rabbits. Sources

IFI 16 localizes within the nucleolus and the nucleoplasma of human cells by indirect immunofluorescence microscopy (IIF) and by immunoblotting of nuclear proteins using human or rabbit anti-IFI 16 which was made monospecific by use of the recombinant antigen. The mode of expression of IFI 16 is cell specific. In myeloid cells such as HL-60, nuclear expression can be induced by IFNy; whereas, lymphoid cells (Trapani et al., 1992) as well as HeLa and HEp-2 cells constitutively express the protein. Upon addition of IFN~, to HeLa and HEp-2 cells, the constitutive expression is augmented considerably. In nonstimulated HEp-2 cells, most of IFI 16 is within the nucleolus but nucleoplasmic fluorescence increases especially with IFN~, treatment (Figure 2). Various other human cells such as endothelia, pancreas acinus epithelia, keratinocytes, fibroblasts, epithelia of adrenal cortex and others also constitutively express IFI 16 in their nuclei (unpublished data). Although suggested to be associated somehow with

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9

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E3

i

E5 1.7 , , , \ \ \ \ \ \ \ ~

~,

E4

E5

i

nucleotides 1 ' amino acids

1"

E6

E7

i

500

' 300

E8

E9

i

1000

' 200

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3.5

2000

' 500

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1500

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I SedThr/Pro I

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9v

9

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vT

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v

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= helix predicted regions = amino acid subs~tutions = SPXX TPXX

Figure 1. The top of the figure showsthe intron-exon organization of Ifi16 (according to Trapani et al., 1994), exons (El, E2, etc.) are indicated as striped boxes and the length of introns is given in kilobases. The middle part shows aligned exons, nucleotides and amino acids. The lower part reveals a schematic representation of the IFI 16 protein deduced from HeLa cell complementary DNA. The main features of the 729 amino acids (aa) containing protein are characterized by two regions, A (aa 207--369) and B (aa 524--684), which constitute imperfect repeats with an overall homology of 44%. There are 13-sheetspredicted in A and B. The region localized between the large repeats (aa 380--520) shows a high content of serine, threonine and proline; a helix is predicted in the proline-poor middle part (aa 448-462) which is flanked by two smaller repeats, C (aa 415-442) and C' (aa 471-498) that form loops. For the lysine-rich N-terminal part both a helical structure and DNA binding can be predicted. This region is followed by a long unstructured region (aa 100--195) which is predicted to form loops. Triangles show additional possible DNA-binding motifs (TPXX and/or SPXX). However, these motifs are embedded in regions of high S+T or S+T+P content which may impede DNA binding. Solid circles show aa substitutions compared to the IFI 16 protein derived from the T-cell complementary DNA used by Trapani et al. (1992).

human myeloid cell differentiation (Trapani et al., 1992; 1994), biological function of IFI 16 in other cells is unknown. IFI 16 contains Ser(Thr)Pro xx motifs that are thought to occur in proteins involved in gene regulation and to contribute to their DNA binding (Suzuki, 1989). The lysine-rich N-terminus is also a potential site for DNA binding. The nucleolar and nucleoplasmic localization of IFI 16 suggests a primary role in the regulation of transcription events or in the processing of peribosomal or premessenger RNA.

product of gene lfll 6. P16 or 16P may be misleading because the molecular weight of IFI 16 is within the range of 90 to 100 kd. Anti-IFI 16 antibodies can be detected by immunoblot only in those human sera which manifest ANA by IIF with an appropriate IFI 16-expressing human substrate (e.g., HEp-2 cells). Antibody titers range up to 1:100,000 in some patients. The prevalent antibody isotype is IgG anti-IFI 16; concomitant IgM anti-IFI 16 is seen in some sera.

Pathogenetic Role THE AUTOANTIBODIES Terminology The previously used synonyms 16P, protein 16P (Trapani et al., 1992) or (IFNT-inducible protein) P16 (Seelig et al., 1994) should be replaced by IFI 16 which characterizes the protein more precisely as

Anti-IFI 16 was found in 29% of well-documented SLE (n = 374) patients and in a lower frequency (p < 0.02) in patients without anti-dsDNA antibodies (23.8%) than in those with anti-dsDNA (35%). No significant correlation was seen between the titer of anti-IFI 16 and the presence or absence of antidsDNA nor between the presence of anti-IFI 16 and anti-RNP, anti-Sm, anti-SSA (Ro) or anti-SSB (La) in

437

Figure 2. Reaction of HEp-2 cells with anti-IFI 16. Antibodies were produced against recombinant IFI 16 and affinity-purified with the recombinant protein. A: HEp-2 cells grown under normal conditions. B: HEp-2 cells treated with IFNy. Expression of IFI 16 is considerably enhanced in IFNT-treated cells and the nucleolar staining is concealed by an intensified nucleoplasmic fluorescence. these patients (Table 1). Further studies are needed to clarify whether anti-IFI 16-positive SLE patients comprise a distinctive subgroup with regard to genetic background, clinical symptoms or response to therapy. No significant associations of anti-IFI 16 with nephritis or sicca symptoms are observed. Anti-IFI 16 occur in diseases other than SLE, including 3.8% of patients with scleroderma/polymyositis (n = 26) but not in rheumatoid arthritis (n = 30). Antibodies were also found in 28% of sera (n = 14) harboring anticentromere antibodies, indicative of a diagnosis of CREST syndrome (Calcinosis cutis, Raynaud's phenomena, Esophageal dismotility, Sclerodactyly, Telangiectasia). Anti-IFI 16 seen in some ANA-positive patients with concomitant antimitochondrial antibodies of anti-M2 specificity suggest a diagnosis of primary biliary cirrhosis. Some IFI 16positive, ANA-positive patients harbor antithyroglobulin and/or antithyroid peroxidase antibodies suggestive of autoimmune thyroiditis. Low titer anti-IFI 16 (1:200 -- 1:400) can be seen in persons with positive ANA tests without significant clinical symptoms.

Detection Methods For detection of anti-IFI 16 in human sera, recombinant IFI 16 can be used in immunoblots or ELISA. Native protein can be isolated from nuclear extracts of cells which constitutively express the antigen. The

438

extent of expression can be pretested by means of monospecific anti-IFI 16 sera. At present, no information is available concerning the immunoreactivity of native IFI 16 with sera from patients of various disease groups. Determination of anti-IFI 16 is indicated only in those patients presenting positive ANA. It is notable that some substrates used for routine ANA testing (other than HEp2) do not express IFI 16 and sera monospecific for anti-IFI 16 can therefore erroneously be considered as negative.

CLINICAL UTILITY Whether anti-IFI 16 is a useful additional marker of SLE can be answered only when other well-defined, ANA-positive disease groups are evaluated. At present, considerations about the diagnostic importance of anti-IFI 16 are premature. In human cells of myeloid and lymphoid lineage as well as in endothelia, which can all be involved in inflammatory reactions, the expression of IFI 16 can be induced or increased by IFN~, which itself is an important mediator of a variety of immunoregulatory effects (Schattner, 1994) such as activation of macrophages, monocytes and B cells, expression of MHC class II molecules or highaffinity IgG receptors. Therefore, possible linkages between an ongoing stimulation of IFNy in chronic inflammatory processes and an overproduction of a

Table 1. Coexistence of Anti-IFI 16 With Other Autoantibodies in 374 Patients with Systemic Lupus Erythematosus Antibody Specificity*

No. of Sera

No. (%) anti-IFI 16-positive

ANA (IIF)

374

107 (28.7)

dsDNA-negative

214

51 (23.8)?

dsDNA-positive

160

56 (35.0)?

RNP (70K, A, C)

105

28 (26.6)

RNP (anti-dsDNA-negative)

34

8 (23.5)

70K

12

1 (8.3)

A

22

4 (18.2)

C

ll

1 (9.1)

70K + A

3

1

70K + C

2

1

70K + A + C

28

11 (39.2)

Sm (D)

38

12 (31.5)

Ro/La

117

32 (28.2)

Ro 52 kd

79

20 (25.3)

Ro 60 kd

88

26 (29.5)

Ro

104

26 (25.0)

La

55

19 (34.5)

ANA = antinuclear antibodies (by indirect immunofluorescence (IIF)); anti-dsDNA = anti-double-stranded DNA. *Anti-RNP includes at least one of the specificities 70K, A or C. ?p < 0.02. Median of titer 1:3,500 for anti-dsDNA-negative and 1"3,000 for anti-dsDNA-positive.

possibly altered antigen triggering the formation of these autoantibodies should be considered. Whether mouse strains prone to develop autoimmune diseases may also develop antibodies to gene products of their Ifi201-204 gene cluster is as yet unknown.

CONCLUSION Antibodies to human-specific, IFNy inducible nuclear protein (IFI 16) are found in up to 35% of SLE

REFERENCES Briggs JA, Burrus GR, Stickney BD, Briggs RC. Cloning and expression of the human myeloid cell nuclear differentiation antigen: regulation by interferon alpha. J Cell Biochem 1992;49:82--92. Burrus GR, Briggs JA, Briggs RC. Characterization of the human myeloid cell nuclear differentiation antigen: relation-

patients. Clinical and immunopathological significance of anti-IFI 16 in these patients must be evaluated in more detail as must the possible associations of antiIFI 16 with other diseases by testing other groups of clinically well-characterized patients. The IFI 16 antigen/antibody system is of special interest in autoimmunity because the expression of IFI 16 in cells involved in inflammatory reactions can be induced or increased by IFNy, an important mediator of i m m u n e response.

ship to interferon-inducible proteins. J Cell Biochem 1992; 48:190--202. Choubey D, Snoddy J, Chaturvedi V, Toniato E, Opdenakker G, Thakur A, Samanta H, Engel DA, Lengyel P. Interferons as gene activators. Indications for repeated gene duplication during the evolution of a cluster of interferon-activatable genes on murine chromosome 1. J Biol Chem 1989;264: 17182-17189.

439

Dawson MJ, Trapani JA, Briggs RC, Nicholl JK, Sutherland GR, Baker E. The closely linked genes encoding the myeloid nuclear differentiation antigen (MNDA) and IFI 16 exhibit contrasting haemopoietic expression. Immunogenetics 1995;41:40-43. Engel DA, Samanta H, Brawner ME, Lengyel P. Interferon action: transcriptional control of a gene specifying a 56000Da protein in Ehrlich ascites tumor cells. Virology 1985; 142:389-397. Engel DA, Snoddy J, Toniato E, Lengyel P. Interferons as gene activators: close linkage of two interferon-activatable murine genes. Virology 1988;166:24-29. Kingsmore SF, Snoddy J, Choubey D, Lengyel P, Seldin MF. Physical mapping of a family of interferon-activated genes, serum amyloid P-component, and alpha-spectrin on mouse chromosome 1. Immunogenetics 1989;30:169-174. Opdenakker G, Snoddy J, Choubey D, Toniato E, Pravtcheva DD, Seldin MF, Ruddle FH, Lengyel P. Interferons as gene activators: a cluster of six interferon-activatable genes is linked to the erythroid alpha-spectrin locus on murine chromosome 1. Virology 1989; 171:568--578.

440

Samanta H, Engel DA, Chao HM, Thakur A, Garcia-Blanco MA, Lengyel P. Interferon as gene activators. Cloning of the 5' terminus and the control segment of an interferon activated gene. J Biol Chem 1986;261:11849--11858. Schattner A. Short analytical review. Lymphokines in autoimmunity- a critical review. Clin Immunol Immunopathol 1994;70:177--189. Seelig HP, Ehrfeld H, Renz M. Interferon-y-inducible protein p16. Arthritis Rheum 1994;37:1672--1683. Suzuki M. SPXX, a frequent sequence motif in gene regulatory proteins. J Mol Biol 1989;207:61--84. Trapani JA, Browne KA, Dawson MJ, Ramsay RG, Eddy RL, Shows TB, White PC, Dupont B. A novel gene constitutively expressed in human lymphoid cells is inducible with interferon-gamma in myeloid cells. Immunogenetics 1992;36: 369-376. Trapani JA, Dawson M, Apostolidis VA, Browne KA. Genomic organization of IFI 16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization. Immunogenetics 1994;40:415-424.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

ISLET CELL A U T O A N T I B O D I E S William A. Hagopian, M.D., Ph.D. and Ake Lemmark, Ph. D.

University of Washington, R.H. Williams Laboratory, Department of Medicine, Seattle, WA 98195-7110, USA

varies from donor to donor (Landin-Olsson, 1990) which hampers progress in assay standardization. Ever since the first demonstration that IDDM sera immunoprecipitate specific islet proteins (Baekkeskov et al., 1982) major efforts have been made to identify the autoantigens which explain the ICA reaction. In 1987, attempts were made to determine the presence of islet cell antibodies which would bind to surface antigens rather than to intracellular antigens of insulinproducing cells (Lemmark et al., 1987). The initial study on ICA was followed by the demonstration that IDDM sera also contain autoantibodies which bind to the plasma membrane of normal insulin-producing 13 cells (Lemmark et al., 1978). Such islet cell surface antibodies (ICSA) which bind to yet unidentified surface antigens, were also found to mediate complement-dependent cytotoxicity (Dobersen et al., 1980) and to inhibit glucose-stimulated insulin release (Kanatsuna et al., 1983; Ziegler and Lemmark, 1982). Islet cell surface antibodies can also mediate antibody-dependent cellular cytotoxicity (ADCC) (Charles et al., 1983). That IDDM IgG can interfere with the initial uptake of glucose in the pancreatic islet 13 cells (Johnson et al., 1990) might

HISTORICAL NOTES Islet cell antibodies (ICA) detected by indirect immunofluorescence on frozen sections of human blood group O pancreas were described in 1974 (Bottazzo et al., 1974; MacCuish et al., 1974) using sera from patients with insulin-dependent diabetes mellitus (IDDM) in addition to another organ-specific autoimmune disease (Table 1). The long sought after ICA were eventually demonstrated because of improved epiflourescence microscopes and methodological investigations of suitable human pancreas. In a comprehensive investigation, the high diagnostic sensitivity and specificity of ICA for IDDM were subsequently demonstrated. These studies were confirmed by numerous investigations, but it was not until 1985 that the first international workshop demonstrated poor inter-laboratory concordance (Gleichmann and Bottazzo, 1987) which led to development of a world standard for ICA (Juvenile Diabetes Foundation (JDF) Standard) (Bonifacio et al., 1988). The JDF standard is now widely used to standardize ICA assays throughout the world (Greenbaum et al., 1992b). However, the quality of human pancreas Table 1. Islet Cell Antibody Assays Antibody

Test

Controls

New I D D M

Reference

ICA

Indirect immunofluorescence on frozen human pancreas sections

2--4%

75--86%

Greenbaum et al., 1994

CF-ICA

Indirect immunofluorescence for complement factor

2--4%

65--70%

Bottazzo et al., 1980

ICSA

Indirect immunofluorescence on dispersed islet cells

1--3%

30-70%

Lernmark et al., 1978

C'AMC

Complement-dependent antibody-mediated cytotoxicity on dispersed islet cells

1-3%

30--70%

Dobersen et al., 1980

441

reflect a direct interaction between IDDM immunoglobulin and GLUT-2, the [3-cell glucose-transporter protein (Inman et al., 1993). Autoantibodies to insulin (IAA) in non-insulintreated IDDM patients were reported in 1989 (Palmer et al., 1982). The IAA assay using labeled human or porcine insulin was subsequently standardized in several international workshops (Greenbaum et al., 1992b). A major outcome of the workshops was the demonstration that a reliable and reproducible diagnostic sensitivity and specificity of IAA for IDDM is observed in radiobinding assays; whereas, solid-phase assays such as ELISA failed (Greenbaum et al., 1992a). After the demonstration that the 64K antigen (Baekkeskov et al., 1982) had glutamate decarboxylase activity (Baekkeskov et al., 1990), several GAD preparations including brain extracts were tested until human islet GAD was cloned and shown to represent GAD65, a novel isoform of GAD (Karlsen et al., 1991). Because IDDM sera detect conformational GAD65 epitopes (Baekkeskov et al., 1990), recombinant human islet GAD65 is now used in radiobinding assays (Grubin et al., 1994; Petersen et al., 1994; Hagopian et al., 1993). The First GAD Antibodies Workshop demonstrated that GAD65 antibodies have high diagnostic sensitivity and specificity for IDDM using radiobinding but not solid-phase ELISA assays (Schmidli et al., 1994). This brief historical background illustrates that understanding of islet cell antibodies has improved from their first detection in a relatively crude indirect immunofluorescence test without molecular resolution to subsequent identifications of individual autoantigens which contribute to but do not yet fully explain the ICA reaction.

AUTOANTIGENS

Identification of the individual target antigens is needed to refine the predictive value of islet cell autoantibody tests, to elucidate specific T-cell and HLA mechanisms of ~-cell-directed autoreactivity and to allow more reasoned and specific interruption of the autoimmune process. Use of patient blood as a source of disease-related antibodies or T cells, together with isolated islets or }-cell-derived cell lines has allowed a large number of possible diabetes-related antigens to be described. The few relevant antigens sequenced at the DNA level illustrate the rapid progress that can be made once

442

recombinant antigen is available (see Table 2, sections B and C). An important criterion for a potential antigen is its tissue distribution. Autoimmunity in IDDM is directed exclusively to the ~ cell, but diabetes-related antigens are often expressed in other tissues. Further, ICA reactivity is clearly directed to all islet cells and not just the ~ cell. A useful way to classify tissue distribution is by the hierarchy: (1) [3cell-specific, (2) islet-specific, (3) neuroendocrine specific, or (4) more widely distributed. Many investigators target cell-surface antigens in their search, reasoning that these are more accessible to the immune system and thus more involved in the pathogenesis. However, in IDDM and in a number of other cell-mediated autoimmune diseases, sequestered intracellular molecules are also major antigens. For example, the intracellular enzyme thyroid peroxidase is a major antigen in Hashimoto's disease. Similarly, the intracellular enzyme glutamic acid decarboxylase (GAD) is a major IDDM antigen with demonstrable autoantibodies and T-cell reactivity in rodent models and in man. Many insulin secretory granule antigens are intracellular, but are expressed on the [3-cell exterior due to exocytosis of the insulin secretory granule. Examples include carboxypeptidase H as well as insulin itself. However, GAD may not be expressed on the cell surface (Vives-Pi et al., 1993; AguilarDiosdado et al., 1994). Similarly, the portion of the cell surface protein antigen ICA512 (IA2) antigen with greatest autoantibody reactivity is the intracellular portion (Passini et al., 1995). Attempts to define the antigen responsible for the primary autoimmune response.in IDDM are difficult due to the long prodrome and limited years of prospective follow-up possible in family studies, because a sibling's greatest risk of developing IDDM is within the first 5 years after diagnosis of the proband. It is clear that GAD and insulin can each be the earliest autoantibody reactivity detected. On the other hand, autoantibodies to ICA512 and to the 37K tryptic fragment (Christie et al., 1992) occur or at least increase in titer within 2 years before disease onset. This illustrates the concept of intermolecular antigen spreading in which additional islet cell-specific antigens are targets of the immune response later in the pathogenesis. This is distinct but likely related to the intramolecular spreading of T-cell epitopes (Lehmann et al., 1992; 1993). Most studies of prediabetes do not include the initial pathogenetic period. When seroconversion to ICA positivity occurs, the role of specific antigens in the primary response cannot be

Table 2. Some Diabetes-related Antigens Antigen

Tissue Dist.

Assay Type

Reference

Controls

New IDDM

n

%

n

%

A. ICA

islet

IF

many

1212

4

500

83

B. IAA

[3

IMP/RIA

Palmer, 1987

>500

1

500

46--70

GAD65ab

NE

IMP/RIA

Baekkesov et al., 1990

>500

1-4

>500

76-82

ICA512 (IA2)

NE

RIA or WB

Rabin et al., 1994

80

2

(preDM)35 74

37K/41K

NE

IMP/PAGE

Christie, 1992

200

0

80

71

GAD67ab

N

IMP/RIA

Hagopian et al., 1995

>100

4

190

10--20

9

IMP/PAGE

Baekkesov et al., 1993

86

0

130

17

155K

9

RIN cell

Thomas et al., 1990

1800

5

>500

92

CPH

NE

WB

Castano et al., 1991

20

0

(preDM)25 64

p69W

WB

Pietropaolo et al., 1993

72

1

(preDM)51 57

p52?

WB

Karounos et al., 1993

154

5

105

58

HSP62

W

WB

Jones et al., 1990

11

23

13

92

gangliosides

NE

TLC

Dotta et al., 1993

25

4

(preDM)21 62

sulfatides

NE

TLC

Buschard et al., 1991

135

0

57

C. 38K

-

--

-

88

Abbreviations for tissue distribution include: 13= beta-cell-specific; NE = neuroendocrine; N = neural; W = widely distributed; 9 = unknown. Abbreviations for assay type include: IMP = immunoprecipitation; RIA = radioimmunoassay, IB, immunoblotting; PAGE = polyacrylamide gel electrophoresis; TLC = thin layer chromatography; RIN cell = rat insulinoma cell surface assay. PreDM denotes assays on serum from prediabetic subjects.

evaluated. Further, because IDDM appears to be a heterogeneous disorder with multiple distinct HLA genotypes contributing to susceptibility, different antigens might be primary in different individuals. That multiple antigens are responsible for the classic ICA signal was demonstrated comparing the ICA immunofluorescence of new IDDM sera adsorbed with excess recombinant GAD65 versus that which was untreated (Marshall et al., 1994; Chaillous et al., 1994). The m a x i m u m blocking obtainable at high concentrations of glutamate decarboxylase (5 micrograms/mL) is 36% (median, range 24--61%). The conclusion is that the majority of the ICA signal is to antigens other than GAD. A partial list of diabetesrelated antigens is provided in Table 2. Pathogenetic

Role

Much interest centers around defining the role of ~cell-directed autoantibodies in the pathogenesis of IDDM. Plasmapheresis of children with new onset

IDDM to remove potentially pathogenic antibodies had little effect on C-peptide levels or disease course, but removal was necessarily incomplete and the stage of the disease was late (Ludvigsson, 1983). Adoptive transfer of only T lymphocytes in a number of mouse and rat models of autoimmune diabetes is fully able to transfer diabetes. Conversely, human neonates with maternal ICA (from transplacental transfer) do not develop IDDM after birth. However, these observations, though telling, do not exclude a role for ~-cell antigen receptors ([3-cell surface antibodies) in antigen uptake and presentation to T cells in spontaneous diabetes in rodents or in man. Indeed, high-affinity IgG antibodies to antigens such as GAD (Richter et al., 1995) suggest that such uptake and presentation occur in human IDDM in an antigen-driven fashion. Soluble antibodies might also aid in 1) antibodymediated, complement-dependent cellular cytotoxicity, 2) natural killer (NK) cell-mediated cytotoxicity, and 3) cell-surface complement fixation and leukocyte chemotaxis. However, antigens expressed and recog-

443

nizable on the cell surface are necessary for these three processes. The ability of circulating antibodies to recognize surface antigens on human islet cells is controversial for several reasons, including the absence of immunoglobulins (Vives et al., 1992; Petersen and Dyrberg, 1992) on the surface of [3 cells undergoing destruction in twin transplant studies (Sutherland et al., 1984). In the later stages of the pathogenesis, after intermolecular antigen spreading, a number of [3-cell surface antigens such as insulin and secretory granule membrane proteins can be recognized. In fact, autoantibodies from new-onset IDDM patients can directly inhibit ~-cell insulin secretion (Kanatsuna et al., 1983; Ziegler and Lernmark 1982; Svenningsen et al., 1983). However, a role for autoantibodies interacting directly with the cell in the early stages of the immune response is unlikely. Methods of Detection In the classical cytoplasmic ICA assay which uses thin sections of human pancreas in the indirect immunofluorescence assay, a large number of antigens are potentially recognized. Cell surface and intracellular antigens are accessible, and protein, lipid and glycosylated antigens are all present. The cumbersome nature of the assay and the variability of human pancreas (Landin-Olsson, 1990) preclude detailed studies on antibody binding and endpoint titrations. In addition to a number of second antibody systems (peroxidase, alkaline phosphatase, biotin-avidin, etc.), bound antibodies can also be detected by their ability to activate complement (Bottazzo et al., 1980). This sandwich-type of reaction is based on detecting the antibody-associated C3 complex with a fluorescent antibody to C3. Whether CF-ICA are involved in the pathogenesis of IDDM or simply reflect [3-cell destruction is still unclear, especially because they tend to correlate with high titer standard IgG-ICA (Greenbaum et al., 1992b). As is the case with many antibody assays on sections, tissue fixation affects antibody reactivity. Hence, ICA of significant diagnostic sensitivity and specificity are detectable on frozen but not on sections of formalin- or Bouin-fixed human pancreas (Gleichmann and Bottazzo, 1987). Because tissue fixation alters the molecular structure of antigens and because human immunoglobulins poorly penetrate cell membranes, IDDM sera can be tested on living non-insulin-producing human insulinoma cells (Maclaren et al., 1975) or suspensions of normal

444

rat islet cells (Lernmark et al., 1978) to detect islet cell surface antibodies. Islet cell surface antibodies (ICSA) In assays to detect islet cell surface antibodies, dispersed cell suspensions are prepared from collagenase-isolated islets of Langerhans from rat, mouse or human pancreas. The dispersed cells are kept in tissue culture to allow recovery of the enzyme-digested cell surface before incubation with human serum from IDDM patients or controls. Cell surface-bound antibodies are detected with a variety of second antibody reagents including fluorescent or radioactive antibodies as well as 125I-protein A (Lernmark, 1987). The ICSA assays are cumbersome and require proficiency in islet isolation and preparation of cell suspensions from the isolated islets. Standardization workshops have yet to be attempted. Although the presence of ICSA may be of pathogenic importance, the diagnostic sensitivity is less than 50% and the specificity 95--99%; this poor sensitivity combined with the cumbersome nature of the assay and the conflicting results (Vives et al., 1992) preclude further detailed ICSA studies. In functional assays, IDDM sera positive for ICSA inhibit glucose-stimulated insulin release from isolated islet cells (Kanatsuna et al., 1983), insulin release from perfused mouse pancreas following passive transfer (Svenningsen et al., 1983), and glucose transport into rat islet cells (Johnson et al., 1990). Surface bound antibodies, however, are also able to activate complement with resultant killing of the cells. Complement-dependent antibody-mediated cytotoxicity (C'AMC) is a sensitive in vitro assay for ICSA. The diagnostic sensitivity for IDDM is reported to be 30--50% and the specificity is 96--98% (Lernmark, 1987). As many as 30% of first degree relatives to IDDM patients are positive for C'AMC (Dobersen et al., 1980). Because it is as cumbersome and difficult to standardize as the assay for ICSA, the C'AMC assay is not widely used. Use of cell lines mandates careful controls for cross-species reactions; animal sera often used as the source of complement make the assay a sensitive measure of cell surfacebound antibodies rather than an assay of pathogenetically relevant antibodies. To what extent C'AMC occurs in vivo is still controversial. Surface antibodies can also mediate antibodydependent cellular cytotoxicity (ADCC) (Charles et al., 1983). Whether the ADCC in vitro reaction has an

in vivo counterpart is still unclear. The slow progress in understanding C'AMC and ADCC is due to the absence of suspensions of normal human [3 cells to be used in these in vitro tests. Autoantigens with which ICA, ICSA and C'AMC react are becoming defined. These components in the islet [3 cells are referred to as "IDDM-related autoantigens".

CLINICAL U T I L I T Y Disease Associations

The classic ICA assay has a high predictive value which is defined in a large number of excellent family studies (Bonifacio et al., 1990; Bingley et al., 1994). The reliability of these studies can be verified since the laboratories participate in international workshops (Greenbaum et al., 1992b) and express results in JDF Units. Given that the classic ICA assay represents autoantibody recognition of multiple islet antigens, it is not surprising that sensitivity of the ICA assay is higher than that for antibodies to GAD, insulin and other single antigens. Sensitivity in new IDDMs is usually over 80% (Landin-Olsson et al., 1989). Also not surprising is that specificity is lower with ICA than with antibody tests to single antigens. Populations with a higher prevalence of IDDM tend to have more healthy individuals with ICA. For example, the prevalence of ICA in healthy schoolchildren was 4.1% in Sweden (Hagopian et al., 1995), 2.8% in England (Bingley et al., 1993) 1.8% in France (Levy-Marchal et al., 1991) and only 0.35% in Spain (Bergua et al., 1987); these frequencies parallel those of IDDM in these countries (LaPorte et al., 1985). However, regardless of the cohort tested, certain features are associated with a higher risk of IDDM among ICA-positive subjects. For example, higher ICA titers (for example, above 20--40 JDF units) mark a higher risk of progression (Bonifacio et al., 1990;

Riley et al., 1990). A younger subject age imparted up to 15-fold higher diabetes risk to ICA positivity when comparing those under 14 years old to those above 40 years of age (Bingley et al., 1994). Thus, in one study 10/25 ICA-positive subjects who were <13.2 years of age at study entry developed diabetes, while only 4/25 aged 13.2-19 years did so, and only 4/57 aged over 19.5 years (Bingley et al., 1994). Family members with ICA were at higher risk of progression to IDDM, the greater the number of high risk HLA DQ alleles they carried. In one study, following ICA-positive relatives for 75 months, those with 4, 2, 1 and 0 highrisk DQ alleles had a 60%, 21%, 11% and 0% risk of developing clinical IDDM, respectively (Lipton et al., 1992; Drash et al., 1991). Subjects with both ICA and other autoantibodies such as GAD antibodies or IAA, have a three- to five-fold higher risk of developing IDDM versus ICA alone, and specificity is greatly improved (Table 3) (Hagopian et al., 1995; Bingley et al., 1994; Landin-Olsson et al., 1992). Subjects with higher ICA titers (for example, over 20 JDF units) had more persistent ICA (Ziegler et al., 1990) which in itself has a much higher risk than fluctuating or transient ICA positivity. In one study, three of four persistently ICA-positive relatives developed IDDM, while 0/7 with "fluctuating ICA" did so (Kajio et al., 1995). Finally, ICA reactivity not restricted to only [3 cells within the islet, nor to human but not mouse pancreas, marked a risk of progression to IDDM, while the less-common, restricted ICA were not nearly as predictive (Gianani et al., 1992; Bjork et al., 1993; Ujihara et a1.,1994). As with any predictive test, positive predictive value depends not only on test sensitivity and specificity, but on prevalence in the group tested. For example, persons diagnosed with apparent NIDDM or family members of current IDDM patients have 3--9% risk of autoimmune IDDM. A positive ICA test among these subjects is tenfold more predictive than a similar finding in the general population. Thus, positive predictive value of ICA positivity >3 JDF

Table 3. Risk for Developing Clinical IDDM Sensitivity

Specificity

Predictive Value

ICA

84%

95.9%

3%

ICA + GAD Ab

62%

99.0%

9%

ICA + IAA

47%

99.2%

9%

Note: Predictive values are for the general population, and would be higher for family studies (Hagopian et al., 1995).

445

units was 4% among Swedish schoolchildren (prevalence = 0.2%) but jumps to 20% among new NIDDM patients with occult IDDM (prevalence = 3%) despite a lower specificity in this population. The well-defined predictive value of ICA was important in its choice as the primary screening assay for the large DPT-1 trial of immunosuppression (Skyler and Marks, 1993). ICA-positive relatives are screened by subsequent ~ cell function testing to identify candidates for immunointervention therapy. Results of this study are pending.

CONCLUSION Insulin-dependent diabetes mellitus is strongly associated with islet cell antibodies during the prodrome of subclinical disease, at clinical onset and during the subsequent life-long period of insulin therapy. The islet cell antibodies detected in crude assays such as on sections of frozen human pancreas (ICA) or

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

Ku AND Ki AUTOANTIBODIES Westley H. Reeves, M.D., Minoru Satoh, M.D., Lovorka Stojanov, M.D. and Jingsong Wang, M.D.

Departments of Medicine and Microbiology and Immunology, Thurston Arthritis Research Center and UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7280, USA

HISTORICAL NOTES

THE AUTOANTIGENS

The Ku antigen is a nonhistone nuclear antigen producing a precipitin line first reported in serum from a patient (designated Ku) with sclerodermapolymyositis overlap syndrome (Mimori et al., 1981). Although originally thought to be relatively specific for this unusual subset of autoimmune disease, autoantibodies to Ku (anti-Ku) are also found in sera of some patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), scleroderma, polymyositis, Graves' disease and primary pulmonary hypertension (Reeves, 1985; Yaneva and Arnett, 1989; Chan et al., 1989; Isern et al., 1992). Thus, serum autoantibodies specific for Ku cannot at present be regarded as diagnostic markers for any particular autoimmune disease subset. There is confusion in the literature regarding the relationship of the Ku and Ki autoantigens (Francoeur et al., 1986; Fritzler, 1992; Wilson, 1987). Antibodies to Ki (anti-Ki) antibodies were originally described as a distinctive precipitin line in double immunodiffusion associated with SLE or overlap syndrome (Tojo et al., 1981). Although autoantibodies to Ki and Ku may be present in the same serum (Francoeur et al., 1986; Mimori et al., 1990b), the Ki antigen is a 29.5 kd protein that is immunologically and biochemically unrelated to Ku (Figure 1) but identical to the SL and PL-2 antigens (Bernstein et al., 1986; Nikaido et al., 1990).

Definition/Nomenclature/Sequence Information Ku and p350 Antigens. Biochemical characterization and electron microscopy reveal that the Ku antigen is a 1:1 heterodimer consisting of 70 kd and 80--86 kd protein subunits (Figure 1) located in the nuclei and nucleoli of most primate cells (Yaneva et al., 1985; de Vries et al., 1989). The 70 and 80 kd subunits are disulfide-linked and can be dissociated by high concentrations of NaC1 plus detergents (Wang et al., 1994). The subunits are generally referred to as p70 (Ku70) and p80 (Ku80), respectively, and the dimer as p70/p80. The Ku antigen binds specifically to the termini of double-stranded DNA (Mimori and Hardin, 1986; Paillard and Strauss, 1991) and serves as the DNA- binding component of a 350 kd catalytic subunit with DNA-dependent protein kinase activity (Lees-Miller et al., 1990; Carter et al., 1990; Gottlieb and Jackson, 1993). The catalytic subunit, termed p350 (Figure 1), is actually closer to 450 kd in size (Blunt et al., 1995). The complex of p350 and Ku is referred to as the DNA-dependent protein kinase (DNA-PK); its components play an important role in repairing double-stranded DNA breaks and in V(D)J recombination (Smider et al., 1994; Taccioli et al., 1994). Human and murine p70 Ku (Chan et al., 1989; Reeves and Sthoeger, 1989; Porges et al., 1990) and p80 Ku (Yaneva et al., 1989; Mimori et al., 1990a; Falzon and Kuff, 1992) cDNAs are cloned, but the amino acid sequence of p350 is not yet reported; sequencing of cDNA clones is in progress in several laboratories. The predicted molecular weights of the

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Figure 1. Immunoprecipitation of radio-labeled Ku, p350 and Ki antigens by autoimmune sera. K562 cells were labeled with 35Smethionine and cysteine; extract was cleared by centrifugation and immunoprecipitated using human autoimmune sera as described (Wang et al., 1994; Satoh et al., 1995). Immunoprecipitates were washed with buffer containing 0.5 M NaC1 in order to dissociate the Ku antigen (p70/p80 dimer) from p350 (Satoh et al., 1995), and radioactive proteins were analyzed by SDS-PAGE and autoradiography. Immunoprecipitates With rabbit anti-p350 polyclonal antiserum (gift of Dr. Carl Anderson) and murine anti-Ku monoclonal antibody 162 (Reeves, 1985) are shown on the left. Lanes 1--5, sera with anti-Ku; lanes 6--8, sera with anti-Ku plus anti-p350; lanes 8--11, sera with anti-Ki. Note that serum used in lane 8 contains anti-Ki plus anti-Ku and anti-p350 activity; whereas, the sera used in lanes 9-11 contain anti-Ki without anti-Ku or anti-p350 activity. N = normal human serum. Positions of p350, p80 Ku, p70 Ku and Ki antigens are indicated. U 1-A protein (indicated by upper "x") migrates similarly to the Ki antigen but can be distinguished by the presence of other U1 snRNP proteins in the immunoprecipitates (lower "x").

human p70 and p80 proteins are 69.8 and 82.7 kd, respectively. Neither protein exhibits significant sequence similarities to other proteins in the Genbank or European Molecular Biology Laboratory (EMBL) databases. Murine and human p70 are 83% identical at the amino acid level, and the human and murine p80 sequences also are very similar. Ku homologues are present in insects and yeast (Jacoby and Wensink, 1994; Feldmann and Winnacker, 1993). For reasons

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that are unclear, primate cells contain 40-to-60-fold more Ku antigen than rodent cells; whereas, the concentration of Ku in bovine cells is intermediate between primate and rodent cells. Thus, primate cells are the richest source of Ku and p350 antigens (Celis et al., 1987; Wang et al., 1993). Ku and p350 are detected in all human cell types and tissues examined to date except for mature neutrophils (Ajmani et al., 1995). Recombinant human p70 and p80 proteins

were expressed in bacteria (Reeves et al., 1991; AbuElheiga and Yaneva, 1992; Suwa, 1990), and recombinant p70/p80 dimer was assembled and purified using recombinant vaccinia virus (Wang et al., 1994) and baculovirus (Ono et al., 1994) vectors. Although a number of linear and conformational autoepitopes recognized by human autoantibodies are identified (Yaneva and Arnett, 1989; Reeves et al., 1991; Suwa et al., 1992), the majority of human autoantibodies are specific for conformational determinants of the native p70/p80 heterodimer. Antibodies recognizing epitopes dependent on the quaternary structure of the p70/p80 dimer (Wang et al., 1994) and of the p350-Ku complex (Satoh et al., 1995) emphasize further the preferential recognition of the native antigens.

Ki Antigen. The Ki antigen is a nuclear protein of unknown function with a mobility of-~32 kd by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1) that can form a multimer (Bernstein et al., 1986; Sakamoto et al., 1989) of which no other components besides the -32 kd protein are yet identified. Molecular cloning of the cDNA encoding Ki antigen reveals a predicted molecular weight of 29.5 kd. The protein sequence is conserved highly among species and contains a sequence similar to the nuclear localization signal of SV40 large T antigen (Nikaido et al., 1990).

AUTOANTIBODIES

Pathogenetic Role and Methods of Detection Anti-Ku. Limited evidence suggests that the amounts of anti-Ku reflect disease activity in a subset of patients (Reeves et al., 1989), but additional studies are needed. There is no standard assay for anti-Ku at present. The most widely used tests are double immunodiffusion (ID) (Mimori et al., 1981), radioimmunoprecipitation (Reeves, 1985) and antigen-capture enzymelinked immunosorbent assay (ELISA) (Satoh et al., 1993). Although most high titer anti-Ku sera contain precipitating antibodies, ID is considerably less sensitive than immunoprecipitation or ELISA for detecting anti-Ku activity. Initial reports utilized calf thymus as a source of antigen, but primate cells are a richer source of Ku (Wang et al., 1993), and certain anti-Ku display selective binding to human Ku (Porges et al., 1990). Thus, the analytical sensitivity of the

ID assay is enhanced by using antigenic extracts derived from human cells. Anti-Ku can also be detected by immunoblot assay (Francouer et al., 1986). Although simple to perform, the usefulness of the immunoblot assay is limited by the fact that most sera containing autoantibodies to Ku recognize the native form of the antigen preferentially (Reeves et al., 1991). The immunoprecipitation assay, utilized to detect autoantibodies specific for native Ku antigen, permits the identification of antibodies to epitopes dependent on the quaternary structure of Ku (Wang et al., 1994). For this reason, the immunoprecipitation assay is a standard technique for detecting anti-Ku. However, unless the immunoprecipitates are washed stringently, anti-dsDNA, anti-histone and antibodies to other DNA-binding proteins can cause false-positive results for anti-Ku in this assay (Satoh et al., 1995). The antigen-capture ELISA based on native Ku antigen correlates well with immunoprecipitation, and if performed with stringent washing, may have comparable analytical sensitivity and specificity. Compared with immunoprecipitation, antigen-capture ELISA is 79% sensitive and 94% specific (Satoh et al., 1993) for detection of anti-Ku in patients with autoimmune diseases. Anti-Ku titers as high as 107 by ELISA are reported (Reeves et al., 1989). At these levels, autoantibodies to Ku can be detected by ID, ELISA, immunoprecipitation or, in many cases, immunoblot. There is limited comparative information regarding the sensitivity and specificity of other assays compared with the standard immunoprecipitation assay. In one study of ten immunoprecipitation-positive sera having ELISA results of anti-Ku antibodies >1: 12,500, only three (30%) were reactive with p70 or p80 by immunoblot (Porges et al., 1990). Thus, the analytical sensitivity of the immunoblot assay is low, due to the preferential reactivity of anti-Ku antibodies with conformational determinants. The only method for detecting anti-p350 examined thus far is immunoprecipitation (Satoh et al., 1995). Two types of autoantibodies can be identified. The first type stabilizes the interaction between Ku and p350, possibly due to recognition of the quaternary structure of DNA-PK. These antibodies do not recognize biochemically purified p350. The second class of autoantibodies recognizes biochemically purified p350. Both types of autoantibodies were associated strongly with Ku antibodies; autoantibodies recognizing the purified p350 protein were associated with "stabilizing" type autoantibodies (Figure 2). The applicability

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of other techniques to the detection of anti-p350 antibodies remains to be determined. Anti-Ki. Studies in Japanese populations suggest an association of anti-Ki with persistent arthritis, pericarditis, pulmonary hypertension, anti-Sm antibodies and lupus erythematosus cells (LE cells) (Tojo et al., 1981), or with CNS involvement (Sakamoto et al., 1989). However, two studies in Europe failed to confirm these associations (Bernstein et al., 1986; Riboldi et al., 1987). The titer of anti-Ki may reflect disease activity in some cases (Yamanaka et al., 1992). However, the role of anti-Ki in the pathogenesis of disease is unknown. Anti-Ki can be detected by ID (Tojo et al., 1981), immunoprecipitation (Figure 1) or ELISAs based on biochemically purified rabbit Ki antigen (Sakamoto et al., 1989) or the recombinant bovine protein (Yamanaka et al., 1992). An ELISA employing rabbit Ki as antigen appears to offer high sensitivity, but the specificity of this assay remains to be determined.

CLINICAL UTILITY Disease Association

Anti-Ku. Autoantibodies to Ku are detected in a variety of autoimmune conditions, but large scale studies of the clinical associations are unavailable, and the clinical usefulness of anti-Ku in differential diagnosis, monitoring disease activity or defining prognosis is unclear. In Japanese patients (Table 1), anti-Ku are associated most frequently with polymyositis-scleroderma overlap syndrome (Mimori et al., 1981). Precipitating antibodies to Ku were found in

six of 11 sera (55%) from this subset, compared with three of 319 Japanese patients (1%) with other connective tissue diseases (one of whom had SLE-scleroderma-polymyositis overlap). The high frequency of precipitating autoantibodies in the Japanese polymyositis-scleroderma overlap subset is confirmed in subsequent studies (Hirakata et al., 1992; Suwa et al., 1992; Satoh et al., 1993). However, by immunoprecipitation and ELISA, nonprecipitating autoantibodies to Ku are also seen in Japanese patients with SLE or scleroderma (Satoh et al., 1993). In contrast to the results obtained by double immunodiffusion, anti-Ku are detected by immunoprecipitation (Table 1) or ELISA (Satoh et al., 1993) at comparable frequencies in Japanese and American SLE patients (5--10%). In Americans, anti-Ku are associated most strongly with overlap syndromes and SLE (Table 1) but are also seen in scleroderma, polymyositis, Sj6gren's syndrome, rheumatoid arthritis, Graves' disease and primary pulmonary hypertension as well (Yaneva and Arnett, 1989; Chan et al., 1989; Isern et al., 1992). Anti-Ku were detected in 9/16 Graves' disease sera (Chan et al., 1989) and in 23% of sera from patients with primary pulmonary hypertension, however, these high frequencies were not confirmed in subsequent studies (Satoh et al., 1993; Stojanov et al., unpublished data). Information regarding HLA associations of anti-Ku is limited. In one study, the class II allele DQwl was present in 17 of 19 (89%) anti-Ku-positive patients, compared with 58% and 61% in white and black controls, respectively (relative risk 5.8) (Yaneva and Arnett, 1989). However, the number of patients was small, and HLA-DQwl is a common allele found at increased frequency in white SLE patients. Anti-p350 are relatively unusual but are reported

Table 1. Frequency of Ku Autoantibodies in Patients with Systemic Autoimmune Disease* Diagnosis

Japanese

American

SLE

5/78 (6%)

5/105 (5%)

Scleroderma

1/34 (3%)

0/46 (0%)

Polymyositis/dermatomyositis

1/26 (4%)

0/7 (0%)

Rheumatoid arthritis

0/30 (0%)

0/24 (0%)

Sj6gren's syndrome

0/12 (0%)

0/3 (0%)

13/44 (30%)

3/11 (27%)

0/7 (0%)

0/7 (0%)

MCTD/overlap syndromes Healthy controls *Determined by immunoprecipitation assay.

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in 4% of mixed connective tissue disease/overlap syndrome patients and 0.6% of SLE patients, but not in patients with scleroderma, polymyositis, rheumatoid arthritis or Sj6gren's syndrome (Satoh et al., 1995). Anti-Ki. Anti-Ki detected by ID were first considered relatively specific for SLE (11.8%) and overlap syndrome (19.4%) (Tojo et al., 1981). Subsequent studies, while confirming a frequency of 6--12% in SLE and 2--14% in MCTD by immunodiffusion or CIE, suggest that these antibodies are also associated with scleroderma, rheumatoid arthritis, Sj6gren's syndrome and other autoimmune diseases (Sakamoto et al., 1989; Riboldi et a1.,1987; Yamanaka et al., 1992). A frequency as high as 20% is reported in SLE by ELISA (Yamanaka et al., 1992), but further studies are necessary to establish the clinical significance of anti-Ki in SLE.

Figure 2. Diagram of the occurrence of anti-Ku, anti-p350 and CONCLUSION Autoantibodies to the Ku and p350 antigens are directed against DNA-PK, a multiprotein complex consisting of a DNA-binding component (Ku p70/p80 heterodimer) and a catalytic subunit (p350). Autoantibodies reactive with p70, p80 and p350 and antibodies that stabilize the quaternary structure of DNAPK constitute a linked set (Figure 2). The DNA-PK antigen is of considerable biological interest, in view of recent studies indicating that Ku antigen and p350 are involved in DNA repair and V(D)J recombination. Autoantibodies specific for Ku are intriguing because they are associated strongly with an unusual disease subset in Japanese patients with systemic autoimmunity; whereas, they are associated primarily with SLE and overlap syndromes in Americans. The initial confusion regarding the relationship of the Ki and Ku antigens is now clarified: the two antigens are unrelated. Initial reports that the antigens

REFERENCES Abu-Elheiga L, Yaneva M. Antigenic determinants of the 70kDa subunit of the Ku autoantigen. Clin Immunol Immunopathol 1992;64:145-152. Ajmani AK, Satoh M, Reap E, Cohen PL, Reeves WH. Absence of autoantigen Ku in mature human neutrophils and human promyelocytic leukemia line (HL-60) cells and

anti-Ki. Anti-Ku and anti-Ki represent distinctive specificities, but a small subset of human autoimmune sera contains both specificities. Anti-p350 are relatively unusual and so far are reported only in sera that contain autoantibodies that stabilize the DNA-PK complex (Ku-p350 stabilizing antibodies). Autoantibodies that stabilize DNA-PK, in turn, are so far reported only in sera containing anti-Ku (specific for p70, p80 or both proteins). Thus, anti-p350, anti-Ku and autoantibodies that stabilize DNA-PK constitute a linked set. The best available evidence suggests that autoantibodies to Ki are not associated with anti-Ku/p350/DNA-PK antibodies more frequently than expected by chance alone.

were identical resulted from the presence of anti-Ku in some anti-Ki reference sera. The Ki antigen is a 32 kd nuclear protein of unknown function recognized by autoantibodies that are clearly different from anti-Ku (Figure ,1). Further studies are needed to define more precisely the clinical significance and role in disease pathogenesis of anti-Ku and anti-Ki.

lymphocytes undergoing apoptosis. J Exp Med 1995;181: 2049--2058. Bernstein RM, Morgan SH, Bunn CC, Gainey RC, Hughes GR, Mathews MB. The SL autoantibody-antigen system: clinical and biochemical studies. Ann Rheum Dis 1986;45:353--358. Blunt T, Finnie NJ, Taccioli GE, Smith GC, Demengeot J, Gottlieb TM, Mizuta R, Varghese AJ, Alt FW, Jeggo PA, et al. Defective DNA-dependent protein kinase activity is linked

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to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 1995;80:813-23. Carter T, Vancurova I, Sun I, Lou W, DeLeon S. A DNA-activated protein kinase from HeLa cell nuclei. Mol Cell Biol 1990; 10:6460--6471. Celis JE, Madsen P, Nielsen S, Ratz GP, Lauridsen JB, Celis A. Levels of synthesis of primate specific nuclear proteins differ between growth-arrested and proliferating cells. Exp Cell Res 1987;168:389--401. Chan JY, Lerman MI, Prabhakar BS, Isozaki O, Santisteban P, Kuppers RC, Oates EL, Notkins AL, Kohn LD. Cloning and characterization of a cDNA that encodes a 70-KD novel human thyroid autoantigen. J Biol Chem 1989;264:3651-3654. de Vries E, van Driel W, Bergsma WG, Arnberg AC, van der Vliet PC. HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA-multimeric protein complex. J Mol Biol 1989;208:65--78. Falzon M, Kuff EL. The nucleotide sequence of a mouse cDNA encoding the 80 KDa subunit of the Ku (p70/p80) autoantigen. Nucleic Acids Res 1992;20:3784. Feldmann H, Winnacker EL. A putative homologue of the human autoantigen Ku from Saccharomyces cerevisiae. J Biol Chem 1993;268:12895--12900. Francoeur AM, Peebles CL, Gompper PT, Tan EM. Identification of the Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity. J Immunol 1986; 136:16481653. Fritzler MJ. Antibodies to nonhistone antigens in systemic lupus erythematosus. In: Lahita RG, ed. Systemic Lupus Erythematosus. New York: Churchill Livingstone, 1992:273--291. Gottlieb TM, Jackson SP. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 1993;72:131--142. Hirakata M, Mimori T, Akizuki M, Craft J, Hardin JA, Homma M. Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in Japanese patients with inflammatory muscle disease. Arthritis Rheum 1992;35:449-456. Isern RA, Yaneva M, Weiner E, Parke A, Rohtfield N, Dantzker D, Rich S, Arnett FC. Autoantibodies in patients with primary pulmonary hypertension: association with anti-Ku. Am J Med 1992;93:307--312. Jacoby DB, Wensink PC. Yolk protein factor 1 is a Drosophila homolog of Ku, the DNA-binding subunit of a DNA-dependent protein kinase from humans. J Biol Chem 1994;269: 11484--11491. Lees-Miller SP, Chen YR, Anderson CW. Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol Cell Biol 1990;10:6472-6481. Mimori T, Akizuki M, Yamagata H, Inada S, Yoshida S, Homma M. Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap. J Clin Invest 1981;68:611-620. Mimori T, Ohosone Y, Hama N, Suwa A, Akizuki M, Homma M, Griffith AJ, Hardin JA. Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human

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autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome. Proc Natl Acad Sci USA 1990a;87:1777--1781. Mimori T, Suwa A, Hama N. Clinical significance of anti-Ku antibodies. Nippon Rinsho 1990b;48(suppl):535-538. Mimori T, Hardin JA. Mechanism of interaction between Ku protein and DNA. J Biol Chem 1986;261:10375-10379. Nikaido T, Shimada K, Shibata M, Hata M, Sakamoto M, Takasaki Y, Sato C, Takahashi T, Nishida Y. Cloning and nucleotide sequence of cDNA for Ki antigen, a highly conserved nuclear protein detected with sera from patients with systemic lupus erythematosus. Clin Exp Immunol 1990;79:209--214. Ono M, Tucker PW, Capra JD. Production and characterization of recombinant human Ku antigen. Nucleic Acids Res 1994 ;22:3918--3924. Paillard S, Strauss F. Analysis of the mechanism of interaction of simian Ku protein with DNA. Nucleic Acids Res 1991; 19:5619-5624. Porges A, NgT, Reeves WH. Antigenic determinants of the Ku (p70/p80) autoantigen are poorly conserved between species. J Immunol 1990;145:4222-4228. Reeves WH. Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera. J Exp Med 1985;161:18--39. Reeves WH, Sthoeger ZM, Lahita RG. Role of antigen selectivity in autoimmune responses to the Ku (p70/p80) antigen. J Clin Invest 1989;84:562--567. Reeves WH, Pierani A, Chou CH, Ng T, Nicastri C, Roeder RG, Sthoeger ZM. Epitopes of the p70 and p80 (Ku) lupus autoantigens. J Immunol 1991; 146:2678--2686. Reeves WH, Sthoeger ZM. Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J Biol Chem 1989;264:5047--5052. Riboldi P, Asero R, Origgi L, Crespi S. The SL/Ki system in connective tissue diseases: incidence and clinical associations. Clin Exp Rheumatol 1987;5:29-33. Sakamoto M, Takasaki Y, Yamanaka K, Kodama A, Hashimoto H, Hirose S. Purification and characterization of Ki antigen and detection of anti-Ki antibody by enzyme-linked immunosorbent assay in patients with systemic lupus erythematosus. Arthritis Rheum 1989;32:1554--1562. Satoh M, Langdon J, Reeves WH. Clinical applications of an anti-Ku antigen capture ELISA. Clin Immunol Newsletter 1993;13:23--31. Satoh M, Ajmani AK, Stojanov LP, et al. Autoantibodies that stabilize the molecular interaction of the Ku and p350 subunits of DNA dependent protein kinase. Submitted, 1995. Smider V, Rathmell WK, Lieber MR, Chu G. Restoration of Xray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science 1994;266:288--291. Suwa A. Studies on the antigenic epitopes reactive with autoantibody in patients with PSS-PM overlap syndrome. Keio Igaku 1990;67:865--879. Suwa A, Mimori T, Hama N, et al. Enzyme-linked immunosorbant assay of anti-Ku antibodies using purified recombinant Ku antigens. Jpn J Clin Immunol 1992;15:337-345. Taccioli GE, Gottlieb TM, Blunt T, Priestley A, Demengeot J,

Mizuta R, Lehmann AR, Alt FW, Jackson SP, Jeggo PA. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 1994;265:14421445. Tojo T, Kaburaki J, Hayakawa M, Okamoto T, Tomii M, Homma M. Precipitating antibody to a soluble nuclear antigen "Ki" with specificity for systemic lupus erythematosus. Ryumachi 1981; 21Suppl:129-134. Wang J, Chou CH, Blankson J, Satoh M, Knuth MW, Eisenberg RA, Pisetsky DS, Reeves WH. Murine monoclonal antibodies specific for conserved and nonconserved antigenic determinants of the human and murine Ku autoantigens. Mol Biol Rep 1993;18:15--28. Wang J, Satoh M, Pierani A, Schmitt J, Chou CH, Stunnenberg HG, Roeder RG, Reeves WH. Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers. J Cell Sci 1994;107:3223-3233.

Wilson MR. Antinuclear antibodies and anticytoplasmic antibodies in lupus erythematosus. In: Wallace DJ, Dubois EL, eds. Dubois' Lupus Erythematosus. Philadelphia: Lea & Febiger, 1987:227--243. Yamanaka K, Takasaki Y, Nishida Y, Shimada M, Shibata M, Hashimoto H. Detection and quantification of anti-Ki antibodies by enzyme-linked immunosorbent assay using recombinant Ki antigen. Arthritis Rheum 1992;35:667--671. Yaneva M, Ochs R, McRorie DK, Zweig S, Busch H. Purification of an 86--70 kDa nuclear DNA-associated protein complex. Biochim Biophys Acta 1985;841:22--29. Yaneva M, Wen J, Ayala A, Cook R. cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen. J Biol Chem 1989;264:13407--13411. Yaneva M, Arnett FC. Antibodies against Ku protein in sera from patients with autoimmune diseases. Clin Exp Immunol 1989;76:366-372.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

LIVER CYTOSOL ANTIGEN TYPE 1 AUTOANTIBODIES Jean-Claude Homberg, M.D., Ph.D., Nisen Abuaf, M.D., Ph.D., Catherine Johanet, M.D. and Eric Martini, M.D.

Laboratoire Central d'Immunologie et d'Hdmatologie, HOpital Saint-Antoine, 75571 Paris Cedex 12, France

HISTORICAL NOTES The first serum with anti-liver cytosol antigen type 1 antibodies (anti-LC1) was detected in September, 1981 due to the persistence of Dr. M. Odievre, who requested further evaluation of the serum of a 9-yearold girl with a chronic active hepatitis and islet cell antibody-positive insulin-dependent diabetes mellitus. The re-examination of this serum by indirect immunofluorescence (IIF) showed a strong positivity on rat liver similar to that given by anti-liver/kidney microsome antibody type 1 (anti-LKM-1) but was completely negative on rat kidney. A systematic study of hepatocyte cytoplasmic fluorescence yielded a similar pattern in six additional cases of autoimmune hepatitis during a 5-year period (Martini et al., 1988). The IIF results were complemented by immunodiffusion (ID) showing reaction of identity with a liver cytosolic subcellular fraction (Martini et al., 1988). In testing 2,856 people, the ID technique demonstrated anti-LC 1 masked in IIF by anti-LKM-1 in 14 cases of autoimmune hepatitis type 2 (AIH-2) (Martini et al., 1988). Since 1993, anti-LC1 have been confirmed by two other teams (Lenzi et al., 1993; Han et al., 1995) and by different colleagues (Mrs. P. Codoner-Franch, Barcelona; G. Maggiore, Pavia; and M. Manns, Hanover, personal communications).

THE AUTOANTIGEN(S) The term "liver cytosol antigen type 1" (LC1 Ag) denotes an organ-specific molecule situated in the liver cytosolic subcellular fraction. By IIF, sera strongly positive for anti-LC1 on liver were negative on 27 other rat organs (Martini et al., 1988). Similarly by ID, the cytosolic fraction of kidney, pancreas,

456

stomach, thyroid, thymus and adrenals from rat, mouse, monkey or human does not react with precipitating anti-LC1 (Martini et al., 1988). The liver of the above four species does contain LC1 Ag (Martini et al., 1988), but the partial reaction of identity between human and rat cytosolic fractions indicates epitopes present only in human liver (Abuaf et al., 1992; Han et al., 1995). Reaction of other autoantibodies with the hepatic cytosol indicates the presence of other antigens in this subcellular fraction: LC2 antigen (Abuaf et al., 1993), rat liver/kidney antigen (Abuaf et al., 1993) and soluble liver antigen (Manns et al., 1987); the two latter antigens are non-organ-specific. The nature of LC1 antigen is unknown. Its molecular weight by gel filtration on Sephadex G200 is 240--280 kd for the human antigen and 220--270 kd for the rat (Abuaf et al., 1992). Immunoblotting reveals a unique antigenic peptide toward 60 kd (Abuaf et al., 1992; Han et al., 1995; Lenzi et al., 1995) different from those of 50 kd LKM-1 antigen of the liver microsomal subcellular fraction, which is cytochrome P45011D6.

AUTOANTIBODIES

Terminology Anti-LC-1 antibodies are named according to the terminology of different autoantibodies such as antimitochondrial antibodies (from anti-M1 to antiM9) and antimicrosome antibodies. For an organspecific autoantibody, only the name of the organ or the cell is indicated, e.g., antithyroid microsome antibodies and antiparietal cell antibodies. On the contrary, "anti-liver/kidney microsome antibodies" indicate non-organ-specific antibodies. A number is

added at the end to differentiate various antibodies reacting against the same subcellular fraction.

Methods of Detection Three detection methods including indirect IF, Ouchterlony double ID and immunoblot using rat or human liver detect anti-LC1 with an increasing sensitivity (Han et al., 1995; Lenzi et al., 1995).

gels permit detection of anti-LC 1. Anti-LC 1 react with a unique human antigenic peptide situated at 58 kd (Lenzi et al., 1995), 60 kd (Han et al., 1995) or 62 kd (Abuaf et al., 1992). Compared to ID, IB might (Han et al., 1995) or might not (Abuaf et al., 1992; Lenzi et al., 1995) be more sensitive for detection of antiLC1.

CLINICAL UTILITY Immunofluorescence (IF). The classical three-organ composite block for the detection of autoantibodies present in liver diseases is used. Sera diluted 1:10 or 1:40 in phosphate-buffered saline 0.15 M, pH 7.4 are screened on 4--6 lam cryostat sections of rat liver, kidney and stomach. The characteristic immunofluorescent pattern of anti-LC1 alone corresponds to two criteria: 1) a homogeneous cytoplasmic fluorescence limited to the hepatocytes with a complete negativity of the kidney; and 2) a weaker intensity of hepatocytes adjacent to the central veins called juxtavenous hepatocytes (Figure 1). By this technique, anti-LC1 are hidden by other cytoplasmic antibodies such as anti-LKM-1 which are frequently associated. In the presence of LKM-1 antibodies, the juxtavenous hepatocytes appear less stained only in a third of cases. An absorption of sera containing both anti-LKM-1 and anti-LC1 with a rat liver microsomal subcellular fraction can sometimes eliminate anti-LKM-1 and demonstrate antiLC1, but this time-consuming determination can be impossible when the titer of anti-LKM-1 is high. Immunodiffusion (ID). Separate detection of antiLC1 and anti-LKM-1 is possible with microsomal and cytosolic liver subcellular fractions together with appropriate controls for specificity by reaction of identity with a reference serum. In testing the sera of 2,857 patients with the cytosolic fraction, 49 precipitating antibodies were found, and only 22 gave a reaction identity with anti-LC1 (Abuaf et al., 1993). The other antibodies corresponded to anti-LC2 (three cases), antirat LKC (10 cases) or individual antibodies (14 cases) (Abuaf et al., 1993). Human liver cytosol seems preferable to that of the rat (Han et al., 1995; Lenzi et al., 1995). Some anti-LKM-1 detected by IF are not precipitating (Abuaf et al., 1993); nonprecipitating anti-LC1 might also exist. Immunoblot (IB). Liver cytosolic fractions separated by electrophoresis on dodecyl sulfate-polyacrylamide

Disease Association The scant information available in the literature (Abuaf et al., 1993; Maggiore et al., 1993; Lenzi et al., 1995; Han et al., 1995) are summarized in Table 1.

Autoimmune Hepatitis Type 2 (AIH-2). In the first description (Martini et al., 1988), anti-LC1 were reported as a second marker of AIH-2. Type 1 is associated with antinuclear and/or smooth muscle antibodies and type 2 with anti-LKM-1. However, anti-LC1 and actin antibodies are not yet included in the diagnosis of AIH by the International Autoimmune Hepatitis Group (Johnson and MacFarlane, 1993). AIH-1 associated with antiactin cable antibodies and AIH-2 associated with anti-LKM-1 or anti-LC1 differ by several points. Clinical data of patients with only anti-LC1 were given for 11 children (Maggiore et al., 1993). Other Autoimmune Liver Diseases. Anti-LC1 are reported in smooth muscle antibody-positive AIH and autoimmune cholangitis (Han et al., 1995). Two studies (Martini et al., 1988; Lenzi et al., 1995) yielded negative results for anti-LC1 in 100 and 30 cases of AIH-1, respectively. Smooth muscle antibodies are present in 6% of anti-LKM-1 and/or anti-LCl-positive AIH-2 with titers equal to or greater than 160 but with no actin activity (Authors, unpublished observations). The positive predictive value of smooth muscle antibodies is low compared to the predictive values of anti-LKM-1 or anti-LC1 (Table 2). These cases were included as AIH-2 in Tables 1 and 3. Hepatitis C Virus Infection. This disorder is now well known to be associated with cryoglobulinemia and autoantibodies such as antinuclear, smooth muscle or anti-LKM-1 antibodies. Anti-LC 1 are also found in HCV infection (Abuaf et al., 1993; Lenzi et al., 1993; 457

Figure 1. a: Anti-LC1. b: Heteroantibody against rat juxtavenous hepatocytes.

458

Table 1. Diagnostic Value of Anti-LC1 and Anti-LKM-1 Antibodies Anti-LC 1 alone

anti-LC 1 + anti-LKM- 1

Anti-LKM- 1 alone

21

45

52

Hepatitis C virus infection (>2,000 cases)

4

5

108

Halothane-induced hepatitis (>20 cases)

0

0

6

Graft-versus-host disease (>50 cases)

0

0

9

Miscellaneous disorders (> 100,000 cases)

0

0

7

12

12

124

12 13

5 70

6 0

7 0

Personal results, 1981-1994 (120,936 cases) Autoimmune hepatitis 2 (92 cases)

No clinical information Lenzi et al., 1995 (100 cases with anti-LKM 1) Autoimmune Hepatitis 2 (17 cases) Hepatitis C virus infection (83 cases) Han et al., 1995 (92 cases) Autoimmune liver diseases* (42 cases) Nonautoimmune liver diseases (55 cases)

13 0

*Smooth muscle antibody/antinuclear antibody AIH (15 cases), anti-LKM-l-positive AIH (13 cases), autoimmune sclerosing cholangitis (14 cases).

Pawlotsky et al., 1994). A prospective study of 374 cases yielded 16 anti-LKM-1 (4%) and one anti-LC1 (0.3%) (Abuaf et al., 1993). The association between anti-LKM-1 and anti-LC1 is rare in HCV infection compared to its frequency in AIH-2 (Lenzi et al., 1995; unpublished observations).The differences between AIH-2 and hepatitis C virus infection associated with anti-LKM-1 or anti-LC1 are now well established. Hepatitis C virus infection with antiLKM-1/anti-LC 1 does not respond to immunosuppressive treatment but does respond to interferon; there is no argument that these cases are autoimmune hepatitis type 2b (Michel et al., 1992). In cases of hepatitis C virus infection associated with anti-LKM-1 or antiLC1, these autoantibodies are apparently epiphenomena (Lunel et al., 1992).

Correlation with Disease Activity There is not a clear relation between the anti-LC 1 titer and the disease activity as with the serum transaminases and gamma globulin levels. In some cases, antiLC1 could be detected in the first month of an acute hepatitis, with high levels of serum transaminases; the autoantibodies had very low titer in IF and were sometimes detected only by ID (personal results). The highest values were found in chronic hepatitis or cirrhosis with high activity (Abuaf et al., 1993). Anti-LC1 have no prognostic value for response to corticosteroids and azathioprine contrary to anti-LKM1 (Abuaf et al., 1993). Anti-LKM-1 are good immunologic markers for the clinical course of the disease and its response to therapy (Abuaf et al.,

Table 2. Sensitivity, Specificity, Positive Predictive Value (Positive PV) and Negative Predictive Value (Negative PV) of Markers for Autoimmune Hepatitis Type 1 (AIH-1) and Type 2 (AIH-2) Sensitivity

Specificity

Positive PV

Negative PV

AIH- 1 Antinuclear antibodies Smooth muscle antibodies Actin antibodies

0.40 1 (by selection) 0.98

0.95 0.98 >0.99

0.11 0.27 0.93

0.96 0.96 >0.99

AIH-2 Anti-LKM- 1 Anti-LC 1

0.84 0.48

>0.99 >0.99

0.34 0.88

>0.99 >0.9

459

Table 3. Comparison Between Autoimmune Hepatitis Type 1 (AIH-1) Associated With Antiactin Antibodies and Autoimmune Hepatitis Type 2 (AIH-2) Associated with Anti-LKM-1 and/or Anti-LC1 Antibodies (Personal Results) AIH- 1 (130 cases)

AIH-2 (118 cases)

Clinical circumstances Age: extremes (years) frequent (years) Sex: F/M

2-89 5-20; 45-70 4/1

0.5-39 2-14 3/1

Associated disorders

before 30 years: ulcerative colitis, primary

Vitiligo, Graves' disease, insulindependent diabetes mellitus

sclerosing cholangitis, Crohn's disease after 30 years: SjOgren's syndrome, systemic

scleroderma, rheumatoid arthritis, primary biliary cirrhosis Symptoms

Active and inflammatory hepatitis usually with high values or serum transaminases (xl0) and gamma globulins (>30 g/L)

Lower values of transaminases and gamma globulins but fulminant acute hepatitis (6%), fluctuating evolution and usually cirrhosis in less than two years

Histology

Classical chronic active hepatitis with severe piecemeal necrosis

Moderate piecemeal necrosis with more frequently lobular necrosis and sclerosis

Immunology

Actin antibodies Antinuclear antibodies (44%) Mitochondrial antibodies (4%) C-ANCA (36%)

Low IgA (20%) Anti-LKM- 1 (82%) Anti-LC 1 (56%) Antiparietal cell antibodies (21%)

Evolution and treatment

Usual efficiency of prednisone (2-0.5 mg/kg/ day) with azathioprine (2-0.5 mg/kg/day) Liver transplantation (16%)

More severe prognosis with necessity of cyclosporine (10%) Liver transplantation (16%)

1993). In several cases with both anti-LKM-1 and anti-LC1 antibodies, anti-LC1 can be the only detectable autoantibody in an inactive state induced by treatment (Abuaf et al., 1993).

Sensitivity and Specificity Values for anti-LC1 and other markers for autoimmune hepatitis were obtained over a 14-year period (1981--1994) in which the sera of 120,936 new patients were tested for liver disease autoantibodies (Table 3). Isolated anti-LC1 were found in 36 patients (0.03%), anti-LC 1-associated with anti-LKM- 1 in 65 patients (0.05%) and isolated anti-LKM-1 in 307 patients (0.25%). According to the diagnoses of this group of patients (Table 2), the sensitivity of anti-LC 1 for AIH-2 (0.48) is lower than that of anti-LKM-1 (0.84) for AIH-2, but the positive predictive value for the first antibody is greater (0.88 vs. 0.34). These values vary greatly by region and by the age of patients. Indeed, AIH-2 is found in young patients

460

contrarily to hepatitis C virus infection associated with anti-LKM-1 or anti-LC1 (Lunel et al., 1992). Moreover, the frequencies of the hepatitis C virus infection in blood donors were respectively established to 0.2, 0.7 and 1.5 in Great Britain, France or Germany and Italy.

CONCLUSION One of the two essential markers for AIH-2, anti-LC 1 antibodies can also be found in hepatitis C virus infection; such false-positive results must be eliminated by testing for HCV antibodies, HCV controlled by RIB A or by HCV RNA assay. A careful reading of the hepatocyte cytoplasmic fluorescence is essential for the classical detection of autoantibodies for liver diseases by IIF on rat liver/kidney/stomach. The immunodiffusion technique with liver cytosolic subcellular fraction can confirm the first result and can be used for a suspected autoimmune hepatitis

without markers. The use of immunoblot on human liver cytosol instead of that of rat will improve the anti-LC 1 detection and test sensitivity. See also ACTIN AUTOANTIBODIES, CRYOGLOBULINS SECONDARY TO

HEPATITIS C VIRUS INFECTION, LIVER]KIDNEY MICROSOMAL AUTOANTIBODIES and SMOOTH MUSCLE AUTOANTIBODIES.

REFERENCES

system in type 2 autoimmune hepatitis and hepatitis C virus infection. Gut 1995;36:749-754. Lunel F, Abuaf N, Frangeuf L, Grippon P, Perrin M, Le Coz Y, Valla D, Borotto E, Yamamoto AM, Huraux JM, et al. Liver/kidney microsome antibody type 1 and hepatitis C virus infection. Hepatology 1992; 16:630-636. Maggiore G, Homberg JC, Bernard O. Antiliver cytosol antibody (anti-LC1) identifies a new subgroup of children with autoimmune hepatitis. J Pediat Gastroenterol Nutr 1993;17:376--381. Manns M, Gerken G, Kyriatsoulis A, Staritz M, Meyer zum Bfischenfelde KH. Characterisation of a new subgroup of autoimmune chronic hepatitis by autoantibodies against a soluble liver antigen. Lancet 1987;1:292--294. Martini E, Abuaf N, Cavalli F, Durand V, Johanet C, Homberg JC. Antibody to liver cytosol (anti-LC1) in patients with autoimmune chronic active hepatitis type 2. Hepatology 1988;8:1662-1666. Michel G, Ritter A, Gerken G, Meyer zum Bfischenfelde KH, Decker R, Manns MP. Anti-GOR and hepatitis C virus in autoimmune liver diseases. Lancet 1992;339:267--269. Pawlotsky JM, Abuaf N, Andr6 C, Deforges L, Duval J, Dumont D. Serum antiliver cytosol antibodies type 1 in hepatitis C virus infection. Gastroenterol Clin Biol 1994;21: 480.

Abuaf N, Johanet C, Chretien P, Martini E, Soulier E, Loperche S, Homberg JC. Characterization of the liver cytosol antigen type 1 reacting with autoantibodies in chronic active hepatitis. Hepatology 1992;16:892--898. Abuaf N, Johanet C, Soulie E, Loeper J, Homberg JC. Antiliver cytosol antibodies in hepatology: autoimmune hepatitis, viral hepatitis C and graft versus-host disease. In: Meyer zum Btishenfelde KH, Gutenberg J, Hoofnagle J, Manns M, eds. Immunology and the Liver (Falk Symposium 70). Dordrecht, The Netherlands: Kluwer Academic Publishers, 1993:215226. Han S, Tredger M, Gregorio GV, Mieli-Vergani G, Vergani D. Antiliver cytosolic antigen type 1 (LC1) antibodies in childhood autoimmune liver disease. Hepatology 1995;21: 58--62. Johnson PJ, MacFarlane IG. Meeting report: International Autoimmune Hepatitis Group. Hepatology 1993;18:9981005. Lenzi M, Manotti P, Muratori L, Cataleta M, Ballardini G, Cassani F, Bianchi FB. Diagnostic relevance of LC 1 antigenantibody system in autoimmune hepatitis type 2. J Hepatol 1993; 18(Suppl):S7. Lenzi M, Manotti P, Muratori L, Cataleta M, Ballardini G, Cassani F, Bianchi FB. Liver cytosolic 1 antigen-antibody

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

LIVER/KIDNEY MICROSOMAL AUTOANTIBODIES Michael P. Manns, M.D.

Department of Gastroenterology and Hepatology, Zentrum Innere Medizin, Medizinische Hochschule Hannover, Hannover, Germany

HISTORICAL NOTES First reported in 1973 in patients with chronic active hepatitis, liver/kidney microsomal (LKM) antibodies are characterized using indirect immunofluorescence by their reaction with the cytoplasm of hepatocytes and proximal, but not distal, renal tubules (Rizzetto et al., 1973). By contrast, the antimitochondrial antibodies characteristic of primary biliary cirrhosis do stain distal renal tubules. LKM-1 antibodies, which are serological markers for autoimmune hepatitis type II (Homberg et al., 1987), must be distinguished from LKM-2 antibodies that occur in the drug-induced hepatitis caused by tienilic acid (Homberg et al., 1984). LKM-3 antibodies are found in some patients with chronic hepatitis D (Crivelli et al., 1983). Antigen characterization and identification started with the demonstration that LKM-1 antibodies react in immunoblots with a 50 kd liver microsomal protein (Alvarez et al., 1985). In 1989, human cytochrome P450 II D6 was shown to be the major autoantigen for LKM-1 antibodies (Zanger et al., 1988; Manns et al., 1989; Gueguen et al., 1989) which recognizing a main autoepitope sequence of eight amino acids that is also found in an intermediate early protein of herpes simplex virus type I (Manns et al., 199 l a). Some patients with chronic hepatitis C develop liver/kidney microsomal antibodies with an immunofluorescent pattern typical of LKM-1 (Lenzi et al., 1991; Michel et al., 1992; Lunel et al., 1992). In chronic hepatitis C, however, these LKM-1 antibodies react with different epitopes on cytochrome P450 II D6 (50 kd) or additional proteins at 59 and 70 kd (Durazzo et al., 1995; Duclos-Vallee et al., 1995; Ma et al., 1994). Liver membrane (LM) antibodies reac-

462

tive with cytochrome P450 I A2 (52 kd), which is expressed in periportal hepatocytes, are associated with rare cases of autoimmune hepatitis and, in particular, with drug-induced hepatitis caused by dihydralazin (Bourdi et al., 1990; Manns et al., 1990b; Sacher et al., 1990). Recently the major autoantigen (55 kd) of LKM-3 antibodies associated with hepatitis D was identified as UDP glucuronosyl transferase (Philipp et al., 1994).

THE AUTOANTIGENS Several molecular targets of these LKM and LM antibodies are now known (Table 1). LKM-1 antibodies react with cytochrome P450 II D6 (Zanger et al., 1988; Manns et al., 1989; Gueguen et al., 1989); LKM-2 antibodies react with cytochrome P450 II C9 (Beaune et al., 1987). The major antigen of LKM 3 antibodies associated with chronic hepatitis D is an epitope in the constant region of family 1 UDP glucuronosyl transferases (Philipp et al., 1994). Some sera react with an epitope of family 2 UDP glucuronosyl transferases (Philipp et al., 1994). LM antibodies react with cytochrome P450 1 A2 and are found in dihydralazininduced hepatitis (Bourdi et al., 1990) and autoimmune hepatitis (Manns et al., 1990a). Liver/kidney microsomal autoantigens of 59 and 70 kd are associated with chronic hepatitis C, but are not further characterized (Durazzo et al., 1995). Consistent with a role as the major autoantigen, recombinant cytochrome P450 II D6 is specifically recognized by T lymphocytes cloned from liver biopsies of patients with autoimmune chronic, active hepatitis (Lohr et al., 1991). The other LKM and LM antibodies and their antigens have not been studied to the same extent as cytochrome P450

Table 1. Microsomal Autoantigens in Liver Disease

Autoantibody

Major Autoantigen

MW (kd)

Associated Disease

LKM-1

P450 II D6

50

Autoimmune hepatitis

LKM-2

P450 II C9

50

Drug-induced hepatitis

LKM-3

UGT 1

55

Chronic hepatitis D

LM

P450 I A2

52

Autoimmune hepatitis Dihydralazine hepatitis

Disulfide isomerase

57

Halothane hepatitis

Carboxylesterase

59

Halothane hepatitis

9

59

Chronic hepatitis C

9

64

Autoimmune hepatitis

9

70

Chronic hepatitis C

II D6 concerning their potential pathogenetic role. However, LKM-2 and LM antibodies also inhibit the function of their antigens in vitro. The expression of cytochrome P450 II D6 is regulated by cytokines. As a negative acute-phase protein, the cellular expression of cytochrome P450 II D6 is downregulated by acute-phase mediators ILl, IL6 and tumor necrosis factor ~ (Trautwein et al., 1992). Some patients treated with interferon experience a deterioration of chronic, active hepatitis type II independent of whether they are HCV-negative or HCV-positive (Ruiz-Moreno et al., 1991).

THE AUTOANTIBODIES Pathogenetic Role

LKM and LM autoantibodies are not likely to play a role in pathogenesis of autoimmune hepatitis. For example, although LKM-1 antibodies do inhibit the function of cytochrome P450 II D6 in vitro, there is apparently insufficient penetration of the liver cell membrane in vivo to cause enzyme inhibition, drug metabolism which is dependent on the enzyme activity of this cytochrome is unimpaired (Manns et al., 1990b). Although expression of cytochrome P450 II D6 and other cytochrome P450 proteins was reported on the surface of liver cells (Loeper et al., 1993), such membrane expression was not confirmed (Yamamoto et al., 1993; Trautwein et al., 1993). Genetics

Cytochrome P450 II D6 is known to be genetically

polymorphic; 5--10% of Caucasians do not express the protein in their livers (Gonzales et al., 1988). Studies of the genetic polymorphism of cytochrome P450 II D6 in patients with autoimmune hepatitis type II show that all patients are extensive metabolizers (Manns et al., 1990b) and carry at least one wild-type allele of cytochrome P450 II D6 (Manns et al., 1991b; Yamamoto et al., 1992). The molecular mimicry of the major autoepitope of cytochrome P450 II D6 in autoimmune hepatitis type II with the immediate early 175 protein (IE 175) of herpes simplex virus type I (Manns et al., 1991a) is relevant to a reported set of identical twins; one twin had both autoimmune hepatitis and serological evidence of herpes simplex virus infection; whereas, none of the other family members had either (Manns et al., 1990c). Methods of Detection

The standard, albeit probably not the best, technique for the detection of LKM-1, LKM-2 and LKM-3 antibodies as well as LM antibodies is indirect immunofluorescence using cryostat sections of rodent liver and kidney. It is important to emphasize that LKM antibodies do not stain distal renal tubules, is an important distinction from antimitochondrial antibodies directed against pyruvate dehydrogenase subunit E2 (Storch, 1983). Immunoblot detection of LKM antibodies with isolated human or rodent liver microsomes reveals a band at 50 kd with LKM-1 and LKM-2 sera; whereas, LKM-3 react at 55 kd and LM antibodies stain a protein at 52 kd (Kyriatsoulis et al., 1987; Durazzo et al., 1995; Philipp et al., 1994; Beaune et al., 1987).

463

An ELISA technique can be used to detect conformational epitopes on cytochrome P450 II D6 (Manns et al., 1984). Inhibition of the enzyme activity of cytochrome P450 II D6 can also be used to detect LKM-1 antibodies (Zanger et al., 1988). Finally, LKM-1, LKM-2, LKM-3 and LM antibodies can be detected with recombinant proteins by ELISA or immunoblotting (Manns and Johnson, 1991; Seelig et al., 1993; Philipp et al., 1994; Stragburg et al., 1995; Yamamoto et al., 1993; Manns et al., 1991a). Immunoblot detection using recombinant P450 II D6 is reported to be the most sensitive and specific method for detection of LKM-1 autoantibodies in the clinical laboratory (Seelig et al., 1993).

CLINICAL UTILITY Disease Associations

LKM-1 antibodies directed against cytochrome P450 II D6 are markers of autoimmune hepatitis type II (Homberg et al., 1987). The disease often starts in childhood. There is a predominance of females, low IgA levels, frequent association with extrahepatic autoimmune syndromes and good response to immunosuppression. Autoimmune hepatitis type II can start with acute hepatitis or even fulminant hepatitis. LKM-1 antibodies can also be detected by immunofluorescence in up to 7% of patients with chronic hepatitis C (Reddy et al., 1993; Czaja et al., 1993; Abuaf et al., 1993; Pawlotsky et al., 1994). When associated with chronic hepatitis C virus infection, they are heterogeneous and react with linear or conformational epitopes of 50 kd cytochrome P450 II D6 (Durazzo et al., 1995; Duclos-Vallee et al., 1995) and other unidentified proteins at 59 and 70 kd. Preliminary data suggest that the response to interferon may depend on the reactivity with these different proteins (Durazzo et al., 1995). Single case reports indicate that some patients with hepatitis C and LKM-1 antibodies may have an increased risk to develop a deterioration of their disease, when treated with interferon. Controlled trials are necessary to confirm this observation. In rare cases, LKM-1 antibodies may be associated with halothane hepatitis. LKM-2 antibodies against cytochrome P450 II C9 are closely associated with a drug-induced hepatitis caused by tienilic acid (Homberg et al., 1984; Beaune

464

et al., 1987). This drug was prescribed only in the U.S. and France. LKM-2 antibodies in drug-induced hepatitis due to tienilic acid should no longer occur, because the drug is withdrawn from the market. LKM-3 antibodies occur in 10--15% of patients with chronic hepatitis D and are directed against an epitope on family 1 UDP glucuronosyl transferases (Philipp et al., 1994; Stragburg et al., 1995). Whether this is of clinical relevance is unknown. Some 10% of patients with autoimmune hepatitis type II have serum LKM-3 autoantibodies against UGT proteins, in addition to LKM-1 autoantibodies against cytochrome P450 II D6. In genuine autoimmune hepatitis, autoepitopes of cytochrome P450 proteins and of UGT proteins are linear and small; whereas, the epitopes are larger and heterogeneous in viral hepatitis C and viral hepatitis D (Durazzo et al., 1995). In addition, the LKM autoantibody titers are much higher in genuine autoimmune hepatitis than in viral hepatitis C or D (Durazzo et al., 1995). LM antibodies occur in rare cases of autoimmune hepatitis (Sacher et al., 1990; Manns et al., 1990a) and in drug-induced hepatitis caused by dihydralazin (Bourdi et al., 1990). There is another uncharacterized LKM antigen at 64 kd (Manns et al., 1989; CodonerFranch et al., 1989).

CONCLUSION

Detection of LKM and LM autoantibodies can be used to assess autoimmune hepatitis. For routine purposes, indirect immunofluorescence on cryostat sections of rat liver and kidney is still the screening method of choice. For rigorous confirmation, positive results require immunoblots with human liver microsomes to check for reactivity with proteins between 50 and 55 kd. Positive results can be further validated by testing with recombinant cytochrome P450 II D6, cytochrome P450 II C9, cytochrome P450 I A2 or family 1 UDP glucuronosyl transferases. For controls, one can use mitochondrial proteins containing 48 kd branchedchain ketoacid dehydrogenase (BCKD-E2), 52 kd oxoglutarate dehydrogenase (OGDC-E2) and 74 kd pyruvate dehydrogenase subunit E2. Additional studies are needed to confirm and perhaps extend the role of specific autoantibodies in management of patients with autoimmune hepatic conditions. See also LIVER MEMBRANE AUTOANTIBODIES.

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Kyriatsoulis A, Manns M, Gerken G, Lohse AW, Ballhausen W, Reske K, Meyer zum BiJschenfelde K-H. Distinction between natural and pathological autoantibodies by immunoblotting and densitometric subtraction. Liver-kidneymicrosomal antibody (LKM) positive sera identify multiple antigens in human liver tissue. Clin Exp Immunol 1987;70: 53-60. Lenzi M, Johnson PJ, McFarlane IG, Ballardini G, Smith HM, McFarlane BM, Bridger C, Vergani D, Bianchi FB, Williams R. Antibodies to hepatitis C virus in autoimmune liver disease: evidence for geographical heterogeneity. Lancet 1991;338:277-280. Loeper J, Descatoire V, Maurice M, Beaune P, Belghiti J, Houssin D, Ballet F, Feldman G, Guengerich FP, Pessayre D. Cytochromes P-450 in human hepatocyte plasma membrane: recognition by several autoantibodies. Gastroenterology 1993;104:203-216. Lohr H, Manns M, Kyriatsoulis A, Lohse AW, Trautwein C, Meyer zum Buschenfelde K-H, Fleischer B. Clonal analysis of liver-infiltrating T cells in patients with LKM-1 antibody positive autoimmune chronic active hepatitis. Clin Exp Immunol 1991;84:297-302. Lunel F, Abuaf N, Frangeul L, Grippon P, Perrin M, Le Coz Y, Valla D, Borotto E, Yamamoto AM, Huraux JM, Opolon P, Homberg J-C. Liver/kidney microsome antibody type 1 and hepatitis C virus infection. Hepatology 1992;16:630-636. Ma Y, Peakman M, Lobo-Yeo A, Wen L, Lenzi M, Gaken J, Farzaneh F, Mieli-Vergani G, Bianchi FB, Vergani D. Differences in immune recognition of cytochrome P4502D6 by liver kidney microsomal (LKM) antibody in autoimmune hepatitis and chronic hepatitis C virus infection. Clin Exp Immunol 1994;97:94--99. Manns M, Meyer zum Bfischenfelde K-H, Slusarczyk J, Dienes HP. Detection of liver-kidney microsomal autoantibodies by radioimmunoassay and their relation to antimitochondrial antibodies in inflammatory liver diseases. Clin Exp Immunol 1984 ;57:600-608. Manns MP, Johnson EF, Griffin KJ, Tan EM, Sullivan KF. Major antigen of liver kidney microsomal autoantibodies in idiopathic autoimmune hepatitis is cytochrome P450dbl. J Clin Invest 1989;83:1066--1072. Manns MP, Griffin KJ, Quattrochi LC, Sacher M, Thaler H, Tukey RH, Johnson EF. Identification of cytochrome P450IA2 as a human autoantigen. Arch Biochem Biophys 1990a;280:229--232. Manns M, Zanger U, Gerken G, Sullivan KF, Meyer zum Buschenfelde KH, Meyer UA, Eichelbaum M. Patients with type II autoimmune hepatitis express functionally intact cytochrome P-450 dbl that is inhibited by LKM-1 autoantibodies in vitro but not in vivo. Hepatology 1990b;12:127132. Manns M, Kaletzko S, Lohr H, Borchard F, Rittner C, Meyer zum BiJschenfelde K-H, Eichelbaum M. Discordant manifestation of LKM-1 antibody positive autoimmune hepatitis in identical twins. Hepatology 1990c;12:840. Manns MP, Griffin KJ, Sullivan KF, Johnson EF. LKM-1 autoantibodies recognize a short linear sequence in P450 II D6, a cytochrome P-450 monooxygenase. J Clin Invest

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1991 a;88:1370-1378. Manns M, Scheucher S, Jentsch M, Broly F, Goergen B, Schneider P, Gerken G, Rittner CH, Meyer zum Btischenfelde K-H, Meyer UA. Genetics in autoimmune hepatitis type 2. Hepatology 1991b;14:60A. Manns M, Johnson EF. Identification of human cytochrome P450s as autoantigens. In: Manns MP, Johnson EF, eds. Methods in Enzymology, Volume 206. Orlando: Academic Press Inc., 1991. Michel G, Ritter A, Gerken G, Meyer zum Btischenfelde K-H, Decker R, Manns MP. Anti-GOR and hepatitis C virus in autoimmune liver diseases. Lancet 1992;339:267--269. Pawlotsky JM, Ben Yahia M, Andre C, Voison MC, Intrator L, Roudot-Thoraval F, Deforges L, Duvoux C, Zafrani ES, Duval J, Dhumeaux D. Immunological disorders in C virus chronic active hepatitis: a prospective case-control study. Hepatology 1994;19:841-848. Philipp T, Durazzo M, Trautwein C, Alex B, Straub P, Lamb JG, Johnson EF, Tukey RH, Manns MP. Recognition of uridine diphosphate glucuronosyl transferases by LKM-3 antibodies in chronic hepatitis D. Lancet 1994;344:578--581. Reddy KR, Krawitt EL, Radick J, Chastenay B, de Medina M, Jeffers LJ, Parker T, Poupon R, Opolon P, Beaugrand M, Johanet C, Abuaf N, Gergeois J, Homberg JC, Schiff ER. Absence of LKM-1 antibody in hepatitis C viral infection in the United States. Hepatology 1993; 18:173A. Rizzetto M, Swana G, Doniach D. Microsomal antibodies in active chronic hepatitis and other disorders. Clin Exp Immunol 1973;15:331--334. Ruiz-Moreno M, Rua MJ, Carreno V, Quiroga JA, Manns M, Meyer zum Btischenfelde K-H. Autoimmune chronic active hepatitis type 2 manifested during interferon therapy in children. J Hepatol 1991;12:265-266. Sacher M, Blumel P, Thaler H, Manns M. Chronic active hepa

466

titis associated with vitiligo, nail dystrophy, alopecia and a new variant of LKM antibodies. J Hepatol 1990; 10:364--369. Seelig R, Renz M, Gtinger G, Schrtiter H, Seelig HP. AntiLKM-1 antibodies determined by use of recombinant P450 2D6 in ELISA and Western blot and their association with anti-HCV and HCV-RNA. Clin Exp Immuol 1993;92:373-380. Storch W. [Diagnostic significance of antibodies against cell organelles (mitochondria, endoplasmic reticulum and ribosomes)]. Schweiz Med Wochenschr. 1983;113:47--59. Stragburg CP, Obermayer-Straub P, Alex B, Philipp T, Tukey RH, Manns M. Autoepitopes on UDP-glucuronosyltransferases (LKM-3) in autoimmune hepatitis differ from those in hepatitis D. Gastroenterology 1995;108:A1177. Trautwein C, Ramadori G, Gerken G, Meyer zum Btischenfelde K-H, Manns M. Regulation of cytochrome P450 IID by acute phase mediators in C3H/HeJ mice. Biochem Biophys Res Commun 1992;182:617-623. Trautwein C, Gerken G, Lohr H, Meyer zum Btischenfelde KH, Manns M. Lack of surface expression for the B-cell autoepitope of cytochrome P450 IID6 evidenced by flow cytometry. Z Gastroenterol 1993;31:225-230. Yamamoto AM, Mura C, Morales MG, Bernard O, Krishnamoorthy R, Alvarez F. Study of CYP2D6 gene in children with autoimmune hepatitis and P450 IID6 autoantibodies. Clin Exp Immunol 1992;87:251--255. Yamamoto AM, Mura C, De Lemos-Chiarandini C, Krishnamoorthy R, Alvarez F. Cytochrome P450IID6 recognized by LKM 1 antibody is not exposed on the surface of hepatocytes. Clin Exp Immunol 1993;92:381-390. Zanger UM, Hauri HP, Loeper J, Homberg JC, Meyer UA. Antibodies against human cytochrome P-450dbl in autoimmune hepatitis type II. Proc Natl Acad Sci USA 1988;85: 8256-8260.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

LIVER M E M B R A N E AUTOANTIBODIES Ulrich Treichel, M.D. and Karl-Hermann Meyer zum Btischenfelde, M.D.

Department of Internal Medicine, Johannes Gutenberg University Mainz, 55101 Mainz, Germany

H I S T O R I C A L NOTES Some chronic liver diseases like primary biliary cirrhosis and autoimmune hepatitis are characterized by a specific pattern of autoantibodies. However, the target antigens for these antibodies are mainly located intracellularly (Meyer zum Btischenfelde et al., 1990). The physiological expression of intracellular target antigens on the liver cell surface like the antigen from liver and kidney microsomes (LKM) remains questionable or minimal (Robin et al., 1995). Therefore, liver-specific and membrane-expressed antigens for autoantibodies are of special interest with regard to a putative pathogenetic role for inflammatory liver diseases. Indeed, until recently a crude preparation, so-called "liver-specific membrane lipoprotein" (LSP), is the only antigen shown to induce an autoimmune-like liver disease in experimental models, including rabbits and mice (Meyer zum Btischenfelde et al., 1972; Lohse et al., 1990). Furthermore, antibodies against LSP, in contrast to their counterparts such as antinuclear antibodies or smooth muscle antibodies, can predict the clinical outcome of an autoimmune liver disease in man (McFarlane et al., 1984a). These experimental and clinical data initiated a search for the relevant target antigen(s) in LSP which could induce and perpetuate antiliver membrane autoreactivity. Several different studies were performed, such as mapping of LSP preparations by murine monoclonal antibodies (Poralla et al., 1984), immunoprecipitation (Kakumu et al., 1979) and density gradient centrifugation (Manns and Meyer zum Biischenfelde, 1982). Furthermore, functionally characterized monoclonal anti-LSP antibodies allowed mapping of immunoreactive LSP epitopes including a 43 kd protein which is expressed on the plasma

membrane of liver cells. This target antigen was attacked by an antibody-dependent cell lysis ultimately leading to experimental liver cell damage (Poralla et al., 1987). However, the epitope was never characterized by sequence analysis. A landmark for the characterization of relevant LSP components was the detection of the asialoglycoprotein receptor (ASGPR) in the LSP preparation (McFarlane et al., 1984b). Known since 1968 as a liver-specific receptor mediating endocytosis of galactose-terminating glycoproteins (Morell et al., 1968), the ASGPR is now defined by amino acid composition and structure (Spiess, 1990). A link of antibodies to ASGPR (anti-ASGPR) with acute and chronic liver inflammation was defined in 1986 (McFarlane et al., 1986). The characterization of antibodies directed against the hepatic plasma membrane provided a different view of so-called liver membrane antibodies (LMA) (Hopf et al., 1976). Defined by a specific fluorescence pattern after binding to isolated hepatocytes, these antibodies defined an immunodominant epitope on a 26 kd protein of unknown amino acid composition (Hopf et al., 1990). Use of plasma membrane proteins from isolated liver cells for immunoblot analysis of serial dilutions of defined sera showed widely varying amounts needed to detect antigenic membrane components. A 60 kd immunodominant band from the liver membranes was described (Swanson et al., 1990). To date, this protein is unidentified. Because characterization of the LMA 26 kd protein and the antigens for antiliver plasma membrane antibodies (including those reactive with the 60 kd protein) are incomplete, studies of the prevalence of antibodies to these proteins are not possible, and their roles in diagnosis or prognosis remain to be elu-

467

cidated. On the other hand, the role of anti-ASGPR in inflammatory liver diseases is clearer because they are found mainly in sera from patients with autoimmune hepatitis (AIH) and can, therefore, be helpful for both diagnosis and prognosis of AIH (Poralla et al., 1991). These antibodies should be considered for the differential diagnosis of chronic hepatitis. According to the recommendation of the International Autoimmune Hepatitis Group, assays for anti-ASGPR should be considered for the differential diagnosis of chronic hepatitis (Johnson and McFarlane, 1993).

THE AUTOANTIGENS

Definitions Preparations from different liver sources are used to identify liver membrane-specific antibodies (Table 1). Among these, only the liver-specific ASGPR is well defined as a physiological autoantigenic constituent of the hepatic plasma membrane. A specific immunofluorescence pattern on isolated hepatocytes is characteristic of so-called liver membrane antibodies (LMA) (Hopf et al., 1976). An LMA radioimmunoassay is also available (Wiedmann et al., 1984). The LMA targets are apparently heterogenous and are distinct from LSP epitopes (Meyer zum Btischenfelde et al., 1979). There is some concern on technical grounds about the definition of LMA, i.e., the generation of artificial epitopes (Gerken et al., 1987). A 26 kd protein beating an LMA epitope was identified by immunoabsorption with human antisera but was not further characterized (Hopf et al., 1990).

Origin and Sources/Methods of Purification LSP. The crude liver extract LSP (liver-specific lipoprotein) is prepared by gel filtration chromatography from tissue homogenate after ultracentrifugation with 100,000 x g (Meyer zum Btischenfelde et al., 1972). Detection of antibodies to LSP (anti-LSP) by radioimmunoassay or enzyme immunoassay yields a high rate of false-positive results (McFarlane et al., 1983). LSP is now known to contain the ASGPR (McFarlane et al., 1984b), as well as liver-specific, non-organ-specific and non-species-specific components (Poralla et al., 1984). The wide variety of putative target molecules for liver membrane antibodies in LSP hampers more specific analysis of antibodies reactive with this antigen preparation. However, a mapping analysis with a murine monoclonal raised against LSP detected a liver-specific, 43 kd protein on the basolateral surface (Poralla et al., 1987). Unfortunately, the hybridoma died during longterm cell culture before the target protein could be purified or characterized. In particular, the protein itself was never purified. On liver tissue, the anti 43kd antibody recognized a liver-specific structure on the basolateral surface (Poralla et al., 1987). Human antibodies to isolated plasma membranes from human and animal liver were investigated by enzyme immunoassay (Swanson et al., 1985) and by an extended immunoblotting format after denaturation and separation by SDS-polyacrylamide electrophoresis (Swanson et al., 1990). Among different organ- and non-organ-specific bands characterized, a human-specific, 60 kd protein band was thought to be promising as a relevant candidate autoantigen on the liver cell surface. Again, this protein was not characterized in detail.

Table 1. Liver Membrane Antigens Name

Source

Preparation

Topology, Composition

Epitope Sequence

LSP

animal, human

gel filtration of homogenate

in part liver-specific

not known

43 kd protein

animal

not done

basolateral liver membrane

not known

LMA

animal

partial (26 kd protein)

liver membrane

not known

26 kd protein

animal

affinity chromatography

liver membrane

not known

Anti-HPM

animal

not done

in part liver-specific

not known

60 kd protein

human

not done

human liver membrane

not known

ASGPR

animal, human

affinity chromatography

basolateral liver membrane

in part defined

468

ASGPR. The ASGPR derived from both human and animal liver was studied in detail for its putative role in pathogenesis and also as a possible diagnostic tool (Poralla et al., 1991). Since its detection as a component of LSP, both humoral (McFarlane et al., 1986; Treichel et al., 1990; 1993a; 1994) and cellular (Wen et al., 1990; Vento et al., 1991; Lohr et al., 1990) immune response against this liver-specific receptor was found. A transmembrane molecule specifically expressed on the sinusoidal and basolateral site of the hepatocellular membrane, the ASGPR mediates the uptake and intracellular degradation of desialylated glycoproteins in the liver cell. Its biochemical properties are well known, including the DNA-sequences of two major subunits named H1 and H2 which were characterized in human hepatoma cell line HEpG2 (Harford and Ashwell, 1982; Spiess, 1990). The assembly of these subunits in heteromers is required for receptor-promoted endocytosis of asialoglycoproteins into hepatocytes (Shia and Lodish, 1989). However, the exact physiological role of the ASGPR is not yet defined. Preparation of ASGPR from liver is usually achieved by affinity chromatography on ligand resins from detergent-lysed liver. Using such a purification method, up to 30% of receptor activity can be recovered from human liver (Treichel et al., 1995). Cellular and humoral activity for the immune response against the ASGPR are available. T-cell responses are measured by migration inhibition and cell proliferation (Poralla et al., 1991); antibodies against the ASGPR were described by using radioimmunoassay, solidphase enzyme immunoassay and by immunoblotting (McFarlane et al., 1986; Treichel et al., 1990). Immunoblotting is less sensitive, and not suitable for screening detection of anti-ASGPR antibodies. Interestingly, however, a collaborative study shows good correlation between the different assays (Treichel et al., 1994). Experimental and clinical evidence suggests a variety of potential ASGPR epitopes: sera which are enzyme immunoassay positive and immunoblot negative presumably recognize conformational epitopes (Treichel et al., 1992). Some sera are able to inhibit receptor function in vitro (Treichel et al., 1992), perhaps corresponding to the hyperasialoglycoproteinemia observed in some patients with chronic liver diseases (Sawamura et al., 1984). Finally, the glycoside portion of a 10 kd cyanogen bromide fragment of human ASGPR contains an immunodominant epitope (Treichel et al., 1993b).

AUTOANTIBODIES M e t h o d s of Detection

Anti-LMA, anti-LSP and, lately, anti-ASGPR were investigated both as putative pathogens and clinical markers. LMA were originally detected by a linear immunofluorescence pattern on isolated hepatocytes (Hopf et al., 1976). The definition of the 26 kd protein has not lead to the development of an easy, reliable assay for LMA. Anti-LSP were detected by radioimmunoassay (McFarlane, 1984) and antiASGPR by radioimmunoassay, enzyme immunoassay and immunoblots in different laboratories (McFarlane et al., 1984a; Treichel et al., 1990). Although enzyme immunoassay for anti-LSP has high false-positive rates (McFarlane et al., 1983), assays for the detection of anti-ASGPR appear to be reliable: A blind study comparing an antirabbit ASGPR radioimmunoassay versus an anti-human-ASGPR enzyme immunoassay indicated a high number of congruent results even on the titer level (Treichel et al., 1994) (Figure 1). Currently under development is a commercial, recom1:2000

...........................................................................................................................................................

9 ...................

1 "1500 < rr r'r"

a. (9

r = 0,64

1 "1000

|

.121 .(3

~

1:500

t.,

o o

1:0

1:-500

1 "1 O0

1:400

1 : 1600

anti-human-ASGPR EIA

Figure 1. In a blindly conducted study, 37 coded sera were

analyzed for the presence of anti-ASGPR by radioimmunoassay (RIA) with rabbit substrate and enzyme immunoassay (EIA) with human substrate. Overall, the antibody titers demonstrated good correlation. However, the human substrate appears to be more sensitive, recognizing high concentrations in six sera exclusively. 469

binant-based, indirect immunofluorescence assay which yields results similar to recent published data (Pfeifer et al., 1994).

Pathogenetic Role Reactions of autoantibodies (nuclear, smooth muscle, . microsomal, soluble liver antigen) with their antigens is apparently not important for ' pathogenesis of the chronic liver disease. Antibodies against liver membrane-specific target antigens are found in sera from patients with different liver and nonhepatic diseases (McFarlane, 1984; McFarlane et al., 1986; Treichel et al., 1990). Frequencies of anti-LSP, anti-LMA and anti-ASGPR are compared in Table 2. Although not disease-specific, anti-LMA as well as anti-LSP occur most often in chronic inflammatory liver diseases of both viral and autoimmune origin. Indeed, in some studies, all patients with autoimmune hepatitis are positive for anti-LMA or anti-LSP (McFarlane, 1984). In patients with nonhepatic diseases, liver-specific antibodies are found less frequently.

Liver membrane antibodies and autoimmune hepatitis. Whereas, antibodies to LMA or LSP are not linked to a specific entity, antibodies against the liverspecific ASGPR are more disease specific, and the specificity rises if human substrate is used (Treichel

et al., 1990). However, some patients with nonhepatic diseases or nonautoimmune liver diseases are antiASGPR-positive, in particular, patients with hepatitis B and acute hepatitis. A prospective study showed that anti-LSP are associated with inflammatory activity in autoimmune hepatitis (McFarlane et al., 1984a). A retrospective study demonstrated that antiASGPR are linked to inflammatory activity and that all patients after confirmed successful immunosuppressive treatment show decreased or nondetectable titers of circulating anti-ASGPR (Treichel et al., 1993a) (Figure 2).

Immunological features of liver membrane antibodies. The occurrence of antibodies against liver membrane structures is presumably the result of a complex immune response. The induction of antiASGPR is in part T-cell dependent, because ASGPRspecific T-cell clones in vitro induce anti-ASGPR secretion by peripheral blood lymphocytes (Lohr et al., 1992). Earlier studies showed that the T-cell response against ASGPR is HLA restricted (Poralla et al., 1991). In patients, a correlation between any liver membrane antibody (LMA, LSP or ASGPR) and a certain HLA haplotype has so far not been described. Moreover, family studies are not available. The occurrence of anti-ASGPR-positive autoimmune hepatitis in a young boy with healthy anti-ASGPR-

Table 2. Frequencies of Liver-Membrane Antibodies Disease Normal individuals

Anti-LMA 0%

Anti-LSP 0%

Anti-ASGPR very rare

Autoimmune hepatitis, active

37--100%

50-100%

83--87%

Autoimmune hepatitis, remission

not done

not done

0-41%

Acute viral hepatitis

0-17%

11--93%

Chronic viral hepatitis B

0-16%

28--93%

6--57%

0-10%

2--12%

Chronic viral hepatitis C/NANB

90%

14%

Cryptogenic cirrhosis

0-61%

20-38%

not done

Primary biliary cirrhosis

0-42%

33-51%

14%

not done

not done

0%

Alcoholic liver disease

0-27%

0-36%

8%

Other liver diseases

0-2%

0-17%

0%

Nonhepatic autoimmunopathies

0-4%

0-18%

0--11%

Nonhepatic inflammatory diseases

not done

not done

3%

Malignancies in the liver

not done

not done

11%

Primary sclerosing cholangitis

470

In a different approach, the effect of anti-ASGPRpositive sera on the ASGPR-function in vitro on hepatoma HEpG2-cells was assessed (Treichel et al., 1994); some but not all sera inhibited the uptake of asialoglycoproteins via ASGPR. Despite the low number of individuals studied, a tendency toward a predominant inhibitory activity in sera from patients with autoimmune hepatitis was observed.

Figure 2. The anti-ASGPR antibody titer were measured in a group of patients with confirmed autoimmune hepatitis for one year beginning with the day of initiation of immunosuppressive treatment and in seven normals. All anti-ASGPR antibodypositive patients (n = 20) demonstrated a sharp decrease of antiASGPR antibody titer; in a subgroup (n = 13) the titer fell below the detection level (Treichel et al., 1993a).

negative twin siblings and parents (Huppertz et al., 1995) suggests a special pathway for the induction of anti-ASGPR.

Effect of liver membrane antibody binding on target autoantigen. Among the different targets for liver membrane antibodies as listed (Table 1) only the effect of antibody-binding to the 43 kd protein and the ASGPR was investigated. A murine monoclonal antibody which recognizes the 43 kd protein detected a structure specific for the basolateral liver membrane (Poralla et al., 1987) and caused damage in vitro. Interpretation of results showing liver damage is difficult, because the murine antibody was raised against rabbit LSP. Use of a xenogeneic system to induce experimental liver cell death was also described for experimental autoimmune hepatitis in rabbits injected with human liver proteins (Meyer zum Btischenfelde et al., 1972). Efforts to establish liverspecific cell lysis in an autologous system are needed.

The putative pathophysiological significance of liver membrane antibodies. When injected into rabbits, xenogeneic LSP, the liver-related antigens shown to cause experimental autoimmune hepatitis in animals (Meyer zum Bt~schenfelde et al., 1972; Lohse et al., 1990), induces lesions in the liver architecture which are similar to the characteristic piecemeal necrosis seen in man. The rabbits also developed antiLSP. In a later investigation, the administration of autologous LSP-like liver protein (so-called supernatant S-100) into mice induced transient lesions in the liver, and the disease could be adoptively transferred to naive animals by splenocytes (Lohse et al., 1990). From the clinical point of view, antibodies against LSP are said to be a useful prognostic ally for treatment withdrawal in chronic AIH (McFarlane et al., 1984a). A similar association between anti-LSP and disease activity was observed (unpublished data). Anti-ASGPR were retrospectively found to be correlated to the disease activity (Figure 2). Therefore, the pathogenic potency of LPS appears to be well established. However, as demonstrated in Table 2, anti-LSP lack disease specificity. The role of welldefined ASGPR for the pathogenesis of chronic hepatitis has to be clarified in the future. Factors in Pathophysiology Taken together, the mechanism(s) for the induction of anti-ASGPR and other liver membrane antibodies remains to be defined. Among these antibodies antiLMA were characterized as immunoglobulin class IgG (Hopf et al., 1976). Isotypes, subclasses, idiotypes were further investigated concerning anti-ASGPR (Treichel et al., 1993b). Both anti-ASGPR IgG and IgM were found. Interestingly, the anti-ASGPR IgM were undetectable in patients with clinical remission of autoimmune hepatitis induced by immunosuppressive therapy; whereas, in untreated patients IgM and the IgG2 isotype were found. In contrast, in viral hepatitis, anti-ASGPR belong to the IgM and predominantly to the IgG4 isotype.

471

CLINICAL UTILITY

CONCLUSION

Disease Association

The characterization of different target antigens on the hepatic plasma membrane shows a polyvalent and heterogenous humoral immune response against the liver surface. Long assumed to have a pathophysiological role in the induction and/or maintenance of chronic inflammatory liver diseases, liver membrane antibodies can induce liver cell damage in vitro, including dysfunction of the target antigen. The autoimmune responses against these organ-specific structures are almost certain to be pathogenically relevant. Among the established liver membrane-related autoantigens, the asialoglycoprotein receptor (ASGPR) represents the only component of the hepatic plasma membrane which is fully characterized to date. Assays for the detection of anti-ASGPR are reliable. The clinical data support the utility of measuring antiA S G P R for the differential diagnosis of patients with chronic inflammatory liver disease (prevalence >80% in autoimmune hepatitis) and during their follow-up (downregulation or elimination during response to immunosuppressive therapy).

Chronic liver inflammatory diseases are associated to a greater or lesser extent with a heterogenous group of autoantibodies (Table 2). Hepatotropic and non-organspecific viruses as well as other pathogens can cause a chronic hepatitis which often leads to progressive liver dysfunction and cirrhosis. Thought to account for up to 15% of chronic liver inflammatory diseases, suspected autoimmune diseases of the liver includes three distinct entities: 1. Autoimmune hepatitis with very good response to immunosuppressive therapy. 2. Primary b!liary cirrhosis with disease-specific mitochondrial antibodies and without response to immunosuppressive therapy. 3. Primary sclerosing cholangitis characteristically with a close association to inflammatory bowel disease. The classical autoimmune disease, i.e., autoimmune hepatitis, is characterized by certain age, sex and HLA-associations (young female with HLA A1, B8, DR3 or DR4) as well as by the occurrence of autoantibodies (nuclear, smooth muscle, microsomal, soluble liver antigen antibodies) (Czaja, 1995).

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Kakumu S, Arakawa Y, Goji H, Kashio T, Yata K. Occurrence and significance of antibody to liver-specific membrane lipoprotein by double-antibody immunoprecipitation method in sera of patients with acute and chronic liver diseases. Gastroenterology 1979;76:665--672. Lohr H, Treichel U, Poralla T, Manns M, Meyer zum Btischenfelde KH, Fleischer B. The human hepatic asialoglycoprotein receptor is a target antigen for liver-infiltrating T cells in autoimmune chronic active hepatitis and primary biliary cirrhosis. Hepatology 1990;12:1314--1320. Lohr H, Treichel U, Poralla T Manns M, Meyer zum Btischenfelde KH. B Liver-infiltrating T helper cells in autoimmune chronic active hepatitis stimulate the production of autoantibodies against the human asialoglycoprotein receptor in vitro. Clin Exp Immunol 1992;88:45--49. Lohse AW, Manns M, Dienes HP, Meyer zum Btischenfelde KH, Cohen IR. Experimental autoimmune hepatitis: Disease induction, time course and T-cell reactivity. Hepatology 1990; 111:24-30. Manns M, Meyer zum Btischenfelde KH. Fractionation of the liver membrane lipoprotein (LSP) and characterization of its antigenic determinants by autoantibodies and heterologous antiserum. Gut 1982;23:14-20. McFarlane IG, Tolley P, Major G, Williams BM, Williams R. Development of a micro enzyme-linked immunosorbent assay

for antibodies against liver-specific membrane lipoprotein. J Immunol Methods 1983;64:215--225. McFarlane IG. Autoimmunity in liver disease. Clin Sci 1984; 67:569--578. McFarlane IG, Hegarty JE, McSorley CG, McFarlane BM, Williams R. Antibodies to liver-specific protein predict outcome of treatment withdrawal in autoimmune chronic active hepatitis. Lancet 1984a;2:954--956. McFarlane IG, McFarlane BM, Major GN, Tolley P, Williams R. Identification of the hepatic asialo-glycoprotein receptor (hepatic lectin) as a component of liver specific membrane lipoprotein (LSP). Clin Exp Immunol 1984b;55:347-354. McFarlane BM, McSorley CG, Vergani D, McFarlane IG, Williams R. Serum autoantibodies reacting with the hepatic asialoglycoprotein receptor protein (hepatic lectin) in acute and chronic liver disorders. J Hepatol 1986;3:196-205. Meyer zum Btischenfelde KH, Kossling FK, Miescher PA. Experimental chronic active hepatitis in rabbits following immunization with human liver proteins. Clin Exp Immunol 1972; 11:99-108. Meyer zum Btischenfelde KH, Manns M, Hutteroth T, Hopf U, Arnold W. LM-Ag and L S P - two different target antigens involved in the immunopathogenesis of chronic active hepatitis? Clin Exp Immunol 1979;37:205-212. Meyer zum Btischenfelde KH, Lohse AW, Manns M, Poralla T. Autoimmunity and liver disease. Hepatology 1990;12:354-363. Morell AG, Irvine RA, Sternlieb I, Scheinberg IH, Ashwell G. Physical and chemical studies on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J Biol Chem 1968;243:155--159. Pfeifer K, Decker R, Czaja A, Vallari A, Michel G. Frequency of antibodies to ASGP-R in patients with autoimmune hepatitis, detected by RIFA. Hepatology 1994;20:387A. Poralla T, Manns M, Dienes HP, et al. Analysis of liver-specific protein LSP using monoclonal antibodies. Eur J Clin Invest 1984;17:360-367. Poralla T, Ramadori G, Dienes HP, Manns M, Gerken G, Dippold W, Hutteroth TH, Meyer zum Buschenfelde KH. Liver cell damage caused by a monoclonal antibody against an organ-specific membrane antigen in vivo and in vitro. J Hepatol 1987;4:373--380. Poralla T, Treichel U, Lohr H, Fleischer B. The asialoglycoprotein receptor as target structure in autoimmune liver disease. Semin Liver Dis 1991 ;11:215--222. Robin MA, Maratrat M, Loeper J, Durand-Schneider AM, Tinel M, Ballet F, Beaune P, Feldman G, Pessayre D. Cytochrome P4502B follows a vesicular route to the plasma membrane in cultured rat hepatocytes. Gastroenterology 1995; 108:111 0 1123.

Sawamura T, Nakada H, Hazama H, Shiozaki Y, Sameshima Y, Tashieo Y. Hyperasialoglycoproteinemia in patients with chronic liver diseases and/or liver cell carcinoma. Asialoglycoprotein receptor in cirrhosis and liver cell carcinoma.

Gastroenterology 1984;87:1217-- 1221. Shia MA, Lodish HF. The two subunits of the human asialoglycoprotein receptor have different fates when expressed alone in fibroblasts. Proc Natl Acad Sci USA 1989;86:158-162. Spiess M. The asialoglycoprotein receptor: a model for endocytic transport receptors. Biochemistry 1990;29:10009-10018. Swanson NR, Bartholomaeus WN, Reed WD, Joske RA. An enzyme-linked immunosorbent assay for the detection of hepatocyte plasma membrane antibodies. J Immunol Methods 1985;85:203-216. Swanson NR, Reed WD, Yarred LJ, Shilkin KB, Joske RA. Autoantibodies to isolated human hepatocyte plasma membranes in chronic active hepatitis. II. Specificity of antibodies. Hepatology 1990;11:613--621. Treichel U, Poralla T, Hess G, Manns M, Meyer zum Btischenfelde KH. Autoantibodies to human asialoglycoprotein receptor in autoimmune-type chronic hepatitis. Hepatology 1990; 11:606--612. Treichel U, Poralla T, Christmann M, Meyer zum Btischenfelde K-H, Stockert RJ. Structural and functional differentiation of epitopes recognized by autoantibodies on the asialoglycoprotein receptor. Hepatology 1992; 16:205A. Treichel U, Gerken G, Rossol S, Rotthauwe HW, Meyer zum Btischenfelde KH, Poralla T. Autoantibodies against the human asialoglycoprotein receptor: effects of therapy in autoimmune hepatitis and virus-induced chronic active hepatitis. J Hepatol 1993a;19:55--63. Treichel U, Stockert RJ, Meyer zum Bfischenfelde K-H. A 10 kD glycosylated fragment is the immunodominant epitope from autoantibodies on the asialoglycoprotein receptor (ASGPR). Hepatology 1993b;18:172A. Treichel U, McFarlane BM, Seki T, Krawitt EL, Alessi N, Stickel F, McFarlane IG, Kiyosawa K, Futura S, Freni MA, et al. Demographics of antiasialoglycoprotein receptor autoantibodies in autoimmune hepatitis. Gastroenterology 1994;107: 799--804. Treichel U, Schreiter T, Meyer zum Btischenfelde K-H, et al. High yield purification and characterization of human asialoglycoprotein receptor. Protein Expr Purif 1995;6:255259. Vento S, Garofano T, Di Perri G, Dolci L, Concia E, Bassetti D. Identification of hepatitis A virus as a trigger for autoimmune chronic hepatitis type 1 in susceptible individuals. Lancet 1991;337:1183--1187. Wen L, Peakman M, Lobo-Yeo A, McFarlane BM, Mowat AP, Mieli-Vergani G, Vergani D. T-cell directed hepatocyte damage in autoimmune chronic active hepatitis. Lancet 1990;336:1527--1530. Wiedmann KH, Bartholomew TC, Brown DJ, Thomas HC. Liver membrane antibodies detected by immunoradiometric assay in acute and chronic virus-induced and autoimmune liver disease. Hepatology 1984;4:199--204.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

LUPUS ANTICOAGULANT Douglas A. Triplett, M.D.

Department of Hematology, Ball Memorial Hospital, Muncie, IN 47303, USA

HISTORICAL NOTES The seminal description of a circulating anticoagulant (synonym: inhibitor) was presented in 1952 in two patients with systemic lupus erythematosus (SLE) (Conley and Hartmann, 1952). Earlier articles suggested a similar anticoagulant (Mueller et al., 1951), and a subsequent report described an association between a biological false-positive Wassermann test and a circulating anticoagulant which could be adsorbed by the Kahn reagent used in serologic tests for syphilis (Laurell and Nilsson, 1957). Thus, the anticoagulant activity was linked to an antibody which reacted with a lipid antigen (Laurell and Nilsson, 1957). Subsequently, "lupus anticoagulant" (LA) was proposed for this antibody (Feinstein and Rapaport, 1972), although this term is a misnomer because the vast majority of patients with LA do not have SLE. Initially, LA were regarded as a laboratory nuisance because they often resulted in cancellation of surgery when coagulation screening procedures (Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT)) were found to be unexpectedly prolonged. There was no correlation between the in vitro inhibition of coagulation and an in vivo predisposition to hemorrhage. The paradoxical relationship between LA and a predisposition to thrombosis was first identified in 1963 (Bowie et al., 1963). Recent studies clearly identified LA as a risk factor for both venous and arterial thromboembolic events (Rosove and Brewer, 1992).

anionic phospholipids (Pengo et al., 1987), recent evidence supports specificity of LA for proteinphospholipid complexes (Vermylen and Arnout, 1992). Approximately two-thirds of LA plasmas have antibodies which express anticoagulant activity only in human plasma (Galli et al., 1992; Bevers et al., 1991). These LA are species-specific and recognize an induced epitope (neotope) of human prothrombin. This neotope is expressed when human prothrombin binds to anionic phospholipids or other surfaces (e.g., microtiter plate). The ~2 glycoprotein I-phospholipid complex also contains a neotope(s) for LA. Prothrombin-dependent LA activity can be separated from ~2 glycoprotein I-dependent LA activity by use of cardiolipin-containing liposomes which adsorb anticardiolipin antibodies and ~2glycoprotein I-dependent LA but not prothrombin-dependent LA (Galli et al., 1992). The remarkable range of antigenic specificities of LA include other potential protein components of protein-phospholipid complexes such as annexin V (placental anticoagulant protein-I), protein S, protein C, thrombomodulin and high molecular weight kininogen (Matsuda et al., 1994; Oosting et al., 1993; Sugi et al., 1993). The expression of neotopes by these proteins is not dependent upon the presence of phospholipids (Matsuura et al., 1994; Roubey, 1994). The spectrum of potential protein targets offers an opportunity to subclassify these heterogeneous antibodies and potentially to establish more meaningful clinicalpathologic correlations.

THE AUTOANTIGEN

AUTOANTIBODIES

Definition/Origin

Terminology

Although early work suggested LA were specific for

LA are immunoglobulins (usually IgG, IgM, IgA or

474

mixtures) which interfere with in vitro phospholipiddependent tests of coagulation (e.g., PT, APTT, dilute Russell Viper Venom Time (dRVVT)). LA belong to the antiphospholipid-protein antibody (APPA) family (Triplett, 1995). Other family members include: anticardiolipin antibodies (aCL) and reagin. These antibodies are defined by various laboratory tests, including serologic tests for syphilis for identification of reagin, phospholipid-dependent coagulation tests to identify LA, and solid phase radioimmunoassay (RIA) or ELISA for identification of aCL (Harris et al., 1983). Encountered in a wide variety of clinical settings, LA can be classified as either autoimmune or alloimmune (Table 1). Autoimmune LA are more commonly associated with clinical complications than alloimmune antibodies. Pathogenetic Role

Initially, LA were thought to be either coincidentally linked or a consequence of thrombosis (Triplett and Brandt, 1988). Recent studies in animal models and prospective clinical studies provide support for LA as a cause of thrombosis (Bakimer et al., 1992; Blank et al., 1991; Branch et al., 1990; Smith et al., 1990). Both arterial and venous thromboembolic events are linked to LA. Given the heterogeneity of LA, more than one pathogenic mechanism is highly probable. Clinically, there is remarkable fidelity of site of recurrent thrombosis in patients with LA (venous events are followed by venous events, arterial by arterial) (Rosove and Brewer, 1992). The three most probable pathophysiologic mechanisms are: inhibition of activated protein C (APC), activation of platelets and perturbation of endothelial

heparan sulfate-AT III system (Shibata et al., 1994). Inhibition of activated protein C has been identified by several groups (Smirnov et al., 1995). Potentially, LA may interfere with phosphatidylethanolamine (PE)-dependent expression of APC (Smirnov et al., 1995) activity, inhibit the down regulation of Va (Lin and Zehnder, 1994) or prevent assembly of the APC/protein S/Va complex on phospholipid surfaces. Alteration of the endothelial platelet balance (thromboxane Ajprostacyclin) offers the most likely explanation for arterial thrombosis. Methods of Detection

The identification of LA requires a coordinated, systematic evaluation (Triplett, 1995). The most critical step in accentuating sensitivity of various test systems is proper preparation of platelet-poor plasma. The residual platelet count in platelet-poor plasma should be less than 10,000 mm3; an excess of platelets may provide a means of "masking" the LA. There are three sequential steps necessary to establish the presence of LA. The first involves a sensitive screening procedure (i.e., APTT, dRVVT, Kaolin Clotting Time (KCT) or dilute PT). There is a wide range of sensitivities to LA among commercial APTT reagents. If there is a strong clinical suggestion of possible LA, it is imperative the laboratory use at least two screening procedures. If either or both are positive, the second step, which involves demonstration of an inhibitor, requires the mixing of patient and normal plasma in various ratios (e.g., 1:1, 4:1 (patient: normal)). In the presence of an inhibitor, the addition of normal plasma will not result in correction of the prolonged screening tests. The third step requires demonstration of phospholipid dependence of the

Table 1. Lupus Anticoagulant: Classification Autoimmune

Alloimmune

PAPS Secondary APS SLE Other CTD Drug-Induced Chlorpromazine Procainamide Quinidine Quinine

Infectious Diseases Viral (e.g., HIV) Bacterial Protozoal Fungal Malignancies Hairy Cell Leukemia Lymphoproliferative Diseases

Note: PAPS = Primary Antiphospholipid Antibody Syndrome; APS = Antiphospholipid Antibody Syndrome; CTD = Connective Tissue Disease; HIV = Human Immunodeficiency Virus.

475

inhibitor. In most cases, laboratory tests are designed to increase the amount of phospholipid in order to "neutralize" or "bypass" LA activity. Examples of such procedures include: the platelet neutralization procedure and employment of hexagonal-phase phospholipid (Staclot LA | (Triplett et al., 1993). As part of the laboratory evaluation of any patient for LA, solid-phase ELISA assays for aCL should also be performed. There is concordance of LA and aCL positivity in approximately 60% of cases. In the remaining patients, one of the two assays will be positive in the setting of the APS.

CLINICAL UTILITY Disease Association The concept of an antiphospholipid antibody syndrome (APS) was elaborated in 1983 (Boey et al., 1983; Harris et al., 1983; Hughes, 1983) as an association of thrombosis and other clinical findings such as recurrent spontaneous abortion, cerebrovascular events and thrombocytopenia with positive laboratory studies for LA and/or aCL. The majority of LA encountered in a routine coagulation lab are transient and unassociated with clinical complications. Most of these transient LA occur in the setting of convalescence from viral or bacterial infections. Autoimmune LA tend to be persistent and are associated with APS. Approximately 40% of patients

REFERENCES Bakimer R, Fishman P, Blank M, Sredni B, Djaldetti M, Shoenfeld Y. Induction of primary antiphospholipid syndrome in mice by immunization with a human monoclonal anticardiolipin antibody (H3). J Clin Invest 1992;89:1558-1563. Bevers EM, Galli M, Barbui T, Comfurius P, Zwaal RF. Lupus anticoagulant IgG's (LA) are not directed to phospholipids only, but to a complex of lipid-bound prothrombin. Thromb Haemost 1991;66:629--632. Blank M, Cohen J, Toder V, Shoenfeld Y. Induction of antiphospholipid syndrome in naive mice with mouse lupus monoclonal and human polyclonal anticardiolipin antibodies. Proc Natl Acad Sci USA 1991;88:3069-3073. Boey ML, Colaco CB, Gharavi AE, Elkon KB, Loizou S, Hughes GR. Thrombosis in systemic lupus erythematosus: striking association with the presence of circulating lupus anticoagulant. Br Med J 1983;287:1021--1023. Bowie EJW, Thompson JH, Pascuzzi CA, Owen CA. Throm476

with SLE have either aCL or LA (Love and Santoro, 1990). Approximately 50% of patients on long-term chlorpromazine therapy have LA (Canoso and Sise, 1982). Although initial studies suggested drug-induced LA were not associated.with clinical complications, several subsequent reports demonstrate an association between drug-induced LA and clinical complications (Triplett et al., 1988; Walker et al., 1988).

CONCLUSION LA are a group of immunoglobulins which appear to react with various proteins which express neotopes in the presence of phospholipids or other surfaces. Convincing evidence exists for such reactions with ~2 glycoprotein I, prothrombin protein C and protein S. Found in a variety of clinical conditions, LA are most frequently encountered in the setting of convalescence from infectious diseases. In autoimmune disease, LA correlate strongly with clinical thromboembolic events, recurrent spontaneous abortions and thrombocytopenia. Patients with LA who require long-term oral anticoagulant therapy should be maintained at a higher intensity of anticoagulation (International Normalized Ratio >3.0) (Rosove and Brewer, 1992; Khamashta et al., 1995). See also Bz-GLYCOPROTEIN I AUTOANTIBODIES, PHOSPHOLIPID AUTOANTIBODIES CARDIOLIPIN and PHOSPHOLIPID AUTOANTIBODIES PHOSPHATIDYLSERINE.

bosis in systemic lupus erythematosus despite circulating anticoagulants. J Lab Clin Med 1963;62:416--430. Branch DW, Dudley DJ, Mitchell MD, Creighton KA, Abbott TM, Hammond EH, Daynes RA. Immunoglobulin G fractions from patients with antiphospholipid antibodies cause fetal death in BALB/c mice: a model for autoimmune fetal loss. Am J Obstet Gynecol 1990;163:210-216. Canoso RT, Sise HS. Chlorpromazine-induced lupus anticoagulant and associated immunologic abnormalities. Am J Hematol 1982;13:121-129. Conley CL, Hartmann RC. A hemorrhagic disorder caused by circulating anticoagulants in patients with disseminated lupus erythematosus. J Lab Clin Med 1952;31:621--622. Feinstein DI, Rapaport SI. Acquired inhibitors of blood coagulation. Prog Hemost Thromb 1972;1:75-95. Galli M, Comfurius P, Barbui T, Zwaal RF, Bevers EM. Anticoagulant activity of beta 2-glycoprotein I is potentiated by a distinct subgroup of anticardiolipin antibodies. Thromb Haemost 1992;68:297--300. Harris EN, Gharavi AE, Boey ML, Patel BM, Mackworth-

Young CG, Loizou S, Hughes GR. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;2: 1211--1214. Hughes GR. Thrombosis, abortion, cerebral disease, and the lupus anticoagulant. Br Med J 1983;287:1088-1089. Khamashta MA, Caudrado MJ, Mujic F, Taub NA, Hunt BJ, Hughes GR. The management of thrombosis in the antiphospholipid antibody syndrome. N Engl J Med 1995;332: 993--937. Laurell AB, Nilsson IM. Hypergamma-globulinaemia circulating anticoagulant, and biologic false positive Wassermann reaction: a study of 2 cases. J Lab Clin Med 1957;49:694-707. Lin RZ, Zehnder JL. Acquired activated protein C resistance caused by factor Va antibody: a possible mechanism of increased thrombosis in the antiphospholipid antibody syndrome. Blood 1994;84(Suppl I):83a. Love PE, Santoro SA. Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systematic lupus erythematosus (SLE) and in non-SLE disorders. Prevalence and clinical significance. Ann Intern Med 1990;112:682--698. Matsuda J, Saitoh N, Gohchi K, Gotoh M, Tsukamoto M. Antiannexin V antibody in systemic lupus erythematosus patients with lupus anticoagulant and/or anticardiolipin antibody. Am J Hematol 1994;47:56-58. Matsuura E, Igarashi Y, Yasuda T, Triplett DA, Koike T. Anticardiolipin antibodies recognize 132-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med 1994;179:457--462. Mueller JF, Ratnoff O, Henile RW. Observations on the characteristics of an unusual circulating anticoagulant. J Lab Clin Med 1951 ;38:254--261. Oosting JD, Derksen RH, Bobbink IW, Hackeng TM, Bouma BN, De Groot PG. Antiphospholipid antibodies directed against a combination of phospholipids with prothrombin, protein C or protein S: an explanation for their pathogenic mechanism? Blood 1993;81:2618--2625. Pengo V, Thiagarajan P, Shapiro SS, Heine MJ. Immunological specificity and mechanism of action of IgG lupus anticoagulants. Blood 1987;70:69--76.

Rosove MH, Brewer PM. Antiphospholipid thrombosis: clinical course after the first thrombotic event in 70 patients. Ann Intern Med 1992;117:303--308. Roubey RA. Autoantibodies to phospholipid-binding plasma proteins: a new view of lupus anticoagulants and other "antiphospholipid" autoantibodies. Blood 1994;84:28542867. Shibata S, Harpel PC, Gharavi AR, Fillit H. Autoantibodies to heparin from patients with antiphospholipid antibody syndrome inhibits formation of antithrombin III-thrombin complexes. Blood 1994;83:2537--2540. Smirnov M, Triplett DT, Comp PC, Esmon NL, Esmon CT. Role of phosphatidylethanolamine in inhibition of activated protein C activity by antiphospholipid antibodies. J Clin Invest 1995;95:309--316. Smith, HR, Hansen CL, Rose R, Canoso RT. Autoimmune MRL -- lpr/lpr mice are an animal model for the secondary antiphospholipid syndrome. J Rheumatol 1990; 17:911--915. Sugi T, Vanderpuye OA, Mc Intyre JA. Partial purification of an antiphosphatidylethanolamine antibody ELISA cofactor. Thromb Haemost 1993;69:596. Triplett DA, Brandt JT. Lupus anticoagulants: misnomer, paradox, riddle, epiphenomenon. Hematol Pathol 1988;2: 121-143. Triplett DA, Brandt JT, Musgrave KA, Orr CA. The relationship between lupus anticoagulants and antibodies to phospholipid. JAMA 1988;259:550--554. Triplett DA, Barna LK, Unger GA. A hexagonal (II) phase phospholipid neutralization assay for lupus anticoagulant identification. Thromb Haemost 1993;70:787--793. Triplett DA. Antiphospholipid-protein antibodies: laboratory detection and clinical relevance. Thromb Res 1995;78:1--31. Vermylen J, Arnout J. Is the antiphospholipid syndrome caused by antibodies directed against physiologically relevant phospholipid-protein complexes? J Lab Clin Med 1992:120: 10-12. Walker TS, Triplett, DA, Javed N, Musgrave K. Evaluation of lupus anticoagulants: antiphospholipid antibodies, endothelium associated immunoglobulin, endothelial prostacyclin secretion, and antigenic protein S levels. Thromb Res 1988;51:267--281.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

LYMPHOCYTOTOXIC AUTOANTIBODIES Antonius J.G. Swaak, M.D., Ph.D.

Department of Rheumatology, Dr. Daniel den Hoed Clinic, 3085 EA Rotterdam, The Netherlands

HISTORICAL NOTES

AUTOANTIGENS

Recognition of autoimmune hemolytic anemia led to investigations of the occurrence of antileukocyte antibodies in patients with systemic lupus erythematosus (SLE) and leukocytopenia. Because different immunological techniques such as agglutination, complement fixation, antiglobulin consumption and cytotoxicity were used (Quismorio and Friou, 1970), comparisons of specificity and sensitivity are difficult. In addition to their presence in lupus, such antibodies against leukocytes are also a consequence of multiple pregnancies and/or blood transfusions. On the other hand, the high frequency of positive results obtained with the direct antiglobulin consumption on leukocytes was shown to result from autoantibodies reacting with surface antigens or with immune complexes adherent to surfaces of white cells or platelets (Miesscher, 1969). False-positive results in the direct antiglobulin consumption tests also resulted from use of preparations of damaged leukocytes, which made other antigens accessible to antinuclear, anticytoplasmatic or other antibodies present in the sera of transplant patients or patients with SLE. A wide variety of antibodies against white cell antigens specifically reactive for granulocytes and/or lymphocytes were substantially recognized. For the detection of antibodies to lymphocytes, the lymphocyte cytotoxicity test which was developed for histocompatibility testing become the standard method with acceptable reproducibility for measurement of specific lymphocytotoxic autoantibodies (LCA) (Mittal et al., 1970). The occurrence of two classes of LCA became evident: the so-called natural LCA and the LCA which developed after immunization.

Definition

478

Although a wide range of specificities for different cell types are well defined, it is not yet known how these antibodies are stimulated, which target autoantigens they bind to and whether or not they are of immunoregulatory or pathological importance in these states. To assess the potential role of LCA, the antigens to which they bind must be defined. T Cells. Initial studies focused on the relative specificity for T cells. Fetal thymocytes were the most reactive targets. In different studies LCA affected Tcell function (Lies et al., 1973) but the target antigens are still obscure. Cell specificity of LCA is claimed for T cells as well as T-cell subsets (T7 cells, T-non- 7 cells, T suppressor cells, NK cells), B cells, cells sharing specificity with lymphocytes as monocytes, erythrocytes and brain neuroblastoma cells (Osman and Swaak, 1994). A common major problem in studying autoantibodies is the likelihood that absorption of autoantibodies in vivo leaves only low titer or low avidity autoantibodies for study. Such residual antibodies make target antigen characterization difficult. CD45. One of the possible target antigens of LCA is CD45, a transmembrane protein expressed at high levels on hematopoietic cells and endothelial cells. That CD45 might be a target was first suggested by a preferential reactivity of LCA in SLE patients with CD45RA + CD4 cells in contrast to CD45RO CD4 + cells (Morimoto et al., 1984). In blotting experiments, 25% of LCA-positive SLE sera stained different isoforms of CD45. In the initial immunoblot analysis,

a Jurkat cell line was used as antigen, however, with CD45, as expressed by B cells or by resting normal T cells, no binding could be observed with SLE sera. CD45 from mitogen-activated peripheral T cells, including the p 180 CD45RO isoform, was stained by the majority of SLE sera tested (Winfield and Czyzyk, 1995).

lymph nodes. Although such different B-cell specificities are demonstrable, the extent to which IgG binding might actually represent immune complexes bound to complement receptors or Fc receptors is unclear.

T-Cell Receptor (TCR). LCA in certain SLE sera react with T cells bearing either t~ ~ or Y o TCRs, but not with T cells that do not express TCRs; these data suggest that SLE autoantibodies are directed against TCR (Marchalonis et al., 1994).

Terminology

[32-Mieroglobulin (~2-m). The observation that LCA titers can be reduced by the presence of platelets suggests that they can have a specificity for class I major histocompatibility complex (MHC) determinants. The introduction of as little as 1 ng free ~2-m into the microcytotoxic assay, inhibits LCA activity in 50% of patients with SLE and with LCA (Revillhard et al., 1979). However, no inhibition by ~2-m was observed with LCA-positive sera of leprosy patients (Rasheed et al., 1991).

Class I and II (MHC) Antigens. LCA directed against class I (MHC) determinants can be observed in transplantation and/or multiparous patients. In 11 sera from sensitized, multiparous patients, all contained LCA to over 70% of a lymph panel from 24 donors. Inhibition of cytotoxic activity against paternal lymphocytes by monoclonal antibodies to HLA framework determinants indicated that all sera contained LCA to paternal class I antigens. In addition, five sera contained LCA to paternal class II antigens (Propper et al., 1991). In corneal transplant patients who had or developed LCA, the most frequently identified antibodies were against the antigens of the A1, A2, A9, B5, B7 and B17 cross-reactive epitope groups (CREGs) (Hahn et al., 1995). However, SLE sera tested in a well-defined panel of target cells of known HLA phenotype revealed no clear-cut HLA specificity. Because F(ab')2 fragments of a heterologous ( r a b b i t ) - antibodies to ~2-m the lymphocytoxicity of SLE sera, the LCA SLE might be directed to an antigenic determinant associated with HLA antigens (Messner et al., 1980). B Cells. In addition to T-cell antigens, LCA-positive sera also contain antibodies to B cells from a variety of sources including peripheral blood, tonsils and

THE AUTOANTIBODIES

LCA are a heterogeneous group of antibodies, including two major classes: 1) LCA associated with immunization, pregnancy, blood transfusions and skin, liver, heart and kidney grafting are warm reactive (37~ and require short incubation times (1 hour). They are primarily directed against HLA antigens present on lymphocyte surfaces, invariably belonging to the IgG class in contrast to the naturally occurring LCA. 2) Naturally occurring LCA are found in many diseases and are mostly IgM and cold reactive with an optimal temperature of 15~ in contrast to LCA which were found after immunization. As first described in patients with infectious diseases like mononucleosis, measles and rubella (Mottironi et al., 1970), the convalescent sera are weaker in LCA activity than acute sera. LCA are also present in other viral diseases (mumps, influenza, herpes) as well as Mycoplasma, Rickettsia and chronic parasitic infections. LCA are also sometimes found in malignancies, multiple sclerosis after a variety of immunizing procedures including vaccination and in high titers in autoimmune diseases like SLE and rheumatoid arthritis (Osman and Swaak, 1994).

Pathogenetic Role There are several potential mechanisms by which LCA could alter lymphocyte function, but insight into the pathogenetic role of LCA remains unclear (Table 1).

Elimination of Lymphocytes. The correlation with lymphopenia and LCA strongly suggests that LCA can eliminate lymphocytes. Although data are not available, LCA might have an effect on migration. In vitro experiments show that LCA can mediate lysis of lymphocytes in the presence of complement (Packer and Loque, 1980). Both lysis of lymphocytes and altered migration might have an effect on the depletion of T-cell subsets in the circulation. Altered 479

Table 1. Lymphocytotoxic Autoantibodies Naturally Occurring

Acquired

Association

SLE, RA, viral illnesses, etc.

Derived from form of immunization; pregnancy, transplantation.

Target antigens

Probably T cells, B cells, monocytes. At this moment, not characterized.

MHC Determinants

Antibody characteristics

IgM, cold reactive (15~

IgG, warm reactive (37~

Clinical significance

Associated with lymphopenia, other associations with diseases of CNS, abortion, altered functions of lymphocytes are doubtful.

Negative effect on graft survival.

CD4/CD8 ratios are described in patients with SLE and LCA; in SLE patients with high CD4/CD8 ratios, LCA are predominantly reactive with CD8-positive cells (Morimoto et al., 1984). That this mechanism might be important is further demonstrated in renal transplant patients in whom administration of the monoclonal OKT3 causes a decrease of OKT3 + T cells, and the appearance of OKT4+/T3 - and OKT8+/T 3 - c e l l s (Chatenod et al., 1982). B cells are also lysed by LCA (Sur~nyi et al., 1985).

Modulation of Surface Determinants (Receptors). LCA can modulate membrane determinants by promoting or shedding from the cell surface or capping (Minota and Winfield, 1988). Methods of Detection As used in early studies, the microcytotoxicity test involves incubation of target lymphocytes with test serum at relatively low temperature (15~ and requires the addition of a heterologous source of complement in order to complete the cytotoxic killing of target cells (Terasaki and McCleland, 1964). Various methods are described with different incubation temperatures, sources of complement, target cells, definitions of a positive test and dyes to determine viability. Temperature variations can result in measuring different antibody classes. Another important factor responsible for the variability is the source of complement. Rabbit sera, which is toxic for most lymphocytes from heterologous species (Terasaki et al., 1971), also contains an IgM antilymphocyte antibody which reacts with human leukocytes, producing cytotoxicity and enhancing activity of LCA present in the sera (Mittal et al., 1973). In some cases, killing of lymphocytes results from addition of auto-

480

logous flesh serum as complement source; thus the assay system is very dependent on the source of complement. The definition of a positive test is also nonuniform. A positive cytotoxic test is commonly defined as 10% or more dead cells, but 20% is also used. Binding of LCA antigens from T-cell surface membranes can also be measured by indirect immunofluorescence or immunoperoxidase techniques (Okudaira et al., 1979; MacPherson and Kottmeyer, 1977). Cell sorting provides a reproducible procedure for the isolation and staining of the cells and allows demonstration of LCA on autologous lymphocytes (Sintnicolaas et al., 1991).

Family Studies LCA occur in high titer in SLE patients and to a lesser extent in RA. Because LCA are common in viral illnesses, the concept has arisen that viruses might be of etiological importance in SLE. LCA are increased in both consanguineous and nonconsanguineous relatives of patients with SLE (De Horatius and Messner, 1975) also in RA patients (Taneja et al., 1991).

CLINICAL U T I L I T Y Disease Association Large amounts of data are available demonstrating that naturally occurring LCA have an influence on the immune system (Yamada and Winfield, 1984; Morimoto et al., 1984) in addition to their well-known association with disease.

Lymphopenia. Lymphopenia in SLE is reported in

28--90% of untreated patients (Osman and Swaak, 1994). Although the cause of the lymphopenia is unclear, LCA might be involved, as manifest by the correlation between their presence and the presence and/or history of leukopenia (Nies et al., 1974). Lymphopenia is also related to avidity and to the concentration of LCA (Winfield et al., 1975) and to the activity of SLE (Rivero et al., 1978).

Central Nervous System. LCA might cross-react with brain tissue (Bluestein and Zvailer, 1976); the frequency and titers of LCA are greater in SLE patients with CNS disease than in SLE patients with other manifestations. LCA in sera of SLE patients are cytotoxic for neuronal and glial cell lines (Bluestein, 1978). Despite the cross-reactivity of LCA or neuronal cell lines and lymphocytes, absorption studies using a lymphoblastoid cell line did not remove the antineuronal activity; this demonstrates the complexity of the antibody system(s) and the need for definition of the antigens involved. Correlations of LCA with seizures and diffuse CNS disease were reported, but no correlations were demonstrated with focal neurologic involvement and/or psychosis (Wilson et al., 1979). Therefore, whether LCA are truly specific for CNS involvement in SLE is unknown; a pathophysiologic role is nevertheless possible in CNS lupus. Overall contradictory results are reported (Hanly et al., 1993). Spontaneous Abortion. LCA were also implicated in the spontaneous abortions seen in SLE (Breshnihan et al., 1977). Pregnancy has long been known to be associated with the occurrence of LCA, but in general, these are HLA-directed warm-reactive LCA. Some, however, have no relationship to HLA antigens and are polyspecific (Tongio et al., 1972). Still, no relationship of warm reactive LCA to complications of pregnancy are noted, only with cold reactive LCA. Nephritis. LCA are found in most cases of lupus nephritis and are also reported in other forms of glomerulonephritis, like membranoproliferative glomerulonephritis, minimal change nephrotic syndrome, IgA glomerulonephritis, membranous glomerulonephritis and poststreptococcal glomerulonephritis (Nakabayashi et al., 1985). Among the various forms of glomerulonephritis, the titers and frequencies are highest in membranous glomerulonephritis and minimal change nephrotic syndrome.

Hypogammaglobulinemia. Sera of patients with a hypogammaglobulinemia contain LCA; the LCA activity is related to IgM antibodies, appears in a 19S peak on sucrose density gradients and is abrogated by absorption with anti-IgM (MacDonald et al., 1982). LCA Caused by Immunization. Previous pregnancies, blood transfusions and early donor transplantations can cause LCA. In most transplantation cases, these LCA recognize HLA antigens and have a deleterious effect on the graft survival as shown in corneal transplantation (Hahn et al., 1995), liver transplantation (Furuya et al., 1992) and renal transplantation (Barocci et al., 1991). Interaction with the Production of Cytokines. Purified IgG fractions of SLE sera inhibit IL-2 production at two distinct phases of the IL-1-dependent production of IL-2: (1) by binding to adherent cells and probably inhibiting IL-1 production by macrophages in response to anti-HLA-DR antibodies, and (2) by binding to T cells and blocking the interaction of IL-1 and T cells (Miyagi et al., 1989). In other experiments LCA (Ig fractions) were able to induce interferon y release (Ramirez et al., 1986). Activation of T and B Cells by Cross-linking of a Receptor. Cross-linking of receptors can stimulate cellular function, as shown by OKT3 which stimulates T cells to proliferate (van Wauwe et al., 1980). F(Ab') 2 fragments of IgG from SLE serum enhance Ig secretion in vitro by B cells (Takeuchi et al., 1982). LCA can suppress the T,cell response to tetanus toxoid (Yamada and Winfield, 1984).

CONCLUSION Since 1970 naturally occurring LCA were reported in a wide variety of diseases. Most attention was paid to LCA found in SLE. Frequencies of LCA and SLE varied from 28--90%. Correlations between LCA and various clinical parameters such as disease activity, lymphopenia and CNS involvement are claimed, but insight into the pathogenetic role of LCA remains unclear. Whether all described effects of LCA are specifically caused by LCA and not attributable to immune complexes, or other nonspecific factors is unknown. More conclusive answers concerning the significance of LCA will require systematic analysis of the anti-

481

body specificities. The possible relationship between L C A and functional effects on T and B cells is

intriguing, but still not conclusively linked to L C A as opposed to other factors present in patient sera.

REFERENCES

relatives. Reactivity with the HLA antigenic molecular complex. Arthritis Rheum 1973;23:265--272. Miesscher PA. Antibodies against thrombocytes and leukocytes. In: Miesscher PA, Eberhard HJ, eds. Textbook of Immunology. Vol II. Grune and Stratton, 1969:500--506. Minota S, Winfield JB. Nature of IgG antilymphocyte autoantibody-reactive molecules shed from activated T-cells in systemic lupus erythematosus. Rheumatol Int 1988;8:165- 170. Mittal KK, Ferrone S, Mickey R, Pelligrino MA, Reisfeld RA, Terasaki PI. Serologic characterization of natural antihuman lymphocytotoxic antibodies in mammalian sera. Transplantation 1973;16:287--294. Mittal KK, Rossen RD, Sharp JT, Lidsky MD, Butler WT. Lymphocyte cytotoxic antibodies in systemic lupus erythematosus. Nature 1970;225:12555-12556. Miyagi J, Minato N, Sumiya M, Kasahara T, Kano S. Two types of antibodies inhibiting interleukin-2 production by normal lymphocytes in patients with systemic lupus erythematosus. Arthritis Rheum 1989;32:1356-- 1364. Morimoto C, Reinherz EL, Distaso JA Steinberg AD, Scholssman SF. Relationship between systemic lupus erythematosus T cell subsets, anti-T-cell antibodies, and T cell functions. J Clin Invest 1984;73:689--700. Mottironi VD, Terasaki PI. Lymphocytotoxins in disease I infectious mononucleosis, rubella and measles. In: Terasaki PT, ed. Histocompatibility Testing. Copenhagen: Munskgaard, 1970:301. Nakabayashi K, Arimura Y, Yoshida M, Nagasawa T. Anti-T cell antibodies in primary glomerulonephritis. Clin Nephrol 1985;23:74-80. Nies KM, Brown JC, Dubois EL, Quismorio FP, Friou GJ, Terasaki PI. Histocompatibility (HLA) antigens and lymphocytotoxic antibodies in systemic lupus erythematosus (SLE). Arthritis Rheum 1974;17:397--402. Okudaira K, Nakai H, Hayakawa T, Kashiwado T, Tanimoto K, Horiuchi Y, Juji T. Detection of antilymphocyte antibody with two-color method in systemic lupus erythematosus and its heterogeneous specificities against human T-cell subsets. J Clin Invest 1979;64:1213-1220. Osman C, Swaak AJG. Lymphocytotoxic antibodies in SLE: a review of the literature. Clin Rheumatol 1994;13:21-27. Packer SH, Loque GL. Quantitation of warm reactive IgG antilymphocyte autoantibodies in systemic lupus erythematosus. Clin Immunol Immunopathol 1980;17:515-529. Propper DJ, Leheny WA, Urbaniak SJ, Catto GR, Macleod AM. Lymphocytotoxins in sera from highly sensitized multiparous dialysis patients: antibody class, relationship with the HLA and with paternal antigens. Clin Sci (Colch) 1991;80:87-93. Quismorio FP, Friou GJ. Serologic factors in systemic lupus erythematosus and their pathogenetic significance. CRC Crit Rev Clin Lab Sci 1970; 1:639-684. Ramirez F, Williams RC Jr., Sibbitt WL Jr., Searles RP.

Barocci S, Valente U, Gusmano R. Perfumo F, Cantarello S, Leprini A, Icardi A, Nocera A. Autoreactive lymphocytotoxic IgM antibodies in highly sensitized dialysis patients waiting for a kidney transplant: identification and clinical relevance. Clin Nephrol 1991;36:12-20. Bluestein HG, Zvailer NJ. Brain-reactive lymphocytotoxic antibodies in the serum of patients with systemic lupus erythematosus. J Clin Invest 1976;57:509--516. Bluestein HG. Neurocytotoxic antibodies in serum of patients with systemic lupus erythematosus. Proc Natl Acad Sci USA 1978;75:3975--3979. Breshnihan B, Grigor RR, Oliver M, Lewkonia R, Hughes, Lovins RE, Faulk WP. Immunological mechanisms for spontaneous abortion in systemic lupus erythematosus. Lancet 1977 ;2:1205-1207. Chatenoud L, Baudrihaye MF, Kreis H, Goldstein G, Schindler J, Bach JF. Human in vivo antigenic modulation induced by the anti-T-cell OKT3 monoclonal antibody. Eur J Immunol 1982;12:979--983. DeHoratius RJ, Messner RP. Lymphocytotoxic antibodies in family members of patients with systemic lupus erythematosus. J Clin Invest 1975;55:1254--1258. Furuya T, Murase N, Nakamura K, Woo J, Todo S, Demetris AJ, Starzl TE. Performed lymphocytotoxic antibodies: the effect of class titer and specificity on liver or heart allografts. Hepatology 1992;16:1415-1422. Hahn AB, Foulks GN, Enger C, Kink N, Stark WJ, Hopkins KA, Sanfilipo F. The association of lymphocytotoxic antibodies with corneal allograft rejection in high risk patients. The Collaborative Corneal Transplantation Studies Research Group. Transplantation 1995 ;59:21--27. Hanley JG, Walsh NM, Fisk JD, et al. Cognitive impairment and autoantibodies in systemic lupus erythematosus. Br J Rheumatol 1993;32:291--296. Lies RB, Messner RP, Williams RC. T-cell specificity of lymphotoxins from patients with systemic lupus erythematosus. Arthritis Rheum 1973;16:369--375. MacDonald S, Webster AD, Platt-Mills TA. An analysis of the lymphocytotoxic activity found in sera from patients with hypogammaglobulinaemia. Scand J Immunol 1982; 15:379387. MacPherson BR, Kottmeyer ME. Detection of antilymphocyte antibodies using the immunoperoxidase antiglobulin technique. Am J Clin Pathol 1977;68:347-350. Marchalonis JJ, Schluter SF, Wang E, Dehghanpisheh K, Lake D, Edmundson AB, Winfield JB. Synthetic autoantigens of immunoglobulins and T-cell receptors: their recognition in aging, infection, and autoimmunity. Proc Soc Exp Biol Med 1994;207:129--147. Messner RP, De Horatius RJ, Ferrone S. Lymphocytotoxic antibodies in systemic lupus erythematosus patients and their

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Immunoglobulin from systemic lupus erythematosus serum induces interferon release by normal mononuclear cells. Arthritis Rheum 1986;29:326-336. Rasheed FN, Locniskar M, McCloskey DJ, Hasan RS, Chiang TJ, Rose P, de Soldenhoff R, Festenstein H, McAdam KP. Specificity oflymphocytotoxic autoantibodies (LCAbs) found in the serum of leprosy patients: class I MHC antigens. Lepr Rev 1991;62:13--20. Revillhard JP, Vincent G, Rivera S. Anti-J3-2 microglobulin lymphocytotoxic autoantibodies in systemic lupus erythematosus. J Immunol 1979;122:614--618. Rivero SJ, Diaz-Jouanen E, Alarcon-Segovia D. Lymphopenia in systemic lupus erythematosus. Arthritis Rheum 1978;21: 295--305. Sintnicolaas K, de Vries W, van der Linden R, Gratama JW, Bolhuis RL. Simultaneous flow cytometric detection of antibodies against platelets, granulocytes and lymphocytes. J Immunol Methods 1991;142:215-222. Sur~nyi P, Matyus L, Sonkoly I, Szegedi G. Subset specificity of lupus antilymphocyte antibodies studies by two-colour microfluorimetry. Immunol Lett 1985; 10:91-93. Takeuchi T, Abe T, Kiyotaki M, Toguchi T, Koide J, Morimoto C, Homma C. In vitro immune response of SLE lymphocytes. The mechanism involved in B-cell activation. Scand J Immunol 1982;16:369-377. Taneja V, Mehra NK, Singh RR, Anand C, Malaviya AN. Occurrence of lymphocytototoxins in multicase rheumatoid arthritis families: relation to HLA. Clin Exp Immunol

1991;86:87-91. Terasaki PI, Esail ML, Cannon JA et al. Destruction of lymphocytes in vitro by normal serum from common laboratory animals. J Immunol 1971;83:383-395. Terasaki PI, McCleland JD. Microdroplet assay of human serum cytotoxins. Nature 1964;204:998-- 1000. Tongio MM, Berrebe A, Mayer S. A study of lymphocytotoxic antibodies in multiparous women having had at least four pregnancies. Tissue Antigens 1972;2:378-388. van Wauwe JP, de Mey JR, Goossens JG. OKT3: a monoclonal antihuman T lymphocyte antibody with potent mitogenic properties. J Immunol 1980;124:2708--2713. Wilson HA, Winfield JB, Lanita RQ, Koffier D. Association of IgG antibrain antibodies with central nervous system dysfunction in systemic lupus erythematosus. Arthritis Rheum 1979;22:459--462. Winfield JB, Winchester RJ, Kunkel HG. Association of coldreactive antilymphocyte antibodies with lymphopenia in systemic lupus erythematosus. Arthritis Rheum 1975;18: 587--594. Winfield JB, Czyzyk J. Pathogenetic significance of antilymphocyte autoantibodies in systemic lupus erythematosus. In: Bijlsma JWJ, van der Linde SM, eds. Rheumatology in Europe. Amsterdam: 1995:220-223. Yamada A, Winfield JB. Inhibition of soluble antigen-induced T cell proliferation by warm reactive antibodies to activated T cells in systemic lupus erythematosus. J Clin Invest 1984 ;74:1948-- 1960.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

Mi-2 AUTOANTIBODIES Ira N. Targoff, M.D.

University of Oklahoma Health Sciences Center, Veterans Affairs Medical Center, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA

HISTORICAL NOTES Anti-Mi antibodies (anti-Mi), the first autoantibodies whose primary association was with myositis (without overlap) were described in 1976 in the prototype patient ("mi"), whose serum fixed complement when mixed with calf thymus extract (Reichlin and Mattioli, 1976). To look for the same antibodies in others, sera were papain-digested to produce Fab fragments, which were tested for blocking of the CF reaction of serum Mi. Complete inhibition was seen with 5/11 dermatomyositis (DM) and 1/6 polymyositis (PM) sera but 0 of 69 control normal or muscle or connective tissue disease sera (although a few showed partial inhibition). Serum Mi showed two precipitin lines by immunodiffusion (ID) against calf thymus extract: one seen only at low concentrations of extract (Mi-1) and the other only at high concentrations (anti-Mi-2). Purified Mi-1 antigen showed a 150 kd protein with apparent 75 kd subunits (Nishikai and Reichlin, 1980). An RIA demonstrated this antibody in only one other DM patient (2 of 19), but in none of 39 PM or 113 other control sera. Mi serum did not fix complement with Mi-1 antigen, and thus anti-Mi-1 was not the original Mi antibody. Mi-1 antigen was found to react with antibodies to bovine IgG, but its subunit size was different, and it was apparently localized to the nucleus. A later study supported the identification of Mi-1 as bovine IgG; the antibody was not myositisspecific, being more frequent in SLE than in DM (Targoff et al., 1983). The Mi-2 antigen as first purified in 1985 by biochemical and immunoaffinity methods (Targoff and Reichlin, 1985) reacted by ELISA with all sera that had anti-Mi-2 by ID. ELISA results were consistent

484

with CF inhibition for most sera and antibodies to Mi2 fix complement, leading to the conclusion that this represented the original Mi antibody. The antibody was found in that study to be specific for myositis and much more frequent in DM than PM, and subsequent studies have confirmed the strong association with DM. More recently, substantial progress has been made in defining its molecular structure and identifying the major antigenic protein.

THE AUTOANTIGEN

Description In the original study SDS-PAGE of purified, antigenically active Mi-2 from bovine thymus revealed proteins of 53 and 61 kd, neither of which reacted by immunoblot (IB) with anti-Mi-2 (Targoff and Reichlin, 1985). Recent studies, however, show that all IDpositive anti-Mi-2 sera immunoprecipitate a major protein that migrates at approximately 235--240 kd, along with other weaker, smaller proteins (Nilasena et al., 1995; Seelig et al., 1995). Study of HeLa cell extract defined at least seven smaller proteins of 200, 150, 72, 65, 64, 50 and 34 kd (Nilasena et al., 1995) (Figure 1). The pattern seen with HEp-2 cells differed somewhat (Seelig et al., 1995). The relationship of these proteins to one another is not yet clear. They may all be individual components of a macromolecular complex; some may be degradation products of the 240 kd (or other) band; or they may represent more than one independent antigen carrying shared epitopes. A preparation of bovine thymus Mi-2 using methods similar to the original study but eliminating the gel filtration step did reveal high-MW proteins of 250,

Figure 1. Immunoprecipitation with anti-Mi-2 positive sera from 35S-methionine-labeled HeLa extract, in 8% SDS-PAGE. Lanes 1--5 of the GEL section show the typical anti-Mi-2 band pattern with prominence of the 240 kd protein. The NITROCELLULOSE section shows immunoprecipitates after transfer; the 240 kd is less prominent. The Mi-2 components are labeled on the left. The section marked IPP shows immunoprecipitates prepared without cross-linking the antibody, so that the IgG heavy chain artifact is seen at -50 kd. Numbered anti-Mi-2 sera are from the same sample (3a and 3b are different serum concentrations). The methods were as in Nilasena et al., 1995. Component MWs in kd: a-240; a'=200; b-150; c=75; d-65; e=63; f=50; g-34 (Reproduced with permission from: Nilasena et al., 1995).

240 and 145 kd as in the immunoprecipitates, but the low-MW bands were much more intense than the high-MW bands (Nilasena et al., 1995). The prolonged preparation time, using tissue, might have resulted in greater proteolytic degradation. That the 240 kd protein is the major antigenic component is suggested by its prominent immunoprecipitation (IP) band. This has now been confirmed by studies showing that all anti-Mi-2 sera react with this protein (Ge et al., 1995; Seelig et al., 1995). Some sera appear to react with one or more other components, but these components react with no more than 50% of sera. Native vs. Denatured vs. Recombinant Antigen Performance About 50% (24/47) of ID-positive anti-Mi-2 sera reacted by IB with the 240 kd protein when anti-Mi-2

immunoprecipitates were used as antigen (Nilasena et al., 1995) (Figure 2); whereas, 77% (10) of 13 IDpositive sera reacted with a similar (235 kd) protein when HEp-2 cell nuclear extracts were used. All antiMi-2 sera studied to date, however, specifically react with recombinant fragments of the 240 kd major antigenic protein; >60 were tested against a fusion protein carrying a 40 kd fragment (Ge et al., 1995) (Figure 3), and 13 against a 55 kd fragment (Seelig et al., 1995). The epitope(s) on the 40 kd fragment are conformational, but the 55 kd recombinant fragment is active after denaturation. The titer of reactivity with this fragment does not correlate with A N A titer, suggesting that it is not a quantitative reflection of the anti-Mi-2 response. However, recombinant antigen is potentially as effective as natural antigen for antibody detection. There are no recombinant forms of the other Mi-2 proteins available. Because only sera that react strongly by IB with

485

Figure 2. Immunoblot of anti-Mi-2 immunoprecipitates, all prepared with serum Mi and transferred as in Fig. 1, then each lane blotted with an individual serum. Lanes 1, 5, 6, 7=normal; lane 29=Mi-2 negative myositis; other lanes=anti-Mi-2 sera from different patients. The strong staining of the 63--65 kd region is not specific for the antibody, and is considered an artifact. Staining of 240 kd is seen in 24 lanes; staining of 200 in lanes 2 and 8; staining of 150 in lanes 11, 18, 20, 26, 31, 35, 46; staining of 75 kd in lanes 11, 16, 19, 22, 33, 38, 39, 51; and staining of 50 kd in lanes 13, 17, 18, 34, 36 (Reproduced with permission from: Nilasena et al., 1995).

the 240 kd protein also stain the 200 kd band, the 200

of 240 kd. Other specific reactions s h o w n in Figure 2

kd m a y be a d e g r a d a t i o n p r o d u c t or alternative form

include: seven sera reactive with the 150 kd protein

Figure 3. Immunoreactivity of anti-Mi-2 sera with plaques of Mi-2 recombinant phage. A 1:1 mixture of Mi-2 recombinant and wildtype plaques is adsorbed to each nitrocellulose. Each numbered section was developed with a different serum at 1:500. Sections 1-22 on each strip were developed with anti-Mi-2 sera; 23--33 with normal sera; and 34-44 with anti-Mi-2 negative disease sera. The antiMi-2 sera used for disk A all were immunoblot positive in Fig. 2, while those used for disk B all were immunoblot negative. Reaction, defined as significant staining of 50% of plaques (indicating specificity for recombinant), is seen with all anti-Mi-2 but no patient sera (Reproduced with permission from: Ge et al., 1995). 486

(four of which were 240 kd-negative); eight with the 75 kd (three of which were 240 kd-negative); and five with the 50 kd (Nilasena et al., 1995). One anti-Mi-2negative control serum reacted with the 150 kd, but none with the 240, 75 or 50 kd proteins. Sources Indirect immunofluorescence (IIF) and/or ID studies demonstrate the Mi-2 antigen in all cell lines and tissues tested from all mammalian species thus far examined (mouse, rat, rabbit, bovine, human). No data are available in nonmammalian species. Calf (or rabbit) thymus extract is a better source of antigen for ID or purification than bovine or rat liver, and nuclear extracts are enriched for the antigen (Nishikai and Reichlin, 1980; Targoff and Reichlin, 1985). HeLa cells (Nilasena et al., 1995) and HEp-2 cells (Seelig et al., 1995) are good sources for IP, and HEp-2 cells are good sources for IIF. Indirect immunofluorescence, which confirms the nuclear localization, usually shows a very strong, finely speckled, nuclear pattern with sparing of the nucleolus and complete absence of cytoplasmic staining. Methods of Purification Mi-2 antigen can be purified from calf thymus extract by a combination of biochemical (DEAE-cellulose ion-exchange chromatography and Sepharose 6B gel filtration) and immunoaffinity methods (eluting with 4 M MgCI2) (Targoff and Reichlin, 1985). SDSPAGE shows the 53 and 61 kd proteins with only weak 25--30 kd additional proteins present. Adequate antigen for ELISA can be obtained using batch DEAE separation (0.1--0.2 M NaC1) followed by immunoaffinity chromatography (Nilasena et al., 1995).

or raised by immunization of a rabbit, cross-reacted with the 235 kd protein. The predicted full protein had 1,912 amino acids and a calculated MW of 218 kd. Although it was a novel sequence, it contained seven motifs (including a "DEAD/H" box and nucleotide binding sites) that are characteristic of the SNF2/ RAD54 family of nuclear helicases, such as the hSNF2L protein, a global activator of transcription. Other helicases in this family are involved in replication, nucleotide excision repair or chromosome segregation. However, as with hSNF2L, no classic DNAbinding motifs were found in this sequence. A similar role for Mi-2 was, therefore, proposed (Seelig et al., 1995). rMi-2 included five of the helicase motifs, but it is not known if they are antibody binding sites. Other motifs noted were N-glycosylation and N-myristoylation sites and several nuclear targeting sites. A 1.6 kb sequence encoding a 60 kd fragment of the Mi-2 major antigen has also been described (Ge et al., 1995). This fragment was specifically reactive with anti-Mi-2 sera, and affinity-purified patient antibodies and raised rabbit antiserum to the fragment cross-reacted with the Mi-2 240 kd protein. Unlike rMi-2, which reacted with all sera after denaturation, reaction of most sera with this fragment was conformation dependent (not formed by in vitro translation). A 40 kd reactive portion of the 60 kd fragment did show areas of similarity to DNA-binding motifs, including two sets of two potential zinc fingers; the two sets showed some sequence similarity to each other. However, no nucleic acid is immunoprecipitated by anti-Mi-2 sera (Nilasena et al., 1995). Several charged regions are also seen, including the region between the sets.

AUTOANTIBODIES

Commercial Sources

Pathogenetic Role

None available.

Human Disease. The role of anti-Mi-2 in DM is unknown. There is no direct evidence supporting or excluding a role for anti-Mi-2 in disease pathogenesis. In favor of a role is the marked disease specificity and the association with DM (rather than PM). The major pathogenetic mechanism of DM appears to be complement-mediated vasculopathy (Targoff, 1993). Antibodies are presumably involved in the local activation of complement, but their specificity is unknown. Against a pathogenic role is the lack of anti-Mi-2 in the majority of DM patients.

Sequence Information A sequence encoding the full-length of the Mi-2 major antigen was recently described (Seelig et al., 1995). Its identity with the 235--240 kd protein was demonstrated by expression of a 1.5 kb fragment of the central portion ("rMi-2"); rMi-2 reacted specifically with anti-Mi-2 sera, and antibodies to the 55 kd product, that were affinity-purified from patient serum

487

Animal Models. No animal models of the production of anti-Mi-2 are available. Rabbits immunized with recombinant fragments of the major antigen produced antibodies to those portions (Ge et al., 1995; Seelig et al., 1995), but no disease in the rabbits was described. Genetics Anti-Mi-2 is associated with a significantly increased frequency of HLA-DR7. In one study, DR7 was found in 75% of anti-Mi-2 patients but only 16% of myositis-specific autoantibody (MSA)-negative controls (p<0.001); all had DRw53, and DQAl*0201 was later associated (Love et al., 1991; Miller, 1993). In a preliminary report (Mierau et al., 1993), DR7 was found in seven of eight (87.5% vs. 25% of normals, p=0.0088), with five apparent homozygotes (0 of 85 normals, p<0.0001). No families or twin pairs have been studied.

Factors Involved in Pathogenicity and Etiology The very high disease specificity of the antibodies suggests an important relationship to fundamental etiologic mechanisms, but whether anti-Mi-2 are a marker of an etiologic event or a mediator of tissue injury is unknown (Plotz et al., 1995). There is no information regarding the isotypes, idiotypes, subclasses or avidity. The cloned 40 kd fragment of the 240 kd protein reacts with all anti-Mi2 sera (Ge et al., 1995); because most or all recognize exclusively conformational epitope(s), there might be a common epitope. The charged regions in this fragment might enhance antigenicity. This region appears to be separate from that which is recognized by IB. As noted, the recombinant 55 kd fragment was active by IB against all anti-Mi-2 patients (Seelig et al., 1995), but the ELISA titer against rMi-2 did not correlate with ANA titer. This is consistent with the possibility that the major binding site is not represented on this fragment. The relation of these findings to pathogenicity is unclear. The indications of a shared epitope suggest the possibility of molecular mimicry in the generation of anti-Mi-2, but specific potential sites with similarity or cross-reaction with proteins of infectious agents are unknown. There are no data regarding polyclonal activation in anti-Mi-2 patients; coexisting autoantibodies (anti-Ro/SSA, anti-U1RNP, etc.) are unusual, but occasionally seen. Cellular autoimmunity to this antigen has not been demonstrated.

488

Methods of Detection The optimal method for accuracy of detection of antiMi-2 is 35S-methionine-labeled IP, which is highly sensitive, yet very specific when an anti-Mi-2 standard is used (Table 1). The multiple bands of Mi-2 increase the specificity of IP by creating a characteristic pattern. With HeLa cells, the 75, 50 and 34 kd bands are not always visible, but the 240, 150, 65 and 63 kd should be present with the 240 kd usually being most intense (Nilasena et al., 1995). In studies with HEp-2 cells, some of these bands were not seen (Seelig et al., 1995). The usual method for clinical detection of anti-Mi2 is ID. Concentrated (>100 mg protein/mL) calf thymus (or rabbit) extract, which is commonly used as antigen, can be enriched for Mi-2 by fractionation by ammonium sulfate (30-60%), DEAE (0.1--0.2 M NaC1) and/or preparation of nuclei. Concentrated cultured-cell lysate can also be used. ID is almost (>95%) as sensitive qualitatively as IP, and because ID is much quicker, simpler and less expensive, it is the most practical method of detection for routine clinical purposes at this time. Counterimmunoelectrophoresis can also be used. ELISAs for detection of anti-Mi-2 using affinitypurified antigen require highly purified antigen to reduce the background and allow a low threshhold for positive results. Several low-level false-positives (could not be inhibited by antigen) were noted using affinity-purified antigen (Targoff and Reichlin, 1985; Targoff et al., 1990). This is a problem because the main value of anti-Mi-2 is its disease specificity, and false-positives could lead to misdiagnosis. Other sera showed apparent true binding but were negative by ID; the significance of these is unclear. At this time the ELISA should be reserved for initial screening (with confirmation of positives by ID or IP), quantitation, or research. Recombinant protein might improve the ELISA. Eleven of twelve known anti-Mi-2 sera were clearly positive against the "rMi-2" recombinant fragment (Seelig et al., 1995); whereas, >99% of 1,355 control samples (from normals or patients with positive ANAs, SLE, or RA) were negative. Among controls, the seven borderline sera were IB-negative, but the three with definite elevation were IB-positive. One was confirmed by IP and ID; the other two were IPnegative. These had the lowest elevations of positiverange sera, and either had very low titer or were falsepositives. The single false-negative anti-Mi-2 serum

Table 1. Methods of Detection of Mi-2 Antibodies Method

Antigen 1

ID 3

Calf thymus extract

IP

ELISA

IB

IIF

Specificity

Finding

Comments

95%

100%

Weak to moderate precipitin line

Requires highly concentrated extract; most practical current method

35S-HeLa or Hep-2 extract

>99%

>99%

_>8 bands, 34--240 kd 240 usually strongest

Best method, but long and difficult; should be compared to standard; no associated nucleic acid

1) Purified calf thymus antigen 2) Purified Rec fragment (rMi-2)

>98%

=90--95% 4

High binding

92%

=99%

Borderline: >mean+=3SD Definite: >mean+4.4SD

Sens/spec depends on quality of antigen Sens tested against 1,355 controls; borderlines should be confirmed by blot

50--55%

High 6

Staining of 240 kd; Less often other bands Staining of 235 kd Staining of 55 kd band

Not reliable, even with purified antigen

Fine-speckled nuclear; spares nucleoli

Useful for excluding, but pattern not specific

1) HeLa 5

Sensitivity 2

2) Hep-27 3) rMi-2

77% 100% 8

High High

Hep-2 cells

100%

Low

ID: double immunodiffusion; IP: immunoprecipitation; ELISA: enzyme linked immunosorbent assay; IB: immunoblotting; IIF: indirect immunofluorescence; sens: sensitivity; spec: specificity. 1 Antigen listed is most often used in reports. Others may be equally effective. e Sensitivity and specificity are for detection of antibody in serum, not for detection of disease. 3 CIE can detect anti-Mi-2, but sensitivity and specificity have not been reported. 4 Sensitivity and specificity of ELISA varies with purity of antigen, technique, confirmatory studies used, etc. 5 Crude whole HeLa extract is usually ineffective as antigen. Partial purification is recommended; immunoaffinity methods can be used for this purpose. 6 Specificity depends on purity of antigen and on technique, and has not been thoroughly studied; at least one false-positive reaction with the 150 kd component has been observed. 7 Reaction was seen with 235 kd protein when extract of isolated nuclei was used. 8 Based on 13 ID-positive sera tested.

may reflect a difference in epitope reactivity; further study is needed using full-length protein or other fragments. Immunoblotting against natural Mi-2 antigen is unreliable for detection of anti-Mi-2. Anti-Mi-2 shows no IB reaction with unfractionated HeLa extract. IB against HeLa anti-Mi-2 immunoprecipitates can detect binding (Nilasena et al., 1995), but almost 50% are still negative against the 240 kd protein. In a study using HEp-2 cell nuclear extracts, IB binding to the 235 kd protein was found with 10 of 13 sera (Seelig et al., 1995). All 13, however, reacted significantly with the recombinant protein by IB. This difference may relate to the concentration of antigen. Low sensitivity prevents recommendation of IB against natural antigen or extracts for detection of anti-Mi-2, but IB against recombinant antigen is more promising. Anti-Mi-2 sera should show a positive ANA, commonly in high-titer (> 1:1000). The nuclear pattern is not specific, but can be distinguished from the coarse-speckled pattern of anti-U1RNP, the most

common other defined antibody giving a high-titer nuclear ANA in PM/DM (Reichlin and Arnett, 1984).

CLINICAL UTILITY

Application Confirm. The very high specificity of anti-Mi-2 for inflammatory myopathy makes it a valuable aid to diagnosis when present (Table 2). Anti-Mi-2 are associated with DM, which is usually easier to diagnose than PM due to the presence of skin lesions. Some DM patients can satisfy the Bohan and Peter criteria (Bohan and Peter, 1975) without a muscle biopsy. However, diagnosis of DM is difficult in some cases. The skin manifestations can be mild, equivocal, or atypical; the CK may not be elevated; or the biopsy or EMG can be normal or without definitive findings. A test that can firmly establish the diagnosis could provide confirmation crucial to increasing the con-

Table 2. Clinical Summary of Mi-2 Antibodies CLINICAL ASSOCIATION: % anti-Mi-2 with: DM ~ PM no PM/DM

=97% ---3% <1% 2

FREQUENCY (U.S.): % with anti-Mi-2 of: Adult DM Juvenile DM PM All PM/DM

=15--25% =10--15% <1% --5-- 10%

GENETICS" % with HLA DR73:

Ethnic association: Associated antibody: CLINICAL USE: Presence: Absence: Predictions:

Anti-Mi-2 pts: 75% Ab-negative: 16% Apparent 1" in Hispanic patients Usually none (but ANA should be positive)

Very strong evidence of PM/DM. Strongly suggests DM. Does not provide help in diagnosis. Rash often florid. Reported patients have had relatively good prognosis, but severe disease may occur.

1 Includes DM rash without myositis. 2 Only patient with definite anti-Mi-2 without PM/DM had SLE (Seelig et a1.,1995). 3 Data from Love et al., 1991.

490

fidence in the diagnosis. Objective documentation can also be helpful for physicians assuming care later in the course, after the rash has resolved. Patients with weakness, elevated CK, an EMG suggestive of PM/DM and a classic DM rash might not need further testing. If the rash is not diagnostic, the CK is normal or there are other atypical features, a biopsy might be advisable, but if such patients have anti-Mi-2 (by ID or IP), further evaluation may be unnecessary. AntiMi-2 would be particularly helpful in "amyopathic DM," in which cutaneous DM lesions are present for 22 years without evidence of myopathy (Euwer and Sontheimer, 1994), because the usual DM criteria cannot be applied to such patients. Anti-Mi-2 has not been reported in definite amyopathic DM, but one patient had a compatible rash treated shortly after its onset, without clinical myositis ever appearing. ELISA screening of a large population recently found one confirmed anti-Mi-2-positive sample (from 901 in the positive ANA group) (Seelig et al., 1995). This patient had a diagnosis of SLE with anti-doublestranded DNA, but further clinical details were not presented. Ninety-four known SLE patients were negative in that study, and numerous other SLE patients (without myositis) have been anti-Mi-2 negative. Two patients with elevated ELISA samples from this group were IP-negative and did not have PM/DM, but their antibody status is uncertain. Overall, the anti-Mi-2 antibody remains useful in confirmation of the diagnosis.

from DM overall. The frequency of the features associated with the "anti-synthetase syndrome" (interstitial lung disease, arthritis, Raynaud' s, etc.) was not increased compared to other myositis patients (Targoff et al., 1990). Presentation tends to be acute, but the prognosis for survival is good (Love et al., 1991).

Differentiate. Among patients with evidence of myopathy, the presence of anti-Mi-2 is strong evidence for DM (or occasionally PM); anti-Mi-2 have not been reported in any other form of muscle disease.

Demographics Anti-Mi-2 occur in both adult and juvenile DM. Although they are slightly less frequent in juvenile DM, they are more common than anti-Jo-1 in children. They are the most common MSA among children with DM without overlap syndromes, although still present in only a minority. The female:male ratio of anti-Mi-2 patients (1.2) is not statistically different from that of PM/DM (2.0) or DM (2.3) overall (Love et al., 1991). Anti-Mi-2 has been seen in Caucasian, African-American, Asian and Hispanic patients. There are indications that anti-Mi-2 may be more common in Hispanic than U.S. Caucasian patients and are particularly frequent among a group of patients from Guatemala (Rider et al., 1994).

Antibody Frequency in Disease Exclude. Because the prevalence of anti-Mi-2 is low even in DM, its absence does not exclude the diagnosis of DM and should not be taken as evidence against the diagnosis.

Subclassify. Almost all myositis patients found to have anti-Mi-2 have had DM (=97%); only two patients without compatible rash have been recognized (Table 2). The rash also is more prominent or more of a problem in adult DM patients with anti-Mi-2. Although only a small number of patients has been studied, anti-Mi-2 patients, compared to antibodynegative DM patients, tend to have a higher proportion with the "V" sign (involvement of the V of the neck, 100% of 10 patients v s . 36% of 70), "shawl" sign (upper back and shoulders, 56% v s . 22%) or cuticular overgrowth (100% v s . 30%) (Love et al., 1991). Although anti-Mi-2 children also have typical rashes, these signs are not increased. The subgroup defined by anti-Mi-2 has no other apparent differences

Correlation with Disease Activity. Anti-Mi-2 has been present in the earliest samples tested in all patients described (it has not arisen in patients who were previously negative). In general, anti-Mi-2 persist throughout the course, and usually do not change much in titer, although there are few longitudinal data reported. Effect of Various Therapies. Although careful studies have not been performed, the effect of treatment on the antibody seems to be limited. However, patient response to treatment tends to be good and often complete, commonly to corticosteroids alone, and generally comparable to those without MSAs (Joffe et al., 1993; Rider et al., 1994). However, antiMi-2-associated DM can be severe, including one recent patient who died of sepsis and another with profound weakness and significant residual incapacity which was resistant to treatment. Several anti-Mi-2

491

patients have had malignancy, including breast or colon cancer and one with thymoma (Targoff and Reichlin, 1985).

disease (such as weakness or CPK). A negative test is not helpful.

Varieties of Disease Associations. Children with antiMi-2 have typical juvenile DM, and cannot be distinguished clinically from those without the antibodies. Adults with anti-Mi-2 often have classic presentations, but do not show distinctive features. Overlap syndromes are quite uncommon.

CONCLUSION

Sensitivity/Specificity As noted, ID and IP are highly specific tests, with very few false-positives (with regard to the presence of the antibody) when properly performed. Both are also very sensitive for detection of anti-Mi-2, with IP only slightly higher. ELISA is equally sensitive but has more false-positives. Assuming accuracy of testing, the presence of antiMi-2 has low sensitivity for disease detection, present in about 5--10% of PM/DM, 15--25% of adult DM (Duncan et al., 1990), and ~-10--15% of juvenile DM. However, anti-Mi-2 are almost completely specific for myositis, and >96% specific for DM. Even with the one exception noted above, the positive predictive value for PM/DM would still be high and would be higher in a population of patients with signs of muscle

REFERENCES Bohan A, Peter JB. Polymyositis and dermatomyositis. Parts 1 and 2. N Engl J Med 1975;292:344--347, 403--407. Duncan AG, Richardson JB, Klein JB, Targoff IN, Woodcock TM, Callen JP. Clinical, serologic, and immunogenetic studies in patients with dermatomyositis. Acta Derm Venereol (Stockh) 1990;71:312-316. Euwer RL, Sontheimer RD. Dermatologic aspects of myositis. Curr Opin Rheumatol 1994;6:583--589. Ge Q, Nilasena DS, O'Brien CA, Frank MB, Targoff IN. Molecular analysis of a major antigenic region of the 240 kd protein of Mi-2 autoantigen. J Clin Invest 1995;96:17301737. Joffe MM, Love LA, Left RL, Fraser DD, Targoff IN, Hicks JE, Plotz PH, Miller FW. Drug therapy of the idiopathic inflammatory myopathies: predictors of response to prednisone, azathioprine, and methotrexate and a comparison of their efficacy. Am J Med 1993;94:379--387. Love LA, Left RL, Fraser DD, Targoff IN, Dalakas M, Plotz PH, Miller FW. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups.

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Anti-Mi-2 are unique in their primary association with DM (rather than PM, PM/DM, or PM/overlap), in being the only defined MSA directed at a nuclear antigen, and in being the only MSA (excluding antiPM-Scl) with a frequency in children comparable to that in adults. Molecular analysis is beginning to uncover the role of the Mi-2 antigen in the cell, with the finding that the 240 kd major antigen is a member of the helicase family of proteins and may be involved in transcriptional activation. The availability of recombinant antigen should facilitate testing for the antibody. The antibody can be of significant clinical value because of its very high disease specificity. Testing should be considered in patients with suspected myositis who have a positive ANA, especially when there are cutaneous findings. See also AMINOACYL-tRNA HISTIDYL (JO-1) SYNTHETASE AUTOANTIBODIES, AMINOACYL-tRNA (OTHER THAN HISTIDYL) SYNTHETASE AUTOANTIBODIES, PM-SCL AUTOANTIBODIES and SIGNALRECOGNITIONPARTICLE AUTOANTIBODIES.

Medicine 1991;70:360-374. Mierau R, Dick T, Genth E. Anti-Mi-2 antibody positive dermatomyositis is associated with HLA-DR7 [Abstract]. Rev Esp Reum Enferm Osteoartic 1993; 20(Supp):41. Miller FW. Myositis-specific autoantibodies: touchstones for understanding the inflammatory myopathies. JAMA 1993; 270:1846--1849. Nilasena DS, Trieu EP, Targoff IN. Analysis of the Mi-2 autoantigen of dermatomyositis. Arthritis Rheum 1995;38: 123--128. Nishikai M, Reichlin M. Purification and characterization of a nuclear nonhistone basic protein (Mi-1) which reacts with anti-immunoglobulin sera and the sera of patients with dermatomyositis. Mol Immunol 1980;17:1129--1141. Plotz PH, Rider LG, Targoff IN, Raben N, O'Hanlon TP, Miller FW. Myositis: immunologic contributions to understanding cause, pathogenesis, and therapy. Ann Intern Med 1995;122:715--724. Reichlin M, Mattioli M. Description of a serological reaction characteristic of polymyositis. Clin Immunol Immunopathol 1976;5:12--20. Reichlin M, Arnett FC. Multiplicity of antibodies in myositis sera. Arthritis Rheum 1984;27:1150-1156.

Rider LG, Miller FW, Targoff IN, Sherry DD, Samayoa E, Lindahl M, Wener MH, Packman LM, Plotz PH. A broadened spectrum of juvenile myositis. Myositis-specific autoantibodies in children. Arthritis Rheum 1994;37:1534- 1538. Seelig HP, Moosbrugger I, Ehrfeld H, Fink T, Renz M, Genth E. The major dermatomyositis-specific Mi-2 autoantigen is a presumed helicase involved in transcriptional activation. Arthritis Rheum 1995;38:1389--1399. Targoff IN, Raghu G, Reichlin M. Antibodies to Mi-1 in SLE: relationship to other precipitins and reaction with bovine

immunoglobulin. Clin Exp Immunol 1983;53:76--82. Targoff IN, Reichlin M. The association between Mi-2 antibodies and dermatomyositis. Arthritis Rheum 1985;28:796-803. Targoff IN, Nilasena DS, Trieu EP, Arnet FC, Pachman LM, Callen JP, Miller FW. Clinical features and immunologic testing of patients with anti-Mi-2 antibodies [Abstract]. Arthritis Rheum 1990;33:$72. Targoff IN. Humoral immunity in polymyositis/dermatomyositis. J Invest Dermatol 1993;100:116S--123S.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

MITOCHONDRIAL AUTOANTIBODIES Peter S.C. Leung, Ph.D. a, Ross L. Coppel, M.D. Ph.D. b and M. Eric Gershwin, M.D. a

aDivision of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, Davis, CA 95616, USA; bDepartment of Microbiology, Monash University, Clayton, Victoria, 3168, Australia

HISTORICAL NOTES The presence of antimitochondrial autoantibodies (AMA) in patients with primary biliary cirrhosis (PBC) was first reported in 1965 (Walker et al., 1965). AMA are detected in over 95% of patients with PBC. The trypsin-sensitive molecules localized in the inner mitochondrial membrane (Berg and Klein, 1987; 1989). The molecular identities of mitochondrial autoantigens in PBC were revealed in 1988 when two groups independently reported on the E2 subunits of 2-oxo acid dehydrogenase complex (Van de Water et al., 1988; Fussey et al., 1988; Yeaman et al., 1988). Subsequently, extensive molecular and immunological studies in PBC, including cloning of mitochondrial autoantigens (Coppel et al., 1988; Surh et al., 1989a; 1989b), antigen-specific isotype studies (Surh et al., 1988), mapping of T- and B-cell epitopes (Jones et al., 1995; Leung et al., 1995; Van de Water et al., 1995), analysis of murine and human monoclonal antibodies (Surh et al., 1990a; Leung et al., 1992a; Cha et al., 1993; 1994) and identification of autoantibody gene usage (Pascual et al., 1994) provided valuable reagents for understanding the immunopathogenesis of PBC as well as for the development of accurate and specific diagnostic tests based on recombinant autoantigens (Van de Water et al., 1989; Leung et al., 1992b). Recently, immunohistochemical studies detected the pyruvate dehydrogenase complexE2 (PDC-E2)-specific murine monoclonal antibody at the luminal region of bile duct epithelials of PBC patients but not in controls (Tsueneyama et al., 1995; Joplin et al., 1995). The pathology of PBC at the intrahepatic bile ducts and the distinct staining of bile duct epithelial cells in PBC livers by the PDC-E2-

494

specific monoclonal antibodies suggest that selective targeting against a molecule cross-reactive with PDCE2 or with an altered form of PDC-E2 (Van de Water et al., 1993) is a possible mechanism for this liverspecific autoimmune disorder.

THE AUTOANTIGENS Definition/Characteristics

Mitochondrial autoantigens are subunits of the 2-oxo acid dehydrogenase complexes (Table 1) and include: (1) Dihydrolipoamide acetyltransferase (EC 2.2.1.12), the E2 subunit of pyruvate dehydrogenase (PDC-E2) (Van de Water et al., 1988; Yeaman et al., 1988); (2) The E2 subunit of branched chain 2-oxo acid dehydrogenase complex (BCOADC-E2) (Yeaman et al., 1988); (3)The E2 subunit of 2-oxo-glutarate dehydrogenase complex (OGDC-E2) (Fussey et al., 1988); (4) Protein X (Surh et al., 1989a); (5) PDC-Ela and PDC-E113 (Fregeau et al., 1990). The mitochondrial autoantigens are located in the inner mitochondrial membrane of eukaryotic cells. Indeed, 2-oxo acid dehydrogenase complexes are ubiquitous and present in all species, ranging from bacteria to mammalian cells. The primary function of the 2-oxo acid dehydrogenase complexes is channeling electrons via oxidative decarboxylase reactions. The 2-oxo acid dehydrogenase complexes are similar in that each of the enzyme complexes consists of three nuclear-coded subunits, El, E2 and E3. Both PDC and BCOADC have E1 subunits, namely Elo~ and E I~ (Yeaman, 1986). The PDC, E 1 subunit decarboxylates pyruvate, releasing CO 2 and an acetyl group; the E2 subunit,

Table 1. Summary of the Autoantigens of Primary Biliary Cirrhosis Antigen

MW (kd)

Complex

cDNA Cloned

B epitope identified

PDC-E2

74

PDC

+

+

BCOADC-E2*

52

BCOADC

+

+

OGDC-E2

48

OGDC

Protein X (E3BP)

56

PDC

PDC-EI~

41

PDC

+

+

PDC-EI[~

36

PDC

dihydrolipoamide acetyltransferase transfers the acetyl group by a lipoic acid cofactor to coenzyme A (Co A) which enters the tricarboxylic acid cycle. The E3 subunit, a flavin adenine dinucleotide (FAD)-containing dihydrolipoamide, reoxidizes lipoic acid, forming reduced nicotine amide adenine dinucleotide (NADH). Similarly, OGDC decarboxylases 2-oxo-glutarate to succinyl CoA, and BCOADC catalyses the catabolism of branched chain amino acids, leucine, valine and isoleucine (Gershwin and Mackay, 1991).

Recombinant Antigens/Commercial Sources The cDNAs of PDC-E2 (Gershwin et al., 1987; Thekkumkara et al., 1987), BCOADC-E2 (Lau et al., 1988; Griffin et al., 1990), OGDC-E2 (Nakano et al., 1991; 1993) and PDC-Ela (Dahl et al., 1987; Koike et al., 1988; De Meirleir et al., 1988) have been cloned (Table 1). Recombinant proteins of PDC-E2, BCOADC-E2, PDC-E 1c~ expressed in a glutathione Stransferase expression vector, pGEX, (Leung et al., 1990; 1995) can be affinity purified by glutathione agarose. E. coli expression clones containing these plasmids can be induced by isopropylthiogalactopyranoside (IPTG), producing recombinant fusion proteins. Alternatively, the native form of the 2-oxo acid dehydrogenase can be biochemically purified (Roche and Cate, 1977) and commercial preparations of porcine heart PDC, OGDC are available (Sigma, St. Louis, MO). Preparations of individual subunits of the 2-oxo acid dehydrogenase complexes are not commercially available. Recombinant proteins of rat and human PDC-E2 (Leung et al., 1990; Surh et al., 1990b), bovine BCOADC-E2 (Leung et al., 1995), rat OGDC-E2 (Leung et al., unpublished data) and human PDC-EI~ (Iwayama et al., 1991) are effective and specific tools for the detection of AMA using either immmunoblot-

ting or ELISA, and the results are comparable with those using commercially available mitochondrial preparations or biochemically purified mitochondrial fractions (Provenzano et al., 1993). Because the cDNA of protein X is not available, detection of protein X is reliant on immunoblots using mammalian mitochondrial protein preparations.

AMA Epitopes of the 2-Oxo Acid Dehydrogenase Complexes. The E2 subunits of the 2-oxo acid dehydrogenase complex are structurally similar in their lipoic acid-binding domain, E3-binding unit and inner E2 core (Yeamen et al., 1986; Leung et al., 1994). StiJdies, with oligopeptides (Van de Water et al., 1988) and recombinant fusion proteins of PDC-E2 (Surh et al.,1990b) show that the epitope of PDC-E2 is uniquely located in the lipoyl domain of PDC-E2 but not in other domains of PDC-E2. PBC patient sera recognize specifically recombinant peptides corresponding to the outer lipoyl domain and inner lipoyl domain of human PDC-E2 (Surh et al., 1990a), as shown by studies with various recombinant peptides spanning the entire human PDC-E2 cDNA. Moreover, most of the reactivity is confined to amino acid residues 128--227 within the inner lipoyl domain; a recombinant peptide containing amino acid residues 160--227 was not reactive. Deletion of amino acid residues beyond residue 221 at the C-terminus abolishes reactivity with PBC sera. Recombinant peptide containing amino acid residues 146--221 is required for detectable binding, and amino acid residues 128--227 are required for strong binding. The data strongly suggest the existence of a conformational epitope between amino acid residues 128 and 227 in PDC-E2. Likewise, when expression clones spanning the entire bovine BCOADC-E2 are tested for their immunoreactivity against PBC sera, immunoreactivity is primarily localized in the lipoic acid binding 495

domain and amino acid residues 1--227 is important for strong binding. Together, it appears that AMA to PDC-E2, BCOADC-E2 recognize conformational epitopes, and the lipoic acid domain is necessary for strong antibody binding (Leung et al., 1995). Interestingly, except for PDC-E2 and protein X, AMA against the E2 subunits of the 2-oxo acid dehydrogenase complexes are not cross-reactive (Surh et al., 1989a). In addition, several studies of the role of lipoic acid in AMA recognition (Quinn et al., 1993) suggest that AMA are capable of binding to both lipoylated PDC-E2 and unlipoylated PDC-E2. The epitope of PDC-Elc~ is mapped within a 300 amino acid region that contains the enzyme functional site, namely the phosphorylation site and TPP binding site (Iwayama et al., 1991).

T-Cell Epitopes Recently extensive studies on cloned T-cell lines derived from peripheral blood mononuclear cells and liver from PBC patients demonstrated the heterogeneity of the T-cell receptor V~ repertoire usage. Furthermore, epitope mapping analysis shows that peripheral T cells from patients with PBC are directed to two entirely separate regions of PDC-E2; T cells from 54 and 36% of PBC patients responded to the outer lipoyl domain and inner lipoyl domain, respectively. Interestingly, T cells from almost half of the patients also responded to PDC-E1; T cells from nonPBC patients do not react to PDC at all (Van de Water et al., 1995). Similarly, peripheral T cells from patients with PBC responded to PDC-E2/X more significantly in precirrhotic PBC patients (stages I--III) than cirrhotic stage (stage IV) (Jones et al., 1995). Moreover, these T-cell clones were restricted by the MHC molecule, HLA DRB4 0101 (Shimoda et al., 1995). Thus, the presence of activated T-cell infiltrate in the portal tract and PDC-E2-specific peripheral T cells also play a significant role in the pathogenesis of PBC.

AUTOANTIBODIES Pathogenetic Role Serial sections of PBC liver stained with a nearly identical frequency for the presence of PDC-E2 and IgA on bile ducts and luminal staining on the bile duct epithelium (Van de Water et al., 1993). More-

496

over, the presence of PDC-E2 antibodies in bile of PBC patients is consistent with a pathological role in the development of PBC. Because IgA is capable of binding intracellular antigens during normal trafficking through epithelial cells, PDC-E2-specific IgA antibodies are hypothesized to bind to PDC-E2 in bile duct epithelium and prevent the normal transportation of PDC-E2 into the mitochondria, resulting in metabolic dysfunction and cell death. However, the existence of PDC-E2-specific IgA antibodies and their participation in the disease process remains to be demonstrated. Because selective targeting of bile duct epithelial cells by AMA is a major characteristic of PCB, needle biopsy specimens from either stage I or stage II PBC patients were simultaneously stained with antibodies to PDC-E2, MHC class II and BB l/B7. In the activation of T cells, MHC class II delivers the first message through its interaction with T-cell receptor; the BB1/B7 present on antigen-presenting cells provide the second signal to T cells through CD28. Study of the order of expression of these molecules by staining liver sections shows that few early stage PBC patients express the HLA-DR antigen and the BB l/B7 antigens (Tsuneyama et al., 1995). A previous report showing a high rate of HLA-DR antigen expression in PBC did not distinguish between early and late stage disease (Nakanuma and Kono, 1991). Taken together, these data suggest that the appearance of PDC-E2 or a PDC-E2 cross-reactive molecule precedes the expression of HLA-DR and the expression of the BB l/B7 co-stimulatory molecule. Idiotype-anti-idiotype interactions in PBC were demonstrated with a PDC-E2-specific mouse monoclonal antibody, CPZ674 (Zhang et al., 1993). Antiidiotypic antibodies in PBC are detected either by their specific binding to CPZ674 by ELISA or by detection of idiotype-anti-idiotype complexes' precipitation. Moreover, these anti-idiotypic antibodies are able to inhibit the binding of AMA to PDC, but not to other mitochondrial autoantigens. However, whether these anti-idiotypic antibodies are physiologically involved in the control of AMA (Zhang et al., 1993) and whether there are also anti-idiotypic antibodies to other mitochondrial autoantigens are unknown. Despite vigorous attempts to induce an animal model with PBC by immunization with antigens (Krams et al., 1989a) and by injection of peripheral blood lymphocytes from patients with PBC into SCID mice (Krams et al., 1989b), no definitive animal model of PBC is now available.

AMA can be detected by: (1) indirect immunofluorescence (Walker et al., 1965); (2)immunoblotting (Leung et al., 1995); (3) ELISA (Charles, 1993); or (4) inhibition of catalytic activity of the enzyme (Teoh et al., 1991). Immunofluorescence is by far the most commonly used clinical test for AMA, using HEp-2 cells or tissue sections from rat. However, immunofluorescence only detects the presence or absence of AMA and lacks the ability to detect specific antigens; whereas, the use of specific recombinant antigens can detect the presence of antigen-specific AMAs with high specificity and sensitivity. Background levels which are caused by nonspecific bonding seem to be the most common problem encountered in indirect immunofluorescence. Therefore, accurate detection of AMA by immunofluorescence requires a serial dilution of sera (usually 1:40, 1:80, 1:160, 1:320 and 1:640) and also an optimal dilution of FITC-labeled antihuman Ig. The precise optimal dilution of secondary FITC antibody may also vary from batch to batch. In addition, thorough washing steps and changes of washing solutions help in minimizing the background level.

autoimmune disease never reported in children; its incidence gradually rises with age. The incidence and prevalence of PBC varies quite widely: highest among northern European populations, lower in Japan and other parts of Asia. Available annual incidence data include 128 million population in Sweden, 128.5 million in England, 23 million for several western European countries, 23 million in Canada and 5.16 million in Japan (Lofgren et al., 1987; Myszor and James, 1990; Triger et al., 1984; Witt-Sullivan et al., 1990; Yasutoshu, 1989; Iwayama et al., 1992). Thus far, limited study has focused on the familial association of PBC and the data suggest a weak (if any) genetic predisposition to PBC (Cladwell et al., 1992). Drug therapy in PBC is based on the use of corticosteroids and immunosuppressive drugs which, however, have not shown a significant benefit to the natural history of the disease. Similar observations were reported with methotrexate and cyclosporine (Lombard et al., 1993). Moreover, corticosteroids can increase the incidence and severity of osteoporosis. Although several studies have suggested a beneficial effect of colichicine, prednisone and ursodeoxycholic acid, further observations are needed to determine the long-term effect of these drugs on PBC (Shibata et al., 1992; Kowdley and Kaplan, 1993; Lindor, 1994; Wolfhagen et al., 1994; Floreani et al., 1994). However, the effects of drug therapy on AMA levels are limited. Ursodeoxycholic acid, when added to culture of AMA-positive patients is reported to inhibit the production of Ig and anti-PDC antibodies in vitro (Ishikawa et al., 1991). Patients treated with ursodeoxycholic acid had significant improvements in serum levels of AMA titer (Poupon et al., 1991). The effect and mechanism of ursodeoxycholic acid in regulating AMA production still remains to be elucidated.

CLINICAL UTILITY

CONCLUSION

Disease Association

The application of molecular biology in clinical medicine dramatically changed our understanding of the immunobiology of PBC. The molecular characterization and identification of the mitochondrial autoantigens and the expression of cloned antigens greatly facilitated the development of reliable assays for mitochondrial autoantibodies. Previously, a number of nonspecific clinical laboratory assays such as the measurement of serum alkaline phosphatase, serum bilirubin, serum lipid levels and serum ceruloplasmin

Factors in Pathogenicity Studies using a recombinant clone of PDC-E2 determined that AMA against PDC-E2 are predominately IgG3 and IgM (Surh et al., 1988). Similar studies of A M A isotypes and subclasses reactive with other mitochondrial autoantigens in PBC are not available. Despite the high titer and specificity of AMA in PBC, their immunopathological role in the disease process is still unclear. Methods of Detection

Clinical diagnosis of PBC requires precise serological, immunological and histopathological data. Although a small number (5%) of PBC patients are AMAnegative, the determination of AMA is a powerful diagnostic tool in PBC. PBC is a disease affecting women as much as 10 to 20 times as frequently as men (Sherlock and Scheur, 1973; Kaplan, 1987) and is perhaps the only

497

were employed in the diagnosis of PBC. Application of recombinant proteins provides an efficient, specific and accurate alternative method for detection of AMA (Van de Water et al., 1989; Iwayama et al., 1991; Leung et al., 1992b) by ELISA and immunoblotting. The use of the appropriate recombinant proteins will eventually become a routine approach to detection of AMA. Through the rapid development of technology and refinement of research reagents, such as the molecular cloning and expression of mitochondrial autoantigens, the identification of B-cell and T-cell epitopes, the generation of antigen-specific antibodies

and the distinct immunohistochemical staining pattern of bile duct epithelial cells in PBC liver sections, questions can now be addressed about the role of AMA in the immunopathological process of PBC. Although AMA concentrations do not correlate with disease stages and prognosis, the specific reactivity of AMA, their ability to inhibit enzyme functions, the unique antibody gene usage and distinct staining pattern of bile duct epithelial cells in patients with PBC suggest that AMA play a definitive role in the immunopathological process of PBC.

REFERENCES

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Myszor M, James OF. The epidemiology of primary biliary cirrhosis in north-east England: an increasingly common disease? Q J Med 1990;75:377-385. Nakano K, Matuda S, Yamanaka T, Tsubouchi H, Nakagawa S, Titani K, Ohta S, Miyata T. Purification and molecular cloning of succinyltransferase of the rat c~-ketoglutarate dehydrogenase complex. Absence of a sequence motif of the putative E3 and/or E1 binding site. J Biol Chem 1991;266: 19013-19017. Nakano K, Matuda S, Sakamoto T, Takase C, Nakagawa S, Ohta S, Ariyama T, Inazawa J, Abe T, Miyata T. Human dihydrolipoamide succinyltransferase: cDNA cloning and localization on chromosome 14q24.2-q24.3. Biochim Biophys Acta 1993;1216:360-368. Nakanuma Y, Kono, N. Expression of HLA-DR antigens on interlobular bile ducts in primary biliary cirrhosis and other hepatobiliary diseases: an immunohistochemical study. Hum Pathol 1991;22:431-436. Pascual V, Cha S, Gershwin ME, Capra JD, Leung PS. Nucleotide sequence analysis of natural and combinatorial anti-PDCE2 antibodies in patients with primary biliary cirrhosis. Recapitulating immune selection with molecular biology. J Immunol 1994;152:2577-2585. Poupon RE, Balkau B, Eschwege E, Poupon R, UDCA-PBC Study Group. A multicenter, controlled trial of ursodiol for the treatment of primary biliary cirrhosis. N Engl J Med 1991 ;324:1548--1554. Provenzano G, Diquattro O, Craxi A, Almasio P, Pinzello G, Marino L, Fiorentino G, Rinaldi F, Pagliaro L. Immunoblotting as a confirmatory test for antimitochondrial antibodies in primary biliary cirrhosis. Gut 1993;34:544-548. Quinn J, Diamond AG, Palmer JM, Bassendine MF, James OF, Yeaman SJ. Lipoylated and unlipoylated domains of human PDC-E2 as autoantigens in primary biliary cirrhosis: significance of lipoate attachment. Hepatology 1993;18:13841391. Roche TE, Cate RL. Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties. Arch Biochem Biophys 1977;183: 664--677. Sherlock S, Scheur PJ. The presentation and diagnosis of 100 patients with primary biliary cirrhosis. N Engl J Med 1973;289:674--678. Shibata J, Fujiyama S, Honda Y, Sato T. Combination therapy with ursodeoxycholic acid and colchicine for primary biliary cirrhosis. J Gastroenterol Hepatol 1992;7:277--282. Shimoda S, Nakamura M, Ishibashi H, Hayashida K, Niho Y.. HLA DRB4 0101-restricted immunodominant T cell autoepitope of pyruvate dehydrogenase complex in primary biliary cirrhosis: evidence of molecular mimicry in human autoimmune diseases. J Exp Med 1995;181:1835-1845. Surh CD, Copper AE, Coppel RL, Leung P, Ahmed A, Dickson R, Gershwin ME. The predominance of IgG3 and IgM isotype antimitochondrial autoantibodies against recombinant fused mitochondrial polypeptide in patients with primary biliary cirrhosis. Hepatology 1988;8:290-295. Surh CD, Danner DJ, Ahmed A, Coppel RL, Mackay IR, Dickson ER, Gershwin ME. Reactivity of primary biliary

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cirrhosis sera with a human fetal liver cDNA clone of branched-chain o~-keto acid dehydrogenase dihydrolipoamide acyltransferase, the 52 kD mitochondrial autoantigen. Hepatology 1989a;9:63--68. Surh CD, Roche TE, Danner DJ, Ansari A, Coppel RL, Prindiville T, Dickson ER, Gershwin ME. Antimitochondrial autoantibodies in primary biliary cirrhosis recognize crossreactive epitope(s) on protein X and dihydrolipoamide acetyltransferase of pyruvate dehydrogenase complex. Hepatology 1989b;10:127-133. Surh CD, Ansari AA, Gershwin ME. Comparative epitope mapping of murine monoclonal and human autoantibodies to human PDH-E2, the major mitochondrial autoantigen of primary biliary cirrhosis. J Immunol 1990a;144:2647-2652. Surh CD, Coppel RL, Gershwin ME. Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis. Implication for a conformational autoepitope. J Immunol 1990b;144:33673374. Teoh KL, Rowley MJ, Mackay IR. An automated microassay for enzyme inhibitory effects of M2 antibodies in primary biliary cirrhosis. Liver 1991;11:287-291. Thekkumkara TJ, Jesse BW, Ho L, Raefsky C, Pepin RA, Javed AA, Pons G, Patel MS. Isolation of a cDNA clone for the dihydrolipoamide acetyltransferase component of the human liver pyruvate dehydrogenase complex. Biochem Biophys Res Comm 1987;2:903--907. Triger DR, Berg PA, Roder J. Epidemiology of primary biliary cirrhosis. Liver 1984;4:195--200. Tsuneyama K, Van de Water J, Leung PS, Cha S, Nakanuma Y, Kaplan M, De Lellis R, Coppel R, Ansari A, Gershwin ME. Abnormal expression of the E2 component of the pyruvate dehydrogenase complex on the luminal surface of biliary epithelium occurs before major histocompatibility complex class II and BB1/B7 expression. Hepatology 1995;21:1031--1037. Van De Water J, Gershwin ME, Leung P, Ansari A, Coppel RL. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site

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of dihydrolipoamide acetyltransferase. J Exp Med 1988;167: 1791--1799. Van de Water J, Cooper A, Surh CD, Coppel R, Danner D, Ansari D, Dickson R, Gershwin ME. Detection of autoantibodies to recombinant mitochondrial proteins in patients with primary biliary cirrhosis. N Engl J Med 1989;320:1377-- 1380. Van de Water J, Turchany J, Leung PS, Lake J, Munoz S, Surh CD, Coppel R, Ansari A, Nakanuma Y, Gershwin ME. Molecular mimicry in primary biliary cirrhosis. Evidence for biliary expression of a molecule cross-reactive with pyruvate dehydrogenase complex-E2. J Clin Invest 1993 ;91:2653-2664. Van de Water J, Ansari A, Prindiville T, Coppel RL, Ricalton N, Kotzin BL, Liu S, Roche TE, Krams SM, Munoz S. Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis. J Exp Med 1995;181:723--733. Walker JG, Doniach D, Roitt IM, et al. Serological tests in the diagnosis of primary biliary cirrhosis. Lancet 1965;1:827-831. Witt-Sullivan H, Heathcote J, Cauch K, Blendis L, Ghent C, Katz A, Milner R, Pappas SC, Rankin J, Wanless IR. The demography of primary biliary cirrhosis in Ontario, Canada. Hepatology 1990;12:98--105. Wolfhagen FH, van Buuren HR, Schalm SW. Combined treatment with ursodeoxycholic acid and prednisone in primary biliary cirrhosis. Neth J Med 1994;44:84--90. Yasutoshi N. Manual of Hepatitis Research Committee of Japan (in Japanese). 1989:8-10. Yeaman SJ. The mammalian 2-oxo-acid dehydrogenases. A complex family. Trends Biochem Sci 1986;11:293-296. Yeaman SJ, Fussey SP, Danner DJ, James OF, Mutimer DJ, Bassendine MF. Primary biliary cirrhosis: identification of two major M2 mitochondrial autoantigens. Lancet 1988;1:1067-1069. Zhang L, Jayne DR, Oliveira DBG. Anti-idiotype antibodies to antimitochondrial antibodies in sera of patients with primary biliary cirrhosis. J Autoimmun 1993;1:93--105.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

MITOTIC SPINDLE APPARATUS AUTOANTIBODIES Jerome B. Rattner, Ph.D. a and Marvin J. Fritzler, M.D., Ph.D. b

Departments of aMedical Biochemistry and bMedicine, The University of Calgary, Calgary, Alberta, T2N 4N1 Canada

HISTORICAL NOTES For more than a century, the mitotic spindle apparatus (MSA) was recognized as a unique structure that participated in the directed movement of chromosomes during cell division. However, it was not until the successful isolation of the MSA by Mazia and his colleagues that an understanding of the complexity of this organelle was appreciated (reviewed in Kane, 1965).

THE AUTOANTIGENS

Definition Mitotic Spindle. A dynamic structure composed of microtubules and a subset of proteins that is organized from the centrosomes during prophase and is visualized as bundles of microtubules in the intercellular bridge at telophase.

Intercellular Bridge. A membrane-bound channel filled with microtubule elements from the mitotic spindle midzone formed as a consequence of cytokinesis. The bridge connects daughter cells following the completion of cell division. In some cell types this structure can persist into the G1 period of interphase. Midbody. A structure found in the middle of the intercellular bridge. This structure represents the localized accumulation of electron-dense material around microtubules within the intercellular bridge. The mitotic spindle is made up of microtubules that are composed of tubulin. A variety of proteins that associate with these microtubules are collectively

known as MAPS (Microtubule Associated Protein S). A few MAPS act as antigens in autoimmune disease (Table 1). Many spindle autoantigens are designated MSA (Mitotic Spindle Apparatus) proteins and technically speaking, centrioles, centrosomes, metaphase chromosomes and kinetochores are also part of the MSA. For purposes of this chapter, however, the discussion is limited to the mitotic spindle, the midbody and the stembody that form during the cell cycle. The most commonly reported mitotic spindle autoantigen is the Nuclear Mitotic Apparatus (NuMA) protein. Cytologically, antibodies to this antigen produce a distinct "<"-shaped pattern at the apex of the spindle culminating at the centrosome and a finely speckled pattern of staining in interphase cells (Figures 1 and 2A). In contrast, less common autoantigens such as MSA-35 produce a more general staining pattern of equal intensity throughout the spindle (Figure 2B) and nuclear reactivity is not seen. Thus, NuMA reactivity can be reliably based on cytological criteria. Other autoantibodies bind to spindle antigens that are prevalent during the latter stages of cell division. Commonly, these autoantigens are detected in metaphase at the spindle midzone and at anaphase are found within the intercellular bridge. Indeed, autoantibodies reactive with autoantigens that map to several regions within the intercellular bridge are useful in revealing seven distinct zones within the intercellular bridge including the midbody (see Rattner 1992 for review). The term "midbody", regretfully used rather loosely to include staining anywhere within the intercellular bridge, should be restricted to staining that co-localizes with the central phase dot structure within the bridge. There are only a few reports of

501

Table 1. Spindle-Associated Proteins Identified with Autoantibodies Protein/ mw

Cellular Location

NuMA

Additional Information

Suggested Function

Disease Association

References

centrosome at mitosis also called SPN, and interphase nucleus centrophilin, SP-H

organization of chromatin architecture and role in spindle function

Sj6gren's syndrome, primary biliary cirrhosis, neoplastic liver disease

Price et al., 1984; Webb et al., 1985; Hansen et al., 1991; Chan and Andrade, 1992

MSA 35 35 kd

throughout mitotic spindleas well as intercellular bridge

role in spindle function

undifferentiated connective tissue disease

Rattner et al., 1992a

MSA 36 36 kd

chromosomes spindle associated with midzone the centromere

unknown

scleroderma & autoimmune liver disease

Rattner et al., 1992b

JB

spindle midzone midbody

unknown

scleroderma

Fritzler et al., 1987; Kingwell et al., 1987

RMSA-1 47 kd

mitotic spindle

spindle assembly

discoid lupus

Yeo et al., 1994

SPA1

spindle poles

chromosome segregation

scleroderma

Calarco-Gillam et al., 1983; Snyder and Davis, 1988

TD60 60 kd

chromosomes spindle midzone intercellular bridge

cytokinesis

unspecified a u t o immune disease

Andreassen et al., 1991

p330d 330 kd

nuclear matrix in Sresembles CENP-F phase kinetochore and spindle midzone in mitosis

unknown

unspecified autoimmune disease

Casiano et al., 1993

CENP-F 372 kd

nuclear matrix kinetochore spindle midzone intercellular bridge

unknown

Raynaud's syndrome

Rattner et al., 1993

activated by phosphorylation by cdc2

revealed a structure called the telophase disc

autoantigens that are confined to the midbody structure (Rattner, 1992). The dynamic nature of the spindle should be taken into account when evaluating MSA patterns. In general, the spindle is composed of two sets of microtubules, those that extend from a pole towards the opposite pole and those that extend from the pole to the kinetochore at the primary constriction of each metaphase chromosome. While these two sets of microtubules can not be distinguished at metaphase, the shortening of the kinetochore microtubules at anaphase allows the visualization of both microtubule sets. Thus, by analyzing the pattern of reactivity within anaphase figures, it is possible to determine if the autoantibody in question is reacting with an antigen that is associated with one or both sets of microtubules.

502

The use of antibodies to study the distribution of spindle-associated proteins during cell division reveals that many antigens move to different locations within the spindle as division precedes. One such set of antigens found to associate with the surface of the chromosome and the relocate to the spindle are designated "chromosomal passenger proteins" (Earnshaw and Bernat, 1995).

THE AUTOANTIBODY Methods of Detection Autoantibodies directed against the mitotic spindle give rise to characteristic patterns of staining of metaphase cells (Figures 1 and 2). Because there are

Figure 1. Human autoantibodies to Nuclear Mitotic Apparatus (NuMA) are characterized by a finely speckled staining pattern of interphase cells and intense staining of the spindle of HEp-2 cells.

Figure 2. Human autoantibodies reactive with mitotic spindle antigens in metaphase cells produce different patterns of reactivity. Antibodies to NuMA characteristically stain the proximal spindle fibers (A). Autoantibodies to MSA-35 produce a more extensive staining of relatively equal intensity throughout the spindle (B). Other autoantibodies stain spindle fibers at later stages of the cell cycle and are localized to the intercellular bridge (C). 503

Table 2. Diseases Associated with Autoantibodies to Mitotic Spindle Apparatus* Disease

References

Sj6gren' s syndrome

Price et al., 1984; Webb et al., 1985; Hansen et al., 1991; Chan and Andrade, 1992

Primary biliary cirrhosis and other autoimmune liver disease

Hansen et al., 1991

Raynaud's phenomenon

Rattner et al., 1993; Fritzler et al., 1987

Scleroderma

Fritzler et al., 1987; Kingwell et al., 1987; Auer-Grumbach and Achleitner, 1994; Price et al., 1984; Rattner et al., 1992b

Autoimmune liver disease

Rattner et al., 1992b; Hansen et al., 1991

Undifferentiated or mixed connective tissue disease

Price et al., 1984; McCarty et al., 1981; Rattner et al., 1992a; Auer-Grumbach and Achleitner, 1994

Polymyositis

Webb et al., 1985

Rheumatoid arthritis

Webb et al., 1985; Auer-Grumbach and Achleitner, 1994

Polyarteritis nodosa

Webb et al., 1985

Systemic and discoid lupus erythematosus

Price et al., 1984; Yeo et al., 1994

Vitiligo & Hashimoto's thyroiditis

Auer-Grumbach and Achleitner, 1994

Epidermolysis bullosa acquisita

Auer-Grumbach and Achleitner, 1994

Melanoma

Auer-Grumbach and Achleitner, 1994

Dilated cardiomyopathy

Auer-Grumbach and Achleitner, 1994

Mycoplasma and other infection

Lind et al., 1988; Auer-Grumbach and Achleitner, 1994

Osteoarthritis

McCarty et al., 1981

*Includes reports of autoantibodies that reacts with the MSA, stembody and midbody.

numerous components of the MSA, it is likely that autoantibodies of varying specificity will give slightly different staining patterns. The IIF staining pattern produced by anti-NuMA characteristically is seen as a finely speckled staining of the interphase nucleus and intense staining of the proximal polar spindle fibers at metaphase (Figure 1). The staining pattern associated with autoantibodies directed against various MSA components suggests that it may be possible to subclassify patients on the basis of different MSA autoantibodies.

CLINICAL UTILITY

Disease Associations (Tables 1 and 2) Anti-MSA antibodies were found in 4/2500 (0.16%) unselected sera from female blood donors (Fritzler et

504

al., 1985). In another study, anti-MSA were found in 2/323 (0.06%) normal sera and in 3/887 (0.03%) of "pathological" sera (Webb et al., 1985). Antibodies to MS A are reported in carpal tunnel syndrome, Raynaud's phenomenon, systemic sclerosis, Sj6gren's syndrome, rheumatoid arthritis, polymyositis and polyarteritis (McCarty et al., 1981; Rattner et al., 1992a; 1993; Fritzler et al., 1987; Price et al., 1984; Webb et al., 1985; Auer-Grumbach and Achleitner, 1994; Yeo et al., 1994). In one study, a remarkably high frequency (71%) of IgG anti-MSA autoantibodies was noted in patients with cold-agglutinin-positive Mycoplasma pneumoniae (Lind et al., 1988). AntiMSA were also found in 2/32 (6%) of PBC, 1/50 (2%) Sj6gren's syndrome and 4/356 (1.2%) of autoimmune chronic liver disease sera (Hansen et al., 1991). In the latter group of autoimmune liver disease, 2/4 (50%) had primary biliary cirrhosis, and when the seven anti-MSA patients in all diagnostic categories

were evaluated, four had PBC, one had autoimmune chronic active hepatitis and two had liver neoplasia. In one of the few studies that immunoblotted purified proteins, anti-NuMA were identified as the most common autoantigenic target in sera that demonstrated the typical MSA staining pattern (Price et al., 1984). The two patterns of M S A staining include one pattern identified as MA-I which gives finely speckled nucleoplasmic staining sparing the nucleolus, and a rim at the centrosome and bright staining of the proximal spindle. These sera react with two protein of --200 kd. This description suggests that MA-I is similar to NuMA. The other pattern, referred to as MA-II, gives staining of the metaphase spindle from the poles to the region of the metaphase plate and recognizes an antigen o f - - 1 0 0 kd. This staining pattern resembles MSA-35. Of interest, 8/16 (50%) MA-I positive sera had Sjrgren's syndrome. In another study (Auer-Grumbach and Achleitner, 1994), 4/11 patients with anti-MSA also had antibodies to SS-A/Ro. The association of M S A antibodies with

REFERENCES Andreassen PR, Palmer DK, Wener M, Margolis R. Telophase disc: a new mammalian mitotic organelle that bisects telophase cells with a possible function in cytokinesis. J Cell Sci 1991;99:523-534. Auer-Grumbach P, Achleitner B. Epidemiology and clinical associations of NuMA (nuclear mitotic apparatus protein) autoantibodies. J Rheumatol 1994;21:1779-- 1781. Callarco-Gillam PD, Siebert MC, Hubble R, Mitchison T, Kirschner M. Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material. Cell 1983;35:621-629. Casiano CA, Landberg G, Ochs RL, Tan EM. Autoantibodies to a novel cell cycleregulated protein which accumulates in the nuclear matrix during S phase and localizes to kinetochores and spindle midzone during mitosis. J Cell Sci 1993; 106:1045-- 1056. Chan EKL, Andrade LEC. Antinuclear antibodies in Sjogren syndrome. Rheum Dis Clin North Am 1992;18:551-570. Earnshaw WC, Bernat RL. Chromosomal passengers: toward an integrated view of mitosis. Chromosoma 1995;100:139-- 146. Fritzler MJ, Pauls JD, Kinsella TD, Bowen TJ. Antinuclear, anticytoplasmic and anti-Sjogren syndrome antigen-A (SSA/Ro) antibodies in female blood donors. Clin Immunol Immunopathol 1985;36:120--128. Fritzler MJ, Ayer LM, Gohill J, O'Connor C, Laxer RM, Humbel R. An antigen in metaphase chromatin and the midbody of mammalian cells binds to scleroderma sera. J Rheumatol 1987;14:2914. Hansen BU, Eriksson S, Lindgren S. High prevalence of

autoimmune liver disease (Hansen et al., 1991) is also interesting because scleroderma and Sjrgren's syndrome patients are known to be at risk of this liver disease. Taken together, these studies suggest that a subset of patients with MSA antibodies may have Sjrgren' s syndrome.

CONCLUSION An association of MSA autoantibodies with specific disease states is not clearly established. This might be expected since studies of large numbers of sera and the correlation between clinical features and specific MSA targets have not been done. Many studies refer to MSA antibodies as anti-NuMA without direct proof by immunoblotting or immunoprecipitation that the 236 kd N u M A protein is the target of the autoantibody response. As noted above, certain criteria applied to the interpretation of staining patterns may help subclassify anti-MSA responses.

autoimmune liver disease in patients with multiple nuclear dot, anticentromere, and mitotic spindle antibodies. Scand J Gastroenterol 1991;26:707-713. Kane RE. The mitotic apparatus. Physical-chemical factors controlling stability. J Cell Biol 1965;25:137-144. Kingwell B, Decoteau J, Fritzler MJ, Rattner JB. The identification and characterization of a protein associated with the stembody using autoimmune sera from patients with systemic sclerosis. Cell Motil Cytoskeleton 1987;8:360--367. Lind K, Hoier-Madsen M, Wiik A. Autoantibodies to the mitotic spindle apparatus in Mycoplasma pneumoniae disease. Infect Immunol 1988;56:714-715. McCarty GA, Valencia DW, Fritzler MJ, Barada FA. A unique antinuclear antibody staining only the mitotic spindle apparatus. N Engl J Med 1981;305:703. Price CM, McCarty GA, Pettijohn DE. NuMA protein is a human autoantigen. Arthritis Rheum 1984;27:774--779. Rattner JB. Mapping the mammalian intercellular bridge. Cell Motil Cytoskel 1992;223:231--235. Rattner JB, Wang T, Mack G, Martin L, Fritzler MJ. MSA-35: a protein identified by human autoantibodies that co-localizes with microtubules. Biochem Cell Biol 1992a;70:1115--1122. Rattner JB, Wang T, Mack G, Fritzler MJ, Martin L, Valencia D. MSA-36: a chromosomal and mitotic spindle-associated protein. Chromosoma 1992b;101:625-633. Rattner JB, Rao A, Fritzler MJ, Valencia DW, Yen TJ. CENP-F is a ca. 400 kd kinetochore protein that exhibits a cell-cycle dependent localization. Cell Motil Cytoskel 1993;26:214222. Snyder M Davis RW. SPA 1: a gene important for chromosome segregation and other mitotic functions in S. cerevisiae. Cell

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1988;54:743-754. Webb J, Maule P, Wells JV. Antibody to mitotic spindle apparatus. J Rheumatol 1985;12:623. Yeo J-P, Forer A, Toh B. A homologue of the human regulator

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of mitotic spindle assembly protein (RMSA-1) is present in crane fly and is associated with meiotic chromosomes. J Cell Sci 1994;107:1845-1851.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

MOLECULAR MIMICRY Robert S. Fujinami, Ph.D.

Department of Neurology, University of Utah, Salt Lake City, UT 84132, USA

HISTORICAL NOTES The observation that micro-organisms share common determinants is not new. Over fifty years ago, investigators described cross-reacting epitopes between microbes and the host they invade. Another designation for this cross-reactivity is molecular mimicry. The term "molecular mimicry" was first used in 1964 to describe antigenic epitopes of microbial origin which resemble host determinants; a major tenet was that this semblance was such that the parasite did not elicit the production of antibodies by the infected host and that microbes could initiate infection without elimination by the host (Damian, 1964). From that time to the 1980s, the term "molecular mimicry" was not used and apparently forgotten. Over a decade ago, the term was introduced to describe common immunologic epitopes between self- and viral proteins in the context of autoimmunity (Fujinami et al., 1983). At the time monoclonal antibodies generated against viral proteins such as measles virus and herpes virus were found to react with host-cell proteins as well as viral proteins, and molecular mimicry was suggested to have the potential for inducing autoimmunity (Fujinami et al., 1983). The field has grown significantly and has been extended by many investigators around the world.

Similarity vs. Homology Occasionally, a discussion or definition of common determinants or sequence similarity versus homology arises. Homology is properly used to refer to a gene or gene product present in the microbe and host which originated from a common ancestral gene. Similarity is best used to refer to common immunologic determinants which may be comprised of a particular

amino acid sequence or conformational determinants recognized by an antibody or T cell in the context of the appropriate major histocompatibility (MHC) molecules.

Forms of Molecular Mimicry Molecular mimicry or the occurrence of common determinants between a microbe and the host can take several forms (Table 1).

Form 1. The first form is a complete identity of a protein between a virus or micro-organism and self. For example, measles virus virion contains the cellular protein, actin (Udem, 1984). This cytoskeletal element is also an important structural component of all eukaryotic cells. In this scenario, actin filaments are incorporated into the virion during viral maturation; immune response against the virion could induce antibodies against the actin filaments (Fagraeus et al., 1983). A second example of this type of mimicry (i.e., the incorporation of host components into the virion) is Semliki Forest virus. This virus acquires host-cell lipids by budding from infected cells as one of the last maturational steps in virus assembly. These lipids are incorporated into the virus envelope. Acquisition of particular glycolipids can potentiate the virus' disease and enhance its ability to induce an immunemediated encephalomyelitis. Semliki Forest virus passaged in vivo in mouse brain or brain-cell cultures can infect mice. Mice infected with brain-derived virus produce autoantibodies to galactocerebroside but not when infected with non-brain-passaged virus (Webb et al., 1984). The brain-passaged virus has an increased demyelinating potential (Khalili-Shirazi et al., 1986). Thus, cellular proteins and host glycolipids

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Table 1. Four Forms of Molecular Mimicry Type

Example Complete identity of a protein between a micro-organism and the host not encoded by the micro-organism.

Measles virus

2.

Homologousor related protein with the host.

Cytomegalovirus Epstein-Barr virus

3.

Commonor similar amino acid sequences.

Hepatitis B virus Klebsiella pneumoniae

Conformational similarities between the micro-organism and host protein.

Theiler's (encephalomyelitis) virus

are associated with this type of mimicry. In this case, the microbe does not code for the homologous protein or host component. F o r m 2. In a second level of molecular mimicry, the virus actually encodes a protein homologous or related to the host. This appears to have a selective advantage for the virus. For example, human cytomegalovirus (CMV) codes for a protein which is homologous to the major histocompatibility complex (MHC) class I heavy chain. Transcripts of this viral region encode a class I-like molecule. MHC class I heavy chain normally associates with ~2-microglobulin and is expressed on the surface of most cells. This heterodimer is an important restricting and recognition element for CD8 + T cells. In many viral infections, these T cells are important in viral clearance. Coexpression of the viral class I-like molecule and 132microglobulin in the same cell shows that the two molecules can associate (Browne et al., 1990). Infection of cells by CMV might lead to a decrease of cellular class I expression, possibly due to the association of ~2-microglobulin with the viral-encoded class I homologue (Browne et al., 1990). If all the I]2microglobulin were bound to the viral class I-like molecule, it would be unavailable for binding to host class I, thus leading to less expression of endogenous MHC class I. Another example of this type of mimicry is viral IL-10 or BCRF1. This molecule is homologous to human and mouse IL-10. The viral IL-10 is encoded by the Epstein-Barr virus (EBV) (Swaminathan and Kieff, 1995). BCRF1 (EBV-encoded) shares 71% nucleotide homology with human IL-10 and 78% sequence homology at the amino acid level. Activated human T cells have the ability to produce IL-10. The IL-10 receptor is expressed mostly on hematopoietic

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cells. In addition to stimulating B cells, thymocytes and mast cells, IL-10 promotes Th2 CD4 + T-cell responses and inhibits Thl-like cytokine responses. Thl T cells can be referred to as "inflammatory T cells" and Th2 T cells as "helper T cells." Thl T cells are responsible for delayed-type hypersensitivity responses and Th2 T cells aid in B-cell activation. This may be one way a virus can determine what type of immune response is generated against itself and favor its survival. F o r m 3. In a third level of molecular mimicry involving common epitopes or immunological determinants, a virus or microbe might share a similar sequence of amino acids or epitope rather than an entire protein (Fujinami and Oldstone, 1985). This would reflect a similarity rather than a homology. In this instance, portions of proteins comprise the common epitope which is identical or similar between the microbe and host. One example is the sharing of a disease-inducing epitope between myelin basic protein (MBP) and the human hepatitis B virus polymerase. A search of the sequences contained within an amino acid data base reveals a common core of amino acids between the encephalitogenic site of MBP and hepatitis B virus polymerase (Fujinami and Oldstone, 1985). The disease-inducing epitope or encephalitogenic site is a rabbit-specific epitope which will induce experimental allergic encephalomyelitis (EAE), an autoimmune demyelinating disease of the central nervous system (CNS). Prediction that injection of rabbits with the viral peptide would lead to the development of an EAE-like disease was validated by injection of rabbits with the viral peptide, with resultant induction of autoantibodies cross-reactive with MBP. Peripheral blood mononuclear cells isolated from immunized rabbits proliferated in response to MBP, and some of

the animals developed lesions in the CNS consistent with EAE (Fujinami and Oldstone, 1985). This was one of the first examples of molecular mimicry disease induced by a viral peptide (Fujinami and Oldstone, 1985). Another well-studied example of this type of molecular mimicry is the determinant which is shared Klebsiella pneumoniae and HLA B27 (Ebringer, 1992). Infection of genetically susceptible humans with Klebsiella can be associated with the development of ankylosing spondylitis and Reiter' s syndrome. In the early 1980s, cross-tolerance was suggested to be responsible for or involved in the pathogenesis of spondyloarthropathies (Ebringer, 1983) due to a relationship between the Klebsiella microbe and the HLA B27 molecule. Using similar search techniques, as described above for MBP and hepatitis B virus, a 6 amino acid region common to the Klebsiella nitrogenase and HLA B27 was identified (Schwimmbeck and Oldstone, 1989). Immunization of rabbits and rats with the common region from the HLA B27 molecule or the similar peptide region from the Klebsiella nitrogenase yielded polyclonal antibodies reactive with either protein and peptides from HLA B27 and Klebsiella nitrogenase. The peptides cross-inhibit the antibody binding to the appropriate regions. Substituting different amino acids within the peptides significantly reduces antibody binding to the respective peptide, suggesting a linear epitope. Antibodies raised to this similar region from either HLA B27 or the Klebsiella nitrogenase bind to joint tissue from patients with ankylosing spondylitis or Reiter's syndrome and not to control joint tissue, this indicates that the epitope is present in the inflamed synovial tissue, reacts with the antibodies and is actively induced at the sites of disease (Husby et al., 1989). The monoclonal antibody (Ye-2) is highly reactive with amino acids 63--84 of HLA B7 and with amino acids 226--244 of bovine carbonic anhydrase (Yong et al., 1989). Even though only three amino acids in the peptide stretch are identical, the region may reflect a similar linear epitope or a conformationally similar determinant comprised of key amino acids. All these data are consistent with the prediction that primary, secondary and tertiary structures of proteins serve as vital contributors to molecular mimicry (Dyrberg and Oldstone, 1986). Studies of peptide epitopes of the acetylcholine receptor (~ chain) showed similarity with the polyomavirus middle T antigen, herpes simplex virus glycoprotein D, and parvovirus H1 and the VP2 proteins with

amino acid 160--167 of the acetylcholine receptor. The amino acids vary in the number of common amino acids comprising the epitope, i.e., in sequence similarity. Antibodies raised to the viral peptides can be assayed by ELISA for reaction with the acetylcholine receptor peptides. When the acetylcholine receptor peptide and/or the polyoma virus T-antigen peptide are coupled to keyhole limpet hemocyanin at either the amino or carboxy end of the peptide for immunization of rabbits, the resulting antisera differ markedly in their ability to bind to the respective peptides (Dyrberg and Oldstone, 1986). The context of the epitope is now well known to be important in the resulting immune response (Sercarz et al., 1993). The monoclonal antibody Ye-2 described above binds not only to HLA B27 but also to OmpA protein of Escherichia coli (Yu et al., 1991). Although there is no sequence similarity between the two proteins, studies of Ye-2 with overlapping peptides showed reaction with several arginine-rich regions consistent with binding to key or common amino acids. Form 4. The fourth level of mimicry includes conformational similarities. For example: A monoclonal antibody to Theiler's murine encephalomyelitis virus (TMEV) (Fujinami et al., 1988a) causes an acute polioencephalomyelitis and a chronic CNS demyelinating disease in mice which is similar to human multiple sclerosis (Yamada et al., 199 l a). This monoclonal antibody (H8) effectively neutralizes the infectivity of TMEV (Fujinami et al., 1988a) and reacts with the viral capsid protein, VP-1 by immunoblot analyses. H8 binds to lysates of TMEVinfected cells but not to uninfected control cells; it also reacts with purified galactocerebroside ([GC], a prominent component of myelin and produced by oligodendrocytes) and can be detected by immunofluorescence in GC-positive oligodendrocytes (Fujinami et al., 1988a). Mice were immunized with MBP to develop EAE and were also given H8. In the H8treated mice, the demyelination is much more extensive; whereas, in mice given a control MAb monoclonal antibody, little or no demyelination is evident (Yamada et al., 1990). The MAb H8 is able to enhance or potentiate the extent of demyelination in areas of inflammation in EAE-induced mice. TMEVinfected mice develop antibodies with similar specificity to MAb H8. Thus, during TMEV infection, these types of antibodies could potentiate the demyelination observed in inflammatory sites in the CNS as observed in the EAE H8 treated mice.

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A second example: An antistreptococcal monoclonal antibody reactive with N-acetyl-~-D- glucosamine (GlcNAc) and peptides derived from a variety of proteins (including coxsackie virus amino acid sequence, RRKLEFF) reacts with the coxsackie virus sequence and neutralizes the infectivity of the virus (Shikhman et al., 1993). This appears to be an example of an antibody which reacts with a carbohydrate structure and a peptide. Several antistreptococcal monoclonal antibodies derived from human B cells of patients react with cytokeratin found in human skin (Shikhman and Cunningham, 1994), seemingly recognizing peptide structures in the keratin protein which are known not to be present as linear sequences in streptococci. Thus, similar conformational determinants with limited or no common linear peptides or sugars can be recognized by antibodies. There is an interesting parallel with the IgGbinding bacterial proteins. Protein G, a cell-wall protein found in bacteria, can bind human IgG. Three unrelated genes encode different bacterial proteins which bind to the same region of the IgG molecule (Frick et al., 1992). Perhaps this reflects convergent evolution in which different bacteria evolve proteins which can bind to the same region on human IgG. The reason for this is unclear. However, the protein G can also bind to human albumin, but the sites of binding of IgG and albumin on the bacterial G protein are different. Binding to plasma proteins might aid in the spread of the microbe or may aid in the bacterium entering and replicating in a phagocytic cell (Frick et al., 1994).

CLINICAL UTILITY Following these early examples of molecular mimicry, the definition of molecular mimicry has been extended (Fujinami and Oldstone, 1985). For example, a peptide from the immediate early region (IE-2) of CMV shares an epitope with the HLA-DR 13 chain. The epitope comprises five amino acids embedded between amino acids 79-88 of the CMV IE-2 and 50--59 of HLA-DR 13 chain. Rabbits immunized with the viral peptide developed antibodies reactive not only with the CMV peptide but also with the HLADR ~ chain. IE-2 peptide significantly inhibits the binding of. the antibody with the DR molecule (Fujinami et al., 1988b). Thus, infection with CMV can generate antibodies to the common epitope resulting in antibodies to self-DR molecules. This may be

510

related to the immunosuppressive nature of this virus. Similarly, almost all patients with neurological complications of AIDS have antibodies to a region of HIV gp-41 which encompasses amino acid sequence 598--609 (Gnann Jr. et al., 1987a; 1987b). Monoclonal antibodies reactive with a 12 amino acid peptide from HIV gp-41 bind to reactive, activated astrocytes of human and/or mouse origin (Yamada et al., 1991b); the binding is specifically inhibited by peptide. By immunoblotting analyses, the anti-gp-41 monoclonal antibody reacts with a 43 kd CNS protein from uninfected tissue. Cerebrospinal fluid from some AIDS patients with CNS alterations contains antibodies directed against the same epitope on astrocytes (Yamada et al., 1991b). Cross-reactive responses generated during infection might lead to CNS reactivity with the complication of AIDS dementia or CNS dysfunction. Recombinant viruses containing self-proteins or epitopes are now being generated to study presentation of self-determinants to the host's immune system by viruses. The rationale of these experiments is to study virus-encoding self-determinants; the model employed is EAE. Using a recombinant vaccinia virus constructed to contain the coding region for proteolipid protein (PLP) (Barnett et al., 1993), in vitro-infected cells express the cytoplasmic PLP protein. Mice infected with this recombinant virus or a control vaccinia virus do not develop EAE. When these mice are then challenged with an encephalitogenic peptide of PLP (139--151 amino acids), enhanced disease develops with earlier and more severe clinical signs. Examination of brains and spinal cords of these animals reveals extensive demyelinating lesions versus control virus-infected mice. Mice develop antibodies to PLP detectable by ELISA (Barnett et al., 1993). Thus, a virus encoding a self-protein cannot, by itself, initiate autoimmune disease. Additional factors are involved in the development of autoimmunity. However, under these conditions, virus infection can lead to an enhanced disease, once the pathogenic mechanisms are initiated. Two transgenic mouse models employ viruses that can start what may be an autoimmune process. Viral genes can be inserted into the mouse under various promoters. For example, transgenic mice expressing lymphocytic choriomeningitis virus (LCMV) proteins use the rate insulin promoter to drive the expression of the LCMV proteins and to target the viral protein to the islet cells in the transgenic mice (Kyburz et al., 1993; Oldstone et al., 1991). When transgenic mice

expressing the LCMV glycoprotein (LCMV GP) in islet cells of the pancreas (Ohashi et al., 1991) are bred to H-2D b transgenic mice (Pircher et al., 1989), the majority of the T cells of the progeny are specific for LCMV GP and are H-2D b restricted. Mice transgenic for both the T-cell receptor and for the LCMV GP succumb to diabetes when subsequently infected with LCMV. The development of disease is dependent on the generation of CD8 + T cells which recognize LCMV GP in the context of H-2D b presented by the islet cells. This suggests that a CD8 + T-cell-mediated autoimmune disease results from viral infection (Ohashi et al., 1991). Transgenic mice expressing the glycoprotein or the nucleoprotein genes of LCMV develop a cytotoxic Tcell response to the viral epitopes and also diabetes in a model that mirrors some of the pathologic features of human diabetes (Oldstone et al., 1991). The development of diabetes is dependent on CD8 + T cells and not on active CD4 + T-cell participation (Laufer et al., 1993).

REFERENCES Barnett LA, Whitton JL, Wada Y, Fujinami RS. Enhancement of autoimmune disease using recombinant vaccinia virus encoding myelin proteolipid protein. J Neuroimmunol 1993;44:15--25. Browne H, Smith G, Beck S, Minson T. A complex between the MH class I homologue encoded by human cytomegalovirus and ~2-microglobulin. Nature 1990;347:770-772. Damian RT. Molecular mimicry: antigen sharing by parasite and host and its consequences. Am Naturalist 1964;98: 129--149. Dyrberg T, Oldstone MB. Peptides as probes to study molecular mimicry and virus-induced autoimmunity. Curr Top Microbiol Immunol 1986;130:25-37. Ebringer A. The cross-tolerance hypothesis, HLA-B27 and ankylosing spondylitis. Br J Rheumatol 1983;22(4 Suppl 2):53--66. Ebringer A. Ankylosing spondylitis is caused by Klebsiella. Evidence from immunogenetic, microbiologic, and serologic studies. Rheum Dis Clin North Am 1992;18:105--121. Fagraeus A, Orvell C, Norberg R, Norrby E. Monoclonal antibodies to epitopes shared by actin and vimentin obtained by paramyxovirus immunization. Exp Cell Res 1983;145: 425-432. Frick IM, Wikstrom M, Forsen S, Drakenberg T, Gomi H, Sjobring U, Bjorck L. Convergent evolution among immunologlobulin G-binding bacterial proteins. Proc Natl Acad Sci USA 1992;89:8532--8536. Frick IM, Akesson P, Cooney J, Sjobring U, Schmidt KH, Gomi H, Hattori S, Tagawa C, Kishimoto F, Bjorck L.

Transgenic mice in which the majority of murine T cells express functionally reactive, MBP-specific Tcell receptors develop EAE when immunized with myelin-basic protein and adjuvant (Goverman et al., 1993). These mice spontaneously develop EAE when housed in non-pathogen-free conditions but do not develop EAE when kept under specific pathogen-free conditions (Goverman et al., 1993). These data strongly suggest that the flora in these mice contribute to the development of EAE and that the myelin-basic protein T cells are not clonally deleted in the thymus during development.

CONCLUSION Molecular mimicry is a viable mechanism for autoimmune disease. While there is still debate about its involvement in human disease, there is strong evidence to indicate that rheumatic heart disease and ankylosing spondylitis are examples of molecular mimicry.

Protein H - a surface protein of Streptococcus pyogenes with separate binding sites for IgG and albumin. Mol Microbiol 1994;12:143-151. Fujinami RS, Oldstone MB, Wroblewska Z, Frankel ME, Koprowski H. Molecular mimicry in virus infection: crossreaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments. Proc Natl Acad Sci USA 1983;80:2346--2350. Fujinami RS, Oldstone MB. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science 1985;230:1043-- 1045. Fujinami RS, Zurbriggen A, Powell HC. Monoclonal antibody defines determinant between Theiler's virus and lipid-like structures. J Neuroimmunol 1988a;20:25-32. Fujinami RS, Nelson JA, Walker L, Oldstone MB. Sequence homology and immunologic cross-reactivity of human cytomegalovirus with HLA-DR~ chain: a means for graft rejection and immunosuppression. J Virol 1988b;62:1001-1005. Gnann JW Jr, McCormick JB, Mitchell S, Nelson JA, Oldstone MB. Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections. Science 1987a;237:1346--1349. Gnann JW Jr, Schwimmbeck PL, Nelson JA, Truax AB, Oldstone MB. Diagnosis of AIDS by using a 12-amino acid peptide representing an immunodominant epitope of the human immunodeficiency virus. J Infect Dis 1987b;156: 261-267. Goverman J, Woods A, Larson L, Weiner LP, Hood L, Zaller DM. Transgenic mice that express a myelin basic proteinspecific T cell receptor develop spontaneous autoimmunity. Cell 1993;72:551-560.

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Husby G, Tsuchiya N, Schwimmbeck PL, Keat A, Pahle JA, Oldstone MB, Williams RC Jr. Cross-reactive epitope with Klebsiella pneumoniae nitrogenase in articular tissue of HLAB27 + patients with ankylosing spondylitis. Arthritis Rheum 1989;32:437--445. Khalili-Shirazi A, Gregson N, Webb HE. Immunological relationship between a demyelinating RNA enveloped budding virus (Semliki Forest) and brain glycolipids. J Neurol Sci 1986;76:91-103. Kyburz D, Aichele P, Speiser DE, Hengartner H, Zinkernagel RM, Pircher H. T cell immunity after a viral infection versus T cell tolerance induced by soluble viral peptides. Eur J Immunol 1993;23:1956--1962. Laufer TM, von Herrath MG, Grusby MJ, Oldstone MB, Glimcher LH. Autoimmune diabetes can be induced in transgenic major histocompatibility complex class II-deficient mice. J Exp Med 1993;178:589--596. Ohashi PS, Oehen S, Buerki K, Pircher H, Ohashi CT, Odermatt B, Malissen B, Zinkernagel RM, Hengartner H. Ablation of "tolerance" and induction of diabetes by virus infection in viral antigen transgenic mice. Cell 1991;65:305-317. Oldstone MB, Nerenberg M, Southern P, Price J, Lewicki H. Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of antiself (virus) immune response. Cell 1991;65:319-331. Pircher H, Burki K, Lang R, Hengartner H, Zinkernagel RM. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 1989;342:559-561. Schwimmbeck PL, Oldstone MB. Klebsiella pneumoniae and HLA B27-associated diseases of Reiter's syndrome and ankylosing spondylitis. Curr Top Microbiol Immunol 1989;145:45--56. Sercarz EE, Lehmann PV, Ametani A, Benichou G, Miller A, Moudgil K. Dominance and crypticity of T cell antigenic determinants. In: Paul WE, Fathman CG, Metzger H, eds. Annual review of immunology. Palo Alto: Annual Reviews, Inc., 1993:729--766. Shikhman AR, Greenspan NS, Cunningham MW. A subset of mouse monoclonal antibodies cross-reactive with cytoskeletal

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proteins and group A Streptococcal M proteins recognizes Nacetyl-13-D-glucosamine. J Immunol 1993;151:3902--3913. Shikhman AR, Cunningham MW. Immunological mimicry between N-acetyl-~-D-glucosamine and cytokeratin peptides. J Immunol 1994;152:4375--4387. Swaminathan S, Kieff E. The role of BCRF1/vIL-10 in the life cycle of Epstein-Barr virus In: McFadden G, ed. Viroceptors, virokines and related immune modulators encoded by DNA viruses. Austin: R.G. Landes Company, 1995:111-125. Udem SA. Measles virus: conditions for the propagation and purification of infectious virus in high yield. J Virol Methods 1984;8:123--136. Webb HE, Mehta S, Gregson NA, Leibowitz S. Immunological reaction of the demyelinating Semliki Forest virus with immune serum to glycolipids and its possible importance to central nervous system viral autoimmune disease. Neuropathol Appl Neurobiol 1984;10:77--84. Yamada M, Zurbriggen A, Fujinami RS. Monoclonal antibody to Theiler's murine encephalomyelitis virus defines a determinant on myelin and oligodendrocytes, and augments demyelination in experimental allergic encephalomyelitis. J Exp Med 1990; 171:1893-- 1907. Yamada M, Zurbriggen A, Fujinami RS. Pathogenesis of Theiler's murine encephalomyelitis virus. In: Maramorosch K, Murphy FA, Shatkin AJ, eds. Advances in virus research. Orlando: Academic Press, Inc., 1991 a:291-320. Yamada M, Zurbriggen A, Oldstone MB, Fujinami RS. Common immunologic determinant between human immunodeficiency virus type 1 gp41 and astrocytes. J Virol 1991b;65: 1370--1376. Yong Z, Zhang JJ, Schaack T, Chen S, Nakayama A, Yu DT. A monoclonal anti-HLA-B27 antibody which is reactive with a linear sequence of the HLA-B27 protein is useful for the study of molecular mimicry. Clin Exp Rheumatol 1989;7: 513--519. Yu DT, Hamachi T, Hamachi M, Tribbick G. Analysis of the molecular mimicry between HLA-B27 and a bacterial Omp A protein using synthetic peptides. Clin Exp Immunol 1991;85:510--514.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

MYELIN-ASSOCIATED GLYCOPROTEIN AUTOANTIBODIES Hans Link, M.D., Ph.D.

Division of Neurology, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden

HISTORICAL NOTES Polyneuropathy (PN) is characterized by the diffuse involvement of the peripheral nervous system (PNS). PN is common, and the prevalence increases with age. PN is a manifestation of many systemic diseases including diabetes mellitus, alcohol abuse and deficiency states of, for example, vitamin B12 and folic acid (secondary PN). A less frequent but clinically important form of secondary PN is associated with antibodies to myelin-associated glycoprotein (antiMAG); MAG is a minor glycoprotein component of both the PNS and the central nervous system (CNS). Anti-MAG were the first autoantibodies related to PN (Latov, 1981; Baldini et al., 1994).

THE AUTOANTIGEN Definitions

MAG contributes <1% of PNS myelin protein, the major proteins being the P0 glycoprotein, P1 (equal to myelin basic protein) and P2. P0 is truly specific for PNS myelin, while P1 and P2 are present in the CNS as well. Characteristics

MAG has a molecular weight of 100,000 and consists of a cytoplasmic C-terminus, a transmembrane protein and oligosaccharide components. These components form 30% of the mass of MAG and project on the extracellular surface. The carbohydrate moiety of MAG constitutes the predominant epitope that is recognized by anti-MAG. The carbohydrate antigenic epitope (sulfate-3 glucuronate) of MAG is shared by

the PNS-specific components, P0 glycoprotein (Bollensen et al., 1988) and the acidic glycolipids sulfoglucuronylparagloboside (SGPG) and sulfoglucuronyllactosaminylparagloboside (SGLPG) (Chou et al., 1985; Latov et al., 1988; Fredman et al., 1993). The occurrence of P0, SGPG and SGLPG only in the PNS and not in the CNS myelin may explain why the clinical syndrome associated with anti-MAG is a predominantly peripheral neuropathy sparing the CNS. Epitope sharing between MAG and CNS neurons is documented (Gregson and Leibowitz, 1985). However, differences in autoantibody fine specificity for MAG and SGPG exist, and some antibodies are MAG- or SGPG-specific with no cross-reactivity (Ilyas et al., 1986; Brouet et al., 1992; Yu et al., 1990; Miller et al., 1987). MAG also shares an epitope with human natural killer cells defined by the monoclonal antibody HNK-1 (Leu7) (Quarles, 1988). This indicates that the HNK-1/sulfoglucuronyl moiety has at least two antigenic epitopes; other carbohydrate, ceramide (or lipid) and peptide epitopes are also possible (Field et al., 1992; Burger et al., 1992a; 1992b). MAG is related in structure to some of the adhesion molecules, e.g., the intercellular adhesion molecules (ICAMs) and lymphocyte function-associated antigen3 (LFA-3), which together with antigen receptors for T and B cells, the co-receptors CD4, CD8 and CD 19 and the invariant domains of MHC molecules are members of the immunoglobulin superfamily. MAG probably functions as an adhesion molecule as does P0 (Griffith et al., 1992) and mediates cell-cell interactions (Quarles, 1989). Methods of Purification

To isolate MAG, human or bovine brain myelin is prepared (Norton and Poduslo, 1973), followed by the

513

lithium diiodosalicylatephenol (LIS-phenol) method and subsequent gel filtration (Quarles, 1988). Purity is checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) which shows characteristic 90-100 kd bands. The inconsistencies reported in some studies between results obtained by immunoblot and enzyme-linked immunosorbent assay (ELISA; see below) to detect anti-MAG IgM in human serum might be due to contamination with other proteins present even when SDS-PAGE shows a pure 100 kd band. A concordance between the two methods for antibody detection of 85 to 90% can be achieved after additional purification of the MAG (Pestronk et al., 1994). Additionally, an endogenous neutral protease can convert MAG into a truncated version (dMAG) which may account for some loss of MAG autoreactivity (Inuzuka et al., 1984). Reactivity to other myelin adhesion molecule epitopes may also account for some assay variation. MAG is also phosphorylated, and SDS-PAGE destroys any putative phosphate epitopes (Bambrick and Braun, 1991). Various control antibody preps can be used to standardize the immunoblot (Ishiguro et al., 1993).

Methods of Detection There are at present no generally accepted and standardized principles in use to demonstrate and quantitate the elevated serum anti-MAG IgM antibody concentrations that are important in PN. The following procedures, either alone or in combinations, are currently used: hnmunoblot. Bovine or human CNS myelin is separated by SDS-PAGE, followed by protein transfer to nitrocellulose sheets. The strips are incubated with diluted test serum. After washing, the binding of immunoglobulin can be visualized by incubation with goat antihuman IgM linked to e.g., horseradish peroxidase and stained with diaminobenzidine (Li et al., 1991) or by incubation with a primary antibody (rabbit antihuman IgM), a secondary antibody (biotinylated goat antirabbit IgG) and avidin-biotin-peroxidase complex followed by immunostaining with 3-amino-9-ethylcarbazole (Cruz et al., 1991). The presence of IgM binding to 90--100 kd bands consistent with MAG is assessed. Reference serum with known M protein of IgM isotype and anti-MAG reactivity must be examined in parallel. Reactivity with MAG is always associated with similarly intense reactivity with the low molecular weight nerve glycoproteins in the range of P0. The procedure can be used for semiquantitative measurements of anti-MAG IgM by additional serial twofold dilutions of the test serum until negative (NobileOrazio et al., 1994) (Figure 1).

AUTOANTIBODIES Application Serum electrophoresis, preferably performed on agarose and combined with immunofixation to uncover small M proteins as well (Keren et al., 1988; Vrethem et al., 1993), should be included in the laboratory screening of PN, in particular when a cause of the PN such as diabetes mellitus or alcohol abuse is not obvious. In rare instances, an M protein can be demonstrated even in patients with a PN secondary to another cause. Agarose electrophoresis combined with immunofixation should be considered in the laboratory evaluation of every patient with clinical PN. Immunoelectrophoresis is less suitable for the demonstration of monoclonal IgM since the high molecular weight IgM diffuses slowly and precipitates poorly (Riches, 1986). In those instances where an M protein of IgM isotype is found, serum should be analyzed for antiMAG antibodies. ELISA can be used, but positive results should be confirmed by immunoblot. The sensitivity and specificity of the assays used by the laboratory must be known by the referring physician.

514

.

ELISA. This assay method provides semiquantitative data regarding titers and affinity of anti-MAG IgM. False-positive results are not rare and may be due to antibody binding to a contaminant of the antigen preparation, nonspecific binding of lower affinity antibodies at low serum concentrations or omission of positive and negative control wells for each serum tested (Pestronk et al., 1994; Latov, 1995). These could be the reasons why some of the studies that employ only ELISA fail to find any specific clinical correlates of anti-MAG (Gosselin et al., 1992; Suarez and Kelly, 1993) or detect anti-MAG in unusually high frequency in heterogenous patient groups (Peter et al., 1992). The specificity of ELISA to diagnose a consistent anti-MAG antibody-associated syndrome can be increased by using 1) a modified ELISA methodol-

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Patients' Groups

Figure 1. Anti-myelin-associated glycoprotein (MAG) IgM

antibody titers by immunoblot after sodium dodecyl sulfate polyacrylamide gel electrophoresis (gradient, 4-20%) of CNS myelin in patients with IgM monoclonal gammopathy with or without neuropathy. Patients with high titers of IgM antibodies to the other antigens tested are also represented. The number of patients in each group is given in brackets. Reprinted with permission from Nobile-Orazio et al. Ann Neurol 1994;36: 416--214. ogy that employs highly purified MAG, and 2) immunoblot confirmation of anti-MAG sera that cross-react strongly with histone H3 since sera with high titers of binding to both MAG and H3 identified by ELISA rarely stain MAG on immunoblot (Pestronk et al., 1994). The parallel determination of antibodies to MAG and SGPG antigen by ELISA has also been claimed to be more specific than determination of anti-MAG antibodies alone (McGinnis et al., 1988). 3. H i g h - P e r f o r m a n c e T h i n - L a y e r C h r o m a t o g r a p h y

(HPTLC). Overlay HPTLC is used to demonstrate reactivity to the PNS glycolipids SGPG and/or SGLPG, since they both bear the target epitope of MAG. The acid glycolipid fractions containing SGPG and SGLPG are separated on HPTLC. After incubation with patient serum and positive control serum, IgM reactivity is detected by immunostaining. Reactivity to MAG found by immunoblot is regularly associated with reactivity with SGPG

and SGLPG detected by overlay HPTLC (NobileOrazio et al., 1994). P a t h o g e n e t i c Role

Although the relevance of anti-MAG antibodies in the pathogenesis of PN is still debated, the clinical improvement observed in some patients after removal of the M protein with plasma exchange supports a pathogenetic role. Deposits of anti-MAG M proteins and complement are found on affected myelin sheaths (Monaco et al., 1990) and anti-MAG IgM antibodies trigger complement-mediated demyelination (Monaco et al., 1995). Demyelination can be induced by passive transfer of patient's serum or M protein with anti-MAG and SGPG activity, together with complement, into sciatic nerve of the cat (a species which shares the same antigenic epitope as human MAG, unlike rodent MAG which does not) (Hays et al., 1987; Willison et al., 1988). Systemic administration of anti-MAG antibodies into the chicken causes neuropathy and demyelination with the characteristic separation of the myelin lamellae at the minor dense line, similar to that seen in human disease (Tatum, 1993). Whether anti-MAG antibodies binding to MAG are responsible for the effects, or a binding to the P0 protein or to any of the two glycosphingolipids SGPG or SGLPG, which all share sulfated glucuronic acidbearing oligosaccharides that bind the anti-MAG antibodies, is not known. The human monoclonal IgMs with anti-MAG activity use a diverse repertoire of V H and V L genes which exhibit somatic mutations, possibly indicating an antigen-driven, T-cell-dependent process (Lee et al., 1994). Sera, purified monoclonal IgM and supernatants from anti-MAG-antibody-secreting cell lines react with bacterial polypeptides, suggesting molecular mimicry as a possible mechanism of PN (Brouet et al., 1994). A majority of anti-MAG IgM antibody-expressing cells in PN with IgM M protein are CD5-positive B cells (Lee et al., 1991). Such cells secrete polyreactive autoantibodies of IgM isotype. Anti-MAG IgM antibodies exhibit relatively low intrinsic affinities to the oligosaccharide antigen, but relatively high affinity to intact MAG (Ogino et al., 1994). While highaffinity antibodies are rapidly removed from the circulation by the normal route via the reticuloendothelial system, low-affinity IgM antibodies tend to form large antigen-antibody complexes, rendering their opsonization for phagocytosis more difficult.

515

Such complexes could be deposited in tissues, causing inflammation with the release of antigen, which could stimulate autoantibody production. Whether such mechanisms are involved in PN associated with antiMAG antibodies remains to be clarified. Cells secreting anti-MAG IgM as well as IgA or IgG antibodies have been demonstrated in the cerebrospinal fluid from patients with PN associated with IgM M protein (Baig et al., 1991). Whether these locally produced antibodies are of any pathogenetic importance is not known. MAG-reactive T cells have also been enumerated (Olsson et al., 1993).

CLINICAL UTILITY Clinical Associations

Anti-MAG IgM antibodies in PN patients almost always occur in association with monoclonal IgM gammopathies (paraproteinemia, M protein). The M protein binds to myelin and to the oligosaccharide determinant of MAG (Vital et al., 1989). Elevated anti-MAG antibodies are rare in patients with PN associated with IgG or IgA monoclonal gammopathy or in other PN patients. About 50% of patients with PN-associated with monoclonal IgM gammopathy have elevated anti-MAG IgM antibody concentrations in serum (Latov et al., 1988; Nobile-Orazio et al., 1994). Many of the remainder react with glycolipids or gangliosides. Monoclonal IgM gammopathies are characterized by the proliferation of a single B-cell clone producing IgM which migrates as a single band on electrophoresis. Waldenstr6m' s macroglobulinemia (WM) is a malignant form, and monoclonal gammopathy of uncertain significance (MGUS) is a nonmalignant form. However, during follow-up over several years, 10-20% of MGUS cases progress to WM or multiple myeloma malignity. MGUS is about 200 times more common than malignant gammopathy (Radl, 1985). The prevalence of monoclonal gammopathies of IgM, IgA and IgG isotypes in the normal population increases with age from about 0.1% in the third decade to about 3% in the eighth decade (Kyle et al., 1972). These frequencies are based on the demonstration of monoclonal gammopathy by serum electrophoresis and immunoelectrophoresis. A simple, reliable and sensitive alternative for demonstration of serum M proteins is agarose electrophoresis combined with immunofixation (Keren et al., 1988; Vrethem et al., 1993). This method has not yet been utilized in

516

population-based prevalence studies of serum or plasma M proteins. The prevalence of PN in patients with WM disease is not known. Frequencies between 7--50% are described (Nobile-Orazio et al., 1994). Also in MGUS, few prevalence data for PN are available, and frequencies between 16--71% are reported (Kahn et al., 1980; Nobile-Orazio et al., 1992). In one study, 16 of 56 patients (29%) with MGUS had polyneuropathy; of these, seven had severe demyelinating PN and all had MGUS of IgM isotype (Kahn et al., 1980). The PN in patients with anti-MAG IgM is usually a slowly progressive, symmetrical, distal PN, frequently with a predominant sensory impairment. Distal paraesthesia may be prominent and out of proportion with sensory loss. Most patients are male in middle or old age. The course is usually extremely insidious, evolving over years, but sometimes causing considerable disability. Electrophysiologic studies typically show demyelination, or demyelination and axonal degeneration. Sural nerve biopsy often shows demyelination with widely spaced myelin lamellae being seen on electron microscopy. There is no clear correlation between the anti-MAG IgM antibody titer and the severity of polyneuropathy (Kelly et al., 1988; Nobile-Orazio et al., 1994). This observation is in line with observations in autoimmune diseases such as myasthenia gravis in which, however, the role of antiacetylcholine receptor antibodies is firmly established. A small proportion of patients with PN associated with M protein and raised titers of anti-MAG IgM antibodies have in parallel elevated concentrations of antisulfatide IgM antibodies (Ilyas et al., 1992; van den Berg et al., 1993; Nobile-Orazio et al., 1994). Clinically, the PN in these patients is not different from that with anti-MAG IgM antibodies exclusively, but only a few patients have been studied. Monoclonal gammopathies with anti-MAG activity are also reported to develop following the onset of PN or in Charcot-Marie-Tooth disease (Valldeoriola et al., 1993). Nonparaproteinemic anti-MAG IgM antibodies are detected in rare patients with multiple sclerosis, Guillain-Barr6 syndrome, chronic polyneuropathy and myasthenia gravis (Sato et al., 1986). Whether these antibodies contribute to the neurological dysfunction is not known. Anti-P0 autoantibodies were also detected in GBS where molecular mimicry between P0 and varicellazoster virus glycoprotein and reovirus protein has been postulated (Adelmann and Linington, 1992; Khalili-Shirazi et al., 1993).

Effect of Therapy

CONCLUSION

In peripheral neuropathy associated with IgM paraproteinemia, removal of M protein by plasma exchange as well as immunosuppressive medication and intravenous immunoglobulins is associated with improvement in some patients (Haas and Tatum, 1988; Latov et al., 1988; Nobile-Orazio et al., 1988; Cook et al., 1990). To counteract the rebound effect on the B-cell clone induced by apheresis, combination with high-dose prednisolone + cyclophosphamide is initiated, and repetitive plasmapheresis added after a few weeks. In some cases, the disease is too insidious to require treatment or too advanced to make interference worthwhile.

A B-cell response to MAG, a minor glycoprotein of both PNS and CNS myelin, can be demonstrated in a variety of neurological diseases. Of clinical relevance are the elevated serum concentrations of anti-MAG IgM antibodies found in about half of the patients with a progressive demyelinating PN associated with IgM monoclonal gammopathy occurring in MGUS and Waldenstr6m's macroglobulinemia in particular. Such patients may benefit from treatment with immunosuppressive medication combined with plasmapheresis. Determination of anti-MAG IgM antibodies is recommended in patients with PN associated with monoclonal IgM gammopathy. ELISA followed by confirmative immunoblot has high sensitivity and specificity for anti-MAG IgM antibodies if highly purified MAG, appropriate controls and standardized methodology are used.

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Adelmann M, Linington C. Molecular mimicry and the autoimmune response to the peripheral nerve myelin P0 glycoprotein. Neurochem Res 1992;17:887--891. Baig S, Jiang YP, Olsson T, Cruz M, Link H. Cells secreting anti-MAG antibody occur in cerebrospinal fluid and bone marrow in patients with polyneuropathy associated with M component. Brain 1991;114:573--583. Baldini L, Nobile-Orazio E, Guffanti A, Barbieri S, Carpo M, Cro L, Cesana B, Damilano I, Maiolo AT. Peripheral neuropathy in IgM monoclonal gammopathy and Waldenstr6m's macroglobulinemia: a frequent complication in elderly males with low MAG reactive serum monoclonal component. Am J Hematol 1994;45:25--31. Bambrick G, Braun P. Phosphorylation of myelin-associated glycoprotein in cultured oligodendrocytes. Dev Neurosci 1991;13:412--416. Bollensen E, Steck AJ, Schachner M. Reactivity with the peripheral myelin glycoprotein P0 in serum from patients with monoclonal IgM gammopathy and polyneuropathy. Neurology 1988;38:1266--1270. Brouet J, Mariette X, Chevalier A, Hauttecoeur B. Determination of the affinity of monoclonal IgM for myelin-associated glycoprotein and sulfated glucuronic paragloboside. J Neuroimmunol 1992;36:209--215. Brouet J-C, Mariette X, Gendron M-C, Dubreuil M-L. Monoclonal IgM from patients with peripheral demyelinating neuropathies cross-react with bacterial polypeptides. Clin Exp Immunol 1994;96:466--469. Burger D, Perruisseau G, Simon M, Steck A. Comparison of the N-linked oligosaccharide structures of the two major human myelin glycoproteins MAG and P0: assessment and relative occurrence of oligosaccharide structures by serial

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Griffith L, Schmitz B, Schachner M. L2/HNK-1 carbohydrate and protein-protein interactions mediate the homophilic binding of the neural adhesion molecule P0. J Neurosci Res 1992;33:639-648. Haas DC, Tatum AH. Plasmapheresis alleviates neuropathy accompanying IgM anti-myelin-associated glycoprotein paraproteinemia. Ann Neurol 1988;23:394--396. Hays AP, Latov N, Takatsu M, Sherman WH. Experimental demyelination of nerve induced by serum of patients with neuropathy and an anti-MAG IgM-M protein. Neurology 1987;37:242--256. Ilyas A, Dalakas M, Brady R, Quarles R. Sulfated glucuronyl glycolipids reacting with anti-myelin-associated glycoprotein monoclonal antibodies including IgM paraproteins in neuropathy: species distribution and partial characterization of epitopes. Brain Res 1986;385:1--9. Ilyas AA, Cook SD, Dalakas MC, Mithen FA. Anti-MAG IgM paraproteins from some patients with polyneuropathy associated with IgM paraproteinaemia also react with sulphatide. J Neuroimmunol 1992;37:85--92. Inuzuka T, Sato S, McIntyre L, Quarles R. Effects of trypsin and plasmin treatment of myelin on the myelin-associated glycoprotein and basic protein. J Neurochem 1984;43:582-585. Ishiguro H, Inuzuka T, Fujita N, Sato S, Nakano R, Tamura A, Kirino T, Miyatake T. Expression of the large myelinassociated glycoprotein isoform in rat oligodendryocytes around cerebral infarcts. Mol Chem Neuropathol 1993;20: 173--179. Kahn SN, Riches PG, Kohn J. Paraproteinaemia in neurological disease: incidence, associations, and classification of monoclonal immunoglobulins. J Clin Pathol 1980;33:617--621. Kelly JJ, Adelman LS, Berkman E, Bhan I. Polyneuropathies associated with IgM monoclonal gammopathies. Arch Neurol 1988;45:1355-1359. Keren DF, Warren JS, Lowe JB. Strategy to diagnose monoclonal gammopathies in serum: High-resolution electrophoresis, immunofixation and kappa/lambda quantification. Clin Chim Acta 1988:34:2196--2201. Khalili-Shirazi A, Atkinson P, Gregson N, Hughes P. Antibody responses to P0 and P2 myelin proteins in Guillain-Barr6 syndrome and chronic idiopathic demyelinating polyradiculoneuropathy. J Neuroimmunol 1993;46:245-252. Kyle RA, Finkelstein S, Elveback LR, Kurland LT. Incidence of monoclonal proteins in a Minnesota community with a cluster of multiple myeloma. Blood 1972;40:719-724. Latov N. Plasma cell dyscrasia and peripheral neuropathy: identification of the myelin antigens that react with human paraproteins. Proc Natl Acad Sci USA 1981;78:7139-7142. Latov N, Hays AP, Sherman WH. Peripheral neuropathy and anti-MAG antibodies. Crit Rev Neurobiol 1988;3:301--332. Latov N. Pathogenesis and treatment of neuropathies associated with monoclonal gammopathies. Ann Neurol 1995;37(Suppl 1):$32-$42. Lee G, Ware RR, Latov N. Somatically mutated member of the human V lambda VIII gene family encodes antimyelin associated glycoprotein (MAG) activity. J Neuroimmunol 1994;51:45--52.

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Lee KW, Inghirami G, Spatz L, Knowles DM, Latov N. The Bcells that express anti-MAG antibodies in neuropathy and nonmalignant IgM monoclonal gammopathy belong to the CD5 subpopulation. J Neuroimmunol 1991 ;31:83--86. Li F, Pestronk A, Griffin J, Feldman EL, Cornblath D, Trotter J, Zhu S, Yee WC, Phillips D, Peeples DM, Winslow B. Polyneuropathy syndromes associated with serum antibodies to sulfatide and myelin-associated glycoprotein. Neurology 1991;41:357--362. McGinnis S, Kohriyama T, Yu RK, Pesce MA, Latov N. Antibodies to sulfated glucuronic acid containing glycosphingolipids in neuropathy associated with anti-MAG antibodies and in normal subjects. J Neuroimmunol 1988;17:119--126. Miller S, Kahn S, Perussia B, Trinchieri G. Comparative binding of murine and human monoclonal antibodies reacting with myelin-associated glycoprotein of myelin and human lymphocytes. J Neuroimmunol 1987;15:229-242. Monaco S, Bonetti B, Ferrari S, Moretto G, Nardelli E, Tedesco F, Mollness TE, Nobile-Orazio E, Manfredini E, Bonazzi L, et al. Complement mediated demyelination and polyneuropathy. N Engl J Med 1990;322:649--652. Monaco S, Ferrari S, Bonetti B, Moretto, G, Kirshfink M, Nardelli E, Nobile-Orazio E, Zanusso G, Rizzuto N, Tedesco F. Experimental induction of myelin changes by anti-MAG antibodies and terminal complement complex. J Neuropathol Exp Neurol 1995;54:96-104. Nobile-Orazio E, Baldini L, Barbieri S, Marmiroli P, Spagnol G, Francomano E, Scarlato G. Treatment of patients with neuropathy and anti-MAG IgM M-proteins. Ann Neurol 1988;24:93--97. Nobile-Orazio E, Barbieri S, Baldini L, Marmiroli P, Carpo M, Premoselli S, Manfredini E, Scarlato G. Peripheral neuropathy in monoclonal gammopathy of undetermined significance: prevalence and immunopathogenetic studies. Acta Neurol Scand 1992;85:383--390. Nobile-Orazio E, Manfredini E, Carpo M, Meucci N, Monaco S, Ferrari S, Bonetti B, Cavaletti G, Gemignani F, Durelli L et al. Frequency and clinical correlates of antineural IgM antibodies in neuropathy associated with IgM monoclonal gammopathy. Ann Neurol 1994;36:41 6-424. Norton WT, Poduslo SE. Myelination in rat brain: method of myelin isolation. J Neurochem 1973;21:749-757. Ogino M, Tatum AH, Latov N. Affinity studies of human antiMAG antibodies in neuropathy. J Neuroimmunol 1994;52: 41--46. Olsson T, Sun J, Solders G, Xiao BG, Hojeberg B, Ekre HP, Link H. Autoreactive T and B cell responses to myelin antigens after diagnostic sural nerve biopsy. J Neurol Sci 1993;117:130--139. Pestronk A, Li F, Bieser K, Choksi R, Whitton A, Kornberg AJ, Goldstein JM, Yee W-C. Anti-MAG antibodies: major effects of antigen purity and antibody cross-reactivity on ELISA results and clinical correlation. Neurology 1994;44: 1131--1137. Peter JB, Sutjita M, Dalakas M, Munsat TL, Engel WK, Tourtellotte WW, Baloh RW, Haapike JF. Rapid method for detection of myelin-associated glycoprotein (MAG) antibodies. Neurology 1992;42(Suppl 3):333.

Quarles RH. Myelin-associated glycoprotein: functional and clinical aspects. In: Marangos PJ, Campbell I, Cohen EN, eds. Neuronal and Glial Proteins: Structure, Function and Clinical Applications. Petaluma, CA: Academic Press, 1988:295--320. Quarles RH. Myelin-associated glycoprotein in demyelinating disorders. Crit Rev Neurobiol 1989;5:1--28. Radl J. Benign monoclonal gammopathy. In: Melchers F, Potter M, eds. Mechanisms in B-cell Neoplasia. Berlin: Springer Verlag, 1985:221--224. Riches PG. Immunological investigation and identification of the paraprotein. In: Delamore IW, ed. Multiple Myeloma and Other Paraproteinaemias. Edinburgh: Churchill Livingstone, 1986:56--74. Sato S, Baba H, Inuzuka T, Miyatake T. Anti-myelin-associated glycoprotein antibody in sera from patients with demyelinating diseases. Acta Neurol Scand 1986;74:115-120. Suarez GA, Kelly Jr JJ. Polyneuropathy associated with monoclonal gammopathy of undetermined significance: further evidence that IgM-MGUS neuropathies are different than IgG-MGUS. Neurology 1993;43:1304--1308. Tatum AH. Experimental paraprotein neuropathy; demyelination by passive transfer of human IgM anti-myelin-associated glycoprotein. Ann Neurol 1993;33:502--506.

Valldeoriola F, Graus F, Steck AJ, Munoz E, de la Fuente M, Gallart T, Ribalta T, Bombi JA, Tolosa E. Delayed appearance of anti-myelin-associated glycoprotein antibodies in a patient with chronic demyelinating polyneuropathy. Ann Neurol 1993;34:394--396. van den Berg LH, Lankamp CL, de Jager AE, Notermans NC, Sodaar P, Marrink J, de Jong HJ, Bar PR, Wokke JH. Antisulphatide antibodies in peripheral neuropathy. J Neurol Neurosurg Psychiatry 1993;56:1164--1168. Vital A, Vital C, Julien J, Baquey A, Steck AJ. Polyneuropathy associated with IgM monoclonal gammopathy. Immunological and pathological study in 31 patients. Acta Neuropathol 1989;79:160-167. Vrethem M, Larsson B, von Schenck H, Ernerudh J. Immunofixation superior to plasma agarose electrophoresis in detecting small M-components in patients with polyneuropathy. J Neurol Sci 1993; 120:93--98. Willison HJ, Trapp BD, Bacher JD, Dalakas MC, Griffin JW, Quarles RH. Demyelination induced by intraneural injection of human antimyelin-associated glycoprotein antibodies. Muscle Nerve 1988;11:1169-1176. Yu R, Ariga T, Kohriyama T, Kusunoki S, Maeda Y, Miyatani N. Autoimmune mechanisms in peripheral neuropathies. Ann Neurol 1990;29(Suppl):S30-S35.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

MYELIN BASIC PROTEIN AUTOANTIBODIES Shuguang Li, M.D., Ph.D.

Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES Autoimmunity is considered to be an important mechanism for myelin destruction in diseases of the central nervous system (CNS). Since the identification of myelin basic protein (MBP) as the antigen causing experimental allergic encephalomyelitis (EAE; Kies and Alvord, 1959), much research has focused on its possible pathogenic role as a target for the immune attack in multiple sclerosis (MS). In 1980, antibodies to MBP (anti-MBP) were first detected in CSF of patients with MS (Panitch et al., 1980). Elevated antiMBP titers were subsequently observed in the cerebrospinal fluid (CSF) of a majority of patients experiencing first relapses of MS (Warren and Catz, 1989) as well as in most patients with first attacks of optic neuritis (Warren et al., 1988; 1994). Anti-MBP are synthesized intrathecally (Catz and Warren, 1986) and anti-MBP IgG is expressed within the CNS tissues of MS patients (Warren and Catz, 1993a; 1993b). Anti-MBP IgG has been purified from CSF of MS patients and patients with optic neuritis by affinity chromatography (Warren and Catz, 1991). B cells secreting anti-MBP are persistently present in relatively high numbers in the CSF of MS and optic neuritis patients (Link et al., 1990; S6derstr6m et al., 1993).

THE AUTOANTIGENS Definition/Origin MBP is a component of the myelin sheath, a multilayered structure surrounding axons and produced by oligodendrocytes and Schwann cells in the CNS and peripheral nervous systems, respectively. MBP comprises 30% of all CNS myelin proteins (Lees and

520

Brostoff, 1984); it is a highly cationic membraneassociated protein that interacts with negatively charged phospholipids in the myelin membrane. Human MBP is present as five isoforms arising from alternative splicing of its primary transcript (Kamholz et al., 1988). Human 18.5 kd MBP is composed of 170 amino acid residues (Carnegie, 1971). Anti-MBP purified from CSF of patients with acute optic neuritis or acute relapses of MS shows specificity for selected synthetic peptides between residues 84 and 95 (Warren and Catz, 1992; 1993b). Several T-cell-recognized immunodominant epitopes of MBP have been identified. The major T-cell responses to MBP are directed to two regions between residues 84 and 106 and between 143 and 172 in MS patients carrying the human leukocyte antigen HLADR2 (about two-thirds of patients) (Hailer and Weiner, 1995). The immunodominant MBP peptide 88--99 is critical for MHC class II binding and for T-cell receptor recognition by HLA-DR2- and HLA-DQ 1-restricted T cell clones (Wucherpfennig and Strominger, 1995). Although peptide sequences derived from common pathogens, such as herpes simplex virus, Epstein-Barr virus, influenza viruses, have quite distinct structure as compared to residues 85--99 of MBP, they can activate MBP-specific T-cell clones from MS patients efficiently (Wucherpfennig and Strominger, 1995). The evidence that a single T-cell receptor can recognize structurally related peptides from multiple pathogens may be important to understanding the pathogenesis of autoimmunity. Other myelin sheath components such as proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG) and myelin-associated glycoprotein (MAG) also trigger humoral and cellular immune responses in MS patients (Steinman et al., 1995). In

addition, nonmyelin encephalitogenic autoantigens that trigger T-cell immune responses include the astroglial calcium-binding protein S 100[~ (Hohlfeld et al., 1995; Hailer and Weiner, 1995), transaldolase, 2', 3'-cyclic / nucleotide 3-phosphodlesterases (Steinman et al., 1995) and ~B-crystallin (van Noort et al., 1995). Thus, an initial T-cell response against myelin or nonmyelin autoantigens might trigger a subsequent T- and B-cell response against other CNS autoantigens, a phenomenon defined as "epitope spreading" (Lehmann et al., 1992; Kaufman et al., 1993; Tisch et al., 1993). Myelin obtained from MS patients is arrested at the stage of the first growth spurt (within the first six years of life) (Moscarello et al., 1994). It is postulated that developmentally immature myelin might be more susceptible to degradation, thereby providing the initial antigenic materials to the immune system. However, immaturity might also be a consequence of myelin regeneration after the demyelination process, and the assembly of the unstable myelin sheath might make myelin antigens more vulnerable to the immune system.

Methods of Purification Several purification methods are available (Table 1). The major problem encountered with MBP purified from brain or spinal cord is the presence of MBP-like peptides produced from MBP by endogenous protease activities. One advantage of recombinant MBP over MBP purified from protease-rich CNS tissues is the absence of such proteolytically derived fragments (Oettinger et al., 1993).

THE AUTOANTIBODIES Pathogenetic Role Although the etiology of MS is not known, evidence suggests the involvement of both cellular and humoral immune responses in the pathogenesis of demyelinating disease. EAE is an experimental, demyelinating autoimmune disease mediated by CD4 + T cells specific for

Table 1. Methods for Purification of Myelin Basic Protein Methods

Tissue Origin

Reference

Comments Advantages

Disadvantages

Acid extraction, precipitation; ion-exchange chromatography

Brain: guinea pig, human, monkey, rabbit Spinal cord: bovine, guinea pig, monkey

1. Large scale 2. Relatively pure

1. Presence of contaminating proteolytically derived peptides 2. Tedious

Deibler et al., 1972

Ammonium sulfate/acetone precipitation; Cation exchange chromatography

1. Relatively pure Brain: bovine, chicken, guinea pig, horse, human, monkey, pig, rabbit, sheep, turtle Spinal cord: bovine

1. Presence of contaminating proteolytically derived peptides 2. Tedious

Eylar et al., 1974

High-performance Brain: human Liquid Chromatography

1. 2. 3. 4.

Recombinant human 18.5 kd MBP; precipitation; solubilization;

1. High purity 2. Free of contaminating peptides 3. Consistent sources 4. High yield

Human MBP expressed in E. coli

Rapid Good yield High purity Minimizes the exposure of MBP to proteases

Giegerich it al., 1990

Oettinger et al., I993

521

MBP. EAE can be effectively induced in healthy rodents (mice and rats) by the transfer of cloned, MBP-specific T-cell lines or clones. Antibodies against MBP and PLP are present in EAE sera with demyelinating activity (Bernard and de Rosbo, 1991); however, polyclonal antibodies raised against preparations of MBP, PLP and MAG do not cause demyelination in cultured CNS tissue (Glynn and Linington, 1989). Passive transfer of MBP-specific antibodies alone does not directly produce disease in the EAE model. However, the role of B cells as antigen-presenting cells (APC) is demonstrated in B-cell-depleted mice where EAE cannot be effectively induced by a combination of antigen/complete Freund's adjuvant immunization and adoptive transfer with MBP-specific CD4 + T cells unless mice are primed with B cells prior to immunization (Myers et al., 1992). Furthermore, the efficiency of EAE induction in B-celldepleted mice ~an be enhanced if MBP-specific antibodies are administrated simultaneously. This study shows that B cells can function as both antigenpresenting cells in the initial primary and subsequent expansion of MBP-specific T cells and as anti-MBPproducing cells. Therefore, B cells may play a role in the induction of EAE. Antibodies against other myelin antigens may participate in demyelination (Bernard and de Rosbo, 1991). Immunoglobulins isolated from CNS white matter of MS, but not control humans, can mediate MBP degradation of purified human myelin (Kerlero de Rosbo and Bernard, 1989). In a chronic relapsing experimental autoimmune encephalomyelitis (CREAE) model, rats injected with a single dose of myelin oligodendrocyte glycoprotein (MOG) develop a relapsing, remitting neurologic disease with extensive plaque-like demyelination (Johns et al., 1995). Immunoglobulins isolated from these animals react specifically with MOG and stimulate a myelin-associated protease activity that leads to MBP degradation. To the contrary, immunoglobulins isolated from animals with EAE induced by MBP poorly stimulate the myelin-associated protease activity. In addition, a mouse monoclonal antibody specific for MOG demonstrated the ability to induce relapses and demyelination in CNS (Bernard and de Rosbo, 1991), suggesting that as a new demyelinating antigen, MOG elicits humoral immune responses that initiate the autoimmune-mediated demyelination process in Lewis rats. This new autoimmune demyelinating model may provide additional insight into the pathogenesis of MS. 522

Epidemiologic studies suggest the involvement of infectious agents in the pathogenesis of MS (Kurtzke, 1993). Viral infection of cells within the CNS might initiate the events leading to focal demyelination (Allen and Brankin, 1993). Several diverse pathogens that possess epitopes which mimic MBP peptides 85--99 can stimulate MBP (85--99)-specific T cell clones (Wucherpfennig and Strominger, 1995). This observation suggests that a group (rather than a single) of common viral pathogens, in particular the herpes virus family (HHV6, EBV, herpes simplex and cytomegalovirus), influenza virus and papilloma viruses might be involved in initiating the autoimmune process (Steinman, 1995; Challoner et al., 1995). Such viruses that cause latent and/or persistent infection might cause chronic antigenic stimulation of autoreactive T-cell clones. Genetics

Genetic susceptibility or resistance to MS is thought to be associated with genes within or close to the HLA-DR-DQ subregion located on the short arm of the sixth chromosome (Hillert and Olerup, 1993). HLA-Dw2 and-DR2 specificities are genetic markers for susceptibility to MS in Caucasian Europeans and North Americans. An immunodominant region of MBP (amino acid residues 84-103) binds to most purified DR molecules, and with high affinity to DR15 molecules (Lin et al., 1991). Recent studies suggest the existence of B-cell immunodominant epitopes in the mid-region of the MBP molecule (Cash et al., 1992; Martina et al., 1991; Warren and Catz, 1992; 1993b). Anti-MBP responses in patients with optic neuritis also are influenced by the HLA because the production of antibodies to the synthetic MBP peptide sequence of 63--88 is predominantly associated with patients with HLA-DR15 genotype (Sellebjerg et al., 1994). These data raise the important question of whether the observed B-cell immunodominance in the DR15-positive patients may be of pathogenetic relevance, or should be viewed merely as an epiphenomenon. Methods of Detection

The most commonly used methods for detecting antiMBP are solid-phase RIA (Randolph et al., 1977) and enzyme-linked immunosorbent assay (ELISA) (Maimone et al., 1994). Immunoblot (Singh et al., 1992; 1993), passive hemagglutination, double diffusion in

agar, the globulin consumption test, immunoadherence, antibody-dependent lymphocyte cytotoxicity, complement fixation, micro precipitin tests, radioimmunologic determination with gel filtration and immunofluorescence can also be used (Leibowitz, 1972). Two forms (free and bound) of anti-MBP can be detected in CSF of MS patients with a solid-phase radioimmunoassay (RIA). CSF immune complexes composed of MBP and anti-MBP can be dissociated with acid treatment. Free (unbound) anti-MBP can be detected without acid dissociation. Total anti-MBP (free plus bound) are determined after the acid dissociation and the bound fraction are calculated by subtracting the free values from the corresponding total of anti-MBP (Warren and Catz, 1987; 1989). Acid dissociation of CSF and serum samples is essential for the reliable detection of total anti-MBP (Warren and Catz, 1986; Maimone et al., 1994). Free and bound anti-MBP concentrations are highly dependent on the time of CSF sampling. CSF anti-MBP IgG is mostly in the complexed form in patients with the chronic progressive form of MS (Warren and Catz, 1986; 1987). Patients infected with HIV-1 can exhibit a steady, rather than phasic, MBP release and/or anti-MBP IgG synthesis (Maimone et al., 1994). Under such conditions, antigen and antibody concentrations might reach optimal ratios, leading to maximal formation of immune complexes. As patients enter into clinical remission, both free and bound antiMBP concentrations generally decline. The free fraction typically declines faster than the bound fraction. Methods are available for detecting secretion of anti-MBP and antibodies to peptides of MBP (Link et al., 1990; S6derstr6m et al., 1993; Sellebjerg et al., 1994). Monitoring antibody-producing cells in vitro avoids the problems of in vivo absorption of antibodies to myelin. The assay is referred to as a nitrocellulose immunospot assay or a solid-phase enzymelinked immunospot (ELISPOT) assay. MBP or MBP peptide antigens are coated on the bottom of nitrocellulose membrane microtiter plates, and CSF cells or PBL cells are added into individual wells. After culturing overnight, followed by removal of the cells, anti-MBP and anti-MBP-peptide antibodies are detected with biotinylated goat antihuman Ig. Spots formed by cells secreting anti-MBP are then counted via a dissection microscope. Cells secreting antibodies against myelin, MBP, PLP and MOG are persistently present in high numbers in the CSF of MS patients

(Link et al., 1990; S6derstr6m et al., 1993; Sellebjerg et al., 1994).

CLINICAL UTILITY Disease Association Antibodies to myelin-associated proteins are associated with the demyelination characteristic of neurological diseases (Gould and Swanborg, 1993). AntiMBP antibodies, in particular, are detected in high frequencies in the CSF and/or sera of patients with MS, acute idiopathic optic neuritis and less frequently in patients with other neurological diseases such as Guillain-Barr6 syndrome, acute idiopathic optic neuritis, subacute sclerosing panencephalitis, chronic relapsing polyradiculoneuritis, carcinomatous polyneuropathy, parainfectious encephalomyelitis, spinal cord tumors and cervical spondylosis (Panitch et al., 1980). Increased CSF anti-MBP in both free (F) and bound (B) forms are associated with MS (Warren and Catz, 1986). Anti-MBP are found predominantly in free form during acute relapse and predominantly in bound form when the disease is insidiously progressive (Warren and Catz, 1986; 1987). It is, therefore, hypothesized that a combination of total anti-MBP concentration and F/B anti-MBP ratio may be of use for differentiation of patients with acute relapse, chronic progression or stable disease (Warren and Catz, 1986; 1987). Alzheimer's disease (AD) is a disorder primarily affecting neurons with secondary involvement of myelin. The high prevalence of anti-MBP in AD patients might reflect a humoral immune response to MBP with the progression of the disease (Singh et al., 1992). In patients with AIDS dementia complex, antiMBP levels were significantly higher than HIV-1 infected patients without neurological disorders (Mastroianni et al., 1991; Maimone et al., 1994). Correlation with Disease Activity Although not disease specific, elevations of CSF antiMBP are strongly associated with disease activity. MS patients with acute exacerbations or chronically progressive disease have significantly elevated, intrathecally synthesized anti-MBP antibodies (Warren and Catz, 1989). Increased CSF anti-MBP are found 96% (173/180) of patients in acute MS relapse and in 96% of progressive MS patients (111/116) (Warren

523

Table

2. Prevalence of anti-MBP Antibodies in Demyelination-associated Disorders

Disease Subgroups

Sample Source/Methods

Acute Relapsing* MS Chronic Progressive MS Relapsing and Remitting MS Total MS

CSF/RIA

96 96 17 79

Warren et al., 1994

Optic neuritis

CSF/RIA

89

Warren et al., 1994

Mild ADC Moderate ADC Severe ADC Total ADC

CSF/ELISA

50 63 100 74

Mastroianni et al., 1991

CSF, Serum/ELISA

Maimone et al., 1994

Total

100 78 42 64

MS NINE

50 0

HIV- 1 infection:

II--III IV B IV C-IV D

AD (patients 57--95) Controls:

Serum/IB

Healthy elderly Healthy adults Parkinson' s disease IMR DS

89

Reference

Singh et al., 1992

12 8 17 11

0 8

Total Autistic Behavior (patients < 10 yrs.)

% Positive

Serum/IB

Controls

58

Singh et al., 1993

9

AD: Alzheimer's disease; ADC" AIDS Dementia Complex; DS: Down's syndrome; IMR: Idiopathic Mental Retardation; MS" Multiple Sclerosis; NINE: Noninflammatory Neurological Disease. ELISA: Enzyme-linked immunosorbent assay; IB: Immunoblot; RIA: Radioimmunoassay.

et al., 1994) (Table 2). That only 17% of the MS patients (15 of 87 patients) had elevated CSF antiMBP in the remission phase of MS (Table 2), suggests that anti-MBP production is reduced during the inactive phase of MS (Warren et al., 1994). Anti-MBP autoantibodies are also detectable in 89% of patients with optic neuritis (Warren et al., 1994). A follow-up study of patients infected with HIV-1 showed that increase of anti-MBP titers is associated with the progression of encephalopathy (Maimone et al., 1994).

CONCLUSION MBP is a major protein component of CNS myelin. Intrathecal production of anti-MBP is significantly increased during the acute relapse phase and disappears

524

during the remission phase of MS (Catz and Warren, 1986; Warren and Catz, 1989). Sensitive and specific methods must be developed to detect anti-MBP antibodies in serum specimens or other body fluids if such an assay is to be useful for patient management. Measurement of free, bound and total anti-MBP antibodies might provide useful information regarding MS disease activities and disease stages. Presently, there are no sensitive and specific laboratory methods to differentiate MS patients into chronic progressive or relapsing and remitting groups. Any method providing such information would complement independent clinical evaluation. Likewise, at present there are no reliable laboratory methods for predicting which patients will respond to which forms of treatment. Assays for anti-MBP are of promising, but as yet un-

proved clinical utility. Because in demyelinating disease, such as MS, myelin sheaths are lost; whereas, axons are preserved, numerous myelin c o m p o n e n t s have been evaluated as possible i m m u n o l o g i c a l l y relevant target antigens. Autoantibodies to M B P are found in various neurological disorders associated with demyelination and are not specific for MS. Limited studies show that anti-MBP titer is associated with the disease activities

in certain demyelinating diseases such as MS, optic neuritis, and HIV-1 encephalopathy. A n t i - M B P are potentially useful markers in assessing the disease activity and the progression of demyelinating diseases. However, the pathogenetic and prognostic significance of the high prevalence of anti-MBP in A l z h e i m e r ' s patients is undefined and further investigations to address this question are needed.

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munity in multiple sclerosis. Immunol Today 1995;16:259-261. Johns TG, de Rosbo NK, Menon KK, Abo S, Gonzales MF, Bernard CC. Myelin oligodendrocyte glycoprotein induces a demyelinating encephalomyelitis resembling multiple sclerosis. J Immunol 1995;154:5536--5541. Kamholz J, Toffenetti J, Lazzarini RA. Organization and expression of the human myelin basic protein gene. J Neurosci Res 1988;21:62--70. Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GS, Robinson P, Atkinson MA, Sercarz EE, Tobin AJ, Lehmann PV. Spontaneous loss of cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993;366:69--72. de Rosbo NK, Bernard CC. Multiple sclerosis brain immunoglobulins stimulate myelin basic protein degradation in human myelin: a new cause of demyelination. J Neurochem 1989;53:513--518. Kies MW, Alvord FC Jr., eds. Allergic Encephalomyelitis. Springfield: Charles C. Thomas, 1959. Kurtzke JF. Epidemiologic evidence for multiple sclerosis as an infection. Clin Microbiol Rev 1993;6:382--427. Lees MB, Brostoff SW. Proteins of myelin. In: Morell P, ed. Myelin. New York: Plennum Press, 1989; 197--224. Lehmann PV, Forsthuber T, Miller A, Sercarz EE. Spreading of T cell autoimmunity to cryptic determinants of an autoantigen. Nature 1992;358:155--157. Leibowitz S. Immunology of multiple sclerosis. In: Adams CW, Leibowitz S, eds. Research on Multiple Sclerosis. Springfield: Charles C. Thomas, 1972:149--168. Lin R-H, Mamula MJ, Hardin JA, Janeway CA, Jr. Induction of autoreactive B cells allows priming of autoreactive T cells. J Exp Med 1991;173:1433--1439. Link H, Baig S, Olsson O, Jiang Y-P, Hojeberg B, Olsson T. Persistent antimyelin basic protein IgG antibody response in multiple sclerosis cerebrospinal fluid. J Neuroimmunol 1990;28:237--248. Maimone D, Annuziata P, Cioni C, Leonardi A, Guazzi GC. Intrathecal synthesis of antimyelin basic protein IgG in HIV1+ patients. Acta Neurol Scand 1994;90:285--292. Martina G, Olsson T, Fredrikson S, Hojeberg B, Kostulas V, Grimaldi LM, Link H. Cells producing antibodies specific for myelin basic protein region 70--89 are predominant in cerebrospinal fluid from patients with multiple sclerosis. Eur J Immunol 1991;21:2971--2976.

Allen I, Brankin B. Pathogenesis of multiple sclerosis -- the immune diathesis and the role of viruses. J Neuropathol Exp Neurol 1993;52:95--105. Bernard CC, de Rosbo NK. Immunopathological recognition of autoantigens in multiple sclerosis. Acta Neurol 1991 ;13:171-178. Carnegie PR. Amino acid sequence from the encephalitogenic protein from human myelin. Biochem J 1971;123:57--67. Cash E, Weerth S, Voltz R, Kornhuber M. Cells of cerebrospinal fluid of multiple sclerosis patients secrete antibodies to myelin basic protein in vitro. Scand J Immunol 1992;35: 695--701. Catz I, Warren KG. Intrathecal synthesis of autoantibodies to myelin basic protein in multiple sclerosis. Can J Neurol Sci 1986;13:21--24. Challoner PB, Smith KT, Parker JD, MacLeod DL, Coulter SN, Rose TM, Schultz ER, Bennett JL, Garber RL, Chang M, Schad PA, Stewart PM, Nowinski RC, Brown JP, Burmer GC. Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci USA 1995;92;74407444. Deibler GE, Martenson RE, Kies MW. Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep Biochem 1972;2:139-- 165. Eylar EH, Kniskern PJ, Jackson JJ. Myelin basic proteins. Meth Enzymol 1974;32B:323--341. Giegerich G, Pette M, Fujita K, Wekerle H, Epplen JT, Hinkkanen A. Rapid method based on reversed-phase high-performance liquid chromatography for purification of human myelin basic protein and its thrombic and endoproteinase Lys-C peptides. J Chromatography 1990;528:79--90. Gould KE, Swanborg RH. T- and B-cell responses to myelin basic protein and encephalitogenic epitopes. J Neuroimmunol 1993:46:193--198. Glynn P, Linington C. Cellular and molecular mechanisms of autoimmune demyelination in the central nervous system. CRC Crit Rev Neurobiol 1989;4:367-385. Hailer DA, Weiner H. Immunologic mechanism and therapy in multiple sclerosis. Immunol Rev 1995;144:75--107. Hillert J, Olerup O. Multiple sclerosis is associated with genes within or close to the HLA-DR-DQ subregion on a normal DR15, DQ6, Dw2 haplotype. Neurology 1993;43:163--168. Hohlfeld R, Londei M, Massaesi L, Salvetti M. T-cell autoim-

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Mastroianni CM, Liuzzi GM, Jirillo E, Vullo V, Delia S, Riccio P. Cerebrospinal fluid markers for the monitoring of AIDS dementia complex severity: usefulness of antimyelin basic protein antibody detection. AIDS 1991 ;5:464--465. Moscarello MA, Wood DD, Ackerley C, Boulias C. Myelin in multiple sclerosis is developmentally immature. J Clin Invest 1994;94:146-- 154. Myers KJ, Sprent J, Dougherty JP, Ron Y. Synergy between encephalitogenic T cells and myelin basic protein-specific antibodies in the induction of experimental autoimmune encephalomyelitis. J Neuroimmunol 1992;41:1--8. Oettinger HF, A1-Sabbagh A, Jingwu Z, LaSalle JM, Weiner HL, Hailer DA. Biological activity of recombinant human myelin basic protein. J Neuroimmunol 1993;44:157-162. Panitch HS, Hooper CJ, Johnson KP. CSF antibody to myelin basic protein. Measurement in patients with multiple sclerosis and subacute sclerosing panencephalitis. Arch Neurol 1980;37:206--209. Randolph DH, Kibler RF, Fritz RB. Solid-phase radioimmunoassay for detection of antibodies to myelin basic protein. J Immunol Methods 1977;18:215--224. Sellebjerg F, Frederiksen JL, Olsson T, Link H, Madsen HO, Ryder LP, Svejgaard A. Peptide specificity of antimyelin basic protein antibodies in patients with acute optic neuritis and the HLA system. Scand J Immunol 1994;39:575-580. Singh VK, Yang Y-Y, Singh EA. Immunoblot detection of antibodies to myelin basic protein in Alzheimer's disease patients. Neurosci Lett 1992;147:25--28. Singh VK, Warren RP, Odell JD, Warren WL, Cole P. Antibodies to myelin basic protein in children with autistic behavior. Brain Behav Immun 1993;7:97--103. S6derstr/Jm M, Link H, Xu Z, Fredriksson S. Optic neuritis and multiple sclerosis: Anti-MBP and anti-MBP peptide antibodysecreting cells are accumulated in CSF. Neurology 1993;43: 1215--1222. Steinman L. Multiple sclerosis: Presenting an odd autoantigen. Nature 1995;375:739--740. Tisch R, Yang X-D, Singer SM, Liblau RS, Fugger L, McDevitt HO. Immune response to glutamic acid decarboxylase correlates with insulitis in nonobese mice. Nature 1993;

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366:72--75. van Noort JM, van Sechel AC, Bajramovic JJ, el Ouagmiri M, Polman CH, Lassman H, Ravid H. The small heat-shock protein czB-crystallin as candidate autoantigen in multiple sclerosis. Nature 1995;375:798--801. Warren KG, Catz I. Diagnostic value of cerebrospinal fluid antimyelin basic protein in patients with multiple sclerosis. Ann Neurol 1986;20:20-25. Warren KG, Catz I. A correlation between cerebrospinal fluid myelin basic protein and antimyelin basic protein in multiple sclerosis patients. Ann Neurol 1987;21:183-- 189. Warren KG, Catz I, Bauer C. Cerebrospinal fluid antibodies to myelin basic protein in acute idiopathic optic neuritis. Ann Neurol 1988;23:297--299. Warren KG, Catz I. Cerebrospinal fluid autoantibodies to myelin basic protein in multiple sclerosis patients. Detection during first exacerbations and kinetics of acute relapses and subsequent convalescent phases. J Neurol Sci 1989;91:143-151. Warren KG, Catz I. Purification of autoantibodies to myelin basic protein by antigen specific affinity chromatography from cerebrospinal fluid IgG of multiple sclerosis patients. Immunoreactivity studies with human myelin basic protein. J Neurol Sci 1991; 103:90-96. Warren KG. Catz I. Synthetic peptide specificity of antimyelin basic protein purified from multiple sclerosis cerebrospinal fluid. J Neuroimmunol 1992;39:81--97. Warren KG, Catz I. Autoantibodies to myelin basic protein within multiple sclerosis central nervous system tissue. J Neurol Sci 1993a;115:169-176. Warren KG, Catz I. Increased synthetic peptide specificity of tissue and CSF-bound anti-MBP in multiple sclerosis. J Neuroimmunol 1993b;43:87-96. Warren KG, Catz I, Johnson E, Mielke B. Antimyelin basic protein and antiproteolipid protein specific forms of multiple sclerosis. Ann Neurol 1994;35:280-289. Wucherpfennig KW, Strominger JL. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 1995;80: 695--705.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

MYOCARDIAL AUTOANTIBODIES Ahvie Herskowitz, M.D. a, David A. Neumann, Ph.D. b and Aftab A. Ansari, Ph.D. c

alschemia Research and Education Foundation, San Francisco, CA 94134; blLSI Risk Science Institute Washington, DC 20036; and CDepartment of Pathology, Winship Cancer Center, Emory University School of Medicine, Atlanta, GA 30322, USA

HISTORICAL NOTES Beginning in the 1950s heart-reactive antibodies were described in patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM) as well as rheumatic carditis and Dressler's syndrome (Limas et al., 1990; Neumann et al., 1990; Schulz et al., 1990; Caforio et al., 1992; Maisch et al., 1979). The possible autoimmune origin of cardiomyopathy associated with Chagas' disease is predicted on the basis of antibodies that cross-react with Trypanosoma cruzi, human cardiac conductive tissue and myocardium (Rose et al., 1992). Myocardial cardiac autoantibodies are present in a variety of cardiac disorders. In rheumatic heart disease, the streptococcal M protein shares epitopes with cardiac myosin, as well as with other (xhelical coiled-coil proteins such as tropomyosin (Fenderson et al., 1989). Early studies used indirect immunofluorescence (IF) to demonstrate circulating muscle-specific antibodies in each of these entities. Two predominant IF staining patterns were 1) antisarcolemmal antibody bound to the myocyte sarcolemmal sheath (Figure 1A) and 2) antifibrillary antibody bound to cytoplasmic contractile proteins, usually myosin (Maisch et al., 1983) (Figure 1B). Experimental studies, particularly in mouse models of postviral myocarditis, show that cardiotropic viral infections or autoimmunization with cardiac antigens can elicit myocardial injury leading to cardiomyopathy, with the common development of heart-specific humoral and cellular autoimmune responses (Lawson et al., 1992; Neumann et al., 1994). The animal models and clinical observations in humans have yet to establish that myocarditis and IDCM are linked and that the former is the inducer of a chronic autoim-

mune-mediated syndrome. Nor is there unequivocal evidence that the presence of autoantibodies is related to the clinical manifestations of the disease.

THE AUTOANTIGENS Definition

In 1987, a putative autoantigen reactive with sera from IDCM patients was identified as the 31--32 kd adenine nucleotide translocator (ANT) protein isolated from the inner mitochondrial membrane (Shultheiss et al., 1987). The ANT, the predominant protein found in human cardiac mitochondria occurs in at least three isoforms (ANT1, ANT2 and ANT3), of which only ANT1 and ANT2 are expressed in cardiac tissue. Cloning and full-length sequencing of the three isoforms of ANT (Neckelmann et al., 1987) allowed for the identification of two peptide sequences of the ANT that react with IDCM sera (Oakley et al., 1993). Sera from IDCM patients also react with a biochemically purified 31--32 kd mitochondrial protein, which is presumably native ANT (Ansari et al., 1994). However, sera from IDCM patients do not react with the recombinant ANT full-length proteins which are present in both cardiac and skeletal muscle (Ansari et al., 1993). In addition, fusion proteins of cloned human cardiac ANT1 cDNA, expressed by the bacterial vector pGEX2T, do not react with sera from IDCM patients nor with rabbit anti-ANT sera (Ansari et al., 1993). The antibody reactivity is most likely against a conformational sequence which is expressed by the native form but not the recombinant form of human ANT. However, this polyvalent rabbit anti-

527

immunized with cardiac myosin develop myocarditis that is indistinguishable from that induced by experimental infection with Coxsackie virus B3 (CVB3) (Rose et al., 1992). Among several other proteins reactive with sera from myocarditis and IDCM patients, the best-characterized include a mitochondrial branched chain ~ketoacid dehydrogenase (BCKD-E2) protein (Ansari et al., 1994), cardiac ~-adrenoreceptor proteins (Limas et al., 1990) and heat shock protein (HSP)-60 (Latif et al., 1993). In acute rheumatic fever, molecular mimicry or epitope similarity between group A Streptococci M proteins, myosin and tropomyosin may contribute to the presence of heart reactive antibodies (Fenderson et al., 1989). Distinct autoantigens have not yet been identified in Dressler's syndrome.

AUTOANTIBODIES Pathogenetic Role

Figure 1. Representative immunofluorescence staining of normal human cardiac tissue incubated with serum from a patient with active myocarditis. a: Cross-section of myocytes with intense membrane fluorescence which is known as the antisarcolemmal antibody (ASA) pattern. b: Longitudinally oriented myocytes highlighting both an ASA pattern and an antifibrillary (AFA) pattern of fluorescence. The AFA pattern is characterized by linear thick bands of fluorescence within the cytoplasm, perpendicular to the long axis of the myocytes.

serum to ANT cross-reacts with Ca ++ channel proteins on the plasma membrane of guinea pig myocytes, resulting in increased Ca ++ uptake followed by myocyte injury (Morad et al., 1988). The cross-reactivity of the rabbit anti-ANT sera with a plasma membrane calcium channel protein is consistent with a role for such antibodies in pathogenesis. Similar demonstrations with monoclonal antibodies would be very useful. In addition to ANT, sera from IDCM patients react with cardiac myosin; the ~ and ~ cardiac myosin heavy chain isoforms might be immunodominant autoantigens in IDCM (Caforio et al., 1992); mice

528

H u m a n Disease. Because most of the proteins identified as cardiac autoantigens are intracellular, it is difficult to envision how antibodies to these intracellular antigens might induce pathology leading to heart disease. That circulating autoantibodies to intracellular antigens might cross-react with native cardiac tissue membrane proteins is supported by the cross-reactivity of antibodies to the ANT protein with myocyte sarcolemmal calcium channel proteins. The failure of the same antisera to react with the recombinant form of the ANT (as discussed above) raises questions regarding the specificity of such antisera. Because most of the proteins identified as cardiac autoantigens are intracellular, it is difficult to envision how antibodies to these intracellular antigens induce pathology and contribute to heart disease. One possibility is that such autoantibodies might cross-react with cardiac tissue membrane proteins, as exemplified by the cross-reactivity of rabbit anti-ANT antibody with myocyte sarcolemmal calcium channel proteins. Another possibility is that myocytes may, in certain circumstances, acquire functional capability to process and present intracellular cardiac tissue proteins in the form of peptides associated with MHC molecules and expressed on the cell surface membrane. Such peptide-MHC complexes initiate T-cell activation. Myocytes initiate autoimmune reactivity in the setting of myocardial inflammation such as that found in viral myocarditis. Immune responses against the viral pep-

tides-MHC complex lead to IFN-y secretion by T cells which, in turn, induces MHC expression by myocytes, initiating the cascade which leads to chronic immune responses and myocyte damage. Once myocyte damage occurs, intracellular myocyte proteins may be processed and presented by resident or infiltrating macrophages/dendritic cells to autologous T cells, also perpetuating autoimmune T-cell responses. These concepts are important to consider since myocytes have been traditionally considered "inert" and not capable of inducing immune responses due to the virtual absence of MHC expression by this cell lineage in normal cardiac muscle. However, several laboratories, including ours, have shown a diffuse expression of MHC antigens by myocytes in endomyocardial biopsies of patients with myocarditis and in focal areas in patients with IDCM (Herskowitz et al., 1990; Ansari et al., 1991) supporting the above views. In addition, intracellular antigens such as ANT and BCKD may be aberrantly expressed on the surface of myocytes (Ansari et al., 1991). Animal Models. The paradigm relating postviral myocarditis to dilated cardiomyopathy is exemplified by murine models of CVB3 and cytomegalovirus (CMV) myocarditis in which an acute viremic stage is associated with inflammation and focal necrosis of myocytes. In the second phase, infectious virus can no longer be isolated by culture, but in situ hybridization and polymerase chain reaction studies demonstrate the presence of residual virus mRNA in the myocardium (Klingel et al., 1992). Heart-specific autoantibodies are seen only in strains of mice that develop the second phase of disease which is associated with pathologic changes in dilated cardiomyopathy (Rose et al., 1992). Susceptibility to virus-induced murine myocarditis is determined by non-H-2-background gene; whereas, H-2-encoded gene products control the severity of the autoimmune response. Even with the well-characterized CVB3 murine model, numerous mechanisms can be identified, reflecting diverse genetic predisposition to early phase myocarditis. In CVB3-infected BALB/C mice, cytotoxic T-cells (CD8 +) are primarily responsible for a cell-mediated cardiac injury. In other strains of mice (DBA/2), humoral immunity plays a more dominant role with production of heart-specific autoantibodies and a pathogenic population of antigen-specific CD4 + helper T cells. CVB3-infected mice with ongoing myocarditis frequently produce circulating heartreactive IgG autoantibodies specific for the cardiac

myosin heavy chain (Rose et al., 1992). Similarly, infected mice also develop antibodies to ANT and BCKD (Neumann et al., 1994), and cardiac-bound IgG eluted from the myocardium demonstrates reactivity with myosin, ANT and BCKD (Neumann et al., 1991). A number of serologic markers can be used to identify individuals with immune-associated heart disease; however, the relationships among the antibodies, the induction of autoantibodies and the clinical features of myocardial disease in both humans and mice are unresolved.

Factors Involved in Pathogenicity and Etiology Molecular Mimicry. Although antigenic cross-reactivity between myosin and Coxsackie virus in CVB3induced myocarditis is not known, sequence similarity between CVB4 and cardiac myosin was suggested (Beisel et al., 1991). A role for molecular mimicry in valvular injury in rheumatic heart disease is supported by recent observations that human heart valves have numerous antigenic sites that are recognized by both antistreptococcal antibodies and acute rheumatic fever sera (Gulzia et al., 1991). Similarly, in Chagasic myocarditis and cardiomyopathy, antisera directed against Trypanosoma cruzi cross-react with sarcolemmal proteins, which may have a direct pathogenic role in cardiac contractile dysfunction (Acosta et al., 1983; Sadigursky et al., 1989). Cellular Autoimmunity. Studies with an SV40 large T-antigen gene-transformed human cardiac myocyte cell line suggest that cardiac myocytes cannot process and present antigens via the class II MHC pathway and have limited ability to process and present via the class I MHC pathway (Ansari et al., 1993). Whether the autoimmune response is due to processing and presentation by other lineages of cells (e.g., resident dendritic cells), infiltrating macrophages or perhaps endothelial cells is unknown. Cytokines can alter myocyte contractility (Finkel et al., 1992), and treatment with IL-1 and TNF-o~ exacerbates CVB3-induced myocarditis and increases formation of autoantibodies in mice ordinarily resistant to CVB3-induced myocarditis (Lane et al., 1993).

Genetic Studies In a meta-analysis involving 361 patients with IDCM, an increased frequency of HLA-DR4 was found (odds 529

ratio 2.1, 95% confidence interval 1.61-2.65, p < 0.0001) (Carlquist et al., 1991). Patients expressing the DR4-DQw4 halotype also bear an indeterminately high risk of disease (relative risk 6.1 compared with controls, p < 0.005). Genetic predisposition influences cardiac autoantibody production: 72% of HLA-DR4positive patients had anti-B receptor antibodies; whereas, 21% of HLA-DR4-negative patients had antibodies of the same specificity (p < 0.001) (Limas et al., 1990).

Methods of Detection Screening tests typically utilize indirect immunofluorescence antibody (IFA) techniques to detect serum autoantibodies to cardiac tissue (Table 1). These assays are now widely available. Because results from such studies can be confounded by unresolved questions of specificity, IFA techniques which utilize fresh-frozen human cardiac tissue should be used whenever possible. The presence of cross-reactive antibodies to skeletal muscle should be determined by also using fresh-frozen human skeletal muscle tissue, although the clinical implications of distinguishing

between cardiac-specific v s . skeletal cross-reactive antibodies are not yet fully known (Caforia et al., 1992). In addition, although the negative predictive value of a negative result by IFA is high (93%), it does not rule out the presence of cardiac-specific autoantibodies that may require more quantitative and protein-specific assays for detection (Table 1). Immunoblotting separates the soluble components of heart tissue based on their molecular mass and permits the identification of antibodies to individual components (Neumann et al., 1990; Caforio et al., 1992). Immunoblot assays, however, are only available in large academic centers. Other immunoassays (e.g., ELISA) employ well-defined constituents of cardiac tissue that permit quantitative measurement of serum autoantibodies to ANT, BCKD, myosin and other cardiac proteins; these are not yet clinically available. (Caforio et al., 1992; Ansari et al., 1994) (Table 2). Once available, these quantitative assays can be fully evaluated for the diagnosis of autoimmune heart disease, the monitoring of response to immunosuppressive therapy and possibly, the prediction of long-term prognosis.

Table 1. Human Heart-Reactive Antibody Titers in the Sera of IDCM Patients and Patients with Other Forms of Cardiomyopathies* as Determined by an Indirect Fluorescent Antibody (IFA) Technique* No. of Sera Positive/Total No. Tested Source of Sera

No. of Sera Tested

IDCM

46

Myocarditis

12

<1:10

1:40

1:80

>1:160

2/46

6/46

10/46

28/46 12/12

Postpartum cardiomyopathy

4

Adriamycin cardiomyopathy

3

Hypertrophic cardiomyopathy

12

9/12

3/12

Alcohol cardiomyopathy

24

10/12

2/12

Coronary artery disease

32

26/32

4/32

Normal

24

24/24

--

--

1/4

3/4

--

3/3

2/32

Sera were obtained from patients with idiopathic dilated cardiomyopathy (IDCM), patients following acute myocarditis (presumably viral), pediatric cases, patients with other forms of cardiomyopathies and normal adult healthy laboratory volunteers. Patients with coronary artery disease include those with myocardial infarction (N = 14) and without myocardial infarction (N = 18). The IFA technique utilized serial sections of fresh-frozen human cardiac tissue (obtained at autopsy from a patient who died of other than cardiac disease). Tissue sections were incubated with 0.1 mL of normal rabbit serum for 30 min to block nonspecific binding. After decanting, two-fold dilutions of the sera to be tested were added, and following incubation for 30 min at 4 ~ C, the sections were washed with PBS pH 7.4. This was followed by the addition of 0.1 mL of a fluoresceinconjugated F(ab') 2 fraction of rabbit anti-human IgG diluted 1/100. The sections were washed and then immediately scored for reactivity.

530

Table 2. Autoantibodies to Mitochondrial and Structural Proteins as Determined by ELISA Frequency (% positive)* DCM* (N = 57)

Alcoholic CM (N = 28)

Sera from CAD (N = 41)

RA (N = 48)

SLE (N = 44)

Mitochondrial Proteins ANT

71

4

8

0

0

BCKD

75

7

5

0

0

Myosin

93

73

82

65

41

Actin

68

77

78

71

81

Alpha-actinin

24

35

43

24

29

Tropomyosin

49

27

27

28

37

Vinculin

21

10

9

11

10

Desmin

37

18

27

34

33

Keratin

84

87

92

92

94

Collagen type I

72

82

92

88

93

Collagen type II

75

73

89

81

88

Collagen type III

72

83

93

91

87

Laminin

53

39

49

43

50

Structural Proteins t

* t

Sera diluted 1/40 were considered positive if reactivity was greater than 2 S.D. above background control. Structural proteins were purchased commercially, and positive controls consisted of monoclonal and or heterologous commercially available antisera. DCM = dilated cardiomyopathy, alcoholic CM = alcoholic cardiomyopathy, CAD = coronary artery disease with myocardial infarction (N=26) and without myocardial infarction (N=15), RA = rheumatoid arthritis, and SLE = systemic lupus erythematosus.

CLINICAL UTILITY

Application Identification of heart-specific autoantibodies, along with results from endomyocardial biopsy, can assist clinicians in determining whether patients are likely to have an autoimmune component to their heart disease. The presence of human heart-reactive serum antibody titers of > 1:40 by the IFA method is 96% sensitive and 81% specific for the diagnosis of IDCM (88% positive predictive value and 93% negative predictive value: IDCM v s . coronary artery disease (Table 1). By ELISA, the presence of either ANT or BCKD antibody titers of > 1:40 is 72--75% sensitive and 93--95% specific for the diagnosis of IDCM (93--96% positive predictive value and 7 0 - 7 4 % negative predictive value: IDCM v s . coronary artery disease (Table 2).

Although the clinical significance of circulating cardiac autoantibodies, when identified, is as yet unresolved, several general concepts have emerged: 1) the presence of cardiac-specific autoantibodies in patients with active myocarditis might portend a better short-term prognosis (Mason et al., 1995); and 2) although published studies examining the effect of immunosuppressive or immunomodulatory therapy on circulating cardiac antibodies are not available, quantitative assays do reveal significant reductions in titers to cardiac-specific proteins and associated clinical improvement after immunosuppressive therapy.

Effect of Therapy On the other hand, the largest prospective study to date showed no clinical benefit from immunosuppressive treatment of patients with active myocarditis

531

(with or without autoantibodies) (Mason et al., 1995). Clinical benefit from corticosteroid therapy was reported in a subgroup of IDCM patients with evidence of "immune reactivity" as defined by those who had fibroblastic or lymphocytic infiltration or immunoglobulin deposition on endomyocardial biopsy, a positive gallium scan or an elevated erythrocyte sedimentation rate (Parillo et al., 1989). Ongoing, prospective studies are examining the role of immunosuppressive therapy in IDCM patients with serologic evidence of cardiac autoimmunity. Over long periods of follow-up, reduced serum antibody titers may be associated with disease progression to end-stage dilated cardiomyopathy.

REFERENCES Acosta AM, Sadigursky M, Santos-Buch CA. Antistriated muscle antibody activity produced by Trypanasoma cruzi. Proc Soc Exp Biol Med 1983;172:364--369. Ansari AA, Wang YC, Danner DJ, Gravanis MB, Mayne A, Neckelmann N, Sell KW, Herskowitz A. Abnormal expression of histocompatibility and mitochondrial antigens by cardiac tissue from patients with myocarditis and dilated cardiomyopathy. Am J Pathol 1991;139:337--354. Ansari AA, Neckelmann N, Wang YC, Gravanis MB, Sell KW, Herskowitz, A. Immunologic dialogue between cardiac myocytes, endothelial cells, and mononuclear cells. Clin Immunol Immunopathol 1993;68:208-214. Ansari AA, Neckelmann N, Villinger F, Leung P, Danner DJ, Brar SS, Zhao S, Gravanis MB, Mayne A, Gerschwin ME, Herskowitz, A. Epitope mapping of the branched chain alpha-ketoacid dehydrogenase-E2 (BCKD-E2) protein that reacts with sera from patients with idiopathic dilated cardiomyopathy. J Immunol 1994;153:4754-4765. Beisel KW, Srinivasappa J, Prabhakar BS. Molecular cloning of a heart antigen that cross-reacts with a neutralizing antibody to coxsackie virus B-4. Eur Heart J 1991;12(Suppl D):60--64. Caforio ALP, Grazzini M, Mann JM, Keeling PJ, Bottazzo GF, McKenna WJ, Schiaffino S. Identification of alpha and betacardiac myosin heavy chain isoforms as major autoantigens in dilated cardiomyopathy. Circulation 1992;85:1734-1742. Carlquist JF, Menlove RL, Murray MB, O'Connell JB, Anderson JL. HLA class II (DR and DQ) antigen associations in idiopathic dilated cardiomyopathy. Validation study and meta-analysis of published HLA association studies. Circulation 1991;83:515-522. Fenderson PG, Fischetti VA, Cunningham MW. Tropomyosin shares immunologic epitopes with group A streptococcal M protein. J Immunol 1989;142:2475--2481. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, 532

CONCLUSION From a practical perspective, the presence of circulating autoantibodies will likely remain the simplest method of identifying patients with myocarditis, IDCM, rheumatic fever and Dressier' s syndrome, who have a possible immune disorder. It is critical, though, to develop sensitive quantitative assays that focus on autoantibodies that are not only cardiac-specific, but disease-specific. Such ideal assays would not only be well suited for the identification of subgroups of cardiac patients of putative autoimmune etiology, but would also allow cardiac-specific antibody titers to be followed as markers of disease progression as well as response to new immunotherapies. See also AUTOANTIBODIES THAT PENETRATE INTO LIVING CELLS and BETA ADRENERGIC RECEPTOR (AND OTHER HORMONE RECEPTOR) AUTOANTIBODIES.

Simmons RL. Negative inotropic effects of cytokines on the heart medicated by nitric oxide. Science 1992;257:387-389. Gulizia JM, Cunningham MW, McManus BM. Immunoreactivity of antistreptococcal monoclonal antibodies to human heart valves. Evidence for multiple cross-reactive epitopes. Am J Pathol 1991;138:285-301. Herskowitz A, Ansari AA, Neumann DA, Beschorner WE, Rose NR, Soule LM, Burek CL, Sell KW, Baughman KL. Induction of major histocompatibility complex (MHC) antigens within the myocardium of patients with active myocarditis: A nonhistologic marker of myocarditis. J Am Coil Card 1990;15:624--632. Klingel K, Hohenadl C, Canu A, Albrecht M, Seemann M, Mall G, Kandolf R. Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: Quantitative analysis of virus replication, tissue damage and inflammation. PNAS USA 1992;89:314--318. Lane JR, Neumann DA, Lafond-Walker A, Herskowitz A, Rose NR. Role of IL-1 and tumor necrosis factor in coxsackie virus-induced autoimmune myocarditis. J Immol 1993;151: 1682--1690. Latif N, Baker CS, Dunn MJ, Rose ML, Brady P, Yacoub MH. Frequency and specificity of antiheart antibodies in patients with dilated cardiomyopathy detected using SDS-PAGE and immunoblotting. J Am Coll Cardiol 1993;22:1378--1384. Lawson CM, O'Donoghue HL, Reed WD. Mouse cytomegalovirus infection induces antibodies which cross-react with virus and cardiac myosin: a model for the study of molecular mimicry in the pathogenesis of viral myocarditis. Immunology 1992:75:513-519. Limas CJ, Limas C, Kubo SH, Olivari MT. Anti-beta-receptor antibodies in human dilated cardiomyopathy and correlation with HLA-DR antigens. Am J Cardiol 1990;15:65:483-487. Maisch B, Berg PA, Kochsiek K. Clinical significance of immunopathologic findings in patients with postpericardiotomy syndrome. I. Relevance of antibody pattern. Clin

Exp Immunol 1979;38:189--197. Maisch B, Deeg P, Liebau G, Kochsiek K. Diagnostic relevance of humoral and cytotoxic immune reactions in primary and secondary dilated cardiomyopathy. Am J Cardiol 1983;52: 1072--1078. Mason JW, O'Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME, Moon TE, Myocarditis Treatment Trial Investigators. A randomized study of the efficacy of immunosuppressive therapy in patients with myocarditis. N Engl J Med 1995;333:269--275. Morad M, Davies NW, Ulrich G, Schultheiss HP. Antibodies against AD8-ATP carrier enhance CA ++ current in isolated cardiac myocytes. Am J Physiol 1988;255(4 pt. 2):H960H964. Neckelmann N, Li K, Wade RP, Shuster R, Wallace DC. cDNA sequence of a human skeletal muscle ADP/ATP translocator: lack of a leader peptide divergence from a fibroblast translocator cDNA, and coevolution with mitochondrial DNA genes. Proc Natl Acad Sci USA 1987;84:7580-7589. Neumann DA, Burek CL, Baughman KL, Rose NR, Herskowitz A. Circulating heart-reactive antibodies in patients with myocarditis or cardiomyopathy. J Am Coil Card 1990;16: 839--846. Neumann DA, Lane JR, Lafond-Walker A, Allen GS, Wulff SM, Herskowitz A, Rose NR. Heart-specific autoantibodies can be eluted from the hearts of Coxsackie virus B3-infected mice. Clin Exp Immunol 1991;86:405--412. Neumann DA, Rose NR, Ansari AA, Herskowitz A. Induction

of multiple heart autoantibodies in mice with coxsackie virus B3- and cardiac myosin-induced autoimmune myocarditis. J Immunol 1994;152:343--350. Oakley CM, Gravanis MB, Ansari AA. The Cardiomyopathies. In: Gravanis MB ed. Cardiovascular Disorders. Pathogenesis and Pathophysiology. St. Louis: Mosby-Year Book, Inc. 1993:210--253. Parrillo JE, Cunnion RE, Epstein SE, Parker MM, Suffredini AF, Brenner M, Schaer GL, Palmeri ST, Cannon RO 3rd, Ailing D, Wittes J, Rerrane VJ, Rodrigues ER, Sauci AJ. A prospective, randomized, controlled trial of prednisone for dilated cardiomyopathy. N Engl J Med 1989;321:1061-- 1068. Rose NR, Neumann DA, Burek CL, Herskowitz A. Autoimmune Heart Disease. In: Rose NR and Mackay RI, eds. The Autoimmune Diseases II. San Diego:Academic Press, 1992:303--314. Sadigursky M, von Kreuter BF, Ling PY, Santos-Buch CA. Association of elevated antisarcolemma, anti-idiotype antibody levels with the clinical and pathologic expression of chronic Chagas myocarditis. Circulation 1989;80:1269-- 1276. Schultheiss HP, Schulze, D, Kulh U, Ulrich G, Klingenberg M. The ADP/ATP carrier as a mitochondrial autoantigen- Facts and perspectives. Ann N Y Acad Sci 1987;488:44-64. Schulze K, Becker BF, Schauer R, Schultheiss HP. Antibodies to ADP-ATP c a r r i e r - an autoantigen in myocarditis and dilated cardiomyopathy- impair cardiac function. Circulation 1990;81:959-969.

533

9 1996 Elsevier Science B.V. All fights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

NATURAL AUTOANTIBODIES Jacob George, M.D. and Yehuda Shoenfeld, M.D.

Department of Medicine "B", Research Unit of Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel

HISTORICAL NOTES First introduced in 1965, the term Natural Autoantibodies (NAA) attributes the production of these distinct autoantibodies to pre-existing subclinical infections possibly acting as "antigenic burdens" to which the immune system gradually responds (Boyden, 1965). NAA are now generally considered to be self-reacting serum antibodies (Table 1), which do not require antigenic stimuli for their production (Shoenfeld and Isenberg, 1992). Theories about the significance of NAA to the functions of the immune system are dominated by two opposing notions. The first assumes that NAAs are side products of an immune system actively and continuously engaged in the processing of numerous amounts of antibodies, thereby regarding them as mere epiphenomena. The second theory suggests, from the teleological point of view, that the human body in general and specifically the immune system, would not "allow" for the production of molecules not deemed necessary to the normal functions of the organism, and therefore justified in evolutionary terms. NAA exist in healthy animals and individuals

(Fritzler et al., 1985; Pandey et al., 1979; Ericsson et al., 1979; Goodwin et al., 1982; Hawkins et al., 1980) (Table 2), in first degree healthy relatives of patients with autoimmune diseases (Isenberg et al., 1985) and among elderly persons (Walford, 1969; Tomer and Shoenfeld, 1988). Some of these antibodies (e.g., thyroid microsome antibodies) can be predictive of development of disease in a subpopulation of healthy individuals (Hawkins et al., 1980). Additionally, patients with monoclonal gammopathies (multiple myeloma, Waldenstr6m's macroglobulinemia and benign monoclonal gammopathy) can harbor significant activity against self-antigen determinants. Antigenic components against which NAA can be detected include: Ro and La (Buskila et al., 1992; Sestak et al., 1987), DNA (Shoenfeld et al., 1986), histones (Shoenfeld et al., 1987), Sm and RNP (AbuShakra et al., 1989), mitochondria (Guilburt et al., 1995) and other cellular constituents (Watts et al., 1989; Davidson et al., 1987; Eng et al., 1987). As is the situation with healthy individuals, NAA in patients with monoclonal gammopathies are generally not associated with corresponding clinical manifestations (Table 3).

Table 1. Support for the Existence of NAA Induction of autoimmunity by injection of organ extracts The diversity of autoantibodies in various populations Autoantibody activity of monoclonal gammopathy Secretion of autoantibodies by hybridomas derived from normal subjects Autoantibody production resulting from in vivo stimulation of peripheral blood lymphocytes of normal individuals Autoantibody production due to in vivo injection of lipopolysaccharides

534

Table 2. The Frequency of NAA in the Sera of Healthy Individuals Autoantibody

No. of Tested Sera

Anti-Ro

2500

Positivity (%)

Reference

0.5

Fritzler et al., 1985

5

Yadin et al., 1989"

Anti-Ro and anti-La

506

Anti-mitochondrial

2500

2.5

Fritzler et al., 1985

ANA

2500

4

Fritzler et al., 1985

ANA

1284

13

Pandey et al., 1979

2-27

Ericcson et al., 1979

98

4

Goodwin et al., 1982

93

3

Goodwin et al., 1982

6.7

Hawkins et al., 1980

Antithyroglobulin

140

Rheumatoid factor Lymphocytotoxic antibody Antimicrosomal

2838

Anti-ssDNA

506

4

Yadin et al., 1989

Anti-dsDNA

506

6

Yadin et al., 1989

Anticardiolipin (aCL)

506

4

Yadin et al., 1989

Anti-Sm

506

5.5

Yadin et al., 1989

Anti-RNP

506

5.5

Yadin et al., 1989

* Only healthy women screened.

Table 3. Reactivity of Sera of Patients with Monoclonal Gammopathies against Autoantigens Autoantigen

No. of Sera

Actin

612

Tubulin

612

Anti-DNA Id (16/6 Id)

265

Histone

249

ssDNA

265

6.4

Shoenfeld, 1987

dsDNA

265

6

Shoenfeld, 1987

5.3

Watts et al., 1989 Davidson et al., 1987

Rheumatoid factor

75

Positivity (%) 5.2 <1 8.7 14

Reference Dighiero et al., 1983 Dighiero et al., 1983 Shoenfeld, 1986 Shoenfeld, 1987

DNA

697

4

RNP

141

23

Abu-Shakra et al., 1989

Sm

141

16

Abu-Shakra et al., 1989

Ro

143

3.5

Sestek et al., 1987

La

143

0

Sestek et al., 1987

Cold-agglutinin (I-Ag)

308

14

Crisp et al., 1982

Smooth-muscle

75

2.6

Watts et al., 1989

Acetylcholine receptor

149

9

Eng et al., 1987

Mitochondria

100

6

Guilbert et al., 1995

535

PROPERTIES NAA are classified as distinct autoantibodies by virtue of several unique properties: 1. NAAs are principally IgM, but a minority are IgG and IgA (Guilbert et al., 1982; Rodman and Pruslin, 1990). 2. NAA exhibit reactivity towards self and nonself (bacterial components serving as potential antigens) (Avrameas et al., 1988). 3. NAA concomitantly express polyreactivity and fine specificities (Ternynck et al., 1986). Indeed, the majority of NAA are capable of reacting with three or more self or nonself antigens. A small group of NAA is considered monoreactive with specificity for a given antigen. 4. NAA can be of low affinity (monovalent binding of NAA to the epitope) and high avidity (multivalent binding to a molecule carrying similar epitopes). This property might shed light on the teleological significance with regard to their possible role in preservation of body hemostasis. 5. NAA are produced by CD5-positive B cells (Raveche, 1990) which constitute 10--25% of the circulating and splenic B-cell repertoire (Casali et al., 1987).

POTENTIAL SIGNIFICANCE Solid evidence supports a role for NAA in biologic regulation (Table 4). This notion, as initially introduced in 1983, viewed NAA as vectors by which harmful self or foreign substances are disposed (Grabar, 1983). The clearing process was postulated to evolve through the direct binding of NAA to the substance destined for disposal, followed by its

opsonization and phagocytosis. Several subsequent studies lend additional support to this concept. For example, NAA reactive with (zgalactosyl residues of human red blood cells are important among healthy subjects in the phagocytosis of defective or senescent red blood cells. The presence of circulating immune complexes consisting of autologous IgG and erythrocyte proteins suggests a possible role for NAA in the clearance of cellular debris (Heegaard, 1990). Natural antiband-3 protein antibodies are important in the elimination of senescent erythrocytes from the circulation (Lutz et al., 1987). During the process of aging, erythrocytes expose on their surfaces higher levels of band-3 protein; once a certain local concentration of band 3 is reached by antiband-3 protein IgG NAA bind with resultant phagocytosis of the complex by monocytes (Lutz et al., 1987). Natural IgG antikeratin antibodies induce the elimination of keratin after keratinocyte death, which results in local accumulation of high concentrations of keratin (Hinter et al., 1987). The subsequent binding of IgG facilitates phagocytosis of the resultant immune complexes. The concept of "physiological NAA" now includes a role in the induction of self-tolerance in which low affinity, nonspecific binding of NAA to self-antigens blocks a vigorous binding with reactive clones (Cohen and Cooke, 1986). This putative "buffer effect" of NAA might prevent the development of autoimmune states following antigenic stimuli, including those with a component of molecular mimicry. The above-described mechanisms are probably more complex, because NAA also display extensive interactions among themselves, possibly via idiotypic determinants. The delicately tuned interaction of the idiotype and anti-idiotype network serves either to

Table 4. Functions of NAA Keratin elimination by antikeratin antibodies Elimination of senescent erythrocytes by IgG natural antiband 3 protein Increased resistance to tumors by IgM NAA Inhibition of interferon by binding to cell surfaces Inhibition of NK cell activity Proteolytic properties of some NAA for vasoactive intestinal peptide Protection against viral infections by natural antitrinitrophenyl antibodies Increased titers of NAA during pathological states (autoimmune states) and following infections

536

upregulate or downregulate the immune response. Indeed, injection of anti-idiotypic NAA into mice causes diminished concentrations of the corresponding idiotype (Vakil and Kearney, 1986). Further evidence for idiotypic connectivities includes the fact that natural IgM autoantibodies present in the sera of normal mice inhibit the binding of IgG autoantibodies to self-antigens by interacting with natural IgG autoantibodies through the F(ab')2 domain (Adib et al., 1990). Autoreactive B cells p e r s e are also engaged in immunoregulatory functions as manifest by the fact that CD5-positive clones (not found to produce autoantibodies) from patients with chronic lymphocytic leukemia suppress the synthesis of immunoglobulins by B cells (Paglieroni et al., 1988). NAA are often present in high titers in patients with various viral, bacterial and parasitic infections (Abu-Shakra and Shoenfeld, 1991; Michel et al., 1990). Whether these NAA exert a protective or a housekeeping effect in infections is as yet unknown (Navin et al., 1989; Gonzalez et al., 1989; Michel et al. 1990). The predominant occurrence of CD-5-positive Bcell clones and IgM polyreactive antibodies with low affinity for viral agents and self-antigens might signify a role for NAA in the first phase of the immune response (Casali and Notkins, 1989). The second stage, namely, the antigen-specific immune response, consists of CD5-negative B cells, which produce monospecific IgG.

Pathogenicity The possible pathogenic properties of NAA are best exemplified by animal models, which demonstrate that end-organ damage can result from immunization with NAA. For example; the 16/6 idiotype (an anti-DNA idiotype produced from a patient with cold agglutinin disease) was used to immunize mice with the resultant appearance of serologic markers and clinical manifes-

REFERENCES Abu-Shakrah M, Krupp M, Argov S Buskila D, Slor H, Shoenfeld Y. The detection of anti-Sm/RNP activity in the sera of patients with monoclonal gammopathies. Clin Exp Immunol 1989;75:349--353. Abu-Shakrah M, Shoenfeld Y. Parasitic infection and autoimmunity. Autoimmunity 1991;9:337--344.

tations of SLE (elevated erythrocyte sedimentation rate, leukopenia, thrombocytopenia, proteinuria and alopecia) (Mendlovic et al., 1988; Blank et al., 1990). These data add to previous reports of detection of pathogenic NAA within areas of autoimmune damage. In the second example of end-organ damage induced by immunization with NAA, a monoclonal NAA with anticardiolipin antibody activity (termed H3 in human monoclonal aCL) led to the elaboration of high titers of aCL when injected to BALB/c mice (Bakimer et al., 1992). Subsequently, a clinical picture consistent with the antiphospholipid syndrome developed in the mice (prolonged activated partial thromboplastin time, lower fecundity rate, higher fetal resorption rate and a decline in the average weight of the embryos) (Cohen et al., 1994).

CONCLUSION NAA are indeed a distinct group of autoantibodies characterized by unique properties. Contradictory reports regarding their existence and participation in either the normal homeostasis or pathogenic states does not as yet permit firm conclusions as to their precise roles. NAA play an active role in the preservation and perpetuation of the normal balanced immune response in several ways by virtue of their unique biochemical and physical properties. The relatively recent evidence attributing pathogenic effects to NAA adds further interest to their yet unsettled roles and further complicates the picture. Studies are needed to delineate properties of NAA and to evaluate further what may now seem to be a "double-edged sword." Sufficient insights should lead to better understanding of the global functions of the immune system and possibly to new treatment modalities. See also ALPHA-GALACTOSYL (ANTI-GAL) AUTOANTIBODIES, IDIOTYPES AND ANTI-IDIOTYPIC ANTIBODIES and MOLECULAR MIMICRY.

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537

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HG, Patel Y, Mackay IR, Welborn TA. Diagnostic significance of thyroid microsomal antibodies in randomly selected population. Lancet 1980;2:1057-- 1059. Heegaard NH. Immunochemical characterization of interactions between circulating autologous immunoglobulin G and normal human erythrocyte membrane proteins. Biochim Biophys Acta 1990;1023:239--246. Hintner H, Romani N, Stanzl U, Grubauer G, Fritsch P, Lawley TJ. Phagocytosis of keratin filament aggregates following opsonization with IgG-antikeratin filament autoantibodies. J Invest Dermatol 1987;88:176--182. Isenberg DA, Shoenfeld Y, Walport M, Mackworth Y, Dudeney C, Todd-Pokropek A, Brill S, Weinberger A, Pinkas J. Detection of cross-reactive anti-DNA antibody idiotypes in the serum of systemic lupus erythematosus patients and of their relatives. Arthritis Rheum 1985;28:999--1007. Lutz HU, Bussolino F, Flepp R, Fasler S, Stammler P, Kazatchkine MD, Arese P. Naturally occurring antiband-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes. Proc Natl Acad Sci USA 1987 ;84:7368--7372. Mendlovic S, Brocke S, Shoenfeld Y, Ben-Bassat M, Meshorer A, Bakimer R, Mozes E. Induction of systemic lupus erythematosus-like disease in mice by a common human antiDNA idiotype. Proc Natl Acad Sci USA 1988;85:2260--2264. Michel C, Gonzales R, Bonjour E, Avrameas S. A concurrent increasing of natural antibodies and enhancement of resistance to furunculosis in rainbow trout. Ann Rech Vet 1990;21:211--218. Navin TR, Krug EC, Pearson RD. Effect of immunoglobulin M from normal human serum on Leishmania donovani promastigote, complement-mediated killing, and phagocytosis by human monocytes. Infect Immunol 1989;57:1343-1346. Paglieroni T, Caggiano V, Mackenzie M. CD5 positive immunoregulatory B cell subsets. Am J Hematol 1988;28:276--278. Pandey JP, Fudenberg HH, Ainsworth SK, Loadholt CB. Autoantibodies in healthy subjects of different age groups. Mech Ageing Dev 1979; 10:394--404. Raveche ES. Possible immunoregulatory role for CD5 + B cells. Clin Immunol Immunopathol 1990;56:135--150. Rodman TC, Pruslin FH. Identification of low-affinity subset of protamine-reactive IgM antibodies present in normal, deficient in AIDS, sera: implication for HIV latency. Clin Immunol Immunopathol 1990;57:430--440. Sestak AL, Harley JB, Yoshida S, Reichlin M. Lupus/Sj6gren' s autoantibody specificities in sera with paraproteins. J Clin Invest 1987;80:138-144. Shoenfeld Y, Ben-Yehuda O, Napartstek Y, Wilner Y, Frolichman R, Schattner A, Lavie G, Joshua H, Pinkhas J, Kennedy RC, et al. The detection of a common idiotype of anti-DNA antibodies in the sera of patients with monoclonal gammopathies. J Clin Immunol 1986;6:194--204. Shoenfeld Y, el-Roiey A, Ben-Yehuda O, Pick AI. Detection of antihistone activity in sera of patients with monoclonal gammopathies. Clin Immunol Immunopathol 1987;42:250-258. Shoenfeld Y, Isenberg DA, eds. Natural autoantibodies. Boca Raton: CRC Press, Inc., 1992:550.

Ternynck T, Avrameas S. Murine natural monoclonal autoantibodies: a study of their polyspecificities and their affinities. Immunol Rev 1986;94:99-112. Tomer Y, Shoenfeld Y. Aging and autoantibodies. Autoimmunity 1988;1:141-149. Vakil M, Kearney JF. Functional characterization of monoclonal auto-anti-idiotype antibodies isolated from the early B cell repertoire of BALB/c mice. Eur J Immunol 1986;16: 1151--1158.

Walford RL. The immunologic theory of aging. Copenhagen: Munskgaard, 1969:248. Watts RA, Williams W, Le-Page S, Norden A, Soltys A, Swana G, Addison I, Hay FC, Isenberg DA. Analysis of autoantibody reactivity and common idiotype PR4 expression of myeloma proteins. J Autoimmun 1989;2:689-700. Yadin O, Sarov B, Naggan L, Slor H, Shoenfeld Y. Natural autoantibodies in the serum of healthy w o m a n - a five year follow-up. Clin Exp Immunol 1989;75:402--406.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

NEPHRITIC FACTOR AUTOANTIBODIES Roger E. Spitzer, M.D. a, Ann E. Stitzel, M.S. a and George C. Tsokos, M.D. b

aDepartment of Pediatrics, SUNY Health Science Center at Syracuse, Syracuse, NY 13210; and bDepartment of Clinical Physiology, Bldg. 40, Room 3078, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA

HISTORICAL NOTES

THE AUTOANTIGEN

The first "nephritic factor" was described in 1969 (Spitzer et al, 1969) in the serum of patients with membranoproliferative glomerulonephritis (MPGN). When mixed with normal serum, this factor mediated C3 consumption and was, therefore, given the trivial name of C3 Nephritic Factor or C3NeF. Originally, C3NeF was only detected in the serum of patients with MPGN but subsequently was noted in patients with partial lipodystrophy (Peters et al., 1973) and in an occasional patient with acute poststreptococcal glomerulonephritis (Spitzer et al., 1992a). C3NeF was thought to be responsible for the persistent hypocomplementemia seen in MPGN and to interact somehow with the alternative pathway of complement. Later, C3NeF was shown to be an immunoglobulin directed against a neoantigen exposed on the Bb portion of the alternative pathway C3/C5 convertase, C3bBb (Daha et al., 1976). Although originally found in a patient with type 2 MPGN, C3NeF is now recognized in all three types of MPGN. C3NeF stabilizes C3bBb and prolongs its half-life by preventing both intrinsic and extrinsic decay dissociation of C3bBb with resultant continued activation of C3-C9 (Daha et al., 1976). Among other nephritic factors with slightly different mechanisms of action, one which apparently works on C3 is properdin-dependent (Spitzer and Stitzel, 1988), and another which acts on C4 is found primarily in patients with systemic lupus erythematosus (Hiramatsu and Tsokos, 1988). Still other nephritic factors are properdin-dependent or properdinindependent (Tanuma et al., 1990) as well as fast or slow-acting depending on the assay temperature and length of time of incubation (Jackson et al., 1987).

Origin/Sources

540

C3NeF appears to react with a neoantigen exposed on the Bb portion of the C3 convertase, (C3bBb) and does not react with native Factor B or with the Bb portion of C3bBb after dissociation from the complex (Bbi) (Daha et al., 1976). The epitope(s), then, appear to be conformational and exposed on Factor B by virtue of its binding to C3b (Daha and Van Es, 1981). The antigen has never been isolated and sequenced. Toward that end, several recent advances document the existence of the epitope(s) on the Factor B portion of the C3/C5 alternative pathway convertase. Limited proteolysis of Factor B with trypsin or pepsin yields a 33 kd fragment which binds to C3b (Lambris and Muller-Eberhard, 1984). Immunoblotting with labeled C3NeF shows binding of C3NeF to the 33 kd fragment in the absence of C3b. In addition, some antiidiotypic antibodies to C3NeF, isolated from patients with MPGN, bear an internal image of the antigenic peptide. Immunization of rabbits or mice with these anti-idiotypes yields circulating C3NeF 2--4 weeks after the third immunization (Spitzer et al., 1990a). Further, combinatorial libraries made from lymphocytes obtained from patients with MPGN yield an IgG molecule which binds monoclonal C3NeF and which also binds goat antibody raised against the Bb portion of Factor B. These reactivities suggest a sequence similarity present in the anti-id antibody which resembles both the antigen for C3NeF as well as a structure in Factor B. Immunization of mice with this IgG yields C3NeF as well as several antibodies to human IgG (Spitzer and Stitzel, 1995).

Although C3NeF production and the onset of MPGN can be associated with meningococcal (Hulton et al., 1992) and hepatitis B infections (Strife et al., 1974), there is no reactivity between C3NeF and cell wall structures related to each of these organisms (Spitzer et al., 1990b). In addition, C3NeF does not react with antibodies to meningococci or a variety of other antigens. While the exact structure for the C3NeF antigenic peptide is not known, it is probably located in the first 259 amino acids of the Bb portion of Factor B. Use of overlapping peptides or site-directed mutagenesis will probably provide a definite structure for the epitope(s).

AUTOANTIBODIES Terminology Among several designations for nephritic factor, such as NF, the preferred terminology remains C3NeF. Much is known about the C3NeF which occurs in MPGN; whether similar characteristics typify other nephritic factors (properdin dependent and independent, slow acting) is not known. Subsequent studies may well subclassify C3NeF on the basis of its activity and reactivity.

Factors Involved in Pathogenicity C3NeF originates from germline genes; the ability to make C3NeF is present in everyone from the time of birth (Tsokos et al., 1989). Numerous experiments show that C3NeF can be produced in pokeweed mitogen-stimulated lymphocyte cultures taken from newborns, normal adults and patients with MPGN. Both IgM and IgG C3NeF are specific for C3bBb (Tsokos et al., 1989). These nephritic factors, including the IgMs, are all of very high affinity (Spitzer et al., 1992b). The Ka values range from 108--1011 L/mol; there are no differences between IgG and IgM moieties or the autoantibodies from normal newborns, adults or patients. The C3NeF produced in culture by cells from normal individuals represents a significant fraction of the total IgG and IgM elaborated (Spitzer et al., 1990c). While cultures from infants make less IgG, the proportion of IgG that is C3NeF is similar to that of adults. In fact, the fraction of IgG that is C3NeF is in the range of the fraction of specific antibody after

immunization (-~5%) (Stevens and Saxon, 1979). Why C3NeF cannot be found in normal plasma in view of this feature is not clear. The range of IgM C3NeF is in keeping with other studies of specific IgM production. A likely explanation for the increased production of both IgG and IgM C3NeF is that C3bBb is constantly formed in the circulation, leading to continuous endogenous immunization (Daha et al., 1978). Since the formation of the complement system occurs early in fetal life, it is likely that the neoantigen for C3NeF is also produced for many months in utero. The nephritic factors found in culture (and in serum) probably affinity matured to this chronic lowgrade immunization occurring in the plasma of all individuals. Indeed, sequences of C3 nephritic factor obtained from monoclonal C3NeF produced by EBVtransfected B cells from patients with MPGN, as well as combinatorial libraries from patients and normal individuals, utilize V.III or VHIV germline gene segments and are strongly mutated (95% homology to germline gene segments) (Victor et al., 1993). A variety of J segments are used with minimal or no changes from the germline and the D segments are extremely variable. Both kappa and lambda light chains are used, but the published sequences are not sufficient to determine any enhanced propensity for specific gene segment utilization. C3NeF is strongly associated with the idiotypic network (Spitzer et al., 1992c). When anti-id antibodies to C3NeF are incubated with variable concentrations of different C3NeF preparations, the amount of each C3NeF preparation which binds 50% of the anti-id antibody can be used as an indicator of avidity of the anti-id interaction. Multiple C3NeF preparations from a variety of sources bind significant amounts of the anti-id indicating a strong idiotypic commonality. Direct determinations by ELISA of the affinity constants between the standard anti-id antibody and the various C3NeF preparations support these conclusions (Spitzer et al., 1992c). Both Ab2o~ and Ab2~3 preparations can be identified among anti-id antibodies obtained from patients with MPGN using a three-column technique for isolation and purification (Spitzer et al., 1992c). When all of these different anti-id antibodies were tested against an equally varied array of C3NeF preparations, all anti-id antibodies, regardless of the source, inhibited the function of al___[1C3NeF preparations from a wide variety of unrelated donors. These results suggest that the C3NeF preparations share or have the same idiotype located near or in the C3bBb binding site.

541

Methods

of Detection

In the original method for detecting C3NeF (Spitzer et al., 1969), equal parts of serum from a patient suspected of having C3NeF is incubated for 60 minutes at 37~ with normal human serum, and C3 consumption is determined by total hemolytic complement, specific C3 hemolytic titrations or loss of the B antigen from C3 (Spitzer et al., 1969; Vallota et al., 1970). A more specific and much more sensitive method relies on precipitation of IgG from the patient's serum and addition of that IgG fraction to sheep cells bearing C3bBb (EC3bBb) (Daha et al., 1976; Tsokos et al., 1989; Spitzer et al., 1992b). Decay dissociation of EC3bBb (which can be made several different ways) is prevented by C3NeF. Stabilization of EC3bBb cells is the gold standard for measuring C3NeF. An ELISA assay utilizing plate bound C3bBb (Seino et al., 1993) and a microassay based on the peroxidase-like activity of heme groups (Matin et al., 1990) are also available. Although cumbersome and difficult, the stabilization assay is the only tried and true method for measuring C3NeF activity. Pathogenetic

Role

Anti-idiotypic antibody levels in normal individuals, as well as in patients, vary considerably between Ab2o~ which is directed against the idiotype on C3NeF and Ab213 which contains the internal image of the neoantigen on Factor B. The paratope of Ab213 may be directed either against the idiotope on C3NeF or against some other antigen. Normal individuals have no circulating C3NeF and typically have more Ab2cz than Ab213 (Spitzer et al., 1992c). Patients who are in remission with MPGN have similar findings although C3NeF often does not disappear completely. At the time of diagnosis or at relapse, high titers of C3NeF, as well as high levels of Ab213, are typical. To investigate this variation and fluctuation of Ab2c~ and Ab[3, rabbits were immunized with C3NeF or with Ab213 (Spitzer et al., 1992c) (Figure 1). Immunization with C3NeF yielded high titer Ab2cz, followed some time later by a rise in Ab213. Immunization with Ab213 yielded Ab3 (C3NeF), followed by Ab4c~ and finally by Ab413. The antibodies which result from these immunizations resemble the anti-id antibodies isolated from normal individuals. Patients, on the other hand, have the converse with high levels of Ab213 and high titers of C3NeF. Whether this

542

Figure 1. Scheme for anti-id production. B, B-cells; T H, T helper cells. Heavy arrows represent stimulation and broken arrows represent inhibition.

reflects a defect in the patient's idiotypic network remains to be determined. The finding of C3NeF in the circulation, therefore, reflects a variety of factors including overproduction as well as poor control by the idiotypic network (Figure 2).

CLINICAL UTILITY There is no known relationship between the severity

1.5.

Ab4a

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1.0.

o--- Ab4[~ 0.5,

0.5.

@ W @ 4.a .N

o.o~_

0

.

--

I~)

,

20

9

r

30

-

9

~

40

50

60

Days

2. C3NeF and anti-C3NeF production in rabbits. White New Zealand rabbits (2-4 kb) were injected subcutaneously at multiple sites with 50 ng of human Ab213 in Ribi adjuvant on the days indicated by arrows. Rabbit C3NeF (Ab3) and Ab4c~ and Ab413 were separated before the assay of each. Z (hemolytic sites/cell) refers to the number of sites/cell remaining after 15 min of decay of EC3bBb cells treated with C3NeF. In all cases, isolated rabbit C3NeF (Ab3) was added at a final concentration of 90-100 ng/mL.

Figure

of MPGN and levels of C3 or C3NeF (Jackson et al., 1987). There does, however, seem to be an association between levels of C3NeF and levels of C3. The presence of C3NeF in a patient with glomerulonephritis suggests a diagnosis of MPGN, but confirmation by a renal biopsy is required. Although C3NeF is most common in type 2 MPGN, circulating C3NeF is found in all three types of MPGN. Slow acting C3NeF is reportedly associated with type 3 MPGN (Jackson et al., 1987). In patients with partial lipodystrophy C3NeF, levels and hypocomplementemia may precede the onset of MPGN by many years (Peters et al., 1973). C3NeF has also been found in four cases of partial lipodystrophy and/or type 2 MPGN (all of whom went on to develop features of SLE and produce anti-Ro antibodies) (Walport et al., 1994) and in six cases of C3NeF was associated with anti-Ro antibodies. Others found C3NeF, together with C4 nephritic factor, in patients with MPGN (Ohi and Yasugi, 1994). Finally, C3NeF can also be found in MPGN with normal C3. Apparently, the C3NeF in normocomplementemic MPGN will not prevent the extrinsic decay dissociation of C3bBb by Factors H and I while C3NeF in the hypocomplementemic variety of MPGN will inhibit both the intrinsic and extrinsic decay of the C3 convertase (Ohi et al., 1992). C3NeF has also been reported with meningococcal meningitis (Hulton et al., 1992) as well as with hepatitis B (Strife et al., 1974). The familial nature of C3NeF can be manifest in patients with and without renal disease. C3NeF, partial lipodystrophy and membranoproliferative glomerulonephritis, either singly or in combination, can coexist in members of a single family spanning two generations (Power et al., 1990). This suggests that the pathogenesis of these conditions may be linked and that genetically determined factors may contribute to disease susceptibility. Likewise, patients with MPGN, both with and without C3NeF, may have similar extended completypes but no distinctive HLA or DR associations (Welch et al., 1986).

Effect of Therapy Long-term therapy with prednisone is known to eliminate C3NeF from the serum (Jackson et al., 1987). C3NeF also disappears (Fremeaux-Bacchi et al., 1994) several months after renal transplantation

and bilateral nephrectomy, although an earlier study had indicated persistence of C3NeF 2 weeks after a bilateral nephrectomy (Vallota et al., 1971). Perhaps some type of antigenic stimulus from the patient's native kidneys yields the autoantibodies and an antigen-driven expansion of self-reactive B cell clones occurs in response to a specific disease process in the kidney. Taken together, these clinical data show that C3NeF production is associated with MPGN. The exact role that C3NeF plays in this disorder is, however, unclear and the presence of C3NeF has little or no specificity, sensitivity or positive predictive value at the present time.

CONCLUSION Data accumulated over the past 25 years strongly suggest that C3 nephritic factor is a typical antibody. Germline genes are involved in the production of C3NeF and the ability to make C3NeF is apparently present in everyone from the time of birth. Elaboration of C3NeF may approximate an antibody response after immunization, and C3NeF antibody, both IgG and IgM, can be produced in large amounts with high affinity and narrow specificity. One can only speculate on how this situation arose. Because the autoantigen of C3NeF is present in early fetal life and because C3 and Factor B interact continuously in the circulation to generate C3bBb, it is possible that this constant antigenic stimulus gives rise to clones of cells producing C3NeF which are specific and of high affinity, even in the newborn. Thus, the unusual situation exists whereby affinity maturation may have occurred in utero leading to the type of IgG antibody present at birth. C3NeF has the characteristics of a mature antibody in the secondary immune response in normal individuals; this makes it an unusual autoantibody. Indeed, its characteristics are those of a pathologic autoantibody but under normal conditions. These data also suggest that under normal conditions, the idiotypic network may play an important part in control of this autoantibody. Presently there are no firm data relating C3NeF to activity in MPGN; this issue must be fully explored as a basis for understanding the immunobiology of the nephritic factors.

543

REFERENCES Daha MR, Fearon DT, Austen KF. C3 nephritic factor (C3NeF) stabilization of fluid phase and cell-bound alternative pathway convertase. J Immunol 1976; 116:1-7. Daha MR, Austen KF, Fearon DT. Heterogeneity, polypeptide chain composition and antigenic reactivity of C3 nephritic factor. J Immunol 1978;120:1389--1394. Daha MR, Van Es LA. Stabilization of homologous and heterologous cell-bound amplification convertases, C3bBb, by C3 nephritic factor. Immunology 1981;43:33--38. Fremeaux-Bacchi V, Weiss L, Brun P, Kazatchkine MD. Selective disappearance of C3NeF IgG autoantibody in the plasma of a patient with membranoproliferative glomerulonephritis following renal transplantation. Nephrol Dial Transplant 1994;9:811--814. Hiramatsu M, Tsokos GC. Epstein-Barr virus transformed B cell lines derived from patients with systemic lupus erythematosus produce a nephritic factor of the classical complement pathway. Clin Immunol Immunopathol 1988;46:91--99. Hulton SA, Risdon RA, Dillon MJ. Mesangiocapillary glomerulonephritis associated with meningococcal meningitis, C3 nephritic factor and persistently low complement C3 and C5. Pediatr Nephrol 1992;6:239--243. Jackson EC, McAdams AJ, Strife CF, Forristal J, Welch TR, West CD. Differences between membranoproliferative glomerulonephritis types I and III in clinical presentation, glomerular morphology, and complement perturbation. Am J Kidney Dis 1987;9:115--120. Lambris JD, Muller-Eberhard HJ. Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity. J Biol Chem 1984;259: 12685-12690. Matin MA, Domingo A, Fontan G. Lopez-Trascasa M. Determination of C3 nephritic factor activity by a microassay based on the peroxidase-like activity of the heme group. Clin Biochem 1990;23:497-499. Ohi H, Watanabe S, Fujita T, Yasugi T. Significance of C3 nephritic factor (C3NeF) in nonhypocomplementaemic serum with membranoproliferative glomerulonephritis (MPGN). Clin Exp Immunol 1992:89:479-484. Ohi H, Yasugi'T. Occurrence of C3 nephritic factor and C4 nephritic factor in membranoproliferative glomerulonephritis (MPGN). Clin Exp Immunol 1994;95:316--321. Peters DK, Charlesworth JA, Sissons JG, Williams DG, Boulton-Jones JM, Evans DJ, Kourilsky O, Morel-Maroger L. Mesangiocapillary nephritis, partial lipodystrophy and hypocomplementaemia. Lancet 1973;2:535-538. Power DA, Ng YC, Simpson JG. Familial incidence of C3 nephritic factor, partial lipodystrophy and membranoproliferative glomerulonephritis. Q J Med 1990;75:387-398. Seino J, van der Wall Bake AW, Van Es LA, Daha MR. A novel ELISA assay for the detection of C3 nephritic factor. J Immunol Methods 1993;159:221--227. Spitzer RE, Vallota EH, Forristal J, Sudora E, Stitzel AE, Davis NC. Serum C'3 lytic system in patients with glomerulonephritis. Science 1969;164:436-437. Spitzer RE, Stitzel AE. On the origin and control of C3NeF. In

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Vivo 1988;2:79-82. Spitzer RE, Stitzel AE, Tsokos GC. Human anti-idiotypic antibody responses to autoantibody against the alternative pathway C3 convertase. Clin Immunol Immunopathol 1990a;57:19--31. Spitzer RE, Stitzel AE, Tsokos GC. Production of IgG and IgM autoantibody to the alternative pathway C3 convertase in normal individuals and patients with membranoproliferative glomerulonephritis. Clin Immunol Immunopathol 1990b;57: 10-18. Spitzer RE, Stitzel AE, Tsokos GC. Evidence that production of autoantibody to the alternative pathway C3 convertase is a normal physiologic event. J Pediatr 1990c; 116:S 103-S 108. Spitzer RE, Stitzel AE, Tsokos GC. On the origin of C3 nephritic factor (Antibody to the alternative pathway C3 convertase): evidence for the Adam and Eve concept of autoantibody production. Clin Immunol Immunopathol 1992a;64:177-183. Spitzer RE, Stitzel AE, Tsokos GC. Autoantibody to the alternative pathway C3/C5 convertase and its anti-idiotypic response: a study in affinity. J Immunol 1992b; 148:137-- 141. Spitzer RE, Stitzel AE, Tsokos GC. Study of the idiotypic response to autoantibody to the alternative pathway C3/C5 convertase in normal individuals, patients with membranoproliferative glomerulonephritis, and experimental animals. Clin Immunol Immunopathol 1992c;62:291-294. Spitzer RE, Stitzel AE. Can a defect in Ab213 production mediate autoantibody proliferation. FASEB J 1995;9:A811. Stevens RH, Saxon A. Differential synthesis of IgM and IgA antitetanus toxoid antibody in vitro following in vivo booster immunization of humans. Cell Immunol 1979;45:142--150. Strife CF, McAdams AJ, McEnery PT, Bove KE, West CD. Hypocomplementemic and normocomplementemic acute nephritis in children: a comparison with respect to etiology, clinical manifestations, and glomerular morphology. J Pediatr 1974:84:29-38. Tanuma Y, Ohi H, Hatano M. Two types of C3 nephritic factor: properdin-dependent C3NeF and properdin-independent C3NeF. Clin Immunol Immunopathol 1990;56:226--238. Tsokos GC, Stitzel AE, Patel AD, Hiramatsu M, Balow JE, Spitzer RE. Human polyclonal and monoclonal IgG and IgM complement 3 nephritic factors: evidence for idiotypic commonality. Clin Immunol Immunopathol 1989;53:113-122. Vallota EH, Forristal J, Spitzer RE, Davis NC, West CD. Characteristics of a non-complement-dependent C3-reactive complex formed from factors in nephritic and normal serum. J Exp Med 1970;131:1306-1334. Vallota EH, Forristal J, Spitzer RE, Davis NC, West CD. Continuing C3 breakdown after bilateral nephrectomy in patients with membranoproliferative glomerulonephritis. J Clin Invest 1971;50:552-558. Victor KD, Pascual V, Stitzel AE, Tsokos GC, Capra JD, Spitzer RE. Nucleotide sequence of a human autoantibody to the alternative pathway C3/C5 convertase (C3NeF). Hybridoma 1993;12:231-237. Walport MJ, Davies KA, Botto M, Naughton MA, Isenberg DA, Biasi D, Powell RJ, Cheung NT, Struthers GR. C3

nephritic factor and SLE: report of four cases and review of the literature. Q J M 1994;87:609--615. Welch TR, Beischel L, Balakrishnan K, Quinlan M, West CD.

Major-histocompatibility-complex extended haplotypes in membranoproliferative glomerulonephritis. N Engl J Med 1986:314:1476-- 1481.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

NEURONAL AUTOANTIBODIES Judah A. Denburg, M.D.

Division of Clinical Immunology and Allergy, McMaster University, Hamilton, Ontario, L8N 3Z5 Canada

HISTORICAL NOTES Autoantibodies against nervous system antigens, generically referred to as neuronal autoantibodies (NA), were described during the last few decades. Identification of common thymus/brain antigens (Golub, 1971) led to a series of observations that lymphoid elements and nervous system tissues may share certain antigenic markers, including CD4, CD8, myelin-associated glycoproteins and other undefined brain cross-reactive lymphocyte specificities (Denburg and Temesvari, 1983). In 1976 a seminal paper on the presence of brain cross-reactive lymphocytotoxic antibodies in systemic lupus erythematosus (SLE) (Bluestein and Zvaifler, 1976) paved the way for further investigation on the role of NA in SLE and related conditions associated with nervous system complications (Denburg S et al., 1993; Denburg J e t al., 1993; Carbotte et al., 1992a).

AUTOANTIGENS

Definition/Structure No single autoantigen fulfills the definition of the prime target of NA. Rather, a number of relevant antigens are found on peripheral and central neurons, neuronal and neuroblastoma cell lines, oligodendrocytes and astroglia; some of these antigens are shared with various T-cell subpopulations (Bluestein, 1993; Minota and Winfield, 1987a; Pischel et al., 1988; Hanly et al., 1989; Denburg and Behmann, 1994). At present, for all these described specificities, there are no recombinant forms of antigen available, and no sequence information exists on specific autoantigens targeted by NA. Included among the antigens described in some detail, are a 50 kd molecule on 546

neuroblastoma cells (Hanson et al., 1992) and a surface-expressed neuronal cell-line antigen which is one of the ribosomal P proteins (Koren et al., 1992). Several protein, glycoprotein and lipid antigens which react in NA or related assays are found in brain tissue, peripheral and central myelin, in neurofilaments at the neuromuscular junction (Zhu et al., 1994; Marx et al., 1992; Dropcho and King, 1994; Smith et al., 1994; Kaneko et al., 1993; Sher et al., 1993; Spezialetti et al., 1993; Zanone et al., 1993; Kiessling et al., 1993) and on lymphocyte membranes by immunoblotting (Minota and Winfield, 1987a; 1987b), including 97--98, 54--55, 50-52 and 32 kd antigens (Denburg et al., 1994; Denburg and Behmann, 1994). No formal proof of the identity of these antigenic activities with known reactivities, such as U1 RNP or SS-B (Ro) is available, and the possibility some are identical may, in fact, be obscured by the multiple antibodies present in SLE sera (Denburg and Behmann, 1994). Neuronal autoantibodies are described in association with diverse clinical conditions, including autonomic neuropathies (Zanone et al., 1993), movement disorders (Kiessling et al., 1993), paraneoplastic syndromes (Posner and Furneaux, 1990; Dropcho and King, 1994), myasthenia gravis (Drachman, 1994; Marx et al., 1992), amyotrophic lateral sclerosis, dementia (Kaneko et al., 1993), Sj6gren's syndrome and lupus (Denburg and Behmann, 1994). Of interest, some neuronal antigens described in association with paraneoplastic syndromes are shared by various tumor tissues (Posner and Furneaux, 1990; Lennon, 1994).

AUTOANTIBODIES

Terminology The various NA are also termed "brain cross-reactive

lymphocytotoxic antibodies" (Bluestein and Zvaifler, 1976; Denburg S e t al., 1988; Long et al., 1990; Denburg and Behmann, 1994; Minota and Winfield, 1987a; 1987b), "brain-reactive antibodies" or more specifically according to reactivities found in various in vitro assays (Spezialetti et al., 1993; Denburg and Behmann, 1994). Pathogenetic Role

At present there is no formal proof of a pathogenetic role for NA in any disease, including the nervous system complications of lupus, Sj6gren's syndrome, Alzheimer's disease, various demyelinating diseases and diseases of nerves. This contrasts with the myasthenic syndromes and paraneoplastic syndromes in which some of the pathogenetically relevant autoantigens are well characterized. Factors Involved in Pathogenicity

The association of serum and CSF NA with disease manifestations in patients with SLE varies greatly. Animal models in which brain-reactive autoantibodies occur, include most strains of autoimmune mice, which typically show a very high frequency of autoantibodies with NA-type profiles (Sakic et al., 1993a; 1993b; Lal and Forster, 1988). Both in humans and in animals, most of the autoantibodies mentioned are of the IgG isotype, but some are also IgM, especially those with lymphocytotoxic activity (Minota and Winfield, 1987a; 1987b). Details of subclass, idiotype and avidity/affinity are not commonly available. Methods of Detection

The autoantibodies are identified by binding assays to brain tissue or neuroblastoma cell lines, 51Cr-release assays or radioimmunoassays (How et al., 1985; Spezialetti et al., 1993; Bluestein, 1993). Sometimes, antibodies characterized as NA react with lymphocyte subpopulations (Minota and Winfield, 1987a; 1987b; Winfield et al., 1993; Denburg and Behmann, 1994; Long et al., 1990) and can be removed by absorption with brain or neuroblastoma cells and related nervous system tissue (Denburg and Behmann, 1994). Thus, the autoantibodies, like the autoantigens, represent a heterogeneous group of reactivities with a rather loosely defined common name. The absence of recombinant antigens or at least readily available, well-characterized native antigens limits progress in this area.

CLINICAL UTILITY Disease Associations

Diagnostic sensitivity and specificity of NA in SLE, including neuropsychiatric SLE, and related conditions are highly controversial. Reports of close to 100% sensitivity and specificity (Hanson et al., 1992) are countered by other studies which show very little sensitivity or specificity and high numbers of falsepositive and false-negative results (Spezialetti et al., 1993). The key point to be made is that different studies are examining different specificities, have different criteria for selection of patients for a given disease manifestation such as neuropsychiatric SLE, and may base the findings upon assays of different biological fluids (serum vs. cerebrospinal fluid) (Denburg S et al., 1993; Carbotte et al., 1992a; Hanly et al., 1989). Some data suggest that only CSF assays have a high degree of specificity in the diagnosis of neuropsychiatric SLE (Spezialetti et al., 1993). Although direct comparisons are not available, the analytical sensitivity of the CSF assay which is based on 12SI-radiolabeling (Bluestein and Woods, 1982), is very likely to be higher than that using a mixed hemadsorption assay which has a very high degree of specificity but extremely low sensitivity for NA (Kelly and Denburg, 1987). Moreover, the CSF profile attending the detection of NA is also controversial; some studies show abnormal albumin quotients but normal IgG indices (Kelly and Denburg, 1987); whereas, others show oligoclonal bands and increased local production of IgG. Whether NA detected in the CSF arise from within the brain parenchyma, or are found there because of passive leakage through a damaged blood-CSF barrier is still unresolved. Nonetheless, and despite poor agreement on sensitivity and specificity, a number of studies indicate clinical associations which may be of utility, including: (1) the relationship of NA to cognitive abnormalities and/or emotional distress (Denburg J et al., 1987; 1993; Denburg S et al., 1993; Carbotte et al., 1992a); (2) the relationship of specificities of cross-reactive lymphocytotoxic antibodies with visualspatial cognitive abnormalities (Denburg S et al., 1988); (3) the distinction between "diffuse" and "focal" neuropsychiatric SLE by differential use of neurofilament, neuronal and antiphospholipid antibody assays (Bell et al., 1991); and (4) the apparent diagnostic specificity in very small groups of patients for an anti-50 kd reactivity with neuropsychiatric SLE 547

Table 1. Clinical Associations of Neuronal Antibodies Neuropsychiatric SLE especially "diffuse" presentation and depression Cognitive dysfunction in SLE especially visual-spatial abnormalities and emotional distress Paraneoplastic syndromes Myasthenic syndromes

(Hanson et al., 1992). As far as is known, none of the NA described have a direct relationship to disease activity or severity in SLE, with the possible exception of a relationship to cognitive dysfunction (Table 1). Neuronal autoantibodies are known to decrease in individual patients after cytotoxic or immunosuppressive therapy (Haply et al., 1989), and the decreases in some instances relate to resolution of clinical and radiological abnormalities associated with nervous system complications (Carbotte et al., 1992b). In the more well-defined paraneoplastic syndrome of cerebellar dysfunction associated with cancer and antiYo/anti-Purkinje cell antibodies, it is clear that removal of the antibodies is not commonly attended by improvement in the neurologic condition. Injection of antibodies with activity against caudate nuclei leads to chorea; (Husby et al., 1976); the effect of therapy upon these antibodies is not known.

CONCLUSION Neuronal autoantibodies represent a heterogeneous group of reactivities against various nervous system antigens, at least some of which are common to other tissues. NA with proved clinical relevance and their antigens targeted in a pathogenic manner are yet to be

REFERENCES Bell CL, Partington C, Robbins M, Graziano F, Turski P, Kornguth S. Magnetic resonance imaging of central nervous system lesions in patients with lupus erythematosus: correlation with clinical remission and antineurofilament and anticardiolipin antibody titers. Arthritis Rheum 1991;34:432-441. Bluestein HG, Zvaifler NJ. Brain-reactive lymphocytotoxic antibodies in the serum of patients with systemic lupus erythematosus. J Clin Invest 1976;57:509--516. Bluestein HG, Woods VL Jr. Antineuronal antibodies in 548

identified. The association of NA with T-cell surface antigenic specificity and cross-reactivity for neuronal antigens is promising in relation to cognitive abnormalities (Denburg and Behmann, 1994; Carbotte et al, 1992a). Likewise, the isolation of a specific and novel, recently cloned brain antigen in lupus mice (Moore et al., 1994) may lead to the development of an understanding of how NA might play a pathogenic role in animal models of neuropsychiatric SLE (Sakic et al., 1994; 1993a, 1993b). Autoantibodies with specificities for lymphocyte antigens which are highly correlated with cognitive dysfunction (Denburg and Behmann, 1994) can now be isolated and injected into animals to test whether an experimental model of neuropsychiatric SLE can be induced. For now, the clinical utility of NA needs to be studied carefully in a prospective fashion, by comparing serum versus CSF in carefully characterized and monitored patients, by defining specificities in relation to specific clinical syndromes and by pooling resources from different centers to assure validity and reliability. Until this is done, NA will remain a loosely used test for a heterogeneous group of reactivities against a wide variety of antigens, which has questionable clinical significance. See also NEURONAL NUCLEAR AUTOANTIBODIES, TYPE 1 (HU), PURKINJE CELL AUTOANTIBODIES, TYPE 1 (YO) and RIBOSOMAL P PROTEIN AUTOANTIBODIES.

systemic lupus erythematosus. Arthritis Rheum 1982;25: 773--778. Bluestein HG. Antibodies to neurons. In: Wallace DJ, Hahn BH, eds. Dubois' Lupus Erythematosus, 4th edition. Philadelphia: Lea & Febiger, 1993:260-263. Carbotte RM, Denburg SD, Denburg JA. Cognitive dysfunction and systemic lupus erythematosus. In: Lahita RG, ed. Systemic Lupus Erythematosus. New York: Churchill Livingstone, 1992a:865--881. Carbotte RM, Denburg SD, Denburg JA, Nahmias C, Garnett ES. Fluctuating cognitive abnormalities and cerebral glucose metabolism in neuropsychiatric systemic lupus erythematosus.

J Neurol Neurosurg Psychiatry 1992b;55:1054-1059. Denburg JA, Temesvari P. The pathogenesis of neuropsychiatric lupus. Can Med Assoc J 1983;128:257--260. Denburg JA, Carbotte RM, Denburg SD. Neuronal antibodies and cognitive function in systemic lupus erythematosus. Neurology 1987;37:464--467. Denburg JA, Carbotte R, Denburg S. Central nervous system lupus. Rheumatol Rev 1993;2:123-132. Denburg JA, Behmann SA. Lymphocyte and neuronal antigens in neuropsychiatric lupus: presence of an elutable, immunoprecipitable lymphocyte/neuronal 52 kd reactivity. Ann Rheum Dis 1994;53:304--308. Denburg SD, Carbotte RM, Long AA, Denburg JA. Neuropsychological correlates of serum lymphocytotoxic antibodies in systemic lupus erythematosus. Brain Behav Immunol 1988;2:222--234. Denburg SD, Denburg JA, Carbotte RM, Fisk JD, Hanly JG. Cognitive deficits in systemic lupus erythematosus. Rheum Dis Clin North Am 1993;19:815--831. Denburg SD, Behmann SA, Carbotte RM, Denburg JA. Lymphocyte antigens in neuropsychiatric systemic lupus erythematosus: relationship of lymphocyte antibody specificities to clinical disease. Arthritis Rheum 1994;37:369-375. Drachman DB. Myasthenia gravis. N Engl J Med 1994;330: 1797-1810. Dropcho EJ, King PH. Autoantibodies against the Hel-N1 RNA-binding protein among patients with lung carcinoma: an association with type I antineuronal nuclear antibodies. Ann Neurol 1994;36:200-205. Golub ES. Brain-associated theta antigen: reactivity of rabbit antimouse brain with mouse lymphoid cells. Cell Immunol 1971;2:353--361. Hanly JG, Behmann S, Denburg SD, Carbotte RM, Denburg JA. The association between sequential changes in serum antineuronal antibodies and neuropsychiatric systemic lupus erythematosus. Postgrad Med J 1989;65:622--627. Hanson VG, Horowitz M, Rosenbluth D, Spiera H, Puszkin S. Systemic lupus erythematosus patients with central nervous system involvement show autoantibodies to a 50-kD neuronal membrane protein. J Exp Med 1992;176:565--573. How A, Dent PB, Liao SK, Denburg JA. Antineuronal antibodies in neuropsychiatric systemic lupus erythematosus. Arthritis Rheum 1985;28:789--795. Husby G, van de Rijn I, Zabriskie JB, Abdin ZH, Williams RC Jr. Antibodies reacting with cytoplasm of subthalamic and caudate nuclei neurons in chorea and acute rheumatic fever. J Exp Med 1976;144:1094--1110. Kaneko K, Seki K, Ishida K, Uchihara T, Kanda T, Inuzuka T, Miyatake T. Circulating autoantibody to mature neurons and astrocytes of humans and some mammals present in a demented patient with autoimmune disorder. Eur Neurol 1993;33:450-453. Kelly MC, Denburg JA. Cerebrospinal fluid immunoglobulins and neuronal antibodies in neuropsychiatric systemic lupus erythematosus and related conditions. J Rheumatol 1987;14: 740-744. Kiessling LS, Marcotte AC, Culpepper L. Antineuronal antibodies in movement disorders. Pediatrics 1993;92:39-43.

Koren E, Reichlin MW, Koscec M, Fugate RD, Reichlin M. Autoantibodies to the ribosomal P proteins react with a plasma membrane-related target on human cells. J Clin Invest 1992:89:1236-1241. Lal H, Forster MJ. Autoimmunity and age-associated cognitive decline. Neurobiol Aging 1988;9:733--742. Lennon VA. Paraneoplastic autoantibodies: the case for a descriptive generic nomenclature. Neurology 1994;44:22412246. Long AA, Denburg SD, Carbotte RM, Singal DP, Denburg JA. Serum lymphocytotoxic antibodies and neurocognitive function in systemic lupus erythematosus. Ann Rheum Dis 1990;49:249--253. Marx A, Kirchner T, Greiner A, Muller-Hermelink HK, Schalke B, Osborn M. Neurofilament epitopes in thymoma and antiaxonal autoantibodies in myasthenia gravis. Lancet 1992;339:707--708. Minota S, Winfield JB. Identification of three major target molecules of IgM antilymphocyte autoantibodies in systemic lupus erythematosus. J Immunol 1987a;139:3644-3651. Minota S, Winfield JB. IgG antilymphocyte antibodies in systemic lupus erythematosus react with surface molecules shared by peripheral T cells and a primitive T cell line. J Immunol 1987b;138:1750-1756. Moore PM, Joshi I, Ghanekar SA. Affinity isolation of neuronreactive antibodies in MRL/lpr mice. J Neurosci Res 1994; 39:140-147. Pischel KD, Bluestein HG, Woods VL. Platelet glycoproteins Ia, Is, and Iia are physicochemically indistinguishable from the very late activation antigens adhesion-related proteins of lymphocytes and other cell types. J Clin Invest 1988;81: 505--513. Posner JB, Furneaux HM. Paraneoplastic syndromes. In: Waksman BH, ed. Immunologic Mechanisms in Neurologic and Psychiatric Disease. New York: Raven Press, 1990:187219. Sakic B, Szechtman H, Denburg S, Carbotte R, Denburg JA. Spatial learning during the course of autoimmune disease in MRL mice. Behav Brain Res 1993a;54:57--66. Sakic B, Szechtman H, Denburg S, Carbotte RM, Denburg JA. Brain-reactive antibodies and behavior of autoimmune MRLlpr mice. Physiol Behav 1993b;54:1025--1029. Sakic B, Szechtman H, Talangbayan H, Denburg SD, Carbotte RM, Denburg JA. Disturbed emotionality in autoimmune MRL lpr mice. Physiol Behav 1994;56:609-617. Sher E, Carbone E, Clementi F. Neuronal calcium channels as target for Lambert-Eaton myasthenic syndrome autoantibodies. Ann N Y Acad Sci 1993;681:373-381. Smith RG, Alexianu ME, Crawford G, Nyormoi O, Stefani E, Appel SH. Cytotoxicity of immunoglobulins from amyotrophic lateral sclerosis patients on a hybrid motoneuron cell line. Proc Natl Acad Sci USA 1994;91:3393-3397. Spezialetti R, Bluestein HG, Peter JB, Alexander EL. Neuropsychiatric disease in Sj6gren's syndrome: antiribosomal P and antineuronal antibodies. Am J Med 1993;95:153--160. Winfield JB, Mimura T, Fernsten PD. Antilymphocyte autoantibodies. In: Wallace DJ, Hahn BH, editors. Dubois' Lupus Erythematosus, 4th edition. Philadelphia: Lea & Febiger,

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1993:254--259. Zanone MM, Peakman M, Purewal T, Watkins PJ, Vergani D. Autoantibodies to nervous tissue structures are associated with autonomic neuropathy in type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1993;36:564--569.

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Zhu J, Link H, Weerth S, Livington C, Mix E, Qiao J. The B cell repertoire in experimental allergic neuritis involves multiple myelin proteins and GM 1. J Neurol Sci 1994; 125: 132--137.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

NEURONAL NUCLEAR AUTOANTIBODIES, TYPE 1 (Hu) Henry M. Furneaux, Ph.D.

Laboratory of Molecular Neuro-Oncology, Program in Molecular Pharmacology and Therapeutics, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA

HISTORICAL NOTES

Paraneoplastic encephalomyelitis and paraneoplastic sensory neuronopathy are infrequent neurological disorders associated with small-cell lung cancer (Henson et al., 1965). Although these disorders are rare, accurate diagnosis is important in that it may prompt the search for an occult lung tumor (Henson and Urich, 1982; Younger et al., 1993). The prevailing hypothesis is that an immune response primarily directed against lung tumor antigens cross-reacts with similar antigens expressed normally in the central or peripheral nervous system. (Rosenblum, 1993; Allermatt et al., 1991). This hypothesis stems from two observations. First, the affected nervous system shows extensive inflammatory infiltrates associated with the area of neuronal loss (Brashear et al., 1991; Dalmau et al., 1991). Second, a serum autoantibody (initially called anti-Hu but now called antineuronal nuclear antibodies, type 1; anti-ANNA-1) which reacts specifically with neurons and tumor has been detected in serum and CSF (Graus et al., 1986; Furneaux et al., 1990).

THE AUTOANTIGENS

ANNA-1 sera react with a group of basic proteins of 35--40 kd that are specifically expressed in neurons and the associated tumor (Graus et al., 1986; Dalmau et al., 1992a). Molecular cloning studies reveal that the antigens in this group are encoded by three distinct genes which are called HuD, HuC/ple21 and Hel-N1 (King et al., 1994; Sakai et al., 1994; Liu et

al., 1995). The HuD gene maps to lp34 (Muresu et al., 1994). HuC/ple21 and Hel-N1 localize to chromosomes 19 and 9, respectively (Furneaux and Sikela, unpublished observations). Hu antigens are the human homologues of Elav, a Drosophila gene required for neuronal differentiation (Yao et al., 1992; Good, 1995). Elav is expressed on termination of the neuroblast cell-cycle (Robinow and White, 1991). Mutation in Elav results in the abrogation of neuronal differentiation and the unrestricted proliferation of neuroblasts. Considering their similar patterns of expression during vertebrate neurogenesis, the Hu proteins are probably required for the development and maintenance of neurons (Marusich et al., 1994; Barami et al., 1995). The most striking feature of the Hu proteins is the presence and organization of three RNA-binding domains. This motif, termed "RRM," consists of 80--90 conserved amino acids containing two stretches of eight and six highly conserved residues called RNP1 and RNP2, respectively (Burd and Dreyfuss, 1994). In most RNA-binding proteins, the RRM is associated with auxiliary domains. In the case of the HuD, HuC and Hel-N 1, two tandemly arranged RRMs are connected to the third RRM by a positively charged segment called the basic domain. In vitro binding studies show that HuD and Hel-N1 bind to a regulatory element in the 3' untranslated region of transiently expressed mRNAs (Levine et al., 1993; King et al., 1994; Liu et al., 1995). Thus, the Hu proteins are important regulators of mRNAs that are required for neural differentiation, proliferation and maintenance.

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AUTOANTIBODIES

Pathogenetic Role Although classified as autoimmune, the initiating event in these disorders is an antitumor response. Thus, this immune response is not strictly autoimmune but is more likely to be triggered by a "foreign" version of the Hu proteins. The simplest hypothesis is that the Hu genes are mutated in tumor cells and that this mutation yields a selective growth advantage, and thus, all tumor cells contain the mutated version. There is also the possibility that overexpression of the Hu protein may result in loss of tolerance and an antigen-driven humoral response. This is unlikely, since the level of the Hu protein in tumor cells is not significantly greater than that in neurons (Dalmau et al., 1992b). It remains possible that these patients are susceptible to viral infection and that the immune response is a cross-reaction to a similar but as yet unknown viral nucleoprotein. The Hu proteins are attractive candidates for the targets of an antitumor immune response. Studies on Drosophila show that disruption of Elav results in loss of nervous system function. Prolonged in vitro exposure of cultured neurons to high concentrations of

ANNA-1 (anti-Hu) sera results in cell death (Greenlee et al., 1993). Thus, inhibition of Hu function by an anti-Hu immune response could cause the neurological disorder. Unsuccessful attempts to generate animal models include injection of anti-Hu sera and vaccination with recombinant antigens. Although HuD, HuC and Hel-N1 are clearly the major antigens recognized by ANNA-1 sera, the disorder could be caused by an immune response directed against minor antigens. Indeed, some anti-Hu sera also harbor antibodies against components of voltage-gated calcium channels (Lennon et al., 1995).

Methods of Detection Originally discovered by an immunofluorescent technique using brain tissue as a source of antigen (Graus et al., 1986), ANNA-1 react specifically with the nuclei of neurons and not with astroglial cell nuclei. Because there are many neuron-specific nuclear proteins, sera should be confirmed as positive by their reaction with antigens of the defined molecular weight (35--40 kd) (Dalmau et al., 1992b). There is still the possibility that false-positives may arise by virtue of reactivity with other neuron-specific proteins of the same size. Thus, reactivity with the recom-

Figure 1. Immunoblot analysis of the reactivity of Normal Human Serum (lanes 1-7) and a typical anti-Hu serum (lanes 8--12) with recombinant HuD protein. 552

binant antigen is the most fastidious assay (Figure 1) (Szabo et al., 1991). Use of an ELISA assay has also been reported (Dropcho and King, 1994). The abundance of anti-E. Coli antibodies in human serum, however, mandates confirmation by immunoblot.

C L I N I C A L UTILITY

Disease Association Detection of ANNA-1 is very useful in clinical diagnosis. Although the well-informed clinician can easily diagnose the more common neurological complications of cancer, accurate diagnosis of paraneoplastic sensory neuronopathy and paraneoplastic encephalomyelitis often requires the skills of an experienced neurologist. A positive Hu test is also useful to oncologists as it is well correlated (r = 0.97) with the presence of an underlying tumor (Dalmau et al., 1992b). In the majority of cases (78%), this tumor is a small-cell lung cancer, but in other cases can be associated with neuroblastoma (Fisher et al., 1994), prostate cancer (Baloh et al., 1993) and sarcoma

REFERENCES Altermatt H, Rodriguez M, Scheithauer B, Lennon V. Paraneoplastic anti-Purkinje and type 1 antineuronal nuclear autoantibodies bind selectively to central, peripheral and autonomic nervous system cells. Lab Invest 1991;165:412--420. Baloh R, DeRossett S, Cloughesy T, Kuncl RW, Miller NR, Merrill J, Posner JB. Novel brainstem syndrome associated with prostate cancinoma. Neurology 1993;43:2591-2596. Barami K, Iversen K, Furneaux HM, Goldman S. Hu protein as an early marker of neuronal pheonotypic differentiation by subependylmal zone cells of the adult songbird forebrain. J Neurobiology 1995; 28:82--101. Brashear HR, Caccamo DV, Heck A, Keeney PM. Localization of antibody in the central nervous system of a patient with paraneoplastic encephalomyeloneuritis. Neurology 1991;41: 1583--1587. Burd CG, Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science 1994;265:615-621. Dalmau, J, Furneaux HM, Rosenblum MK, Graus F, Posner JB. Detection of the anti-Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy. Neurology 1991; 42:1757--1764. Dalmau J, Furneaux HM, Cordon-Cardo C, Posner JB. The expression of the Hu (paraneoplastic encephalomyelitis/ sensory neuronopathy) antigen in human normal and tumor tissues. Am J Pathol 1992a;41:1--6.

(Vershuuren et al., 1994). ANNA-1 have also been detected in paraneoplastic opsoclonus/myoclonus (Hersh et al., 1994). Thus, the finding of ANNA-1 should prompt the clinician to vigorously search for an underlying tumor. Studies at Memorial Sloan Kettering Cancer Center have indicated that treatment of the patient with immunosuppressive agents is of little utility (Dalmau et al., 1992b). Such a course should, however, be considered if the disease is detected early, before irreversible neurological damage has occurred.

CONCLUSION A pathogenic role for ANNA-1 has yet to be unequivocally established. Definitive detection of these antibodies, however, provides an invaluable clinical tool for diagnosis of paraneoplastic encephalomyelitis or sensory neuronopathy and for detecting an underlying tumor. See also CALCIUM CHANNEL AND RELATED PARANEOPLASTIC DISEASE AUTOANTIBODIES and PURKINJE CELL AUTOANTIBODIES, TYPE 1 (YO).

Dalmau J, Graus F, Rosenblum MK, Posner JB. Anti-Huassociated paraneoplastic encephalomyelitis/sensory neuronopathy: a clinical study of 71 patients. Medicine 1992b;71: 59--72. Dropcho EJ, King PH. Autoantibodies against the Hel-N1 RNA-binding protein among patients with lung carcinoma: an association with type I antineuronal antibodies. Ann Neurol 1994;36:200- 205. Fisher P, Wechsler D, Singer H. Anti-Hu antibody in a neuroblastoma-associated paraneoplastic syndrome. Pediatr Neurol 1994;10:309--312. Furneaux HM, Reich L, Posner JB. Central nervous system synthesis of autoantibodies in paraneoplastic syndromes. Neurology 1990;40:1085--1091. Good P. A conserved family of elav-like genes in vertebrates. Proc Natl Acad Sci USA 1995;92:4557-4561. Graus F, Elkon KB, Cordon-Cardo C, Posner JB. Sensory neuronopathy and small cell lung cancer: antineuronal antibody that also reacts with the tumor. Am J Med 1986; 80:45--52. Greenlee J, Parks T, Jaeckle K. Type IIa ('anti-Hu') antineuronal antibodies produce destruction of rat cerebellar granule neurons in vitro. Neurology 1993;43:2049--2054. Henson RA, Hoffman HL, Urich H. Encephalomyelitis with carcinoma. Brain 1965;88:449--464. Henson RA, Urich H, editors. Cancer and the Nervous System. Oxford: Blackwell Scientific, 1982. Hersh B, Dalmau J, Dangond F, Gultekin S, Geller E, Wen PY. Paraneoplastic opsoclonus-myoclonus associated with anti-Hu 553

antibody. Neurology 1994;44:1754-1755. King PH, Levine TD, Fremeau RT, Keene JD. Mammalian homologues of Drosophila ELAV localized to a neuronal subset can bind in vitro to the 3' UTR of mRNA encoding the Id transcriptional repressor. J Neurosci 1994;4:19431952. Lennon VA, Kryzer TJ, Griesmann GE, O'Suilleabhain Pe, Windebank AJ, Woppmann A, Miljanich GP, Lambert EH. Calcium-channel antibodies in the Lambert-Eaton syndrome and other paraneoplastic syndromes. N Engl J Med 1995; 332:1467--1474. Levine TD, Gao DF, King PH, Andrews LC, Keene JD. HelN 1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs. Mol Cell Biol 1993;13:3494--3504. Liu J, Dalmau J, Szabo A, Rosenfeld M, Huber J, Furneaux H. Paraneoplastic encephalomyelitis antigens bind to the AUrich elements of mRNA. Neurology 1995;45:544--550. Marusich HM, Furneaux HM, Henion P, Weston JA. Hu neuronal proteins are expressed in proliferating neurogenic cells. J Neurobiol 1994;25:143--155. Muresu R, Baldini A, Gress T, Posner JB, Furneaux HM, Siniscalco M. Mapping of the gene coding for a paraneoplastic encephalomyelitis antigen (HuD) to human chromosome site lp34. Cytogen Cell Genetics 1994;65:177-178. Robinow S, White K. Characterization and spatial distribution

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of the ELAV protein during Drosophila melanogaster development. J Neurobiol 1991;22:443--461. Rosenblum M. Paraneoplasia and autoimmunologic injury of the nervous system: the anti-Hu syndrome. Brain Pathol 1993;3:199-212. Sakai K, Gofuku M, Kitagawa Y, Ogasawara T, Hirose G, Yamazaki M, Koh CS, Yanagisawa N, Steinman L. A hippocampal protein associated with paraneoplastic neurologic syndrome and small cell lung carcinoma. Biochem Biophys Res Commun 1994;199:1200-1208. Szabo A, Dalmau J, Manley G, Rosenfeld MR, Wong E, Henson J, Posner JB, Furneaux HM. HuD, a paraneoplastic encephalomyelitis antigen contains RNA-binding domains and is homologous to ELAV and Sex-Lethal. Cell 1991;67: 325--333. Verschuuren J, Twijnstra A, De Baets M, Thunnissen F, Dalmau J, van Breda Vriesman P. Hu antigens and anti-Hu antibodies in a patient with myxoid chondrosarcoma. Neurology 1994;44:1551--1552. Yao K-M, Samson M-L, Reeves R, White K. Gene elav of Drosophila melanogaster: A prototype for neuronal-specific RNA binding protein gene family that is conserved in flies and humans. J Neurobiol 1992;24:723--739. Younger D, Dalmau J, Inghirami G, Sherman WH, Hays AP. Anti-Hu-associated peripheral nerve and muscle microvasculitis. Neurology 1993;44:181-- 183.

9 Elsevier Science B.V. All fights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

NEUTROPHIL AUTOANTIBODIES Kip R. Hartman, M.D.

Department of Hematology, Walter Reed Army Institute of Research, Washington, DC 20307, USA

HISTORICAL NOTES Like erythrocytes and platelets, neutrophils and their precursors in the bone marrow can be targets for autoimmune reactions that lead to a depletion of these cells from the blood. Neutropenia caused by such reactions can be an isolated manifestation of autoimmunity or part of a complex of signs and symptoms in idiopathic, multisystem autoimmune disorders, in association with drug reactions or in primary lymphoid diseases. Many of the conditions associated with neutropenia involve disturbances of immunoregulation, and the immune basis for many cases of neutropenia is clearly established (Wright, 1983; Shastri and Logue, 1993; Dale and Giuerry, 1979; Bux and Mueller-Eckhardt, 1992). Therapies directed at modulating the immune response are sometimes successful in moderating the neutropenia and in reducing associated morbidity.

THE AUTOANTIGENS Definition Molecules expressed on the surface membranes of

mature blood neutrophils, and on the surface of myeloid precursor cells in the bone marrow, contain the antigens associated with immune neutropenia. Origin/Structure Two polymorphic forms of the Fc receptor type III (FcRIII, CD16) differ in their apparent molecular masses and in their reactivity with antisera that recognize the antigenic determinants NA1 and NA2 (Huizinga et al., 1990) (Table 1). The molecular basis for this polymorphism was determined by study of cDNAs encoding the two forms of FcRIII. The cDNA that encodes the NA1 isoform differs from that encoding the NA2 isoform by five nucleotides predicting four amino acid substitutions. As a result, NA1 FcRIII has four potential N-linked glycosylation sites, compared with six for NA2 FcRIII (Ory et al., 1989). This allelic specificity is found in 10% of patients with autoimmune neutropenia of infancy, (Lalezari et al., 1986) and in many cases of neonatal alloimmune neutropenia. The neutrophil-specific antigen NB 1 is clinically important in some cases of leukocyte-mediated transfusion reactions (Van Buren et al., 1990) and in neonatal neutropenia. The NB 1 antigen is located on

Table 1. Molecular Targets of Naturally Occurring Antineutrophil Antibodies Fc Receptor Type III (CD16; NA1/NA2 antigen) Complement Receptor Type 3 (CR3, Mac-1, ~m152integrin) "TSH-receptor-like" molecule Actin-like molecule NB 1 antigen: 58--64 kd protein Myeloid precursor cell antigens

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a 58--64 kd glycoprotein that is anchored to the neutrophil surface membrane with a glycosyl-phosphoinositol linkage. Removal of carbohydrates reduces the apparent molecular mass to 45 kd but does not remove reactivity with NB 1 antisera. Surface expression of NB1 is slightly increased by stimulation of living neutrophils with the chemotactic peptide f-metleu-phe, and NB1 antigen is detected in purified secondary granules but not in primary granules by immunoblotting (Skubitz et al., 1991; Stroncek et al., 1990). The neutrophil adhesion glycoprotein complex CD 1 lb/CD 18 (CR3, Mac- 1, am~32 integrin) can be identified by antibodies from some patients with autoimmune neutropenia (Hartman and Wright, 1991). When sera from patients known to have antineutrophil IgG antibodies were evaluated by an immunobead "antigen capture" assay, anti-CD 1 lb/CD 18 autoantibodies were detected in 15% of sera positive for antineutrophil antibodies. The patient with the highest levels of anti-CD1 lb/CD 18 suffered recurrent skin infections and cellulitis, and died of respiratory failure during one of multiple episodes of pneumonia. Purified IgGs from several of these patients demonstrated effects on adhesion and/or opsonin receptor-mediated functions when tested with intact neutrophils in vitro (Hartman and Wright, 1991). Apparently these autoantibodies can, in some cases, interfere with neutrophil function, thereby amplifying the risk of infection associated with neutropenia. Some patients with Graves' disease and neutrope-

nia have autoantibodies to neutrophils that can be displaced by thyroid stimulating hormone (TSH), which binds to neutrophils rapidly, specifically and reversibly (Weitzman et al., 1985). Autoantibodies to TSH receptors in some patients with Graves' disease might bind to a cross-reactive "TSH-receptor-like" epitope on neutrophils. TSH inhibits the ability of serum from patients with Graves' disease to opsonize normal test neutrophils (Weitzman et al., 1985). These findings suggest a basis for the association of Graves' disease with neutropenia. Other antigens identified in some cases of immune neutropenia include actin-like molecules on neutrophil cell surfaces (Hartman et al., 1990) and molecules present on myeloid precursor cells (Hartman et al., 1994).

AUTOANTIBODIES

Terminology The term "antineutrophil antibodies" is commonly used to refer to antibodies that are found in the sera of patients with neutropenia and that are directed towards neutrophil membrane components (Table 2). These autoantibodies differ from "antineutrophil cytoplasmic antibodies" (ANCA) that are detected in the sera of patients with vasculitic disorders, as discussed elsewhere.

Table 2. Disorders Associated with Antineutrophil Antibodies Isolated autoimmune neutropenia Systemic autoimmune disease

RA, SLE, Hashimoto's thyroiditis, Graves' disease, Sj6gren's syndrome

Other cytopenias

ITP, autoimmune hemolytic anemia

Viral infections

EBV, HIV disease

Lymphoproliferative disorders

Hodgkin's disease, Non-Hodgkin's lymphoma, immunodeficiency syndromes

Bone marrow transplantation Autoimmune neutropenia of infancy and childhood Neonatal alloimmune neutropenia Drug ingestion

Penicillins, phenothiazines, antithyroid drugs (propylthiouracil), hydantoins, antiarrhythmics (procainamide), sulfa drugs (sulphasalazine), nonsteroidal anti-inflammatory agents (ibuprofen), levamisole

*Alloimmune neonatal neutropenia is caused by transplacental passage of maternal IgG antibodies against the infant's neutrophils and is also known as "isoimmune" neonatal neutropenia.

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phil cell surface (Hartman et al., 1990; McCallister et al., 1979). Indirect immunofluoresence microscopy involves the incubation of target neutrophils with serum, followed by detection of IgG or IgM binding using a fluoresceinated antihuman immunoglobulin (Verheugt et al., 1978). By flow cytometric techniques utilizing indirect immunofluorescence, antineutrophil antibodies are detected in about 35% of sera from patients with clinically suspected immune neutropenia (Maher and Hartman, 1993). Additional tests for the presence of immune complexes may need to be done, and some laboratories include a high-speed centrifugation step to remove immune complexes. Also, monoclonal antibodies to neutrophil Fc receptors may be used to block immune complex binding (McCullough et al., 1988). A cell:cell recognition assay has been used to detect antineutrophil opsonic activity in serum. This assay is based on the observation that normal neutrophils recognize other neutrophils opsonized with antibody and respond with an increase in glucose oxidation. Normal neutrophils are incubated with test serum, then exposed to "indicator" neutrophils, and enhanced glucose oxidation is measured by release of radioactive CO 2 from radiolabeled glucose (Weitzman et al., 1985). Autoantibodies directed against neutrophils can

Methods of Detection

Antineutrophil antibodies with anti-NB1 or antiCD1 l b/CD18 specificities cross-react in the ANCA indirect immunofluorescence test (CD1 l b/CD18 can be in granules also) (Stroncek et al., 1993b). Methods for detecting antineutrophil antibodies are reviewed extensively elsewhere (McCullough et al., 1988) and are presented briefly below. Agglutination assays detect aggregation of neutrophils on microscope slides or in chambers using light microscopy (Lalezari and Bernard, 1964). Unlike red blood cell agglutination assays, granulocyte agglutination procedures do not require the addition of an antiglobulin reagent, but rather rely on an energydependent mechanism of neutrophil self-association. Neutrophil-specific allelic antigens were defined by neutrophil agglutination assays using sera from mothers of infants with alloimmune neutropenia. Neutrophil-specific antigens identified in this fashion are described by the following nomenclature: NA1/ NA2, NB 1/NB2, NC1, ND 1, NE1, 9a (McCullough et al., 1988). Several methods detect antibody bound to the surface of intact neutrophils. Radioactive labeling with 125I-Staphylococcal protein A and 125I-anti-human IgG can both be used to detect IgG binding to the neutro80A

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Neutropenic Patients with IgG Antibodies to Bone Marrow CD34 Cells

F i g u r e 1. IgG antineutrophil antibodies can inhibit the growth of myeloid progenitor cells in clonal assay. The dashed lines represent the 95 % confidence interval for the mean CFU-GM for normal controls. IgG from some patients with antineutrophil antibodies (patients A, B, C) showed inhibition of CFU-GM, and IgG from some patients with antibodies to primitive bone marrow CD34 cells (patients D and E) strongly suppressed CFU-GM growth. (Reprinted with permission. Hartman et al., 1994).

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also bind to myeloid precursor cells in normal bone marrow (Harmon et al., 1984; Hartman et al., 1994). Sera from patients with progressively more severe neutropenia show a trend towards binding to more immature myeloid precursors, as well as to mature forms. In vitro clonal assays for hematopoietic progenitor cells document selective, antibody-mediated inhibition of myeloid progenitor cell growth in sera from patients with severe neutropenia (Levitt et al., 1983; Hartman et al., 1994) (Figure 1). Techniques directed at identifying the specific molecular targets of antineutrophil antibodies include immunoblotting, immunoprecipitation and antigen capture assays. The antigen capture technique uses monoclonal antibodies to immobilize neutrophil membrane molecules on polystyrene beads or multiwell plates. The immobilized molecules are incubated with sera, and specific antibodies are detected with anti-immunoglobulin reagents (Hartman and Wright, 1991).

CLINICAL UTILITY Disease Association

Multisystem autoimmune disorders often accompanied by autoimmune neutropenia include systemic lupus erythematosus (Hadley et al., 1987), Sj6gren's syndrome (Yamato et al., 1990), Graves' disease (Weitzman et al., 1985) and mixed autoimmune disorders (Table 2). Lymphoproliferative disorders can exhibit neutropenia that is not explained by tumor invasion of the bone marrow or by cytotoxic chemotherapy. An immune mechanism might explain certain cases of neutropenia associated with Hodgkin's disease, nonHodgkin's lymphomas and chronic lymphocytic leukemia (Chandor, 1988). Viral infections may be accompanied by immune neutropenia, in particular infections with Epstein-Barr virus (Schooley et al., 1984) and human immunodeficiency virus (HIV) (Klaassen et al., 1990; Stroncek et al., 1992). Autoantibodies to neutrophils appear in the asymptomatic stage of HIV infection and the prevalence of these autoantibodies further increases in the symptomatic stages. Autoimmune neutropenia may also occur following bone marrow transplant (Klumpp et al., 1992; Stroncek et al., 1993a). Drugs capable of inducing neutropenia secondary to immunologic reactions involving the bone marrow, include penicillins, phenothiazines, antithyroid drugs

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(e.g., propylthiouracil), hydantoins and antiarrhythmic agents such as procainamide (Wright, 1983; McCullough et al., 1988). Drug-associated antineutrophil antibodies capable of opsonizing normal neutrophils are dependent on drug concentration in patients taking semisynthetic penicillins (Weitzman and Stossel, 1978). Complement-dependent opsonization of neutrophils, as well as IgM-dependent opsonic activity, and IgG dependent/complement-independent opsonic activity, can be demonstrated in the sera of patients receiving drugs (Weitzman and Stossel, 1978). Extensive studies of quinine-dependent antibodies have identified drug-dependent epitopes on molecules of 60 kd and 85 kd on neutrophils (Stroncek, 1993c). Immunologic reactions involving drugs are usually spontaneous (they may occur in patients who have taken the drug previously without adverse effect) and are reversible upon discontinuation of the drug. Neutropenia of infancy and childhood is clearly an autoimmune syndrome (Lalezari et al., 1986). The median age at which neutropenia occurs is 8 months, with a range of 3--33 months, and a female/male ratio of 3:2. Mild, recurrent infections, commonly accompany a severe neutropenia (absolute neutrophil count 0 to 500); monocytosis and eosinophilia may be present. The median duration of disease is 20 months, and spontaneous resolution occurs in most patients by 5 years of age. Ten percent of patients have antibodies with specificity for the neutrophil antigens NA1 or NA2 (Lalezari et al., 1986). Neonatal neutropenia caused by transplacental passage of maternal antineutrophil antibodies can be accompanied by life-threatening infections. Spontaneous resolution, which occurs by about 2 months of age, is associated with the physiologic loss of maternal antibody. Two forms of immune neonatal neutropenia are described: (1) "alloimmune neonatal neutropenia", caused by alloantibody directed against neutrophil-specific antigens of the infant and of the father, and (2)"transitory neonatal neutropenia", in newborn infants of women who have an autoimmune neutropenia, due to maternal autoantibody that is cross-reactive with the infant's neutrophils (McCullough et al., 1988). Other cytopenias such as immune thrombocytopenic purpura (ITP) or autoimmune hemolytic anemia (AHA) may be associated with autoimmune neutropenia (Linker et al., 1980). The combination of ITP and AHA is commonly known as Evans' syndrome (Evans et al., 1951); patients who carry this diagnosis frequently have autoimmune neutropenia as well, and

the term "immunopancytopenia" is more appropriate for this condition (Pui et al., 1980). In childhood, the association of other cytopenias in addition to neutropenia implies a worse prognosis than isolated immune neutropenia (Lalezari et al., 1986) and patients with demonstrable antibodies against multiple hematopoietic cell types can suffer a chronic relapsing course over several years (Miller et al., 1983). These patients usually do not recover spontaneously and require more aggressive therapy than the common form of autoimmune neutropenia of infancy. Combined immunosuppressive therapy can be successful in inducing transient or persistent remissions, including normalization of the peripheral blood and disappearance of antibodies directed against the affected cell lines (Wiesneth et al., 1985). Despite the variety and ingenuity of techniques to detect antineutrophil antibodies, the usefulness of these methods in patient management remains limited. Some assays are not specific for antineutrophil antibodies, because immunoglobulins attach naturally to neutrophil Fc receptors, and positive results may not distinguish between immune complexes attached to these receptors and immunoglobulin fixed to specific antigenic sites. Moreover, certain techniques, such as

the agglutination assay, measure phenomena that do not depend necessarily on cell-specific immunoglobulin. Positive assay results may not correlate with the presence of neutropenia, particularly among patients with underlying autoimmune disease. Furthermore, many patients with apparently immune-mediated neutropenia have negative results in one or more of these assays. Thus, these techniques do not consistently provide information that determines specific approaches for management of individual patients. Further studies are clearly needed to define the molecular specificities of antineutrophil antibodies.

REFERENCES

autoimmune neutropenia. Blood 1994;84:625-631. Huizinga TW, Kleijer M, Tetteroo PA, Roos D, von dem Borne AE. Biallelic neutrophil Na-antigen system is associated with a polymorphism on the phospho-inositol-linked Fc gamma receptor III (CD16). Blood 1990;75:213--217. Klaassen RJ, Mulder JW, Vlekke AB, Eeftinck Schattenkerk JK, Weigel HM, Lange JM, von dem Borne AE. Autoantibodies against peripheral blood cells appear early in HIV infection and their prevalence increases with disease progression. Clin Exp Immunol 1990;81:11-- 17. Klumpp TR, Herman JH, Macdonald JS, Schnell MK, Mullaney M, Mangan KF. Autoimmune neutropenia following peripheral blood stem cell transplantation. Am J Hematol 1992;41:215--217. Lalezari P, Bernard GE. Improved leukocyte antibody detection with prolonged incubation. Vox Sang 1964;9:664-672. Lalezari P, Khorshidi M, Petrosova M. Autoimmune neutropenia of infancy. J Pediatr 1986;109:764-769. Levitt LJ, Ries CA, Greenberg PL. Pure white-cell aplasia. Antibody-mediated autoimmune inhibition of granulopoiesis. N Engl J Med 1983;308:1141--1146. Linker CA, Newcom SR, Nilsson CM, Wolf JL, Shuman MA. Combined idiopathic neutropenia and thrombocytopenia: evidence for an immune basis for the syndrome. Ann Intern Med 1980;93:704--707. Maher GM, Hartman KR. Detection of antineutrophil autoanti-

Bux J, Mueller-Eckhardt C. Autoimmune neutropenia. Semin Hematol 1992;29:45--53. Chandor SB. Autoimmune phenomena in lymphoid malignancies. Clin Lab Med 1988;8:373--384. Dale DC, Guerry D 4th, Wewerka JR, Bull JM, Chusid MJ. Chronic neutropenia. Medicine (Baltimore) 1979;58:128--144. Evans RS, Takahashi K, Duane RT, Payne R, Lie CK. Primary thrombocytopenic purpura and acquired hemolytic anemia. Arch Intern Med 1951;87:48--65. Hadley AG, Byron MA, Chapel HM, Bunch C, Holburn AM. Antigranulocyte opsonic activity in sera from patients with systemic lupus erythematosus. Br J Haematol 1987;65:61--65. Harmon DC, Weitzman SA, Stossel TP. The severity of immune neutropenia correlates with the maturational specificity of antineutrophil antibodies. Br J Haematol 1984;58:209-215. Hartman KR, Mallet MK, Nath J, Wright DG. Antibodies to actin in autoimmune neutropenia. Blood 1990;75:736-743. Hartman KR, Wright DG. Identification of autoantibodies specific for the neutrophil adhesion glycoproteins CD1 l b/ CD18 in patients with autoimmune neutropenia. Blood 1991 ;78:1096--1104. [-Iartman KR, LaRussa VF, Rothwell SW, Atolagbe TO, Ward FT, Klipple G. Antibodies to myeloid precursor cells in

CONCLUSION Antineutrophil antibodies can be detected in association with neutropenia in a variety of clinical conditions, and detection of these antibodies may help to establish the diagnosis of autoimmune neutropenia in many cases. The large number of negative antineutrophil antibody studies, in patients with apparent autoimmune neutropenia, limits the use of these assays as a predictor of outcome in many cases.

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bodies by flow cytometry: use of unfixed neutrophils as antigenic targets. J Clin Lab Anal 1993;7:334--340. McCallister JA, Boxer LA, Baehner RL. The use and limitation of labeled staphylococcal protein A for study of antineutrophil antibodies. Blood 1979;54:1330-- 1337. McCullough J, Clay M, Press C, Kline W, eds. Granulocyte Serology. Chicago: ASCP Press, 1988. Miller B A, Schultz Beardsley D. Autoimmune pancytopenia of childhood associated with multisystem disease manifestations. J Pediatr 1983;103:877-881. Ory PA, Clark MR, Kwoh EE, Clarkson SB, Goldstein IM. Sequences of complementary DNAs that encode the NA1 and NA2 forms of Fc receptor III on human neutrophils. J Clin Invest 1989;84:1688--1691. Pui CH, Wilimas J, Wang W. Evans syndrome in childhood. J Pediatr 1980;97:754-758. Schooley RT, Densen P, Harmon D, Felsenstein D, Hirsch MS, Henle W, Weitzman S. Antineutrophil antibodies in infectious mononucleosis. Am J Med 1984;76:85-90. Shastri KA, Logue GL. Autoimmune neutropenia. Blood 1993 ;81:1984-- 1995. Skubitz KM, Stroncek DF, Sun B. Neutrophil-specific antigen NB 1 is anchored via a glycosyl-phosphatidylinositol linkage. J Leukoc Biol 1991;49:163--171. Stroncek DF, Skubitz KM, McCullough JJ. Biochemical characterization of the neutrophil-specific antigen NB1. Blood 1990;75:744--755. Stroncek DF, Kline WE, Clay ME, Plachta LB, Stricker RB, Hollander H, Greenspan JS, Shubitz KM. Antibodies to granulocytes in patients infected with human immunodeficiency virus. J Lab Clin Med 1992;119:724--731. Stroncek DF, Shapiro RS, Filipovich AH, Plachta LB, Clay ME. Prolonged neutropenia resulting from antibodies to neutrophil-specific antigen NB 1 following marrow transplantation. Transfusion 1993a;33:158--163.

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Stroncek DF, Egging MS, Eiber GA, Clay ME. Neutrophil alloantibodies react with cytoplasmic antigens: a possible cause of false-positive indirect immunofluorescence assays for antibodies to neutrophil cytoplasmic antigens. Am J Kidney Dis 1993b;21:368-373. Stroncek DF, Shankar RA, Herr GP. Quinine-dependent antibodies to neutrophils react with a 60-kd glycoprotein on which neutrophil-specific antigen NB 1 is located and an 85kd glycosyl-phosphatidylinositol-linked N-Glycosylated plasma membrane protein. Blood 1993c;81:2758--2766. Van Buren NL, Stroncek DF, Clay ME, McCullough J, Dalmasso AP. Transfusion-related acute lung injury caused by an NB2 granulocyte-specific antibody in a patient with thrombotic thrombocytopenic purpura. Transfusion 1990;30:42--45. Verheugt FW, von dem Borne AE, van Noord-Bokhorst JC, Engelfriet CP. Autoimmune granulocytopenia: the detection of granulocyte autoantibodies with the immunofluorescence test. Br J Haematol 1978;39:339--350. Weitzman SA, Stossel TP. Drug-induced immunological neutropenia. Lancet 1978;1:1068--1072. Weitzman SA, Stossel TP, Harmon DC, Daniels G, Maloof F, Ridgway EC. Antineutrophil autoantibodies in Graves' disease. Implications of thyrotropin binding to neutrophils. J Clin Invest 1985;75:1119-123. Wiesneth M, Pflieger H, Frickhofen N, Heimpel H. Idiopathic combined immunocytopenia. Br J Haematol 1985;61:339-348. Wright DG. Autoimmune leukopenia. In: Lichtenstein LM, Fauci AS, eds. Current Therapy in Allergy and Immunology. St. Louis: BC Decker, 1983:277--281. Yamato E, Fujioka Y, Masugi F, Nakamura M, Tahara Y, Kurata Y, Ogihara T. Autoimmune neutropenia with antineutrophil autoantibody associated with Sj6gren' s syndrome. Am J Med Sci 1990;300:102--103.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

NUCLEAR ENVELOPE PROTEIN AUTOANTIBODIES Konstantin N. Konstantinov, M.D., Ph.D.

W.M. Keck Autoimmune Disease Center, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA

HISTORICAL NOTES The subset of antinuclear antibodies yielding peripheral or rim staining of the nucleus in immunofluorescence microscopy has been recognized for a long time. Although ring-like staining of the nucleus initially was specified as anti-DNA (Casals et al., 1964), this pattern is now reserved for antibodies to nuclear envelope components only. In 1983, serum from a 24-year-old female patient with linear scleroderma was shown to react specifically with highly conserved determinants of nuclear lamins A and C (McKeon et al., 1983). Subsequently, several other nuclear envelope antigens targeted by autoantibodies were evaluated.

AUTOANTIGENS Definition The nuclear envelope (NE) forms the boundary of the nucleus in eukaryotic cells. It has three main components: inner and outer nuclear membranes enclosing a lumenal space, the nuclear pore complexes (NPCs) and the nuclear lamina (Dessev, 1992). Some of the major nuclear envelope proteins and their membrane topology are shown in Figure 1.

Origin/Structure The nuclear pore complexes are large supramolecular structures that span the NE at regions where the inner and outer nuclear membranes are joined (Rout and Wente, 1994). They provide passageways between the nucleus and cytoplasm, through which proteins and

RNAs are actively transported by selective, signalmediated mechanisms. The NPC contains a number of distinct substructures, including a central "spoke" assembly attached to peripheral "rings", framing its cytoplasmic and nuclear sides. Attached to this generally symmetrical framework are sets of fibrils emanating from both the nucleoplasmic and cytoplasmic rings, while a central "plug" or putative "transporter" is located in the middle of the spoke assembly. Currently, only a relatively small number of the estimated 100-200 NPC polypeptides are well characterized. In vertebrates, these include a set of at least eight peripheral membrane polypeptides modified with O-linked N-acetylglycosamine, some of which appear to assist mediated transport across the NE. A particularly abundant transmembrane glycoprotein (gp210) with a single membrane-spanning segment located near its C-terminus is thought to provide a membrane anchor involved in NPC assembly (Greber et al., 1990). More recently, a large coiled coil protein (Tpr) localized exclusively to the cytoplasmic surface of the NPCs was described (Byrd et al., 1994). Tpr, previously detected in oncogenic fusions with the kinase domains of the Trk, Met and Raf proto-oncogenes, is a component of the cytoplasmic fibrils of the NPCs and may be involved in ligand docking. The outer nuclear membrane of NE contains attached ribosomes and, at many points, is continuous with the endoplasmic reticulum; the inner nuclear membrane has a unique protein composition and is lined by a filamentous meshwork called the nuclear lamina. The nuclear lamina contains a polymeric assembly of nuclear lamins, members of the intermediate filament protein superfamily (Georgatos et al., 1994), as well as a number of biochemically minor lamina-

561

Figure 1. Diagram of the eukaryotic nuclear envelope. Shown are the major architectural components and autoimmune antigens, their membrane topology and binding characteristics. See text for discussion. Adapted with modifications from Gerace and Foisner, 1994. Permission to use and modify the published figure by Gerace and Foisner has been granted.

associated polypeptides. Mammalian lamins are classified into A and B subtypes, based on their sequence properties and state of membrane association during mitosis. A type lamins (lamins A and C) are identical throughout most of their sequences and are likely to be alternatively spliced products of the same gene. They are expressed only during or following terminal differentiation in most cells and are not membrane-associated during mitosis. B type lamins (B 1 and B2), which are products of separate genes, are present in somatic cells throughout development and remain membrane-attached during M-phase. Both Aand B-type lamins bind to chromatin or DNA. Thus, lamins are thought to provide a structural framework for NE and an anchoring site at the nuclear periphery for interphase chromosomes. Currently, little is known about the molecular basis for the lamina-inner nuclear membrane interaction. Because lamins are peripheral membrane proteins that lack membrane-integrated regions, the interaction of lamins with membranes must be indirect. Certain lamin isotypes (lamin B 1 and B2) are found to be

562

posttranslationally modified by farnesylation of the Cterminal "CaaX" sequence, and this covalently bound lipid is potentially important for membrane association of these proteins. However, by analogy with other membranes it also is likely that integral membrane proteins are involved in association of the lamina with the inner nuclear membrane. A number of unique integral membrane proteins, restricted to the nuclear membrane are able to bind to lamins in vitro and therefore might be involved in mediating the lamina-membrane interaction (Gerace and Foisner, 1994). These integral membrane proteins include lamina-associated polypeptides (LAPs) 1 and 2, and a 54/58 kd polypeptide found in avian and human cells, proposed to act as "lamin B receptor". LAP 1, of which three related isoforms exist, interacts with both A- and B-type lamins, while LAP 2 binds only to lamin B. Interestingly, LAP 2 also binds to mitotic chromosomes in a phosphorylation-regulated manner. Together, these data suggest that integral membrane proteins have a key role in attaching lamins to the NE and in NE reassembly.

AUTOANTIBODIES Autoantibodies to NE components are relatively infrequent in routine ANA serology and only a few antigens recognized by these antibodies are characterized. Antibodies against Nuclear Lamins. Antibodies to nuclear lamins are reported in some patients with autoimmune and inflammatory diseases. Because of similarities in the structure of lamins A and C, autoantibodies against lamin A generally recognize lamin C. The antibodies specific for lamin C found in some patients with chronic hepatitis delta virus infection appear to recognize an epitope in the carboxy-terminal region of lamin C, which contains only six amino acids not found in the primary structure of lamin A (Wesierska-Gadek et al., 1990; Konstantinov et al., 1990). In SLE patients, both the initiation and maintenance of lamin B antibody production is independent of the level of polyclonal B-lymphocyte activation (Chou et al., 1991). In addition, the antibodies recognize highly specific lamin B epitopes (Chou and Reeves, 1992). These findings, together with the restricted heterogeneity of lamin B autoantibodies (Reeves and Ali, 1989), suggest that "self' lamin B molecules are responsible for the generation of lamin B autoantibodies by specifically stimulating only one or a few lymphocyte clones. Recently, the fourth minor lamin polypeptide (lamin B2) has also been described as a target of autoimmune recognition (Brito et al., 1994). There is uncertainty about the prevalence and specificity of antilamin antibodies which can occur as either natural antibodies, cross-reacting anti-intermediate filament antibodies or antigen-driven autoantibodies (Senecal et al., 1993). In general, despite the fact that results vary considerably depending on what method is used for detection, the lamin autoantibodies in autoimmune disease patients are usually at high titer and of the IgG isotype. Antibodies to gp210. Between 10--25% of patients with primary biliary cirrhosis (PBC) have autoantibodies against gp210 (Courvalin et al., 1990a; Lassoued et al., 1990). These are highly specific and are sometimes present in patients without mitochondrial antibodies (Nickowitz et al., 1994). Autoantibodies from patients with PBC recognize at least two different epitopes. The first one is located

in the cytoplasmic carboxy-terminal tail of gp210, where the epitope is contained within a stretch of 15 amino acids (Nickowitz and Worman, 1993). The recognized amino acid sequence is similar to a portion of E. coli mutY gene product, suggesting that antigp210 antibodies may arise by molecular mimicry of a bacterial antigenic determinant (Nickowitz and Worman, 1993). The second epitope is positioned within the glycosylated amino-terminal domain of gp210, and it seems that the carbohydrate residues are an essential part of this epitope (Wesierska-Gadek et al., 1995). Antibodies to Tpr/p265. In a collection of 55 human autoimmune sera that react with the NE by indirect immunofluorescence (IIF) (Konstantinov et al., 1995), nine sera reacted specifically with a 265 kd band (Tpr) as well as a series of 175--265 kd bands. The multiple reactive bands are likely to be proteolytic products of p265/Tpr (Byrd et al., 1994). By IIF staining of cultured cells, antibodies to Tpr react exclusively with the nuclear envelope in a punctate pattern. A 180 kd antigen of the NE is detected in a variety of vertebrate cells with human autoantibodies (Wilken et al., 1993). In immunoelectron microscopy of isolated NEs of Xenopus oocytes, this antigen is localized to the cytoplasmic ring and associated fibrils of the NPC, similar to the localization described for Tpr. In view of the properties of this 180 kd antigen, including the possible existence of higher molecular mass cross-reacting forms, this 180 kd antigen probably corresponds to a proteolytic product of Tpr. Antibodies to p58. Sera from some patients with PBC recognize p58 of avian erythrocyte NEs, an integral protein of the inner nuclear membrane that selectively binds lamin B, as well as its rat (p61) and human (p59) homologues (Courvalin et al., 1990b). Furthermore, p58 autoantibodies are anti-idiotypic to some lamin B autoantibodies, this suggests that the epitope recognized by these antibodies could be the binding site of lamin B to p58 (Lassoued et al., 1991). Antibodies to LAP 1 and LAP 2. Compared to lamins, LAP 1 and LAP 2 are relatively minor NE components, since they are approximately 2--4% as abundant as individual lamins. Recently, a study analyzing sera selected for NE IIF labeling of cultured cells showed that antibodies to LAP 2 were present in 29% of all sera, compared to frequencies of 31 and

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14% for lamins A/C and B1/B 2, respectively (Konstantinov et al., 1995). LAP 1 autoantibodies were found in only 9% of these sera. These results suggest that LAP 2 may be among one of the most prominent autoantigens of the NE. Methods of Detection The most widely used screening method for detection of antibodies to NE is IIF which reveals characteristic NE staining patterns (Figure 2). Because nuclei in HEp-2 cells (as well as in many other cultured cells) are not spherical structures but rather are flattened against the plane of the substratum, most focal planes that image the nucleus will include the dorsal or ventral nuclear surface as well as the nuclear edge. Hence, antigens that are restricted to the nuclear envelope will present a pattern of relatively uniform

Figure 2. Localization of nuclear envelope (NE) antigens by immunofluorescence microscopy on HEp-2 cells. Shown are labeling obtained with human autoantibodies to lamins A/C (continuous, rim-like pattern. A: and human autoantibodies to Tpr (B-C). Images in B and C represent two separate focal planes within the same field. In optical sections containing the nuclear surface, the antibodies to Tpr (and NPC in general) give discontinuous punctate labeling of the NE (panel C), while images of the equatorial regions of nuclei demonstrate better the nuclear rim reactivity (B, arrow). 564

nuclear labeling superimposed, to various extents, on peripheral rim labeling. The latter is usually described as continuous (lamins, LAPs) and discontinuous (NPCs) rim-like staining of the nuclear periphery in interphase cells (Martin et al., 1995). Staining of chromosomes in mitotic cells excludes antibodies to dsDNA and histones which can simulate peripheral nuclear labeling. Cryostat rat liver sections used for routine ANA testing do not discriminate between antibodies to dsDNA, NPCs, lamins or LAPs. IIF analysis on cells treated in situ with DNase I and/or RNase A, nonionic detergents and salt is most helpful in cases in which the serum contains multiple reactivities (Brito et al., 1994). Removing most of the cytoplasmic intermediate filaments and nuclear proteins makes evident the NE-associated fluorescence, which is otherwise difficult to detect. NE antigens can be readily identified by immunoblotting using NEs isolated from rat liver (Figure 3) or NE-enriched fraction from cultures cells (Martin et al., 1995). Following the fractionation behavior of an autoantigen with immunoblotting during extraction of NEs with nonionic detergents and salts is a very useful procedure, which could determine whether the

Figure 3. Immunoblot analysis of autoantibodies to nuclear envelope (NE) components. Salt-washed NEs from rat liver nuclei were probed with patient sera reacting with lamin A/C (lane 1), lamin B (lane 2), LAP 2 (lane 3), gp210 (lane 4), Tpr (lane 5). Molecular weight markers are designated on the left.

target antigen is peripheral or integral membrane protein (Konstantinov et al., 1995). By operational criteria, the integral membrane proteins are resistant to extraction by alkaline pH or chaotropic agents, presumably due to a membrane-integrated domain(s). In general, the IIF staining pattern can sometimes be difficult to interpret, and only approximates an antibody titer. ELISA is rapid and relatively simple and allows detection of NE autoantibodies in an interference-free specific assay. ELISA assays for gp210 (Tartakovsky and Worman, 1995) and lamin B 1 (Uthman et al., 1994) using human recombinant proteins were used successfully to screen large groups of patients.

brain or skin vasculitis (Lassoued et al., 1988). AntiLAP 1/LAP 2 and anti-Tpr antibodies also lack specific disease correlation. Whether some of these autoantibodies may have value in diagnosis or patient" classification remains to be tested by examination of specific disease groups. Likewise, some of the antibodies to NEs might be a component of autoantibody profiles, which correlate better with specific diseases. Two recent studies found combined occurrence of antilamin and antiphospholipid antibodies (Konstantinov et al., 1992; Uthman et al., 1994) in some patients with SLE and others with primary antiphospholipid syndrome.

CONCLUSION CLINICAL UTILITY

Disease Associations To date, a clinical association is known only for antibodies to gp210 and p58, which appear in a subset of primary biliary cirrhosis patients (Courvalin et al., 1990a; 1990b). Antibodies to nuclear lamins are reported in diverse disease conditions. Most of these fall into the broad category of systemic rheumatic diseases (Reeves et al., 1987; Konstantinov et al., 1992), chronic active hepatitis (Wesierska-Gadek et al., 1988) and a peculiar connective tissue disease subset characterized by steroid-responsive blood cytopenia, hepatitis and

REFERENCES Brito J, Biamonti G, Caporali R, Montecucco C. Autoantibodies to human nuclear lamin B2 protein. Epitope specificity in different autoimmune diseases. J Immunol 1994;153:2268-2277. Byrd DA, Sweet DJ, Pante N, Konstantinov KN, Guan T, Saphire A, Mitchell PJ, Cooper CS, Aebi U, Gerace L. Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex. J Cell Biol 1994;127:1515-1526. Chou CH, Ali SA, Roubey R, Buyon J, Reeves WH. Onset and regulation of antilamin B autoantibody production is independent of the level of polyclonal activation. Autoimmunity 1991;8:297-305. Chou CH, Reeves WH. Recognition of multiple epitopes in the coiled-coil domain of lamin B by human autoantibodies. Mol Immunol 1992;29:1055--1064. Casals S, Friou G, Myers L. Significance of antibody to DNA

The autoantibodies to NE components constitute a large family and subsequent studies are likely to identify new members. Some of these autoantibodies are clinically useful in diagnosis/classification of patients with liver diseases. For others, the clinical association remains to be established. Analysis of the autoimmune response to NE antigens may give some insight in the nature of autoimmunity. Moreover, because autoantibodies frequently target highly conserved, functionally important regions of the proteins, they may provide valuable tools to understand structural and functional aspects of their cognate antigens.

in systemic lupus erythematosus. Arthritis Rheum 1964;7: 379-390. Courvalin JC, Lassoued K, Bartnik E, Blobel G, Wozniak RW. The 210-kd nuclear envelope polypeptide recognized by human autoantibodies in primary biliary cirrhosis is the major glycoprotein of the nuclear pore. J Clin Invest 1990a;86: 279--285. Courvalin JC, Lassoued K, Worman H, Blobel G. Identification and characterization of autoantibodies against the nuclear envelope lamin B receptor from patients with primary biliary cirrhosis. J Exp Med 1990b;172:961-967. Dessev, G. Nuclear envelope structure. Curr Opin Cell Biol 1992;4:430-435. Georgatos SD, Meier J, Simos G. Lamins and lamin-associated proteins. Curr Opin Cell Biol 1994;6:347-353. Gerace L, Foisner R. Integral membrane proteins and dynamic organization of the nuclear envelope. Trends Cell Biol 1994;4:127--131. Greber UF, Senior A, Gerace L. A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide 565

with a large lumenal domain and a small cytoplasmic tail. EMBO J 1990;9:1495--1502. Konstantinov KN, Galcheva-Gargova Z, Hoier-Madsen M, Wiik A, Ullman S, Halberg P, Vejlsgaard G. Autoantibodies to lamins A and C in sera of patients showing peripheral fluorescent antinuclear antibody pattern on HEp-2 cells. J Invest Dermatol 1990;95:304--308. Konstantinov KN, Halberg P, Wiik A, Hoier-Madsen M, Wantzin P, Ullman S, Galcheva-Gargova Z. Clinical manifestations in patients with autoantibodies specific for nuclear lamin proteins. Clin Immunol Immunopathol 1992;62:112118. Konstantinov KN, Foisner R, Byrd D, Liu FT, Tsai WM, Wiik A, Gerace L. Integral membrane proteins associated with the nuclear lamina are novel autoimmune antigens of the nuclear envelope. Clin Immunol Immunopathol 1995;74:89--99. Lassoued K, Guilly MN, Danon F, Andre C, Dhumeaux D, Clauvel JP, Brouet JC, Seligman M, Courvalin JC. Antinuclear antibodies specific for lamins. Characterization and clinical significance. Ann Intern Med 1988;108:829-833. Lassoued K, Brenard R, Degos R, Courvalin JC, Andre C, Danon F, Brouet JC, Zine-el-Abidine Y, Degott C, Zafrani S, et al. Antinuclear antibodics directed to a 200 kD polypeptide of the nuclear envelope in primary biliary cirrhosis. Clinical and immunological study of a series of 150 patients with primary biliary cirrhosis. Gastroenterology 1990;99:181-186. Lassoued K, Danon F, Brouet JC. Human autoantibodies to lamin B receptor are also anti-idiotypic to certain antilamin B antibodies. Eur J Immunol 1991;21:1959--1962. Martin L, Crimaudo C, Gerace L. cDNA cloning and characterization of lamina-associated polypeptide 1C (LAP1C), an integral protein of the inner nuclear membrane. J Biol Chem 1995 ;270: 8822-8828. McKeon FD, Tuffanelli DL, Fukuyama K, Kirschner MW. Autoimmune response directed against conserved determinants of nuclear envelope proteins in a patient with linear scleroderma. Proc Natl Acad Sci 1983;80:4374-4378. Nickowitz RE, Worman HJ. Autoantibodies from patients with primary biliary cirrhosis recognize a restricted region within

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the cytoplasmic tail of nuclear pore membrane glycoprotein gp210. J Exp Med 1993;178:2237--2242. Nickowitz R, Wozniak R, Schaffner FR, Worman H. Autoantibodies against integral membrane proteins of the nuclear envelope in patients with primary biliary cirrhosis. Gastroenterology 1994;106:193--199. Reeves WH, Chaudhary N, Salerno A, Blobel G. Lamin B autoantibodies in sera of certain patients with systemic lupus erythematosus. J Exp Med 1987;165:750-762. Reeves WH, Ali SA. Preferential use of lamda chain in lamin B autoantibodies. J Immunol 1989; 143:3614--3618. Rout MP, Wente SR. Pores for thought: nuclear pore complex proteins. Trends Cell Biol 1994;4:357--365. Senecal JL, Ichiki S, Girard D, Raymond Y. Autoantibodies to nuclear lamins and to intermediate filament proteins: natural, pathologic or pathogenic? J Rheumatol 1993 ;20:211--219. Tartakovsky F, Worman HJ. Detection of gp210 autoantibodies in primary biliary cirrhosis using a recombinant protein containing the predominant autoepitope. Hepatology 1995; 21:495-500. Uthman I, Guimond M, Labbe P, Raynauld JP, Raymond Y, Senecal JL. Clinical significance of IgG autoantibodies to nuclear lamin B1. Arthritis Rheum 1994;37:S174. Wesierska-Gadek J, Penner E, Hitchman E, Saucrmann G. Antibodies to nuclear lamins in autoimmune liver disease. Clin Immunol Immunopathol 1988; 19:107-115. Wesierska-Gadek J, Penner E, Hitchman E, Sauermann G. Antibodies to nuclear lamin C inchronic hepatitis delta virus infection. Hepatology 1990;12i 1129-1133. Wesierska-Gadek J, Hohenauer H, Hitchman E, Penner E. Autoantibodies from patients with primary biliary cirrhosis preferentially react with amino-terminal domain of nuclear pore complex glycoprotein gp210. J Exp Med 1995;182: 1159--1162. Wilken N, Kossner U, Senecal JL, Scheer U, Dabauvalle MC. Nup 180, a novel nuclear pore complex protein localizing to the cytoplasmic ring and associated fibrils. J Cell Biol 1993;123:1345--1354.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

NUCLEOLAR AUTOANTIBODIES Marc Monestier, M.D., Ph.D.

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA

HISTORICAL NOTES

Antinucleolar antibodies (ANoA) include several autoantibody populations directed against molecules that are uniquely or predominantly found in the nucleolus. The recognition that autoantibodies can react with the nucleolus came in the early 1960s when ANoA were detected by indirect immunofluorescence (IIF) in the sera of patients with autoimmune disease (especially scleroderma/systemic sclerosis), but not in normal individuals (Beck et al., 1962). The relationship between ANoA and scleroderma is amply confirmed. ANoA can occasionally be found in other systemic autoimmune diseases and in some cancer patients (Reimer and Tan, 1993). Like other antinuclear antibodies, ANoA are instrumental in the identification and the characterization of their autoantigenic targets. The antinucleolar specificities that are associated with autoimmunity are individually described below.

THE AUTOANTIGENS

The nucleolus, a membraneless organelle in the interphase nucleus, is the site of ribosome biogenesis (Hernandez-Verdun, 1991; Melese and Xue, 1995). Depending upon their level of activation, cells can contain from zero to five nucleoli. In metazoan cells, three morphological domains can be microscopically defined within the nucleolus: (1) several lightly stained "fibrillar centers" (FC) are surrounded and interconnected by (2) densely stained regions known as "dense fibrillar components" (DFC); (3) the entire fibrillar region is embedded within a "granular component" (GC). How these domains relate to the

various phases of ribosome formation is still controversial. In the first step, the genes that code for the precursor molecules of ribosomal RNA (rRNA) are transcribed by RNA polymerase I. The resulting prerRNA is processed into mature rRNA by several small nucleolar ribonucleoprotein complexes (snoRNP) composed of nucleolar proteins bound to RNA molecules. The exact compositions and roles of the various snoRNP are not yet fully elucidated. Next; rRNA and ribosomal proteins are assembled into ribosomes with the help of several nucleolar nonribosomal proteins.

THE AUTOANTIBODIES Methods of Detection/Disease Associations Antinucleolar Antibodies. Like other antinuclear specificities, ANoA are routinely detected by IIF using a variety of cell substrates (Fritzler, 1992). Estimates of ANoA prevalence range from 8--43% in scleroderma patients (Reimer et al., 1988; Bernstein et al., 1982). The discrepancies among the studies stem in part from differences in IIF procedures such as the cell substrate utilized. HEp-2 cells are particularly suitable, because each cell possesses several large nucleoli. Diagnostic kits with slides of HEp-2 cells are available from several manufacturers. Three different types of nucleolar staining correlate with the three most frequent antinucleolar specificities in scleroderma (Bernstein et al., 1982) which may or may not be associated with concomitant nuclear staining: 1. The speckled or punctate pattern consists of small and distinct speckles in the nucleolus which are often separated by a dark halo from the nuclear

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staining. The nucleolar-organizing regions appear as fluorescent dots in mitotic cells. This pattern is evocative of reactivity with RNA polymerase I. 2. In the homogeneous pattern, the nucleoli are evenly stained. This reactivity is usually associated with antibodies to the PM-Scl antigen. 3. The clumpy nucleolar staining consists of densely clustered granules which are larger than the abovementioned nucleolar speckles. Chromosomal staining can also be observed in metaphase cells, and this pattern evokes the presence of antibodies to snoRNP antigens. Treatment with actinomycin D or 5,6-dichloro-1-~-Dribofuranosyl-benzimidazole results in the segregation of the nucleolus fibrillar component from the granular component (Reimer et al., 1987). The treated cells can be used as IIF substrates for further characterization of the nucleolar reactivity, but this technique is not routinely utilized. A more commonly used procedure is immunoprecipitation (IP) which involves preparation of cell extracts, incubation of the extracts with ANoA linked to a carrier (such as protein G-agarose), electrophoretic separation of the molecules bound by the ANoA and detection of these molecules by several possible techniques (Craft and Hardin, 1992). Metabolic labeling of the cells with 35S-methionine allows visualization of immunoprecipitated proteins; RNA can be detected either by silver staining or, more sensitively by 3'-end labeling with 32p-cytidine 3',5'biphosphate (Monestier et al., 1994). Some antinucleolar specificities can also be visualized by immunoblotting (IB) (Chan and Pollard, 1992), although detection of certain nucleolar antigens such as fibrillarin is difficult. Recent studies of ANoA involve more novel approaches such as the use of recombinant nucleolar antigens in immunoassays (Kasturi et al., 1995) or of antisense riboprobes for snoRNA (Verheijen et al., 1994). Anti-RNA Polymerase I Antibodies. Located in the FC region, the RNA polymerase I enzyme complex is responsible for the transcription of genes that code for the precursor molecules of rRNA. Anti-RNA polymerase I antibodies immunoprecipitate a complex of at least 13 proteins (including several phosphoproteins) with molecular weights ranging from 12.5 kd to 210 kd (Reimer et al., 1987). No RNA is apparently associated with this complex. These autoantibodies are found in about 4% of scleroderma patients and appear restricted to this autoimmune disease. In a limited

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study, most patients with anti-RNA polymerase I antibodies suffered from the more severe diffuse form of scleroderma and were at risk of developing renal complications (Reimer et al., 1988). Anti-PM-Scl, Antibodies. Anti-PM-Scl antibodies recognize a multimolecular complex of unknown function located in the GC region. The complex precipitated by IP is formed of 11 to 16 proteins and is apparently not associated with any nucleic acid. By IB, most anti-PM-Scl sera react with a 100 kd protein, half of them also react with a protein migrating at about 75 kd and a few with a 37 kd protein (Ge et al., 1994). Two cloned cDNA corresponding to the 100kd protein show some sequence similarity to serine/ threonine kinases (Bluthner and Bautz, 1992; Ge et al., 1992). A full-length cDNA has also been cloned for the 75 kd protein which has a predicted molecular weight of 39 kd, but an aberrant electrophoretic migration is due to a stretch of charged residues in its carboxy terminus (Alderuccio et al., 1991). There is no cross-reactivity between antibodies to the 100kd and the 75 kd proteins and, accordingly, these two proteins do not share any sequence similarity. Fifty to seventy percent of patients with anti-PM-Scl antibodies present with an overlap of scleroderma and polymyositis or dermatomyositis. They also have a high frequency of Raynaud' s phenomenon, interstitial lung disease and arthritis. Most patients with anti-PMScl antibodies are HLA-DR3-positive and do not have other myositis- or scleroderma-specific antibodies, further indicating that anti-PM-Scl antibodies help define a distinct group of autoimmune patients (Oddis et al., 1992). ANTI-snoRNP Antibodies. snoRNP are complexes of RNA and proteins mostly localized in the DFC or GC of the nucleolus. The U3 RNP is the most abundant snoRNP in the nucleolus where it plays a role in pre-rRNA processing. The U3 RNP complex is composed of a 217 nucleotide RNA and of several proteins, including fibrillarin and at least five unidentified proteins. By IB, a large fraction of anti-snoRNP antibodies in scleroderma are directed against fibrillarin, a 34 kd basic protein rich ih dimethylarginine and glycine which is found in the DFC and is a component of the U3 RNP. In the nucleolus, fibrillarin is also a component of several other less abundant snoRNP, known as U8, U 13, U 14, U 15 and Y, which are also immunoprecipitated by antifibrillarin antibodies. Since fibrillarin is the only characterized

protein associated with these snoRNP, it is possible that other proteins or the snoRNA themselves are the targets of some anti-snoRNP autoantibodies. Anti-U3 RNP/fibrillarin antibodies are present in 6% of all scleroderma patients, but are more frequent in North American black scleroderma patients (40%) (Kuwana et al., 1994). Clinically, the presence of these autoantibodies is associated with an increase in skeletal muscle disease and primary pulmonary hypertension (Okano et al., 1992). Antifibrillarin antibodies are also reported in a few patients with hepatocellular carcinoma (Imai et al., 1992). Some scleroderma patients possess antibodies (known as anti-To, anti-Th or anti-Wa) to a nucleolar 40 kd protein (referred to as To or Th antigen) which is a component of the 7-2/MRP RNP (Okano and Medsger, Jr. 1990). Most of the 7-2/MRP RNP is found in the GC region of the nucleolus and <1% of this snoRNP appears located in the mitochondria. The 7-2/MRP RNP is an endoribonuclease which can cleave RNA in a sequence-specific manner. In addition, sera from patients with anti-Th antibodies immunoprecipitate RNaseP (also referred to as 8-2 RNP), a cytoplasmic endoribonuclease that processes precursor transfer RNA transcripts to generate their mature 5' termini. Whether the To antigen itself or a cross-reactive protein is associated with the RNaseP RNA is still unclear. The 7-2/MRP RNA and the RNaseP RNA each possess a stretch of nucleotides forming similar secondary structures that are predicted to be To antigen-binding domains (Liu et al., 1994). Four percent of scleroderma patients (most of them with limited cutaneous involvement) have anti-To antibodies. The presence of anti-To antibodies in these patients is associated with a shorter survival, probably due to pulmonary arterial hypertension. Anti-To antibodies are also reported in a few patients with primary Raynaud's phenomenon (Okano and Medsger, Jr. 1990).

Anti-NOR-90 Antibodies. Genes coding for rRNA are clustered at the secondary constrictions of chromosomes, called nucleolus organizer regions (NOR). The NOR are the sites where nucleoli reform after mitosis. Antibodies to a 90 kd protein associated with NOR (NOR-90) can be identified by IB in a few patients with scleroderma, systemic lupus erythematosus, rheumatoid arthritis or hepatocellular carcinoma (Rodriguez-Sanchez et al., 1987; Imai et al., 1992). By IF, these sera display a speckled pattern of nucleolar reactivity and staining of several pairs of dots in

cells undergoing mitosis. The NOR-90 protein is the human upstream binding factor, a nucleolar transcription factor involved in the regulation of rRNA transcription (Imai et al., 1994). Although Raynaud's phenomenon is present in about half of patients with anti-NOR-90 antibodies, the clinical relevance of these autoantibodies is limited because of their low prevalence.

Antinucleophosmin/B23 Antibodies. Nucleophosmin (also called B23, numatrin, ribocharin or No38) is a 37 kd RNA-associated phosphoprotein located in the GC. Nucleophosmin, a member of the nucleoplasmin family, is involved in the later stages of ribosome assembly and may be a shuttle protein involved in transport from the nucleolus to the cytoplasm. Antinucleophosmin antibodies are present in a few patients with various systemic autoimmune syndromes (including scleroderma), chronic graft-versus-host (GVH) disease reaction or various carcinomas (KindasMugge, 1989; Wesierska-Gadek et al., 1992; Imai et al., 1992). One study reported the presence of antinucleophosmin antibodies correlating with anticardiolipin antibodies, although the significance of this association is unknown (Li et al., 1989). Antinucleolin Antibodies. Nucleolin (also called C23 or 100k) is a 110 kd phosphoprotein present in the DFC and GC which appears to have several functions in the nucleolus, including ribosome assembly. Nucleolin indeed displays sequence similarities with various RNP proteins and can associate with several molecules, including DNA, histone H1, RNA polymerase I and topoisomerase I. Antinucleolin antibodies (mostly IgM) are reported in patients with systemic lupus erythematosus, acute hepatitis A, infectious mononucleosis and chronic GVH (Minota et al., 1991; Wesierska-Gadek et al., 1992). The prevalence and the significance of these antibodies remain to be determined. Animal Models. Mice that are heterozygotes for the tight-skin mutation display a phenotype with similarities to scleroderma including cutaneous hyperplasia resulting from increased collagen production. These mice also produce autoantibodies of the type associated with human scleroderma, such as antibodies to the 190 kd subunit of polymerase I (Shibata et al., 1993). Administration of parental lymphocytes into adult F1 hybrid (semiallogeneic) mice induces chronic GVH. In certain mouse strains, the resultant autoim-

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mune disease can clinically and serologically mimic various syndromes, including scleroderma. For instance, (BALB/c x A/J)F1 mice injected with A/J lymphocytes produce antibodies that immunoprecipitate the U3 RNP and recognize fibrillarin by IB (Gelpi et al., 1988). Likewise, anti-NOR-90 antibodies are reported in (C57BL/10 x DBA/2)F1 mice injected with DBA/2 lymphocytes (Gelpi et al., 1994). An intriguing experimental model of ANoA can be induced by the administration of subtoxic doses of mercuric chloride (Monestier et al., 1994). This syndrome is genetically restricted since only mice carrying H-2 s histocompatibility antigens are susceptible. Within 10 days after mercury injection, the mice develop an autoimmune lymphoproliferative syndrome that includes the production of ANoA (Figure 1). Monoclonal ANoA obtained from these mice immunoprecipitate several snoRNP, including U3 and U8 RNP. Some but not all the monoclonal antisnoRNP antibodies from these mice react with fibrillarin suggesting that additional self-epitopes are present on snoRNP (Monestier et al., 1994).

CLINICAL UTILITY Because of the low prevalence of most antinucleolar specificities, their clinical associations in scleroderma or other autoimmune diseases remain tentative (Table 1).

Figure 1. Nucleolar IF staining of HEp-2 cells by ASWA1 monoclonal antibody. ASWA1 is an antifibrillarin IgG monoclonal antibody obtained from a mercury-injected mouse (Monestier et al., 1994).

CONCLUSION The presence of ANoA is mostly correlated with scleroderma or related conditions such as primary Raynaud's phenomenon. It is likely that the true prevalence of ANoA is currently underestimated since the routinely used IIF technique is rather insensitive. Moreover, the concomitant presence of antinuclear antibodies can mask the antinucleolar reactivity. Newly developed techniques using recombinant nucleolar proteins or antisense riboprobes suggest a greater prevalence of ANoA in scleroderma and their presence in other systemic autoimmune diseases (Kasturi et al., 1995; Verheijen et al., 1994). In addition, some nucleolar components that are targets of systemic autoimmunity probably remain to be identified. Indeed, several sera recognizing unknown nucleolar proteins are reported (Hernandez-Verdun, 1991). Not all scleroderma patients actually develop

570

ANoA, but it is "noteworthy that other targets of autoimmunity in scleroderma, such as centromeres, are also associated with the nucleolus (Ochs and Press, 1992). The observation that the nucleolus is a central autoimmunogenic element in scleroderma is consistent with the view that systemic autoimmune diseases are targeted against antigens that are functionally associated within certain macromolecular structures. See also FIBRILLARIN AUTOANTIBODIES, PM-ScL AUTOANTIBODIES and RNA POLYMERASE I-III AUTOANTIBODIES.

ACKNOWLEDGEMENT The author's work has been supported by NIH Grant AI-26665.

Table 1. Nucleolar Autoantibodies Nucleolar Antigen

Structure

Function

IIF Pattern

Associations in Scleroderma

Other Clinical Associations

RNA polymerase I

complex of 13 proteins

rRNA transcription

speckled

diffuse form; cardiac and renal involvement

not reported

PM-Scl

complex of 11-16 proteins

unknown

homogeneous

myositis-scleroderma overlap syndrome; arthritis; scleroderma; interstitial lung disease; HLA-DR3

some cases of polymyositis or dermatomyositis without scleroderma overlap

fibrillarin

34 kd protein, component of several snoRNP

pre-rRNA processing

clumpy

black patients; skeletal muscle disease; pulmonary hypertension

some hepatocellular carcinomas

To (or Th)

40 kd protein, component of the 7-2/MRP RNP

sequence-specific endoribonuclease

limited cutaneous involvement; pulmonary hypertension; shorter survival

primary Raynaud's phenomenon

NOR-90

90 kd protein (human upstream binding factor)

nucleolar transcription factor

speckled; nucleolus organizer regions stained as dots in mitosis

Raynaud' s phenomenon

Raynaud's phenomenon; some cases of systemic lupus erythematosus, rheumatoid arthritis, hepatocellular carcinoma

nucleophosmin (B23)

37kd phosphoprotein

late stages of ribosome assembly

homogeneous

some cases

some systemic autoimmunity cases; chronic GVH; some carcinomas; anticardiolipin antibodies

nucleolin

i 10 kd phosphoprotein

ribosome assembly

not reported

not reported

some cases of systemic lupus erythematosus, acute hepatitis A, infectious mononucleosis, chronic GVH

, homogeneous

REFERENCES Alderuccio F, Chan EK, Tan EM. Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75-kD acidic protein of the nucleolar complex. J Exp Med 1991;173:941--952. Beck JS, Anderson JR, McElhinney AJ, Rowell NR. Antinucleolar antibodies. Lancet 1962;2:575-577. Bernstein RM, Steigerwald JC, Tan EM. Association of antinuclear and antinucleolar antibodies in progressive systemic sclerosis. Clin Exp Immunol 1982;48:43--51. Bluthner M, Bautz FA. Cloning and characterization of the cDNA coding for a polymyositis-scleroderma overlap syndrome-related nucleolar 100-kD protein. J Exp Med 1992:176:973-980. "Chan EK, Pollard KM. Autoantibodies to ribonucleoprotein particles by immunoblotting. In: Rose NR, de Macario EC, Fahey JL, Friedman H, Penn GM, eds. Manual of Clinical Laboratory Immunology. Washington, DC: American Society for Microbiology, 1992:755--761. Craft J, Hardin JA. Immunoprecipitation assays for the detection of soluble nuclear and cytoplasmic nucleoproteins. In: Rose NR, de Macario EC, Fahey JL, Friedman H, Penn GM, eds. Manual of Clinical Laboratory Immunology. Washington, DC: American Society for Microbiology, 1992:747--754. Fritzler MJ. Immunofluorescent antinuclear antibody test. In: Rose NR, de Macario EC, Fahey JL, Friedman H, Penn GM, eds. Manual of Clinical Laboratory Immunology. Washington, DC: American Society for Microbiology, 1992:724--729. Ge Q, Frank MB, O'Brien C, Targoff IN. Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoantigen. J Clin Invest 1992;90:559--570. Ge Q, Wu Y, Trieu EP, TargoffIN. Analysis of the specificity of anti-PM-Scl autoantibodies. Arthritis Rheum 1994;37: 1445-1452. Gelpi C, Rodriguez-Sanchez JL, Martinez MA, Craft J, Hardin JA. Murine graft vs host disease. A model for study of mechanisms that generate autoantibodies to ribonucleoproteins. J Immunol 1988;140:4160-4166. Gelpi C, Martinez MA, Vidal S, Targoff IN, Rodriguez-Sanchez JL. Autoantibodies to a transfer RNA-associated protein in a murine model of chronic graft versus host disease. J Immunol 1994;152:1989--1999. Hernandez-Verdun D. The nucleolus today. J Cell Sci 1991 ;99: 465-471. Imai H, Ochs RL, Kiyosawa K, Furuta S, Nakamura RM, Tan EM. Nucleolar antigens and autoantibodies in hepatocellular carcinoma and other malignancies. Am J Pathol 1992;140: 859--870. Imai H, Fritzler MJ, Neri R, Bombardieri S, Tan EM, Chan EK. Immunocytochemical characterization of human NOR-90 (upstream binding factor) and associated antigens reactive with autoimmune sera. Two MR forms of NOR-90/hUBF autoantigens. Mol Biol Rep 1994;19:115--124. Kasturi KN, Hatakeyama A, Spiera H, Bona CA. Antifibrillarin autoantibodies present in systemic sclerosis and other connective tissue diseases interact with similar epitopes. J . .

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Exp Med 1995;181:1027--1036. Kindas-Mugge I. Human autoantibodies against a nucleolar protein. Biochem Biophys Res Commun 1989;163:1119-1127. Kuwana M, Okano Y, Kaburaki J, Tojo T, Medsger TA Jr. Racial differences in the distribution of systemic sclerosisrelated serum antinuclear antibodies. Arthritis Rheum 1994:37:902--906. Li XZ, McNeilage LJ, Whittingham S. Autoantibodies to the major nucleolar phosphoprotein B23 define a novel subset of patients with anticardiolipin antibodies. Arthritis Rheum 1989:32:1165--1169. Liu MH, Yuan Y, Reddy R. Human RNaseP RNA and nucleolar 7-2 RNA share conserved "To" antigen-binding domains. Mol Cell Biochem 1994;130:75-82. Melese T, Xue Z. The nucleolus: an organelle formed by the act of building a ribosome. Curr Opin Cell Biol 1995;7:319-324. Minota S, Jarjour WN, Suzuki N, Nojima Y, Roubey RA, Mimura T, Yamada A, Hosoya T, Takaku F, Winfield JB. Autoantibodies to nucleolin in systemic lupus erythematosus and other diseases. J Immunol 1991;146:2249--2252. Monestier M, Losman MJ, Novick KE, Aris JP. Molecular analysis of mercury-induced antinucleolar antibodies in H-2 s mice. J Immunol 1994:152:667--675. Ochs RL, Press RI. Centromere autoantigens are associated with the nucleolus. Exp Cell Res 1992;200:339--350. Oddis CV, Okano Y, Rudert WA, Trucco M, Duquesnoy RJ, Medsger TA Jr. Serum autoantibody to the nucleolar antigen PM-Scl. Clinical and immunogenetic associations. Arthritis Rheum 1992;35:1211--1217. Okano Y, Steen VD, Medsger TA Jr. Autoantibody to U3 nucleolar ribonucleoprotein (fibrillarin) in patients with systemic sclerosis. Arthritis Rheum 1992;35:95--100. Okano Y, Medsger TA Jr. Autoantibody to Th ribonucleoprotein (nucleolar 7-2 RNA protein particle) in patients with systemic sclerosis. Arthritis Rheum 1990;33:1822--1828. Reimer G, Rose KM, Scheer U, Tan EM. Autoantibody to RNA polymerase I in scleroderma sera. J Clin Invest 1987;79:65-72. Reimer G, Steen VD, Penning CA, Medsger TA Jr, Tan EM. Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma). Arthritis Rheum 1988;31:525--532. Reimer G, Tan EM. Systemic sclerosis (scleroderma) and mixed connective tissue disease. In: Lachmann PJ, Peters K, Rosen FS, Walport MJ, eds. Clinical Aspects of Immunology Boston:Blackwell Scientific Publications, 1993:1241-1258. Rodriguez-Sanchez JL, Gelpi C, Juarez C, Hardin JA. AntiNOR 90. A new autoantibody in scleroderma that recognizes a 90-kDa component of the nucleolus-organizing region of chromatin. J Immunol 1987;139:2579--2584. Shibata S, Muryoi T, Saitoh Y, Brumeanu TD, Bona CA, Kasturi KN. Immunochemical and molecular characterization of anti-RNA polymerase I autoantibodies produced by tight skin mouse. J Clin Invest 1993;92:984-992. Verheijen R, Wiik A, De Jong BA, Hoier-Madsen M, Ullman S, Halberg P, Van Venrooij WJ. Screening for autoantibodies

to the nucleolar U3- and Th(7-2) ribonucleoproteins in patients' sera using antisense riboprobes. J Immunol Methods 1994;169:173-182.

Wesierska-Gadek J, Penner E, Hitchman E, Kier P, Sauermann G. Nucleolar proteins B23 and C23 as target antigens in chronic graft-versus-host disease. Blood 1992;79:1081-1086.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

NUCLEOSOME-SPECIFIC AUTOANTIBODIES Jo H.M. Berden, M.D. Ph.D. a and Ruud J.T. Smeenk, Ph.D. b

aDivision of Nephrology, Academic Hospital St. Radboud, NL-6525 GA Nijmegen; and bDepartment of Autoimmune Diseases, C.L.B., NL-1066 CX Amsterdam, The Netherlands

HISTORICAL NOTES Antibodies to nucleosomes were the first autoantibodies described in association with systemic lupus erythematosus (SLE). The morphological characteristics of the first serological marker for SLE "the LE cell phenomenon" (Holman and Kunkel, 1957) evolve after opsonization of "LE cell factors" complexed to DNA and histones. The "LE cell factors" were soon identified as autoantibodies, and nucleosomes were shown to inhibit the formation of the "LE cell phenomenon" in contrast to free dsDNA or histones (Rekvig and Hannestad, 1981). Therefore, the "LE cell phenomenon" is related to antinucleosome autoantibodies. The possibility that nucleosomes might be important antigens in generating antinuclear antibodies was suggested in 1986 (Hardin, 1986), but not much attention was given to the nucleosome as autoantigen in systemic autoimmune diseases until recently when an important role for nucleosomes in the etiopathogenesis of SLE was recognized (Tax et al., 1995). The formation of nucleosome-specific antibodies was well documented during analysis of monoclonal antibodies derived from lupus mice (Losman et al., 1993a; Kramers et al., 1994; Monestier and Novick, 1995; Losman et al., 1992; Losman et al., 1993b; Jacob et al., 1989). A similar autoantibody reactivity in SLE patients was reported in 1987 (Faiferman and Koffier, 1987). The formation of these nucleosome-specific antibodies precedes the formation of other antinuclear specificities (Burlingame et al., 1993; Amoura et al., 1994) and ultimately develops in 80% of MRL/Ipr mice (Amoura et al., 1994). Their nephritogenic potential is illustrated by the identification of this antigen specificity in glomerular eluates of MRL/lpr

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mice (Amoura et al., 1994). Antinucleosome antibodies are detected in 84--88% of patients with SLE (Burlingame et al., 1994; Chabre et al., 1995).

THE AUTOANTIGENS Structure

As the basic structure of chromatin, the nucleosome is important in the compaction of DNA in the nucleus. It consists of pairs of the four core histones H2A, H2B, H3 and H4 forming the histone-octamer around which 146 bp of DNA are wound twice (Figure 1). Histone H1 interacts with the nucleosome and together with linked-DNA connects neighboring nucleosomes. This architecture is responsible for the characteristic appearance of chromatin in electron microscopy as "beads-on-a-string." The four core histones are single polypeptide chains with molecular weights between 11 and 15 kd. Due to basic residues, which are clustered at the N-terminal regions of the core-histones and, therefore, located at the outside of the nucleosome, regions are present with a strong positive charge. These positive charges on the cylindrical surface of the histone octamer appear as a left-handed spiral situated in the path of the DNA (Arents and Moudrianakis, 1993) and are partly involved in the binding of DNA. In addition to histones, nucleosomes contain the so-called "high mobility group" (HMG) proteins. Although autoantibodies directed against HMG-14 and HMG-17 proteins (which are associated with the histone-octamer) are known (Tzioufas et al., 1993), this aspect will not be addressed further in this chapter.

molecules retain their native structure after preparation; this is important because several autoantibodies bind to these macromolecular complexes and not to the individual components. At present, no reliable nucleosome or subnucleosome complex preparations are commercially available. As will be outlined later, (H2A-H2B)-DNA can be used for screening purposes as a substitute for measuring antinucleosomal reactivity. To this end, commercially obtained H2A and H2B (Boehringer Mannheim) can be mixed in equal amounts after checking for purity on PAGE-analysis. This dimer is then coated to ELISA plates with the subsequent addition of calf thymus dsDNA after pretreatment with S1 nuclease, proteinase K and phenol-extraction (Rubin et al., 1992).

Sequence Information

Figure 1. Topography of the nucleosome. The octamer is formed out of four homo-dimers of H2A-H2B, H3 and H4. Around this octamer, two superhelical turns of 146 bp of DNA are wrapped twice (Tax et al., 1995). Methods of Purification Several purification procedures for the preparation of nucleosomes for antibody tests (Lutter, 1978; Kornberg et al., 1989) use micrococcal nuclease digestion which causes an internucleosomal cleavage. All eukaryotic cells can serve as starting material, but most frequently calf thymus, the mouse erythroleukemia cell line L 1210 or rat liver are used. After enzymatic digestion, the fragments are purified by sucrose gradient centrifugation. Depending on the conditions used, oligonucleosomes, mononucleosomes or histoneoctamers can be obtained. After preparation, the purity and the homogeneity should be checked both for the histones by PAGE analysis and for DNA by extraction and analysis on agarose gels. From the nucleosome preparation, different subnucleosomal structures can be isolated, including (H2A-H2B)-DNA, (H3H4)z-DNA, H2A-H2B dimers or H3-H4 tetramers (Burlingame and Rubin, 1990). In general, these

Lupus-derived, nucleosome-specific monoclonal autoantibodies recognize a multitude of epitopes within the nucleosome (Kramers et al., 1995b). All recognize the (H2A-H2B)-DNA complex and to a lesser extent (H3-H4)z-DNA; whereas, they have (by definition) almost no or very low reactivity towards H2A-H2B, H3-H4, DNA, individual core-histone proteins or synthetic histone peptides. Linear epitopes are apparently not recognized by these nucleosomespecific monoclonal antibodies which are directed against conformational epitopes created by the interaction between DNA and the different core-histones. Similar binding characteristics are found in some human and murine lupus-derived polyclonal antibodies.

AUTOANTIBODIES Terminology Antinucleosome antibodies are defined as antibodies which are directed exclusively or predominantly against nucleosomes or subnucleosomal complexes consisting of core histones and DNA. By definition, they have no or very low reactivity against individual histones or native-nonprotein complexed DNA. In the recent literature, these antibodies are called either "nucleosome-restricted" (Amoura et al., 1994; Chabre et al., 1995), "nucleosome-specific" (Losman et al., 1993a) or "antichromatin" antibodies (Burlingame et al., 1993). We prefer the term "nucleosome-specific" autoantibodies, which will be used in this chapter.

575

Pathogenetic Role Before considering the evidence that nucleosomespecific autoantibodies play a role in SLE, the significance of apoptosis in SLE will be reviewed. Apoptosis is a process of programmed cell death starting with the internucleosomal cleavage of chromatin. This process initiates condensation and fragmentation of the nucleus, which ultimately leads to the appearance of blebs at the cell surface, the socalled "apoptotic bodies." During apoptosis, changes at the cell surface (including expression of phosphatidylserine and thrombospondin receptors) lead to rapid phagocytosis of the apoptic cell by macrophages to prevent leakage of phlogistic cell constituents into the microenvironment. However, quantitative and/or qualitative differences in apoptosis can lead to the systemic release of nucleosomes (Franek and DolnNovfi, 1991; Emlen et al., 1994). The DNA in the circulation of SLE patients is present in the form of (oligo)nucleosomes (Rumore and Steinman, 1990), consistent with origination from apoptotic cells. In several (MRL/lpr and gld) but not all (NZBWF1) lupus mouse strains, apoptosis is disturbed either by a deficient expression of the Fas (APO-1; CD95) receptor or its ligand (Singer et al., 1994). Activation of this Fas receptor leads to apoptosis. In human SLE, the expression of the Fas system is normal, but increased concentrations of soluble Fas, increased expression of the apoptosis inhibitor bcl-2 and increased rate of apoptosis of lymphocytes in vitro point to abnormalities in apoptosis in human SLE (Tax et al., 1995). Very recently, a defective expession of Fas was also described in humans. It was, as in MRL/Ipr mice, associated with lymphoproliferation and autoimmunity (Fisher et al., 1995; Rieuxlaucat et al., 1995). Disturbances in apoptosis can contribute in at least two different ways to the pathogenesis of systemic autoimmunity (Figure 2), including persistence of autoreactive T cells (Marrack et al., 1993) and the release of nucleosomes (Casciola-Rosen et al., 1994). Due to the abnormal apoptosis, these nucleosomes may be altered (becoming more immunogenic) by qualitative changes in the intracellular oxidative processes operative during apoptosis (Casciola-Rosen et al., 1994). Decreased phagocytosis, which is often present in SLE (Salmon et al., 1984; Meryhew et al., 1991), might enhance the magnitude of nucleosome release. This abnormal release of nucleosomes has two important consequences: First, nucleosomes can act as polyclonal B-cell activators (Bell et al., 1990), which 576

Dysregulation of apoptosis

r- Persistence of autoreactive T cells

Decreased phagocytosis? 1 Quantitative/qualitative k , changes in nucleosomes Anti-nucleosome Ab, anti-DNA Ab In situ binding of / " nucleosomes to GBM,(HS?)

Depositionof circulating nucleosome-Ab complex

Nucleosome-mediated Ab-binding to GBM Activation of complement, glomerulonephrifis

Figure 2. Schematic diagram of the proposed etiopathogenesis of lupus nephritis (Tax et al., 1995).

may be relevant for the initial phase of the disease. But more importantly, because SLE is an antigendriven disease, nucleosomes are autoantigens recognized by pathogenic T-helper cells in SLE. In the lupus mouse strain SNF 1, 50% of the pathogenic Thelper cells respond to nucleosomes (Mohan et al., 1993). These nucleosome-specific T-helper cells not only induce production of nucleosome-specific autoantibodies by syngeneic B cells, but also induce the formation of anti-DNA and antihistone antibodies. The central and dominant role of nucleosomes in precipitating the antinuclear autoantibody response in SLE is buttressed by the observation that the formation of nucleosome-specific antibodies not only is antigendriven but also precedes the development of anti-DNA and antihistone antibodies in MRL/Ipr mice (Burlingame et al., 1993). Support for the causative relationship between apoptosis, nucleosomes and nucleosomespecific antibodies can be derived from several observations including the fact that nucleosomes clustered in apoptotic bodies can act as autoantigens (Casciola-Rosen et al., 1994). Similarly, human lupusderived, nucleosome-specific antibodies are bound to nucleosomes released from apoptotic mononuclear cells (Suenaga and Abdou, 1995). Finally, because Creactive protein (CRP) is thought to facilitate the removal of nuclear material released from apoPtotic cells, the influence of CRP on the clearance of nucleosomes was evaluated in normal BALB/c mice (Du Closet al., 1994). Unexpectedly, CRP does not alter the clearance and organ localization of nucleosomes. In NZBWF 1 mice, however, CRP produces a transient decrease in antinuclear antibody production and prolonged survival.

How nucleosome-specific antibodies might induce disease manifestations in lupus is being clarified (van Bruggen et al., 1994; Tax et al., 1995). Both monoclonal and polyclonal anti-DNA antibodies are known to bind to heparan sulfate (HS), an intrinsic constituent of the glomerular basement membrane (GBM) (Faaber et al., 1986) which is responsible for the charge-dependent permeability of the GBM. The binding of anti-DNA antibodies to HS is due to DNA/histone complexes (nucleosomal material) bound to the antibody (Termaat et al., 1990). In vivo sequential perfusion of rat kidneys with histones, DNA and noncomplexed, purified monoclonal anti-DNA antibodies yield deposition of the antibodies in subendothelial or subepithelial locations depending on the perfusion protocol (Termaat et al., 1992). Control experiments using histones, DNA and nonrelevant monoclonal antibodies, histones and anti-DNA monoclonal antibodies or anti-DNA monoclonal antibodies alone reveal no glomerular binding. Because histones and DNA do not exist as separate entities in vivo, perfusion of antinucleosomal antibodies complexed to nucleosomes was performed; this also yielded binding along the GBM with subsequent complement activation (Kramers et al., 1994). In contrast to nucleosomespecific and anti-DNA antibodies, antihistone monoclonal antibodies do not localize in the GBM after being complexed with nucleosomes (van Bruggen et al., unpublished observation). Apparently, the specificity of the antibody bound to the nucleosome is a critical determinant for the nephritogenic potential of the complex. Perhaps binding of antinucleosome or anti-DNA antibodies to nucleosomes leads to a decrease of the negative charges in the complex, tipping the balance to an overall cationic charge which allows firm interaction of the complex with the anionic charges (mainly HS) in the GBM; binding of antihistone antibodies to nucleosomes might have the opposite effect, thereby decreasing the nephritogenicity. This view can explain the low frequency of renal disease in drug-induced lupus in which antihistone antibodies are commonly found. Apart from the targetifig of autoantibodies to the GBM by nucleosomes, an additional mechanism involves binding of nucleosomes to mesangial cells (Coritsidis et al., 1995). This observation provides a mechanism for in situ immune complex formation in the mesangium, where abundant deposits of immune complexes are generally found in SLE nephritis. Several lines of evidence support the involvement of nucleosomes and nucleosome-specific antibodies in

SLE nephritis in vivo. First, elution studies from isolated glomeruli of lupus mice reveal not only antiDNA but also antinucleosome antibodies. In fact, these nucleosome-specific antibodies are deposited earlier and to a greater extent in the early phases of the disease than anti-DNA antibodies, which accumulate later (van Bruggen et al., 1995c). Nucleosomes deposited in the GBM can presumably act as planted antigens for subsequent anti-DNA binding. Secondly, histone- and nucleosome-specific monoclonal antibodies stain deposits along the GBM in 100% and 35%, respectively, of human kidney biopsies which show diffuse proliferative lupus nephritis (Kramers et al., 1995a). The higher frequency of histone deposits than nucleosome deposits might reflect masking of nucleosome-specific epitopes in vivo by bound antibodies in contrast to epitopes recognized by antihistone antibodies. This observation confirms and extends the detection of histones in glomerular deposits reported earlier (Schmiedeke et al., 1992; St6ckl et al., 1994). Thirdly, the absence of staining of GBM HS by HS-specific monoclonal antibodies in human (van den Born et al., 1993) and murine lupus nephritis (van Bruggen et al., 1995a) is due not to a decreased HS content of the GBM but to the binding of nucleosomecomplexed antinuclear antibodies with resultant masking of HS (van Bruggen et al., 1995a). Fourthly, treatment with heparin or noncoagulant heparinoids considerably delays the onset of nephritis in MRL/lpr mice; presumably these polyanionic drugs, which resemble HS, can prevent the deposition of autoantibodies in the GBM (van Bruggen et al., 1995b). And finally, an HS ELISA, which detects nephritogenic nucleosome-complexed autoantibodies, shows that onset (Termaat et al., 1990) and flares (Kramers et al., 1993) of lupus nephritis are significantly associated with anti-HS reactivity. Lupus manifestations other than glomerulonephritis might reflect a similar mechanism because HS and other glycosaminoglycans like chondroitin sulfate, dermatan sulfate and keratan sulfate are abundantly present at sites generally afflicted in SLE, including vessel walls, joints and skin. Taken together, these data point to the pathogenetic significance of nucleosomes and nucleosomespecific antibodies in SLE. Genetics

"

As yet the formation of nucleosome-specific antibodies cannot be related to certain MHC genes, to other genetic factors or to familial preponderance. For 577

the nucleosome-specific monoclonal antibodies so far analyzed, Sm7, Q52, J558 or 7183 Vi_ifamily and 4/5, 9 or 10 V L family are used (Losman et al., 1993a; Monestier and Novick, 1995). This gene usage resembles that in anti-DNA and antihistone antibodies. Relevant for their binding properties, the V regions of these nucleosome-specific monoclonal antibodies contain an increased amount of cationic residues within the CDR3 region (like anti-DNA antibodies) and an increase in anionic residues in the CDR2 region (as in antihistone antibodies). Therefore, these nucleosome-specific autoantibodies seem to harbor antigen-binding characteristics for both anionic DNA and cationic histone epitopes.

interaction of the complexed nuclear antigens with DNA or histones. This reactivity can be removed by purification with DNAse and high-salt conditions (Kramers et al., 1994). Finally, (H2A-H2B)-DNA is a good "surrogate" antigen for nucleosomes, because all nucleosome-specific antibodies are positive on (H2A-H2B)-DNA. However, using this substrate, the same pitfalls arise as described for nucleosomespecific assays. Further research should define whether an epitope exists within the nucleosome which is uniquely recognized by nucleosome-specific antibodies and not by anti-DNA or antihistone antibodies. The availability of such an epitope will make the assessment of nucleosome-specific antibodies much easier.

Factors in Pathogenicity

CLINICAL UTILITY In general, nucleosome-specific autoantibodies belong to the IgG class, consistent with the idea that this autoimmune response is antigen driven. Further support for this assumption is provided by the fact that the murine antibodies predominantly belong to the IgG2a and IgG2b subclasses, which are generally induced by T-dependent antigens (Burlingame et al., 1993). Methods of Detection

Assays for nucleosome-specific antibodies with nucleosomes as substrate will also detect anti-dsDNA and antihistone antibodies. Therefore, these latter antibodies should be removed by absorption on DNAcellulose beads (Sigma or P-L Biochemicals Inc, Milwaukee, WI) and histones coupled to Sepharose (Amoura et al., 1994; Kramers et al., 1994; Chabre et al., 1995). Such absorption studies indicate that in the presence of anti-DNA antibodies, 25--65% of the antnucleosome reactivity in nonabsorbed plasma is due to anti-dsDNA antibodies; whereas, in the presence of antihistone antibodies only 10--20% of the antinucleosome reactivity is caused by antihistone antibodies (Masa et al., 1994; Burlingame et al., 1994). For screening purposes, a less laborious approach can be chosen by simultaneous testing in ELISA on nucleosomes, dsDNA and histones. A much higher reactivity in the nucleosome ELISA suggests the presence of nucleosome-specific antibodies. However, this approach has one major caveat. Nucleosomespecific antibodies complexed to nucleosomes display anti-dsDNA and antihistone reactivity due to the

578

Disease Association

In murine lupus, nucleosome-specific antibodies develop in 80% of MRL/1 mice (Amoura et al., 1994). Their formation precedes the development of other antinuclear autoantibodies (Burlingame et al., 1993, Amoura et al., 1994) and is associated with glomerulonephritis (Amoura et al., 1994). Nucleosome-specific antibodies are found in 84-88% of SLE patients (Burlingame et al., 1994; Chabre et al., 1995). As observed for nucleosome-specific monoclonal antibodies from lupus mice, these polyclonal nucleosomespecific antibodies from SLE mice and patients always react with the H2A-H2B/DNA complex but in only 33% with (H3-H4)z-DNA or dsDNA (Burlingame et al., 1994). Only the reactivity with nucleosomes and the (H2A-H2B)-DNA complexes is associated with nephritis (Burlingame et al., 1994). The resemblance of the antigen reactivity of human polyclonal nucleosome-specific autoantibodies with those found in murine SLE models suggests that overlapping or identical epitopes are recognized. At this time, the loss of tolerance for the nucleosome is considered an early event triggering other antinuclear autoantibody formation; the stimulus evoking these antibodies appears similar or identical in murine and human lupus (Burlingame et al., 1994). Antibody Frequencies in Disease 9

Studies relating serum anti-dsDNA activity to disease manifestations show that the anti-dsDNA titer rises before onset or flares of disease manifestations and

sharply drops after initiation of immunosuppressive treatment (ter Borg et al., 1990). Similar data on the association between nucleosome-specific antibodies and disease flares and/or certain disease manifestations are presently lacking or conflicting (Suenaga and Abdou, 1995; Massa et al., 1994). For example, of five patients with active lupus nephritis, three had nucleosome-specific antibodies; patients with active nonrenal SLE manifestations were not studied (Suenaga and Abdou, 1995). In children with SLE, no correlation was found between nephritis and nucleosomespecific antibodies (Massa et al., 1994). Whetl~er these nucleosome-specific autoantibodies occur in other systemic autoimmune diseases such as MCTD, scleroderma or Sj6gren's syndrome is not known except that antibodies to the (H2A-H2B)-DNA complex in the absence of anti:dsDNA antibodies are more common in scleroderma than in SLE (Wallace et al., 1994). Nucleosome-specific and antihistone antibodies in the absence of anti-dsDNA antibodies are also reported in HIV infection (Viard et al., 1994). The reasons for this different autoantibody profile compared to SLE patients is not clear but suggests a different induction of the autoantibodies. The sensitivity, specificity and predictive values of detection of nucleosome-specific antibodies are unknown, but should soon be available. If nucleosomes are the driving autoantigen in lupus, nucleosome-specific antibodies might be clinically more powerful tools for monitoring than are anti-DNA antibodies.

persistence of autoreactive T cells and to the formation of nucleosome-specific autoantibodies in genetically susceptible individuals. A central role of nucleosomes in the antigen-driven autoantibody repertoire of lupus is supported by the formation of these antibodies prior to other antinuclear specificities, including anti-dsDNA and antihistone. Nucleosome-specific antibodies seem to play an important role in the pathogenesis of disease manifestations, especially glomerulonephritis. The histone components of nucleosomes facilitate binding to the anionic sites of basement membranes either as planted antigen enabling formation of immune complexes in situ, or via deposition of circulating immune complexes consisting of nucleosomes and nucleosome-specific antibodies. In both cases, increased permeability and inflammatory response are the results. Still, the very recent identification of the nucleosome as a major autoantigen in SLE leaves many questions to be answered. Which epitopes of the nucleosome are recognized by Thelper cells? Do different epitopes dictate the formation of nucleosome-specific antibodies, anti-dsDNA antibodies or antihistone antibodies? Are genetic factors important? What is the clinical significance of nucleosome-specific antibodies in relation to disease activity, disease manifestations and in the differential diagnosis of the various systemic autoimmune diseases? These questions and others surely will be answered in the future. See also DsDNA AUTOANTIBODIES and HISTONE (H2A-H2B)-DNA AUTOANTIBODIES.

CONCLUSION

ACKNOWLEDGEMENTS

Nucleosomes are now considered a major autoantigen in SLE. The disturbed apoptosis present in both murine and human lupus might contribute both to the

Research of the authors has been generally supported by grants from the Dutch Kidney Foundation and the Dutch League Against Rheumatism.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

OTHER AUTOANTIBODIES TO NUCLEAR ANTIGENS Hans Peter Seelig, M.D.

Institute of Immunology and Molecular Genetics, D-76133 Karlsruhe, Germany

HISTORICAL NOTES The large number of antinuclear antibodies (ANAs) identified as recognizing a wide range of intranuclear targets continues to increase. Many chapters of this book are dedicated to those ANAs which have attained special clinical, immunopathological and diagnostic interest, but several less well known and often poorly characterized ANAs exist. These antibodies either occur less frequently in human disease, or their disease associations are still unknown. The biochemistry and molecular biology of the corre-

ABBREVIATIONS Methodologies C I E - Counterimmunoelectrophoresis C T E - Calf thymus extract DB -- Dot Blot ELISA- Enzyme-linked immunosorbent assay I B - Immunoblot I D - Immunodiffusion I I F - Indirect immunofluorescence I P - Immunoprecipitation n A g - Natural antigen P H A - Phytohemagglutinin r A g - Recombinant antigen R T E - Rabbit thymus extract 35SM- 35S methionine 35SMCP- 35S methionine-labeled cell proteins

Diseases A I D - Autoimmune disease

582

sponding antigens and the role these antigens play in the biology of nuclear processes vary greatly. For some of the antibodies, only a variant nuclear fluorescence pattern has been described; whereas, in others, well-advanced information about their biochemistry and biology has been obtained. Some knowledge of this group of ANAs is mandatory for differential diagnosis in determining ANA subspecificities in routine laboratory work and in uncovering and characterizing new ANA specificities. Therefore, a condensed review of these additional ANAs in alphabetical order is given in the following tables.

A V - Allergic vasculitis C A H - Chronic active hepatitis C T D - Connective tissue disease D L E - Drug-induced lupus erythematosus D M - Dermatomyositis I T P - Immune thrombocytopenia L E D - Lupus erythematosus discoides MCTD- Mixed connective tissue disease NAID- Nonautoimmune disease O A - Osteoarthritis P B C - Primary biliary cirrhosis P M - Polymyositis P s - Psoriasis p S S - Primary Sji3gren's syndrome RA/JRA- Rheumatoid arthritis/juvenile rheumatoid arthritis R P - Raynaud's phenomenon S C L - Progressive scleroderma S L E - Systemic lupus erythematosus S S - Sj6gren's syndrome S S c - Systemic sclerosis s S S - Secondary Sj6gren's syndrome UCTD- Undifferentiated connective tissue disease

ANA specificity

Antigen/Autoantibody descriptions

Autoantibody assays

Alu RNA Protein

68 kd protein which complexes with Alu RNA by binding to the 5' half of Alu RNA. Other autoantibodies prevailing in sera containing anti-Alu RNA protein antibodies were anti-Sm, anti-RNP, anti-SS-A/Ro, anti-SS-B/La. (Kole et al., 1985).

12% of randomly selected CTD IP of RNPs containing patients: SLE, LED, MCTD, SCL. Alu RNA transcripts. 35SM or 3H-uridine labeled HeLa cell extracts. Not detected by IB.

Annexin Xl 56K antigen

Annexin XI consisting of 505 amino acids (calculated Mr 54K) is a member of the IB, ELISA annexin family of Ca2+-dependent phospholipid binding proteins. C-terminus of annexin (nAg, rAg) is homologous in all family members and contains phospholipid binding sites; Nterminus is unique for every particular annexin and confers specificity of function. Anti-56K antibodies recognize epitopes localized on the N-terminus of annexin XI. Most annexins are believed to be localized underneath the plasma membrane, but the 56K autoantigen shows cytoplasmic as well as nuclear localization (no staining after conventional methanol/acetone fixation). Disease-associated anti-annexin XI antibodies are prevalent mostly in high titers and belong to the IgG isotype. (Misaki et al., 1995).

CB Antigen

A 40 kd DNA binding protein of unknown function, seems to be expressed in peripheral lymphocytes at a higher level than in thymocytes or bursal lymphocytes. (Juarez et al., 1988).

IP (35SMCP) ELISA (nAg)

IIF PS0 Coilin P80 coilin consists of 576 amino acids, with a calculated Mr of 62K, but shows a ELISA, IB (nAg, rAg) Coiled bodies (CBs) slower mobility in SDS-PAGE. P80 coilin contains a nuclear location signal sequence and is encoded by an about 25 kb spanning gene with 7 exons located on chromosome 17q22-23. The protein is abundant in nuclear CBs with diameters of 0.3--1.0 ~m and composed of coiled fibrils. CBs assemble and disassemble during the cell cycle. Some small CBs (3--8) appear at the mid and late G1 phase, condense and enlarge in the S and G2 phase (1--2), and disassemble during mitosis where the overall cellular level of p80 coilin remains the same as in interphase nuclei. P80 coilin switches from a soluble pool in mitosis and early G1 phase to an insoluble CB-associated compartment in later interphase. Assembly of CBs follows that of nucleoli with a phase lag. Many RNAassociated proteins (e.g., U1 nRNPs, Sm, MaS, see below) are enriched but not solely localized in CBs (storage sites?) which also may be involved in pre-mRNA metabolism (splicing sites?). Monospecific anti-p80 coilin antibodies stain CBs of culture cells used for routine ANA screening, but differentiation of antibody specificity in patient sera may become difficult because of prevalence of other antibodies. (Andrade et al., 1993; Chan et al., 1994).

Disease associations

SLE RA SS PM SSc RP

8% 10% 7% 10% 8% 4%

4% of randomly selected ANApositive sera of patients with SSc, SS, AV, RA, NAID. Patients with features of AID. No figures available with regard to frequency and disease associations.

4~

ANA specificity

Antigen/Autoantibody descriptions

Autoantibody assays

Disease associations

DA1, DA2

Nuclear proteins sensitive to trypsin digestion contained in 30-60% ammonium sulfate fractions of spleen nuclear extracts. Monospecific antibodies show homogenous nucleoplasmic fluorescence and staining of chromosomes without nucleolar staining, Identity to MA (see below) suggested but not proved. (Asero et al., 1988).

ID, CIE Human spleen nuclear extract

anti-DA1 anti-DA2 SLE 5%* 22%** * More severe disease. ** Higher incidence of lymphopenia. No correlation with disease activity.

DNA-dependent ATPase

Nuclear ATPases dependent on DNA binding hydrolyse ATP only when DNA is ELISA (nAg) present. Some participate in DNA replication, recombination or restriction by utilizing the energy of ATP hydrolysis to separate the strands of a DNA complex or to bind to a single strand and to move in relation to it during their action. One enzyme of the large family of DNA-dependent ATPase with a molecular weight of about 67 kd as revealed by SDS-PAGE was used as antigen for antibody screening by ELISA. (Astaldi Ricotti et al., 1987).

DNA Polymerase c~ Nuclear multisubunit enzyme consists of four subunits displaying molecular weights of

ANA (+) aged persons and SLE. No figures available with regard to frequency and disease associations.

ELISA (nAg)

ANA (+) aged persons and SLE. No figures available with regard to frequency and disease associations.

Nuclear protein of 1215 amino acids (calculated Mr 132K), with an electrophoretic mobility corresponding to about 160 kd. The proteins feature a serine-rich region localized in the center of the molecule and a putative nuclear localization sequence, which is localized within a stretch of 80 amino acids constituting the main autoepitope(s). Protein of unknown function. Monospecific anti-Ge-I antibodies induce a speckled nuclear staining pattern. (Bloch et al., 1994).

IB (nAg, rAg)

Single patient (GE) with SjOgren's syndrome.

Acidic nuclear protein of 69 kd of unknown function. Monospecific, cold-reactive antiGN-1 antibodies of IgM isotype show an exclusively nuclear fluorescence with speckled pattern on mouse liver sections. Antibodies were present in sera of patients with IgA nephropathy and their consanguineous relatives (genetic disposition?). (Nomoto et al., 1986).

IIF, IB (4~ Purified thymus or spleen extracts

IgA nephropathy (83%) and their consanguineous relatives (53%). Antibody titer may reflect disease activity.

(DNA primase complex)

195,000 and 180,000 for the DNA synthesizing ~-subunit, of 68,000 for the B-subunit, and of 55,000 and 48,000 for the primase-carrying Y- and 8-subunit, respectively. Now called DNA polymerase ~-DNA primase complex. Involved in nuclear DNA replication of the lagging strand. The antigen used for ELISA was composed of two proteins of about 50 and 42 kd as shown in SDS-PAGE possibly belonging to the y- and 8-subunit but primase activity of the antigen was not demonstrated. (Astaldi Ricotti et al., 1987).

Ge-1

GN-1

ANA specificity

Antigen/Autoantibody descriptions

HCC-1 Nuclear protein of 530 amino acids (calculated Mr 59 K) with an electrophoretic Hepatocellular mobility corresponding to 64 kd. The protein contains an arginine/serine-rich domain, carcinoma antigen 1 three ribonucleoprotein consensus sequence domains and two classes of motifs present in several splicing factors. HCC-1 may be associated with splicing activities. Monospecific anti-HCC-1 antibodies reveal a speckled nuclear immunofluorescence pattern and colocalize with U snRNP (anti-m3G) and non-snRNP splicing factor SC35 (Imai et al., 1993).

HMG-1 HMG-2 HMG-17 High mobility group proteins

Autoantibody assays

Disease associations

IB, ELISA (rAg)

Single patient with hepatocellular carcinoma.

HMGs are nonhistone proteins in nuclei of higher eukaryotes. Five main types of HMG IB, ELISA (nAg, rAg) are H M G - 1 , - 2 , - 1 4 , - 1 7 , and HMG I/Y. HMG-1 (25 kd) and HMG-2 (24 kd) are associated with internucleosomal DNA in condensed chromatin and may function in chromosomal replication and transcription as general transcription factors for class II genes. In HMG-1 there are sequence motifs homologous to NOR-90 (hUBF, see below). HMG-14 (11 kd) and HMG-17 (9 kd) bind to nucleosomal core histones of transcriptionally active chromatin. A major epitope recognized by 70% of anti-HMG-17 positive JRA sera is a proline and lysine rich octapeptide which shows homologies to nucleotide sequence of Sindbis virus, Bordetella pertussis, Salmonella typhimurium, and E. coli (molecular mimicry?). (Neuer et al., 1994; Tzioufas et al., 1993; Vlachoyiannopoulos et al., 1994).

anti-HMG- 1/2 anti-HMG- 17 SLE 6% 34--76% DLE 2% 67% MCTD -10% SCC 30-40% 17--40% SS 12% RP 23% 29% RA 5% JRA 40% (47%)* (16%)** * ANA (+) patients with pauciarticular-onset and ** polyarticular onset.

L7

The L7 protein (29 kd) associates with the large ribosomal subunit, but presumably is not an integral structural component of ribosomes. It binds specifically to sites of mRNA and 28S rRNA. Protein L7 resides within the nucleus and cytoplasm, not in the nucleolus. (Neu et al., 1995).

ELISA (rAg)

SLE 34% SSc 41%* * Not associated with lung fibrosis. Antibodies do not define clinically distinct subpopulations.

Locus 93D Antigen

Antibodies stain a particular antigen localized at the 93D locus of polytene chromosomes of Drosophila melanogaster. Polytene chromosomes are actively transcribed interphase chromosomes and highly active genes are visualized as unfolded bands (chromosome puffs). Heat shock activates the genes of locus 93D and therefore induces either a de novo accumulation of antigens (e.g., RNP particles) at this locus or structural transitions which render local antigens accessible for antibodies. Antibodies contained in the sera from patients with ankylosing spondylitis selectively stained for 93D chromosomal puffs; antibodies contained in sera of SLE and MCTD patients also stained additional chromosomal puffs. No correlation was seen between anti-93D and HEp-2 cell staining patterns of antibodies. (Lakomek et al., 1984).

IIF (Chromosomal puff preparations)

Ankylosing spondylitis 39%. No correlation with patient's age, duration or activity of disease.

MA

Acidic nuclear protein showing a peripheral immunofluorescence pattern. No biochemical or biological data about the antigen are available. (Winn et al., 1979).

ID (CTE)

SLE 18%. Subset with more severe disease?

(MA-1)

L~h

c~

ANA specificity

Antigen/Autoantibody descriptions

Mas

Single RNA species running slower than most tRNAs was precipitated from a serum of RNA precipitation a myositis patient (4S RNA precipitating antibody?). No associated proteins were coprecipitated by the serum, which also did not produce precipitations with calf thymus nuclear extract. Biological function of the antigen unknown. (Bernstein et al., 1984a)

Single patient with myositis

MaS Autoantigen p150

Nuclear protein of 150 kd localized within the nucleoplasm with elevated concentration IP of U4/U6 snRNPs in coiled bodies (CBs, see above) but excluding nucleoli. The protein is associated with from HeLa cell nuclear U4/U6 snRNP, 20SU5 RNP, U4/U6.U5 tri-snRNP and 17SU2 snRNPs, but not with extracts U1 snRNP. Anti p150 antibodies selectively immunoprecipitate a subpopulation of U4/U6 snRNPs in which the m3G-cap structure is marked. P150 is preferentially associated with U6 snRNA in the U4/U6 particle. Anti-P150 antibodies coprecipitate proteins corresponding to 120, 50, 46 and 34 kd. Anti MaS (autoantigen p150) antibodies specifically recognize mammalic U4/U6 through an associated protein. (Blencowe et al., 1993).

Single patient with SSc

Me

Anti-Me antibodies recognize four nuclear proteins of 100 kd, 65 kd, 21 kd, and 16 kd. The 100 kd and 21 kd proteins are regarded as specific for Me. Me antigen revealed some similar physicochemical characteristics similar to Sm antigen in nuclear extracts. Biological function of Me proteins is not known. (Treadwell et al., 1987).

ID, PHA (RNase treated CTE)

SLE 2% MCTD 7% UCTD 21% 20% of anti-Me positive sera were from SLE patients, 20% from patients with MCTD and 60% from patients with UCTD.

NOR-90 Nucleolus organizer region Human upstream binding factor (hUBF)

94 and 97 kd proteins of nucleolus organizer region produced by alternative splicing of a single gene. Recognize ribosomal RNA promoters and activate RNA polymerase I. NOR constitutes the locus where the nucleolus reforms after mitosis. Human cells possess ten NORs located on chromosomes 13, 14, 15, 21, 22. (Dick et al., 1995; Fritzler et al., 1995).

IIF, IB, ELISA (nAg, rAg) IP with in vitro translated protein

SLE, SSc, RA, SS, PBC, hepatocellular carcinoma, melanoma. Systemic rheumatic diseases in children (<1%). Not specific for SSs but association with HLA-DR1.

NSPI Nuclear speckled type I NSPII Nuclear speckled type II

Two forms of speckled nuclear staining pattern (NspI, NspII) have been described that visually resemble anticentromere pattern (pseudocentromere pattern) and therefore may be misinterpreted as anticentromere antibodies (ACA). Differentiation from ACA can be achieved by staining patterns seen in mitotic cells. Anti-NspI antibodies (IgG and IgM isotype) do not stain metaphase chromosomes as do ACA and anti-NspII antibodies (IgG isotype) do not show the discrete speckled interphase nuclear staining. NspI antigen seems to be a soluble nuclear protein but NspII antigen seems to be more tightly bound within the nucleus. Biological function of the two proteins is unknown. (Fritzler et al., 1994).

IIF Culture cells used for routine ANA screening

Anti-NspI: two patients with CAH. Anti-NspII: three patients with RA and SS.

(System C)

Autoantibody assays

Disease associations

--,..I

Autoantibody assays

Disease associations

ANA specificity

Antigen/Autoantibody descriptions

Nuclear Helicase

A nuclear ~rotein consisting of 1912 amino acids (calculated Mr 218K) and containing ELISA, IB (rAg) several functional domains which are characteristic for helicases have been shown to be precipitated by anti-Mi-2 sera. The protein encoding gene has been localized on human chromosome 12p13. The 218 kd nuclear helicase may represent one of the main antigen specificities recognized by anti-Mi-2 sera. (Seelig et al., 1995).

SLE 0.5% RA 0% DM: 12 of 12 patients with anti-Mi2 antibodies as revealed by ID.

Nucleolar 128 p antigen

A nuclear protein consisting of 1094 amino acids (calculated Mr 128 K). About 8% of sera positive for antinucleolar antibodies in immunofluorescence also react with this protein in ELISA and immunoblot. Protein is characterized by coiled-coil structures (heptad repeats). (Seelig et al., unpublished data).

IB, ELISA (rAg)

No figures yet available concerning frequency and disease associations.

Nucleosides Nucleotides Adenosine Guanosine Cytidine Thymidine Uracil

All bases contained in DNA or RNA as well as their nucleosides have been shown to ELISA, PHA constitute targets of autoantibodies. Antibodies often cross react with ssDNA and RNA. Nucleosides (tides) They may specifically recognize a particular nucleotide but cross-reactions between covalently bound to different nucleotides also have been reported. Low titered antinucleoside (tide) albumin antibodies of IgM isotype have been described in the majority of healthy persons; whereas, in patients with connective tissue diseases, high titered antibodies of IgG isotype prevail. Most frequently seen were antiguanosine antibodies in SLE and druginduced LE with higher titers in active disease. (Munns et al., 1987; Yee et al., 1985).

SLE 40-80%* DLE 83%**

c-myc Oncogene Product

Nuclear 65 kd phosphoprotein, binding to ssDNA and dsDNA, localizes with snRNPs in the interchromatinic areas of the nucleus. Modulation of its expression has been implicated in control of cell growth and differentiation. Alterations in expression of the c-myc gene by amplification, translocation, and increased transcription has also been associated with a number of human tumors and tumor cell lines. There is no significant correlation between presence of antibodies and kind of disease activity and presence of other autoantibodies. (Deguchi et al., 1988; LaFond et al., 1992).

ELISA, IB Synthetic peptide fragments of human c-myc protein

SLE 35% DM 40% SSc 40% SS 20% African Burkitt's lymphoma and normal Ghanians

Poly(ADP-ribose)

Poly(ADP-ribose) is a biopolymer which is made up of repeating ADP-ribose units and synthesize d by poly(ADP-ribose) polymerase (synthetase) within the cell nuclei. The highly immunogenic polymer can be reproducibly synthesized in vitro in pure form. Antipoly (ADP-ribose) antibodies found in SLE patients were regarded as a specific marker for that disease. High titer antibodies have been reported in SLE patients with obstetric complications (abortion, premature delivery). Comparative studies, however, revealed a lower sensitivity of antipoly(ADP-ribose) than of anti-dsDNA antibodies (64% sensitivity compared to anti-dsDNA antibodies) for SLE patients and also a lower specificity because of their prevalence in drug-induced LE. Cross reactions of antipoly(ADP-ribose) antibodies with synthetic polynucleotides but not with dsDNA have been reported. Presence of antibodies is not correlated with antibodies to poly(ADP-ribose) polymerase. (Dudeney et al., 1986; Kanai et al., 1989).

IB radiolabeled poly(ADP-ribose) ELISA (synthetic antigen)

SLE 42--58% Consanguineous relatives of SLE patients 44% DLE 50--90%* MCTD 10% SSC 44% * Depending on disease activity

* Depending on kind of nucleoside (tide) and activity of disease. ** Antiguanosine antibodies.

L~ O0

ANA specificity

Antigen/Autoantibody descriptions

Autoantibody assays

Disease associations

Poly(ADP-ribose) Polymerase

PARP (Ec 2.4.2.30) is a chromatin-bound, DNA-dependent enzyme of 113 kd containing two zinc fingers for recognition of double and single strand breaks and a nuclear location signal and catalyzing the ADP-ribosylation of nuclear acceptor proteins by using NAD as a substrate. The resulting homopolymer poly(ADP-ribose) has a branched structure containing phosphodiester and ribose bounds. Enzymes (e.g., topoisomerase I and II, DNA polymerase ~ and 13, RNA polymerase II) that are covalently modified by poly(ADP-ribosyl)ation generally are inhibited. Poly(ADPribosyl)ation of nuclear proteins has been correlated with DNA repair, gene expression, cell differentiation. PARP also catalyze the transfer of ADP-ribose residues to nonenzyme acceptor proteins e.g., histones, high mobility group proteins, lamins. (Muller et al., 1994).

ELISA, DB (nAg, rAg or synthetic peptide of second zinc finger domain). DB yields considerably more positive results than ELISA.

SLE Ps sSS

Poly(A) Polymerase

Nuclear enzyme which synthesizes the poly(A) tail of eukaryotic mRNA. This poly(A) tail is added in a 3' end-processing reaction after endonucleolytic cleavage of premRNA and may consist of (A)250 and more. Antibodies against liver type (rat) and tumor type (rat hepatoma cell nuclei)poly(A) polymerase have been described in patients with connective tissue disorders. (Stetler et al., 1987).

RIA (purified enzymes from rat liver (L) and rat hepatoma cells (H)

SLE RA SS SSc

Protein Kinase Nil

Nuclear enzyme (140 kd), consisting of subunits of 42 kd and 25 kd, respectively. Activates RNA polymerase I. Antiprotein kinase II antibodies have been found in patients with connective tissue disorders and are always associated with anti-RNA polymerase I antibodies. Antibodies inhibit the activity of nuclear protein kinase II but not that of cytoplasmic cyclic AMP-dependent protein kinase. (Stetler et al., 1984).

RIA (purified enzyme autophosphorylated with [~2p]ATP.

SLE MCTD

Pro-T(z constitutes a highly acidic protein of 12 kd with suspected nuclear localization because of its nuclear location sequence. Pro-T~ is synthesized not only within the thymus but also in other tissues especially in the spleen. Pro-T~ is involved in differentiation and maturation of thymocytes. (Vlachoyiannopoulos et al., 1989).

ELISA (nAg)

SLE

18%

IIF, ID (EBV-infected cells and extracts) ELISA (synthetic peptide p62)

RA

60%

PARP

Prothymosin Pro-T~

RANA RANA constitutes epitopes of Epstein-Barr nuclear antigen 1 (EBNA-1). The main Rheumatoid arthritis epitope is localized on a stretch of 20 amino acids of the p62 peptide which nuclear antigen corresponds to part of the internal repeat sequence (IR3) of EBNA-1. This epitope is recognized by anti-RANA antibodies in ELISA and IIF, but other epitopes may be recognized also in immunoprecipitation reactions. Anti-RANA antibodies may represent a specific immune response to EBV-encoded antigen in patients with rheumatoid arthritis. Anti-RANA (p62) also cross-react with keratin, actin and collagen and may be found in healthy persons at low titer. (Venables et al., 1988).

35% (57%*) 42% (50%*) 56% (60%*)

* Results obtained with synthetic peptides. Considerably lower incidence in LED, RA, SSc, infectious diseases. No correlation with prevalence of antipoly (ADPribose) antibodies. 60% 90% 40% 50%

(L,H) (L,H) (L)80% (H) (L)90% (H)

56% (n=9)

100% (n=4)

ANA specificity

Antigen/Autoantibody descriptions

Autoantibody assays

RD Gene Product

RD gene is localized between Bf and C4A genes in the human MHC class III region. ELISA, IB (rAg) IP (35SMCP) The gene encodes a nuclear protein of 381 amino acids (calculated Mr 42K) which shows homologies especially to human U1 snRNP 70K protein. Antibodies against the recombinant protein have been found in particular in patients with Sj6gren's syndrome. (Cheng et al., 1993).

SS No figures available concerning frequency and disease associations.

RMSA-1 Regulator of mitotic spindle assembly

Chromosomal protein of 418 amino acids (calculated Mr 47K) containing conserved nuclear location sequences and consensus motifs for phosphorylation by Cdc2 kinase. Protein is phosphorylated by Cdc2 kinase at the interphase/mitosis transition. Protein is possibly complexed with polypeptides of 31 kd, 67 kd, and 200 kd, respectively, and constitutes a chromatin factor necessary for mitotic spindle assembly as revealed by mitotic arrest induced by antisense DNA. Monospecific anti-RMSA-1 antibodies stain nuclei in prometaphase and metaphase but not interphase nuclei. (Yao et al., 1994).

Single patient suffering from LED

IB (rAg)

Replication Protein RPA protein A (RPA) a conserved single-stranded DNA binding protein complex is IB (rAg) A composed of three protein subunits of 70, 32 and 14 kd (RPA-70, RPA-32, RPA-14), RPA-70 respectively. It plays a central role in eukaryotic DNA replication, recombination and RPA-32 repair. The function of RPA-70 as DNA binding protein is well established. RPA-32 is phosphorylated in S and G2 phase, after binding to single stranded DNA within the replication initiation complex. RPA-70 and RPA-32 are localized in the nucleus (giving a dense speckled pattern with monospecific antibodies) as well as within the cytoplasm. (Garcia-Lozano et al., 1995).

Disease associations

Two patients with SS and SLE

o

ANA specificity

Antigen/Autoantibody descriptions

Autoantibody assays

Disease associations

RNA

Antigens used for demonstration of anti-RNA antibodies included various double or single stranded (ds, ss) polyribonucleotides (e.g., poly A-poly U, poly I-poly C, poly Gpoly C, poly A, poly U, poly(ADP-ribose)) and native viral, bacterial, fungal and eukaryotic ds- or ssRNAs. Antibodies against these less well characterized antigens were searched for by assays of various sensitivity and specificity such as agargel double diffusion, counter current electrophoresis, passive hemagglutination, RIA and ELISA, resulting in incomparable figures of antibody prevalence in a particular disease and of association with clinical activity and course of disorder. Therefore, anti-RNA antibodies have drawn less attention as diagnostic means. Native RNAs, however, comprise many discrete molecules as are ribosomal RNAs, tRNAs (see Mas), U snRNAs, and hY5-Ro RNA with unique sequences as well as unique secondary and tertiary structures. Functional domains and unique conformation epitopes of these RNAs also have been shown to be targets of autoantibodies (e.g., GTPase-associated center of 28S rRNA, anticodon loop of tRNA Ala, stem loops II and IV of U1 snRNA, and 2,2,7 trimethylguanosine cap of U snRNAs. The precise characterization of these antibody RNA interactions require more sophisticated analytical procedures; for example, immunoprecipitation of radiolabeled RNAs, competition assays, RNase protection assays, and RNA sequencing may yield clues to the nature of the antigens, their mode of autoantibody induction and their diagnostic role. (Hoet and van Venrooij, 1992; Shirai et al., 1991).

IB, CIE, PHA, CF, RIA, ELISA IP of radiolabeled RNAs. Competition and RNase protection assays. RNA sequencing

Synthetic ds/ss RNAs SLE 18--91% RA 14--87% JRA 31--87% OA 92-- 100% LED 42% MCTD 20% SCC 100% 20% SS CAH 27% Defined RNA epitopes 28S rRNA SLE 75%*; 8%** 4S RNA PM SLE, CAH, PBC tRNA tRNA Ala PM 3% tRNA Met PM SLE 4% U1 RNA Overlap 65% -TMG-cap SCC hY5-Ro RNA SLE * with and ** without anti-P antibodies

Nuclear protein of 254 amino acids with a calculated molecular weight of Mr 29.5 K and an electrophoretic mobility of 32 kd not associated with RNA. A stretch of 16 amino acids shows homology with the nuclear localization sequence of the SV40 large T antigen. A corresponding synthetic peptide of this region was immunoreactive in ELISA with SLE sera. Antibodies are suggested as a marker of disease activity. There is no significant association with sicca syndrome. Antigens are different from the Ku/Ki-system. (Harmon et al., 1981" Tojo et al., 1981" Yamanaka et al., 1992).

ID, CIE (nAg) IP (3SSMCP) ELISA (nAg, rAg, synthetic antigens)

SLE 7*--37%** MCTD 3% SS 8% PM/DM 3% SSc 7% RA 3% ITP 3% PBC 3% * CIE, ** synthetic peptide ELISA, recombinant antigen.

Polyribonucleotides

SL/Ki SL (Sicca/Lupus, Harmon et al., 1981) Ki (Tojo et al., 1981) PL-2

.

ANA specificity

Antigen/Autoantibody descriptions

Spl00

The acidic nuclear protein, Spl00 consists of 480 amino acids with a calculated Mr of IIF, IB, ELISA (nAg, 53K but a considerably slower electrophoretic mobility corresponding to 95--100 kd. rAg) One main reactive domain of 142 amino acids recognized by all sera is localized within the C-terminal half of the molecule and contains a sequence which shows homologies to HIV nef protein. Latter sequence is recognized by 60% of the sera (molecular mimicry with retroviruses expressing nef related proteins?). This 142 aa domain either may contain multiple linear epitopes or discontinuous conformation epitopes. Sp 100 synthesis can be induced by IFNo~, f3 and ~, up to 8 times the basal level. Its functional role is still obscure, it may act as transcription regulator protein and may be involved in induction of an antiviral state. Spl00 localizes in distinct nuclear spots (dots), which can be seen by IIF on routine HEp-2 cell substrates. The spots vary in size and number (5--30), are spread over the nucleoplasm with exception of the nucleoli. In endometrial cells the immunoreactive dots can be induced by diethylstilbestrol (DES). A protein of 55 kd (NP55) was also associated with nuclear dots. (Guldner et al., 1992; Szostecki et al., 1990).

PBC SSc SLE SS MCTD

Su MoS

Possibly chromatin-associated nuclear protein (100/102 kd) and possibly complexed with a 200 kd protein to form a 10S particle. Sera containing anti-Su antibodies can be negative for immunofluorescent antinuclear antibodies using routine assay procedures. Therefore, the nuclear localization of the antigens has to be confirmed unequivocally. 37% anti-Su positive sera also contain anti-Ku. Anti-Su antibodies can be induced in BALB/c mice by injection of 2,6,10,14-tetramethylpentadecane (pristane). (Satoh et al., 1994a; b; Treadwell et al., 1991).

ID (CTE) IP (3SSMCP) Capture ELISA

SLE MCTD Overlap PM/DM SCC SS RA

Th To RNase P

The Th and To autoantigens are associated with 7-2 RNA (MRP RNA) and 8-2 RNA (HI RNA). MRP RNA is associated with the enzyme RNase MRP, a site specific RNP endoribonuclease which processes mitochondrial substrates (mitochondrial RNA processing). H1 RNA copurifies with RNase P which cleaves precursor tRNA to produce mature 5'-ends. Both enzymes are associated with the Th/To autoantigen complex and will be precipitated by the same autoantibody species contained in antiTh/To sera. Anti-Th/To sera recognize a 40 kd protein (Th40) which interacts with RNA and constitutes an integral part of both the enzymes mentioned above. (Verheijen et al., 1994; Yuan et al., 1991).

25% IP of Th/To RNA from SLE 4%* cell extracts and probing SSc * In patients with limited cutaneous with radiolabeled involvement 8.4% antisense RNA

Transcription Factor TFIIIA

Nuclear proteins contained in HeLa cell extract with Mr of 37K (p37) and 32K (p32), respectively, noncovalently associated with 5SRNA and sedimenting as 7-10S particles immunologically related to TFIIIA transcription factor. Protein p37 binds in vitro to 5SRNA. (Lagaye et al., 1988).

IP (3SSMCP)

Nuclear dots MND-ANA Multiple nuclear d o t s - ANA

Autoantibody assays

Disease associations 13--44%* 7% 2% 9 9

* Especially in connection with SS. Specificity for PBC questionable because only 42% of Ab positive sera were from PBC patients ( 1 4 % Ai-CAH, 11% other liver disorders, 33% various immunological disorders).

17--21% 14% 22--40% 8% 13--20% 8% 7%

Two patients with autoimmune disorders

L~h t,O

ANA specificity

Antigen/Autoantibody descriptions

Autoantibody assays

Disease associations

XH

XH antigen is present in human spleen extract and resistant to digestion by RNase and trypsin. Neither biochemical or biological data have been reported nor has antigen localization within the nucleus been demonstrated. (Bernstein et al., 1984b).

CIE Human spleen extract

PBC 6% CAH (HBsAg negative) 2%

XR

XR antigen is present in rabbit thymus extracts and susceptible to digestion by RNase and trypsin. Neither biochemical nor biological data have been reported concerning this antigen, nor has its localization within the nucleus been demonstrated. (Bernstein et al., 1984b).

CIE Rabbit thymus extract

PBC 10% CAH (HBsAgnegative) 24%

REFERENCES Andrade LE, Tan EM, Chan EK. Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation. Proc Natl Acad Sci USA 1993;90:1947--1951. Asero R, Origgi L, Bertetti E, D'Agostino P, Riboldi P. Detection of two associated precipitating autoantibodies (DA1 and DA2) in sera from patients with systemic lupus erythematosus. J Clin Lab Immunol 1988;26:63--66. Astaldi Ricotti GC, Pazzaglia M, Martelli AM, Cerino A, Bestagno M, Caprelli A, Riva S, Pedrini MA, Facchini A. Autoantibodies to purified nuclear proteins related to DNA metabolism during ageing and in SLE patients. Immunology 1987;61:375--381. Bernstein RM, Neuberger JM, Bunn CC, Callender ME, Hughes GR, Williams R. Diversity of autoantibodies in primary biliary cirrhosis and chronic active hepatitis. Clin exp Immunol 1984a;55:553-560. Bernstein RM, Bunn CC, Hughes GR, Francoeur AM, Mathews MB. Cellular protein and RNA antigens in autoimmune disease. Mol Biol Med 1984b;2:105-120. Blencowe BJ, Carmo-Fonseca M, Behrens SE, Luhrmann R, Lamond AI. Interaction of the human autoantigen p 150 with splicing snRNPs. J Cell Sci 1993;105:685--697. Bloch DB, Rabkina D, Quertermous T, Bloch KD. The immunoreactive region in a novel autoantigen contains a nuclear localization sequence. Clin Immunol Immunopathol 1994;72:380-389. Chan EK, Takano S, Andrade LE, Hamel JC, Matera AG. Structure, expression and chromosomal localization of human p80-coilin gene. Nucleic Acids Res 1994;22:449. Cheng J, Macon KJ, Volanakis JE. cDNA cloning and characterization of the protein encoded by RD, a gene located in the class III region of the human major histocompatibility complex. Biochem J 1993;294:589--593. Deguchi Y, Negoro S, Kishimoto S. Autoantibody to human cmyc oncogene product in autoimmune patients' sera. Int Arch Allergy Appl Immunol 1988;87:313--316. Dick T, Mierau R, Sternfeld R, Weiner EM, Genth E. Clinical relevance and HLA association of autoantibodies against the nucleolus organizer region (NOR-90). J Rheumatol 1995;22:67--72. Dudeney C, Shoenfeld Y, Rauch J, Jones M, Mackworth Young C, Tavassoli M, Shall S, Isenberg DA. A study of antipoly(ADP-ribose) antibodies and an anti-DNA antibody idiotype and other immunological abnormalities in lupus family members. Ann Rheum Dis 1986;45:502--507. Fritzler MJ, Valencia DW, McCarty GA. Speckled pattern antinOclear antibodies resembling anticentromere antibodies. Arthritis Rheum 1994;27:92--96. Fritzler MJ, von Muhlen CA, Toffoli SM, Staub HL, Laxer RM. Autoantibodies to the nucleolar organizer antigen NOR90 in children with systemic rheumatic diseases. J Rheumatol 1995;22:521--524. Garcia-Lozano R, Gonzalez-Escribano F, Sanchez-Roman J, Wichmann I, Nunedan A. Presence of antibodies to different subunits of replication protein A in autoimmune sera. Proc Natl Acad Sci USA 1995;92:5116-5120.

Guldner HH, Szostecki C, Grotzinger T, Will H. IFN enhance expression of Spl00, an autoantigen in primary biliary cirrhosis. J Immunol 1992;149:4067-4073. Harmon C, Peebles C, Tan EM. S L - a new precipitating system. Arthritis Rheum 1981;24:S122. Hoet RM, van Venrooij WJ. B-cell epitopes of RNA autoantigens. Mol Biol Rep 1992;16:199-205. Imai H, Chan EK, Kiyosawa K, Fu X-D, Tan EM. Novel nuclear autoantigen with splicing factor motifs identified with antibody from hepatocellular carcinoma. J Clin Invest 1993 ;92:2419-2426. Juarez C, Agusti M, Gelpi C, Vila JL, Amengual MJ, Rodriguez JL. Characterization of the CB antigen, a DNAbinding protein recognized by autoantibodies and which has a differential expression in lymphoid cells. J Immunol 1988; 141:3841-3846. Kanai Y, Isonishi S, Terashima Y. Antibody to poly(ADPribose) is an indicator of obstetric complications in pregnant patients with systemic lupus erythematosus. Immunol Lett 1989;21:217--222. Kole R, Fresco LD, Keene JD, Cohen PL, Eisenberg RA, Andrews PG. Alu RNA-protein complexes formed in vitro react with a novel lupus autoantibody. J Biol Chem 1985;260:11781-11786. LaFond R, Eaton RB, Watt RA, Villee CA, Actor JK, Schur PH. Autoantibodies to c-myc protein: elevated levels in patients with African Burkitt's lymphoma and normal Ghanians. Autoimmunity 1992;13:215-224. Lagaye S, Barque JP, le Maire M, Denis H, Larsen CJ. Characterization by human antibodies of two HeLa cell proteins which are related to Xenopus laevis transcription factor TFIIIA. Nucleic Acids Res 1988;16:2473--2487. Lakomek HJ, Will H, Zech M, Kruskemper HL. A new serologic marker in ankylosing spondylitis. Arthritis Rheum 1984 ;27:961-997. Misaki Y, van Venrooij WJ, Pruijn GJ. Prevalence and characteristics of anti-56K/annexin XI autoantibodies in systemic autoimmune diseases. J Rheumatol 1995;22:97-102. Muller S, Briand J-P, Barakat S, LaGueux J, Poirier GG, de Murcia G, Isenberg DA. Autoantibodies reacting with poly(ADP-ribose) and with a zinc-finger functional domain of poly(ADP-ribose) polymerase involved in the recognition of damaged DNA. Clin Immunol Immunopathol 1994;73:187--196. Munns TW, Freeman SK, Liszewski MK, Kaine JL. Distribution and specificity of nucleotide-reactive autoantibodies in human SLE. J Immunol 1987; 139:393--399. Neu E, von Mikecz AH, Hemmerich PH, Peter H-H, Fricke M, Deicher H, Genth E, Krawinkel U, Members of the SLE Study Group. Autoantibodies against eukaryotic protein L7 in patients suffering from systemic lupus erythematosus and progressive systemic sclerosis: frequency and correlation with clinical, serological and genetic parameters. Clin Exp Immunol 1995; 100:198--204. Neuer G, Bautz FA, Bustin M, Michels H, Truckenbrodt H. Sera from JRA patients contain antibodies against a defined epitope in chromosomal protein HMG-17. Autoimmunity 1994;17:23--30.

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Nomoto Y, Suga T, Miura M, Nomoto H, Tomino Y, Sakai H. Characterization of an acidic nuclear protein recognized by autoantibodies in sera from patients with !gA nephropathy. Clin Exp Immunol 1986;65:513-519. Satoh M, Reeves WH. Induction of lupus-associated autoantibodies in BALB/c mice by intraperitoneal injection of pristane. J Exp Med 1994a; 180:2341--2346. Satoh M, Langdon JJ, Chou CH, McCauliffe DP, Treadwell EL, Ogasawara T, Hirakata M, Suwa A, Cohen PL, Eisenberg RA, Reeves WH. Characterization of the Su antigen, a macromolecular complex of 100/102 and 200-kDa proteins recognized by autoantibodies in systemic rheumatic diseases. Clin Immunol Immunopathol 1994b;73:132-141. Seelig HP, Moosbrugger I, Ehrfeld H, Fink T, Renz M, Genth E. The major dermatomyositis specific Mi-2 autoantigen is a presumed helicase involved in transcriptional activation. Arthritis Rheum 1995:In press. Shirai M, Watanabe S, Nishioka M. Autoantibody specific for transfer ribonucleic acid (tRNA) in patients with autoimmune chronic active hepatitis and primary biliary cirrhosis. Hepatogastroenterology 1991 ;38:543-546. Stetler DA, Rose KM, Wenger ME, Berlin CM, Jacob ST. Antiprotein Kinase NII antibodies in rheumatic autoimmune diseases. J Biol Chem 1984: 259:2077-2079. Stetler DA, Reichlin M, Berlin CM, Jacob ST. Antibodies against nuclear poly(A)polymerases in rheumatic autoimmune diseases. J Clin Immunol 1987;7:24-28. Szostecki C, Guldner HH, Netter HJ, Will H. Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis. J Immunol 1990;145:4338-4347. Tojo T, Kaburaki J, Hayakawa M, Okamoto T, Tomi M, Homma M. Precipitating antibody to a soluble nuclear antigen "Ki" with specificity for systemic lupus erythematosus. Ryumachi 1981 ;21:129-- 134. Treadwell EL, Bock AM, Kovacs SA, Chen JT, Wang RJ, Sharp GC, Agris PF. The autoimmune antigen Me is distinct and related to undifferentiated connective tissue disease. Arthritis Rheum 1987;30:1239--1246. Treadwell EL, Muller UR, Volkman A. Extraction and differentiation of the Su autoantigen from calf thymus nuclei.

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J Immunol Methods 1991;142:157--167. Tzioufas AG, Boumba VA, Seferiadis K, Tsolas O, Moutsopoulos HM. Autoantibodies to HMG-17 nucleosomal protein in autoimmune rheumatic diseases. Correlation with systemic lupus erythematosus clinical activity and with antibodies to double-stranded DNA. Arthritis Rheum 1993;36:955--961. Venables PJ, Pawlowski T, Mumford PA, Brown C, Crawford DH, Maini RN. Reaction of antibodies to rheumatoid arthritis nuclear antigen with a synthetic peptide corresponding to part of Epstein-Barr nuclear antigen 1. Ann Rheum Dis 1988;47:270-279. Verheijen R, Wiik A, de Jong BA, Ullman S, Halberg P, van Venrooij WJ. Screening for autoantibodies to the nucleolar U3- and Th(7-2) ribonucleoproteins in patients' sera using antisense riboprobes. J Immunol Methods 1994;169:173-182. Vlachoyiannopoulos PG, Frillingos S, Tzioufas AG, Seferiadis K, Moutsopoulos HM, Tsolas O. Circulating antibodies to prothymosin alpha in systemic lupus erythematosus. Clin Immunol Immunopathol 1989;53:151-- 160. Vlachoyiannopoulos PG, Boumba VA, Tzioufas AG, Seferiadis C, Tsolas O, Moutsopoulos HM. Autoantibodies to HMG-17 nucleosomal protein in patients with scleroderma. J Autoimmun 1994;7:193-201. Winn DM, Wolfe JF, Harmon D, Sharp GC. Characterization of a distinct nuclear acidic protein antigen (MA) and clinical findings in systemic lupus erythematosus patients with MA antibodies. J Clin Invest 1979;64:820-823. Yamanaka K, Takasaki Y, Nishida Y, Shimada K, Shibata M, Hashimoto H. Detection and quantification of anti-Ki antibodies by enzyme-linked immunosorbent assay using recombinant Ki antigen. Arthritis Rheum 1992;35:667--671. Yao J-P, Alderuccio F, Toh B-H. A new chromosomal protein essential for mitotic spindle assembly. Nature 1994;367:288-291. Yee WS, Weisbart RH. The fine specificity of IgG antiguanosine antibodies in systemic lupus erythematosus. Clin Immunol Immunopathol 1985;36:161--167. Yuan Y, Tan E, Reddy R. The 40-kilodalton To autoantigen associates with nucleotides 21 to 64 of human mitochondrial RNA processing/7-2 RNA in vitro. Mol Cell Biol 1991;11:5266--5274.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

p53 AUTOANTIBODIES Thierry Soussi, Ph.D. and Richard Lubin, M.D.

Unitd 301 INSERM, Institut de Gdndtique Moldculaire, 75010 Paris, France

HISTORICAL NOTES The humoral response of mice to some methylcholanthrene-induced tumor cells such as MethA is directed to the p53 protein (DeLeo et al., 1979). In animals, several types of tumors elicit an immune response specific for p53 (Kress et al., 1979; Melero et al., 1979; Rotter et al., 1980). In 1982, antibodies against human p53 protein were described in 9% of breast cancer sera (Crawford et al., 1982); no significant clinical correlation was reported, and at that time, no information was available concerning mutations of the p53 gene. Such antibodies are also present in sera of children with a wide variety of cancers at an average frequency of 12%; this figure increased to 20% in Burkitt lymphoma (Caron de Fromentel et al., 1987). Those studies, performed in the early eighties, were virtually ignored for more than 10 years due to the lack of interest in p53. In the early nineties, mutations of the p53 gene were found to be the most common target for molecular alteration in every type of human cancer. Subsequent studies of the p53 protein and its function in normal and transformed cells led to the rediscovery of this humoral response which is found in cancer patients. All the studies described below concern p53 autoantibodies detected in patients at the time of diagnosis, prior to any treatment such as surgery or chemotherapy.

THE AUTOANTIGEN(S) Definition/Origin The tumor suppressor gene p53 is a phosphoprotein

protein expressed at very low, barely detectable levels in the nucleus of normal cells. Following DNA damage, particularly that induced by UV or gamma radiation, p53 can arrest cell cycle progression in the late G1 phase thus allowing the DNA to be repaired prior to its replication. This function is achieved by the transcriptional regulation properties of p53, which activate a series of genes involved in cell cycle arrest (Donehower and Bradley, 1993). In cancer cells bearing a mutant p53, this protein can not induce G1 arrest in response to DNA damage, resulting in inefficient DNA repair and the emergence of genetically unstable cells. The most common change of p53 in human cancers is a point mutation within the coding sequences of the p53 gene which give rise to an altered protein (Caron de Fromentel and Soussi, 1992; Soussi et al., 1994). Mutations in the p53 gene are found in all major histogenetic groups, including cancers of the colon, stomach, breast, lung, brain and esophagus. Mutations of p53 are the most frequent genetic event in human cancers occurring in more than 50% of cases. More than 90% of the point mutations reported so far are clustered between exons 4 and 9 and are localized in the DNA-binding domain of the p53 protein (Caron de Fromentel and Soussi 1992). One of the most striking features of the inactive mutant p53 protein is its increased stability. The mutant p53 protein has an abnormal conformation, is more stable than the wild type (half-life of several hours compared to 20 min for the wild type p53), accumulates in the nucleus of neoplastic cells and is thus immunologically detectable. Therefore, positive immunostaining is indicative of abnormalities of the p53 gene and its product (Dowell et al., 1994).

595

THE AUTOANTIBODIES Pathogenetic Role The discovery that p53 mutations can lead to the accumulation of the p53 gene product in tumor cells has shed new light on the earlier discovery of p53 autoantibodies in sera of patients with breast cancer. The relationships among the p53 gene mutation, the p53 accumulation and the p53 humoral response are now being explored. A new series of studies confirms that p53 autoantibodies can be found in sera of patients with various types of cancer; whereas, the prevalence of such antibodies in the normal population is very low (Angelopoulou et al., 1994; Davidoff et al., 1992; Green et al., 1994; Labrecque et al., 1993; Lubin et al., 1993; Marxsen et al., 1994; Schlichtholz et al., 1992; 1994; Volkmann et al., 1993; Winter et al., 1992). Studies correlating the presence of p53 autoantibodies with the p53 mutation and/or p53 accumulation suggest that most patients with p53 antibodies have a p53 mutation which leads to p53 accumulation (Davidoff et al., 1992; Guinee et al., 1995; Lubin et al., 1993; Preudhomme et al., 1994; Volkmann et al., 1993; Wild et al., 1995; Winter et al., 1992). Nevertheless, exceptions exist (Guinee et al., 1995; Preudhomme et al., 1994; Wild et al., 1995), i.e., certain patients have p53 autoantibodies, yet no p53 mutation is found in the tumor. On the other hand, not all patients with a p53 alteration develop p53 autoantibodies. The type of mutation is thought to influence the production of p53 antibodies (Davidoff et al., 1992) but given more recent results (Guinee et al., 1995; Preudhomme et al., 1994; Wild et al., 1995), this hypothesis requires further investigation. It is also possible that for an identical mutation the humoral response is dependent on the major histocompatibility complex (MHC) class I or II molecule specific to each individual. In general, 30 to 40% of patients with an alteration in the p53 gene develop p53 antibodies (Lubin et al., 1995a). Although originally thought to be directed to the central region of the p53 protein, which is the target for the various mutations, these p53 autoantibodies actively bind both wild-type and mutant p53 (Labrecque et al., 1993; Lubin et al., 1993; Schlichtholz et al., 1992; 1994). As determined with truncated p53 or synthetic peptides, the epitopes recognized by the p53 autoantibodies are mainly located in the amino and carboxy terminal regions of the protein, regions which 596

are not in the "hot spot" areas for the p53 mutation (Lubin et al., 1993; Schlichtholz et al., 1992; 1994). These immunodominant epitopes are also detected in the sera of mice and rabbits hyperimmunized with wild-type p53 (Legros et al., 1994). Taken together: 1) the presence of immunodominant epitopes outside the hot spot region of the p53 mutation, 2) the correlation between p53 accumulation (and p53 gene mutation) in tumor cells and p53 antibody responses, 3) the similarity of humoral responses in patients independent of the cancer type and 4) the similarity of antigenic site profiles in patients and hyperimmunized animals all suggest that p53 accumulation is a major determinant of the humoral response in patients with cancer. Such accumulation might lead to the appearance of p53 antibodies, because the low level of p53 proteins in a normal organism is expected to correlate with very weak (or absent) tolerance to endogenous p53.

Factors in Pathogenicity Autoantibodies to p53 are mainly IgG1 and IgG2. Some patients exhibited a predominantly IgA response (Lubin et al., 1995a; 1995b), and some patients also had IgM, but none had p53 IgM as the only isotype. No IgG3 or IgG4 were detected. Again, this result strengthens the hypothesis of an active humoral response to p53. The mechanism by which p53 is presented to the immune system is unknown. Although possibly released following cell necrosis, p53 is not found in human sera (Hassapoglidou et al., 1993; Winter et al., 1992). Either the p53 protein is very rapidly eliminated from sera, or else other mechanisms of p53 presentation are involved. So far, neither free p53 nor p53/anti-p53 has been detected in sera.

Sequence Information Although its p53 tertiary structure is not entirely known at present, the primary sequence of p53 from 9 different species provides a clue as to its organization (Soussi et al., 1990). The polypeptide chain of p53 is subdivided into three regions: a highly charged acidic amino-terminal region that comprises the first 80 amino acids, a hydrophilic charged basic region at the carboxy terminus comprising residues 319 to 393 and a central part of the protein having a low charge density and containing a high number of hydrophobic residues (Soussi et al., 1990). Recent structural analyses of the p53 protein confirm such a model with

CLINICAL UTILITY

the amino (residues 1 roughly 95) and carboxy terminal (residues 300 to 393) regions of p53, which are highly exposed and accessible at the protein surface; whereas, the central region seems to be buried in the interior of the molecule (Bargonetti et al., 1993).

Application The p53 mutation/accumulation (assessed either by molecular or immunohistochemical analysis) is an independent predictive factor for unfavorable prognosis in patients with breast (Barnes et al., 1993) and colon cancer (Hamelin et al., 1994), i.e., more aggressive tumors and poorer survival. Confirmation of these results by serological analysis strengthens the relationship between p53 autoantibodies and the p53 gene alteration (Peyrat et al., 1995; Schlichtholz et al., 1992) (Figure 1).

Methods of Detection Until recently, the methods most frequently used to detect p53 autoantibodies were, immunoblot and immunoprecipitation using transformed cell extract as a source of antigen. Two different ELISAs have been described. One of them uses a sandwich method and mutant p53 as antigen (Angelopoulou et al., 1994); whereas, the second one is a direct ELISA using wildtype p53 as antigen (Lubin et al., 1995b). This use of very different methods of detection leads to sharp variations in the frequency of p53 autoantibodies in a given cancer. In breast carcinomas, which are extensively studied, the frequency varies: 1% (Vojtesek et al., 1995), 5% (Angelopoulou et al., 1994), 9% (Crawford et al., 1982), 14% (Peyrat et al., 1995; Schlichtholz et al., 1992), 25.6% (Mudenda et al., 1994) (Figure 1). This discrepancy is partially explained by the various techniques (ELISA, immunoprecipitation or immunoblot) used in these studies, but it might also reflect unsuspected bias in the choice of patients (difference in clinical status, environmental or geographical factors).

60"

Disease Associations Detection of p53 alterations is now important as a clinical marker of poor prognosis and also as a potential target for cancer treatment. Although p53 autoantibodies are not systematically associated with a p53 alteration, serological analysis of the p53 alteration offers some unique advantages over DNA sequencing (i.e., analysis (ELISA) is simple, tumor tissue is not needed and the antibodies can be monitored during treatment of the patient). In addition, assay for p53 antibodies is a global approach to assessing p53 alterations, which does not depend on sampling of the tumor, the composition of which may be heterogeneous. Molecular analysis of tumor tissues

II [~

p53 Antibodies (Lubin et ah, 1995) p53 Antibodies (Angelopoulou et al., 1994)

9 p53 mutation

o~ >.

(,~

40

Z Ill O

UJ LL 20

LUNG

COLON

OVARY

PANCREAS BLADDER BREAST

THYROID LEUKEMIA PROSTATE BLOOD DONNOR

CANCER Figure 1. Comparison of the frequency of p53 mutations and p53 antibodies in various types of cancer. Data were compiled from the work of Lubin et al. (1995) and Angelopoulou et al. (1994). ND; not determined. 597

or biopsies corresponds to local analysis of p53 status and may be misleading if the tumor is too heterogeneous or is too dispersed in normal tissue. Furthermore, mutation is not necessary for p53 accumulation (Andersen et al., 1993; Moll et al., 1992), and p53 antibodies can be detected in such patients. Recently, p53 antibodies were detected in sera of two patients who were heavy smokers without diagnosed lung malignancy (Lubin et al., 1995a; 1995b). Both of these patients developed invasive squamous lung cancer 5 and 15 months after detection of serum p53 antibodies. In the second patient, p53 overexpression was detected in tumor cells from bronchial biopsy specimens. Because p53 alterations are the most common, earliest genetic changes in lung carcinogenesis, detection of p53 antibodies might be a new and sensitive tool for detection of preneoplastic and

REFERENCES Andersen TI, Holm R, Nesland JM, Heimdal KR, Ottestad L, Borresen AL. Prognostic significance of TP53 alterations in breast carcinoma. Br J Cancer 1993;68:540-548. Angelopoulou K, Diamandis EP, Sutherland DJ, Kellen JA, Bunting PS. Prevalence of serum antibodies against the p53 tumor suppressor gene protein in various cancers. Int J Cancer 1994;58:480-487. Bargonetti J, Manfredi JJ, Chen X, Marshak DR, Prives C. A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53-protein. Genes Dev 1993;7:2565-2574. Barnes DM, Dublin EA, Fisher CJ, Levison DA, Millis RR. Immunohistochemical detection of p53 protein in mammary carcinoma: an important new independent indicator of prognosis? Hum Pathol 1993;24:469-476. Caron de Fromentel C, May-Levin F, Maorisesse H, Lemerle J, Chandrasekaran K, May P. Presence of circulating antibodies against cellular protein p53 in a notable proportion of children with B-cell lymphoma. Int J Cancer 1987;39:185-189. Caron de Fromentel C, Soussi T. TP53 tumor suppressor gene: a model for investigating human mutagenesis. Genes Chromosom Cancer 1992;4:1-15. Crawford LV, Pim DC, Bulbrook RD. Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer. Int J Cancer 1982;30:403--408. Davidoff AM, Iglehart JD, Marks JR. Immune response to p53 is dependent upon p53/HSP70 complexes in breast cancers. Proc Natl Acad Sci USA 1992;89:3439-3442. DeLeo AB, Jay G, Appella E, Dubois GC, Law LW, Old LJ. Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse. Proc Natl Acad Sci USA 1979;76:2420-2424. 598

microinvasive bronchial lesions in patients with a high risk of lung cancer, i.e., heavy smokers.

CONCLUSION Serological analysis of p53 alterations is still in its infancy and will require some standardization before a clear picture can emerge concerning the true frequency of p53 autoantibodies. Commercial ELISA kits will soon be available and will enable large scale analysis, with comparisons in various populations. This should further clarify the clinical utility of these assays for detection of p53 alterations as well as their predictive value for patients with high risk of lung cancer.

Donehower LA, Bradley A. The tumor suppressor p53. Biochim Biophys Acta 1993;1155:181--205. Dowell SP, Wilson PO, Derias NW, Lane DP, Hall PA. Clinical utility of the immunocytochemical detection of p53 protein in cytological specimens.-Cancer Res 1994;54:2914-2918. Green JA, Mudenda B, Jenkins J, Leinster SJ, Tarunima M, Green B, Robertson L. Serum p53 autoantibodies: incidence in familial breast cancer. Eur J Cancer 1994;30:580-584. Guinee DG, Travis WD, Trivers GE, De Benedetti VM, Cawley H, Welsh JA, Bennett WP, Jett J, Colby RV, Tazelaar H, et al. Gender comparisons in human lung cancer: analysis of p53 mutations, anti-p53 serum antibodies and C-erbB-2 expression. Carcinogenesis 1995; 16:993-1002. Hamelin R, Laurent-Puig P, Olschwang S, Jego N, Asselain B, Remvikos Y, Girodet J, Salmon RJ, Thomas G. Association of p53 mutations with short survival in colorectal cancer. Gastroenterology 1994;106:42--48. Hassapoglidou S, Diamandis EP, Sutherland DJ. Quantification of p53 protein in tumor cell lines, breast tissue extracts and serum with time-resolved immunofluorometry. Oncogene 1993;8:1501--1509. Kress M, May E, Cassingena R, May P. Simian virus 40-transformed cells express new species of proteins precipitable by antisimian virus 40 tumor serum. J Virol 1979;31:472-483. Labrecque S, Naor N, Thomson D, Matlashewski G. Analysis of the anti-p53 antibody response in cancer patients. Cancer Res 1993;53:3468--3471. Legros Y, Lafon C, Soussi T. Linear antigenic sites defined by the B-cell response to human p53 are localized predominantly in the amino and carboxy-termini of the protein. Oncogene 1994;9:2071-2076. Lubin R, Schlichtholz B, Bengoufa D, Zalcman G, Tredaniel J, Hirsch A, de Fragmental CC, Preudhomme C, Fenaux P, Fournier G, et al. Analysis of p53 antibodies in patients with various cancers define B-Cell epitopes of human p53:

distribution on primary structure and exposure on protein surface. Cancer Res 1993;53:5872--5876. Lubin R, Schlichtholz B, Teillaud JL, et al. p53 antibodies in patients with various types of cancer: assay, identification and characterization. Clinical Cancer Res 1995a; 1:in press. Lubin R, Zalcman G, Bouchet L, et al. Serum p53 antibodies as early markers of lung cancer. Nature Med 1995b;1:701-702. Marxsen J, Schmiegel W, Roder C, Harder R, Juhl H, HenneBruns D, Kremmer B, Kalthoff H. Detection of the anti-p53 antibody response in malignant and benign pancreatic disease. Br J Cancer 1994;70:1031--1034. Melero JA, Stitt DT, Mangel WF, Carroll RB. Identification of new polypeptide species (48--55K) immunoprecipitable by antiserum to purified large T antigen and presel~t in SV40infected and -transformed cells. Virology 1979;93:466-480. Moll UM, Riou G, Levine AJ. Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc Natl Acad Sci USA 1992;89:7262-7266. Mudenda B, Green JA, Green B, Jenkins JR, Robertson L, Tarunina M, Leinster SJ. The relationship between serum p53 autoantibodies and characteristics of human breast cancer. Br J Cancer 1994;69:1115-- 1119. Peyrat JP, Bonneterre J, Lubin R, Vanlemmens L, Fournier J, Soussi T. Prognostic significance of circulating p53 antibodies in patients undergoing surgery for locoregional breast cancer. Lancet 1995;345:621--622. Preudhomme C, Lubin R, Lepelley P, Vanrumbeke M, Fenaux P. Detection of serum anti p53 antibodies and their correlation with p53 mutations in myelodysplastic syndromes and acute myeloid leukemia. Leukemia 1994;8:1589-1591. Rotter V, Witte ON, Coffman R, Baltimore D. Abelson murine leukemia virus-induced tumors elicit antibodies against a host

cell protein, P50. J Virol 1980;36:547--555. Schlichtholz B, Legros Y, Gillet D, Gaillard C, Marty M, Lane D, Calvo F, Soussi T. The immune response to p53 in breast cancer patients is directed against immunodominant epitopes" unrelated to the mutational hot spot. Cancer Res 1992;52: 6380-6384. Schlichtholz B, Tredaniel J, Lubin R, Zalcman G, Hirsch A, Soussi T. Analyses of p53 antibodies in sera of patients with lung carcinoma define immunodominant regions in the p53 protein. Br J Cancer 1994;69:809-816. Soussi T, Caron de Fromentel C, May P. Structural aspects of the p53 protein in relation to gene evolution. Oncogene 1990;5:945--952. Soussi T, Legros Y, Lubin R, Ory K, Schlichtholz B. Multifactorial analysis of p53 alteration in human cancer: a review. Int J Cancer 1994;57:1--9. Vojtesek B, Kovarik J, Dolezalova H, Nenutil R, Havlis P, Brentani RR, Lane DP. Absence of p53 autoantibodies in a significant proportion of breast cancer patients. Br J Cancer 1995;71:1253--1256. Volkmann M, Muller M, Hofmann WJ, Meyer M, Hagelstein J, Rath U, Kommerell B, Zentgraf H, Galle PR. The humoral immune response to p53 in patients with hepatocellular carcinoma is specific for malignancy and independent of the alpha-fetoprotein status. Hepatology 1993;18:559--565. Wild CP, Ridanpaa M, Anttila S, Soussi, T, Husgafvel-Pursiainen K, Vainio H. p53 antibodies in the sera of lung cancer patients: comparison with p53 mutation in the tumour tissue. Int J Cancer 1995;64:176--181. Winter SF, Minna JD, Johnson BE, Takahashi T, Gazdar AF, Carbone DP. Development of antibodies against p53 in lung cancer patients appears to be dependent on the type of p53 mutation. Cancer Res 1992;52:4168--4174.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PARIETAL CELL AUTOANTIBODIES Paul A. Gleeson, Ph.D., Ian R. van Driel, Ph.D. and Ban-Hock Toh, M.B.B.S, Ph.D.

Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria 3181, Australia

HISTORICAL NOTES

Native vs. Recombinant Antigen Performance

Circulating autoantibodies to gastric parietal cells were first detected in patients with pernicious anemia by a complement fixation test (Irvine et al., 1962) and subsequently by immunofluorescence staining of the cytoplasm of gastric parietal cells (Taylor et al., 1962). The antibodies do not bind to tissues or cell types other than gastric mucosa, but do bind parietal cells of most species. The parietal cell autoantigen(s) was localized to the secretory canaliculi of gastric parietal cells and to gastric microsomes (Hoedemaeker and Ito, 1970). Subsequent biochemical and molecular cloning studies identified the autoantigens as the (xand ~3-subunits of the gastric H/K ATPase (Gleeson and Toh, 1991). The membrane-bound gastric H/K ATPase is a proton pump responsible for the acidification of the stomach lumen.

The autoantibodies to the H/K ATPase are subunitspecific. There are differences in the binding of autoantibodies to the individual subunits of the H/K ATPase. The H/K ATPase (x-subunit autoantibodies recognize the denatured antigen by immunoblotting of reduced gastric membrane extracts: To avoid aggregation of the multiple-membrane spanning (x-subunit, membrane extracts should notbe boiled prior to SDSPAGE (Jones et al., 1991b; Callaghan et al., 1993). The (x-subunit, specific antibodies recognize recombinant bacterial fusion proteins (Callaghan et al., 1993) and the rat H/K ATPase (x-subunit protein expressed in insect cells (unpublished data). The (x-subunitspecific autoantibodies can probably recognize the native autoantigen, as indicated by H/K ATPase inhibition experiments (Burman et al., 1989), although formal proof is lacking. Autoantibodies to the H/K ATPase 13-subunit can recognize the native antigen as demonstrated by immunoprecipitation of detergent extracts of gastric membranes and by immunofluorescence of frozen sections (Goldkorn, 1991). In addition, the [3-subunitspecific autoantibodies immunoblot the antigen from gastric extracts; however, and in contrast to (x-subunit autoantibodies, optimal reactivity is observed when the gastric samples are nonreduced and boiled (Callaghan et al., 1993; Goldkorn et al., 1989). Binding of the autoantibodies ,to the [3-subunit is dependent on both the carbohydrate and protein moieties of the autoantigen as treatment with peptide; N-glycosidase F or reduction of disulfide bonds reduces autoantibody binding (Goldkorn et al., 1989). Partial deglycosylated ~-subunit, or recombinant [3-subunit bearing high mannose N-glycans, fails to bind the autoantibodies,

THE AUTOANTIGENS Definition

The gastric H/K ATPase (EC 3.6.1.3) is a hydrogentransporting enzyme, or proton pump, responsible for acid secretion (Rabon and Reuben, 1990). It belongs to the family of electroneutral P-type ATPases which also include the Na/K and Ca ATPases (Pedersen and Carfoli, 1987). The gastric H/K ATPase consists of two subunits, an 8-10 transmembrane catalytic (xsubunit of 1033 amino acids and a heavily glycosylated associated (x-subunit with a 294 amino acid core (Figure 1). On SDS-PAGE the apparent molecular mass of the (x-subunit is 92 kd and the ~-subunit 60-90 kd.

600

C l Mf

[3subunit

Extracellular

fifififi UUUU

fifififi UUVU C

N"

N

Cytoplasm

subunit 7 Figure 1. A model of the gastric H/K ATPase. The ~ subunit contains 10 transmembrane domains with the ATP binding site localized on the cytoplasmic, hydrophilic domain between transmembrane domains 4 and 5. The [3 subunit contains a short amino terminal cytoplasmic tail, transmembrane domain, and the luminal carboxy terminal domain. Seven potential N-glycosylation sites on the luminal domain of the 13subunit are indicated.

indicating the requirement for a full complement of the native carbohydrate structures (Callaghan et al., 1993).

Origin, Sources, Organs, Tissue, Cells The gastric H/K ATPase is localized to specialized intracellular and apical membranes of parietal cells of the gastric mucosa (Mercier et al., 1989; Smolka et al., 1983; Pettitt et al., 1995). This H/K ATPase is found in the gastric mucosa of all mammals examined, and shows a high degree of conservation in amino acid sequence across species (van Driel and Callaghan, 1995). The native antigens must be obtained from stomach tissue as parietal cell lines are not available.

Methods of Purification The ATPase ~-subunit glycoprotein specifically reacts with lectins derived from tomato and potato (Callaghan et al., 1990). These lectins bind polylactosamine carbohydrate sequences. Tomato lectin chromatography allows the rapid purification of the parietal cell autoantigen from pig stomach in high yield (Callaghan et al., 1992). The pig autoantigens are purified as an

active H/K ATPase a- and [3-subunit complex. Tomato lectin chromatography can be employed to isolate the autoantigen complex from other species including dog (Chuang et al., 1992), mouse and rabbit (unpublished data).

Commercial Sources There are no commercial sources of the gastric H/K ATPase.

Sequence Information The protein sequences of the ATPase subunits were deduced from cDNA sequences from a variety of species, for the c~-subunit human, mouse, rat, dog, pig and rabbit (Genbank codes HUMHKATPC, MMU 17282, RATATPASEZ, DOGHKATP, PIGATPHK and OCATPRNA, respectively) and for the ~-subunit, pig, rabbit, rat and mouse (Genbank codes PIGGASBAA, RABGHKAB, RATHKATPB, MUSATPB05, respectively). The epitopes are not yet mapped on either subunit; however, studies with modified native antigens and recombinant fusion proteins suggest that the autoepitopes of the [~-subunit are located on the luminal domain (Goldkorn et al., 1989); whereas, ,at

601

least some ~-subunit autoepitopes are located within the catalytic cytosolic domain of the molecule (Callaghan et al., 1993; Burman et al., 1989).

AUTOANTIBODIES

Terminology Antiparietal cell autoantibody is often abbreviated to PCA.

Pathogenetic Role Human Disease. Parietal cell autoantibodies are unlikely to have a role in the pathogenesis of pernicious anemia, because the H/K ATPase is localized only to the intracellular membranes and to the apical surface of parietal cells and is absent from the basolateral surface (Callaghan et al., 1990). Further, at least some of the autoepitopes of the H/K ATPase ~subunit are localized on the cytoplasmic domains of this membrane protein (Callaghan et al., 1993). Because access of circulating autoantibodies to the H/K ATPase of intact parietal cells is problematic, a direct pathogenic role of these autoantibodies is unlikely. Nevertheless, other as yet undetected autoantigens on the basolateral membrane of parietal cells could be involved. Animal Models. Neonatal thymectomy of certain strains of mice, e.g., B ALB/c and AJ, results in a high frequency of autoimmune gastritis in which circulating autoantibodies to the c~ and ~-subunits of the gastric H/K ATPase are a characteristic feature (Jones et al., 1991a). However, parietal cell autoantibodies are unable to induce the disease when transferred to a nude or SCID mouse recipient; whereas, autoimmune gastritis can be transferred with CD4 + T cells obtained from gastritic mice (Sagaguchi et al., 1985; Smith et al., 1992). Thus, experimental autoimmune gastritis is a cell-mediated not autoantibody-mediated disease. Autoimmune gastritis can also be induced in adult mice by combined thymectomy and cyclophosphamide treatment (Barrett et al., 1995) and by immunization with purified mouse gastric H/K ATPase (unpublished data). Genetics A genetic predisposition to the disease is suggested by

602

familial occurrence of pernicious anemia, the presence of circulating parietal cell autoantibodies and associated type A chronic atrophic gastritis in 20--30% of relatives of patients with pernicious anemia (Strickland, 1990a; Whittingham et al., 1991). In addition, first-degree relatives also have a higher-than-normal frequency of autoantibodies to antigens of other organ-specific endocrinopathies that cluster with pernicious anemia (Whittingham et al., 1991). Limited studies of pernicious anemia in monzyogotic twins support genetic factors in disease susceptibility (Delva et al., 1965; Irvine et al., 1965). Although an increased frequency of a number of MHC alleles is reported in patients compared with control groups, the associations are generally weak and are not observed in all studies (Whittingham et al., 1991).

Methods of Detection Circulating parietal cell autoantibodies are routinely detected in clinical laboratories by immunofluorescence reactivity with gastric parietal cells in frozen sections of mouse stomach. Rat stomach should not be used due to the presence of heteroantibody in human sera (Hawkins et al., 1977; Strickland and Hooper, 1972). The autoantibodies typically show a reticular cytoplasmic staining of parietal cells (Figure 2). The immunofluorescence assay is semiquantitative and requires tissue sections with preserved antigenicity. Currently, the best immunofluorescence results are obtained with sections of frozen mouse stomach. However, the gastric autoantigens are unusual in also being preserved after paraffin fixation. Recently, an ELISA was developed to detect parietal cell autoantibodies using tomato lectin-purified gastric H/K ATPase (Chuang et al., 1992). Autoantibodies to the H/K ATPase subunits can also be detected by immunoblotting using gastric extracts (Callaghan et al., 1993). An advantage of immunoblotting is that the individual subunit specificities can be identified.

CLINICAL UTILITY

Application Pernicious anemia is the most common cause of vitamin B12 deficiency in Western populations. Longitudinal studies suggest that pernicious anemia is the end stage of type A chronic atrophic gastritis (Irvine et al., 1974), a disease characterized by

Figure 2. Indirect immunofluorescent staining of gastric mucosa with parietal cell autoantibody-positive human serum. A typical example of the staining pattern of a paraffin-embedded section of rodent gastric mucosa with parietal cell autoantibodies showing A positive staining cells at the middle and base of the gland and B reticular intracellular staining of parietal cells at higher magnification.

pathological lesions of the fundus and body of the stomach, including gastric mucosal atrophy, selective

loss of parietal and chief cells from the gastric mucosa and submucosal lymphocytic infiltrates (Whitting-

603

ham and Mackay, 1985; Strickland, 1990b). Autoantibodies to pepsinogen secreted by Chief.cells have been detected by ELISA in patients with pernicious anemia (Mardh et al., 1991). Patients with pernicious anemia appear pale, physically tired and mentally depressed. The anemia is a direct consequence of the lack of intrinsic factor, a secreted product of gastric parietal cells that is required for the dietary absorption of vitamin B 12. Laboratory tests are supportive. Detection of parietal cell autoantibodies by immunofluorescence together with a second autoantibody, namely intrinsic factor autoantibody by radioimmunoassay, is diagnostic of pernicious anemia (Strickland, 1990b). The value of parietal cell antibodies in the diagnosis of pernicious anemia has been challenged as differences were found according to age and race (Carmel, 1992). However, rat sections were used as substrate for detection of these antibodies and rat is not the preferred species as these sections may detect heterophil antibodies. When both parietal cell and intrinsic factor autoantibodies are negative, it is unlikely that pernicious anemia is the explanation for low serum vitamin B12 levels (Strickland, 1990a). Whether screening is useful in high-risk groups (for example, relatives of patients with pernicious anemia) even in the absence of vitamin B 12 deficiency, is worthy of study. Parietal cell autoantibodies can also be used to differentiate type A atrophic gastritis from the other forms of nonspecific histological gastritis. These include type B, Helicobacter pylori-associated gastritis, type AB, and reflux gastritis following surgery; all are rarely associated with gastric autoimmune reac- . tions (Strickland, 1990b).

Disease Associations Pernicious anemia is predominantly a disease of middle-age northern white Europeans. The phenotypic blue eyes, blood group A and fair skin are associated with pernicious anemia (Callender et al., 1957). Females have a higher incidence of disease than males. Pernicious anemia is rare among southern Europeans, Blacks, Latin Americans and Asians. The disease appears to occur in Blacks at an earlier age than in northern Europeans (Carmel and Johnson, 1978). Pernicious anemia associates with a number of other diseases. These associated diseases are predominantly organ-specific autoimmune diseases of the endocrine glands in which autoantibodies to other

604

tissue-specific antigens are also present. The associated organ-specific diseases include Hashimoto's thyroiditis, Type 1 diabetes mellitus, and primary Addison's disease (Whittingham and Mackay, 1985). Late stages of pernicious anemia may also be associated with peripheral neuropathy and subacute combined degeneration of the spinal cord due to vitamin B 12 deficiency.

Antibody Frequencies in Disease Early studies consistently found parietal cell autoantibodies in the sera of--90% of patients with pernicious anemia (Whittingham and Mackay, 1985; Strickland, 1990a). However, a recent study, involving mixed racial groups found a lower frequency (-~55%) of parietal cell autoantibodies; this may reflect different racial groups, or the use of rat sections as substrate for detection of parietal cell antibodies or a younger age of the pernicious anemia patients (Carmel, 1992). A correlation between autoantibody titer and severity of gastric atrophy is supported by one study (Wright et al., 1966), but not another (Irvine et al., 1965). Explanations for the seronegative cases in pernicious anemia patients could include: 1) juvenile pernicious anemia prior to the development of autoantibodies, 2) an immunological reaction restricted to a cellular response rather than an antibody response, 3) exhaustion of the autoimmune response as the parietal cell autoantigens are depleted, 4) incorrect diagnosis or 5) unrecognized autoantibodies directed towards highly sensitive epitopes. Treatment with corticosteroid drugs, such as azathioprine, glucocorticoid and prednisolone result in regeneration of gastric parietal cells and/or improved gastric function (Baggett and Welsh, 1970; Jorge and Sanchez, 1973; Wall et al., 1968). Activities of parietal cell autoantibodies, however, showed no correlative change (Wall et al., 1968).

Sensitivity, Specificity, Predictive Value

Positive

and

Negative

By indirect immunofluorescence, parietal cell autoantibodies are detected in up to 90% of pernicious anemia patients and are also detected in 2--5% of the adult population (Whittingham and Mackay, 1985). The recently developed ELISA to detect parietal cell autoantibodies has a sensitivity of 82% and a specificity of 90% (Chuang et al., 1992). There is an agerelated increase in the presence of parietal cell auto-

antibodies in the adult population. In an Australian population the prevalence rose from 2.5% in the third decade to 9.6% in the eighth decade (Hawkins et al., 1979). A study of the relationship between parietal cell autoantibody and gastric' mucosal morphology indicates these parietal cell-positive individuals in a random population may indeed have early type A gastritis (Uibo et al., 1984). Higher prevalence rates (20--30%) of parietal cell autoantibodies have been noted in patients with autoimmune endocrine disorders such as thyrotoxicosis, Hashimoto's thyroiditis and insulin-dependent diabetes mellitus (Whittingham and Mackay, 1985). Histological examination of gastric biopsies reveals that in the majority of cases individuals positive for parietal cell autoantibodies also have a type A gastric lesion (Wangel et al., 1968; Varis et al., 1979).

REFERENCES Baggett RT, Welsh JD. Observations on the effects of glucocorticoid administration in pernicious anemia. Am J Dig Dis 1970;15:871--881. Barrett SP, Toh BH, Alderuccio F, van Driel IR, Gleeson PA. Organ-specific autoimmunity induced by adult thymectomy and cyclophosphamide-induced lymphopenia. Eur J Immunol 1995;25:238--244. Burman P, Mardh S, Norberg L, Karlsson FA. Parietal cell antibodies in pernicious anemia inhibit H+, K+-adenosine triphosphatase, the proton pump of the stomach. Gastroenterology 1989;96:1434-1438. Callaghan JM, Toh BH, Pettitt JM, Humphris DC, Gleeson PA. Poly-N-acetyllactosamine-specifictomato lectin interacts with gastric parietal cells. Identification of a tomato-lectin binding 60--90 x 10(3)Mr membrane glycoprotein of tubulovesicles. J Cell Sci 1990;95:563--576. Callaghan JM, Toh BH, Simpson RJ, Baldwin GS, Gleeson PA. Rapid purification of the gastric H+/(+)-ATPase complex by tomato-lectin affinity chromatography. Biochem J 1992;283: 63--68. Callaghan JM, Khan MA, Alderuccio F, van Driel IR, Gleeson PA, Toh BH. Alpha and beta subunits of the gastric H+/K(+)ATPase are concordantly targeted by parietal cell autoantibodies associated with autoimmune gastritis. Autoimmunity 1993;16:289-295. Callender ST, Denborough MA, Sneath J. Blood groups and other inherited characters in pernicious anaemia. Br J Haematol 1957;3:107-114. Carmel R, Johnson CS. Racial patterns in pernicious anemia. Early age at onset and increased frequency of intrinsic-factor antibody in Black women. N Engl J Med 1978;298:647-650. Carmel R. Reassessment of the relative prevalences of antibodies to gastric parietal cell and to intrinsic factor in patients with pernicious aneamia: influence of patient age and race. Clin Exp Immunol 1992;89:74-77.

CONCLUSION Parietal cell autoantibodies are associated with autoimmune gastritis and pernicious anemia. These autoantibodies recognize the ~- and 13-subunit of the gastric H/K ATPase, a highly specialized proton pump located in the unique intracellular membranes of gastric parietal cells. The identification of the target autoantigens provides an explanation for the cell specificity of these autoantibodies. It is unlikely that these autoantibodies are involved in the pathogenicity of disease but rather are produced as a secondary consequence of a cell-mediated autoimmune response to the gastric mucosa. Nonetheless, the presence of these autoantibodies provides a convenient diagnostic probe for type A chronic atrophic gastritis.

Chuang JS, Callaghan JM, Gleeson PA, Toh BH. Diagnostic ELISA for parietal cell autoantibody using tomato lectin purified gastric H+/K(+)-ATPase (proton pump). Autoimmunity 1992;12:1--7. Delva PL, Macdonald JE, Macintosh OC. Megaloblastic anemia occurring simultaneously in white female monozygotic twins. Can Med Assoc J 1965;92:1129--1131. Gleeson PA, Toh BH. Molecular targets in pernicious anaemia. Immunol Today 1991;12:233--238. Goldkorn I, Gleeson PA, Toh BH. Gastric parietal cell antigens of 60--90, 92, and 100--120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60--90-kDa component. J Biol Chem 1989;264:18768-18774. Goldkorn I. Characterization of gastric parietal cell autoantibodies in chronic atrophic gastritis [Thesis]. Australia: Monash University, 1991. Hawkins BR, McDonald BL, Dawkins RL. Characterisation of immunofluorescent heterophile antibodies which may be confused with autoantibodies. J Clin Pathol 1977;30:299-307. Hawkins BR, Houliston JB, Dawkins RL. Distribution of HLA A, B and C antigens in Australian population. Hum Genet 1979;52:193--201. Hoedemaeker PJ, Ito S. Ultrastructural localization of gastric parietal cell antigen with peroxidase-coupled antibody. Lab Invest 1970;22:184--188. Irvine WJ, Davies SH, Delamore IW, Williams AW. Immunological relationship between pernicious anemia and thyroid disease. Br Med J 1962;2:454--456. Irvine WJ, Davies SH, Teitelbaum S, Delamore IW, Williams AW. The clinical and pathological significance of gastric parietal cell antibody. Am NY Acad Sci 1965;124:657--691. Irvine WJ, Cullen DR, Mawhinney H. Natural history of autoimmune achlorhydric atrophic gastritis. A 1-15-year follow-up study. Lancet 1974;2:482-485. Jones CM, Callaghan JM, Gleeson PA, Mori Y, Masuda T, Toh 605

BH. The parietal cell autoantigens recognized in neonatal thymectomy-induced murine gastritis are the alpha and beta subunits of the gastric proton pump. Gastroenterology 1991 a; 102:287--294. Jones CM, Toh B H, Pettitt JM, Martinelli TM, Humphris DC, Callaghan JM, Goldkorn I, Mu FT, Gleeson PA. Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+-ATPase). An autoantigen targeted in pernicious anaemia. Eur J Biochem 1991b;197: 49-59. Jorge AD, Sanchez D. The effect of azathioprine on gastric mucosal histology and acid secretion in chronic gastritis. Gut 1973;14:104-106. Mardh S, Ma JY, Song YH, Aly A, Henriksson K. Occurance of autoantibodies against intrinsic factor, H,K-ATPase, and pepsinogen in atrophic gastritis and rheumatoid arthritis. Scand J Gastroenterol 1991;26:1089-1096. Mercier F, Reggio H, Devilliers G, Bataille D, Mangeat P. Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin upon stimulation of gastric acid secretion. J Cell Biol 1989;108:441--453. Pedersen PL, Carfoli E. Ion motive ATPases. I. Ubiquity, properties, and significance to cell function. Trends Biochem Sci 1987;12:146-149. Pettitt J, Humphris DC, Barrett SP, Toh BH, van Driel IR, Gleeson PA. Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubules: a new model for parietal cell transformation. J Cell Sci 1995;108:1127-1141. Rabon EC, Reuben MA. The mechanism and structure of the gastric H, K-ATPase. Annu Rev Physiol 1990;52:321--344. Sagaguchi S, Fukuma K, Kuribayashi K, Masuda T. Organspecific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease. J Exp Med 1985; 161: 72--87. Smith H, Lou Y-H, Lacy P, Tung KS. Tolerance mechanism in experimental ovarian and gastric autoimmune diseases. J Immunol 1992;149:2212--2218. Smolka A, Helander HF, Sachs G. Monoclonal antibodies

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against the gastric H++K+ ATPase. Am J Physiol 1983;245: G589--G596. Strickland RG, Hooper B. The parietal cell heteroantibody in human sera: prevalence in a normal population and relationship to parietal cell autoantibody. Pathology 1972;4:259--263. Strickland RG. Chronic gastritis and pernicious anemia. In: Targan SR, Shanahan F, eds. Immunology and Immunopathology of the Liver and Gastrointestinal Tract. Igaku-Shoin, 1990a;535--546. Strickland RG. Gastritis. Springer Semin Immunopathol 1990b; 12:203-217. Taylor KB, Roitt IM, Doniach D, Couchman KG, Shapland C. Autoimmune phenomena in pernicious anaemia: gastric antibodies. Br Med J 1962;2:1347--1352. Uibo R, Krohn K, Villako K, Tammur R, Tamm A. The relationship of parietal cell, gastrin cell, and thyroid autoantibodies to the state of the gastric mucosa in a population sample. Scand J Gastroenterol 1984; 19:1075-- 1080. van Driel IR, Callaghan JM. Proton and potassium transport by H/K ATPases. Clin Exp Pharmacol Physiol 1995;in press. Varis K, Ihamaki T, Harkonen M, Samloff IM, Siurala M. Gastric morphology, function, and immunology in firstdegree relatives of probands with pernicious anemia and controls. Scand J Gastroenterol 1979;14:129-139. Wall AJ, Whittingham S, Mackay IR, Ungar B. Prednisolone and gastric atrophy. Clin Exp Immunol 1968;3:359-366. Wangel AG, Callender ST, Spray GH, Wright R. A family study of pernicious anaemia. II. Intrinsic factor secretion, vitamin B12 absorption and genetic aspects of gastric autoimmunity. Br J Haematol 1968;14:183--204. Whittingham S, Mackay IR. Pernicious anemia and gastric atrophy. In: Rose NR, Mackay IR, eds. The Autoimmune Diseases. New York: Academic Press, 1985:243--266. Whittingham S, Mackay IR, Tait BD. The immunogenetics of pernicious anemia. In: Farid NR, ed. The Immunogenetics of Autoimmune Diseases, Boca Raton, Florida: CRC Press, 1991;2:215--227. Wright R, Whitehead R, Wangel AG, Salem SN, Schiller KF. Autoantibodies and microscopic appearance of gastric mucosa. Lancet 1966; 1:618--621.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PATHOGENIC MECHANISMS Ricard Cervera, M.D. a and Yehuda Shoenfeld, M.D. b

aUnitat de Malalties and Autoimmunes Sistbmatiques, Hospital Clinic I Provincial de Barcelona, 08036 Barcelona, Catalonia, Spain; and bDepartment of Medicine "B", Research Unit of Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel

INTRODUCTION Autoantibodies are immunoglobulins that bind to antigens originated in the same individual or species (autoantigen). Three aspects of the relationship between the autoantigen and the autoantibody are central clues in autoimmunity: i.

Although any self molecule that is bound to an antibody is an antigen, this does not necessarily imply that the antigen is the molecule that induced the production of the antibody. ii. The binding between an autoantigen and an autoantibody may or may not lead to an autoimmune disease. For instance, natural autoantibodies are immunoglobulins that occur in normal individuals and bind to a variety of self proteins producing a beneficial role in helping to clear self molecules from the circulation (Shoenfeld et al., 1992). iii. Autoantibodies can be produced in response to tissue breakdown induced by trauma or infection, such as the antibodies to cardiac myosin that appear after a wide variety of insults to the heart, but these antibodies are usually short lived and their role in the production of autoimmune disease is uncertain (De Scheerder et al., 1989). In light of these considerations, the pathogenic significance of an autoantibody should be evaluated with caution. Indeed, establishing a pathogenic role for autoantibodies requires that they meet rigorous criteria: (1) the autoantibody should be capable of causing the lesions attributed to it in experimental systems; (2) a suitable immunization that leads to the

production of similar autoantibodies should lead to a similar disease process; (3) the autoantibody should be found along with a plausible target antigen at the site of tissue damage; (4) autoantibody levels and disease activation should correlate; and (5) removal of the autoantibody should ameliorate the disease process (Naparstek et al., 1993).

PATHOGENIC MECHANISMS FOR AUTOANTIBODY-MEDIATED INJURY Certain autoantibodies probably have no pathogenic effects, e.g., natural autoantibodies (Guilbert et al, 1982; Shoenfeld et al., 1992) and those produced as a response to tissue injury (De Scheerder et al., 1989). Pathogenic mechanisms of other autoantibodies are unclear, e.g., antiphospholipid antibodies (Cervera et al., 1995; Harris, 1990; Khamashta, et al., 1989) and antibodies to endothelial cells (Cervera et al, 1994, Meroni et al, 1995). A direct role for autoantibodymediated injury is possible or likely for many other autoantibodies. The different established or postulated mechanisms of autoantibody-mediated tissue damage will be reviewed.

Cell Surface Binding and Lysis (Cytotoxicity) Binding to surface membranes and subsequent destruction of the cell is a well established pathogenic mechanism of autoantibodies. This lysis may be mediated by complement (complement-mediated cytotoxicity), by K cells (antibody-dependent cellmediated cytotoxicity) or by enhancing phagocytosis by the mononuclear phagocyte system.

607

Complement-Mediated Cytotoxicity. Complement is the name given to a complex series of some 20 proteins which forms one of the triggered enzyme systems present in plasma. One of the most remarkable functions of the complement is to produce membrane lesions leading to cell lysis. Activation of the complement system leads to the sequential attachment of several compounds (C3b, C5b, C6, C7, C8 and C9) that are capable of insertion into the lipid bilayer of a cell membrane and form an annular structure termed "membrane attack complex." This forms a transmembrane channel fully permeable to electrolytes and water, and due to high internal colloid osmotic pressure of cells, there is a net influx of sodium and water leading to cell lysis (Arnett et al., 1991; Asghar, 1995; Erdei et al., 1991; Fearon et al., 1983; Frank, 1987; Frank et al, 1991; Ochs, 1986; Ohishi et al., 1995; Ruddy, 1985). Autoantibodies bound to cell membrane antigens can activate the complement cascade thus producing cell lysis (complement-mediated cytotoxicity) (Figure 1). Typical examples of these autoantibodies are some antithyroid antibodies of Hashimoto' s disease (Kohno et al., 1993; Bermann et al., 1993; Rose et al., 1981) and antierythrocyte antibodies of autoimmune hemolytic anemia. In the later case, erythrocytes coated with autoantibodies produce complement activation resulting in extravascular hemolysis. Subsequent clearance of destroyed red blood cells from the circulation is produced through phagocytosis by macrophages (Mollnes et al., 1995; Quismorio Jr., 1993; Scott et al., 1994; Terness et al., 1993).

cell attacks the Fc receptor of the K cell, this cell releases hydrogen peroxide and hydroxyl radicals which are powerful cytotoxic agents. The specificity of the killing is determined by the specificity of the involved IgG antibody and not by the mononuclear cell (Garner et al., 1994; Greenberg, 1987; Hellstrand et al., 1994; Lanier et al., 1985; Thiele et al., 1989) (Figure 2). Although antibody-dependent cell-mediated cytotoxicity (ADCC) against a variety of target cells is decreased in vitro in some autoimmune diseases such as systemic lupus erythematosus (SLE) (Schneider et al., 1975, Wright et al., 1981), ADCC can be enhanced in vivo. For instance, antilymphocyte antibodies may bind to lymphocytes through the F(ab')2 fragment and to K cells through their Fc portion, thus producing lymphopenia. In this way, it is possible to explain the detection of antilymphocyte antibodies, lymphopenia and low in vitro ADCC that is common in SLE (Kumagai et al, 1981).

Phagocytosis by the Mononuclear Phagocyte System. The mononuclear phagocyte system (previously included with endothelial cells and phagocytic cells under the term "reticuloendothelial system") is con-

Antibody-Dependent Cell-Mediated Cytotoxicity. This is a lytic mechanism mediated by a population of natural killer (NK) cells that carry receptors for the Fc portion of the IgG, also known as killer (K) cells. When the autoantibody which is bound to the target

Figure 1. Schematic diagram of complement-mediatedcytotoxicity. 608

Figure 2. Schematic diagram of antibody-dependent cellmediated cytotoxicity.

stituted by macrophage cells spread throughout the human body. These cells are derived from bone marrow promonocytes which, after differentiation to blood monocytes, finally settle in the tissue as mature macrophages. The macrophages are long-lived cells that provide a major defense system against hostile elements through phagocytosis and subsequent intracellular destruction of the particle or cell. Before phagocytosis can occur, the hostile element must first adhere to the surface of the macrophage, an event mediated by some rather primitive recognition mechanism likely to involve carbohydrate elements. Once the element is attached to the surface membrane, an actin-myosin contractile system extends pseudopods around it until it is completely enclosed in a vacuole called "phagosome." Then, the macrophage cytoplasmic granules fuse with the phagosome and discharge their contents around the imprisoned element which is killed or destroyed (Athanasou, 1995; Werb, 1987) (Figure 3). In certain autoimmune conditions, activated macrophages carrying Fc receptors in their surface membrane may attach autoantibodies bound to target cells, such as erythrocytes, thus leading to the phagocytosis and subsequent destruction of these cells. For instance, the pathogenesis of red blood cell damage by antierythrocyte antibodies in SLE and autoimmune hemolytic anemia has been extensively investigated

(Matsumoto et al., 1978; Hillyer et al., 1990; Quismorio Jr., 1993). Erythrocytes sensitized with warmreactive IgG antibodies are cleared from the circulation by macrophages in the splenic sinusoids. The macrophage Fc receptors bind erythrocytes with bound IgG antierythrocyte antibodies, causing membrane damage, spherocytosis, and phagocytosis of some red blood cells. Microspherocytes have a shortened life span because of their increased rigidity and increased osmotic fragility. As the amount of surface-bound antibody increases, splenic trapping becomes more efficient, and erythrocyte survival shortens significantly. When the density of bound IgG autoantibody is substantial, complement activation also occurs resulting in extravascular hemolysis. Erythrocytes coated with autoantibodies and complement are cleared by two distinct macrophage receptors, namely C3b and Fc receptors. The IgG subclass of the antierythrocyte antibody is an important determinant in the clearance because splenic macrophages have IgG Fc receptors for IgG1 and IgG3 subclasses. Therefore, erythrocytes with critical quantities of IgG1 and IgG3 antibodies on their surface are destroyed. It has been calculated that erythrocytes coated with IgG1 antibody alone or with additional IgG2 and IgG4 antibodies require at least 2,000 molecules per red blood cell to initiate phagocytosis, while as few as 230 molecules of IgG3 anti-

Figure 3. Schematic diagram of phagocytosis by the mononuclear phagocyte system (from Roitt, 1991, with permission). 609

o

Figure 4. Schematic diagram of modulation of cell surface receptors by autoantibodies.

erythrocyte antibodies per cell are required for binding to macrophages (Zupanski et al., 1986).

Binding to Cell Surface Receptors without Cytolysis Binding to cell surface receptors and subsequent modification (inhibition or stimulation) of cell biological activity, without cytolysis, is another well-established pathogenic mechanism of some autoantibodies (Dawkins et al, 1987, Kirkness et al, 1989; Kuks et al., 1991; Naparstek et al., 1993; Salvi et al., 1988; Tzartos et al., 1991). This cell activity modification may be produced by modulation, blockage or stimulation of cell surface receptors.

Modulation of Cell Surface Receptors. Binding of antibodies to cell surface receptors may produce a reduction in the expression of these receptors. This is due to the aggregation and redistribution of the receptors in the membrane with subsequent disappearance from the outer side of cell surface (Figure 4). This is the mechanism of action by which antiacetylcholine receptor antibodies impair neuromuscular function in myasthenia gravis. Acetylcholine receptors are located at the tips of folds in the postsynaptic membranes of skeletal muscle fibers. They bind acetylcholine released from the nerve ending and, in response, open a cation-specific channel, resulting in local depolarization of the postsynaptic membrane and the triggering of a muscle action potential. Binding of antibodies to the receptor produce an increased receptor degradation with subsequent disturbance of the proper function of the ion channels. Additionally, the binding of complement to these antibodies may result in the lysis of the membrane (Eymard et al., 1991; Hara et al., 1993; Kuks et al., 1991; Liblau et al., 1991; Lindstrom, 1979; Pachner, 1989; Shonbeck et al., 1990; Tzartos et al., 1991; Vincent, 1980). 610

Blockage of Cell Surface Receptor. Binding of antibodies to the receptor may block the binding of the physiological ligand thus leading to inhibition of cell activity (Figure 5). This is the case of type I antiintrinsic factor antibodies. Intrinsic factor is a glycoprotein expressed on the gastric parietal cell that binds to vitamin B12. The presence of these antibodies blocks the attachment of vitamin B12 to the intrinsic factor molecule, thus producing pernicious anemia (Pruthi et al., 1994). Occasionally, antibodies to the thyroid stimulating hormone (TSH) receptor may cause hypothyroidism by blocking the action of TSH on the gland (thyroid blocking antibodies (Michelangeli et al., 1995; Salvi et al., 1988). Stimulation of Cell Surface Receptors. Some autoantibodies may bind to cell surface receptors and activate these through the adenyl cyclase system, thus resulting in stimulation of cell activity (Figure 6). This is the main mechanism of action of the antibodies to TSH receptors. These antibodies mainly appear in Graves' disease and were recognized in studies that attempted to identify the so-called long-acting thyroid stimulating (LATS) factor in the serum of patients with this condition. These autoantibodies are directed against the TSH receptor and mimic the action of the pituitary hormone. Since their production is not subject to the feedback control of the thyroid hormone O

Figure 5. Schematic diagram of blockage of a cell surface receptor by autoantibodies.

Figure 6. Schematic diagram of stimulation of a cell surface receptor by autoantibodies. whose synthesis they stimulate, the gland overproduces the hormone and enlarges under the trophic stimulus delivered through the receptor. These autoantibodies associate strongly with the disease, but they are found in many cases of the closely related Hashimoto's thyroiditis, in which the patient may have normal thyroid function at the time the disease is detected, but may often become hypothyroid. The precise role of the autoantibodies in these cases is not clear, and the disease is thought to result primarily from the action of the T lymphocytes infiltrating the gland (Burman et al., 1985; Fan et al., 1994; FeldtRasmussen et al., 1994; Naparstek et al., 1993). As previously mentioned, antibodies to the TSH receptor can much less commonly cause hypothyroidism by blocking the action of TSH on the gland (thyroid blocking antibodies) (Michelangeli et al., 1995; Salvi et al., 1988).

Immune Complex-Mediated Damage The formation and subsequent removal of immune complexes is a fundamental physiological event primarily concerned with the defense of the host against exogenous pathogens. Immune complexes are rapidly cleared from the circulation by the mononuclear phagocyte system and their formation is thus a normal and usually beneficial expression of the immune response. However, in certain autoimmune conditions, this normally protective mechanism can act inappropriately causing tissue damage (Figure 7). A number of factors regulate the physical characteristics of immune complexes and, therefore, their biologic properties (the ability to fix complement, the efficiency with which they are cleared by the mononuclear phagocyte system and the propensity to deposit in tissues other than the mononuclear phagocyte system, among others). These factors include the nature of the antibody in the complex (e.g., class,

Figure 7. Schematic representation of immune complexmediated damage.

subclass, quantity, avidity, charge), the nature of the antigen (e.g., valence, size, charge, tissue trophism) and the nature of the antigen-antibody interaction (e.g., molar ratio). The characteristics of the antibody are essential factors influencing the properties of the immune complexes. Those formed with IgG or IgM antibody can activate complement by the classical pathway, IgA-containing complexes can activate complement by the alternative pathway, but IgD and IgE cannot activate complement. Furthermore, the IgG1, IgG2 and IgG3 subclasses of IgG fix complement better than IgG4. The quantity of antibody produced to a given antigen will control the amount of immune complexes produced, as well as affect the molar antigen to antibody ratio. The strength with which an antibody binds to antigen (avidity) can affect the nature of the immune complex formed and, in general, low-avidity antibodies are more likely to form small immune complexes. Finally, the net charge of the antibody or the antigen in an immune complex influences binding of the immune complex to specific tissues. For instance, immune complexes containing positively charged antibody or antigen bind to the renal glomerulus to a much greater degree than immune complexes containing neutral antibodies (Emlen, 1993; Gautier et al., 1990). The antigen contained within an immune complex can also markedly affect the properties of the immune complexes. The valence of the antigen is defined as the number of antibody binding sites per molecule. 611

Small antigens with low valence form small immune complexes, while large antigens can bind multiple antibodies, resulting in the formation of large immune complexes. The. lattice of an immune complex is defined as the number of antigen and antibody molecules in a given immune complex. Those with a high degree of lattice are more efficient at fixing complement, are cleared from the circulation rapidly via binding to cellular receptors, and are potent initiators of inflammation. Low-lattice immune complexes may persist in the circulation, but are relatively poor inducers of inflammation (Mannik, 1987). Antigens may also alter the properties of immune complexes independent of their combination with antibody. For instance, certain antigens such as DNA may be specific ligands for their own receptors, and clearance of immune complexes containing these antigens may be mediated not only by Fc receptors, but also by antigen receptors (Emlen, 1988). Antigen may also bind to a specific tissue either on the basis of charge or direct tissue trophism (Emlen, 1993). Finally, the molar ratio of antigen to antibody in an immune complex plays a major role in determining the properties of immune complexes. At molar equivalence, the chances for cross-linking antigen and antibody are maximized, resulting in the formation of large-latticed precipitates. At moderate degrees of antigen excess, soluble immune complexes are formed that are still relatively large latticed, and are therefore active in complement activation and binding to cellular receptors. At extreme antigen or antibody

excess, small immune complexes with low inflammatory potential are formed. The biological effects of immune complexes depend on their ability to interact with the complement via the classical and alternative pathways and trigger cells via Fc and C3b receptors. The major pathological effects of immune complexes are due to inflammatory mediators generated during these processes but, in addition, immune complexes may exert some of their most important effects in autoimmune diseases through their ability to modulate immune responses at both the inductive and effector levels (Lachmann et al., 1984; Theofilipoulos, 1980). The biological activities of immune complexes are summarized in Table 1. Immune complex formation and tissue localization may occur in autoimmune conditions by three distinct mechanisms: (1) antibody reacting with structural antigens in specific tissues, such as in Goodpasture's disease; (2) local formation of immune complexes, as it happens in the farmer's lung; and (3) deposition of circulating immune complexes, as in certain glomerulonephritis cases (Shoenfeld, 1989). Translocation of Intracellular Antigens to Cell Membrane

Several studies have postulated that autoantibodies to intracellular proteins bind to cell surface membranes. The two mechanisms that have been proposed are cross-reaction between the intracellular and the

Table 1. Main Biological Functions of Immune Complexes

Activity

Effect

Complement activation

Immune adherence (C3b-C4b receptors) Chemotaxis (C5a) Anaphylaxis (C3a-C5a) Lysis (C56789) Macrophage activation (Bb) Immune complex solubilization (alternative pathway) Inhibition of precipitation (classical pathway)

Interaction with cells (Fc and/or C3 receptors)

Phagocytosis and lysosomal enzyme release Aggregation and vasoactive amine release Phagocytosis and cytotoxicity Formation and release of type I mediators

Modulation of immune responses

Inhibition Activation Induction of anti-idiotype Blockade of effector functions

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membrane antigens and translocation of the intracellular antigen following injury or activation of the cell.

Cross-Reactions Between the Intracellular and the Membrane Antigens. Observations on the binding of some autoantibodies directed against intracellular antigens with cell membranes have opened up the possibility that these antibodies exert a pathogenic effect by cross-reacting with a protein on the external membrane of cells. This is the case, for instance, of antiribosomal P protein antibodies which are present mainly or exclusively in SLE patients and can be associated with depression and psychosis. Fluorescent and electron microscopic studies show that affinitypurified antiribosomal P protein antibodies bind to the surface of intact cells. Additionally, these antibodies also bind specifically to a protein of the same size as the previously identified target from ribosomes when tested by immunoblotting with a plasma membrane preparation. Therefore, although much prior evidence suggests that these autoantibodies have had their production driven by ribosomes, they may become pathogenically significant only by a cross-reaction with a protein on cell surfaces (Koren et al., 1992). Other cross-reactions between membrane antigens and intracellular compounds are thought to happen in the cases of the lupus-associated membrane protein (LAMP) antigen (Jacob et al., 1987) and sulfated glycolipid or glycosaminoglycan components of the glomerular basement membrane which cross-react with anti-DNA antibodies (Murakami et al., 1991; Termaat et al., 1990). Another example of a similar phenomenon may be the autoantibodies against the intracellular small ribonucleoprotein particles Ro (SS-A) and La (SS-B). These appear mainly in SLE, Sj6gren's syndrome and the apparently transplacentally mediated syndromes, neonatal lupus and congenital heart block, that occur in a small proportion of the infants born to women with these antibodies. Recent studies showed that immunoglobulins containing anti-Ro (SS-A) antibodies from the mothers of children without heart block bound fetal but not adult cardiac tissue and altered transmembrane action potentials (Alexander et al., 1992). Translocation of the Intracellular Antigen Following Injury to the Cell. According to this hypothesis, an autoantibody to an intracellular component exerts a pathogenic role because the component is released, following injury or activation of the cell, into

the extracellular space in a location where the consequences of the local formation of immune complexes are likely to be severe. Although fundamentally an attractive hypothesis, this mechanism was initially criticized because intracellular particles might be released from dead cells and adhere to the surfaces of live cells in the experiments performed in vitro and also because most studies were performed with polyclonal patient sera allowing for the possibility that binding to cell surface membranes can be due to antibodies primarily directed against uncharacterized membrane antigens (Lefeber et al, 1984). However, some recent studies suggest that injury or activation of the cell might certainly translocate a normally intracellular antigen to a site where circulating antibodies could bind to it. This can be the case of the antineutrophilic cytoplasmic antibodies (ANCA) that appear in some primary systemic vasculitis, especially in Wegener's granulomatosis. The most specific of these antibodies, the cytoplasmic ANCA (C-ANCA), is directed against proteinase 3 which is a component of the primary lysosomes of neutrophils and monocytes. In neutrophils, the enzyme is found in the azurophilic granules released with activation of the cell (Goldschmeding et al., 1991; Jennette et al., 1992). In vitro experiments evidenced that if IgG C-ANCA is added to neutrophils "primed" by various cytokines, especially tumor necrosis factor, the neutrophils are "activated" as shown by a rise in superoxide radical formation, by changes in protein kinase C and other second messenger pathway components and by granular release. Apparently, the reaction called "priming" brings the components of the granules to the cell surface. It has been proposed that the exposed antigen is the primary target, and that the encounter with the antibody brings the cells from the primed state to the fully activated state, able to release much more granular material into the medium. This could lead to the local formation of immune complexes, complement fixation and a widening inflammatory cascade (Gross, 1992).

Penetration into Living Cells The evidence for antibody penetration into living cells mainly comes from the classical detection of IgG within epidermal cells on skin biopsies of some patients with SLE (Tan et al, 1966) and within a subpopulation of T lymphocytes in patients with high titer of anti-RNP antibodies (Alarc6n-Segovia et al, 1978). However, the functional effect of autoanti-

613

Figure 8. Schematic representation of penetration of autoantibodies into living cells.

bodies after intracellular uptake is controversial (Figure 8). Binding to Extracellular Molecules Binding to extracellular molecules, especially complex extracellular cascades, is a possible but not confirmed mechanism of action of some autoantibodies. In this case, the autoantibodies do not bind to any cell a n t i g e n - neither surface receptors nor intracellular molecules. This is a postulated pathogenic mechanism for antiphospholipid antibodies, which are related to the development of arterial and venous thrombosis and recurrent fetal losses in patients with SLE and other autoimmune conditions. It is clear from in vitro studies with monoclonal antibodies and from the association with prolonged clotting times that antiphospholipid antibodies can interfere with the intravascular coagulation cascade. However, hemorrhages are uncommon in patients with these antibodies while thromboses are their main clinical complications. Therefore, antiphospholipid antibodies might produce their pathogenic effects through other mechanisms. The identification of beta2-glycoprotein I as the target antigen for the anticardiolipin antibodies, the most representative of the antiphospholipid antibodies, may help in clarifying the mechanism by which they can lead to increased coagulopathy. Beta-2-glycoprotein I interacts with

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various steps of the coagulation pathways, it binds to platelets, inhibits platelet aggregation and inhibits the intrinsic coagulation pathway. Interference with these activities might therefore lead to hypercoagulation (Asherson et al., 1993; Cervera et al., 1995; Harris, 1990; Khamashta et al., 1989). Additionally, antibodies to complement components, especially to C1 and C lq, have been detected. However, their pathogenic effects interfering with the complement cascade are uncertain. Finally, autoantibodies to a circulating hormone, insulin, and to an extracellular protein, type II collagen, have also been described. Antibodies to insulin are most often found in diabetic patients repeatedly injected with insulin, but sometimes these occur spontaneously and may present as hypoglycemia. The mechanism of this paradoxical phenomenon is thought to be either antibody-induced potentiation of insulin's action or a complex interplay of circulating free and antibody-bound hormone (Rodriguez et al., 1992). On the other hand, antibodies to type II collagen appear in several joint diseases. However, such antibodies are not disease-specific and seem to arise nonspecifically in response to joint damage (Stuart et al., 1984; Bari et al., 1989).

CONCLUSION For many autoantibodies, a direct pathogenic effect and the mechanism whereby they cause damage remain to be proven. The most clear-cut way to establish a cause-effect relationship is to passively administer the antibody in question to an experimental animal model and test for the effect. Such an approach is relatively easy in organ-specific autoimmune diseases, but is more difficult in systemic autoimmune conditions since each patient has a variety of autoantibodies. Additionally, some autoantibodies may produce their effects through a diversity of mechanisms (complement-mediated cytotoxicity, ADCC, formation of immune complexes, etc.). There is still a lot to be clarified to ,explain the mechanisms of action of so many autoantibodies. However, important revelations are expected in the next few years from the experimental animal models. See also AUTOANTIBODIES THAT PENETRATE INTO LIVING CELLS, C IQ AUTOANTIBODIES, COLLAGENAUTOANTIBODIES,NATURAL AUTOANTIBODIES and XENOREACTIVEHUMAN NATURAL ANTIBODIES.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PERINUCLEAR FACTOR (PROFILAGGRIN) AUTOANTIBODIES Pierre Youinou, M.D., Ph.D. a, Paul Le Goff, M.D. b and Raya Maran, M.D. c

aLaboratoire d'Immunologie, bDepartment of Rheumatology, Centre Hospitalier Rdgional et Universitaire, Brest, Cedex, France; and CDepartment of Medicine "B", Research Unit of Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel

HISTORICAL NOTES The antiperinuclear factor (APF) was first detected as a previously unknown immunofluorescence pattern denoted by the presence of nucleus-surrounding dots during the search for a convenient substrate for the antinuclear antibody test using human buccal mucosa epithelial cells (Nienhuis and Mandema, 1964). Unexpectedly, most of the sera containing this new factor derived from patients with rheumatoid arthritis (RA). Owing to technical improvements (SondagTschroots et al., 1979), the test has achieved reasonable sensitivity and specificity for RA (Hoet and van Venrooij, 1992; Berthelot et al., 1994a; Youinou and Le Goff, 1994; Youinou and Serre, 1995). Yet it has gradually fallen into disuse. The main reason may be the difficulty in obtaining appropriate substrate material. In an attempt for standardization, five European groups set up a consensus study on the interlaboratory variability of the test and, despite the use of different cells, conjugates and criteria for positivity, obtained comparable results (Feltkamp et al., 1993). The target antigen was recently identified (Sebbag et al., 1995).

THE AUTOANTIGENS

Definition APF binds to cytoplasmic aggregates encircling the nucleus of buccal epithelial cells and presumptively termed keratohyalin granules, on the basis of their rough resemblance to the keratohyalin bodies in the stratum granulosum of human epidermis (Smit et al.,

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1980). The location of the antigens is confirmed by electron microscopy (Vivino and Maul, 1990), and their precise nature was recently established (Sebbag et al., 1995).

Characteristics On immunoblots, APF-positive sera recognize a diffuse protein band with an apparent molecular weight (MW) of 200--400 kd. This has been demonstrated to be profilaggrin (Sebbag et al., 1995), which is the precursor of filaggrins, but recombinant forms of the molecule are not currently available.

Origin/Sources Human buccal mucosa epithelial cells are used as substrate. They can be obtained from a healthy donor, by scraping the inside of the cheek with a wooden tongue depressor. The problem is that the reliability of particular cells is unpredictable (Youinou et al., 1990a), inasmuch as variations are noticed from one donor to another (same serum), one day to another (same donor), one cell to another (samesample) and one site to another (same cell). The proportion of donors giving a good yield of antigenic substrate range from 11% (Johnson et al., 1981) to 69% (Youinou et al., 1990a). Unfortunately, the target antigen for APF is not accessible in cultured cells of the buccal mucosa of positive donors (Hoet et al., 1991 a). In an attempt to define the characteristics of positive and negative donors, the distinguishing features of a large group of volunteers were divided into two subgroups according to the presentation of their buccal cells in the APF test. Though not statistically sig-

give an extract enriched in the neutral/acidic isoform of filaggrin. The extract can be further purified by passing the extract over an anti(pro)filaggrin monoclonal antibody column. Bound filaggrin is eluted with 0.2 M glycine-HC1. The molecule is not Commercially available.

nificant, there was a trend for more of the young donors than of the elderly to be positive. APF titrated in the serum of 10 patients with RA on buccal cell smears from five individuals. Their titers fluctuated from one donor to another. This intriguing observation was substantiated by a systematic analysis of six sera on a panel of 16 randomly selected buccal cell donors. Cells from some donors were recognized by all the sera, cells from other donors were recognized by none of the sera, while the remaining donors gave positive staining with some sera and negative with other sera (Veys et al., personal communication; Hoet et al., 1991b). The possibility of qualitative differences between donors cannot be excluded. Alternate sources, such as human vaginal epithelial cells and cryostat sections of human and rabbit buccal and esophageal mucosa (Smit et al., 1980) are less appropriate than the buccal cells for the assay.

Sequence Information Profilaggrin, the insoluble precursor of filaggrin and the major APF antigen (Dale et al., 1990), is a histidine-rich insoluble protein consisting of 10 to 12 repeats of filaggrin arranged in tandem and separated by a short heptapeptide linker sequence (Figure 1). This accumulates in a nonfunctional and heavily phosphorylated form within the granular layer of keratinizing epithelia, before being dephosphorylated and cleaved by excision of the linker sequence to release the functional and highly basic polypeptide filaggrin. Dephosphorylation, probably the key event in the processing of profilaggrin, is rapidly achieved in vitro and resolves profilaggrin into peptides of lower MW. Profilaggrin aggregation and subsequent processing are likely to depend on the calcium concentration, given that its amino-terminus shows great

Methods of Purification Some of the target antigens can be purified (Simon et al., 1993) by lysis of human epidermis in buffer containing 0.5% Nonidet P-40, precipitation of the proteins in ethanol and suspension in distilled water to

Truncatedfilaggrin

Truncated filaggrin

Linker peptide(7aa)

NH2 I

,i

1 i

--

--

--

i

....

I

I

Illll

10 to 12 repeatsof.completefilaggrin(324aaeach) ~

.....

.~ COOH

processing

NH2

Figure 1. Relationships between profilaggrin and filaggrin. Profilaggrin is the precursor of filaggrin which consists of 10 to 12 repeats arranged in tandem and separated by a short heptapeptide linker sequence.

619

homology with the S-100 family of calcium-binding proteins. Although a complementary DNA clone encoding human filaggrin has been characterized and the gene localized to chromosome region l q21 (McKinley-Grant et al., 1989), the accurate sequence of linear and/or conformational epitopes has yet to be determined.

There is no widely accepted synonym for APF, but, following the identification of one of the target antigens, the term antifilaggrin antibody was coined (Sebbag et al., 1995). Neither is there any current evidence for a pathogenetic role for this autoantibody in RA, given that the molecule is not considered to be expressed by synoviocytes or chondrocytes.

the lymphotropic EBV also binds to oropharynx epithelial cells supports this view. The question, therefore, arises as to whether EBV infection is involved in the APF/PNA system, either through molecular mimicry or by enhancing the immunogenicity of the PNA within the keratohyalin granules. There is modest but significant APF production in patients with acute infectious mononucleosis (Buisson et al., 1994). EBV could also drive significant APF production by induction or enhancement of autoantigens, because some viruses need the cytoskeletal framework for their intracellular replication. Virus material was not found inside the granules (Buisson et al., 1994); nor was the EBV genome found in buccal cells of PNA-expressing or non-PNA-expressing donors. Antibodies to EBV viral capsid antigen and early antigen do not recognize the keratohyalin granules (Hoet et al., 199 lb), suggesting that the PNA reactivity cannot be explained by association with EBV-encoded proteins.

Pathogenetic Factors

Methods of Detection

An increased prevalence of HLA DR4 is reported in RA patients positive for rheumatoid factor (RF) with or without APF and those negative for RF but positive for APF, compared with patients negative for both antibodies (Boerbooms et al., 1990). This finding was not confirmed in HLA-DR4-positive and/or HLADRl-positive RA patients from Israel (Maran et al., submitted for publication). APF is mostly of the IgG isotype (Kataaha et al., 1985); IgG-APF was found in all 16 sera tested, although in four of them there was additional IgM and in three additional IgA activity in another study. IgG antibody was present in all of the four RA sera tested with class-specific conjugates; whereas, weaker staining for IgM was obtained with three sera, and for IgA with one of them (Youinou et al., 1990b). Finally, APF of the IgA isotype has been investigated in 80 sera from patients with active RA and found to be present in 31 sera (Berthelot et al., 1994b).

Human buccal mucosa epithelial cells are still used as the substrate to detect APF in an indirect immunofluorescence (IIF) test. After three washes in phosphate buffered saline (PBS), pH 7.4, these cells are resuspended in PBS containing colimycin and sodium azide and transferred dropwise to multispot slides (roughly 5,000 cells per well). After drying under a fan, the slides are ready to use. Sera diluted 1/80 are applied to the cell smear for 90 min in a moisture chamber. After three washes with PBS and incubation for 30 min with fluorescein-labeled F(ab')2 anti-IgG, a recognizable pattern is produced as denoted by the presence of several brightly fluorescent, bean-shaped, homogeneous 0.5--4.0 ~m diameter, sharply demarcated granules located in the cytoplasm surrounding the nucleus (Figure 2). The serum dilution is critical in the assay, due to a striking prozone phenomenon; with buccal cells as the substrate, a serum dilution of 1/80 used for detecting APF on a routine basis has proved reliable (Youinou et al., 1990a). If positive at this dilution, sera are further diluted to determine the end-point titer. Approximately 200 cells are examined and a serum scored positive when at least 10% of the cells are stained. Sera are recorded as positive by other groups when at least one buccal cell is found to elicit a conspicuous perinuclear fluorescence, but this procedure is associated with the risk that an artifact may be

THE AUTOANTIBODIES Synonyms/Terminology

Pathogenetic Role Epstein-Barr virus (EBV) infection on the one hand, and APF production by the patients and/or perinuclear antigen (PNA) expression by the cell donors on the other, are related by serum APF in over half of the patients with infectious mononucleosis (Kataaha et al., 1985; Westgeest et al., 1989). The demonstration that

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Figure 2. Immunofluorescence pattern produced by the binding of the antiperinuclear factor to the keratohyalin granules surrounding the nucleus of human buccal mucosa epithelial cells.

mistaken for a stained granule. Most authors do not titrate APF, so their interpretation is an "all or nothing" phenomenon.

CLINICAL UTILITY Disease Association

The diagnostic potential of the APF test, i.e., the compromise between sensitivity (49 to 87%) and specificity (73 to 90%) proved to be beneficial in RA (Hoet and van Venrooij, 1992; Youin0u and Le Goff, 1994). The differences between the initial and the subsequent sensitivities of the assay are probably the result of refinements of the IIF technique. The serum dilution of 1/80 used in the assay appears to be a critical parameter. It is suggested that 10% of the cells must be identified by the test serum to be recorded positive (Youinou et al., 1990b). Although the frequency of this autoantibody is higher than that of RF in patients with RA (Boerbooms et al., 1990; Youinou et al., 1990b), there is a rather good correlation between these two markers. The highest frequency of APF-positive sera is found in the same patient groups as the highest frequency of RF, i.e., those with a

variety of related autoantibodies, such as RF, antinuclear antikeratin and antivimentin antibodies (Youinou et al., 1990b). Furthermore, the APF titer is significantly higher in RA patients when RF is present than when not (Youinou et al., 1990b). Technical improvements (including addition of 0.5% Triton-X100 to the washing buffer) (Feltkamp et al., 1993) allow a gain in specificity, without loss of sensitivity (Youinou et al., 1990a; Manera et al., 1994) and indeed APF is uncommon in connective tissue diseases other than RA. In systemic lupus erythematosus (SLE), 23 of 50 sera were reported positive for APF (Vivino and Maul, 1990) but for unknown reasons, this is grossly discrepant with previous studies (Hoet and van Venrooij, 1992). APF is also reported in sera from patients with juvenile RA with an overall diagnostic sensitivity and specificity of 34 and 90%, respectively (Nesher et al., 1992). The test was more frequently positive in children with pauciarticular-onset juvenile RA than in other onset types. APF is occasionally described in primary myxedema (Scherbaum et al., 1984) and primary Sj6gren's syndrome (Youinou et al., 1984a). Nevertheless, the titer of APF is much higher in patients with RA than in those with other connective tissue diseases. APF are also reported, albeit at low titers in

621

40 of 79 patients with primary and 21 of 36 metastatic lung cancer, compared with 12 of 95 sex- and age-matched normal controls (Youinou et al., 1984b). APF frequency and titers correlated well with tumor dissemination, though no relationship could be established with histopathological type. The predictive value of APF in RA is still a matter of debate. APF is thought to separate two subpopulations in a group of RF seronegative patients. The APF-positive subpopulation has a more severe form of the disease, i.e., higher functional class, more extraarticular features (such as rheumatoid nodules, secondary Sj6gren's syndrome and Raynaud's phenomenon and a faster radiological progression than the APFnegative subpopulation (Westgeest et al., 1987). An increased prevalence of extra-articular complications in APF-positive/RF-negative patients was also reported. Another investigation did not confirm both a different functional class and a worse radiological progression in these patients (Manera et al., 1994). In contrast, the relationship between titers of APF and fluctuations in disease activity is accepted unanimously (Manera et al., 1994). The mean APF titer is significantly higher in early than in long-standing RA. Twenty-nine RA patients were examined over a few

months' time: 24 remained positive and one negative throughout the survey; three sera were negative on initial study and later were positive (Manera et al., 1994). Conversely, others could not find any correlation between APF titer and disease activity in RA patients treated with methotrexate or azathioprine and concluded that serial measurements of the APF in the monitoring of such patients do not provide additional information (Kerstens et al., 1994).

REFERENCES

clear factor (APF) test for rheumatoid factor. Clin Exp Rheumatol 1993,11:57--59. Hoet RM, Voorsmit RA, van Venrooij WJ. The perinuclear factor, a rheumatoid arthritis-specific autoantigen, is not present in keratohyalin granules of cultured buccal mucosa cells. Clin Exp Immunol 1991a;84:59--65. Hoet RM, Boerbooms AM, Arends M, Ruiter DJ, van Venrooij WJ. Antiperinuclear factor, a marker autoantibody for rheumatoid arthritis: colocalisation of the perinuclear factor and profilaggrin. Ann Rheum Dis 1991b;50:611--618. Hoet RM, van Venrooij WJ. The antiperinuclear factor and antikeratin antibodies in rheumatoid arthritis. In: Sm01en J, Kalden J, Maini RN, eds. Rheumatoid Arthritis. Berlin: Springer-Verlag, 1992:299--318. Johnson GD. Caravalho A, Holborow EJ, Goddard DH, Russel G. Antiperinuclear and keratin antibodies in rheumatoid arthritis. Ann Rheum Dis 1981;i~0:263--266. Kataaha PK, Mortazavi-Milani SM, Russel G. Holborow EJ. Anti-intermediate filament antibodies, antikeratin antibody, and antiperinuclear factor in rheumatoid arthritis and infectious mononucleosis. Ann Rheum Dis 1985;44:446-449. Kerstens PJ, Boerbooms AM, Jeurissen ME, Westgeest TA, van Erp A, Mulder J, van de putte LB. Antiperinuclear factor and disease activity in rheumatoid arthritis. Longitudinal evaluation during methotrexate and azathioprine therapy. J Rheumatol 1994;21:2190-2194.

Berthelot JM, Vincent C, Serre G, Youinou P. The antiperinuclear factor. In: van Venrooij WJ, Maini RN, eds. Manual of Biological Markers B12. Amsterdam: Kluwer Academic Publishers, 1994a: 1--9. Berthelot JM, Bendaoud B, Maugars Y, Audrain M, Prost A, Youinou P. Antiperinuclear factor of the lgA isotype in active rheumatoid arthritis. Clin Exp Rheumatol 1994b;12: 615--619. Boerbooms AM, Westgeest AA, Reekers P, van de Putte LB. Immunogenetic heterogeneity or seronegative rheumatoid arthritis and the antiperinuclear factor. J Rheumatol 1990:49: 15,17. Buisson M, Berthelot JM, Le Goff P, Chastel C, Lamour A, Seigneurin J-M, Youinou P. Lack of relationship between the Epstein-Barr virus and the antiperinuclear factor, perinuclear antigen, system in rheumatoid arthritis. J Autoimmun 1994;7:485- 495. Dale BA, Resing KA, Haydock PV. Cellular and molecular biology of intermediate filaments. In: Goldman RD, Steinert PM, eds. Filaggrins. New York: Plenum Press, 1990:393-412. Feltkamp TE, Berthelot JM, Boerbooms AM, Geertzen HG, Hoet R, De Keyser F, van Venrooij WJ, Verbruggen G, Veys EM, Youinou P. Interlaboratory variability of the antiperinu-

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CONCLUSION In spite of the inconstancy of the substrate material, the APF test warrants being used on a regular basis. With the simple IIF assay, the prevalence of the autoantibody has been thoroughly evaluated in RA and other connective tissue diseases. Sensitivity as well as specificity of the test are excellent for RA. The recent definition of the APF-targeted antigens should lead to the development of enzyme-linked immunosorbent assays. Characterization of the epitopes recognized by APF on filaggrin may even provide insights into the physiopathology of RA. See also FILAGGRIN (KERATIN) AUTOANTIBODIES.

Manera C, Francheschini F, Cretti L, Braga S, Cattaneo R. Clinical heterogeneity of rheumatoid arthritis and the antiperinuclear factor. J Rheumatol 1994;21:2021--2025. McKinley-Grant LJ, Idler WW, Bernstein IA, Parry DA, Cannizzaro L, Croce CM, Huebner K, Lessin SR, Steinert PM. Characterization of a cDNA clone encoding human filaggrin and localization of the gene to chromosome region lq21. Proc Natl Acad Sci USA 1989:86:4848-4852. Nesher G, Moore TL, Glisanti MW, E1-Najdawi E, Osborn TG. Antiperinuclear factor in juvenile rheumatoid arthritis. Ann Rheum Dis 1992;51: 350-352. Nienhuis RL, Mandena E. A new serum factor in patients with rheumatoid arthritis, the antiperinuclear factor. Ann Rheum Dis 1964;23:202--205. Scherbaum WA, Youinou P, Le Goff P, Bottazo GF. Antiperinuclear and rheumatoid factor in different forms of autoimmune thyroid disease. Clin Exp Immunol 1984:55: 516--518. Sebbag M, Simon M, Vincent C, Masson-Bessierre C, Girbal E, Durieux JJ, Serre G. The antiperinuclear factor and the socalled antikeratin antibodies are the same rheumatoid arthritis-specific autoantibodies. J Clin Invest 1995 ;95:26722679. Simon M, Girbal E. Sebbag M, Gomes-Daudrix V, Vincent C, Salam G, Serre G. The cytokeratin filament-aggregating protein filaggrin is the target of the so-called antikeratin antibodies, autoantibodies specific for rheumatoid arthritis. J Clin Invest 1993;92:1387-1393. Smit JW, Sondag-Tschroots IR, Aaij C, Feltkamp TE, Feltkamp-Vroom TM. The antiperinuclear factor. II. A light microscopical and immunofluorescence study on the antigenic substrate. Ann Rheum Dis 1980;39:381--386. Sondag-Tschroots IR, Aaij C, Smit JW, Feltkamp TE. The

antiperinuclear factor. I. The diagnostic significance of the antiperinuclear factor for rheumatoid arthritis. Ann Rheum Dis 1979;39:248--251. Vivino FB, Maul GG. Histologic and electron microscopic characterization of the antiperinuclear factor antigen. Arthritis Rheum 1990;33:960-969. Westgeest A, van Loon AM, van der Logt JT, van de Putte LB, Boerbooms AM. Antiperinuclear factor, a rheumatoid arthritis specific autoantibody: its relation to the Epstein-Barr virus. J Rheumatol 1989; 16:626--630. Westgeest AA, Boerbooms AM, Jongmans M, Vandenbroucke JP, Vierwinden G, van de Putte LBA. Antiperinuclear factor: indicator of more severe disease in seronegative rheumatoid arthritis. J Rheumatol 1987;14:893--897. Youinou P, Pennec YL, Le Goff P, Ferec C. Morrow WJ, Le Menn G. Antiperinuclear factor in SjOgren's syndrome in the presence or absence of rheumatoid arthritis. Clin Exp Rheumatol 1984;2:5--9. Youinou P, Zabbe C, Eveillaud C, Dewitte JD, Kerbourch JF, Ferec C, Clavier J. Antiperinuclear activity in lung carcinoma patients. Cancer Immunol Immunother 1984;18:80-81. Youinou P, Seigneurin JM. Le Goff P, Dumay A, Vicariot M, Lelong A. The antiperinuclear factor .II. Variabilility of the perinuclear antigen. Clin Exp Rheumatol 1990a;8:265--269. Youinou P, Le Goff P, Dumay A, Lelong A, Fauquert P, Jouquan J. The antiperinuclear factor. I. Clinical and serologic associations. Clin Exp Rheumatol 1990b;8:259--264. Youinou P, Le Goff P. The reliability of the antiperinuclear factor test, despite the inconstancy of the targeted antigens. J Rheumatol 1994;21:1990-1991. Youinou P, Serre G. The antipelinuclear factor and antikeratin antibody systems. Int Arch Allergy Immunol 1995;107:508, 518.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PHOSPHOLIPID AUTOANTIBODIES

CARDIOLIPIN

Munther A. Khamashta, M.D., Ph.D. and Graham R.V. Hughes, M.D. Lupus Arthritis Research Unit, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK

HISTORICAL NOTES

THE AUTOANTIGENS

The study of antiphospholipid antibodies (aPL) began when a serological test for syphilis was introduced in 1906 (Wasserman et al., 1906). In 1941, the active antigenic component in the test was found to be a phospholipid, which was subsequently termed "cardiolipin" (Pangborn, 1941). In the 1950s it became clear that a number of people had positive tests for syphilis without any evidence of the disease. This phenomenon was referred to as the biological false-positive serological test for syphilis. A high prevalence of autoimmune disorders, including systemic lupus erythematosus (SLE) and Sj6gren's syndrome occurred in this group of patients. The presence of circulating anticoagulants in patients with SLE was first documented in 1952 (Conley and Hartmann, 1952) and was associated with an increased risk of paradoxical thrombosis in 1963 (Bowie et al., 1963). The term "lupus anticoagulant" (LA), first used in 1972 (Feinstein and Rapaport, 1972), is clearly a misnomer, because LA is more frequently encountered in patients without lupus and is associated with thrombosis rather than abnormal bleeding. The introduction in 1983 of a radioimmunoassay (Harris et al., 1983) and shortly after of an ELISA to detect and measure anticardiolipin antibodies (aCL) resulted in widespread interest in aPL and in their clinical associations (Harris, 1990). The antiphospholipid syndrome (APS), a syndrome associated with aPL, was described in clinical detail (Hughes, 1983; 1993; Alarcon-Segovia, 1994; Khamashta and Asherson, 1995).

Definition

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aPL are a heterogeneous population of immunoglobulins which were originally thought to recognize anionic phospholipids. In 1990, three groups independently reported that the aCL detected by ELISA are not directed against cardiolipin alone, because purified IgG from aCL-positive patients did not bind to cardiolipin unless a plasma protein cofactor was present (Galli et al., 1990; McNeil et al., 1990; Matsuura et al., 1990). This protein was [~2-glycoprotein I (~2-GPI). It has since become clear that aCL in patients with the APS is dependent on both cardiolipin and ~2-GPI for optimal binding, though the relative importance of the two molecules, or their combination, is uncertain (Ichikawa et al., 1994) (Figure 1). Origin and Sources

~2-GPI is a 50 kd protein present at approximately 200 lag/mL in normal plasma. Although its physiological role is not known, in vitro data suggest that [32GPI may play an anticoagulant role (Roubey, 1994). It has recently been demonstrated that the fifth C terminal domain of ~2-GPI contains the major phospholipid binding site (Cys-281 to Cys-288), a region critical for binding aCL (Hunt et al., 1993; Hunt and Krilis, 1994). This observation was confirmed using monoclonal aCL derived from patients with APS (Wang et al., 1995). Immunization of healthy mice and rabbits with [32-GPI yields high titers of anti-~zGPI and aCL; whereas, cardiolipin alone is not immunogenic (Gharavi et al., 1992). These results

Figure 1. The possible epitope of anticardiolipin antibodies (aCL). A: aCL recognize a cryptic epitope on [32-GPIexposed by binding to CL. B: the epitope recognized by aCL comprises both 132-GPIand CL. C: aCL recognizes the conformational change of CL induced by binding to 132-GPI. suggest that a phospholipid-binding protein may be the key immunogen in APS.

AUTOANTIBODIES Methods of Detection Antiphospholipid antibodies are detected by a variety of laboratory tests; the most useful for identifying patients with the APS are the LA and the aCL tests. These antibodies are distinct and separable immunoglobulins present alone or in combination in the plasma of people with the APS (McNeil et al., 1989). The autoantibodies sometimes bind phospholipids utilized in the Venereal Disease Research Laboratory (VDRL) test; hence, some patients may have a falsepositive test for syphilis. However, the VDRL test is positive in only --5% of individuals with APS and is, thus, of little diagnostic value (Harris et al., 1993).

Anticardiolipin Antibody Test. The most sensitive test for aPL is the aCL test, introduced in 1983 and extensively improved since that time (Khamashta and Hughes, 1993; Harris et al., 1994a). Serum or plasma samples may be used for the aCL assay. The test uses enzyme-linked immunosorbent assay to determine antibody binding to solid plates coated either with

cardiolipin or other phospholipids. Although the original ELISA employed cardiolipin as the target antigen, aCL, owing to cross-reactivity, may bind other negatively charged phospholipids. However, the routine detection of antibodies against other phospholipids, such as phosphatidylinositol or phosphatidylserine are still controversial (Harris and Pierangeli, 1994; Rote et al., 1990; Arnold and Haughton, 1992). The availability of isotype-specific (IgG and IgM) reference sera has greatly improved interlaboratory testing and quantitation of aCL (Harris et al., 1994). IgG and IgM isotype concentrations are expressed as GPL and MPL units, respectively. One unit represents the binding activity of 1 pg/mL of affinity-purified aCL antibody. Results are expressed as low-, mediumand high-positive according to levels below 20 units, between 20 and 80 units and above 80 units, respectively. IgA aCL reference sera are now also available, yet the diagnostic value of IgA aCL is unclear (Lopez et al., 1992). Sensitive kits are commercially available and most laboratories now routinely measure IgG and IgM aCL; some laboratories measure all three isotypes. Decomplementation by heating serum to 56~ gives rise to false-positive results and should be avoided (Hasselaar et al., 1990). Freeze-thawing samples may result in a decrease of aCL binding activity (Triplett, 1994). The commercial source of

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microtiter plates plays an important role in this system (De Moerloose et al., 1990). Recently, a more specific aCL ELISA system has been established (Matsuura et al., 1992). ~2-GPI-dependent aCL can be differentiated by comparing the binding activity of cardiolipincoated wells with and without ~2-GPI on the same plate. In ~2-GPI-coated wells, the cardiolipin binding of APS-associated aCL is higher than that of wells without [32-GPI. In contrast, ~2-GPI depresses the cardiolipin binding of infectious type aCL (Matsuura et al., 1992). This system is now commercially available (Kaburaki et al., 1995). Flow cytometry has also been used to test for aCL (Stewart et al., 1993), including simultaneous measurement of aPL isotypes with different phospholipid specificity. The routine detection of other phospholipids, such as phosphatidylserine, phosphatidylinositol and phosphatidic acid, gives little additional information.

Pathogenetic Role Pregnant mice passively (Blank et al., 1991) and actively (Bakimer et al., 1992) immunized with human or mouse aCL develop pregnancy loss, and aCL increases thrombus size and persistence over time in a mouse model (Pierangeli and Harris, 1994). Both findings argue in favor of a pathogenetic role. Precisely how aPL relate to thrombosis and pregnancy loss is unknown. Possible mechanisms of the prothrombotic nature of the APS include effects of aPL on platelet membranes, on endothelial cells and on clotting components such as antithrombin III, protein C and protein S (Roubey, 1994). Cross-reaction between aCL and oxidized LDL antibodies (Vaarala et al., 1993) and the association of antibodies to oxidized LDL with atherosclerosis suggest that APS might provide clues to the pathogenesis not only of thrombosis but also atherosclerosis.

Genetics aPL-positive families exist, and HLA studies suggest association with DR4, DR7, DRw53, DQw7 and C4 null alleles (Asherson et al., 1992; Wilson et al., 1995).

Factors Involved in Pathogenicity The association of clinical complications with aPL appears to depend on specificity, isotype, level and probably the time during which these antibodies are

626

present (Harris et al., 1993). Apart from SLE and primary APS, aPL are detected in patients with a variety of autoimmune, infectious, malignant and drug-induced disorders, as well as in some apparently healthy individuals. In the latter cases, aPL are usually of low titer, of the IgM isotype, and unassociated with thrombotic events. The specificities of aPL probably differ in various disorders. These differences are demonstrated in several studies of aPL in patients with autoimmune disorders or with infection (Harris et al., 1988; Hunt et al., 1992). Compared with infection-associated aPL, autoimmune aPL have higher titer, are more commonly of the IgG isotype (all subclasses and notably IgG2 and IgG4), have higher avidity and require the presence of a co-factor (Lockshin, 1993). The interaction of autoimmune-type aCL with ~2-GPI is directly associated with thrombosis. Autoimmune aCL are unable to bind ~2-GPI in free solution but have a strong affinity for ~2-GPI bound to phospholipid. This might reflect the fact that most circulating anti-[32-GPI antibodies are of low affinity; the interaction of ~2-GPI and phospholipid in some way proving a more potent substrate for binding (Roubey, 1994).

CLINICAL UTILITY Disease Association The most frequent cause of acquired thrombophilia is the APS. Patients with this disorder have LA and/or aCL in their blood and are predisposed to venous and arterial thrombosis, thrombocytopenia and, in women who conceive, recurrent fetal loss (Hughes, 1993). The unrelated behavior of LA and aCL in the course of disease and in individual patients indicates that both assays are required if all cases with the APS are to be detected (Khamashta and Hughes, 1993). Vessels of all sizes can be affected; the vascular pathology is bland occlusion without inflammatory infiltrate (Lie, 1994). Clinical features widely believed to be associated with aPL are well established (Table 1) as are minimal criteria for the diagnosis of APS (Table 2). Although first described in patients with SLE (Hughes, 1983), aPL are not confined to lupus patients but may well occur frequently (Hughes, 1993) in nonlupus p a t i e n t s - the "primary" APS (Asherson et al., 1989; Alarcon-Segovia and Sanchez-Guerrero, 1989). For research and classification purposes, the term "primary" is useful, even though there are few

Table 1. Clinical Manifestations of the Antiphospholipid Syndrome Venous thrombosis Deep vein thrombosis Pulmonary thromboembolism Budd-Chiari syndrome Renal vein thrombosis Ocular thrombosis Arterial thrombosis Stroke, transient ischemic attacks, amaurosis Myocardial infarction Limb ischemia Recurrent pregnancy loss Thrombocytopenia and hemolytic anemia Other features Livedo reticularis Migraine Epilepsy Chorea Myelopathy Heart valve disease Pulmonary hypertension Addison' s disease Skin ulcers Ischemic necrosis of bone

differences in aPL-related complications or antibody specificity in the presence or absence of SLE (Vianna et al., 1994). aPL are positive in 30-40% of SLE patients, but only one-third of these patients develop clinical features of APS (Love and Santoro, 1990). Neither the LA nor aCL correlate with age, duration of disease or clinical features of SLE, including polyarthritis, vasculitis or serositis. Up to 30% of patients attending an anticoagulation clinic have aPL (Chu et al., 1988; Exner and Koutts, 1988). High levels of aCL are associated with an in-

creased risk of venous thrombosis and pulmonary embolism (Ginsburg et al., 1992). aPL are now recognized as an important risk factor for stroke and may be present in 7% of all patients who have suffered a stroke (Montalban et al., 1991). aPL should be sought especially in young patients with stroke where they may account for up to 18% (Nencini et al., 1992). Recurrent spontaneous pregnancy losses are one of the most consistent complications of the APS. Losses can occur at any stage of pregnancy, though aPLrelated miscarriage are strikingly frequent during the second and third trimester. The rate of miscarriages in aPL-positive patients is still uncertain, although the epidemiology is being studied and, increasingly, aPL testing is becoming a routine investigation in women with recurrent miscarriages. In a large prospective study of 389 primiparous women assessed at study entry and delivery, 24% (93) were aPL-positive, 15.8% (61) of whom had fetal loss compared with 6.5% (19) of antibody-negative mothers (Lynch et al., 1994). The management of patients with APS is largely based on anticoagulant therapy (Hughes, 1993). Steroids or immunosuppressives to reduce antibody activity are not beneficial. Long-term anticoagulant treatment may be needed for patients who have had thrombosis to prevent recurrence (Khamashta et al., 1995).

CONCLUSION Confirmatory evidence that aPL (LA and/or aCL) are associated with an increased risk for thrombosis and recurrent pregnancy loss has lead to increased laboratory requests for identification of these antibodies. Criteria for the definition of the APS are now well

Table 2. Criteria for the Diagnosis of the Antiphospholipid Syndrome* Clinical

Laboratory

Venous thrombosis

IgG aCL (moderate/high titer)

Arterial thrombosis

IgM aCL (moderate/high titer)

Recurrent fetal loss

Positive LA

Thrombocytopenia *Patients with the syndrome should have at least one clinical plus one laboratory finding during their disease, aPL test must be positive on at least two occasions more than 3 months apart.

627

established. Although at present both the pathogenesis and the optimal m a n a g e m e n t of the APS are uncertain, animal models are providing useful clues (Shoenfeld and Fishman, 1994). See also ~2-GLYCOPROTEIN

I AUTOANTIBODIES, BROMELAIN-TREATED ERYTHROCYTE AUTOANTIBODIES, LUPUS ANTICOAGULANT and PHOSPHOLIPID AUTOANTIBODIES PHOSPHATIDYLSERINE.

REFERENCES

glycoprotein I. J Clin Invest 1992;90:1105-1109. Ginsburg KS, Liang MH, Newcomer L, Goldhaber SZ, Schur PH, Hennekens CH, Stampfer MJ. Anticardiolipin antibodies and the risk for ischemic stroke and venous thrombosis. Ann Intern Med 1992;117:997--1002. Harris EN, Gharavi AE, Boey ML, Patel BM, MackworthYoung CG, Loizou S, Hughes GR. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis. Lancet 1983;2:1211--1214. Harris EN, Gharavi AE, Wasley GD, Hughes GRV. Use of an enzyme-linked immunosorbent assay and of inhibition studies to distinguish between antibodies to cardiolipin from patients with syphilis or autoimmune disorders. J Infect Dis 1988; 157:23-31. Harris EN. Special Report. The Second International Anticardiolipin standardization workshop/the Kingston Antiphospholipid Antibody Study (KAPS) Group. Am J Clin Pathol 1990;94:476--484. Harris EN, Khamashta MA, Hughes GRV. Antiphospholipid antibody syndrome. In: McCarty DJ, Koopman WJ, eds. Arthritis and Allied Conditions, 12th edition. Philadelphia: Lea and Febiger, 1993:1201--1212. Harris EN, Pierangeli S. Anticardiolipin antibodies: specificity and function. Lupus 1994;3:217-222. Harris EN, Pierangeli S, Birch D. Anticardiolipin wet workshop report: Vth International Symposium on Antiphospholipid Antibodies. Am J Clin Pathol 1994;101:616-624. Hasselaar PH, Triplett DA, Lame A, Derksen RH, Blokzijl L, Groot PG, Wagenknecht DR, Mclntyre JA. Heat treatment of serum and plasma induces false-positive results in the antiphospholipid antibody ELISA. J Rheumatol 1990;17: 186-191. Hughes GRV. Thrombosis, abortion, cerebral disease and lupus anticoagulant. Br Med J 1983;287:1088--1089. Hughes GRV. The antiphospholipid syndrome: ten years on. Lancet 1993;342:341-344. Hunt JE, McNeil HP, Morgan GJ, Crameri RM, Krilis SA. A phospholipid-132-glycoprotein I complex is an antigen for anticardiolipin antibodies occurring in autoimmune disease but not with infection. Lupus 1992;1:83-90. Hunt JE, Simpson RJ, Krilis SA. Identification of a region of l]2-glycoprotein I critical for lipid binding and anticardiolipin co-factor activity. Proc Natl Acad Sci USA 1993;90:2141-2145. Hunt JE, Krilis S. The fifth domain of 132-glycoprotein I contains a phospholipid binding site (Cys281-Cys288) and a region recognized by anticardiolipin antibodies. J Immunol 1994;152:653-659. Ichikawa K, Khamashta MA, Koike T, Matsuura E, Hughes GRV. 132-glycoprotein I reactivity of monoclonal anticar-

Alarcon-Segovia D, Sanchez-Guerrero J. Primary antiphospholipid syndrome. J Rheumatol 1989;16:482--488. Alarcon-Segovia D. Antiphospholipid syndrome within systemic lupus erythematosus. Lupus 1994;3:289--291. Arnold LW, Haughton G. Autoantibodies to phosphatidylcholine. The murine antibromelain RBC response. Ann N Y Acad Sci 1992;651:354-359. Asherson RA, Khamashta MA, Ordi-Ros J, Derksen RH, Machin SJ, Barquinero J, Outt HH, Harris EN, Vilardell Torres M, Hughes GR. The "Primary" antiphospholipid syndrome: major clinical and serological features. Medicine (Baltimore) 1989;68:366--374. Asherson RA, Doherty DG, Vergani D, Khamashta MA, Hughes GRV. Major histocompatibility complex associations with primary antiphospholipid syndrome. Arthritis Rheum 1992;35:124-125. Bakimer R, Fishman P, Blank M, Sredni B, Djaldetti M, Shoenfeld Y. Induction of experimental antiphospholipid syndrome in mice by immunization with human monoclonal anticardiolipin antibody (H-3). J Clin Invest 1992;89:1558-1563. Bowie EJ, Thompson JH, Pascuzzi CA, Owen CA. Thrombosis in Systemic Lupus Erythematosus despite circulating anticoagulants. J Lab Clin Med 1963;62:416--430. Blank M, Cohen J, Toder V, Shoenfeld Y. Induction of antiphospholipid syndrome in naive mice with mouse lupus monoclonal and human polyclonal anticardiolipin antibodies. Proc Natl Acad Sci USA 1991;88:3069--3073. Chu P, Pendry K, Blecher TE. Detection of lupus anticoagulant in patients attending an anticoagulation clinic. BMJ 1988; 297:1449. Conley CL, Hartmann, RC. A haemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest 1952;31:621-623. De Moerloose P, Reber G, Vogel JJ. Anticardiolipin antibody determination: comparison of three ELISA assays. Clin Exp Rheumatol 1990;8:575-577. Exner T, Koutts J. Autoimmune cardiolipin-binding antibodies in oral anticoagulant patients. Aust NZ J Med 1988;18:669-673. Feinstein DI, Rapaport SI. Acquired inhibitors of blood coagulation. Prog Hemost Thromb 1972;1:75--95. Galli M, Comfurius P, Maasen C, Hemker HC, de Baets MH, van Breda Vriesman PJ, Zwall RF, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein co-factor. Lancet 1990;336:1544--1547. Gharavi AE, Sammaritano LR, Wen J, Elkon KB. Induction of antiphospholipid autoantibodies by immunization with [32-

628

diolipin antibodies from patients with the antiphospholipid syndrome. Arthritis Rheum 1994;37:1453-1461. Kaburaki J, Kuwana M, Yamamoto M, Kawai S, Matsuura E, Ikeda Y. Clinical significance of phospholipid-dependent anti-132-Glycoprotein I (I]2-GPI) antibodies in systemic lupus erythematosus. Lupus, 1995;(in press). Khamashta MA, Hughes GRV. Detection and importance of anticardiolipin antibodies. J Clin Pathol 1993;46:104-- 107. Khamashta MA, Asherson RA. Hughes syndrome- Antiphospholipid antibodies move closer to thrombosis in 1994. Br J Rheumatol 1995;34:493--494. Khamashta MA, Cuadrado MJ, Mujic F, Taub NA, Hunt BJ, Hughes GRV. The management of thrombosis in the antiphospholipid antibody syndrome. N Engl J Med 1995;332: 993--997. Lie JT. Vasculitis in the antiphospholipid syndrome: Culprit or consort ? J Rheumatol 1994;21:397--399. Lockshin MD. Which patients with antiphospholipid antibody should be treated and how? Rheum Dis Clin North Am 1993"19:235-247. Lopez LR, Santos ME, Espinoza LR, La Rosa FG. Clinical significance of immunoglobulin A versus immunoglobulins G and M anticardiolipin antibodies in patients with systemic lupus erythematosus. Correlation with thrombosis, thrombocytopenia, and recurrent abortion. Am J Clin Pathol 1992; 98:449--454. Love PE, Santoro SA. Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and non-SLE disorders: prevalence and clinical significance. Ann Intern Med 1990;112:682--698. Lynch A, Marlar R, Murphy J, Davila G, Santos M, Rutlege J, Emlen W. Antiphospholipid antibodies in predicting adverse pregnancy outcome. Ann Intern Med 1994;120:470-475. Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Koike T. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990;336:177--178. Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Suzuki T, Sumida T, Yasuda T, Koike T. Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor. J Immunol 1992;148:3885-3891. McNeil HP, Chesterman CN, Krilis SA. Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroups with different phospholipid binding characteristics. Br J Haematol 1989;73:506--510. McNeil HP, Simpson RJ, Chesterman CN, Krilis S. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-bindig inhibitor of coagulation: 132-

glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990; 87:4120--4124. Montalban J, Codina A, Ordi J, Vilardell M, Khamashta M, Hughes GRV. Antiphospholipid antibodies in cerebral" ischemia. Stroke 1991;22:750-753. Nencini P, Baruffi MC, Abbati R, Massai G, Amaducci L, Inzitari P. Lupus anticoagulant and anticardiolipin antibodies in young adults with cerebral ischemia. Stroke 1992:23:189193. Pangborn MD. A new serologically active phospholipid from beef heart. Proc Soc Exp Biol Med 1941;48:484--486. Pierangeli SS, Harris EN. Antiphospholipid antibodies in an in vivo thrombosis model in mice. Lupus 1994;3:247--251. Rote NS, Dostal-Johnson D, Branch DW. Antiphospholipid antibodies and recurrent pregnancy loss: correlation between the activated partial thromboplastin time and antibodies against phosphatidylserine and cardiolipin. Am J Obstet Gynecol 1990;163:575-584. Roubey RAS. Autoantibodies to phospholipid-binding plasma proteins: a new view of lupus anticoagulants and other "antiphospholipid" antibodies. Blood 1994;84:2854--2867. Shoenfeld Y, Fishman P. Role of IL-3 in the antiphospholipid syndrome. Lupus 1994;3:259--261. Stewart MW, Etches WS, Russell AS, Percy JS, Johnston CA, Chew CK, Gordon PA. Detection of antiphospholipid antibodies by flow cytometry: rapid detection of antibody isotype and phospholipid specificity. Thromb Haemost 1993 ;70:603--607. Triplett DA. Assays for detection of antiphospholipid antibodies. Lupus 1994;3:281-287. Vaarala O, Alfthan G, Jauhiainen M, Leirisalo-Repo M, Aho K, Palosuo T. Cross-reaction between antibodies to oxidised low-density lipoprotein and to cardiolipin in systemic lupus erythematosus. Lancet 1993;341:923--925. Vianna JL, Khamashta MA, Ordi-Ros J, Font J, Cervera R, Lopez-Soto A, Tolosa C, Franz J, Selva A, Ingelmo M. Comparison of the primary and secondary antiphospholipid syndrome: a European multicenter study of 114 patients. Am J Med 1994;96:3--9. Wassermann VA, Neisser A, Bruck C. Eine serodiagnostische Reaktion bei Syphilis. Deutsche Medizinische Wochenschrift 1906;19:745-746. Wilson WA, Scopelitis E, Michalski JP, Periangeli SS, Silveira LH, Elston RC, Harris EN. Familial anticardiolipin antibodies and C4 deficiency genotypes that co-exist with MHC DOB 1 risk factors. J Rheumatol 1995;22:227--235.

629

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PHOSPHOLIPID AUTOANTIBODIES

PHOSPHATIDYLSERINE

Noori E. Barka, Ph.D.

Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES

A unique in vitro anticoagulant phenomenon was discovered in SLE patients with biological falsepositive results in serological tests for syphilis (BFPSTS) (Conley and Hartman, 1952). This phenomenon, termed "lupus anticoagulant" (LA), is due to antibodies that act as an inhibitor to negatively charged phospholipids at the stage of conversion from prothrombin to thrombin (Feinstein and Rapaport, 1972). The association of BFP-STS and/or LA with SLE is buttressed by the absence of LA in syphilis (Johansson and Lassus, 1974) and the presence of LA in many patients with venous and arterial thrombosis and thrombocytopenia with or without SLE (Boxer et al., 1976; Schleider et al., 1976). In 1983, a solid-phase radioimmunoassay for the detection of antibodies to cardiolipin, a negatively charged phospholipid, revealed a strong correlation among anticardiolipin antibodies (aCL), LA and BFPSTS (Harris et al., 1983). The aCL antibodies found in 61% of SLE patients were associated with venous and arterial thrombosis, thrombocytopenia and recurrent fetal loss (Harris et al., 1983). These seminal observations and the introduction of an enzyme-linked immunosorbent assay (ELISA) for the detection of aCL antibodies in 1985 (Loizou et al., 1985) generated widespread interest in these antibodies and the clinical syndrome associated with their presence. In 1987, the term "antiphospholipid syndrome" (APS) was proposed for the combination of both venous and arterial occlusive events, often accompanied by thrombocytopenia, in the presence of antibodies to negatively charged phospholipids (Harris et al., 1987). The term "primary APS" is used to identify patients without SLE; whereas, the term "secondary APS" is used when APS is secondary to SLE. Diagnostic criteria for APS are based on finding at least .one 630

clinical manifestation of venous or arterial thrombosis, recurrent fetal loss or thrombocytopenia plus at least one laboratory abnormality, i.e., aCL antibodies or LA (Harris, 1990). Detection of aCL antibodies by ELISA became the standard laboratory method for the diagnosis of APS, because in comparison to LA, (1) ELISA is easier, more sensitive and quantitative; (2) can determine specific antibody isotypes (IgG, IgM, IgA); and (3) can be performed on stored sera. Subsequent studies showed that the aCL and LA tests are detecting different subgroups of antibodies and can be discordant in up to 35% of patients (Triplett et al., 1988). Different techniques such as column chromatography with siliconized sand (Exner et al., 1988), ion-exchange gel-filtration and anti-Ig affinity chromatography (McNeil et al., 1989) were used to prove that the two assays can measure IgG, IgM or IgA antibody reactivities to different epitopes. It is clear now that both assays are required to maximize the detection of antibodies to negatively charged phospholipids and to identify most APS cases (Cervera et al., 1990; Jouhikainen et al., 1992; Petri, 1994; Triplett, 1994). Other antibodies to negatively charged phospholipids (aPL) considered and studied by ELISA to determine their sensitivity and clinical utility in the evaluation of APS patients include antiphosphatidylserine (aPS) antibodies (Gharavi et al., 1987; Loizou et al., 1990).' These antibodies were found to be promising markers and were proposed in some studies as an alternative to or in addition to aCL antibodies (Branch et al., 1987; Blank et al., 1994).

THE AUTOANTIGEN

Phosphatidylserine (PS) is a negatively charged phospholipid composed of the three-carbon glycerol

I Serine

o,,, o ~

I OH=-(;.H~H= I I

o

I

oI

coo-

I

o

I

I

I

I

o

CH2-CH-CH21 !

o

o

I

I

C=O C=O

<

0-

I

?H25H-CH2 I

c=o c=o

OH

O-P-O-CHr-CH--CH~--O-P=O I I 0 0 o I

C=O C=O

I

< <

( < a

b

Figure 1. The chemical structure of PS (a) and CL (b).

moiety that has two fatty acids esterified at C I' and C2' carbons forming diacylglycerol. A phosphate group is attached at the C3' position of the diacylglycerol to form a phosphatidyl group to which the hydroxyl serine is linked to yield phosphatidylserine (Figure l a). The phosphatidyl moieties of PS and cardiolipin (CL) are similar. What is unique about CL is having a second glycerol moiety as the head group to which an additional phosphatidyl component is attached (Figure lb). PS antigen from bovine brain is available commercially in a purified form (Sanders, 1967) and at relatively low cost (Sigma, St Louis, MS).

THE A U T O A N T I B O D I E S

Under the term "antiphospholipid antibodies" (aPL) are subsumed antibodies to negatively charged phospholipids, such as CL, LA and PS. Of these, CL is the phospholipid most commonly used as antigen to test for aPL by ELISA; PS is less commonly used. Phosphatidic acid and to a lesser extent phosphatidylglycerol and phosphatidylnositol can also be used to detect aPL (Jones et al., 1995). Studies comparing PS to CL as antigens for the detection of aPL antibodies are inconclusive. PS seems to be more physiologically relevant than CL as the antigen for the detection of aPL antibodies, because unlike CL, which is located on the inner surface of plasma membranes, PS is

found on the outer surface of plasma membranes of platelets and endothelial cells (McNeil et al., 1991). Likewise, PS participates in the coagulation cascade and has a role in clot formation (Bevers et al., 1982). In addition, aPS antibodies correlate better with the presence of LA than do aCL (Branch et al., 1987; Rote et al., 1990; Zuazu-Jausoro et al., 1990; Zacur and Moutos, 1991; Vogt et al., 1992) and can be found in aCL-negative sera from SLE patients (Toschi et al., 1993). PS is reported to recognize affinitypurified aCL antibodies (prepared by passing sera from APS and syphilis patients through a protein-GSepharose column with CL liposomes) from APS patients, but not affinity-purified IgG from patients with syphilis; by contrast, CL was recognized by affinity-purified antibodies from both disease populations (Pierangeli et al., 1994). Animal studies show that purified IgG aPS can induce APS in naive mice by passive infusion of the purified IgG into pregnant ICR mice. These mice showed increased fetal resorption, lower mean weights of placentas and fetuses and prolonged aPTT (Blank et al., 1994). In contrast to these data which support the use of PS instead of or in addition to CL as antigen in assays for aPL, other studies found no advantage in the use of PS (alone or combined with CL). In a study to determine whether detection of aCL or aPS by ELISA can substitute for LA in screening for aPL antibodies, the use of PS neither improved the correlations with the presence of LA nor offered any advantages over

631

CL (Cowchock and Fort, 1994). Similar findings and conclusions were described by others (Gharavi et al., 1987; Exner and McRea, 1990; Harris and Pierangeli, 1994). Consequently, the use of both CL and PS antigens by ELISA, in addition to LA, is not uniformly recommended by all investigators for detection of aPL in patients with suspected APS. To assess the frequency and coincidence of aCL and aPS in patients with suspected primary or secondary APS, a recent study evaluated 2,000 consecutively received sera from patients with suspected APS (Barka et al., 1995). A standard ELISA procedure was used for the detection of specific IgG, IgM and IgA antibodies to CL and PS. aCL results were expressed in phospholipid units (GPL, MPL and APL Units/mL) based on the antiphospholipid international standard using a cut-off of 20 Units/mL. For aPS, an in-house calibrator was used for the calculation of the results based on a cutoff established from the mean + 5SD of 100 sera from healthy individuals. All of the 2,000 sera were tested in duplicate in antigen-coated wells versus non-antigen-coated wells (control wells) to eliminate any nonspecific reaction caused by attachment of some sticky sera to the plastic. The results of this study (Barka et al., 1995) showed that 8% (161/ 2,000) of tested sera were positive, and 92% (1,839/ 2,000) were negative (Table 1). Of the 161 aPLpositive sera, 75% (120/161) showed reactivity for both aCL and aPS antibodies, 14% (23/161) showed reactivity for aCL only, and 11% (18/161) showed reactivity for aPS only. Of the 120 sera positive for both aCL and aPS, 63% (76/120) showed reactivity with more than one isotype (Table 2). When only one test (i.e., aCL or aPS) was positive, the percentage of samples showing reactivity with more than one isotype was 17.4% (4/23) and 16.6% (3/18) for aCL and aPS, respectively. Of the 18 patients positive only for aPS (negative for aCL), 4, 9 and 2 were positive only for IgG aPS, IgM aPS and IgA aPS respectively; 2 and 1 were positive only for IgG and IgA aPS and IgM and IgA aPS. If testing for aCL and aPS were performed for IgG antibodies

only, 29% (35/120) of the sera positive for IgM and/or IgA would have been missed (Table 2). These data emphasize the importance not only of testing for aPS to detect aPL-positive sera missed by testing only for aCL, but also the importance of testing for all three isotypes of aPS and aCL if the detection of aPL is to be maximized. This study confirms the significant cross-reactivity and overlapping between aCL and aPS as previously described (Cowchock and Fort, 1994; Harris and Pierangeli, 1994) and shows that combined testing for aCL and aPS by ELISA as well as testing for all three isotypes along with LA is apparently necessary to achieve the best sensitivity for detection of aPL antibodies in APS patients. Preliminary studies suggest that the clinical characteristics of patients with aPS-restricted antibodies are similar to aCL/LA-positive patients with APS, including recurrent abortion and thrombosis (Barka et al., unpublished). Detailed prospective and retrospective studies of patients with aPS antibodies of one or more isotypes are needed. Laboratory evaluation of patients with APS now also includes the use of ~2-glycoprotein I (~2-GPI) as the target antigen rather than any of the negatively charged phospholipids (Matsuura et al., 1994). [32-GPI, a phospholipid-binding plasma protein, is a co-factor needed for binding of CL by aCL (Galli et al., 1990; McNiel et al., 1990; Matsuura et al., 1990) and for binding of PS by aPS (Keedy et al., 1994; Jones et al., 1995). In addition, [32-GPI antibodies can distinguish aPL found in patients with infectious diseases, such as syphilis, malaria, hepatitis A and infectious mononucleosis from those found in autoimmune diseases (Matsura et al., 1992). In infectious diseases, aPL bind to CL or PS antigens in the absence of 132GPI co-factor; whereas, this co-factor is required for binding of aPL to CL or PS in autoimmune diseases. A recent study suggests that [32-GPI p e r s e is probably a target antigen for aPL because aPL can bind directly to ~2-GPI coated on irradiated polystyrene surfaces in the absence of CL (Matsuura et al., 1994). In addition,

Table 1. Comparison of ELISA Results for 2,000 Sera Tested for aCL and aPS Antibodies aCL

+ aPS

632

120

18

23

1839

Table 2. The Distribution of IgG, IgM and IgA Antibodies to CL and PS in 161 Sera Tested by ELISA aCL Neg

G 8

Neg G

4

M

9

A

2

M

A

2

9

15 16"

GMA

GM

GA

2

1

5

6

4 13"

1

1

MA 1

1

2

GM

1"

30

1"

18

GA

2

3

5

MA

1

1"

2

Total

18

23

20

27

16

15

7

41

23 30

aPS GMA

Total

5

14 13

23 3*

5

21

6

7 161

*Sera positive for IgM and/or IgA to CL and/or PS. These samples represent 29% (35/120) of IgM and IgA positives with negative IgG.

the clinical manifestations of the antiphospholipid syndrome in patients with systemic lupus erythematosus associate more strongly with anti-[32-GPI than with antiphospholipid antibodies (Cabiedes et al., 1994).

CONCLUSION The antiphospholipid syndrome represents a heterogeneous group of diseases characterized by recurrent v e n o u s or arterial thrombosis, recurrent fetal loss and

REFERENCES

Barka N, Reagan K, Agopian M, Peter JB. Frequency of anticardiolipin and antiphosphatidylserine antibodies in patients with suspected antiphospholipid syndrome (Abstract). Clin Chem 1995;41:5--73. Bevers E, Comfrius P, van Rijn J, Hemker C, Zwaal R. Generation of prothrombin-converting activity and the exposure of phosphatidylserine at the outer surface of platelets. Eur J Biochem 1982;122:429--436. Blank M, Tincani A, Shoenfeld Y. Induction of experimental antiphospholipid syndrome in naive mice with purified IgG antiphosphatidylserine antibodies. J Rheumatol 1994;21:100-104. Boxer M, Ellman L, Carvalho A. The lupus anticoagulant. Arthritis Rheum 1976;19:1244-1248. Branch D, Rote N, Dostai D, Scott J. Association of lupus anticoagulant with antibody against phosphatidylserine. Clin Immunol Immunopathol 1987;42:63-75. Cabiedes J, Cabral AR, Alarcon-Segovia D. Identification of

thrombocytopenia. The laboratory diagnosis of APS is based on the presence of antiphospholipid antibodies, such as aCL, aPS and LA. Testing for all three isotypes of aCL and aPS by E L I S A offers greater sensitivity for improved evaluation of patients with suspected APS. Studies of ~2-GPI antibodies are increasing our understanding of the antiphospholipid syndrome and its laboratory evaluation. See also [32GLYCOPROTEIN I ANTIBODIES, LUPUS ANTICOAGULANT and PHOSPHOLIPID AUTOANTIBODIES CARDIOLIPIN.

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Coag Fibrinol 1990; 1:17--21. Feinstein DI, Rapaport SI. Acquired inhibitors of blood coagulation. Prog Hemostasis Thromb 1972;1:75--95. Galli M, Comfurius P, Maasen C, Hemker HC, de Baets MH, van Breda-Vriesman PJ, Barbui T, Zwaal RF, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990;336:1544-1547. Gharavi AE, Harris EN, Asherson R A, Hughes GRV. Anticardiolipin antibodies: isotype distribution and phospholipid specificity. Ann Rheum Dis 1987;46:1--6. Harris EN, Gharavi AE, Boey ML, Patel BM, MackworthYoung CG, Loizou S, Hughes GR. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;2: 1211--1214. Harris EN, Baguley E, Asherson RA, Hughes GRV. Clinical and serological features of the antiphospholipid syndrome (APS). Br J Rheumatol 1987;26:19. Harris EN. A reassessment of the antiphospholipid syndrome. J Rheumatol 1990;17:733-735. Harris EN, Pierangeli S. Anticardiolipin antibodies: specificity and function. Lupus 1994;3:217--222. Johansson EA, Lassus A. The occurrence of circulating anticoagulants in patients with syphilitic and biologically falsepositive antilipoidal antibodies. Ann Clin Res 1974;6:105-108. Jones JV, James H, Mansour M, Eastwood BJ. I]2-glycoproteinI is a cofactor for antibodies reacting with 5 anionic phospholipids. J Rheumatol 1995;22:2009. Jouhikainen T, Julkunen H, Vaarala O, Leirisalo-Repo M, Stephansson E, Vahtera E, Palosuo T, Myllyla G. Antiphospholipid antibodies and thrombosis in SLE: comparison of three lupus assays and anticardiolipin ELISA in 188 patients. Blood Coagul Fibrinolysis 1992;3:407--414. Keedy K, Santos M, Lopez L. Antiphosphatidylserine antibodies require 13-2 glycoprotein I as cofactor in ELISA. Lupus 1994;3:327. Loizou S, McCrea JD, Rudge AC, Reynolds R, Boyle CC, Harris EN. Measurement of anticardiolipin antibodies by an enzyme linked immunosorbent assay (ELISA): standardization and quantitation of results. Clin Exp Immunol 1985; 62:738--745. Loizou S, Mackworth-Young CG, Cofiner C, Walport MJ. Heterogeneity of binding reactivity to different phospholipid of antibodies from patients with systemic lupus erythematosus (SLE) and with syphilis. Clin Exp Immunol 1990;80: 171--176. Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Koike T. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease (Letter). Lancet 1990; 1:177-- 178. Matsuura E, Igarashi Y, Fuiimoto M, Ichikawa K, Suzuki T, Sumida T, Yasuda T, Koike T. Heterogeneity of anticardio-

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lipin antibodies defined by the anticardiolipin cofactor. J Immunol 1992;148:3885--3891. Matsuura E, Igarashi Y, Yasuda T, Triplets DA, Koike T. Anticardiolipin antibodies recognize I]2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med 1994;179:157-162. McNeil HP, Chesterman CN, Krilis SA. Anticardiolipin antibodies and lupus anticoagulants comprise separate antibody subgroup with different phospholipid binding characteristics. Br J Haematol 1989;78:506-513. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor: I]2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990;87: 4120--4124. McNeil HP, Chesterman CN, Krilis SA. Immunology and clinical importance of anticardiolipin antibodies. Adv Immunol 1991;49:193-280. Petri M. Diagnosis of antiphospholipid antibodies. Rheum Dis Clin North Am 1994;20:443--469. Pierangeli S, Goldsmith G, Krnic S, Harris N. Differences in functional activity of anticardiolipin antibodies from patients with syphilis and those with antiphospholipid syndrome. Infect Immun 1994;62:4081-4084. Rote MS, Dostal-Johnson D, Branch WD. Antiphospholipid antibodies and recurrent pregnancy loss: correlation between the activated partial thromboplastin time and antibodies against phosphatidylserine and cardiolipin. Am J Obstet Gynecol 1990;163:575-584. Sanders H. Reparative isolation of phosphatidylserine from brain. Biochim Biophys Acta 1967;144:485--487. Schleider MA, Nachman RL, Jaffe EA, Coleman M. A clinical study of lupus anticoagulant. Blood 1976;48:499-509. Toschi V, Motta A, Castelli S, Gibelli S, Cimminiello C, Molaro GL, Gibelli A. Prevalence and clinical significance of antiphospholipid antibodies to noncardiolipin antigens in systemic lupus erythematosus. Haemostasis 1993;23:275--283. Triplett DA, Brand JT, Musgrave KA, Orr CA. The relationship between lupus anticoagulants and antibodies to phospholipid. JAMA 1988;259:550--554. Triplett DA. Assays for detection of antiphospholipid antibodies. Lupus 1994;3:281--287. Vogt E, Liden T'W, Ng AK, Rote NS. Monoclonal antiphosphatidylserine antibody induces intrauterine growth retardation in BALB/c mice (Abstract). Fifth International Symposium on Antiphospholipid Antibodies. San Antonio, Texas USA, September 1992. Zacur HA, Moutos D. Repeated pregnancy losses. Curr Opin Obstet Gynecol 1991 ;3:197-204. Zuazu-Jausoro A, Oliver I, Monsarrat M, Borrell M, Gaff I, Pich E. Grau y J. Fontcuberta Importance of antiphosphatidylserine antibodies in patients with lupus anticoagulant. Analysis of 30 cases. Med Clin (Barc) 1990;95:210-213.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PLATELET AUTOANTIBODIES Alaa E.E. Ahmed, Ph.D. Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES

PLATELET ALLOANTIGENS

Antiplatelet antibodies are the main cause of immunemediated thrombocytopenia purpura, a relatively common hematological disorder (Ahmed et al., 1994) first reported in 1735 by Werlhof as morbus maculosus hemorrhagicus (Jones and Tocantins, 1933). The immune nature of the disease was suspected when it was observed that thrombocytopenic mothers often gave birth to children with transient thrombocytopenia, suggesting the passage of humoral factors across the placenta. Understanding of immune-mediated thrombocytopenia purpura changed dramatically when Harrington transfused plasma from autoimmune thrombocytopenia patients into several recipients, including himself, and noted an immediate decline in the recipients' platelet counts (Harrington et al., 1951). Since this landmark study, knowledge of platelet autoantigens and the pathophysiology of platelet autoantibodies has burgeoned.

Platelet alloantigens are defined by the presence of single amino acid substitutions within the polypeptide chain of the glycoprotein bearing the alloantigenic epitope. Biochemical and immunochemical characterizations of allele-specific recombinant forms of these platelet glycoproteins show that these amino acid dimorphisms control both the formation and expression of the alloantigenic determinant (Newman, 1994). To date, all the known alloantigens are associated with glycoproteins (GP) IIIa, Ia, Ib and IIb. Some alloantigens are shared with other cells; others are platelet-specific. Of the shared alloantigens, the most important are the human leukocyte antigen (HLA) class I and the glycoconjugates of the ABH, Lewis, I and P systems. The nomenclature of plateletspecific alloantigens is based on a genetic name (human platelet antigen; HPA), numbering the antigens in chronological order of discovery and designating the high frequency antigens (a) and the low frequency antigens (b) (Newman, 1994). There are now eight well-defined, platelet-specific, alloantigen systems (HPA1-8) (Table 1).

AUTOANTIGENS Definition/Characteristics Platelets as lmmunogens. Under the proper condi-

tions, platelet surface antigens stimulate an immune response, leading to production of platelet autoantibodies and subsequent development of autoimmunemediated thrombocytopenia purpura. Autoimmunemediated thrombocytopenia purpura is classified on the basis of the specific autoantigen as neonatal alloimmune, isoimmune (posttransfusion) or autoimmune (and drug-induced) thrombocytopenia purpura (AITP) (Waters, 1992a).

PLATELET AUTOANTIGENS The main target antigens encountered in AITP appear to be GP IIb/IIIa, Ib/IX and V (Kiefel et al., 1992). The specific features of these complexes that make them so prevalent in platelet autoimmunity are yet to be elucidated. GPIa/IIa, GPIV and GPVI are also implicated as target autoantigens in AITP (MuellerEckhardt et al., 1990). Many drugs, including quinine, quinidine, apronal and sulfonamide cause platelet destruction both in vivo

635

and in vitro through an immunological mechanism (McFarland, 1993). Recent studies show that GPIIb/ IIIa and GPIb/IX are the main target antigens in both drug-induced (Christie, 1993) and viral-induced AITP (Kaplan et al., 1992); whereas, platelet factor 4 and heparin complexes are the autoantigens for heparininduced thrombocytopenia (Amiral et al., 1992).

Structure of the Major Platelet Antigens Platelet Glycoprotein IIb/IIla. GPIIb/IIIa (also termed c~IIb[33) is a dimeric glycoprotein with subunits of 125 and 95 kd, the products of separate but homologous genes. The subunits contain 15% carbohydrate with a high content of mannose residues, suggesting the presence of both N-linked and mannose oligosaccharide units. Restricted to platelets and cells with megakaryoblastic potential, GPIIb/IIIa is the most abundant member of the integrin family found in platelets ( 4 x 104 copies/human platelet) where they serve as receptors for several adhesive proteins. Binding of these proteins is mediated in part by the Arg-Gly-Asp sequence, inducing GPIIb/IIIa to form patches on the platelet surface. GPIIb/IIIa also links both the extracellular fibrin matrix and cell-surface fibrinogen to contractile proteins of the cell interior. The former mechanism allows platelet aggregation (induced by specific proteins such as fibrinogen and von Willebrand factor (Phillips et al., 1991), and the latter facilitates clot retraction. Like the alpha subunits of all integrins, GPIIb has putative divalent cation binding sites with sequence similarity to those of calmodulin. Removal of calcium from these sites by chelation causes a loss of GPIIb/IIIa receptor function and, at temperatures below 25~ causes dissociation of GPIib/IIIa into its individual subunits (Phillips et al., 1991). Many monoclonal antibodies against both the intact complex and its individual subunits are commercially available. Inherited deficiencies of GPIIb/IIIa (e.g., Glanzmann's/thrombasthenia) are characterized by the inability of platelets to bind adhesive proteins and subsequent failure to aggregate, leading to a life-long hemorrhagic diathesis (Glanzmann, 1918).

Platelet Glycoprotein Ib/IX. GPIb/IX, an adhesion protein receptor, is a noncovalent, heterotrimeric complex of GPIX (22 kd) and the disulfide-linked (135 kd) and 13 (25 kd) subunits of GPIb. All three polypeptides derive from a common ancestor and are members of a protein family characterized by a

636

leucine-rich motif (Fitzgerald and Phillips, 1989). The complex binds von Willebrand factor, an interaction that normally mediates the adhesion of platelets to damaged blood vessel walls and can initiate the activation of platelets in regions of high shear. The complex is also necessary for platelet aggregation induced by low concentrations of thrombin. When platelets are lysed in the absence of chelating agents such as EDTA, GPIb is converted into a water-soluble derivative, glycocalicin, by an intracellular calciumdependent protease (Fitzgerald and Phillips, 1989). Genetic deficiencies of the GPIb/IX complex (e.g., Bernard-Soulier syndrome) are characterized by defective platelet adhesion to the vascular subendothelium and decreased thrombin-induced platelet activation (Bernard and Soulier, 1948).

Platelet Glycoprotein V. Platelet glycoprotein V (GPV) is a major (--1.2 x 104 molecules/platelet), heavily glycosylated platelet membrane glycoprotein with an apparent molecular weight of 82 kd. Mature GPV is composed of 544 amino acids and consists of a single transmembrane domain, a short cytoplasmic domain and a large extracellular domain with eight potential N-glycosylation sites. The extracellular domain contains 15 tandem repeats of 24 amino acids with sequence similarity to GPIb~, but no hirudin-like sequence (Lanza et al., 1993). During thrombininduced platelet activation, thrombin cleaves a soluble 69 kd fragment from GPV near the carboxy terminus of the extracellular domain at a site with homology to the ~ chain of fibrinogen. Recent studies suggest that GPV can form a noncovalent interaction with the GPIb/IX complex (Fitzgerald and Phillips, 1989).

AUTOANTIBODIES In autoimmune-mediated thrombocytopenia purpura, platelets are targets for autoantibodies, leading to platelet activation and destruction (Ahmed et al., 1994; Tardio et al., 1993). Platelets sensitized by autoantibodies can cause hemorrhagic diathesis or thrombotic episodes, depending on the nature of the antibody class and specificity. All isotypes of immunoglobulins (Ig) are present in immune-mediated thrombocytopenia purpura at the following frequencies: IgG (92%), IgM (65%) and IgA (48%) (Ahmed et al., 1995). The IgG class has received the most attention, because it mediates the interaction between sensitized platelets and the reticulo-endothelial system

(Ahmed et al., 1994) and with IgE are the classes that transfers across the placenta. All IgG subclasses can be present, but IgG1 and IgG3 are the most frequent (Rosse et al., 1980). The specific Fab arms and Fc moeities of IgG autoantibodies cross-link platelets in vitro via antigenic sites and Fcy-RII, respectively, eventually leading to platelet activation, aggregation and local thrombi formation. Alternatively, autoantibodies can cause hemorrhage by inhibiting the binding of von Willebrand factor to the GPIb/IX complex, blocking the binding of platelets to the subendothelium and preventing formation of a platelet "plug" at the site of vascular injury. Activated or nonactivated platelets sensitized by autoantibodies can be destroyed through either of two main pathways, mediated by complement and Fc receptors, respectively.

Complement-Mediated Platelet Destruction Platelets express a receptor for C lq (a subunit of the C1 macromolecule of complement) capable of signal transduction and platelet activation upon binding to a C 1q-IgG-platelet complex. Activation of complement by immune complexes (platelet and autoantibody) leads to the deposition of C3b on the platelets and/or autoantibodies; C3b then mediates platelet lysis by the formation of the membrane attack complex C5b-9. Alternatively, C3b binds to CR2 (complement receptor 2) expressed on macrophages. Platelets are then phagocytized by RES cells (Ahmed et al., 1994).

Fc Receptor-Mediated Platelet Activation and/or Destruction Bound autoantibodies of the IgG class can activate platelets through an interaction with the Fc 7-RII expressed on human platelets. Alternatively, antibodies bound by specific Fab arms can cross-link the target platelet to macrophages expressing Fc receptors. In contrast, human platelets do not express Fc receptors for immunoglobulins of the IgM or IgA classes (Rosenfeld and Anderson, 1989).

CLINICAL UTILITY

Disease Associations Immune Thrombocytopenias. Neonatal Alloimmune Thrombocytopenia Purpura is caused by placental transfer of alloantibodies from mother to fetus fol-

lowing sensitization to fetal platelet antigens during pregnancy. Neonatal alloimmune thrombocytopenia purpura has a frequency between 1:2000 and 1:5000. In about one-half of all cases, neonatal alloimmune TP results from maternal alloimmunization against the HpA-la antigen, present on the platelets o f - 9 8 % of the general population. Sensitization against HpA-la occurs most often in women positive for HLA-DR3, DRw52 and to some extent DR6, DRw52. Maternal alloimmunization against HLA-A, HLA-B and HLA-C antigens occurs in 25--50% of all pregnancies, usually without influencing the fetal platelet count. Neonatal alloimmune TP usually occurs in a first-born infant, with subsequent infants likely to have neonatal alloimmune TP. Affected infants can be asymptomatic, but often they are born with extensive petechiae on the skin and mucosa. Gastrointestinal and urinary tract bleeding is common. Symptomatic infants usually have 0.5-2.5 x 104 platelets/~tL. The typing of maternal and paternal platelets for platelet alloantigens is helpful in evaluation of neonatal alloimmune TP (Table 1).

Posttransfusion Thrombocytopenic Purpura. Platelets transferred during blood transfusion carry alloantigens not present on autologous platelets, This triggers formation of platelet-specific alloantibodies and subsequent acute thrombocytopenia. As in neonatal alloimmune TP, the most common alloantigen in posttransfusion TP is HPA-la, occurring in >90% of cases. There are a number of possible mechanisms by which alloantibodies destroy autologus platelets. Alloantibodies can form immune complexes with transfused platelets via Fc 7-RII, leading to activation and destruction of autologous platelets. Alternatively, platelet fragments and/or soluble antigens present in the transfused blood can trigger cross-reactive antibodies to autologous platelets, or soluble alloantigens can adhere to the platelet nonspecifically, leading to autologous platelet activation by subsequently formed alloantibodies. Autoimmune Thrombocytopenias (AITP). Patients who fulfill all of the following criteria are diagnosed as having AITP: (1) increased platelet destruction manifested by a platelet count of less than 2 x 104/luL; (2) increased number of megakaryocytes in the bone marrow; (3) giant platelets present in peripheral blood smears; (4) presence of platelet antibodies and platelet-associated Ig; (5) absence of splenomegaly; and (6) exclusion of drug-induced thrombocytopenia purpura. 637

Table 1. Platelet-Specific Alloantigens

Allelic Form

Gene Frequency

Serologic Designation

GPIIIaLeu33

0.85

HPA-la (PIA1)

GPIIIapro33

0.15

HPA- 1b (PIA2)

GPIbT~145

0.93

HPA-2a (Kob)

GPIbMet145

0.07

HPA-2b (Ko a)

GPIIbiie843

0.61

HPA-3a (Baka)

GPIIbser843

0.39

HPA-3b (Bakb)

GPIIIaArgl43

0.85

HPA-4a (Pena)

GPIIIaaln143

<0.01

HPA-4b (Penb)

GPIaGluSO5

0.89

HPA-5a (Bra)

GPIaLysSO5

0.11

HPA-5b (Brb)

GPIIIaArg489

0.85

HPA-6a (Ca]Tub)

GPIIIaGln489

<0.01

HPA-6b (Ca/Tua)

GPIIIapro407

0.85

HPA-7a (Mob)

GPIIIaAla407

<0.01

HPA-7b (Mo a)

GPIIIaArg636

0.85

HPA-8a (Srb)

GPIIIacys636

<0.01

HPA-8b (Sra)

Chronic vs. Acute AITP. Patients with acute AITP generally present with platelet counts well under 2 x 104/pL. The acute variant presents in children younger than 10 years of age in most cases, and the malefemale ratio is 1:1. Onset tends to be postinfectious. The highest levels of platelet-associated IgG are found in patients with acute AITP; there are no known HLA associations. In contrast, a patient whose platelet count is persistently low (2--7.5 x 104/~tl) for six months or longer is defined as having chronic AITP. Chronic AITP is usually found in children older than ten years, and females predominate 3:1. Subjects with IgA deficiency seems to be at higher risk for the development of chronic AITP (Kurtzberg and Stochman, 1994). Only moderately high amounts of plateletassociated IgG are present, and HLA associations with A3B7 or A26W16 may be found in patients with chronic AITP. Chronic AITP presents with more insidious signs and symptoms than the acute variety. Idiopathic vs. Secondary Thrombocytopenia. The diagnosis of idiopathic (primary or essential) thrombocytopenia purpura is made only after the exclusion of causes of secondary AITP usually an autoimmune

638

disease such as SLE. Idiopathic TP in children is more common than the adult disease (40% of all patients with idiopathic TP are <10 years old), and both sexes are equally affected (Kurtzberg and Stockman, 1994). Onset is typically abrupt, with severe thrombocytopenia and spontaneous, permanent remission occurring in about 80% of children (Waters, 1992b). In contrast, adult idiopathic TP is a clinically distinct disorder. Seventy-two percent of patients over 10 years old are women; 70% of these women are <40 years old (Kurtzberg and Stockman, 1994). Evans' syndrome (acquired hemolytic anemia and thrombocytopenia), an uncommon condition, is characterized by autoantibodies against red cells and platelets, with resultant autoimmune hemolytic anemia and AITP occurring simultaneously or sequentially. The target antigen in autoimmune hymolytic anemia is usually the erythrocyte Rh antigen. The specificity of the platelet autoantibodies remains to be identified (Ahmed et al., 1994). Secondary AITP syndrome resembles idiopathic TP but bears a clear and identifiable relationship to a primary disorder, usually an autoimmune disease or malignancy. Secondary AITP constitutes the majority of the cases seen in clinical practice.

HIV-Related Autoimmune Thrombocytopenia Purpura. Thrombocytopenia occurs in 5--10% of asymptomatic patients infected with HIV and in 25--45% of patients with AIDS. HIV-induced AITP shares laboratory and clinical features with classic idiopathic TP. However, the amounts of plateletassociated Ig and complement are three to four times higher than those in patients with idiopathic TP, and the concentrations of circulating immune complexes are three to seven times higher than idiopathic TP (Karpatkin, 1990). Sera from patients with HIVrelated autoimmune thrombocytopenia purpura react with normal platelets but not with thrombasthenic platelets deficient in glycoprotein IIb/IIIa. In HIVrelated autoimmune thrombocytopenia purpura there is no direct correlation between platelet-associated Ig and the severity of thrombocytopenia. Indeed, platelet antibodies are found in patients with AIDS who have a normal platelet count. There is conflicting evidence about the involvement of immune complexes, HIV autoantibodies, anti-idiotypes, platelet antibodies and complement in the pathogenesis of HIV-related autoimmune thrombocytopenia purpura (Ahmed et al., 1994). Systemic Lupus Erythematosus-Related Autoimmune Thrombocytopenia Purpura. Mild thrombocytopenia occurs in about one-third of patients with systemic lupus erythematosus; severe thrombocytopenia with purpura has a frequency of only 5%. The nature of the target antigen on the platelet membrane is still a matter of debate (Ahmed et al., 1994). AITP in Pregnancy. AITP in pregnancy is a maternal disorder; fetal consequences occur only rarely. Alloimmune thrombocytopenia purpura, on the other hand, is a fetal platelet disorder with no maternal significance. Chronic autoimmune thrombocytopenia purpura usually occurs in the first half of pregnancy and is one of the most common autoimmune disorders accompanying pregnancy. Autoimmune thrombocytopenia purpura usually occurs as an isolated disorder with no other hematological abnormality (Pillai, 1993). AITP in Antiphospholipid Syndrome. AITP is a frequent complication of antiphospholipid syndrome and typically is associated with high levels of anticardiolipin antibodies. Antiphospholipid syndrome usually is associated with thromboembolic phenomena (Harris et al., 1985). Although antiphospholipid

antibodies cause thrombosis by inducing platelet activation and aggregation, it is notable that the target phospholipids are located on the platelet inner membrane. Thus, before phospholipids are exposed to the antibodies, there must be a change in the platelet membrane environment (Ahmed et al., 1994).

Methods of Detection The assays developed to aid in the diagnosis of immune-mediated thrombocytopenia purpura have advantages and disadvantages (Table 2). Presently, no single assay offers sufficient sensitivity and specificity for immune-mediated thrombocytopenia purpura testing to be performed on a routine basis (Ahmed et al., 1994).

Platelet-Associated Immunoglobulins Increased amounts of platelet-associated Ig are a major feature of immune-mediated thrombocytopenia purpura. Although their clinical significance is not well defined, tests for platelet-associated Ig are commonly used as diagnostic criteria for autoimmune thrombocytopenia purpura. Whether such tests are more useful if surface or total platelet-associated IgG is measured remains a subject of controversy (Sinha and Kelton, 1990). There is a wide normal range for total platelet IgG ( 1 0 2 - > 104 IgG molecules/platelet, depending on the assay used), and on the other hand, platelets are unstable in vitro (Sinha and Kelton, 1990). Also, while detection of platelet-associated Ig is sensitive for autoimmune thrombocytopenia purpura, it lacks specificity. False-positive results are obtained in numerous disorders other than autoimmune thrombocytopenia purpura, including thrombocytopenia that are apparently nonimmune in nature. Thus, although consistent with the diagnosis of autoimmune thrombocytopenia purpura, a positive test for platelet-associated Ig cannot be considered confirmatory.

Specific Assays for Platelet Antibodies The lack of specificity of assays for platelet-associated Ig led to the development of assays to identify and quantitate platelet autoantibodies that react with a specific target alloantigen, isoantigen or autoantigen (Table 2). Assays for serum autoantibodies including immunoblotting, immunoprecipitation, antigen-capture assays and monoclonal antibody immobilization of

639

Table 2. Diagnostic Tests for ITP

Assay

A dvantag es

Disadvantages

Autoantibody-Mediated Response

Simple; uses patient serum

Low sensitivity/specificity; qualitative donorresponse variablility

Platelet-associated Ig

Simple

Platelet instability; wide normal range; low clinincal specificity

Direct and Indirect Binding

Simple; uses monoclonal antibodies; can be Platelet instability; difficult to calibrate; low performed w/different methodologies (RIA, clinical specificity EIA and Flow cytometry)

Immunoblotting (Western Blotting)

Uses patient serum; sensitive; determines the molecular weight of the target antigen

Time-consuming; qualitative; loss of antigen epitopes after denaturation

Immunoprecipitation

Identifies target antigens; sensitive; semiquantitative

Time-consuming; requires radioisotopes; variability of platelet labeling efficiency

Antigen-Capture

Sensitive; specific; simple; rapid; suitable Semiquantitative; limited availability o f for routine testing; detects serum or platelet monoclonal antibodies against the target antibodies antigen

Monoclonal Antibody Sensitive; specific; simple; suitable for Immobilization of Platelet Antigen routine testing; rapid; detects serum and platelet antibodies

Semiquantitative; epitope masking by monoclonal antibodies; interference by human antimouse antibodies; limited availability of antigen-specific monoclonal antibodies

Serologic Allotyping

Sensitive; specific; simple

Qualitative; limited avilability of alloantibodies.

PCR Allotyping

Sensitive; specific; simple; rapid

Used only for known alloantigens

platelet antigen (MAIPA) are used to determine antibody specificity. Recently introduced molecular techniques such as PCR promise to offer enhanced specificity and sensitivity for both neonatal autoimmune TP and PTP.

CONCLUSION Although our knowledge of immune-mediated thrombocytopenia purpura has increased greatly over the past few decades, its specific causes remain unknown.

REFERENCES

Ahmed AE, Peter JB, Shoenfeld Y. Autoimmune thrompocytopenia purpura: Pathogenesis, diagnosis and management. Clin Immunother 1994;1:348-357. Ahmed AEE, Agopian MS, Peter JB. Immmaoglobulin isotype frequency of platelet-specific and platelet-associated antibodies in autoimmune thrombocytopenia [Abstract]. Proceedings of the Clinical Research Meetings. San Diego, 1995. Amiral J, Bridgey F, Dreyfus M, Visocc AM, Fressinaud E, Wolf M, Meyer D. Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia. Thromb Haematol 1992;68:95--96. 640

Research has focused on identifying and measuring the specificity of the autoantibodies involved in autoimmune thrombocytopenia purpura and neonatal autoimmune TP, as the induction of an autoantibody response against platelet antigens is far from understood. Future studies will most likely define the structure-function relationships of platelet surface antigens, which should clarify the underlying mechanisms involved in the pathophysiology of the disease. There is a need for a simple, sensitive and specific diagnostic assay for immune-mediated thrombocytopenia purpura.

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of platelet alloantigen systems. Trans Med Rev 1990;7:93-109. Newman PJ. Nomenclature of human platelet alloantigens: a problem with the HPA system? Blood 1994;6:1447--1451. Phillips DR, Chavo IF, Scarborough RM. GpIIb/IIa: The responsive integrin. Cell 1991;65:359-392. Pillai M. Platelets and pregnancy. Br J Obstet Gynaecol 1993;100:201--204. Rosenfeld SI, Anderson CL. Fc receptors of human platelets. In: Kunicke TJ, George JN, eds. Platelet Immunology. Philadelphia: Lippincott, 1989:337. Rosse WF, Adams JP, Yount WJ. Subclasses of IgG antibodies in immune thrombocytopenia purpura (ITP). Br J Haematol 1980;46:109-114. Sinha RK, Kelton JG. Current controversies concerning the measurement of platelet-associated IgG. Trans Med Rev 1990;4:121--135. Tardio DJ, McFarland JA, Gonzalez MF. Immune thrombocytopenia purpura: current concepts. J General Int Med 1993;8:160--163. Waters AH. Platelets as immunogens. Blood Coag and Fibrinolysis 1992a;3:637--641. Waters AH. Autoimmune thrombocytopenia: clinical aspects. Semin Hematol 1992b;29:18-25.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PM-Scl AUTOANTIBODIES Chester V. Oddis, M.D. a and Ira N. Targoff, M.D. b

aUniversity of Pittsburgh, Department of Medicine, Division of Rheumatology and Clinical Immunology, Pittsburgh, PA 15213-3221; and bUniversity of Oklahoma Health Sciences Center, Oklahoma Medical Research Foundation, Department of Arthritis and Immunology, Oklahoma City, OK 73104, USA

HISTORICAL NOTES Autoantibodies that recognize intracellular antigens are found in up to 90% of patients with polymyositisdermatomyositis (PM-DM). Of the numerous defined specificities, each individual antibody is found in only a small proportion of patients. One of these, anti-PMScl was described in 1977 as a myositis-associated autoantibody. It was called anti-PM-1 antibody when first detected as a weak precipitation reaction by immunodiffusion in 61% of 28 PM-DM patients, including seven of eight with myositis in overlap with systemic sclerosis (SSc) (Wolfe et al., 1977). Subsequent studies showed that more than one antibody was being detected and by exchange of sera between laboratories, a unique specificity, labeled anti-PM-Scl, was defined (Reichlin et al., 1984). This was found in much lower frequency, 8% of 168 myositis patients, but up to 50% of patients with the antibody had an overlap of myositis and SSc, leading to the name of the antibody (Reichlin and Arnett, 1984). Subsequently identified in pure PM-DM and in SSc without myositis, anti-PM-Scl and its associated antigen were extensively studied over the past several years. In addition to its molecular characterization, many clinical and immunogenetic associations are now defined.

THE AUTOANTIGEN Definition The PM-Scl autoantigen is a complex of 11--16 proteins ranging from 20 to 110 kd in reported molecular weight as determined by PAGE (Gelpi et

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al., 1990; Reimer et al., 1986; Oddis et al., 1992) (Figure 1). No RNA or DNA is specifically precipitated by anti-PM-Scl. Known only by its diseaseassociation name, the antigen(s) and its cellular function are unknown. Immunoprecipitation from 35S-methionine-labeled HeLa cell extracts with anti-PM-Scl serum demonstrates numerous antigenic proteins; the most prominent band is seen at 100 kd (Figure 1). The complex also includes a protein with migration variously reported between 68 and 80 kd (usually called 70 or 75), multiple proteins between 20 and 39 kd and possibly other proteins. Phosphorylation of the 70-75 kd protein, the 20 kd protein and possibly the 39 kd protein is reported (Gelpi et al., 1990; Reimer et al., 1986). Of anti-PM-Scl sera (defined by reaction in immunoprecipitation), 90-98% react with the 100 kd protein in immunoblot studies using HeLa PM-Scl antigen (Ge et al., 1994) (Figure 2), while, 50--63% react with the 70--75 kd protein, and 12% react with the 37 kd protein (Ge et al., 1994). No direct immunoblot reactivity with other components of the PMScl complex is known. The 100 and 75 kd proteins are immunologically distinct by immunoblot, recognized independently and without cross-reactivity (Alderuccio et al., 1991; Ge et al., 1992). Recombinant 100 kd protein is equally reactive with denatured natural HeLa 100 kd protein. There is nearly complete concordance between reaction of the recombinant and HeLa PM-Scl antigens with anti-PMScl sera (Ge et al., 1994). The few anti-PM-Scl sera that fail to react with the recombinant 100 kd protein usually fail to react with any HeLa antigen component by blot. Although absorption of anti-PM-Scl sera with recombinant 100 kd protein eliminates reactivity with

Figure 1. Autoradiogram of a 10% polyacrylamidegel fractionation of 35S-methionine-labeledproteins present in protein A-Sepharosefacilitated immunoprecipitates from a total HeLa cell extract, using 9 serum samples from patients with anti-PM-Scl. These antisera consistently immunoprecipitate 11 proteins with molecular weights of 20-110 kd (1). The protein bands near 50 kd (all lanes) are nonspecific precipitates (probably actin), as indicated by their presence in normal human serum (NS). Lane 1 (Mr) contains anti-PM-Scl reference serum; molecular weights are shown to the left.

HeLa 100 kd antigen by immunoblot, absorption with E. coli extract containing bacterially expressed recombinant 100 kd protein does not eliminate reactivity of anti-PM-Scl by indirect immunofluorescence or with the 100 kd protein by immunoprecipitation (Ge et al., 1994). This suggests the existence of additional, apparently conformational epitopes, that may be on the 100 kd, the 75 kd or other antigenic peptides. Nevertheless, the bacterially expressed recombinant PM-Scl antigen will fail to detect only 2--10% of antiPM-Scl sera that are detectable by native, intact (nondenatured) antigen. Sera that exclusively react with the 75 kd protein (by blot) are very rare.

Origin/Sources Calf thymus is commonly used as a source of PM-Scl

antigen for immunodiffusion testing. The antigen is a very small proportion of cell protein, and the extract must be highly concentrated for detection by immunodiffusion (Targoff and Reichlin, 1985; Treadwell et al., 1984). Anti-PM-Scl are detected by indirect immunofluorescence in all tissues and cell lines tested (Reimer et al., 1986) including human (e.g., HeLa, human lymphocytes, MOLT-4 cells) and other mammalian forms (mouse, rat and rabbit liver, mouse kidney, etc.). The presence of PM-Scl in fish, turtle and duck cells suggests that the reactive target protein is conserved and vital to cellular function (Reimer et al., 1986). Commercially available thymus tissue or extracts are good sources for detection of the anti-PMScl by immunodiffusion if prepared in sufficient concentration. There are no known commercial sources for purified PM-Scl antigen.

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Figure 2. Immunoblot with anti-PM-Scl sera, normal sera (NL) and anti-PM-Scl-negative myositis control sera ("Others"). The antigen anti-PM-Scl immunoprecipitates from HeLa cell extract, was prepared with a single serum (that blotted the 100 and 70 kd bands), electrophoresed in 10% SDS-PAGE. All anti-PM-Scl sera shown (1-32) stain the 100 kd band, but only sera 20-32 stain the 70-75 kd band. Sera 28-31 also stain the 37 kd band. All control sera are negative. (From: Ge et al., 1992. Reproduced with permission.) Methods of Purification

All anti-PM-Scl sera examined react intensely with the nucleolus and occasionally the nucleus by indirect immunofluorescence (Figure 3). Affinity-purification of anti-PM-Scl using the cloned 100 kd protein reacts in a similar pattern (Ge et al., 1992). Nucleolar isolation studies demonstrate enrichment of the antigen in nucleolar extract (Targoff and Reichlin, 1985). Immunoelectron microscopic detection of the antigen in the granular component of the nucleolus, suggests a role in ribosomal maturation and/or transport (Reimer et al., 1986). Furthermore, drugs that inhibit ribosomal RNA synthesis have a marked effect on the expression of the PM-Scl antigen in that actinomycin-D-treated cells have significantly reduced immunofluorescence staining by anti-PM-Scl (Reimer et al., 1986) with migration of staining to the nucleoplasm (Gelpi et al., 1990).

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Purification of the PM-Scl antigen is difficult due to its low concentration. The antigen can be enriched for easier detection by isolation of nuclei or nucleoli (Gelpi et al., 1990; Targoff and Reichlin, 1985; Treadwell et al., 1984), and/or by Sepharose 6B chromatography and ammonium sulfate precipitation (Treadwell et al., 1984). As with other autoantigens, purification can be achieved by immunoaffinity methods, e.g., immunoprecipitation from HeLa cell extract for use in immunoblotting (Ge et al., 1994). Purified, concentrated antigens give higher sensitivity for detection of the antibody and facilitate identification of different bands of the PM-Scl complex. Of the two cDNAs encoding the 100 kd protein (Ge et al., 1994; Bluthner and Bautz, 1992), the thymocyte cDNA is 2,739 bp (2580 coding), encodes 860 amino acids and predicts a 98.1 kd protein. The HeLa cDNA is identical except that it contains a 75 bp segment (after amino acid 695) not present in the

Figure 3. Indirect immunofluorescence on HEp-2 cells using anti-PM-Scl serum diluted 1:100. Anti-PM-Scl sera stain the nucleolus intensely, and also show some staining of the nucleoplasm. (Ge et al., 1992. Reproduced with permission.) thymocyte cDNA. This does not appear to affect antigenicity. The sequence has thus far yielded little insight into function. Immunoblot studies against expressed products of restriction fragments (Ge et al., 1994) and PCR-derived fragments (Bluthner and Bautz, 1992) show that almost all anti-PM-Scl sera react with the amino terminal fragment of the 100 kd protein. This reactive fragment between amino acids 156 and 312 (Ge et al., 1994) contains within it a highly hydrophilic area suggesting that it is on the surface of the molecule. Within the amino terminal fragment the major site of reactivity has been further localized and some sera also react with additional areas of the fragment (Ge et al., 1993). A second fragment near the center of the 100 kd protein (between amino acids 507 and 749) also reacts with most anti-PM-Scl sera (Ge et al., 1994; Bluthner and Bautz, 1992), but is not further localized.

Sequence/Information A 1.6 kd, full-length human cDNA encoding the PM-

Scl component that migrates at about 75 kd was isolated (Alderuccio et al., 1991). The predicted protein is only 355 amino acids and 39.19 kd, but migrates aberrantly, possibly due to a highly acidic region. There is no sequence similarity with the 100 kd protein. The initial screening of a MOLT-4 expression library with anti-PM-Scl sera identified a partial cDNA (representing the 3' portion) encoding 138 amino acids that must contain at least one epitope; further epitope localization is not yet available.

AUTOANTIBODIES

Terminology/Pathogenetic Role The preferred name of the autoantibody is anti-PMScl (Reichlin et al., 1984) although the designation, anti-PM-1 is occasionally used. There is no current evidence that anti-PM-Scl play a pathogenic role in connective tissue diseases (e.g., myositis and scleroderma) and the fact that the antibody does not vary in

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titer with disease activity also speaks against a pathogenic role. There is strong evidence for an antigendirected, T-cell-mediated attack on muscle fibers in PM but little support for a direct role for antibody (Engel et al., 1990). Complement-mediated vascular injury is well documented in DM, but the role of autoantibody is undefined, and the specificity of such antibodies, if they exist, is not known (Kissel et al., 1986). No animal model of spontaneous production of anti-PM-Scl is known. Those animal models of myositis induced by viruses that have been tested for autoantibodies (including EMC-induced disease) do not produce anti-PM-Scl. Rabbits immunized with the recombinant 75 or 100 kd proteins of the PM-Scl complex produced antibodies to them (Alderuccio et al., 1991; Bluthner and Bautz, 1992), but illness in the animals was not reported.

Genetics Anti-PM-Scl is among the most closely linked of all autoantibodies to a particular MHC Class II antigen, DR3. In two separate studies (Genth et al., 1990; Marguerie et al., 1992) all 34 patients with anti-PMScl were HLA-DR3-positive. Six of 12 from one study were DR3/4 heterozygotes (Genth et al., 1990). This significantly increased DR3/4 heterozygosity suggests that both HLA haplotypes are involved in generation of the immune response against PM-Scl antigens. In another study of 23 patients with antiPM-Scl, 20 were tested for HLA-DR and DQ antigens (Oddis et al., 1992). Fifteen (75%) of the 20 patients were positive for HLA-DR3 (22% in a local Caucasian control population: relative risk [RR] 10.6, p < 0.001) and three were homozygous for DR3. The five HLA-DR3-negative patients with anti-PM-Scl were the first to be described. Of the DQ specificities, DQw2 was found in 17 of 20 (85%) patients with anti-PM-Scl compared with 40% of controls (RR = 8.5, p < 0.001). DQw2 may therefore demonstrate a stronger immunogenetic association with the anti-PMScl immune response than DR3. However, only two of the five DR3-negative patients had the DQw2 allele. Further analysis of the DNA encoding DQ~ and DQ~ in 10 of the 23 patients including four who were negative for HLA-DR3 revealed DQBI*0201 in 8 of the 10 anti-PM-Scl patients (Oddis et al., 1992). Thus, there are n__o_ounique alleles common to the DR3positive and DR3-negative anti-PM-Scl-positive patients (Oddis et al., 1992). Of class I antigen

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associations determined in 12 anti-PM-Scl-positive patients, eight (67%) were positive for HLA-A1 (RR = 6.1), and 11 (93%) were positive for HLA-B8 (RR = 47.5) (Genth et al., 1990). Myositis is extremely rare in families; indeed, no family has been described in which more than one member had anti-PM-Scl. A preliminary report has described a pair of identical twins in which one of the 53 year old women had scleroderma with anti-PM-Scl and symptoms for >10 years; whereas, the other was healthy and antibody-negative (McHugh et al., 1994). Thus, although genetic background is clearly important for production of anti-PM-Scl, other factors are certainly involved. No information is available regarding V H and V L gene usage for anti-PM-Scl.

Pathogenetic Mechanism The reported high frequency of reaction with the 100 kd polypeptide strongly suggests that this component of the PM-Scl antigen is the primary immunogen (Ge et al., 1994). If molecular mimicry were the mechanism for generation of these antibodies, the almost uniform reactivity with the amino terminal epitope suggests this as a candidate site for cross-reactivity. However, no areas on the 100 kd (or 75 kd) PM-Scl proteins have been proposed as potential sites of molecular mimicry with infective agents by sequence analysis, either in epitope areas or in the molecule as a whole.

Factors in Pathogenicity Studies of the isotypes of anti-PM-Scl are not available, but IgG antibodies are primarily produced. Idiotypes and avidity have not been studied. Anti-PMScl is found in the earliest available serum from all patients in which it is found, but the time course of appearance of the different isotypes has not been studied.

Methods of Detection The usual screening test used for anti-PM-Scl is ouchterlony double immunodiffusion. Most sera that are positive for the antibody by other techniques will be positive by immunodiffusion, despite the low quantitative sensitivity of the test. Calf or rabbit thymus extract was used in several studies as antigen for immunodiffusion (Reichlin et al., 1984; Reichlin and Arnett, 1984; Genth et al., 1990; Blaszczyk et al., 1990).

Immunoprecipitation for protein analysis using 35Smethionine-labeled human cell lines is a specific and effective method of antibody detection. The pattern of 11 bands is easily recognized and, because intact native antigen is used, the technique is sensitive and detects all sera with the antibody. The 100 kd protein is usually intense and easily recognized but the lowmolecular weight bands can be overlooked. Sera with anti-PM-Scl detected by protein Aassisted immunoprecipitation (usually with 35S_labeled cultured cell extracts), but missed by testing against recombinant antigen (presumably because of lack of conformational epitopes) are usually detectable by immunodiffusion methods. Although some anti-PMScl-positive sera can be missed by immunodiffusion due to low antigen amounts in the extract, immunodiffusion is nearly as sensitive as immunoprecipitation when antigen-rich extracts are utilized. Thus, immunodiffusion is the recommended method for routine screening for the anti-PM-Scl antibody. Immunoblotting detects most patients with the antiPM-Scl but a small percentage (5--10%) exclusively reactive with conformational epitopes will be missed. A preliminary report of an ELISA using intact recombinant PM-Scl 100 kd antigen for detection of antiPM-Scl (Ge et al., 1993) was able to quantitate the amount of antibody more easily and with high sensitivity and specificity, but was only slightly more effective than a fragment containing the major Nterminal epitope. A very small percentage of low-level false-positive results was encountered. The recombinant antigen is a very convenient source, but the use of any currently available recombinant antigen in ELISA has similar limitations to immunoblotting, i.e., conformational epitopes might not be detected. The ELISA may be valuable as a rapid and convenient screening method with the positives being confirmed by double immunodiffusion. However, a small per-

centage of anti-PM-Scl-positive sera may be missed by this method. Ouchterlony is an effective technique to detect anti-PM-Scl in some sera not reactive in immunoblotting while ELISA is rapid, convenient and quantitatively sensitive. Anti-PM-Scl produce intense, homogeneous nucleolar staining with weaker nuclear staining by indirect immunofluorescence. Although some sera with homogeneous nucleolar staining do not have antiPM-Scl (Blaszczyk et al., 1990), this pattern, when accompanied by nuclear staining, should raise suspicion for the presence of anti-PM-Scl.

CLINICAL UTILITY Disease Associations

Among all patients with connective tissue disease, anti-PM-Scl are relatively rare, being identified in only 8% of patients with myositis (Reichlin and Arnett, 1984) and approximately 3% of persons with SSc (Reimer et al., 1988). Although detected in some patients with myositis or SSc alone (Reichlin et al., 1984; Oddis et al., 1992; Genth et al., 1990), approximately 50% of anti-PM-Scl patients have the myositis/scleroderma overlap syndrome (Oddis et al., . 1992); only 25% of patients with this specific overlap have anti-PM-Scl. In one large series of 617 patients with various connective tissue diseases screened for the presence of anti-PM-Scl, 23 patients were positive; anti-PM-Scl was detected in 24% of myositis-scleroderma overlap patients and in 0 - 5 % of those with other diagnoses (Table 1). One patient with anti-PMScl had scleroderma in overlap with rheumatoid arthritis (RA) and another had DM in overlap with RA. The reported frequencies of anti-PM-Scl positivity among scleroderma and myositis patients will vary

Table 1. Frequency of Anti-PM-Scl in Patients with Connective Tissue Diseases (adapted from Oddis et al., 1992) Diagnostic Group

Number (% with anti-PM-Scl Antibodies)

Myositis-scleroderma overlap

10/41 (24)

PM-DM only

5/105 (5)

Scleroderma only

6/359 (2)

Other overlap syndromes

2/47 (4)

Primary Raynaud's syndrome

0/64

Total

23/617 (4)

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depending on the referral pattern of the institution surveyed. As seen in a population of connective tissue diseases (including cohorts of pure myositis), detection of anti-PM-Scl antibodies has a low diagnostic sensitivity, but substantial disease association in that nearly all anti-PM-Scl-positive patients have PM-DM, SSc or an overlap of these two major connective tissue diseases. The predictive value depends on the particular assay used for antibody detection and the circumstances of its application. For example, if the ELISA is used for population screening, then the positive predictive value (PPV) would be very low due to rarity of both the antibody and the disease and the occurrence of the occasional false-positive test result. Conversely, if immunodiffusion or immunoprecipitation techniques were employed, then the false-positive rate would diminish and diagnostic sensitivity and PPV would increase. Similarly, use of the antibody screen in patients with proximal weakness and an elevated creatine kinase (CK) would raise the PPV further for PM-DM but probably would not provide clinically useful information. Polymyositis and DM occur about equally in all patients with anti-PM-Scl but limited cutaneous SSc is considerably more common (80%) than diffuse cutaneous disease in antibody-positive patients. As seen with other autoantibodies, anti-PM-Scl are much more frequent in women than in men. These antibodies are found predominantly in Caucasians as only five of 67 patients in three combined series were nonCaucasians (Oddis et al., 1992; Genth et al.,1990; Marguerie et al., 1992). Anti-PM-Scl have not been detected in large groups of Japanese myositis or

overlap patients (Hirakata et al., 1992); interestingly, HLA-DR3 is rare in the Japanese population. Anti-PM-Scl is one of the few connective tissue disease-associated autoantibodies frequently reported in children. Five of 23 patients in one series were children and three of the five had an overlap of myositis with systemic sclerosis and limited skin thickness (Oddis et al., 1992). A larger study of 14 children with anti-PM-Scl classified the patients as having "scleromyositis" (Blaszczyk et al., 1991). In the majority of these children, the disease began with predominantly articular symptoms before 10 years of age and certain characteristic features of systemic sclerosis such as digital pitting scars and ulcers, acroosteolysis, induration of the hands and contractures of the fingers were lacking. Also, muscle weakness was relatively mild and the cutaneous features of DM were only intermittently present. Therapy was less aggressive than for typical childhood scleroderma or childhood DM, and the course of disease in all 14 children was chronic but benign. No other antibodies were detected in these 14 patients, and repeated serologic analysis over several years of follow-up did not demonstrate any relationship of PM-Scl antibody titers to disease severity or course, In addition, anti-PM-Scl did not disappear from the serum in any patient, even if the disease was clinically quiescent. This overlap syndrome of mild scleroderma and myositis in childhood seems to be the most common scleroderma-like illness seen in the pediatric population (Blaszczyk et al., 1991). Adults with anti-PM-Scl can present at any age with a variety of rheumatic complaints. Among the

Table 2. Clinical Features in Patients with Anti-PM-Scl Organ System Involvement

Oddis et al. (n = 23)

Myositis

16 (70)

Arthritis

Marguerie et al. (n = 32)

Blasyczyket al. (n = 27)

Total (%) (n = 94)

6 (50)

28 (88)

27 (100)

77 (82)

19 (83)

7 (58)

31 (97)

27 (100)

84 (89)

Raynaud's phenomenon

15 (65)

11 (92)

32 (100)

16 (59)

74 (79)

Esophageal hypomotility

9 (39)

4 (33)

25 (78)

3 (11)

32 (45)

Interstitial lung disease

7 (30)

5 (42)

25 (78)

4 (15)

41 (44)

Cardiac disease

0

0

Renal disease

0

w0

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Genth et al. (n = 12)

0 (3)

3 (11)

3/94 (3)

0

~ (1)

clinical features in 94 anti-PM-Scl-positive patients reported from four different institutions (Oddis et al., 1992; Genth et al., 1990; Marguerie et al., 1992; Blaszczyk et al., 1990), myositis, defined as proximal muscle weakness and CK elevation was noted in 77 (82%) of 94 patients (Table 2). Several other patients had one but not both of these findings. Arthritis or inflammatory arthralgias at some time during the disease course were observed in 84 (89%) of 94 patients. Joint symptoms appear to be more common in these persons than in those with myositis or scleroderma alone. The arthritis principally involves the small joints of the hand and feet and erosive changes may occur as well (Marguerie et al., 1992). Raynaud's phenomenon is frequently reported, but internal organ involvement is variable. Gastrointestinal complications when present are generally seen in patients with systemic sclerosis and include lower dysphagia with associated distal esophageal hypoperistalsis and reflux esophagitis. Significant cardiac disease is distinctly unusual. Pulmonary involvement in patients with anti-PM-Scl usually takes the form of interstitial lung disease with associated bilateral basilar fibrosis on chest radiograph. Severe respiratory complications are unusual, but one patient developed a sudden decline in pulmonary function with associated pulmonary hypertension (Marguerie et al., 1992). Renal involvement is uncommon or limited to minimal proteinuria, but one case of "scleroderma renal crisis" with minimal skin thickening was described (Zwettler et al., 1993). The classic cutaneous lesions of DM are seen in patients with this myositis subset. The frequency of soft tissue calcification may be increased in patients with anti-PM-Scl antibody compared to myositis, scleroderma and other overlap populations without the antibody (Oddis et al., 1992). "Mechanic' s hands" are classically associated with the Jo-1 antibodies and other aminoacyl-tRNA synthetase autoantibodies, but this unusual finding was observed in 26% of one anti-PM-Scl-positive series (Oddis et al., 1992). Generally, anti-PM-Scl are detected on initial presentation and persist during subsequent follow-up except for a man whose anti-PM-Scl became undetectable by immunodiffusion and immunofluorescence over 4--5 years while taking D-penicillamine (Targoff, unpublished). This occurred in association with complete remission of sclerodermatous and myopathic features. In most patients, however, the anti-PM-Scl remain serologically detectable and the titer of antiPM-Scl varies little with treatment. In particular, no

therapeutic intervention such as immunosuppressive therapy, anticytokines, intravenous gamma globulins or plasmapheresis are effective in eliminating the antibodies or for treating patientS for the clinical manifestations characteristic of the antibodies. All patients with anti-PM-Scl demonstrate a nucleolar staining pattern on routine antinuclear antibody testing. Anti-dsDNA antibodies are extremely rare in these patients (Marguerie et al., 1992) and no other autoantibodies against extractable nuclear antigens (e.g., anti-U1 RNP) are detected (Oddis et al., 1992; Genth et al., 1990; Marguerie et al., 1992). No reports of both anti-PM-Scl and anti-Jo-1 or any other anti-aminoacyl-tRNA synthetase autoantibody have been published, and no SSc-specific autoantibodies are detected in these patients. Considering about 200 patients with anti-PM-Scl have been studied, and considering the frequency of other autoantibodies in selected myositis and scleroderma patients, the absence of anticentromere, anti-Scl-70 (antitopoisomerase), or antisynthetases in any anti-PM-Scl patient clearly indicates that this antibody subset is a discrete subgroup among myositis and scleroderma patients. The outcome of patients with anti-PM-Scl is quite favorable (Oddis et al., 1992; Marguerie et al., 1992; Blaszczyk et al., 1990). In one series, 60% (19/32) were receiving no immunosuppressive agents or were taking prednisolone <7.5 mg daily after a median follow-up of 8 years (Marguerie et al., 1992). Varying degrees of disability are observed, but inadequate data are available to accurately judge morbidity. A series of 23 patients had a cumulative survival rate of 100% at 5 and 10 years after the initial diagnosis of connective tissue disease (Oddis et al., 1992). Most patients with anti-PM-Scl die from nonconnective tissue disease-related problems.

CONCLUSION Anti-PM-Scl represent an uncommon serum autoantibody in patients with connective tissue disease which identify a subset of patients with myositis in overlap with systemic sclerosis (scleroderma). The presence of anti-PM-Scl is a good prognostic sign, unlike the p6or prognosis seen with other myositis- and systemic sclerosis-specific autoantibodies. Although the antigenic structure of the PM-Scl complex of proteins is well studied and fairly well characterized, its exact function remains elusive. Future studies should focus

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on the association of certain clinical features with i m m u n o l o g i c responses to the various protein subunits of the autoantigen. See also AMINOACYL-tRNA HISTYDL (JO-1) SYNTHETASE AUTOANTIBODIES,

AMINOACYL-tRNA (OTHER THAN HISTIDYL) SYNTHETASE AUTOANTIBODIES and 56-KD NUCLEAR PROTEIN AUTOANTIBODIES.

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M. Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in Japanese patients with inflammatory muscle disease. Arthritis Rheum 1992;35:449--456. Kissel JT, Mendell Jr, Rammchan KW. Microvascular deposition of complement membrane attack complex in dermatomyositis. N Engl J Med 1986;314:329-334. Marguerie C, Bunn CC, Copier J, Bemstein RM, Gilroy JM, Black CM, So AK, Walport MJ. The clinical and immunogenetic features of patients with autoantibodies to the nucleolar antigen PM-Scl. Medicine 1992;71:327--336. McHugh NJ, Harvey G, Whyte J, Dorsey K, Silman A. Autoantibodies segregate with disease in monozygotic twin pairs discordant for scleroderma: three further cases. Arthritis Rheum 1994;37:$261. Oddis CV, Okano Y, Rudert WA, Trucco M, Duquesnoy RJ, Medsger Jr. TA. Serum autoantibody to the nucleolar antigen PM-Scl. Clinical and immunogenetic associations. Arthritis Rheum 1992;35:1211-1217. Reichlin M, Maddison PJ, Targoff I, Bunch T, Amett F, Sharp G, Treadwell E, Tan EM. Antibodies to a nuclear/nucleolar antigen in patients with polymyositis overlap syndromes. J Clin Immunol 1984;4:40-44. Reichlin M, Amett Jr FC. Multiplicity of antibodies in myositis sera. Arthritis Rheum 1984;27:1150-1156. Reimer G, Scheer U, Peters JM, Tan EM. Immunolocalization and partial characterization of a nucleolar autoantigen (PMScl) associated with polymyositis/scleroderma overlap syndromes. J Immunol 1986;137:3802--3808. Reimer G, Steen VD, Penning CA, Medsger TA Jr., Tan EM. Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma). Arthritis Rheum 1988;31:525--532. Targoff IN, Reichlin M. Nucleolar localization of the PM-Scl antigen. Arthritis Rheum 1985;28:226--230. Treadwell EL, Alspaugh MA, Wolfe JF, Sharp GC. Clinical relevance of PM-1 antibody and physiochemical characterization of PM-1 antigen. J Rheumatol 1984;11:658-662. Wolfe JF, Adelstein E, Sharp GC. Antinuclear antibody with distinct specificity for polymyositis. J Clin Invest 1977;59: 176--178. Zwettler U, Andrassy K, Waldherr R, Ritz E. Scleroderma renal crisis as a presenting feature in the absence of skin involvement. Am J Kidney Dis 1993;22:53--56.

Alderuccio F, Chan EK, Tan EM. Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75-kD acidic protein of the nucleolar complex. J Exp Med 1991;173:941--952. Blaszczyk M, Jablonska S, Szymanska-Jagiello W, JarzabekChorzelska M, Chorzelski T, Mohamed AH. Childhood scleromyositis: an overlap syndrome associated with PM-Scl antibody. Pediatr Dermatol 1991;8:1--8. Blaszczyk M, Jarzabek-Chorzelska M, Jablonska S, Chorzelski T, Kolacinska-Strasz Z, Beutner EH, Kumar V. Autoantibodies to nucleolar antigens in systemic scleroderma: clinical correlations. Br J Dermatol 1990;123:421--430. Bluthner M, Bautz FA. Cloning and characterization of the cDNA coding for a polymyositis-scleroderma overlap syndrome-related nucleolar 100-kD protein. J Exp Med 1992;176:973--980. Engel AG, Arahata K, Emslii-Smith A. Immune effector mechanisms in inflammatory myopathy. In: Waksman BH, ed. Immunologic Mechanisms in Neurologic and Psychiatric Disease. New York: Raven Press, 1990:141-- 156. Ge Q, Frank MB, O'Brien C, Targoff IN. Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoanfigen. J Clin Invest 1992;90:559--570. Ge Q, Wu Y, Targoff IN. Epitope mapping of the N-terminal portion of the PM-Scl 100 kD antigen. Arthritis Rheum 1993;36:S105. Ge Q, Wu Y, Targoff IN. Detection and quantification of human anti-PM-Scl autoantibodies using recombinant PM-Scl protein. Arthritis Rheum 1993;36:S150. Ge Q, Wu Y, Trieu EP, Targoff IN. Analysis of the specificity of anti-PM-Scl autoantibodies. Arthritis Rheum 1994;37: 1445--1452. Gelpi C, Alguero A, Angeles Martinez M, Vidal S, Juarez C, Rodriguez-Sanchez JL. Identification of protein components reactive with anti-PM/Scl autoantibodies. Clin Exp Immunol 1990;81:59-64. Genth E, Mierau R, Genetzky P, yon Muklen CA, Kaufman S, von Wilmowsky H, Meurer M, Kreig T, Pollman HJ, Hartl PW. Immunogenetic associations of scleroderma-related antinuclear antibodies. Arthritis Rheum 1990;33:657--665. Hirakata M, Mimori T, Akizuki M, Craft J, Hardin JA, Homma

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PROLIFERATING CELL NUCLEAR ANTIGEN AUTOANTIBODIES Gale A. McCarty-Farid, M.D.

Department of Health and Human Services, Primary Care C e n t e r - GWU/FDA, Washington, DC 20036, USA

HISTORICAL N O T E S .

Spontaneous or induced autoantibodies are important markers for many systemic rheumatic diseases, and useful probes for differentiated cell function and division (Yeo and Toh, 1994; Balczon, 1993). A novel autoantibody was identified in 1978 in 2% of systemic lupus erythematosus (SLE) patients by immunodiffusion (ID) and by indirect immunofluorescence (IIF) showed diffuse nuclear speckling which varied in intensity on dividing cells (Miyachi et al., 1978). The antigen could also be induced by mitogens and was thus named "proliferating cell nuclear antigen" (PCNA) (Figure 1). In 1985, a protein called "cyclin" was identified, which controlled the cell cycle in sea urchin eggs, accumulated prior to mitosis but was then degraded (Fairman, 1990). The term "cyclin" now refers to regulatory proteins which accumulate in coordination with specific kinases and are degraded in a cell-cycle related manner (Lewin, 1994). Because it increased during late G1 and S phases with a diffuse nuclear staining pattern by IIF which shifted to a punctate nuclear pattern as DNA synthesis progressed, PCNA was inappropriately termed "cyclin". This confusion was unfortunately propagated in the literature as "PCNA/cyclin" (Lewin, 1994). The correlation of IIF staining patterns for PCNA with DNA synthesis or replication implied a role in DNA regulation (Fairman, 1990). PCNA was subsequently defined as an auxiliary protein for DNA polymerase 5 (Fairman, 1990). In rapid succession, PCNA was purified to homogeneity, cloned, and some of its epitopes identified (Huff et al., 1990). Thus, the stage was set for the advances in epitope mapping, histochemical and clinicopathologic applications, and the elucidation of the roles of this protein in DNA synthesis and excision repair that comprise the re-

mainder of this chapter (Roos et al., 1993; Yu and Filipe, 1993).

THE AUTOANTIGEN(S) Definition

PCNA is encoded by a single gene, has a molecular weight of 36 kd, and, in its active form, is a trimer (Roos et al., 1993). It is evolutionarily highly conserved, as human PCNA has over 90% sequence similarity to rat and 35% to yeast; nevertheless, yeast PCNA can substitute for the human antigen in in vitro studies (Yu and Filipe, 1993). Plant mitogens such as Con A, PWM, and PHA induce late PCNA expression in lymphocytes; whereas, an acid-precipitable fraction of Mycobacterium tuberculosis (but not PHA or antiCD3 antibodies) induces earlier PCNA expression in peripheral blood mononuclear cells (Haftel et al., 1994). Relation to the Cell Cycle

The antigen is very stable, not degraded in cell transition from quiescence to growth, with a half life of 20 hrs; it canbe detected in cells that have stopped dividing 24--48 hr earlier. PCNA expression increases proportionally to DNA synthesis and/or growth in many cells, beginning in late G1, with a two- to threefold increase in S and a decrease at the S/G2 and G2/M interfaces. PCNA gene transcripts are enhanced 9or increased in resting cells after 10 hr of serum stimulation. During S phase, two major populations of PCNA exist, with different IIF patterns reflecting nucleoplasmic and DNA replication sites (Yu and Filipe, 1993). A tightly bound nucleolar form is

651

epitope {V S D Y E M K L M D L V E Q} contribute to its antigenicity. The reactivity of SLE sera to PCNA is related to the 261 N-terminal residues, and most are conformation-dependent as shown by immunoprecipitation. Some SLE sera, however, show additional reactivity with denatured protein as shown by immunoblotting.

AUTOANTIBODIES Terminology/Characteristics

Figure 1. Anti-PCNA staining on HEp-2 cells by indirect immunofluorescence. The classic nuclear speckled pattern of variable intensity is noted in the top 5 cells; whereas, the punctate and nucleolar forms are seen below (400x).

sometimes detected from late G1 phase to the G2/M transition (Lohr et al., 1995) (Figure 1, lower cells). A second diffuse nucleoplasmic form extractable by Triton-containing buffers prior to methanol fixation is present throughout the entire cell cycle, probably as a reserve pool (Waseem and Lane, 1990). PCNA expression increases proportionally to DNA synthesis and/or growth in many cells, beginning in late G1, with a two- to threefold increase in S and a decrease at the S/G2 and G2/M interfaces. Meiotic prophase cells express PCNA, and in damaged cells, PCNA is elevated, reflecting the role of PCNA in excision repair (Chapman and Wolgemuth, 1994).

Relevant Epitopes Purified PCNA, recombinant fusion proteins and synthetic peptides based on the cloned sequence were used to examine epitopes bound by SLE sera or monoclonal antibodies (MAbs) (Roos et al., 1993). As in other autoantigen-autoantibody systems, the epitopes recognized by natural autoantibodies are highly conserved and conformationally dependent domains; whereas, most induced antibodies react with nonconserved domains (Roos et al., 1993). Two dominant PCNA epitopes span residues 111-125 and 181-195. Most residues of the first

652

Nomenclature for the natural anti-PCNAs is based on the initials of the prototype patients (e.g., the two original SLE sera MN and EB which contained only anti-PCNA precipitins, and serum PT, which had antiPCNA and anti-Sin precipitins) (Miyachi et al., 1978). IgG isotypes predominate in the few sera studied. Data on subtypes, idiotypes, genetics, and the maturation of the anti-PCNA response are not available. Recently, a serum KO has been identified which reacts with a novel peptide of 58 kd which binds to the 34 kd PCNA polypeptide (Takasaki et al., 1994). The first induced antibody was a rat anti-PCNA (RAPCNAb) (Ogata et al., 1987). The major murine MAbs are termed "19A2 (IgM), 19F4 (IgG), PC10 (IgG2a) and pS2," and are all commercially available. PC10 is widely used because of its avidity and utility in archival material; whereas, 19A2 is more affected by tissue fixation.

Methods of Detection The IIF method of detection of anti-PCNA shows the characteristic nuclear speckled pattern of varying intensity on human epithelial cells, line 2 (HEp-2) (Figure 1). Commercial IIF and ELISA kits are available, with isotype-specific secondary antibodies and positive and negative control sera. The presence of anti-PCNA is also suggested by the concurrent finding of this IIF pattern in sera which give a precipitin line in ID with ubiquitous antigen sources; confirmatory testing for a line of identity with a positive control serum is then performed. Immunoperoxidase can also be used (Figure 2) and in some instances gives resolution superior to IIF.

Pathogenetic Role Consistent with a pathogenic role for anti-PCNA is

Figure 2. Anti-PCNA staining on HEp-2 cells by immunoperoxidase. The same variety of speckled patterns as noted in IIF are demonstrated (250x).

the inhibition of DNA synthesis by SLE anti-PCNA sera used in in vitro assays and in microinjection studies which show disruption of accessory protein functions, albeit without inhibiting DNA polymerase activity directly (Balczon, 1993; Roos et al., 1993). These results are analogous to several other autoantibody-autoantigen systems in which natural autoantibodies react with functionally important regions of target molecules. The role, if any, of anti-PCNA in spontaneous or induced SLE in animals is unknown.

CLINICAL UTILITY

Disease Associations Anti-PCNA was initially identified by ID in three of 70 sera, and later identified by IIF and confirmed by ID in 7/3000 sera (Fritzler et al., 1983). The frequency in SLE sera by IIF ranges from 2--10%; in one recent study, 6.5% of 71 SLE patients exhibited antiPCNA by more sensitive ELISA or immunoblot methods (Grimaudo et al., 1995). In the first three anti-PCNA patients reported, the clinical features associated with their SLE were not stated. Of the seven subsequent patients, five had SLE

by criteria, one had rheumatoid arthritis, and one had diffuse proliferative glomerulonephritis (DPGN). Arthritis (5/5), DPGN (4/5), and hypocomplementemia (4/5) were common in the SLE group; in addition to anti-PCNA, two patients had anti-Sm and one had anti-DNA. After treatment with steroids or cytotoxic drugs in three patients, anti-PCNA became undetectable. In one patient, anti-PCNA disappeared but later became positive when steroids were discontinued. A case report cited an uncomplicated pregnancy and delivery in a patient with SLE, anti-PCNA, antiDNA, anti-U1 snRNP and anticardiolipin antibody (Auer-Grumbach et al., 1994). In a recent survey of several large University referral laboratories, no distinctive profile of clinical or laboratory features has emerged to delineate anti-PCNA-positive SLE patients (McCarty-Farid, personal communication, 1995).

Histochemical Applications. In rheumatoid synovitis, fibroblast-like synoviocytes show proliferation as measured by anti-PCNA staining to a much greater extent than synovium from patients with osteoarthritis or normals (Qu et al., 1994).

Sensitivity and Specificity When calculated from the largest series addressing

653

frequency of anti-PCNA and other autoantibodies in systemic rheumatic disease patients, both the sensitivity and specificity of anti-PCNA for SLE approaches 100%. These calculations were based on 100 patients with SLE, two of w h o m had anti-PCNA; antiP C N A was not found in patients with other systemic rheumatic diseases, nor in normal controls (Fritzler et al., 1983).

REFERENCES Auer-Grumbach P, Ramschak H, Kainer F. Uncomplicated pregnancy and birth of a healthy child by a woman with systemic lupus erythematosus and anti-U 1 RNP, anti-PCNA/ cyclin, anti-dsDNA and antiphospholipid autoantibodies. Clin Exp Dermatol 1994;19:401--403. Balczon R. Autoantibodies as probes in cell and molecular biology. Proc Soc Exp Biol Med 1993;204:138-154. Chapman DL, Wolgemuth DJ. Expression of proliferating cell nuclear antigen in the mouse germ line and surrounding somatic cells suggests both proliferation-dependent and -independent modes of function. Int J Dev Biol 1994;38:419-- 417. Fairman MP. DNA polymerase 8/proliferating cell nuclear antigen: actions and interactions. J Cell Sci 1990;95:1--4. Fritzler MJ, McCarty.GA, Ryan JP, Kinsella TD. Clinical features of patients with antibodies directed against PCNA. Arthritis Rheum 1983;26:140-145. Grimaudo SA, Guilleron C, Manni JA. Immunoblotting for detection of antiself reactivities in collagen diseases: autoantibody profiles and clinical significance in patients with SLE. Lupus 1995;4(Suppl 2):160. Haftel HM, Chang Y, Hinderer R, Hanash S, Holoshitz J. Induction of the autoantigen prolifei'ating cell nuclear antigen in T lymphocytes by a mycobacterial antigen. J Clin Invest 1994;94:1365-- 1372. Huff JP, Roos G, Peebles CT, Houghten R, Sullivan KF, Tan EM. Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products. J Exp Med 1990;172:419--429. Lewin B, editor. Genes V. New York: Oxford University Press, 1994.

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CONCLUSIONS Anti-PCNA is a rare specificity, but when present alone or in combination with other autoantibodies, should alert the clinician to the presence of defined or evolving SLE. P C N A antigen is important in D N A synthesis and repair, and in the regulation of the cell cycle.

Lohr F, Wenz F, Haas S, Flentje M. Comparison of proliferating cell nuclear antigen (PCNA) staining and BrdUrdlabelling index under different proliferative conditons in vitro by flow cytometry. Cell Prolif 1995:28:93--104. Miyachi K, Fritzler MJ, Tan CK. Autoantibody to a nuclear antigen in proliferating cells. J Immunol 1978;121:22282234. Ogata K, Ogata Y, Takasaki Y, Tan EM. Epitopes on PCNA recognized by human lupus autoantibody and murine monoclonal antibody. J Immunol 1987;139:2942--2946. Qu Z, Garcia CH, O'Rourke LM, Planck SR, Kohli M, Rosenbaum JT. Local proliferation of fibroblast-like synoviocytes contributes to synovial hyperplasia. Results of proliferating cell nuclear antigen/cyclin, c-myc, and nucleolar organizer region staining. Arthritis Rheum 1994;37:212--220. Roos G, Landberg G, Huff JP, Houghten R, Takasaki Y, Tan EM. Analysis of the epitopes of PCNA recognized by monoclonal antibodies. Lab Invest 1993;68:204--210. Takasaki Y, Kogure T, Takahashi T, Yano T, Ando S, Takeuchi K, Yamanaka K, Hashimoto H. Autoantibodies to a novel polypeptide of 58kd associated with PCNA. Arthritis Rheum 1994;37:S171. Waseem NH, Lane DP. Monoclonal antibody analysis of proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form. J Cell Sci 1990;96(Pt 1):121--129. Yeo JP, Toh BH. Cell cycle-associated autoantibodies: markers for autoimmunity and probes for molecular cell biology. Autoimmunity 1994;18:291--300. Yu CC, Filipe MI. Update on proliferation-associated antibodies applicable to formalin-fixed paraffin-embedded tissue and their clinical applications. Histochemical J 1993 ;25:843--853.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

PURKINJE CELL AUTOANTIBODIES, TYPE 1 (Yo) Josep O. Dalmau, M.D., Ph.D. and Jerome B. Posner M.D.

Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA

H I S T O R I C A L NOTES

THE AUTOANTIGEN(S)

The term "Yo" is used to specifically identify the 34 and 62 kd proteins with which Purkinje Cell Autoantibodies, Type 1 (PCA-1) react in immunoblots of human Purkinje cells. The name "Yo" comes from the first two letters of the last name of the first patient whose serum displayed this reactivity. The presence of PCA-1 in patients with paraneoplastic cerebellar degeneration (PCD) was first reported in 1976 (Trotter et al., 1976). Among several associated antibodies, some are highly specific for the Purkinje cells of the cerebellum (i.e., the antibodies detected in patients with PCD and Hodgkin's disease) (Hammack et al., 1992); whereas, others react with neurons of the peripheral and central nervous system, (i.e., antineuronal nuclear autoantibodies, type 1 (Hu) known as ANNA-1 and type 2 (Ri) known as ANNA-2). First described in a patient with PCD and ovarian cancer (Greenlee, 1982), PCA of the anti-Yo type which display granular reactivity with the cytoplasm of Purkinje cells were subsequently identified in the sera and cerebrospinal fluid (CSF) of most patients with PCD and ovarian or breast cancer (Greenlee and Brashear, 1983; Jaeckle et al., 1983; 1985). The antigens identified by these antibodies include 34 and 62 kd proteins expressed in the cytoplasm of Purkinje cells (Cunningham et al., 1986) and in the tumors of all patients with anti-Yo-associated PCD (Fumeaux et al., 1990). The 34 kd antigen (Dropcho et al., 1987) and 62 kd antigen (Sakai et al., 1990; FathallahShaykh et al., 1991) have been cloned.

Characteristics

Using immunohistochemistry of human tissues, the Yo antigens are mainly expressed in the cytoplasm of Purkinje cells as a coarse granular pattern (Greenlee and Brashear 1983; Jaeckle et al., 1985). Low levels of expression of these antigens can sometimes be identified in large neurons of the brainstem. In animal tissues, the Yo antigens are easily detected in other large neurons of the nervous system and spinal cord (Jaeckle et al., 1985). Immunoelectron microscopy shows that Yo antigens are localized to membranebound and free ribosomes (Hida et al., 1994). With antibodies showing Yo-like immunostaining, reactivity is noted with clusters of ribosomes, granular endoplasmic reticulum, and the trans-face of the Golgi complex vesicles in Purkinje cells (Rodriguez et al., 1988). By immunoblot analysis of purified Purkinje cells (Cunningham et al., 1986), the Yo antigens include two proteins of 34 and 62 kd molecular weight. The 34 kd protein (or CDR-34) is encoded in a single exon consisting of tandem repeats of six amino acids (Leu/Phe; Leu; Glu; Asp; MetNal; Asp) (Dropcho et al., 1987). CDR-34 mRNA is preferentially expressed in cerebellum as well as in tumor tissue and cell lines from patients with and without PCA-1 (antiYo)-associated PCD (Dropcho et al., 1987; Dropcho, 1991) and at a low level in brain. The gene encoding the 34 kd protein is located on the long arm of the X chromosome near the fragile X gene (Siniscalco et al., 1991). The mouse gene is larger than the human gene, but as in the human, amino acids 3, 4 and 6 are always glutamate, aspartate and aspartate, respectively (Chen et al., 1990).

655

The cloned 62 kd protein (or CDR-62) (Sakai et al., 1990; Fathallah-Shaykh et al., 1991) contains leucine-zipper and zinc-finger motifs in the predicted open reading frame, suggesting that this protein plays a role in regulation of gene expression (FathallahShaykh et al. (1991). CDR-62 mRNA is expressed not only in cerebellum and brainstem but also in intestinal mucosa as well as in several tumor cell lines derived from adenocarcinoma of the colon, small-cell lung carcinoma, and squamous-cell lung carcinoma (Sakai et al., 1990; 1992). The gene encoding the 62 kd protein is on chromosome 16; a second gene encoding a 62 kd protein highly homologous to CDR 62 has recently been cloned (Fathallah-Shaykh and Posner, unpublished). By immunoblot analysis of ovarian carcinomas, the 34 and 62 kd proteins are expressed in all the tumors from PCA-1 (anti-Yo)-associated PCD patients, and in 34% of ovarian tumors from patients without paraneoplastic symptoms (Liu et al., 1995).

sensitive. Yo false-negatives or false-positives are not identified in tests using recombinant 62 kd protein (Figure 1). If immunoblot of the recombinant 62 kd protein is available, immunohistochemistry is not necessary (Fathallah-Shaykh et al., 1991). Using immunoblots of Purkinje cells, PCA-1 react predominantly with the 62 kd protein, and to a lesser degree with the 34 kd protein. Therefore, due to the absence of false results with the recombinant 62 kd protein, the use of this protein is sufficient to detect PCA-1 (anti-Yo).

Pathogenetic Role The role of the PCA-1 in the pathogenesis of PCD is unknown. Attempts to develop an animal model of PCD, using passive transfer of PCA-1 serum do not reproduce the disease (Tanaka et al., 1994). Injections of IgG from PCA-1 sera into the CSF of animals results in accumulation of IgG in Purkinje cells, but no cell destruction or symptoms (Graus et al., 1991).

AUTOANTIBODIES Methods of Detection Best identified by immunohistochemistry and immunoblot analysis of human tissues (Dalmau and Posner, 1994), PCA-1 in serum or CSF display coarse granular reactivity with the cytoplasm of Purkinje cells (Jaeckle et al., 1985) and to a much lesser degree with some large neurons of the brainstem. Because similar immunostaining may be displayed by other antibodies (Dalmau and Posner, 1994), the immunohistochemical analysis of serum and CSF from patients with suspected PCA-1 should always be complemented with immunoblot analysis of purified Purkinje cells or the recombinant 62 kd protein. Immunoblot analysis of homogenates of cerebellum or cerebral cortex should be avoided due to low sensitivity and possible false-positive results. For example, using immunohistochemistry and immunoblots of cerebellar homogenate, "anti-Hu antibodies" were detected in some patients with Sj6gren's syndrome (Moll et al., 1993). Examination of these sera using immunoblots of either isolated Purkinje cells or recombinant HuD fusion protein demonstrated the absence of antineuronal nuclear autoantibodies, type 1 (Hu) (Sillevis-Smitt et al., unpublished data). Immunoblot analysis of purified Purkinje cells, or the recombinant 62 kd protein are highly specific and

656

Figure 1. Immunoblot analysis of sera from patients with paraneoplastic disease, using purified human Purkinje cells. Lane N corresponds to serum from a normal individual; lane Hu corresponds to ANNA-1 serum from a patient with smallcell lung cancer and paraneoplastic encephalomyelitis; lane Ri corresponds to ANNA-2 serum from a patient with breast cancer and paraneoplastic opsoclonus-ataxia; lane Yo corresponds to serum from a patient with ovarian cancer and PCA1 (anti-Yo)-associated paraneoplastic cerebellar degeneration. Note that the PCA-1 antibodies (lane Yo) react with Purkinje proteins of 34 and 62 kd.

careful breast and pelvic examination, mammography and measurement of the ovarian tumor antigen CA125. Either pelvic CT or MRI may be used in the standard work-up for malignancy. If no malignancy is revealed, repeat mammography, pelvic examination under anesthesia followed by a dilation and curettage are recommended. Depending on the findings, hysterectomy and salpingo-oophorectomy may be considered (Anderson et al., 1988; Peterson et al., 1992; Hammack et al., 1990). In patients with known cancer who develop symptoms of cerebellar dysfunction, detection of high titers of PCA-1 confirms the paraneoplastic nature of the disorder. If the tumor found in these patients is not breast or ovarian cancer or does not express the Yo antigens, a search for a breast and gynecological neoplasm is recommended. PCA-1 are not found in patients with nonparaneoplastic cerebellar dysfunction or in healthy individuals. One percent of patients with ovarian cancer without PCD harbor low titers of PCA-1 in their sera (Liu et al., 1995; Brashear et al., 1989), and 4% of ovarian cancer patients without PCD have anti-Purkinje cell antibodies other than anti-Yo in their serum. The clinical significance of these antibodies is unknown. In rare instances, PCA-1 (anti-Yo)-associated PCD

Animal immunization with the 62 kd recombinant protein, yields high titers of PCA-1 antibodies, but the animals do not develop neurological symptoms or pathological signs of PCD (Tanaka et al., 1994).

CLINICAL UTILITY

Disease Association In two-thirds of the patients with PCD, the neurological symptoms precede identification of the tumor (Posner, 1995) (Table 1). If PCA-1 is detected, the titer is usually high (> 1:1000 by immunoperoxidase techniques) and, relative to the same amount of total IgG, the titer of PCA-1 in CSF is usually higher than the titer in serum. In the initial stages of the disease, the CSF usually demonstrates mild pleocytosis, increased total proteins and IgG, oligoclonal bands and an increased IgG index; all these findings suggest intrathecal synthesis of PCA-1 IgG (Furneaux et al., 1990). Because PCA-1 are highly specific for PCD associated with ovarian and breast cancer, detection of these antibodies should elicit a search for a pelvic or breast tumor. The initial work-up should include a

Table 1. Antibodies Associated with Paraneoplastic Cerebellar Dysfunction Subtype

Sex

Tumor#

Onset*

Clinical Findings

PCA- 1 (anti-Yo)

F

Gynecologic, breast

before

subacute pancerebellar symptoms

PCD-HD (PCAb)

M>F

Hodgkin's

after

may remit, less severe than other subtypes

PCD-LEMS (VGCC Abs)

M=F

SCLC

before

pancerebellar symptoms with proximal weakness; decreased reflexes in legs (LEMS may be overlooked)

ANNA- 1 (Hu)**

F>M

SCLC

before

sensory neuronopathy, encephalomyelitis

ANNA-2 (Ri)

F

Breast

before/after

opsoclonus, truncal ataxia, myoclonus

# * **

Only the most frequently associated tumors are indicated .Onset of neurological symptoms "before or after" tumor diagnosis Anti-Hu-associated encephalomyelitis may develop with predominant cerebellar dysfunction which usually is associated with other symptoms, including sensory neuronopathy, limbic encephalitis, brainstem encephalitis, myelitis, and/or autonomic dysfunction. Patients with SCLC and PCD alone or associated with LEMS may be anti-Hu negative. P C D - paraneoplastic cerebellar degeneration SCLC -- small-cell lung cancer H D - Hodgkin's disease LEMS -- Lambert-Eaton myasthenic syndrome PCAb- Purkinje cell antibody (antigen not defined) VGCC A b s - voltage-gated calcium channel antibodies

657

can be found in patients with tumors other than breast and ovarian cancer. These tumors mainly include other gynecologic cancers (fallopian tube and uterus), although one woman had adenocarcinoma of the lung (Peterson et al., 1992). Except for two men (one with adenocarcinoma of the salivary gland and the other with adenocarcinoma of unknown origin) only women are reported with PCA-1 (anti-Yo)-associated PCD (Felician et al., 1995; Krakawer et al., unpublished). Once symptoms of PCD stabilize, plasmapheresis, immune suppression or treatment of the tumor are not usually effective in reversing the neurological dysfunction (Graus et al., 1992; Peterson et al., 1992; Vega et al., 1994). The pathological basis for this lack of response is the acute and severe degeneration of Purkinje cells in the cerebellum of these patients (Sindic et al., 1993; Verschuuren et al., 1994).

REFERENCES Anderson NE, Rosenblum MK, Posner JB. Paraneoplastic cerebellar degeneration clinical-immunological correlations. Ann Neurol 1988;24:559-567. Brashear HR, Greenlee JE, Jaeckle KA, Rose JW. Anticerebellar antibodies in neurologically normal patients with ovarian neoplasms. Neurology 1989;39:1605-1609. Chen Y-T, Rettig WJ, Yenamandra AK, Kozac CA, Changanti RS, Posner JB, Old LJ. Cerebellar degeneration-related antigen: a highly conserved neuroectodermal marker mapped to chromosomes X in human and mouse. Proc Natl Acad Sci USA 1990;87:3077--3081. Cunningham J, Graus F, Anderson N, Posner JB. Partial characterization of the Purkinje cell antigens in paraneoplastic cerebellar degeneration. Neurology 1986;36:1163--1168. Dalmau J, Posner JB. Neurologic paraneoplastic antibodies (anti-Yo; anti-Hu; anti-Ri): the case for a nomenclature based on antibody and antigen specificity. Neurology 1994;44: 2241--2246. Dropcho EJ, Chen YT, Posner JB, Old LJ. Cloning of a brain protein identified by autoantibodies from a patient with paraneoplastic cerebellar degeneration. Proc Natl Acad Sci USA 1987;84:4552-4556. Dropcho EJ. Expression of the "onconeural" CDR34 gene in human carcinomas. Neurology 1991; 41 (Suppl 1):238. Fathallah-Shaykh H, Wolf S, Wong E, Posner JB, Fumeaux HM. Cloning of a leucine zipper protein recognized by the sera of patients with antibody-associated paraneoplastic cerebellar degeneration. Proc Natl Acad Sci USA 1991;88: 3451-3454. Felician O, Renard JL, Vega F, Creange A, Chen QM, Bequet D, Delattre JY. Paraneoplastic cerebellar degeneration with

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CONCLUSION PCA-1 (anti-Yo) are autoantibodies directed against 34 and 62 kd proteins expressed in the cytoplasm of Purkinje cells and breast and gynecologic tumors. Since symptoms of PCD usually precede the identification of the cancer, detection of PCA-1 is an important marker for the presence of an underlying tumor, usually breast or ovarian cancer. In patients with known cancer who develop symptoms of cerebellar dysfunction, detection of high titers of PCA-1 serves to confirm the paraneoplastic nature of the disorder. If the underlying tumor is not breast or ovarian cancer, or if the tumor does not express Yo antigens, a search for a second neoplasm in the breast or ovary is recommended. See also NEURONAL NUCLEAR AUTOANTIBODIES, TYPE 1 (Hu) and CALCIUM CHANNEL AND RELATED PARANEOPLASTIC DISEASE AUTOANTIBODIES.

anti-Yo antibody in a man. Neurology 1995;45:1226-1227. Fumeaux HM, Rosenblum MK, Dalmau J, Wong E, Woodruff P, Graus F, Posner JB. Selective expression of Purkinje-cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration. N Engl J Med 1990;322:1844-1851. Graus F, Ilia I, Agusti M, Ribalta T, Cruz-Sanchez F, Juarez C. Effect of intraventricular injection of anti-Purkinje cell antibody (anti-Yo) in a guinea pig model. J Neurol Sci 1991;106: 82-87. Graus F, Vega F, Delattre JY, Bonaventura I, Rene R, Arbaiza D, Tolosa E. Plasmapheresis and antineoplastic treatment in CNS paraneoplastic syndromes with antineuronal autoantibodies. Neurology 1992;42:536-540. Greenlee JE. Is paraneoplastic cerebellar degeneration an immune-mediated condition? Detection of circulating antibodies to Purkinje cells in a patient with the disorder. Ann Neurol 1982;12:103. Greenlee JE, Brashear HR. Antibodies to cerebellar Purkinje cells in patients with paraneoplastic cerebellar degeneration and ovarian carcinoma. Ann Neurol 1983;14:609--613. Hammack JE, Kimmel DW, O'Neill BP, Lennon VA. Paraneoplastic cerebellar degeneration: a clinical comparison of patients with and without Purkinje cell cytoplasmic antibodies. Mayo Clin Proc 1990;65:1423-1431. Hammack JE, Kotanides H, Rosenblum MK, Posner JB. Paraneoplastic cerebellar degeneration. II. Clinical and immunologic findings in 21 patients with Hodgkin's disease. Neurology 1992;42:1938--1943. Hida C, Tsukamoto T, Awano H, Yamam0to T. Ultrastructural localization of anti-Purkinje cell antibody-binding sites in paraneoplastic cerebellar degeneration. Arch Neurol 1994;51: 555-558. Jaeckle KA, Houghton AN, Nielsen SL, Posner JB. Demonstra-

tion of serum anti-Purkinje antibody in paraneoplastic cerebellar degeneration and preliminary antigenic characterization. Ann Neurol 1983;14:111. Jaeckle KA, Graus F, Houghton A, Cordon-Cardo C, Nielsen SL, Posner JB. Autoimmune response of patients with paraneoplastic cerebellar degeneration to a Purkinje cell cytoplasmic protein antigen. Ann Neurol 1985;18:592-600. Liu S, Mezfich J, Berk J, Federici M, Dalmau J, Posner JB. Expression of Purkinje-cell antigens in ovarian tumor, and presence of anti-Purkinje cell antibodies in the serum of patients without paraneoplastic cerebellar degeneration. Neurology 1995; 45(Suppl 4):A228--A229. Moll JWB, Markusse HM, Pijnenburg JJJM, Vecht ChJ, Henzen-Logmans SC. Antineuronal antibodies in patients with neurologic complications of primary Sj6gren's syndrome. Neurology 1993;43:2574-2581. Peterson K, Rosenblum MK, Kotanides H, Posner JB. Paraneoplastic cerebellar degeneration. I. A clinical analysis of 55 anfi-Yo antibody-positive patients. Neurology 1992;42:1931-1937. Posner JB, editor. Neurologic Complications of Cancer. Philadelphia: F.A. Davis, 1995. Rodriguez M, Truh LI, O'Neill BP, Lennon VA. Autoimmune paraneoplastic cerebellar degeneration: ultrastructural localization of antibody-binding sites in Purkinje cells. Neurology 1988;38:1380--1386. Sakai K, Mitchell DJ, Tsukamoto T, Steinman L. Isolation of a complementary DNA clone encoding an autoantigen recognized by an antineuronal cell antibody from a patient

with paraneoplastic cerebellar degeneration. Ann Neurol 1990;28:692-698. Sakai K, Negami T, Yoshioka A, Hirose G. The expression of a cerebellar degeneration-associated neural antigen in human tumor line cells. Neurology 1992;42:361-366. Sindic CJ, Andersson M, Boucquey D, Chalon MP, Bisteau M, Brucher JM, Laterre EC. Anti-Purkinje cells antibodies in two cases of paraneoplastic cerebellar degeneration. Acta Neurol Belg 1993;93:65-77. Siniscalco M, Oberle I, Melis P, Alhadeff B, Murray J, Filippi G, Mattioni T, Chen YT, Furneaux H, Old LJ, et al. Physical and genetic mapping of the CDR gene with particular reference to its position with respect to the FRAXA site. Am J Med Genet 1991;38:357-362. Tanaka K, Tanaka M, Onodera O, Igarashi S, Miyatake T, Tsuji S. Passive transfer and active immunization with the recombinant leucine-zipper (Yo) protein as an attempt to establish an animal model of paraneoplastic cerebellar degeneration. J Neurol Sci 1994;127:153-158. Trotter JL, Hendin BA, Osterland CK. Cerebellar degeneration with Hodgkin disease. An immunological study. Arch Neurol 1976;33:660--661. Vega F, Graus F, Chen QM, Schuller E, Poisson M, Delattre JY. Intravenous (IV) immunoglobulin therapy in paraneoplastic neurologic syndromes (PNS) with antineuronal autoantibodies. Neurology 1994;44:A157. Verschuuren J, Rosenblum M, Pryor A, Weldon PH, Dalmau J. Complete absence of Purkinje cells in anti-Yo associated cerebellar degeneration. Ann Neurol 1994;36:294.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RA-33 (HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN COMPLEX) AUTOANTIBODIES Gtinter Steiner, Ph.D. a and Josef S. Smolen, M.D. b

aLudwig Boltzmann-Institutefor Rheumatology and Balneology, Department of Rheumatology, ViennaA-1130; and b2nd Department of Medicine, Lainz Hospital, Department of Rheumatology, University of Vienna, ViennaA-1090, Austria

HISTORICAL NOTES

In 1989, an autoantibody reactive on immunoblots with a nuclear protein of approximately 33 kd was described (Hassfeld et al., 1989). Because such autoantibodies were initially detected almost exclusively in sera from patientswith rheumatoid arthritis (RA), the antigen was given the name "RA33". Later this antigen was shown to be identical with the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex (Steiner et al., 1992). Autoantibodies to the closely related A1 protein of the hnRNP complex (hnRNP-A1) were previously described in sera of patients with RA and other connective tissue diseases (Jensen et al., 1988; Astaldi-Ricotti et al., 1989). Using partially purified hnRNP-A2, autoantibodies to this antigen were also detected in sera from patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) (Steiner et al., 1992; Meyer et al., 1993; Hassfeld et al., 1995). Moreover, these studies demonstrated that most antihnRNP-A2/RA-33-positive sera were also reactive with the hnRNP proteins B1 and B2 (hnRNP-B 1, B2), confirming an earlier observation (Dangli et al., 1988).

processing takes place on a multicomponent nuclear ribonucleoprotein complex called the spliceosome (Sharp, 1994). Major components are the small nuclear ribonucleoproteins (snRNPs) (Ltihrmann et al., 1990), the hnRNPs (Dreyfuss et al., 1993) and a considerable number of additional protein factors (Lamm and Lamond, 1993). Much is known about structure and function of snRNPs, but hnRNPs are still much less characterized. After mild RNase treatment of nuclear extracts, particles can be isolated which sediment at 40S in sucrose gradients (Beyer et al., 1977). In addition to pre-mRNA fragments, these particles contain approximately 30 different proteins which can be immunopurified with a monoclonal antibody to the hnRNP C proteins (Pifiol-Roma et al., 1988). Thus, all proteins precipitated by this antibody are defined as hnRNP proteins. According to their molecular weights and their migration behavior in two-dimensional gels, these proteins are described in alphabetical order from hnRNP-A 1 (34kd) to hnRNPU (120 kd) (Pifiol-Roma et al., 1988). Among these, the hnRNP A and B (hnRNP A/B) proteins form a group of several highly related proteins presumably derived from a single ancestor gene. Native vs. Recombinant Antigen Performance

THE AUTOANTIGEN(S) Definition/Standard Nomenclature

In eukaryotic cells, most pre-mRNAs are transcribed as large precursor molecules which are processed to mature mRNA before leaving the nucleus. This

660

The A1 protein was the first hnRNP protein to be cloned and expressed in bacteria (Cobianchi et al., 1986; Riva et al., 1986). Most data available on structure, function and antigenicity of this protein were obtained with the recombinant form, which appears to be as biologically active as the natural protein (Cobianchi et al., 1988; Montecucco et al.,

1990; Mayeda and Krainer, 1992). Although much less data are available on recombinant hnRNP-A2, there is good evidence that the performance of the recombinant protein does not differ substantially from that of its natural counterpart (Mayeda et al., 1994; Skriner et al., 1994a; unpublished observations).

Origin/Cellular Localization The hnRNP proteins are evolutionarily highly conserved proteins which seem to be contained in all cells and tissues of vertebrates (Dreyfuss et al., 1993). Moreover, several proteins with sequences similar to vertebrate hnRNP A/B proteins can be identified in Drosophila (Matunis et al., 1992). The hnRNP proteins are primarily localized in the nucleus where they are among the most abundant proteins. Functionally, they are involved in packaging and processing of premRNA, and there is strong evidence that hnRNP A/B proteins play an important role in regulating alternative splicing (Mayeda and Krainer, 1992; Yang et al., 1994; Mayeda et al., 1994). Moreover, there is some evidence that these proteins can shuttle between nucleus and cytoplasm, being presumably involved in mRNA transport (Pifiol-Roma and Dreyfuss, 1992).

Methods of Purification Originally hnRNP proteins were isolated from HeLa cells, but other fast growing (human) cell lines (such as MOLT-4 or Jurkat) are also good sources. A semipurified preparation of hnRNP A/B proteins can be easily obtained in a single-step procedure by affinity chromatography on heparin-Sepharose (Steiner et al., 1992; Hassfeld et al., 1995). Briefly, a nuclear extract is applied to a heparin-Sepharose column equilibrated with 20 mM Hepes, 0.3 M NaC1, pH 7.9; after washing the Sepharose with the same buffer, bound protein is first eluted with 20 mM Hepes, 1 M NaC1, pH 7.9 and, finally, with the same buffer containing 6 M urea. The urea eluate is approximately 20-fold enriched for hnRNP proteins and is sufficiently pure for immunoblot analysis. The two hnRNP A proteins are major components of this preparation and can be easily visualized by Coomassie blue staining; whereas, the two hnRNP B proteins are much less abundant. Highly purified hnRNP-A2 can be obtained in good yield by further purification on the weak cation exchanger CM-Sepharose (Kumar et al., 1986). However, the purified protein is almost insoluble in aqueous buffers, and 6 M urea or 1% SDS are re-

quired for solubilization. For purification of hnRNPA 1, hydrophobic interaction chromatography on phenyl-Sepharose is recommended (Mayeda and Krainer, 1992). Alternatively, hnRNP proteins can be purified by affinity chromatography on single-stranded DNA (Cobianchi et al., 1988; Pifiol-Roma et al., 1988; Steiner et al., 1992) or by density gradient centrifugation (Barnett et al., 1990). However, the latter method is not recommended for large-scale purifications.

Commercial Sources None.

Sequence Information The molecular weights of hnRNP-A1 and hnRNP-A2 are 34 and 36kd, respectively. Both are basic proteins with isoelectric points of 8.4 and 9.0; these proteins are partly phosphorylated in vivo (Wilk et al., 1985). The two proteins are highly related and share more than 80% identical amino acids in their N-terminal halves. They contain two conserved RNA-binding domains (RBD, also called RNA recognition motifs (RRM)) which are followed by a glycine-rich section at the carboxy terminus (Figure 1). hnRNP-B1 is identical to hnRNP-A2 except for a 12 amino acid insertion close to the N-terminus generated by an alternative splicing event (Burd et al., 1989); whereas, the structure of hnRNP-B2 is unknown. Based on the extensive cross-reactivity of anti-hnRNP-A2/RA33 antibodies with both hnRNP B proteins (Hassfeld et al., 1995), it is not unlikely that hnRNP-B2 is also generated by alternative splicing. For hnRNP-A1, an alternatively spliced variant has been described (hnRNP-Alb) which contains a 50 amino acid insertion in the C-terminal region (Buvoli et al., 1990). The genes encoding hnRNP-A1 and hnRNP-A2 show similar structures indicating a common ancestor gene (Biamonti et al., 1994). So far, only limited information is available on epitopes recognized by autoantibodies to the hnRNP A/B proteins. The data indicate that major antigenic regions are located in the N-terminal portions of these proteins (Montecucco e t al., 1990, Skriner et al., 1994a). Because these regions contain the two RBDs, the autoimmune response seems to be predominantly directed to functionally important parts of the molecules. Recently, a conformational epitope of hnRNPA2 comprising the complete 2nd RBD was identified (Skriner et al., 1994b).

661

Figure 1. Structural Features of hnRNP A/B Proteins. All hnRNP A/B proteins are characterized by the same modular structure: the N-terminal portion contains two adjacent conserved RNA-binding domains (RBD) of approximately 80 amino acids which are present in many RNA-binding proteins. Black bars symbolize the two most highly conserved sequences within each RBD. The C-terminal section, called auxiliary domain, contains about 50% glycine residues and shows some sequence similarities to other glycine-rich structures, such as collagen, keratin or EBNA-1. The hnRNP-B1 protein is identical with hnRNP-A2 except for a 12 amino acid insertion close to the N-terminus generated by alternative splicing. The primary structure of hnRNP-B2 is unknown but it is assumed to be another alternatively spliced form of hnRNP-A2, hnRNP-A1 shows more than 80% sequence similarity with hnRNP-A2 in the N-terminal region and approximately 40% sequence similarity in the glycine-rich region. An alternatively spliced variant, hnRNP-A1 b, contains a 50 amino acid insertion in the glycine-rich auxiliary domain. The majority of autoantibody reactivities seem to be directed to the N-terminal portions of the antigens. For hnRNP-A2 a conformational epitope which comprises the complete RBD II was identified recently (Skriner et al., 1994b). AUTOANTIBODIES

evidence that these autoantibodies play a pathogenic role in murine lupus.

Name(s) Genetics The proteins are named in alphabetical order beginning with hnRNP-A1, hnRNP-A2, etc. To avoid confusion with antigens associated with snRNPs (such as U I-snRNP and Sm-specific proteins), which are also named in alphabetical order, the full names should always be used. Because the antigen recognized by anti-hnRNP-A2 was initially termed RA33, the synonym anti-RA33 may be used for these antibodies. However, it must be noted that most, if not all, anti-hnRNP-A2 cross-react with the two hnRNP B proteins, and, therefore, the term "anti-RA33" implies reactivities to the three hnRNP proteins A2, B 1, B2 (just as anti-Sm designates reactivities to the snRNP core proteins B, B', D, etc.).

Pathogenetic Role Human Disease. There is no direct evidence that antibodies to the hnRNP A/B proteins are directly involved in the pathogenesis of RA, SLE or MCTD. A correlation between the occurrence of anti-hnRNPA2/RA33 and severe erosive arthritis in British patients with SLE was reported recently (Isenberg et al., 1994).

Animal Models. Antibodies to hnRNP-A1 have been detected in the sera of several autoimmune mouse strains (Jensen et al., 1988), and the presence of antihnRNP-A2/RA33 in lupus-prone mice is suggested (unpublished). As in humans, there is so far no

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There are almost no data available on the genes involved in the generation of autoantibodies to these proteins. Studies recently performed with identical twins discordant for RA showed the presence of antihnRNP-A2/RA33 also in some of the unaffected twins (Williams et al., submitted) arguing for a pronounced genetic background in this autoimmune response.

Factors in Etiology Isotypes, Avidity, Epitopes, Cross-Reactivities. Like most autoantibodies, anti-hnRNP-A/B are predominantly of the IgG isotype. Sera can also contain IgM antibodies, but these are usually of low titer and occur only in conjunction with IgG. The avidity of IgG antibodies appears relatively low with most sera; reactivities are easily detected by immunoblotting at 1:25 serum dilution but disappear at higher dilutions. Nevertheless, higher titers of anti-hnRNP-A2/RA33 (up to 1:1600) are observed in about one-third of positive sera. The major antigenic sites are located in the Nterminal RNA-binding regions. Nevertheless, for antihnRNP-A1 sera, indirect evidence suggests that antibodies of some RA patients bind to the glycinerich C-terminal structure. However, this reactivity was considered due to antikeratin antibodies cross-reacting with an epitope in the C-terminal region (Montecucco et al., 1990). So far, detailed epitope mapping is

available only for hnRNP-A2; this identified a major conformational epitope (Figure 1) which was recognized by most sera from RA patients, by 60% of sera from SLE patients, but by only one-third of sera from MCTD patients (Skriner et al., 1994b). In addition, several minor epitopes seem to exist, some of which may be linear; such epitopes will be investigated in future studies employing synthetic peptides. Despite the close sequence similarities between the two hnRNP A proteins, only a minority of sera contain reactivities to both antigens. And in these sera, cross-reactivity between the two antibody species is seen (Hassfeld et al., 1995). In contrast, cross-reactivity of anti-hnRNP-A2/RA33 antibodies with both hnRNP B proteins is generally observed.

Methods of Detection

protein migrates as doublet in SDS gels (Kumar et al., 1986). Anti-hnRNP-A2/RA33 stain a band of approximately 36 kd as well as a doublet of 37/38 kd corresponding to hnRNP-B l/B2. These characteristic staining patterns considerably facilitate interpretation of immunoblot results (Figure 2). ELISA. The first data on anti-hnRNP-A1 were obtained by ELISA and immunoblotting which employed the purified recombinant antigen (Jensen et al., 1988; Astaldi-Ricotti et al., 1989). No discrepancies between ELISA and immunoblot data were reported. Preliminary ELISA studies employing highly purified natural hnRNP-A2 indicated ELISA to be more sensitive than immunoblotting, particularly for detection o f antibodies in sera of SLE and MCTD patients. However, some of the discrepancies between ELISA and immunoblotting may have been caused by

Immunofluorescence. Immunofluorescence is not a suitable technique for detection of these autoantibodies, because they often occur in sera negative for antinuclear antibodies (ANA) (Steiner, 1994). Moreover, even in sera with strong anti-hnRNP-A2/RA33 reactivity, no characteristic nuclear staining pattern is observed (Hassfeld et al., 1989). Immunodiffusion. This method is not suitable since the antibodies do not form precipitates. Immunoblotting. Antibodies to hnRNP A/B proteins are best detected by immunoblotting which employs semipurified antigens. Unpurified nuclear extracts can also be used, but identification of the relevant bands is sometimes difficult, particularly with sera from patients with SLE. The hnRNP preparation obtained by heparin-Sepharose chromatography is separated on 12% SDS gels and blotted onto nitrocellulose membranes. Sera are diluted 1:25 in 20 mM Tris-HC1 or phosphate-buffered saline, pH 7.4, containing 3% nonfat dried milk and incubated for 40 min with the blotted antigens. Detergents such as Triton-X-100 or Tween 20 should be avoided in the incubation buffer because they may favor false-positive results presumably caused by nonspecific binding of DNA-anti-DNA immune complexes present in SLE sera (unpublished observation). For immunodetection, the use of alkaline phosphatase conjugated antihuman antibodies is recommended because some peroxidase-labeled antibodies may produce nonspecific staining, particularly of the hnRNP-A2 band. Anti-hnRNP-A1 stain a double band of approximately 33/34 kd because this

i~i ii

Figure 2. Autoantibody Reactivities to Partially Purified hnRNP A/B Proteins. (N) normal human serum, (R1) anti-hnRNPA2/RA33 reference serum staining the hnRNP proteins A2, B 1, B2; (R2) anti-hnRNP-A1 reference serum staining the hnRNPA1 double band; (1-6) patient sera. In double positive sera (5, 6), cross-reactivity between the two antibodies was generally observed. 663

differences in epitope exposure and recognition and/or by the poor solubility of the antigen (Steiner et al., 1993). With no commercial assays available at present, immunoblotting is still the recommended technique for detection of these antibodies.

CLINICAL UTILITY

Furthermore, antibodies to hnRNP A/B proteins are not correlated with disease activity or stage; antibody levels, as estimated by immunoblotting, appear relatively stable over periods of several years (Isenberg et al., 1994), indicating that their occurrence is generally not affected by therapy. However, the frequency of anti-hnRNP-A2/RA33 is reduced in RA patients under long-term corticosteroid therapy (Hassfeld et al., 1992).

Application and Disease Association Antibody Frequencies in Disease Antibodies to the hnRNP A/B proteins occur mainly in RA, SLE and MCTD, although, they can be detected with lower frequency in other connective tissue diseases, particularly in progressive systemic sclerosis (PSS) (Astaldi-Ricotti et al., 1989; Meyer et al., 1993; Hassfeld et al., 1995). In SLE, they are significantly associated with antibodies to UI-snRNP and/or Sm, which is especially true for anti-hnRNPA1 (Steiner et al., 1992; Hassfeld et al., 1995). Because antibodies to U 1-snRNP-specific proteins are detectable in 20--30% of patients with SLE and in all patients with MCTD, but usually not in patients with RA (van Venrooij and Sillekens, 1989), anti-hnRNPA/B without concomitant anti-UlsnRNP (or other well-defined antibodies not found in RA patients) seem to be largely confined to RA. Anti-hnRNP-A2/RA33 may appear very early in the course of RA when a clear diagnosis is not yet possible (Hassfeld et al., 1993; Cordonnier et al., 1994) and have been observed even in one pre-illness sample (Aho et al., 1993). Therefore, their presence can provide very useful diagnostic help, particularly in rheumatoid factor-negative sera. The absence of these antibodies does not exclude RA but their presence makes other arthritic diseases, such as psoriatic arthritis, ankylosing spondylitis or osteoarthritis, highly unlikely. Although the usefulness of antihnRNP-A/B for diagnosis of SLE and MCTD is not yet established, they may provide additional diagnostic information in sera with weak or borderline antiU 1snRNP reactivities. The presence of anti-hnRNP-A2/RA33 is independent of age, gender and disease duration (Hassfeld et al., 1989; Meyer et al., 1993; Isenberg et al., 1994). However, their prevalence varies in different populations. Thus, compared to central and West European patients, the prevalence is very low in RA patients from Finland (Aho et al., 1993) and Greece (Dangli et al., 1988) and elevated in non-Caucasian patients with SLE (Isenberg et al., 1994). 664

The frequencies of autoantibodies to hnRNP-A1 andA2 in connective tissue diseases were the subject of several independent studies (Astaldi-Ricotti et al., 1989; Meyer et al., 1993; Hassfeld et al., 1995). When the autoimmune response to the whole group of hnRNP A/B proteins was investigated with purified natural antigens on immunoblots, antibodies to hnRNP-A2 were more frequently, detected than antibodies to hnRNP-A1 (Hasseld et al., 1995) (Table 1). In SLE patients, antibodies to hnRNP-A1 always occurred together with anti-hnRNP-A2/RA33. Thus, at least in this one investigation, anti-hnRNP-A2/ RA33 seemed to be the predominating antibody. Nevertheless, when anti-hnRNP-A1 were investigated by ELISA employing the recombinant antigen (Astaldi-Ricotti et al., 1989), these antibodies were detected in patients with RA, MCTD and SLE with frequencies similar to those reported for anti-hnRNPA2/RA33 (Hassfeld et al., 1995).

Sensitivity and Specificity for RA In two recent studies of anti-hnRNP-A2/RA33 as a diagnostic marker for RA, comparable data were obtained, including sensitivity of 35% and specificities of 85 and 89% (Meyer et al., 1993; Hassfeld et al., 1995). Specificity increased to 96% when only those anti-hnRNP-A2/RA33-positive sera which did not contain concomitant anti-Ul-snRNP were included. For anti-hnRNP-A1 as determined by ELISA (Astaldi-Ricotti et al., 1989), sensitivity for RA was 50% and specificity was 85%. When anti-hnRNP-A1 was determined by immunoblotting, sensitivity was 20% and specificity was 94% (Hassfeld et al., 1995); this value increased to 98% when only anti-hnRNPA 1-positive sera without concomitant anti-U 1-snRNP were considered. Although it is difficult to compare data from two different studies employing different patients and methodologies, these data as well as our

Table 1. Frequencies of Autoantibodies to hnRNP-A2 and hnRNP-A1 in Rheumatic Diseases Disease

n

Anti-hnRNP-A2/RA33

Anti-hnRNP-A1

Total

RA

60

21 (35%)

12 (20%)

26 (43%)

SLE

70

16 (23%)

9 (13%)

16 (23%)*

MCTD

26

10 (39%)

3 (12%)

11 (42%)**

Other CTD

63

2 (3%)

1 (2%)

3 (5%)

PSA

23

0

0

0

AS

10

0

0

0

SN Oligo

27

1 (4%)

0

1 (4%)

OA, Cryst

24

1 (4%)

1 (4%)

2 (8%)

Healthy

30

1 (3%)

1 (3%)

1 (3%)

RA (rheumatoid arthritis); SLE (systemic lupus erythematosus); MCTD (mixed connective tissue disease; Other CTD (connective tissue diseases): progressive systemic sclerosis (n = 16, one anti-hnRNP-A2/RA33 and anti-topoisomerase-pos), CREST syndrome (n = 8); dermato/polymyositis (n = 11, one anti-hnRNP-A2/RA33 and anti-Jol-pos), polymyositis/scleroderma-overlap (n = 4), primary Sj0gren's syndrome (n = 24, one anti-hnRNP-A1 and anti-Ro/anti-La-pos); PSA (psoriatic arthropathy); AS (ankylosing spondylitis); SN Oligo (seronegative oligoarthritis): reactive arthritis (n = 10), Reiter's disease (n = 4), Crohn's disease with arthritis (n = 2) and unclassified oligoarthritis (n = 11, one anti-hnRNP-A2/RA33-pos); OA (inflammatory osteoarthritis) (n = 19, one anti-hnRNP-A2/RA33, one anti-hnRNP-Al-pos) and Cryst (crystal arthropathies) (n = 5).

own preliminary data on anti-hnRNP-A2~A33 indicate that ELISA may be more sensitive but less specific than immunoblotting.

CONCLUSION Autoantibodies to the A/B proteins of the hnRNP complex are newly described serologic markers which seem to be of high value for the diagnosis of RA, especially as they occur independently of rheumatoid factor. Although less frequently detectable than the latter, they appear to be more specific. These antibodies can also be detected in sera from patients with other connective tissue diseases (particularly SLE and MCTD), but there they frequently occur together with antibodies to snRNP-associated antigens. Interestingly, there is a strong functional and structural relationship between snRNPs and hnRNPs during the process of mRNA maturation, which takes place at the spliceosome. It has been known for many years that patients

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with SLE and MCTD generate an autoimmune response to this particle, especially anti-Sm and anti-U 1snRNP. In contrast, autoantibodies to snRNPs are only very rarely observed in patients with RA. Thus, it was assumed that the antispliceosomal autoimmune response was rather specific for SLE and MCTD, and so it was not too unexpected to detect antibodies to hnRNP proteins in sera of such patients. The fact that antibodies to hnRNP-associated components of the spliceosome can also be found in sera from RA patients demonstrates that this disease may be immunologically more closely linked to SLE and MCTD than previously assumed. Thus, apart from being valuable serological markers, these antibodies form a sort of immunological bridge between these three diseases. So far, only hnRNP A/B proteins have been identified as autoimmune targets in rheumatic autoimmune diseases. Future studies should investigate whether the autoimmune response in these diseases is also directed to other components of the hnRNP complex.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RECOMBINANT AUTOANTIGENS E. William St. Clair, M.D.

Department of Medicine, Division of Rheumatology, Allergy and Clinical Immunology, Duke University Medical Center, Durham, NC 27710, USA

HISTORICAL NOTES Autoantibody specificities are commonly defined on the basis of interactions with (native) antigens. A native antigen for the purposes of this review is defined as a natural constituent of a living organism. Studies using native antigens have found that most autoantibodies target structures that are highly conserved among species. These sites are often located in functionally vital regions of the protein (e.g., enzymatic activity) dependent on native structure for biological activity. Native structure in this sense implies that the protein folds into its active conformation. The preference of human autoantibodies for highly conserved sites explains why many different mammalian species of antigen can readily detect these serological responses. Nonconserved sites are also recognized by autoantibodies as suggested by a study of the differential binding of autoantibodies to human and bovine Ro antigen (Reichlin and Reichtin, 1989). Both conserved and nonconserved antigenic sites may present conformational or linear epitopes. A conformational epitope requires specific topographical orientation with elements of secondary, tertiary or quaternary structure and may consist of amino acid residues discontinuous in the primary structure. A linear epitope consists of short stretches of amino acid residues in a sequential arrangement. Crude or partially purified extracts of transformed cells lines or mammalian tissue are reliable sources of native antigens for immunodiffusion, immunoblotting and immunoprecipitation assays. By contrast, ELISA, an increasingly popular method for serological studies, requires pure antigen for optimal sensitivity and specificity. Antigen purification procedures are typically cumbersome, labor-intensive and generally

668

require large quantities of starting material for a sufficient yield of pure protein. Standardization of antigen preparations is problematic if individual laboratories utilize different purification methods. Moreover, autoantigens that exhibit high-affinity interactions with other cellular macromolecules may be difficult to purify to biochemical homogeneity. The development of recombinant DNA technology provided laboratories with a powerful tool to generate abundant quantities of molecularly defined proteins. These methods make possible not only the overexpression of a single protein (or a fragment of that protein) by a clone of cells, but also allow deduction of the amino acid sequence of that protein from the nucleotide sequence of the cloned complementary DNA (cDNA), or transcribed expression unit. Immunologists hoped that molecular cloning would facilitate the analysis of autoimmune specificity and illuminate the role of autoantibodies in disease pathogenesis. In 1985 the isolation of a cDNA encoding the carboxyl terminus of the human La protein marked the beginning of intensive efforts by many laboratories to characterize the structure and biology of an array of human autoantigens (Chambers and Keene, 1985). Recombinant DNA technology yielded the structure of many human autoantigens, including the 70K (Query and Keene, 1987), A (Sillekens et al., 1987) and C (Yamamoto et al., 1988) proteins of the U I' ribonucleoprotein (RNP) complex, as well as B, B' and D (Elkon et al., 1990; Rokeach et al., 1988) of Sm, 60 kd Ro (Deutscher et al., 1988; Ben-Chetrit et al., 1989), La (Chambers et al., 1988; Sturgess et al., 1988), topoisomerase I (D'Arpa et al., 1988), ribosomal P protein (Rich and Steitz, 1987), 80 kd centromere autoantigen (Eamshaw et al., 1987), Ku (Reeves

and Sthoeger, 1989; Mimori et al., 1990), E2 subunit of the pyruvate dehydrogenase complex (Coppel et al., 1988; Fussey et al., 1988), thyroid peroxidase (McLachlan and Rapoport, 1992) and glutamic acid decarboxylase (Karlsen et al., 1991). The list of cloned antigens continues to grow. Recombinant proteins as initially expressed in bacterial systems are often insoluble in aqueous buffers and require dissolution in detergents, strong chaotropic reagents or urea before use in ELISA; such procedures can influence their antigenicity. Other potential disadvantages of bacterial expression include inefficient production/expression of some recombinant proteins, absence of critical posttranslational modifications and improper folding of the protein into a native structure. The recent development of eukaryotic expression systems overcomes some of these deficiencies and promises to broaden the applicability of recombinant DNA technology.

EcoRI

~A ~B

mRNA

rnRNA cDNA

E

F

Transformants

Positive Screen

obtaining recombinant DNA is the isolation of messenger RNA (mRNA) from a cell line or tissue. The m R N A is reverse transcribed into cDNA, which in turn is converted to double-stranded (ds) DNA. The dsDNA is then ligated into a vector (i.e., a carrier which takes the ligated DNA into bacteria [procaryotes] or into nucleated [eukaryotic] cells) such as a bacterial virus (e.g., bacteriophage )~gtl 1) or a plasmid. A population of ligated vectors representing m R N A from a given tissue constitutes a recombinant cDNA library (Figure 1). A particular cDNA can be selected from a cDNA library by oligonucleotide screening or isolated from a cDNA expression library (i.e., a library expressing the cDNA-determined proteins) by antibody screening. Serum autoantibodies are frequently used as probes to isolate cDNA encoding autoantigens from expression libraries.

Figure 1. Creation of a cDNA Expression Library. The first step of this procedure is to prepare the relevant mRNA from tissue or cells. Bulk mRNA from mammalian cells will encode between 10,000 and 30,000 different polypeptides with varied abundance. A) Synthesis of the first strand of cDNA using RNA-dependent DNA polymerase (reverse transcriptase). After completion of first strand cDNA synthesis, the second strand of cDNA is synthesized using E. coli DNA polymerase I (not shown). B) The double-stranded DNA is then digested at specific sites with a restriction enzyme such as EcoRI, creating numerous small DNA fragments. C) The vector DNA is cleaved with the same restriction enzyme, creating ends that are complementary to the double-stranded cDNA molecules. D) The linearized vector DNA and foreign cDNA are annealed by virtue of their cohesive ends and then joined using bacteriophage T4 DNA ligase. This reaction produces a chimeric, circular, double-stranded plasmid encoding the foreign cDNA. E) A special strain of E. coli is then infected with the chimeric vector by a process termed transformation. The bacteria are grown in media containing ampicillin to select for colonies of bacteria infected with the recombinant plasmids. F) Bacterial colonies encoding the cDNA of interest can be identified by hybridization with radiolabeled nucleic acid probes (e.g., oligonucleotides that match a defined nucleic acid sequence). Alternatively, the bacteria may be induced to express the cloned protein (e.g., cDNA expression library) and then screened using antibodies of defined specificity.

Procaryotic Expression Systems. Recombinant cDNAs are often engineered in bacteriophage or plasmids for expression in special strains of E. coli that are modified to efficiently transcribe and translate cloned proteins (Saitta and Keene, 1992). Standard cloning procedures employ a wide variety of host/vector systems. E. coli has been a valuable host for expression of foreign protein because of the advanced knowledge about its genetics and physiology. Foreign genes are usually expressed in plasmids or bacterio-

phage vectors. Bacteriophage )~ (e.g.,)~gtl 1, )~ZAP) has been a popular vector for use in procaryotic expression systems. It exists as a linear, doublestranded DNA in phage particles that forms a circular molecule after entering the host. Phage vectors contain the minimal elements for gene expression. Bacterial plasmids are double-stranded circular DNA molecules that can be exploited to express foregin DNA. Vectors include an origin of replication for initiation of DNA

Methodology Isolation of Cloned Autoantigens. The first step in

669

synthesis, a selection marker for plasmid propagation and a strong promoter of efficient transcription and translation of foreign proteins. Both types of vectors behave inside cells as separate genetic units that utilize the synthetic machinery of their hosts for replication and expression of the gene product. Genetic markers have been engineered into vectors to facilitate identification of recombinant bacteriophages or plasmids. For example, bacteriophages expressing ~-galactosidase ordinarily form dark blue plaques in the presence of a chromogenic substrate. Replacement of most of the [3-galactosidase gene with foreign DNA results in the formation of colorless plaques, thereby allowing for simple and rapid screening of colonies for recombinants. Sophisticated techniques have been developed to manipulate DNA and enable construction of vectors with the necessary elements to express foreign proteins. Restriction enzymes are powerful tools that cleave DNA by recognition of sites adjacent to a specific nucleotide sequence. They are used to move DNA from one genetic context to the next. Many other enzymes aid the molecular biologist in manipulating DNA and support a wide variety of molecular cloning procedures. Efficient gene expression requires a foreign DNA to be inserted downstream of a strong promoter, which is a regulatory DNA element recognized by the host RNA polymerase. Examples of strong promoters for expression of genes in E. coli include the bacteriophage ~, Piac, hybrid trp-lac and bacteriophage T7 promoters. Translation of the rnRNA is a complex series of events and may be influenced by numerous factors, including the sequence of the cloned gene. Many other factors related to the vector/host system may affect the product yield. The lac operon in ~, phage has been widely used to control the complex process of gene expression. Expression vectors using this system can generate large quantities of foreign protein (Studier et al., 1990). This design produces recombinant proteins with [~-gal sequences at the amino terminus and foreign sequences at the carboxyl terminus of the expressed polypeptide (Figure 2). Such hybrid proteins are often called fusion proteins. Expression of the cloned DNA as a fusion protein has several advantages: 1) the fusion protein is usually produced at high levels because initiation of transcription and translation is directed by normal E. coli sequences; 2) fusion proteins are often more stable in bacteria than native foreign proteins; and 3) the large

670

Polycloning site

"

I ~"" NH 2

I ~,o.,~ Protein I COOH

Figure 2. A plasmid vector (pUC18/pUC19) derived from bacteriophage ~,. Vector systems have been developed for the expression of lacZ fusion genes. LacZ codes for the protein ~-galactosidase (13gal). The pUC18/pUC19 series of plasmids contain the bacteriophage ~ promoter (Plac), which is regulated by a temperature-sensitive repressor that strongly antagonizes its activity. The lacI gene codes for the repressor. The repressor functions normally at low temperatures (e.g., 30~ to inhibit transcription, but is inactivated by higher temperatures (e.g., 42~ This temperature-sensitive repressor permits continued growth of the bacteria in the absence of cDNA transcription. The temperature of the growing cells is shifted from 30~ to 42~ after the bacteria have grown to sufficient density to obtain high levels of transcription of the cloned gene. The ampicillin resistance (ampr) gene codes for an enzyme which inactivates ampicillin by hydrolyzing its ~-lactam ring. Only bacteria containing the recombinant plasmid will grow in the presence of ampicillin. Cloning cDNA adjacent to the lacZ gene will produce a hybrid molecule (e.g., fusion protein) with ~-gal sequences at the amino terminus and cloned protein at the carboxyl terminus.

size of the fusion protein allows for its easy separation on gels from E. coli proteins (Sillekens et al., 1987). Recombinant proteins may be produced in linkage with sequences other than ~-gal, such as anthranilate synthetase (TrpE), glutathione-S transferase (GST), or hexahistidine (His x 6). These fusion moieties have been exploited as "tags" for single-step affinity purification (anti-~-galactosidase antibodies for ~-gal; anti-trpE antibodies for TrpE; glutathione for GST; and Ni +2 for His x 6). The main limitation of prokaryotic expression systems is the potential toxicity of the gene product to the bacterial cell. Other disadvantages include protein instability, insolubility and the absence in bacteria of machinery to modify proteins or regulate folding. Sometimes the reasons behind poor expression of a recombinant protein in bacteria are obscure, compelling the investigator sometimes to undertake alternative approaches.

Eukaryotic Expression Systems. Eukaryotic expression systems have a distinct advantage over prokaryotic expression systems in providing some of the protein modification, processing and transport functions intrinsic to mammalian cells. A popular eukaryotic expression systems utilizes baculovirus, a virus which infects invertebrate cell lines. In this system, a cDNA clone is inserted into a baculovirus transfer vector which is subsequently transfected into insect cells (e.g., Spodoptera frugiperda). The cells are grown in culture where they produce large quantities of the cloned protein (O'Reilly et al., 1992). The baculovirus-based system was employed to express the p70 and p86 subunits of the Ku antigen (Ono et al., 1994) which had been previously expressed only in trace amounts in a bacterial expression system (Reeves and Sthoeger, 1989; Mimori et al., 1990). Other recombinant autoantigens expressed in the baculovirus system include the full-length CENPB protein (Stahnke et al., 1994), histidyl-tRNA synthetase (Raben et al., 1994), the extracellular domain of the pemphigus vulgaris antigen (Amagai et al., 1994) and the Goodpasture antigen (Turner et al., 1994).

CLINICAL UTILITY

Basic Assay Methods. An ELISA is sensitive, quantitative and adaptable for analysis of large number of samples at relatively low labor costs. Recombinant proteins have been successfully used as antigens in ELISA for the detection of autoantibodies. They need not be highly pure for optimal ELISA reliability because the potential for detection of autoantibodies to different antigens of mammalian origin is absent when bacterial lysates are employed. However, autoimmune and normal sera exhibit IgG reactivity with E. coli proteins (Gharavi et al., 1988), leading to "false-positive" signals in recombinant-based ELISAs. This unwanted binding activity can be eliminated, albeit inconveniently, by preincubating the test sera with bacterial lysates from the same bacterial strain used to construct the expression system. Recombinant proteins can also be separated from E. coli proteins on polyacrylamide gels, electrophoretically transferred to nitrocellulose and tested for immunoreactivity using the immunoblotting technique. The relevant bands can be distinguished on blots by incubating with autoantibodies or antisera against either the "sequence tag" (e.g., ~-gal in a fusion

protein) or the cloned protein itself. Immunoblotting is a more labor-intensive method than ELISA and only semiquantitative at best, but does not require a highly purified protein preparation to be informative and when positive is, of course, very useful for confirmation of ELISA results.

Antigenicity of Recombinant Proteins. Most recombinant autoantigens have been derived to date from procaryotic expressirn systems. They are for the most part similar to native proteins in their usefulness as antigens for detection of autoantibodies. The implications of using recombinant proteins in this way are difficult to ascertain due to our incomplete understanding of the specificity of autoimmune responses and the conformation of the autoantigen which is subject to assay. For example, it is believed that most autoantibodies bind to native structures. However, most responses include specificities to conformational as well as linear epitopes. The relative pathogenic and clinical significance of autoantibodies to conformational and linear epitopes remains virtually unexplored. Since recombinant antigens may not adopt their active conformation, they potentially differ both qualitatively and quantitatively from native antigens in reactions with autoantibodies. Eukaryotic expression systems have the advantage of more closely recreating the environment prevailing in the mammalian cell, which increases the likelihood that the recombinant protein will fold into its native state. For example, investigators using procaryotic expression systems had been previously unable to produce a functional thyrotropin (TSH) receptor capable of autoantibody binding. The use of a baculovirus vector expression led to the successful generation of a functional TSH receptor extracellular domain protein which could inhibit autoantibody binding to wild-type TSH receptor on the surface of cultured cells (Chazenbalk and Rapoport, 1995). The recombinant TSH receptor derived from the baculovirus system was heavily glycosylated, unlike its counterpart expressed in procaryotic systems. However, the role of glycosylation in TSH receptor binding to autoantibodies remains unanswered. No studies have been done that directly compare autoantibody binding to an autoantigen expressed in both procaryotic and eukaryotic systems. When expressed as fusion proteins, recombinant autoantigens may contain extra bacterial sequences that produce "false-positive" signals in ELISA. For example, autoimmune sera contain low levels of anti671

~-gal antibodies that bind to 13-gal fusion proteins (St. Clair et al., 1988). Human sera less frequently exhibit anti-TrpE activity (Bini et al., 1990). While absorption with ~-galactosidase and TrpE has only been variably employed by investigators, it is prudent to quantify the potential contribution of this nonspecific binding activity. Fusion proteins are often insoluble as expressed and would not be expected to display native epitopes. For example, La-~-gal (St. Clair et al., 1988) and 60 kd Ro-[3-gal (James et al., 1990) are both recombinant fusion proteins that must be solubilized in urea and immobilized on microtiter wells or nitrocellulose before testing for autoantibody reactivity. Nearly all anti-La sera bind to La-13-gal in ELISA (St. Clair et al., 1988); whereas, a significant number of sera positive for anti-Ro by immunodiffusion fail to react with the 60 kd Ro-[3-gal antigen (James et al., 1990). Recombinant 60 kd Ro engineered to express without the extra [3-gal sequences is also produced as an insoluble protein and, like 60 kd Ro-~-gal, is not recognized by a substantial proportion of anti-Ro sera. One interpretation of these findings is that bacterial expression bypasses the normal folding pathways and results in production of an improperly folded, or denatured 60 kd Ro molecule. The cDNA of an autoantigen may be transcribed and translated in a cell-free system. The advantage of this approach is that the protein can be translated as a soluble antigen in the presence of a radiolabeled amino acid such as 35S-methionine. A radiolabeled antigen can be readily tested by immunoprecipitation for antibody reactivity. Immunoprecipitation assays detect autoantibodies to soluble antigens that may express epitopes not presented by solid phase-bound autoantigen in ELISA or immunoblot assays. For example, some sera failing to bind recombinant 60 kd Ro protein in ELISA immunoprecipitate the soluble translation product of 60 kd Ro protein (Saitta et al., 1994; St. Clair et al., 1994). Other autoantibodies also appear to preferentially recognize conformational epitopes. These include autoantibodies to the nuclear/nucleolar particle termed PM-Scl (Alderuccio et al., 1991), fibrillarin (Lapeyre et al., 1990), thyrotropin receptor (McLachlan and Rapoport, 1993), proliferating cell nuclear antigen (Muro et al., 1994) and glutamic acid decarboxylase (Karlsen et al., 1991). Particular attention must be paid in such cases to the form of the antigen for sensitive and quantitative detection of autoantibodies.

672

Diagnostic and Prognostic Utility Assaying serum autoantibodies is valuable in the diagnostic assessment of patients with suspected autoimmune diseases. Several studies have demonstrated that the sensitivity and specificity of recombinant ELISAs are comparable with those of more conventional immunoassays using native antigens (Table 1). Recombinant human 70K, A, and C [3-gal fusion proteins have been successfully used in ELISA for detection of autoantibodies to U1 RNP, which occur primarily in association with mixed connective tissue disease (MCTD) and SLE (Habets et al., 1989; de Rooij et al., 1990; Wagatsuma et al., 1993; Delpech et al., 1993; St. Clair et al., 1990a). Anti-U1 RNP represents a mixture of antibodies to 70K, A and C. Separating these responses has not proven to be of major clinical value, although a higher frequency of serum anti-70K binding has been described in patients with MCTD than SLE and, thus, may have some diagnostic relevance. The recombinant 70K antigen can discriminate between responses to the 70K U1 RNP antigen and that to a 70 kd antigen recognized by anti-Sm autoantibodies (Habets et al., 1989); however, this capability is primarily of research interest. In ELISA, recombinant human SmB TrpE fusion protein detects anti-Sm with high specificity and greater sensitivity than counterimmunoelectrophoresis (CIE) (Hines et al., 1991), underscoring the potential clinical value of recombinant-based assays which is as yet incompletely assessed. Many other recombinant autoantigens have been valuable in the serological evolution of patients with autoimmune disease. Recombinant human La proteins are excellent antigens for characterizing anti-La responses (Wagatsuma et al., 1993; Delpech et al., 1993; Veldhoven et al., 1992). Autoantibodies to recombinant human CENP-B, as expressed in the baculovirus-based system, are a sensitive and specific serological marker of CREST syndrome (Stahnke et al., 1994). Recombinant ELISAs accurately measure serum autoantibodies to topoisomerase I (Verheijen et al., 1992; Seelig et al., 1993), thyroid peroxidase (Kendler et al., 1990) and the E2 complex of pyruvate dehydrogenase (Leung et al., 1992; Van de Water et al., 1989). Caution must be exercised in other cases. A significant proportion of sera positive for anti-Ro by immunodiffusion do not bind the recombinant human 60 kd Ro antigen in ELISA (James et al., 1990; St. Clair et al., 1994; Wagatsuma et al., 1993; Veldhoven et al., 1992). Thus, immunodiffusion

Table 1. Sensitivity and Specificity of Recombinant-Based ELISAs for Detection of Autoantibodies: A Comparison with Conventional Assays Using Native Antigens Autoantibody

Gold-Standard**

Expression System

Sensitivity %**

Anti-U1 RNP Anti-70K Anti-70K Anti-A

CIE*** IB IB

P P P

85 100 82

93 96 97

Anti-SmB

CIE IB

P P

91 89

86 ND

Hines et al., 1991 Wagatsuma et al., 1993

Anti-60 kd Ro

CIE/IB native ELISA

P P

85 79

94 ND

Veldhoven et al., 1992 Wagatsuma et al., 1993

Anti-La

CIE/IB

P

100

98

native ELISA

P

96

ND

St. Clair et al., 1988; Delpech et al., 1993; Veldhoven et al., 1992 Wagatsuma et al., 1993

Topoisomerase I

IB native ELISA

P P

100 98

100 >99

Verheijen et al., 1992 Seelig et al., 1993

CENP-B*

IIF

E

100

>99

Stahnke et al., 1994

Thyroid Peroxidase

native ELISA

E

89-- 100

95

Kendler et al., 1990

PDH-E2*

IB

P P

93--96

* **

Specificity %**

100

References

St. Clair et al., 1990a Habets et al., 1989 Habets et al., 1989

Leung et al., 1992; Van de Water et al., 1989

CENP-B= Centromere protein B; PDH E2=pyruvate dehydrogenase E2 (dihydrolipoamide acetyltransferase and dihydrolipoamide acyltransferase). Sensitivities and specificities of the recombinant ELISAs were calculated based on a comparison of results obtained by this method and those of an accepted "gold standard" assay, including indirect immunofluorescence (IIF), counterimmunoelectrophoresis (CIE), immunoblotting (IB), and native ELISA with native proteins as antigens. *** CIE detects anti-U1 RNP precipitins, which consist of antibodies to the 70K, A and C proteins of the U1 RNP complex. **** The 89% sensitivity in 11% of patient sera in this study is an artifact that resulted from detection of autoantibodies to thyroglobulins contaminating the native antigen preparation. P prokaryotic. E eukaryotic.

ta~

methods for detection of anti-Ro are more sensitive and cheaper than recombinant assays. A central issue to the clinician is whether quantitative ELISAs, apart from their potential to be highly sensitive and specific tools, are preferable to qualitative methods for the detection of autoantibodies. In general, most studies have shown that the levels of anti-U1 RNP, anti-Sm, anti-60 kd Ro, and anti-La responses in patients with connective tissue diseases do not correlate with disease activity (de Rooij et al., 1990; St. Clair et al., 1990a; 1990b). These assays potentially measure autoantibodies of high as well as low avidity. They may also miss detecting autoantibodies to conformational epitopes not represented by antigen adhered to the solid phase. Further studies are needed to examine this issue more closely. At the moment, the existing evidence does not support longitudinal measurement of serum autoantibodies other than anti-DNA for clinical purposes. The advantage of a quantitative ELISA (recombinant or native) may only possess marginally superior accuracy, which must be weighed against the relatively low cost and simplicity of qualitative methods such as CIE.

REFERENCES Alderuccio F, Chan EK, Tan EM. Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA sequence encoding an apparent 75-kD acidic protein of the nucleolar complex. J Exp Med 1991;173:941-952. Amagai M, Hashimoto T, Shimizu N, Nishikawa T. Absorption of pathogenic autoantibodies by the extra cellular domain of pemphigus vulgaris antigen (Dsg3) produced by baculovirus. J Clin Invest 1994;94:59-67. Ben-Chettlt E, Gandy BJ, Tan EM, Sullivan KF. Isolation and characterization of a cDNA clone encoding the 60-kD component of the human SS-A/Ro ribonucleoprotein autoantigen. J Clin Invest 1989;83:1284--1292. Bini P, Chu JL, Okolo C, Elkon K. Analysis of autoantibodies to recombinant La (SS-B) peptides in systemic lupus erythematosus and primary Sj/3gren's syndrome. J Clin Invest 1990;85:325-333. Chambers JC, Keene JD. Isolation and analysis of cDNA clones expressing human lupus La antigen. Proc Natl Acad Sci USA 1985;82:2115-2119. Chambers JC, Kenan D, Martin BJ, Keene JD. Genomic structure and amino acid sequence domains of the human La autoantigen. J Biol Chem 1988;263;18043-18051. Chazenbalk GD, Rapoport B. Expression of the extracellular domain of the thyrotropin receptor in the baculovirus system using a promoter active earlier than the polyhedrin promoter. Implications for the expression of the functional highly 674

CONCLUSION The availability of recombinant human proteins for solid-phase immunoassays represents an important advance in serodiagnosis. However, immunofluorescence and immunodiffusion techniques still predominate in clinical laboratories for detection of most autoantibodies. One reason that recombinant-based ELISAs have not gained broader acceptance in clinical testing is the lack of evidence that quantification of autoantibody responses other than antinative DNA has clinical value. A potential drawback of this technology is that recombinant autoantigens as expressed in procaryotic systems may not be in the proper form for recognition by autoantibodies. This limitation arises if the autoantibodies preferentially target epitopes present only on the native molecule as is true for many responses. Since available ELISA kits utilize recombinant antigens expresses in procaryotic systems, this problem remains a significant issue. The further development and application of eukaryotic expression systems may surmount these weaknesses and expand the utility of recombinant autoantigens for serological diagnosis.

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polymerase chain reaction. J Immunol 1990;145:636--643. Fussey SP, Guest JR, James OF, Bassendine MF, Yeaman SJ. Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis. Proc Natl Acad Sci USA 1988;85: 8654--8658. Gharavi AE, Chu JL, Elkon K. Autoantibodies to intracellular proteins in human systemic lupus erythematosus are not due to random polyclonal B cell activation. Arthritis Rheum 1988;31:1337-- 1345. Habets WJ, Hoet MH, Sillekens PT, De Rooij DJ, Van de Putte LB, van Venrooij WJ. Detection of autoantibodies in a quantitative immunoassay using recombinant ribonucleoprotein antigens. Clin Exp Immunol 1989;76:172--177. Hines JJ, Danho W, Elkon KB. Detection and quantification of huma~n anti-Sm autoantibodies using synthetic peptide and recombinant SmB antigens. Arthritis Rheum 1991;34:572-579. James JA, Dickey WD, Fujisaku A, O'Brien CA, Deutscher SL, Keene JD, Harley JB. Antigenicity of a recombinant Ro (SSA) fusion protein. Arthritis Rheum 1990;33:102--106. Karlsen AE, Hagopian WA, Grubin CE, Dube S, Disteshe CM, Adler DA, Barmeier H, Mathewes S, Grant FJ, Foster D, Lernmark A. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc Natl Acad Sci USA 1991;88:8337--8341. Kendler DL, Martin A, Magnusson RP, Davies TF. Detection of autoantibodies to recombinant human thyroid peroxidase by sensitive enzyme immunoassay. Clin Endocrinol 1990;33: 751-760. Lapeyre B, Mariottini P, Mathieu C, Ferrer P, Amaldi F, Amalric F, Caizergues-Ferrer M. Molecular cloning of Xenopus fibrillarin, a conserved U3 small nuclear ribonucleoprotein recognized by antisera from humans with autoimmune disease. Mol Cell Biol 1990;10:430-434. Leung PS, Iwayama T, Prindiville T, Chuang DT, Ansari AA, Wynn RM, Dickson R, Coppel R, Gershwin ME. Use of designer recombinant mitochondrial antigens in the diagnosis of primary biliary cirrhosis. Hepatology 1992;15:367-372. McLachlan SM, Rapoport B. The molecular biology of thyroid peroxidase: cloning, expression and role as autoantigen in autoimmune thyroid disease. Endocr Rev 1992; 13:192--206. McLachlan SM, Rapoport B. Recombinant thyroid autoantigens: the keys to the pathogenesis of autoimmune thyroid disease. J Intern Med 1993;234:347--359. Mimori T, Ohosone Y, Hama N, Suwa A, Akizuki M, Homma M, Griffith AJ, Hardin JA. Isolation and characterization of cDNA encoding the 80-kDa subunit of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome. Proc Natl Acad Sci USA 1990;87:1777--1781. Muro Y, Tsai WM, Houghten R, Tan EM. Synthetic compound peptide simulating antigenicity of conformation-dependent autoepitope. J Biol Chem 1994;269:18529-18534. O'Reilly DR, Miller LK, Luckow VA, eds. Baculovirus Expression Vectors. New York: W. H. Freeman and Company, 1992. Ono M, Tucker PW, Capra JD. Production and characterization of recombinant human Ku antigen. Nucleic Acids Res

1994 ;22:3918-3924. Query CC, Keene JD. A human autoimmune protein associated with U1 RNA contains a region of homology that is crossreactive with retroviral p30gag antigen. Cell 1987;51:211-220. Raben N, Nichols R, Dohlman J, McPhie P, Sridhar V, Hyde C, Left R, Plotz P. A motif in human histadyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope. J Biol Chem 1994;269:24277-24283. Reeves WH, Sthoeger ZM. Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J Biol Chem 1989;264:5047-5052. Reichlin M, Reichlin MW. Autoantibodies to the Ro/SS-A particle preferentially with the human antigen. J Autoimmun 1989;2:359--365. Rich BE, Steitz JA. Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly. Mol Cell Biol 1987:7:4065--4074. Rokeach LA, Haselby JA, Hoch SO. Molecular cloning of a cDNA encoding the human Sm-D autoantigen. Proc Natl Acad Sci USA 1988;85:4832--4836. Saitta MR, Keene JD. Molecular biology of nuclear autoantigens. Rheum Dis Clin North Am 1992;18:283--310. Saitta MR, Arnett FC, Keene JD. 60-kDa Ro protein autoepitopes identified using recombinant polypeptides. J Immunol 1994; 152:4192-4202. Seelig HP, Schroter H, Ehrfeld H, Renz M. Autoantibodies against topoisomerase I detected with the natural enzyme and overlapping recombinant peptides. J Immunol Methods 1993;165:241--252. Sillekens PT, Habets WJ, Beijer RP, van Venrooij WJ. cDNA cloning of the human U1 snRNA-associated A protein: extensive homology between U1 and U2 snRNP-specific proteins. EMBO J 1987;6:3841-3848. St. Clair EW, Pisetsky DS, Reich CF, Chambers JC, Keene JD. Quantitative immunoassay, of anti-La antibodies using purified recombinant La antigen. Arthritis Rheum 1988;31: 506-514. St. Clair EW, Query CC, Bentley R, Keene JD, Polisson RP, Allen NB, Caldwell DS, Rice JR, Cox C, Pisetsky DS. Expression of autoantibodies to recombinant (U1) RNP associated 70K antigen in systemic lupus erythematosus. Clin Immunol Immunopathol 1990a;54:266-280. St. Clair EW, Burch JA Jr, Ward MM, Keene JD, Pisetsky DS. Temporal correlation of antibody responses to different epitopes of the human La autoantigen. J Clin Invest 1990b; 85:515--521. St. Clair EW, Burch JA Jr, Saitta M. Specificity of autoantibodies for recombinant 60-kd and 52-kd Ro autoantigens. Arthritis Rheum 1994;37:1373--1379. Stahnke G, Meier E, Scanarini M, Northemann W. Eukaryotic expression of recombinant human centromere autoantigen and its use in a novel ELISA for diagnosis of CREST syndrome. J Autoimmun 1994;7:107-118. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 polymerase to direct expression of cloned genes. Methods

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RED CELL A U T O A N T I B O D I E S Dieter Roelcke, M.D.

Ruprecht-Karls-University Heidelberg, Institute for Immunology, 69120 Heidelberg, Germany

HISTORICAL NOTES An autoantibody to red cells (RBCs), now termed Donath-Landsteiner (DL) antibody, was the first human autoantibody to be described (Donath and Landsteiner, 1904). Autoantibodies to RBCs cause autoimmune hemolytic anemia (AIHA). AIHA was the first autoimmune disease in which autoantibodies were proven to cause the disease. Autoantibodies to red cells are termed warm autoantibodies, cold agglutinins and DL-antibodies. Although antiquated, the names designate characteristics that are typical not only for the optimal reaction temperature of the antibodies but also for the clinical events.

THE AUTOANTIGENS Definition/Origin Antigens recognized by red cell autoantibodies are RBC membrane structures.

Warm Autoantibodies. Warm autoantibodies (WAs) detect antigens originally defined by allo(blood group)-antibodies. Almost every blood group system can be a target for WAs (Table 1) but the Rh system seems to predominate. Because they react with RBCs of all individuals except those with Rhnull RBCs lacking all Rh antigens, WAs could recognize a "Rhcore" structure, but the situation may be more complex. Rhnull RBCs not only lack Rh antigens but lack or have reduced numbers of several antigens and ubiquitous membrane protein components, including Rh-related glycoproteins: CD 47 (the LW glycoprotein), glycophorin B and the Fy glycoprotein. Rh

proteins could be associated with these proteins in the membrane forming-clusters (Cartron and Agre, 1995). Consequently, antigens detected by WAs that do not react with Rhnull RBCs could be antigens on Rh proteins only in combination with other components of the complex or even on other components which manifest full antigenicity only in the complex. Most of the protein components carry determinants to which WAs can be directed, e.g., LW, U, Ss, Fy. Because WAs can react with band 3 glycoprotein, band 3 glycoproteins could be defective in Rhnull RBCs (Victoria et al., 1990). Complexes of the Rh family antigens with band 3 protein and of band 3 protein with glycophorin A, carrying MN and En a antigens, are also responsible for WA binding (Leddy et al., 1993). In rare cases, WAs have (preferential) specificity for distinct Rh epitopes, e.g., anti-e. The overwhelming majority of WAs react with peptide epitopes. As a main example, Rh antigens are not glycosylated proteins. Carbohydrate structures including sialyl groups might influence glycophorin autoantigens (Roelcke, 1994).

Cold Agglutinins. The antigens reactive with cold agglutinins (CAs) differ from antigens recognized by WAs. They are not detected by alloantibodies but are limited to the autoantibody group of CAs. They are carbohydrate antigens that are not influenced by the peptide backbone. Three main groups of antigens are defined on a serological and immunochemical basis (Table 2) (Roelcke, 1995). The first group, the Ii antigens, to which the j antigen was recently added (Roelcke et al., 1994), are protease- and sialidase-resistant, developmentally regulated antigens. I antigen is fully expressed on adult, i antigen is fully expressed on newborn RBCs, and j antigen is fully expressed on both RBCs. The i

677

Table 1. Antigens Shown to be Targets for Warm Autoantibodies Blood group system

Antigen

Rh

Rh ("core"), D, C, E, c, e, f

Glycophorin systems

M., N, S, U, Ena, Wrb, Ge

Kell

K, Kpb, K13, other high incidence Kell system antigens

LW

LWa, LWab"

Kidd

Jka, Jkb, Jk3

Duffy

Fyb

ABO

A,B

Others

Xg a, Vel, Scl, Sc3, Co

Modified from Garratty, 1994; Issitt, 1985.

epitope is represented by linear poly-N-acetyllactosamine or type 2 chains, which are converted into I epitopes in the first year after birth by branching. The j antigen is represented by linear and branched type 2 chains. The second group, the Pr and Sa antigens, are not developmentally regulated but are expressed in equal strength on adult and newborn RBCs. Prl, Pr2, Pr3 are destroyed by proteases and sialidases on the RBC surface; whereas, Pra is sialidase-resistant. Pr and Sa antigens are the O-glycans of glycophorins on the human RBC membrane with immunodominant sialyl (NeuNAc) groups. The third group, the Sia-ll, Sia-bl, Sia-lbl (formerly termed Vo, F1, Gd) antigens, are sialidaselabile but protease-resistant on RBCs. Sia-11 and Siab l antigens are differentiation antigens created by sialylation of linear and branched type 2 chains, respectively (Table 2). Represented by linear as well as branched type 2 chains, the Sia-lbl antigen is not developmentally regulated but is expressed equally on adult and newborn RBCs. It is apparent that Sia-11, Sia-bl and Sia-lbl antigens resemble I, i and j antigens. Because sialylation of type 2 chains abolishes (partially) I, i, j antigens, creating Sia-11, Siab l, Sia-lbl antigens, the antigens are biochemically related but are entirely different immunologically. Donath-Landsteiner Antibodies. The antigen recognized by Donath-Landsteiner (DL)-antibodies is the blood group P of the P system antigens (Worlledge and Rousso, 1965). Individuals with the phenotype p lacking P are very rare (random incidence of 1:150.000). The P antigen is the glycosphingolipid

678

globoside with the sugar sequence GalNAc~ 1-3Gal~l4Gal[~ 1-4G 1c. The cell and tissue distribution of autoantigens varies markedly. For example, Rh antigens are restricted to RBCs, whereas Ii antigens show a wide cell and tissue distribution.

THE AUTOANTIBODIES Factors in Pathogenicity Most WAs belong to the IgG class. IgG WAs can be accompanied by IgA or IgM WAs, but this is not common (Petz and Garratty, 1980). Pure IgA WAs are rare; pure IgM WAs are even rarer. The IgG subclass distribution of WAs shows a high preponderance of IgG 1. The subclass of RBC-bound IgG of 746 patients included 94% with IgG1 on their RBCs, 12% with IgG2, 13% with IgG3 and 3% with IgG4 (Engelfriet et al., 1992). Although 74% had only IgG1 on their RBCs, IgG2, 3, 4 were usually combined with other subclasses. The data on classes and subclasses of WAs point to a polyclonal autoimmune response in WA-induced AIHA, although some evidence suggests that WAs can be restricted in the Gm allotype (Litwin et al., 1973) and the light chain type (Leddy and Bakemeier, 1965). In contrast to WAs, CAs are unique among human RBC antibodies in their greatly restricted heterogeneity. Postinfection CAs are of oligoclonal origin. CAs in chronic CA disease are monoclonal and are the only naturally occurring monoclonal antibodies to RBCs in man. CAs are of IgM isotype except for rare

Table 2. Serological and Biochemical Characterization of Antigens Recognized by Human CAs

I

Expression on RBCs

Effect of enzymes

adult

prot.

+

newborn $

i adult $

1"

sialid, 1"

Designation

Structure

endo[3-g. $

(1) 0 - 0

Expression of the 9G4 idiotype on CAs

branched type 2 chains

\ ,o-n-o-

o-n

i

$

+

+

1"

1"

j

+

+

+

t

t

Sia-bl

+

$

,1,

+

(2) 0 q 2 ] - O - D - O -

linear type 2 chains

--/$

(1) and (2)

linear and branched type 2 chains

--/$

(3) Siac~2-30-Ul \ O-i--i-O-

sialylated branched type 2 chains

Fucotl-2 o - n (4) Siac~2-30-Ul-O-Ul-O-

sialylated linear type 2 chains

+

(3) and (4)

sialylated linear and branched type 2 chains

+

+

tetra/trisaccharides of glycophorins, gangliosides

O-glycans?

+

+

+

glycophorins

O-glycans?

+

+

+

$

+

trisaccharides of glycophorins, gangliosides

O-glycans

+

$

+

$

+

Sia-ll

$

+

+

+

Sia-lbl,2

+

+

+

+

Prl,z,3PrIvI*

+

+

Pr a

+

Sa Lud

--

sialylated type 1 chains?

Note: Prot. = Proteases; sialid. = sialidase; endo-13-g. = endo-[3-galactosidase; + = present; = inactivated; 1" = increased; $ = decreased; * = preferential reaction with M+ RBCs at higher temperatures; O = Gall31-4; Ul = GlcNac[31-3; Sia = N-acetylneuraminic acid; NT = not tested.

IgG and very rare IgA examples. ~:-type light chains predominate exceedingly in CAs. Most IgM CAs (and all IgG and IgA CAs described) are ~-monotypic. Because CAs in chronic CA disease are monoclonal antibodies present in high amounts in patients, structural analyses of the variable (V) regions are possible. CAs with anti-I and anti-i specificity utilize essentially heavy chains encoded by the Vrt4-21 gene segment, which is a m e m b e r of the human VH4 family (Leoni et al., 1991; Pascual et al., 1991;

Pascual et al., 1992; Silberstein et al., 1991). They share the idiotype recognized by the anti-idiotypic antibody 9G4 (Stevenson et al., 1986). This idiotype is defined by an amino acid motif at position 23--25 in the FR1 region of Vrt4-21 encoded heavy chains (Potter et al., 1993). The anti-I light chains use predominantly the V ~ I I families, but ~ chains are also described, especially in association with anti-i specificity (Silberstein et al., 1991). CAs with anti-Pr and a n t i - S i a - l l , Sia-bl, S i a - l b l

679

Table 3. Schematic Description Of Erythrocyte Destruction by Autoantibodies

Mechanisms of red cell destruction 1.

C-activ.

2.

1"

3.

-->

C3b --->C5b-9 $

Antibodies -->

intravascular lysis

DL-antibodies CAs

", ,,

macrophages(Kupffer's cells): phagocytosis:intrahepatic lysis

CAs (WAs)

K cells: ADCC: intralineal lysis

WAs

C3b-receptors

E + Ab

[

-->

Fcy 1,3-receptors

Note: E = erythrocyte; CAs = cold agglutinins; Ab = antibody; WAs = warm autoantibodies; C = complement.

specificities do not express the idiotype recognized by the 9G4 antibody (Smith et al., 1995). Anti-Pr CAs use VHI, VHII and VHIII heavy chains and preferentially (4/5) use VKIV chains that have only been detected by sequence analyses of anti-Pr CAs (Wang et al., 1973). Cross-reacting idiotypes among anti-Pr CAs are known (Feizi et al., 1974) but their structures are not yet identified. DL-antibodies belong to the IgG class. Pathogenetic Role

Autoantibodies to RBCs cause AIHA. Binding of the autoantibodies to RBCs initiates complement activation via the classical pathway by binding of C lq to the Fc part of the RBC membrane-bound antibody with consecutive activation of the C cascade. Complement activation may be arrested at C3, when C3bcoated RBCs are sequestered and partially eliminated by Kupffer cells in the liver (Table 3). Complement activation can also result in formation of the membrane attack complex C5b-9 leading to intravascular RBC lysis. The RBC destruction by CAs and probably by DLantibodies, that causes paroxysmal cold hemoglobinuria (PCH), is exclusively mediated by Complement activation. With WAs, antibody-dependent cellular cytotoxicity (ADCC) predominates. ADCC is mediated by Fc receptor-bearing K cells in the spleen that recognize the Fc parts of RBC-bound IgG WA molecules (Table 3). C3b coating additionally to bound IgG WAs is seen in approximately 50% of patients with WA-induced AIHA (Garratty, 1994). The presence of C3b together with IgG on RBCs enhances markedly the phagocytosis of the RBCs by macrophages.

680

Methods of Detection

Autoantibodies to RBCs are detected on the RBCs and/or in the plasma of the patient. WAs are so-called "incomplete antibodies" coating but not agglutinating RBCs. Because WAs react best at 37~ they are demonstrated on the patient's RBCs by the direct antiglobulin (Coombs) test using polyvalent antisera with the main components antihuman IgG plus antihuman C3 and/or with antihuman IgG. In approximately 50% of cases, C3 (C3d) is also demonstrated on the RBCs using anti-C3. WAs in the plasma are detected by the indirect antiglobulin test (with polyvalent antisera). Agglutination of proteinase-treated RBCs is a sensitive test for the detection of WAs, provided that the antigens they recognize are not destroyed by proteinases on RBCs. CAs are "complete antibodies" agglutinating untreated RBCs. They react best at 0~ and the reaction is reversible at 37~ They are demonstrated in vitro in the patient's plasma at 0 to 4~ The direct antlglobulin test is positive with anti-C3, because CAs can react with RBCs in the peripheral circulation where the temperature drops below 37~ thereby activating complement. RBC-bound C3d remains attached to the RBCs if they recirculate at 37~ in contrast to CA IgM molecules, which are removed from the RBCs at 37~ It is important to perform titration assays with CA-containing sera, because lowtiter CAs (titer < 32) are normally present in human sera. Using enzyme-treated adult and newborn RBCs, several CA specificities can be defined as previously described (Table 2). DL-antibodies are also cold reacting. They are demonstrated in the patient's plasma by the biphasic

Table 4. Diseases Commonly Found in AIHA Patients WAs

CAs

DL-antibodies

+

+

Lymphoma benign gammopathy

B cell (Morb. Waldenstr., CLL) T cell Myelodyslplastic syndrome Infections Autoimmune syndrome (SLE) Drugs (methyldopa) Note: + = present.

DL test. In the first phase, DL-antibodies bind at 0 to 4~ in vitro to RBCs, which are lysed in the second phase at 37~ by complement activation.

CLINICAL UTILITY Disease Associations Detection of WAs and DL-antibodies confirms AIHA by these autoantibodies if other parameters of hemolytic anemia (e.g., LDH increase, reticulocytosis, absence/decrease of haptoglobin) are present. With CAs, only high titers (>32) confirm AIHA. Rare exceptions are low-titer CAs with high thermal amplitude. They may react at body temperature despite their low titer and may cause AIHA (Schreiber et al., 1977). Estimated to occur in one per 40,000--80,000 of the population, 75--80% of AIHA are due to WAs and 20--25% are due to CAs. DL-antibodies are rare (approximately 1%). Combined forms of WAs and CAs are also rare. Both WA- and CA-induced AIHA can occur as "primary" or "idiopathic" conditions or secondary to other diseases. With improvements in diagnosis, e.g., of lymphoproliferative disorders, the ratio is shifting in favor of secondary forms. From the clinical conditions commonly found in patients with secondary AIHA (Table 4), the impression emerges that several conditions are capable of inducing a polyclonal WA autoimmune response, possibly on a genetic background as indicated by an increased frequency of HLA-A1, A8 and B8 (Hawkes and Nourse, 1977; Abdel-Khalik et al., 1980). In contrast, the conditions for CA and DL-antibody

induction are limited, possibly indicating a different mechanism of induction. This possibility is emphasized by the finding that CAs in B-cell lymphomas, including benign gammopathy, are the monoclonal products of the B cells; whereas, WAs are polyclonal also in B-cell lymphoma patients. The significant association between carcinomas and the occurrence of RBC autoantibodies including CAs suggest a disturbance in immune homeostasis in carcinoma patients (Sokol et al., 1994). Although various infections are associated with induction of WAs, associations between specific microbes and distinct WA specificities are not known. DL-antibodies were commonly mentioned in syphilis patients in older literature but not during the past 20 years. DL antibodies are now observed particularly in children with viral infections. In postinfection CA patients, infectious agents are well documented, and distinct CA specificities could be assigned to certain agents (Table 5). Among examples of CAs after rubella and varicella infections described in the last few years, all had the relatively rare specificity anti-Pr (Konig et al., 1992; Herron et al., 1993).

Effect of Therapies The course of postinfection and drug-induced AIHA is transient. In the other forms of AIHA, the autoantibodies persist. Therapy of AIHA is generally aimed at treating the underlying disease. The standard therapy for WA-induced AIHA is treatment with corticosteroids. Splenectomy may be necessary. The main management in CA-induced AIHA is avoiding exposure of the patient to the cold. Transfusions with red cell units must be considered very carefully, because compatible blood will not be available for

681

Table 5. Associations Between Infectious Agents and Cold Agglutinin Specificities

CA specificity

CA incidence (%)

Mycoplasma pneumoniae

anti-I, anti-Sia-bl

50--80

Cytomegalovirus

anti-I

rare

Epstein-Barr virus

anti-i

30-50

Rubella virus

anti-Pr

very rare

Varicella virus

anti-Pr

very rare

AIHA patients. Transfusion may, however, be indispensable even in transient postinfection AIHA in rare cases. In childhood, AIHA differs from AIHA in adults by a marked increase of postinfection, transient, acute AIHA with a much higher frequency of DL-antibodies.

CONCLUSION Autoantibodies to red cells cause AIHA. The t h r e e main groups of autoantibodies are WAs, CAs and DLantibodies. Some minor rare types, i.e., warm hemolysins, may exist (Wolf and Roelcke, 1989). The isotypes of RBC autoantibodies and the antigens they recognize are well known, and the mechanisms of

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RBC destruction induced by binding of the antibodies to RBCs are identified. Based on the monoclonality of CAs, structural analyses of the variable regions of CA heavy and light chains and of the epitopes recognized by CAs have been performed, proving CAs to be an excellent model of autoimmune disease in man. Clinical observations in CA disease document a striking association between infectious agents and CA specificities that indicates the agents are responsible not only for the specificity but also for the production of these autoantibodies. Because these specificities are restricted to postinfection CAs and to CAs produced by B-cell lymphomas, both conditions could be connected (Roelcke, 1989); although the autoimmune regulation in both conditions might be different (Terness et al., 1995).

Herron B, Roelcke D, Orson G, Myint H, Boulton FE. Cold autoagglutinins with anti-Pr specificity associated with fresh varicella infection. Vox Sang 1993;65:239--242. Konig AL, Keller HE, Braun RW, Roelcke D. Cold agglutinins of anti-Pr specificity in rubella embryopathy. Ann Hematol 1992:64:277--280. Leddy JP, Bakemeier RF. Structural aspect of human erythrocyte autoantibodies. I. L chain types and electrophoretic dispersion. J Exp Med 1965;121:1-- 17. Leddy JP, Falany JL, Kissel GE, Passador ST, Rosenfeld SI. Erythrocyte membrane proteins reactive with human (warmreacting) antired cell autoantibodies. J Clin Invest 1993;91: 1672-1680. Leoni J, Ghiso J, Goni F, Frangione B. The primary structure of the Fab fragment of protein KAU, a monoclonal immunoglobulin M cold agglutinin. J Biol Chem 1991;266:2836-2842. Litwin SD, Balaban S, Eyster ME. Gm allotype preference in erythrocyte IgG antibodies of patients with autoimmune hemolytic anemia. Blood 1973;42:241--246. Pascual V, Victor K, Lelsz D, Spellerberg MB, Hamblin TJ, Thompson KM, Randen I, Natvig J, Capra JD, Stevenson FK. Nucleotide sequence analysis of the V regions of two

IgM cold agglutinins: evidence that the VH4-21 gene segment is responsible for the major cross reactive idiotype. J Immunol 1991;146:4385--4391. Pascual V, Victor K, Spellerberg MB, Hamblin TJ, Stevenson FK, Capra JD. VH restriction among cold agglutinins. The VH4-21 gene segment is required to encode anti-I and anti-I specificities. J Immunol 1992;149:2337-2344. Petz LD, Garratty G. Acquired Immune Hemolytic Anemias. New York: Churchill Livingstone, 1980. Potter KN, Li Y, Pascual V, Williams Jr RC, Byres LC, Spellerberg M, Stevenson FK, Capra JD. Molecular characterization of a cross-reactive idiotope on human immunoglobulins utilizing the VH4-21 gene segment. J Exp Med 1993;178:1419-1428. Roelcke D. Cold agglutination. Transfus Med Rev 1989;3:140166. Roelcke D. Sialic acid-dependent red blood cell antigens. In: Garratty G, ed. Immunobiology of Transfusion Medicine. New York: Marcel Dekker, 1994:69--95. Roelcke D, Kreft H, Hack H, Stevenson FK. Anti-j: human cold agglutinins recognizing linear (I) and branched (I) type 2 chains. Vox Sang 1994;67:216-221. Roelcke D. Serology, biochemistry, and pathology of antigens defined by cold agglutinins. In: Cartron JP, Rouger P, eds. Blood Cell Biochemistry. Molecular Basis of Major Human Blood Group Antigens. New York: Plenum Press, 1995;6: 117--152. Schreiber AD, Herskovitz BS, Goldwein M. Low-titer cold agglutinin disease. Mechanism of hemolysis and response to corticosteroids. N Engl J Med 1977;296:1490-1494. Silberstein LE, Jefferies LC, Goldman J, Friedman D, Moore JS, Nowell PC, Roelcke D, Pruzanski W, Rouder J, Silverman GJ. Variable region gene analysis of pathologic human

autoantibodies to the related I and I red blood cell antigens. Blood 1991;78:2372--2386. Smith G, Spellerberg M, Boulton F, Roelcke D, Stevenson F. The immunoglobulin VH gene, VH4-21, specifically encodes autoantired cell antibodies against the I or I antigens. Vox Sang 1995;68:231-235. Sokol RJ, Booker DJ, Stamps R. Erythrocyte autoantibodies, autoimmune haemolysis, and carcinoma. J Clin Pathol 1994;47:340-343. Stevenson FK, Wrightham M, Glennie MJ, Jones DB, Cattan Ar, Feizi T, Hamblin TJ, Stevenson GT. Antibodies to shared idiotypes as agents for analysis and therapy for human B cell tumors. Blood 1986;68:430-436. Terness P, Kirschfink M, Navolan D, Dufter C, Kohl I, Opelz G, Roeckle D. Striking inverse correlation between IgG antiF(ab') 2 and autoantibody production in patients with cold agglutination. Blood 1995;85:548--551. Victoria EJ, Pierce SW, Branks MJ, Masouredis SP. IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein. J Lab Clin Med 1990;115:74-88. Wang AC, Fudenberg HH, Wells JV, Roelcke D. A new subgroup of the kappa chain variable region associated with anti-Pr cold agglutinins. Nature New Bio11973 ;243 :126--128. Wolf MW, Roelcke D. Incomplete warm hemolysins. II. Corresponding antigens and pathogenetic mechanisms in autoimmune hemolytic anemias induced by incomplete warm hemolysins. Clin Immunol Immunopathol 1989;51:68--76. Worlledge SM, Rousso C. Studies on the serology of paroxysmal cold haemoglobinuria (PCH), with special reference to its relationship with the P blood group system. Vox Sang 1965;10:293-298.

683

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RETICULIN AUTOANTIBODIES David Joseph Unsworth, Ph.D.

Department of Clinical Immunology, Southmead Hospital, Bristol BSIO 5ND, UK

Doniach, 1973). Collagen type III- and fibronectinspecific antisera give R1-ARA-like patterns of reactivity by indirect immunofluorescence on tissue sections, but neither pure collagen III nor fibronectin absorb R1-ARA (Unsworth et al., 1982). Six purified collagenase-resistant proteins (18.5-37 kd) from human fetal lung absorb IgA R1-ARA and IgA endomysial antibodies (EMA) but not IgA antigliadin antibodies (AGA) (Maki et al., 1991b). Only R1 and R2 react predominantly with fibrillar extracellular connective tissue fibrils, each giving distinct patterns of reactivity (Table 1). The R1 pattern most closely resembles the staining pattern obtained by silver impregnation. The R2 antibody by comparison has a more restricted pattern of reactivity so that R1 and R2 are easily distinguished from one another by their patterns of reactivity, for example, on rat kidney sections (Figures la, lb). The R1 antigen withstands 10 minutes of methanol fixation while the other ARA antigens do not. Rs, R3 and R4 react predominantly with liver sinusoidal mesenchyme. The Rs antibody, the one

H I S T O R I C A L NOTES First subdivided in 1973 according to their pattern of reactivity with intra and/or extracellular connective tissue components in frozen tissue sections (Rizzeto and Doniach, 1973), antireticulin antibodies (ARA) include five subtypes designated R1, R2, Rs, R3 (reacting with Kupffer cells and originally referred to as "KC" reactive) and R4 (originally referred to as sinusoidal adherent cell or "AC" reactive).

THE AUTOANTIGENS

Definition To this day, the identities of both the argyrophilic fibers which histologists recognize as being "reticulin" and the autoantigen(s) in celiac disease/dermatitis herpetiformis (CD/DH) with which the R1-ARA react are not fully defined. Antisera raised against procollagens do not yield R1 or R2 patterns (Rizzetto and

Table 1. Antireticulin Antibody (ARA) Patterns of Reactivity on Rat Tissue Sections Tissue Substrate Kidney

Stomach

Liver

ARA Types Peritubular Blood vessels Periglomerular

Sinusoids BloodVessels

Hairs in Parenchyma

Submucosal Intergastric Gland Fibers

R1

+

+

+/m

+

+

+

+

R2

--

+

+

+

+

+

~

~

+

Rs

R3 R4

684

Figure la. IgA R1-ARA by indirect immunofluorescence on rat kidney. Part of a glomerulus is seen top left. The reactivitiy is with the peritubular and periglomerular connective tissue fibers. The glomerulus including the basement membrane, and the tubule cells are not stained (x400).

Figure lb. Reactivity of R2 antibody on rat kidney. Reactivity is exclusively with the blood vessels (far left-lower comer, and a smaller vessel toward the lower-fight comer. Reactivity with the internal elastic lamina is noted in both vessels. The peritubular and periglomerular reactivity seen with R1 reactivity (Figure la) is absent. Note: the spots of fluorescence scattered across the picture are due to nonspecific conjugate-related staining.

685

most commonly encountered in routine screening for tissue autoantibodies with rodent tissue sections, gives extensive staining of all sinusoids (Figure 2a). Many of the Rs sera simultaneously react with kidney tubule brush border tissue, suggesting a relationship to heterophile antibodies in these cases (Figure 2b). The heterophile antibodies are typically IgG (Hawkins et al., 1977) and, hence, do not interfere in IgA-based autoantibody tests. R3 and R4 antibody types also react with liver sinusoids but give far more restricted patterns of reactivity. R3 react principally with Kupffer cells as demonstrated by testing on sections of liver derived from rats prefed carbon particles to mark Kupffer cells when viewed under phase-contrast microscopy. R4 react with blood-derived, glassadherent mononuclear cells demonstrated by testing on cytocentrifuged leukocyte preparations. These leukocytes are found within occasional sinusoids and sometimes additionally scattered through other tissues. There is some overlap between the patterns of reactivity defined above. Thus, some R1-ARA sera will show some additional Rs-like staining. The R1-ARA pattern (Table 1) stands out from the others and is the only one of diagnostic and pathogenic interest. Only the R1-ARA reacts on human tissues as well as rodent tissues.

Purified gliadin, the wheat protein which is toxic for patients with gluten-sensitive enteropathy (GSE); binds to reticulin in tissue sections to give a pattern of reactivity which is strikingly similar to that given by both R1-ARA EMA and silver impregnation and quite unlike that gi~)en by any of the other ARA types (Unsworth et al., 1981a).

THE AUTOANTIBODIES

Terminology If reticulin is defined by silver impregnation, then only the R 1-ARA is a genuine contender for bona fide "reticulin" reactivity. The other ARA react with tissue antigens located in areas of tissue rich in reticulin but fail to bind to other sites rich in reticulin.

Pathogenetic Role The occurrence of CD in patients with hypogammaglobulinemia (Webster et al., 1981) is often quoted as evidence that R1-ARA does not play a crucial role in the pathogenesis of CD. This contention is tenuous because many antibody-deficient patients manufacture

Figure 2a. Rs reactivity on rat liver. Note that staining is sinusoidal associated throughout the parenchyma with the hepatocytes unstained. 686

Figure 2b. Some Rs antibodies react with renal tubule brush border, as seen here on sections of rat kidney. Note that the glomeruli and more proximal tubules are unstained. small amounts of functional autoantibodies; autoimmune hemolysis, for example, has been reported in association with hypogammaglobulinemia (Hermaszewski and Webster, 1993). Because R1-ARA are very closely associated with untreated GSE and are not seen in non-gluten-sensitive enteropathies such as cow's milk-sensitive enteropathy (Unsworth et al., 1983), R1-ARA are unlikely to be nonspecific consequences of small bowel damage. Several theories are proposed to account for how gluten ingestion in certain predisposed individuals leads to R1-ARA generation (Unsworth et al., 1985). HLA-DR3 may well be a prerequisite (Maki et al., 1991 a). There is no support for a putative cross-reactivity between gluten and reticulin. Attempts to specifically absorb out R1-ARA with wheat protein preparations including pure gliadin have failed (Unsworth et al., 1985; Maki et al., 1991b). Wheat proteins including gliadins bind selectively to reticulin in human and other mammalian tissue sections (Unsworth et al., 1981a). If similar in vivo binding occurs in CD and DH patients, reticulin autosensitization might generate R1-ARA and explain why R1-ARA is dependent on the continued eating of gluten. In vivo gliadin deposits are not, however, detectable in DH skin (Unsworth et al., 198 l a).

Mixing AGA with gliadin in vitro leads de novo to R1-ARA type reactivity due to the generation of gliadin-containing immune complexes capable of depositing on reticulin fibers (Unsworth et al., 1985), but similar immune complexes are not detected in sera from patients with CD or DH, and there is no evidence that the R1-ARA found in sera from patients represent gliadin-containing immune complexes (Unsworth et al., 1985). There is speculation that R1-ARA might account for the pathognomonic IgA detected in association with reticular connective tissue in the dermal papillae of DH skin, but the absence of serum IgA R1-ARA and IgA EMA (Chlorzelski et al., 1984) in many DH cases casts doubt on this hypothesis. IgA elution studies fail to identify antibody specificity largely because the extraction methods used involve covalent bond-breaking reagents which will denature IgA. But even when milder extractants such as 2M NaC1 at pH 2 were successful, eluted IgA did not react in AGA, ARA, nor EMA assays (Jones et al., 1989). The pathogenesis of R1-ARA thus remains ill understood.

Methods of Detection Indirect immunofluorescence is still the method of

687

Figure 3. IgA R1-ARA by indirect immunofluorescence on rat liver. Reactivity is seen with blood vessel wall connective tissue (center top), and with multiple hair-like reticulin fibrils (e.g., bottom right) scattered throughout the parenchyma. The latter feature is only seen with R1-ARA. In this example, the sinusoids are completely unstained (x400). Some Rl-positive sera also show varying degrees of minor sinusoidal reactivity. choice (Seah et al., 1971). Solid-phase assays are badly needed but await the preparation of R1-ARA autoantigen in an ELISA-compatible form which retains all the crucial epitopes required for autoantibody detection. The tissue reactivity of R1-ARA on rat kidney, liver and stomach (Figures l a, 3 and 4) is characteristic, and similar to the distribution of reticulin as defined by silver impregnation (Figure 5). R1-ARA is the only ARA type that stains the reticular connective tissue fibrils around all the glomeruli and tubules (Table 1 and Figure l a). All ARA types react on liver, but only the R1-ARA shows reactivity with hair-like fibrils in the parenchyma (Figure 3).

Relationships Among R1-ARA, EMA and Antijejunal Antibody (AJA) The interrelationship among R1-ARA, EMA and antijejunal autoantibodies has caused confusion (Table 2). First reported in 1984 (Chlorzelski et al.), this "new" antiendomysial antibody (EMA), like the previously reported R1-ARA, stains monkey esophagus sections in tissue sites rich in reticulin as defined by silver impregnation. IgA R1-ARA, eluted off rat tissue 688

sections, when transferred to monkey esophagus sections shows EMA reactivity (Unsworth, 1995, unpublished data). Both EMA and R1-ARA are closely associated with untreated GSE and disappear on a strict gluten-free diet (Hallstrom, 1989). In 1986, AJA were described in DH using fetal human small bowel as substrate (Karparti et al., 1986); a close association of AJA with GSE is now recognized (Karparti et al., 1990). Strong circumstantial evidence suggests that a single anticonnective tissue autoantibody reactivity accounts for all three appearances, which differ simply as a consequence of the choice of tissue substrate. Thus, the so called AJA, which were initially suspected of being organ-specific, and the EMA, are both absorbed by crude reticulin preparations of human or monkey (but not rodent) origin (Karparti et al., 1990). Ultrastructural studies suggest that both EMA and AJA react with the same amorphous connective tissue component which seems to be distinct from the reticular fibers p e r se (Karparti et al., 1992). Evidence that R1-ARA and EMA are probably one and the same includes the fact that patients with IgA R1-ARA will predictably also be positive in an IgA-EMA test

Figure 4. IgA R1-ARA on rat stomach. A high-power view of the base of the gastric glands is shown. Thick bundles of reticular connective tissue are stained around the clusters of unstained gastric parietal cells. The muscularis mucosea is just seen (top left) (x400).

Figure 5. Silver impregnation of reticulin fibrils in rat kidney. Staining of nuclei in tubular cells and mesangial cells (glomerulus top left) is not seen with R1-ARA (Figure 1). Otherwise, the pattern is strikingly similar to that seen with R1 antibody (see Figure la). 689

(Hallstrom, 1989; Unsworth and Brown, 1994), but the converse is not necessarily true, and the observation that both decline in parallel during a strict gluten-free diet (Hallstrom, 1989). Also, both are absorbed by the same extracts of human connective tissue (Hallstrom, 1989; Maki et al., 199 l b). Rodent tissue preparations of reticulin seem to absorb EMA less reliably than primate-derived reticulin (Hallstrom, 1989). The fact that some EMA-positive sera are R1-ARA negative (Table 2) might reflect the superior sensitivity of the EMA test. R1-ARA but not EMA nor AJA show up in a routine autoantibody screen, because monkey esophagus and human jejunal tissue are not routinely employed. IgA-EMA is the superior diagnostic test (Table 2).

CLINICAL UTILITY Disease Associations

The type 1 or "RI" (R1-ARA), is equivalent to the reticulin antibodies first reported in sera from patients with GSE, CD or DH(Seah et al., 1971; 1973). The other ARA types (R2, Rs etc.) are encountered frequently in routine autoantibody screening, but have no known disease associations and, in contrast to the R1-ARA, tend to be IgG and not IgA isotype (Rizetto and Doniach, 1973; Unsworth and Brown, 1994). R1ARA, especially of IgA isotype, are highly specific for GSE, even when subclinical (Maki et al., 1991 a). R1-ARA tend to be accompanied by other serological markers of GSE such as the IgA antigliadin (wheat protein) antibodies (AGA) and IgA-EMA (Table 2). By contrast, GSE-associated AGA and EMA show no relationship with the other ARA types (Unsworth and Brown, 1994). Knowledge of the existence of the other ARA types and of so-called heterophile patterns is required to prevent irrelevant specificities being misidentified as the highly disease-specific R1-ARA. R1-ARA disappear after several months of a strict gluten-free diet and reappear after gluten reintroduction. The best available data for IgA R1-ARA in adults and children with celiac disease clearly show disappearance of antibody within three to 12 months of a strict gluten-free diet (Unsworth et al., 1981b; Maki et al., 1984; Hallstrom, 1989). In DH, R1-ARA are seen in patients with severe small intestinal atrophy rather than cases with milder enteropathy (Kumar et al., 1976) R1-ARA of the IgA isotype are highly specific for GSE (Unsworth and Brown, 1994).

690

R1-ARA are also present in certain other conditions such as type 1 insulin-dependent diabetes and Down's syndrome, but only because there is a strong association between these and susceptibility to GSE. A recent Italian study of Down's syndrome showed biopsyconfirmed celiac disease in five of the 83 cases studied (Lazzari et al., 1994). In these cases, the presence of R1-ARA points to concurrent GSE (Maki et al., 1995). The early mistaken suspicion that R1 ARA are not specific for GSE (Alp and Wright, 1971; Eade et al., 1977) is probably explained by a combination of factors. IgA R1-ARA are far more disease-specific than the IgG isotype (Eade et al., 1977); IgA-based testing has the advantage of eliminating the other ARA types (IgG class) which were reported in many of the earlier studies. In addition, because CD can present atypically, for example with arthralgias and no gastrointestinal symptoms (Collin et al., 1990, Unsworth and Brown, 1994), classification of R1-ARA as false-positive may well be mistaken in patients who lack the expected clinical features. In fact, the R1-ARA is such a reliable marker that all seropositives irrespective of clinical background merit a biopsy to exclude GSE. R1-ARA of IgG class is also considered highly disease specific, but apparently less sensitive than IgA R1-ARA in detecting celiac disease. In untreated, biopsy-proven cases, the percentage of adults positive for IgA/IgG R1-ARA was on the order of 90%/45%, respectively, and for children, 95%/60%, respectively (Maki et al., 1984; Hallstrom, 1989). Note that the IgA R1-ARA i n these reports shows a sensitivity comparable to that of IgA EMA. Most other investigators agree that the IgA R1-ARA is a more sensitive marker than IgG R1-ARA, but report inferior sensitivities as compared with IgA EMA (Ferreira et al., 1992, Lerner et al., 1994), as discussed in Table 2. All the cases quoted in Table 2 were biopsy proven. The issue is further complicated by reports that R1-ARA can be detected in persons with a normal small bowel biopsy, who on follow-up develop full-blown GSE (Maki et al., 1990; Collin et al., 1993). Seropositives with a normal small bowel biopsy who later develop GSE, are said to have "latent" gluten sensitivity, which may require several years follow-up before enteropathy develops (Collin et al., 1993). Large scale serological surveys (Watson et al., 1992; Unsworth and Brown, 1994) suggest that R1ARA (especially IgA isotype) is an excellent marker of GSE, with a disease specificity of close to 100%.

Table 2. Antibodies Showing a Close Association with Gluten-Sensitive Enteropathy Celiacs Normal Diet

GI Controls

IgA-R 1-ARA Sen

Spe

ppv

npv

Sen

Spe

ppv

npv

Sen

Spe

ppv

npv

Sen

Spe

ppv

npv

Karpati et al., 1990

96

53

ND

ND

ND

ND

84

100

100

79

84

100

100

79

74

96

96

67

McMillan et al., 1991

28

68

ND

ND

ND

ND

89

100

100

96

75

100

100

91

100

100

100

100

Ferreira et al., 1992

21

31

91

99

91

99

100

99

91

100

ND

ND

ND

ND

91

85

45

99

Lemer et al., 1994

28

41

65

100

100

77

97

98

97

98

ND

ND

ND

ND

52

94

87

74

Unsworth et al., 1995

19

37

63

100

100

79

95

94

94

97

ND

ND

ND

ND

89

83

74

94

Key: ND Sen Spe ppv npv

= = = = =

not done sensitivity specificity positive predictive value negative predictive value

R1-ARA EMA AJA AGA

= = = =

IgA-EMA

antireticulin antibody antiendomysial antibody antijejunal antibody anti-gliadin antibody

IgA-AJA

IgA-AGA

CONCLUSION R 1 - A R A and E M A are probably one and the same. W h e n detected in an IgA-specific test, R 1 - A R A are highly specific for GSE. However, assay for IgAE M A is currently the serological test of choice for laboratory evaluation of suspected celiac disease.

REFERENCES Alp MH, Wright R. Autoantibodies to reticulin in patients with idiopathic steatorrhea, celiac disease, and Crohn's disease, and their relation to immunoglobulins and dietary antibodies. Lancet 1971 ;2:682-685. Chorzelski TP, Beutner EH, Sulej J, Tchorzewska H, Jablonska S, Kumar V, Kapuscinska A. IgA antiendomysium antibody. A new immunological marker of dermatitis herpetiformis and coeliac disease. Br J Dermatol 1984;111:395-402. Collin P, Hallstrom O, Maki M, Viander M, Keyrilainen O. Atypical celiac disease found with serological screening. Scand J Gastroenterol 1990;25:245-250. Collin P, Helin H, Maki M, Hallstrom O, Karvonen AL. Follow-up of patients positive in reticulin and gliadin antibody tests with normal small-bowel biopsy findings. Scand J Gastroenterol 1993;28:595-598. Eade OE, Lloyd RS, Lang C, Wright R. IgA and IgG antireticulin antibodies in celiac and nonceliac patients. Gut 1977;18:991-993. Ferreira M, Lloyd Davies S, Butler M, Scott D, Clark M, Kumar P. Endomysial antibody; is it the best screening test for celiac disease? Gut 1992;33:1633-1637. Hallstrom O. Comparison of IgA-class reticulin and endomysial antibodies in celiac disease and dermatitis herpetiformis. Gut 1989;30:1225-1232. Hawkins BR, McDonald BL, Dawkins RL. Characterisation of immunofluorescent heterophile antibodies which may be confused with autoantibodies. J Clin Pathol 1977;30:299307. Hermaszewski RA, Webster ADB. Primary hypogammaglobulinemia: a survey of clinical manifestations and complications. Q J Med 1993;86:31-42. Jones, P, Kumar V, Beutner EH, Chlorzelski TP. A simple method for elution of IgA deposits from the skin of patients with dermatitis herpetiformis. Arch Dermatol Res 1989;281: 406--410. Karpati S, Torok E, Kosnai I. IgA class antibody against human jejunum in sera of children with dermatitis herpetiformis. J Invest Dermatol 1986;87:703--706. Karpati S, Burgin-Wolff A, Krieg T, Meurer M, Stolz W, Braun-Falco O. Binding to human jejunum of serum IgA antibody from children with celiac disease. Lancet 1990;336: 1335-1338. Karpati S, Meurer M, Stolz W, Burgin-Wolff A, Braun-Falko O, Krieg T. Ultrastructural binding sites of endomysium antibodies from sera of patients with dermatitis herpetiformis

692

Small bowel biopsy in seropositives is still advisable before r e c o m m e n d i n g a lifelong gluten-free diet. The R 1 - A R A seen in routine autoantibody testing, if confirmed to be of IgA isotype, are useful in detecting atypical presentations of GSE. The significance of the R2, Rs, R3 and R4 is unknown. See also ENDOMYSIAL AUTOANTIBODIES and GLIADIN ANTIBODIES.

and celiac disease. Gut 1992;33:191-193. Kumar VJ, Hemedinger E, Chorzelski TP, Beutner EH, Valeski JE, Kowalewski C. Reticulin and endomysial antibodies in bullous diseases. Arch Dermatol 1987; 123:1179-- 1182. Kumar PJ, Ferguson A, Lancaster-Smith M, Clark ML. Food antibodies in patients with dermatitis herpetiformis and adult celiac disease relationship to jejunal morphology. Scand J Gastroenterol 1976;11:5-10. Lazzari R, Collina A, Arena G, Corvaglia L, Marzatico M, Vallini M, Bochicchio A, Pasetti A, Frassineti S, Forchielli L. Celiac disease in children with Down's syndrome. Pediatr Med Chir 1994;16:467--470. Lerner A, Kumar V, Lancu TC. Immunological diagnosis of celiac disease: comparison between antigliadin, antireticulin, and antiendomysial antibodies. Clin Exp Immunol 1994;95: 78--82. Maki M, Hallstrom O, Vesikari T, Visakorpi JK. Evaluation of a serum IgA-class reticulin antibody test for the detection of childhood celiac disease. J Paediatr 1984;105:901-905. Maki M, Holan K, Koskimies S, Hallstrom O, Visakorpi JK. Normal small bowel biopsy followed by celiac disease. Arch Dis Child 1990,65:1137-1141. Maki M, Holm K, Lipsanen V, Hallstrom O, Viander M, Collin P, Savilahti E, Koskimies S. Serological markers and HLA genes among healthy first-degree relatives of patients with celiac disease. Lancet 1991a;2:1350--1353. Maki M, Hallstrom O, Martinen A. Reaction of human noncollagenous polypeptides with celiac disease autoantibodies. Lancet 1991b;338:724-725. Maki M, Huupponen T, Holm K, Hallstrom O. Seroconversion of reticulin antibodies predicts celiac disease in insulindependent diabetes mellitus. Gut 1995;36:239--242. McMillan SA, Haughton DJ, Biggart JD, Edgar JD, Porter KG, McNeill TA. Predictive value for celiac disease antibodies to gliadin, endomysium, and jejunum in patients attending for jejunal biopsy. Br Med J 1991;303:1163-1165. Rizetto M, Doniach D. Types of reticulin antibodies detected in human sera by immunofluorescence. J Clin Pathol 1973;26: 841--847. t Seah PP, Fry L, Hoffbrand AV, Holborow EJ. Tissue autoantibodies in dermatitis herpetiformis and adult celiac disease. Lancet 1971;i:834-836. Seah PP, Fry L, Holborow EJ, Rossiter M, Doe WF, Maglahes AF, Hoffbrand AV. Antireticulin antibody: incidence and diagnostic significance. Gut 1973;14:311-315. Unsworth DJ, Johnson GD, Haffenden G, Fry L, Holborow JE. Binding of wheat gliadin in vitro to reticulin in normal and

dermatitis herpetiformis skin. J Invest Dermatol 1981a;76: 88--93. Unsworth DJ, Manuel TD, Walker-Smith JA, Campbell CA, Johnson GD, Holborow EJ. A new immunofluorescent blood test for gluten-sensitivity. Arch Dis Child 1981b;56:864868. Unsworth DJ, Scott DL, Walton KW, Walker-Smith JA, Holborow EJ. Failure of R1 antireticulin antibody to react with fibronectin, collagen type III, or the noncollagenous reticulin component (NCRC). Clin Exp Immunol 1982;57: 609--613. Unsworth DJ, Walker-Smith JA, Holborow EJ. Gliadin and reticulin antibodies in childhood celiac disease. Lancet 1983;1:874-875. Unsworth DJ, Walker-Smith JA, McCarthy D, Holborow EJ. Studies on the significance of the R1 antireticulin antibody

associated with gluten sensitivity. Int Archs Allergy Appl Immunol 1985;76:47--51. Unsworth DJ, Brown DL. Serological screening suggests that adult celiac disease is underdiagnosed in the UK and increases the incidence by up to 12%. Gut 1994;35:61-64. Unsworth DJ, Brown DL, Pitcher M, Neale G. Comparison of serology and measurements of abnormal small bowel permeability in patients undergoing small bowel biopsy for possible celiac disease. 1995 (submitted). Watson, RGP, McMillan SA, Dickey W, Biggart JD, Porter KG. Detection of undiagnosed celiac disease with atypical features using antireticulin and antigliadin antibodies. Q J Med 1992;84:713-718. Webster ADB, Slavin G, Shiner M, Platts Mills TAE, Asherson GL. Celiac disease and severe hypogammaglobulinemia. Gut 1981;22:153-157.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RETINAL AUTOANTIBODIES Charles E. Thirkill, Ph.D.

Ophthalmology Research, University of California, Davis Medical Center, Sacramento, CA 95816, USA

HISTORICAL NOTES Inquiries into the pathogenesis of many types of unexplained loss of vision have included the possibility of autoimmune involvement, either as a primary cause or as a contributory secondary effect. Retinopathies of unknown cause and those recognized as being inherited or age related continue to be investigated for evidence of immunologic involvement which might contribute to the degradation process. The phenomenon of sympathetic ophthalmia exemplifies how severe vision loss can result from autoimmune reactions when the host loses tolerance to the sequestered antigens within the eye (Krause-Mackiw, 1990). Belief in the susceptibility of the neurosensory retina to similar immune-mediated damage stems from the early reports on retinal hypersensitivity in which extracts of ocular tissues incited experimental autoimmune ocular inflammation in laboratory animals (Elschnig, 1910). Experimentally induced loss of tolerance to well-defined retinal proteins demonstrates that the retina contains proteins with characteristics comparable to the recognized neurologic autoantigen, myelin basic protein; both share the ability to produce organ-specific autoimmune disease. Uveitis patients, particularly those with Behcet's disease and the Vogt-Koyanagi-Harada syndrome, present with clear indications of immunologic involvement in the form of intraocular leukocytes and a demonstrable response to immunosuppressive treatment (Chan et al., 1985). However, autoimmunity can function at more subtle levels, such as that seen in patients with paraneoplastic retinopathies, often recognized by high concentrations of antiretinal antibodies, with few if any indications of intraocular inflammation (Jacobson et al., 1990; Thirkill et al., 1993a; 1993b; 1993c; Keltner et al., 1992).

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Not all examples of intraocular inflammation involve the retina. Anterior chamber components, the iris and ciliary body, can be damaged by iridocyclitis occurring in diseases such as rheumatoid arthritis (Uchiyama et al., 1989). Vision loss involving the frontal regions of the eye, in the context of recognized or suspected autoimmune disease, naturally falls under suspicion of "guilt by association", but the ocular antigens involved have yet to be identified. This contrasts with the retina where most investigative studies into autoimmune involvement in vision loss focus on a small collection of retinal proteins which exhibit remarkable autoantigenic characteristics.

THE AUTOANTIGEN(S) Definition/Nomenclature There are currently five recognized retinal autoantigens (Table 1). The first to be discovered, the retinal S-antigen identified in experiments on the production of autoimmune retinopathies in guinea pigs (Wacker et al., 1977), is implicated in the ocular inflammation of uveitis. Rhodopsin, recognized as a potent experimental autoantigen (Schalken et al., 1988), is not yet implicated in any form of human retinopathy. Autoantibodies to the interphotoreceptor retinolbinding protein (IRBP), are reported in a variety of human retinopathies (Wiggert et al., 1991; Hoekzema et al., 1990); whereas, phosducin, demonstrably autoantigenic in experimental animals, is not associated with any recognized form of human vision loss (Dua et al., 1992). The 23 kd cancer-associated retinopathy (CAR) autoantigen (Thirkill et al., 1987), is exceptional in that autoantibody reactions with this retinal protein are

Table 1. The Five Recognized Retinal Autoantigens Capable of Inducing Experimental Autoimmune Uveitis in Laboratory Animals Retinal Protein

Molecular Size (kd)

Association with Human Retinal Hypersensitivity

IRBP

135-- 140

Weak correlation with Behcet's disease and other forms of uveitis.

S-antigen

48--50

Incriminated in many forms of uveitis and the vision loss of multiple sclerosis

Rhodopsin*

40

No correlation

Phosducin

33

No correlation

The CAR autoantigen

23

Cancer associated retinopathy

*Rhodopsin mutations are associated with inherited retinopathies, such as autosomal dominant retinitis pigmentosa, but not any recognized autoimmune disease.

found only in cancer patients with severe retinopathies (Polans et al., 1993). Cloning and sequencing the CAR autoantigen identified it as the photoreceptor component recoverin (Thirkill et al., 1992), a protein involved in the cyclic response of rhodopsin to light. Autoantibody reactions with the CAR autoantigen recoverin are recognized as an immunologic marker for one form of paraneoplastic retinopathy, the CAR syndrome, which is associated with a variety of neoplasia, especially small-cell carcinoma of the lung. The high correlation of this retinal autoantibody reaction with cancer-induced retinopathy led to the first commercially available blood test for the early diagnosis of immune-mediated vision loss. Recoverin autoantibody reactions distinguish CAR from other forms of paraneoplastic retinopathy, such as melanoma-associated retinopathy: the MAR syndrome. The cancer-induced vision loss of CAR and MAR occur in association with cancers derived from neuroendocrine tissues. The neuronal origins of the causal carcinomas are implicated in the initiation of the retinal autoimmune reactions which characterize the two syndromes. Retinal bipolar cells are reportedly involved in the autoantibody reactions of MAR, but no single disease-associated protein comparable to the CAR antigen has yet been found (Milam et al., 1993; Weinstein et al., 1994). Other suspected retinal antigen targets for autoimmune reactions in cancer patients include components of neurofilaments and ganglion cells (Kornguth et al., 1986). Early research into the cause of retinal autoantibody reactions in paraneoplastic retinopathy patients produced evidence of the expression of a collection of retinal antigens by lung cancers (Kornguth, 1989). Many forms of paraneoplasia are now considered to be immune-mediated, with pathogenesis emanating

from cancers aberrantly expressing specific autoantigens (Anderson, 1989; Lennon and Lambert, 1989; Furneaux et al., 1989). The discovery of cancer cells actively expressing the 23 kd CAR autoantigen provided tangible evidence of the antigenic stimulation responsible for inducing the autoimmune reactions of the CAR syndrome (Thirkill et al., 1989; Thirkill, 1994).

AUTOANTIBODIES Definition/Characteristics

Suspected and recognized antibody-mediated autoimmune diseases include myasthenia gravis, thyroiditis and several distinct forms of paraneoplasia including the Lambert-Eaton myasthenic syndrome (LEMS), subacute cerebellar degeneration, encephalomyeloneuropathy and paraneoplastic pemphigus (Kim, 1986; Liu et al., 1993). In general, however, all autoimmune diseases described as antibody-mediated, can also be passively transferred with T cells. Indeed, while any given part of the immune system may predominate in the production of autoimmune disease, it will not be alone, but in communication and coordination with the intricate workings of the system as a whole. Evidence supporting autoantibody involvement in vision loss has accumulated from a series of studies in which the actions of immunoglobulins were demonstrated to contribute to retinal malfunctions. Antibodymediated immediate hypersensitivity and the actions of complement play a role in the production of experimental autoimmune uveitis (de Kozak et al., 1985; de Kozak et al., 1981; Faure and de Kozak, 1981 ; Marak et al., 1979).

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The importance of autoantibodies in the production of paraneoplasias has been found in the LambertEaton myasthenic syndrome (LEMS) and paraneoplastic pemphigus. Both these forms of paraneoplasia can be passively transferred to experimental animals, using only the patient's serum antibodies (Drachman, 1990; Liu et al., 1993; Newson-Davis, 1988; Lang et al., 1983; Kim, 1986; Anhalt et al., 1990). Inquiries into the clinical significance of autoantibodies in paraneoplastic retinopathies have accordingly included attempts at passive transfer to experimental animals, using serum antibodies from patients with paraneoplastic retinopathy. These studies are encouraged by the successful experimental production of retinopathic effects in cats with infusions of antibodies reactive with retinal proteins (Kornguth et al., 1982), and alterations in ERG performance induced with antiretinal S-antigen antibodies (Stanford et al., 1992). Data issuing from such studies identified the ability of autoantibodies to influence the workings of the retina and increases suspicion of their involvement in the types of vision loss in which autoantibodies have been demonstrated. Microbial diseases such as onchocerciasis, toxoplasmosis, AIDS and hepatitis often include retinopathies which can be the first indication of occult infection. Although microbial-induced retinopathies involve localization of pathogens within the retina, they are not uncommonly associated with the production of retinal autoantibodies (Zhou et al., 1994). Autoantibodies reactive with retinal antigens are also demonstrable in a variety of human retinopathies such as diabetes, retinitis pigmentosa and age-related macular degenerations (Gurne et al., 1991). In these situations, autoantibodies can contribute to some of these retinopathies by hastening retinal decay but are not implicated as the cause and are better described as epiphenomena stimulated by retinal degradation of unrelated etiology (Reid et al., 1987). Experience teaches that the simple demonstration of autoantibodies in retinal disease does not incriminate immunoglobulins in the pathogenesis of the patient's retinopathy. Their presence may reflect nothing more than a cleaning-up process following unrelated disease, surgery or trauma. Exceptional types of human retinopathies are linked with the actions of autoantibodies, functioning through activation of complement and cytokine and antibody-dependent cell cytotoxicity. The most convincing evidence of autoimmune involvement appears in cancer patients who experience secondary retinopa-

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thies unrelated to intraocular metastasis. With the exception of those resulting from inherited chromosomal abnormalities, paraneoplastic retinopathies exhibit a cause-and-effect relationship not readily apparent in other forms of vision loss. Although rare, cancer-induced ocular degenerations hold the potential to supply much information on the means whereby damage to the eye can result from a disease process located distant from the globe, in some other organ. The quiet retinal degeneration in paraneoplastic retinopathies may prove most informative concerning the culpability of retinal autoantibodies in certain types of vision loss. Histologic characteristics of paraneoplastic retinopathies include intraretinal immune complexes and small foci of leukocyte infiltration (Adamus et al., 1993). Investigations into the pathogenesis and progress of paraneoplastic retinopathies should reveal the means whereby retinal antibodies gain access to the eye and initiate the cascade of events leading to vision loss. Idiopathic retinopathy patients presenting with a history of abnormal electroretinogram readings may be experiencing subclinical allergic reactions which can be identified through immunologic investigations. Antibody assays with retina and cancer-related ocular antigens are recommended for recognized and suspected cancer patients, who lose vision rapidly while complaining of a decrease in color vision, night blindness and flashing lights (Jacobson et al., 1990). Patients presenting with this triad of symptoms may benefit from the simple blood tests now readily available (Athena Diagnostics, Worcester, Massachusetts and San Francisco, California). Disease association of these assays is impressive, supported by examples of the early recognition of occult cancer through the demonstration of related autoantibodies (Lafeuillade et al., 1993).

Pathogenetic Role Access to the retina is a key requirement for autoantibodies to impart any pathological influence. The blood-brain barrier normally prevents entry of components of the immune system to the so-called "immunologically privileged" components of the central nervous system (CNS). Experimental autoimmune retinopathies illustrate that leaks in the barrier can be produced with bacterial toxins, permitting access of an activated immune response to the sequestered antigens of the eye (Gery, 1994; Adamus, 1994). Blood/CNS

barrier leaks are evident in some retinopathy patients through the demonstration of autoantibodies in their cerebrospinal fluid. The possibility exists that such leaks could be a result of secondary bacterial infections which provide the toxins necessary to influence the barrier. Methods of Detection

Methods of detecting antiretinal autoantibody reactions begin with an extract of whole retina, followed by a dissection of the patient's autoantibody reactions to determine if it includes autoantibody reactions with any of the five recognized autoantigens. These more precise secondary procedures utilize molecularly cloned antigens in immunoblot, "dot-blot" and ELISA analyses. Molecular techniques ensure that positive results are occurring with the recognized autoantigens and not other retinal antigens with similar molecular characteristics.

experimental animals and uveitis patients (Weiner et al., 1994; Dick et al., 1993; Gregerson et al., 1993). It is of interest that antigenic epitopes matching those of the S-antigen appear in normal gut flora including yeast cells and Escherichia coli (Singh et al., 1989a, 1989b). Such ubiquitous dissemination of autoantigenic epitopes is relevant to the antigen processing which occurs in gut-associated lymphoid tissue and its influence on the maintenance of immunologic tolerance to recognized autoantigens. The implications of this phenomenon could be extensive, involving the critical maintenance of tolerance necessary for the survival o f the visual system. The successful inhibition of retinopathies by oral and nasal instillations of whole retina extract suggests this approach may function in the suppression of autoimmune retinopathy involving all retinal autoantigens.

CONCLUSION CLINICAL UTILITY The clinical value of autoantibody assays with retina varies. Autoantibody reactions naturally change with the stage of the disease when titers fluctuate considerably. Significance is influenced by the retinal antigen(s) involved, according to their disease relationship, which may increase as clinical data accumulate and new retinal antigens with connections to specific forms of vision loss are identified. Increasing autoantibody titers coupled with their decline following immunomodulation provides substance to suspicion of autoimmune involvement. The S-antigen has been the subject of the most research into the characteristics of uveitogenic retinal proteins and surpasses all others as a suspected cause or contributor to the human retinopathies, including vision loss in multiple sclerosis (Ohguro et al., 1993). No comparable immunologic incrimination has been described for phosducin, rhodopsin or the IRBP. Although each is known to induce experimental autoimmune uveitis in laboratory animals, there is little evidence for association in human ocular ailments. Autoimmune reactions involving the 23 kd CAR autoantigen are cancer-specific, and rare in comparison to those implicating S-antigen involvement. That certain autoantigenic epitopes of the Santigen can abrogate autoimmune reactions is shown by oral and nasal administrations of retinal proteins in

Reports of autoantibodies reactive with specific ocular autoantigens stimulate interest in determining precisely the role of autoimmunity in vision loss in humans. In addition to the recognized benefits of oral and nasal desensitization procedures using retinal extracts, attempts to intercept the autoantibody reactions which characterize some of the human retinopathies using established procedures such as immunoglobulin and anti-idiotype therapy are anticipated. The application of pooled human immunoglobulins to re-establish homeostasis in a patient experiencing autoimmune disease may work by providing a return to the immunologic equilibrium lost in the disease process. Anti-idiotypic reactions almost certainly play a role in whole immunoglobulin treatment which must induce a variety of feedback-suppressive actions resulting from the recipients' immune response to the complex components of the donor antibodies which are recognized as alien and, therefore, antigenic (Hall, 1993). Specific desensitization procedures, applying either the autoantigen(s) or corresponding autoantibodies, provide the opportunity to intercept autoimmune reactions without compromising the host's defensive immune system. These forms of mediation have been demonstrated most effective in the abrogation of Santigen produced experimental uveoretinitis, a predominantly T-cell-induced disease, but which has

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significant autoantibody involvement (de Kozak, 1990; de Kozak and Mirshahi, 1990). Therapeutic intervention using specific autoantigens and autoantibodies

may prove useful in humans when the major antigen(s) involved in the patient's retinal hypersensitivity are recognized.

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Adamus G, Guy J, Schmeid JL, Arendt A, Hargrave PA. Role of antirecoverin autoantibodies in cancer-associated retinopathy. Invest Ophthalmol Vis Sci 1993;34:2626--2633. Adamus G, Ortega H, Witkowska D, Polans A. Recoverin: a potent uveitogen for the induction of photoreceptor degeneration in Lewis rats. Exp Eye Res 1994;59:447--456. Anderson NE. Antineuronal autoantibodies and neurological paraneoplastic syndromes. Aust N Z J Med 1989;19:379-387. Anhalt GJ, Kim SC, Stanley JR, Korman NJ, Jabs DA, Kory M, Izumi H, Rattle H 3rd, Mutasim D, Ariss-Abdo L, et al. Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia. N Engl J Med 1990;323: 1729--1735. Chan CC, Palestine AG, Nussenblatt RB, Roberge FG, Benezra D. Antiretinal autoantibodies in Vogt-Koyanagi-Harada syndrome, Behcet's disease, and sympathetic ophthalmia. Ophthalmology 1985;92:1025-1028. de Kozak Y, Sainte-Laudy J, Benveniste J, Faure JP. Evidence for immediate hypersensitivity phenomena in experimental autoimmune uveoretinitis. Eur J Immunol 1981; 11:612--617. de Kozak Y, Mirshahi M, Sainte-Laudy J, Thillaye B, Faure JP. Experiemental autoimmune uveoretinitis in athymic rats: specific IgE response to retinal S-antigen and disease. Immunol Lett 1985;9:109-- 115. de Kozak Y. Regulation of retinal autoimmunity via the idiotypic network. Curr Eye Res 1990;9:S 193-$200. de Kozak Y, Mirshahi M. Experimental autoimmune uveoretinitis: idiotypic regulation and disease suppression. Int Ophthalmol 1990;14:43--56. Dick AD, Cheng YF, McKinnon A, Liversidge J, Forrester JV. Nasal administration of retinal antigens suppresses the inflammatory response in experimental allergic uveoretinitis. A preliminary report of intranasal induction of tolerance with retinal antigens. Br J Ophthalmol 1993;77:171--175. Drachman DB. How to recognize an antibody-mediated autoimmune disease. In: Waksman BH, ed. Immunologic Mechanisms in Neurologic and Psychiatric Disease. New York: Raven Press, 1990;183-- 186. Dua HS, Lee RH, Lolley RN, Barrett JA, Abrams M, Forrester JV, Donoso LA. Induction of experimental autoimmune uveitis by the retinal photoreceptor cell protein, phosducin. Curr Eye Res 1992;11 :S 107-S 111. Elschnig A. Studien zur sympathischem Ophthalmie. Albrecht von Graefes Arch. Klin Ophthalmol 1910;75:459. Faure JP, de Kozak Y. Cellular and humoral reactions to retinal antigen: specific suppression of experimental uveoretinitis. In: Helmsen RJ, Suran A, Gery I, Nussenblatt RB, eds. Immunology of the Eye. Workshop 2. Washington, DC: Information Retrieval Inc., 1981:33--48. 698

autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180. J Clin Invest 1993;92:2480--2488. Marak GE Jr., Wacker WB, Rao NA, Jack R, Ward PA. Effects of complement depletion on experimental allergic uveitis. Ophthalmic Res 1979;11:97-- 107. Milam AH, Saari JC, Jacobson SG, Lubinski WP, Feun LG, Alexander KR. Autoantibodies against retinal bipolar cells in cutaneous melanoma-associated retinopathy. Invest Ophthalmol Vis Sci 1993;34:91-- 100. Newson-Davis J. Lambert-Eaton myasthenic syndrome: a review. Monogr Allergy 1988;25:116--124. Ohguro H, Chiba S, Igarashi Y, Matsumoto H, Akino T, Palczewski K. Beta-arrestin and arrestin are recognized by autoantibodies in sera from multiple sclerosis patients. Proc Natl Acad Sci USA 1993;90:3241-3245. Polans AS, Burton MD, Halyt TL, Crabb JW, Palczewski K. Recoverin, but not visinin, is an autoantigen in the human retina identified with a cancer-associated retinopathy. Invest Ophthalmol Vis Sci 1993;34:81--90. Schalken JJ, Winkens HJ, van Vugt AH, Bovee-Geurts PH, de Grip WJ, Broekhuyse RM. Rhodopsin-induced experimental autoimmune uveoretinitis: dose-dependent clinicopathological features. Exp Eye Res 1988;47:135--45. Singh VK, Yamaki K, Donoso LA, Shinohara T. Sequence homology between yeast histone H3 and uveitopathogenic site of S-antigen: lymphocyte cross-reaction and adoptive transfer of the disease. Cell Immunol 1989a; 119:211-221. Singh VK, Yamaki K, Abe T, Shinohara T. Molecular mimicry between uveitopathogenic site of retinal S-antigen and Escherichia coli protein: induction of experimental autoimmune uveitis and lymphocyte cross-reaction. Cell Immunol 1989b; 122:262--273. Stanford MR, Robbins J, Kasp E, Dumonde DC. Passive administration of antibody against retinal S-antigen induces electroretinographic supernormality. Invest Ophthalmol Vis Sci 1992;33:30--35. Thirkill CE, Roth AM, Keltner JL. Cancer-associated retinopathy. Arch Ophthalmol 1987;105:372--375. Thirkill CE, Fitzgerald P, Sergott RC, Roth AM, Tyler NK, Keltner JL. Cancer-associated retinopathy (CAR syndrome) with antibodies reacting with retinal, optic-nerve, and cancer cells. N Engl J Med 1989;321:1589-1594. Thirkill CE, Tait RC, Tyler NK, Roth AM, Keltner JL. The

cancer-associated retinopathy is a recoverin-like protein. Invest Ophthalmol Vis Sci 1992;331:2768-2772. Thirkill CE, Keltner JL, Tyler NK, Roth AM. Antibody reactions with retina and cancer-associated antigens in 10 patients with cancer-associated retinopathy. Arch Ophthalmol 1993a; 111:931-937. Thirkill CE, Tait RC, Tyler NK, Roth AM, Keltner JL. The cancer connection: an antigen immunologically related to the retinal CAR antigen is expressed in small cell carcinoma of the lung. In: Dernouchamps JP, ed. Proceedings of the Third International Symposium on Uveitis. Amsterdam: Kugler Publications, 1993b:133-135. Thirkill CE, Tait RC, Tyler NK, Roth AM, Keltner JL. Intraperitoneal cultivation of small-cell carcinoma induces expression of the retinal cancer-associated retinopathy antigen. Arch Ophthalmol 1993c;111:974--978. Thirkill CE. Cancer-associated retinopathy. The CAR Syndrome. Neuro-Ophthalmol 1994;14:297-323. Uchiyama RC, Osborn TG, Moore TL. Antibodies to iris and retina detected in sera from patients with juvenile rheumatoid arthritis with iridocyclitis by indirect immunofluorescence studies on human eye tissue. J Rheumatol 1989;16:1074-1078. Wacker WB, Donoso LA, Kalsow CM, Yankeelov JA Jr., Organisciak DT. Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina. J Immunol 1977;119: 1949--1958. Weiner HL, Friedman A, Miller A, Khoury SJ, al-Sabbagh A, Santos L, Sayegh M, Nussenblatt RB, Trentham DE, Hailer DA. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu Rev Immunol 1994;12:809--837. Weinstein JM, Kelman SE, Bresnick GH, Kornguth SE. Paraneoplastic retinopathy associated with antiretinal bipolar cell antibodies in cutaneous malignant melanoma. Ophthalmology 1994;101:1236--1243. Wiggert B, Kutty G, Long KO, Inouye L, Gery I, Chader GJ, Aguirre GD. Interphotoreceptor retinoid-binding protein (IRBP) in progressive rod-cone degeneration (prcd)-biochemical, immunocytochemical and immunologic studies. Exp Eye Res 1991;53:389--398.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RETROVIRAL ANTIBODIES Martin Herrmann, Ph.D. and Joachim R. Kalden, M.D., Ph.D.

Department of Medicine III, Institute for Clinical Immunology and Rheumatology, Friedrich-Alexander University of Erlangen-Nurenberg, Erlangen 91054, Germany

HISTORICAL NOTES Viruses, especially retroviruses have been suggested as causative agents for autoreactivity in human diseases (Kalden et al., 1991, Krieg and Steinberg, 1990). In ungulates, caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV) or maedi-visna virus are well-known causes of autoimmune diseases (Crawford et al., 1980). In man, lentiviruses are considered candidates for induction of autoimmunity. In some animal models, proteins of endogenous retroviruses are associated with the autoimmune status. Thus, populations of anti-DNA, anti-Sm RNP, and anti-gp70 antibodies appear to constitute a network of autoantibodies in MRL-lpr/lpr mice and a common ancestor of these autoantibodies is discussed (Migliorini et al., 1987). In MRL-lpr/lpr mice, integration of a retrovirus into the fas gene is reported to interfere with T cell apoptosis with resultant survival of autoreactive T cells and an autoimmune phenotype (Mountz and Talal, 1993). Deposits of retroviral proteins in kidneys of humans with systemic lupus erythematosus were first described in 1976 (Mellors and Mellors, 1976). Detection of antibodies to C-type retroviruses in these kidney deposits suggested the presence of immune complexes, including retroviral protein (Mellors and Mellors, 1978). The association of retroviruses with autoimmune rheumatic diseases in humans and animals was recently reviewed (Kalden and Gay, 1994). Humans infected with HIV-1 sometimes present rheumatological symptoms (Kaye, 1989) such as a Sj6gren's syndrome (SS)-like disease with antibodies to Ro and La (Talal, 1991). Furthermore, the depletion of CD 4-positive T cells in HIV infection might be at least partially due to an autoreactive CTL response

700

(Salemi, 1995). Viruses might influence the immune system by modification or release of sequestered cellular proteins or by polyclonal activation of B cells, release of lymphokines and superantigen activity. Anti viral antibodies might also be deleterious to the host by molecular mimicry, anti-idiotypic antibodies or formation of immune complexes (Schattner and Rager-Zisman 1990).

THE AUTOANTIGENS Characteristics

Infectious retroviruses contain at least three proteins: (1) the protein encoded by the pol gene, the reverse transcriptase, is responsible for the viral replication; (2) the env gene products form the viral envelopes; and (3) during virus maturation, the polyprotein encoded by the gag gene (group-specific antigens) is cleaved into small units, which associate with the viral RNA and form the viral core. In the lentiviruses, multiple splice events lead to the production of mRNAs coding for proteins, which are mainly responsible for viral gene regulation. As retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase, the viral genome can be integrated into the host's nuclear DNA. Multiple integration events during evolution of humans have lead to a high copy number of mostly truncated retrovirus-related DNA in the human genome. In contrast to mice and other animals, however, most, if not all the endogenous retroviruses of humans have lost their infectivity (i.e., their ability to be transcribed into infectious particles) and are simply

replicated as part of the human genome. Some rare descriptions of human endogenous retrovirus particles, e.g., the isolation of the (endogenous?) retroviral element LM 7 from a patient with multiple sclerosis (MS) (Perron et al., 1992) have never conclusively proven their infectious potency. Furthermore, the data could not be confirmed by other investigators. Nevertheless, in several human tissues, retrovirus-related transcripts (Herrmann and Kalden, 1994) and even particles can be detected; endogenous retroviral sequences are known to be involved in the immune regulation of mice (Krieg et al., 1989). With a monoclonal antibody against HTLV-1 p19 gag, positive reactions were demonstrated in salivary glands of 12/39 SS patients, 4/17 RA patients, and 3/14 patients with sicca symptoms. The antigen, which is distinct from the human endogenous sequence HRES-1, can be induced in the salivary gland of normal healthy donors by PHA or IFNy (Shattles et al., 1992). By epitope mapping with recombinant and truncated La protein fragments, the most distinct autoepitope (amino acid 81--101) had a striking sequence similarity to a retroviral gag polyprotein (Kohsaka et al., 1990). Using PCR with DNA from peripheral blood mononuclear cells of 21 patients with multiple sclerosis of the chronic progressive type, 6/21 were positive for pol sequences from HTLV-1 and three patients were additionally positive with env PCR. Long terminal redundancy (LTR) and gag-specific DNA could not be detected (Greenberg et al., 1989). Patients with adult T-cell leukemia and tropical spastic paraparesis served as positive controls. Nevertheless these data are still under debate, as these results could not be reproduced by other investigators (Lisby, 1993). The expression of HTLV-1-related antigens in bone marrow-derived cells from multiple sclerosis patients (Sandberg-Wollheim, 1988) and the demonstration of tax DNA in 2/9 patients with Sj6gren's syndrome by PCR (Mariette et al., 1993) led to the hypothesis that an HTLV-l-related endogenous sequence may be involved in the etiopathogenesis of these diseases, because no exogenous HTLV-1 RNA was found by in situ hybridization (Hauser et al., 1986).

CLINICAL UTILITY

Disease Association SjiJgren's Syndrome (SS). The presence of antibodies

against HIV-1 or HLTV-1 in patients with SS is still debated. Reports that sera of about 30% of SS patients, which showed a low reactivity against Ro and La, contained antibodies to p24 gag of HIV-1 (Talal et al., 1990a; Talal et al., 1992) were not confirmed by other investigators using a recombinant p24 ELISA (Hermann et al., 1992). The positive finding might reflect a special group of SS patients in southern USA, but more likely reflects technical difficulties including cross-reactivity of test sera with cellular protein contaminants in the antigen preparation as isolated virions were used as antigen source. Nevertheless, among SS patients negative in the conventional HTLV-1 immunoblot, 32% react with a peptide of HRES- 1/HTLV- 1 related endogenous sequences), which is similar to a HTLV-1 p 19gag peptide (Brookes et al., 1992, Mariette et al., 1993). Furthermore, retroviral particles isolated from T-cell lines after coculturing with salivary gland homogenates from SS patients are antigenically related to HIV (Garry et al., 1992).

Systemic Lupus Erythematosus (SLE). Mapping of the recognition sites from autoantibodies against U1 snRNPs revealed that the epitopes were clustered on the p68 component. The sequence similarity of one of the p68 epitopes to a retroviral gag-encoded protein and the autoantibody specificities argue for an antigen-driven autoimmune response instead of random mutations of the immune globulin genes (Guldner et al., 1988). Antibodies to retroviral proteins, most frequently to HIV p24 gag and p55 gag (immunoblot with virus isolates), were found in 14/22 SLE and 5/8 DLE (discoid LE) patients but not in eight subacute cutaneous LE sera. The best clinical correlation was observed to severe skin lesions and recurrent infections (Ranki et al., 1992). Among SLE patients, 22/61 reacted with p24 gag in conventional immunoblot with HIV-1 isolates as antigen and in 20/22 p24-reactive antibodies, the 4B4 idiotype was demonstrated. This idiotype is often found on anti-Sm autoantibodies. In competition studies, cross-reactivity of anti-Sm with p24 gag of HIV-1 was confirmed (Talal et al., 1990b). A shared proline-rich epitope between p24 and Sm nucleoprotein may be the molecular basis for the described cross-reactivity (Talal et al., 1992). However, the data on p24 antibodies in SLE are apparently dependent on the assay system employed, because other authors did not find a significant antip24 reactivity in SLE sera using a recombinant ELISA (Herrmann et al., 1992), and there is no 701

association between antibodies to retroviral proteins and lupus anticoagulant (Matsuda et al., 1994). Among Sm-positive SLE patients, 20% are reported to have antibodies to a major epitope of HTLV-1 p19 gag peptides, which were cross-reactive with HRES-1 sequences, in absence of reactivity in a diagnostic HTLV-1 virus immunoblot (Brookes et al., 1992). A reported increased antibody of human SLE sera to the baboon endogenous retrovirus correlated with the presence of antibodies to RNP, Sm, and some retroviral env- and gag-derived peptides, which were similar to U1 snRNP. The latter finding correlated with discoid rash and other symptoms (Blomberg et al., 1994).

Other Rheumatic Disorders. In MCTD, the 70kd protein, associated with U1 snRNP has a 23 amino acid region with similarity to p30 gag of the murine leukemia virus. The region was recognized by autoantibodies to U1 snRNP and encompassed the site of immunological cross-reactivity as shown by epitope mapping (Query and Keene 1987). However, the most amino-terminal region of the 70kd RNP, which is similar to p30 gag from murine retrovirus was only seldom recognized by MCTD sera (Nyman et al., 1990). Antibodies to p24 gag and p55 gag native HIV-1 isolates were found in 6/8 MCTD patients (Ranki et al., 1992). Reactivity against gag-related proteins (p 10, p 12, p15, p30, p40 gag and p65 gag) can also be detected in patients with autoimmune connective tissue disorders by immunoblotting, but because some reactivity is also seen in normal sera, cross-reactivity might reflect reaction with cellular proteins (Rucheton et al., 1985). A minority of patients with rheumatological diseases (4/30 patients with rheumatoid arthritis, 3/13 patients with polymyositis/dermatomyositis and 2/5 patients with SLE) showed a weak cross-reactivity of antibodies to antigens of HTLV-1, but PCR did not detect HTLV-1 or HIV-1 DNA (Nelson et al., 1994). The expression of HTLV-1-related antigens in the synovial lining cells of patients with rheumatoid arthritis was confirmed (Trabant et al., 1992). Multiple Sclerosis (MS). In a peptide ELISA, 23% of MS patients had antibodies to a major epitope of HTLV-1 p 19gag, which is cross-reactive to an HRES-1 sequence and 19% reacted with the corresponding peptide of HRES-1. In conventional immunoblots, no serum reacted with HTLV-1 (Brookes et al., 1992). These results were confirmed by HTLV-1 ELISAs for 702

sera and cerebrospinal fluid of patients with MS as compared to healthy controls (Hauser et al., 1986). Because molecular genetic techniques (PCR) do not support a role for HTLV-I-like viruses in MS (Lisby 1993), anti-p24 reactivity reported in sera from patients with MS might reflect difficulties in distinguishing patients with MS from those with HTLV-1associated myelopathy (HAM) or tropical spastic paraparesis (Grimaldi et al., 1988). Immunoblots against virus lysates in Norwegian MS patients failed to detect HTLV-1, HIV-1, HIV-2 and SIV antibodies but revealed distinctive and reproducible reactivity against cellular proteins (Brokstad et al., 1994); the reported seropositivity to HTLV-1 in some MS patients probably reflects reaction with cellular proteins of the host. Nevertheless, isolation of the (endogenous?) retroviral element LM 7 from a patient with MS and reactivity of sera from other MS patients with the isolated proteins may point to an involvement of a novel retrovirus in the pathogenesis of the disease (Perron et al., 1992).

Insulin-Dependent Diabetes Mellitus (IDDM). A subgroup of the IDDM patients is characterized by autoantibodies to insulin. Among sera with such autoantibodies to insulin, 66% react with p73 gag of the murine intracisternal A-type particles, consistent with a molecular mimicry mechanism for the etiology of a subgroup of human IDDM (Hao et al., 1993). Retroviral antibodies in human autoimmune diseases react predominantly with gag-derived proteins or peptides. A possible explanation for this finding is the high degree of conservation of the gag antigen leading to a cross-reactivity of antibodies to the gag derived proteins from various exogenous as well as endogenous retroviruses.

CONCLUSION The clearly defined roles of exogenous and endogenous retroviruses in some animal models of autoimmune diseases led to speculations on the involvement of such viruses in human diseases. Despite intensive research in this field over a long period of time, molecular data on retroviral involvement in human autoimmune disorders are rather scarce. In ungulates, infections with CAEV, EIAV and Maedi-Visna virus cause well-defined autoimmune disease, and the appearance of seropositivity of the

Table 1. Retroviral Target Structures Recognized by Human Autoantibodies Diagnosis

Protein

Gene

Virus

Reference

SS

n.s.

gag

n.s.

Kohsaka et al., 1990

SS

p24

gag

HIV-1

Talal et al., 1990a; 1992

SS

p 19

gag

HTLV I- 1

Brookes et al., 1992; Mariette et al., 1993

SS

n.s.

n.s.

HIV-1

Garry et al., 1992

SLE

p30

gag

C-type oncovirus

Mellors and Mellors, 1976; 1978

SLE

n.s.

gag

n.s.

Guldner et al., 1988

SLE

p24, p55

gag

HIV-1

Ranki et al., 1992

SLE

p24

gag

HIV-1

Talal et al., 1990a; 1992

SLE

p19

gag

HTLV-1

Brookes et al., 1992

SLE

n.s.

env, gag

baboon endogenous RV

MCTD

p30

gag

MuLV

Query and Keene, 1987

MCTD

p30

gag

n.s.

Nyman et al., 1990

MCTD

p24, p55

gag

HIV-1

Ranki et al., 1992

CTD

pl0, p12, p15, p30, p40, p65

gag

MuLV

Rucheton et al., 1985

MS

p19

gag

HTLV-1

Brookes et al., 1992; Hauser et al., 1986

MS

n.s.

n.s.

LM 7

Perron et al., 1992

IDDM

p73

gag

intracisternal A-type particles

Hao et al., 1993

Note: n.s. = not specified; SS = Sj~3gren's syndrome; SLE = systemic lupus erythematosus; MCTD = mixed connective tissue disease; MS = multiple sclerosis; IDDM = insulin-dependent diabetes mellitus.

animals is of diagnostic value. L i k e w i s e , in the M R L l p r / l p r m i c e antibodies against e n d o g e n o u s p70 are i n v o l v e d in k i d n e y destruction and again are therefore suitable for predicting k i d n e y disease. In case of h u m a n a u t o i m m u n e diseases, n o n e of the assays suitable for the d e m o n s t r a t i o n of retroviral antibodies

is useful for diagnostic or differential diagnostic purposes. H o w e v e r , detection of retroviral antibodies in h u m a n a u t o i m m u n e disease by indisputable, vigorous assays with p r o p e r controls is u n d o u b t e d l y important with regard to the search of retroviruses as disease-causing agents (Table 1).

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response to and expression of cross-reactive retroviral gag sequences in autoimmune disease. Br J Rheumatol 1992;31: 735--742. Crawford TB, Adams DS, Cheevers WP, Cork LC. Chronic arthritis in goats caused by a retrovirus. Science 1980;207: 997-999. Garry RF, Fermin CD, Hart DJ, Alexander SS, Donehower LA, Luo-Zhang H. Detection of a human intracisternal A type retroviral particle antigenically related to HIV. Science 1992;250:1127-1129. Greenberg SJ, Ehrlich GD, Abbott MA, Hurwitz BJ, Waldmann TA, Poiesz BJ. Detection of sequences homologous to human

Blomberg J, Nived O, Pipkorn R, Bengtsson A, Erlinge D, Sturfelt G. Increased antiretroviral antibody reactivity in sera from a defined population of patients with systemic lupus erythematosus. Correlation with autoantibodies and clinical manifestations. Arthritis Rheum 1994;37:57-66. Brokstad KA, Kalland KH, Page M, Nyland H, Haaheim L. Serum antibodies from MS patients do not recognize HTLVI, HIV-1, HIV-2 and SIV. APMIS 1994;102:514-520. Brookes SM, Pandolfino YA, Mitchell TJ, Venables PJ, Shattles WG, Clark DA, Entwistle A, Maini RN. The immune

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retroviral DNA in multiple sclerosis by gene amplification. Proc Natl Acad Sci USA 1989;86:2878--2882. Grimaldi LM, Roos RP, Devare SG, Casey JM, Maruo Y, Hamada T, Tashiro K. HTLV-I-associated myelopathy: oligoclonal immunoglobulin G bands contain anti-HTLV I p24 antibody. Ann Neurol 1988;24:727--731. Guldner HH, Netter HJ, Szostecki C, Lakomek HJ, Will H. Epitope mapping with a recombinant human 68 kDa (U1) ribonucleoprotein antigen reveals heterogeneous autoantibody profiles in human autoimmune sera. J Immunol 1988;141: 469-475. Hao W, Serreze DV, McCulloch DK, Neifing JL, Palmer JP. Insulin (auto) antibodies from human IDDM cross react with retroviral antigen p73. J Autoimmun 1993;6:787--798. Hauser SL, Aubert C, Burks JS, Kerr C, Lyon Caren O, de The G, Brahic M. Analysis of human T-lymphotrophic virus sequences in multiple sclerosis tissue. Nature 1986;322:176-177. Herrmann M, Baur A, Nebel-Schickel H, Vornhagen R, Jahn G, Krapf FE, Kalden JR. Antibodies against p24 of HIV-1 in patients with systemic lupus erythematosus? Viral Immunol 1992;5:229-231. Herrmann M, Kalden JR. PCR and reverse dot hybridization for the detection of endogenous retroviral transcripts. J Virol Methods 1994;46:333-348. Kalden JR, Gay S. Retroviruses and autoimmune rheumatic diseases. Clin Exp Immunol 1994;98:1-5. Kalden JR, Winkler TH, Herrmann M, Krapf F. Pathogenesis of SLE: immunopathology in man. Rheumatol Int 1991; 11: 95--100. Kaye BR. Rheumatologic manifestations of infection with human immunodeficiency virus (HIV). Arthritis Rheum 1989;18:225--239. Kohsaka H, Yamamoto K, Fujii H, Miura H, Miyasaki N, Nishioka K, Miyamoto T. Fine epitope mapping of the human SS-B/La protein. Identification of a distinct autoepitope homologous to a viral gag polyprotein. J Clin Invest 1990;85:1566-1574. Krieg AM, Gause WC, Gourley MF, Steinberg AD. A role for endogenous retroviral sequences in the regulation of lymphocyte activation. J Immunol 1989;143:2448--2451. Krieg AM, Steinberg AD. Retroviruses and autoimmunity. J Autoimmun 1990;3:137--166. Lisby G. Search for an HTLV-I-like retrovirus in patients with MS by enzymatic DNA amplification. Acta Neurol Scand 1993;88:385-387. Mariette X, Agbalika F, Daniel MT, Bisson M, Lagrange P, Brouet JC, Morinet F. Detection of human T lymphotropic virus type I tax gene in salivary gland epithelium from two patients with Sj6gren's syndrome. Arthritis Rheum 1993;36: 1423--1428. Matsuda J, Gotoh M, Gohchi K, Tsukamoto M, Saitoh N. Absence of an association between antibodies to retroviral proteins and anticardiolipin antibody and/or lupus anticoagulant in systemic lupus erythematosus. Ann Rheum Dis 1994;53:352--353. Mellors RC, Mellors JW. Antigen related to mammalian type-C RNA viral p30 proteins is located i n renal glomeruli in

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human systemic lupus erythematosus. Proc Natl Acad Sci USA 1976;73:233-237. Mellors RC, Mellors JW. Type C RNA virus-specific antibody in human systemic lupus erythematosus demonstrated by enzymeimmunoassay. Proc Natl Acad Sci USA 1978;75: 2463--2467. Migliorini P, Ardman B, Kaburaki J, Schwartz RS. Parallel sets of autoantibodies in MRL-lpr/lpr mice. An anti-DNA, antiSmRNP, anti-gp70 network. J Exp Med 1987;165:483--499. Mountz JD, Talal N. Retroviruses, apoptosis and autogenes. Immunol Today 1993;14:532--536. Nelson PN, Lever AM, Bruckner FE, Isenberg DA, Kessaris N, Hay FC. Polymerase chain reaction fails to incriminate exogenous retroviruses HTL-I and HIV-I in rheumatological diseases although a minority of sera cross react with retroviral antigens. Ann Rheum Dis 1994;53:749--754. Nyman U, Lundberg I, Hedfors E, Pettersson I. Recombinant 70-kD protein used for determination of autoantigenic epitopes recognized by anti-RNP sera. Clin Exp Immunol 1990;81:52--58. Perron H, Gratacap B, Lalande B, Genoulaz O, Laurent A, Geny C, Mallaret M, Innocenti P, Schuller E, Stoebner P, et al. In vitro transmission and antigenicity of a retrovirus isolated from a multiple sclerosis patient. Res Virol 1992; 143"337--350. Query CC, Keene JD. A human autoimmune protein associated with U1 RNA contains a region of homology that is crossreactive with retroviral p30 gag antigen. Cell 1987; 51:211220. Ranki A, Kurki P, Riepponen S, Stephansson E. Antibodies to retroviral proteins in autoimmune connective tissue disease. Relation to clinical manifestations and ribonucleoprotein autoantibodies. Arthritis Rheum 1992;35:1483-- 1491. Rucheton M, Graafland H, Fanton H, Ursule L, Ferrier P, Larsen CJ. Presence of circulating antibodies against gaggene MuLV proteins in patients with autoimmune connective tissue disorders. Virology 1985;144:468-480. Salemi S, Caporossi AP, Boffa L, Longobardi MG, Barnaba V. HIV gpl20 activates autoreactive CD4-specific T-cell responses by unveilling of hidden CD4 peptides during processing. J Exp Med 1995;181:2253--2257. Sandberg-Wollheim M, Alumets J, Biorklund A, Gay R, Gay S. Bone marrow derived cells express human T-cell lymphotropic virus type I (HTLV-I)-related antigens in patients with multiple sclerosis. Scand J Immunol 1988;28:801--806. Schattner A, Rager-Zisman B. Virus-induced autoimmunity. Rev Infect Dis 1990;12:204--222. Shattles WG, Brookes SM, Venables PJ, Clark DA, Maini RN. Expression of antigen reactive with a monoclonal antibody to HTLV-1 P19 in salivary glands in Sj6gren's syndrome. Clin Exp Immunol 1992;89:46-51. Talal N, Dauphinee MJ, Dang H, Alexander SS, Hart DJ, Garry RF. Detection of serum antibodies to retroviral proteins in patients with primary Sj6grens syndrome (autoimmune exocrinopathy). Arthritis Rheum 1990a;33:774-781. Talal N, Garry RF, Schur PH, Alexander S, Dauphinee MJ, Livas IH, Ballester A, Takei M, Dang H. A conserved idiotype and antibodies to retroviral proteins in systemic

lupus erythematosus. J Clin Invest 1990b;85:1866-1871. Talal N. AIDS and Sj6gren's syndrome. Bull Rheum Dis 1991 ;40:6--8. Talal N, Flescher E, Dang H. Are endogenous retroviruses

involved in human autoimmune disease? J Autoimmun 1992;5:61-66. Trabant A, Gay RE, Gay S. Oncogene activation in rheumatoid synovium. APMIS 1992;100:861--875.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RHEUMATOID FACTORS Marie BCrretzen, M.Sc., Ove J. Mellbye, M.D., Ph.D., Keith M. Thompson, Ph.D. and Jacob B. Natvig, M.D., Ph.D.

Institute of Immunology and Rheumatology, The National Hospital, N-O172 Oslo, Norway

HISTORICAL NOTES Initially described in 1940 as antibodies against gamma globulins (Waaler, 1940), rheumatoid factors (RF) were among the first autoantibodies identified. Although named after the disease they were initially associated with, RFs are found in various other diseases and in the healthy population. The frequent occurrence and strong association with rheumatoid arthritis (RA) led to the hypothesis that RFs have a role in the pathogenesis of RA. RFs in RA patients also react with autologous IgG (Williams and Kunkel, 1963), and are indeed produced by plasma cells in the inflamed synovial tissues (Natvig et al., 1989). Despite decades of study, the role of RF in the pathogenesis of RA and other conditions remains to be fully elucidated.

THE AUTOANTIGENS Definition RFs are autoantibodies directed against the C-terminal part of the constant region of the IgG heavy chain, the IgG Fc. They react with native IgG, but more strongly with aggregated or denatured IgG in immune complexes. This reaction may be kinetically favored by the cross-linking of many antigen epitopes. RFs recognize several determinants distributed among the four subclasses of human IgG on the two Fc domains, CH2 and CH3 (Natvig et al., 1972). These determinants are frequently isotypic determinants, expressed on one or more IgG subclass. Due to polymorphism within the IgG subclass regions, there are also allotypic determinants which can interact with

706

RFs. They also frequently react with IgG from other species, particularly rabbit IgG (Williams and Kunkel, 1963; Tonder, 1962). RFs thus recognize a diverse range of antigenic determinants on native and denatured IgG.

Origin In normals, IgG, the predominant immunoglobulin (Ig) isotype (8-16 mg/mL), diffuses more readily than other Ig classes into extravascular body spaces. High concentrations of antigen are thus available in most compartments of the human body and T cells consequently develop tolerance against the IgG Fc. In normal serum, the predominant (Bogen, 1993) IgG subclass is IgG1 followed by IgG2, IgG3 and IgG4. Elevated concentrations of particular IgG subclasses are found in the synovial membranes of patients with RA (Munthe and Natvig, 1972). Several studies have found elevated levels of IgG3 in the rheumatoid synovium compared to normal blood (Munthe and Natvig, 1972, Hoffman et al., 1982, Mellbye et al., 1990). The subclass of highest concentration in the synovium is IgG1 as in normals (Mellbye et al., 1990). As well as this bias in IgG subclasses, the structure of serum IgG differs in some diseases compared to health, including reported deficiencies of galactose in serum IgG from patients with RA and SLE (Mullinax and Mullinax, 1975). Agalactosylated IgG is also found in patients with a variety of chronic inflammatory diseases, with or without elevated levels of RF (Rademacher et al., 1988). Some monoclonal RFs from patients with RA bind better to agalactosylated than normal IgG (Soltys et al., 1994), but the relation between agalactosylated IgG, RFs and different diseases is still unclear as are the mecha-

nisms that may result in the alteration of IgG. Human IgG, denatured by oxygen-free radicals, has increased reactivity with RF (Lunec et al., 1988); free radicals produced by neutrophils could possibly alter IgG in vivo and increase reactivity with RFs.

Sources Purified IgG and IgG Fc are available from several commercial sources. For further characterization of RF, myeloma proteins of varying IgG subclasses and their fragments including CH domains are valuable in revealing the fine specificity of RFs (Natvig et al., 1972). Human monoclonal IgG from hybridoma cells and genetically engineered antibodies can be used for the same purposes. Four main methods are used to purify IgG: salt fractionation by ammonium sulphate precipitation, size fractionation by gel filtration chromatography or ultracentrifugation, ion-exchange chromatography and affinity chromatography with protein A or protein G (Hudson and Hay, 1989). For further isolation of the Fc and fragments of this, IgG can be enzymatically digested by papain or mild pepsin, followed by ion-exchange or affinity chromatography to separate the fragments (Natvig and Turner, 1993).

AUTOANTIBODIES Terminology The term "rheumatoid factors," which is used for autoantibodies against the IgG Fc fragment, is still the most common name, even though RFs are also found in several nonrheumatoid conditions and normals. Other designations like anti-IgG and anti-y-globulins are perhaps more descriptive but do not limit the reactivity to IgG Fc and are, therefore, broader than RF.

Pathogenetic Role Human Diseases. RFs are found both in the healthy population and several disease conditions (Table 1). In health, RFs or RF-producing B cells have physiological roles in cleating complexes, enhancing the avidity of IgG antibodies or presenting antigen to T cells (Van Snick et al., 1978; Roosnek and Lanzavecchia, 1991). The roles of RFs in various diseases are still under investigation. The diseases commonly asso-

Table 1. Frequency of Rheumatoid Factors in some Diseases Disease

%RF

Rheumatoid Arthritis

50-90

Systemic lupus erythematosus

15-35

SjOgren's syndrome

75-95

Systemic sclerosis

20-30

Polymyositis/Dermatomyositis

5-- 10

Cryoglobulinemia

40-100

MCTD

50-60

This table is modified from Shmerling and Delbanco, 1991.

ciated with high RF concentrations are RA and Sj6gren's syndrome. Much evidence suggests that RFs have a pathogenic role in RA (Table 2). The RFs in RA are mainly of IgM, IgG and IgA isotypes (Procaccia et al., 1987), but IgE and IgD RFs are also reported (Gioud-Paquet et al., 1987; Banchuin et al., 1992). Fluctuation in concentrations of IgA RF in RA patients reportedly correlates with grip strength, erythrocyte sedimentation rate and composite index of clinical parameters (Withrington et al., 1984). IgG RF concentrations are associated with changes in erythrocyte sedimentation rate and grip strength, but levels of IgM RF show only weak association with fluctuation in erythrocyte sedimentation rate. Even in fluctuations of IgM, RFs do not correlate with clinical variables; elevated concentrations of IgM RFs correlate with RA disease activity (Robbins et al., 1986) and vasculitis (Veys et al., 1976) and are considered a risk factor for RA in normal subjects (Walker et al., 1986). In juvenile rheumatoid arthritis (JRA) patients, seropositivity is associated with rheumatoid nodules, vasculitis, HLA-DR4 and a poorer prognosis (Cassidy and Valkenburg, 1967; Vehe et al., 1990). Elevated concentrations of RF are also found in other inflammatory autoimmune diseases such as systemic lupus erythematosus (SLE), Sj6gren's syndrome and mixed connective tissue disease (MCTD).In SLE, IgG RFs significantly associate with the absence of kidney disease; whereas, IgM RFs indicate active disease (Tarkowski and Westberg, 1987). Vasculitis in Sj6gren's syndrome and mixed cryoglobulinemia can he associated with the presence of a monoclonal IgM RF (Fitzgerald et al., 1987; Muller et al., 1988). Paraproteins with RF activity in B cell neoplasias can be associated with vasculitis (Roudier et al., 1990).

707

Table 2. Evidence indicating a pathogenic role of RF in RA

References

RF are found in high frequency in RA serum and synovial fluid

Shmerling and Delbanco, 1991

RF titers correlate with disease activity

Shmerling and Delbanco, 1991

Structural and functional differences between RA RFs and RFs found in other conditions

Thompson et al., 1994 BCrretzen et al., 1994

RF in RA are associated with HLA class II DR4

Nelson et al., 1994

Seropositive RA patients have local and circulating immune complexes that may activate complement.

Winchester et al., 1971 Conn et al., 1976

Animal Models. RFs found in spontaneous and induced animal models may or may not be associated with autoimmune symptoms. In some models, RF production and/or titers are associated with arthritic symptoms like synovitis (Hang et al., 1982; Holmdahl et al., 1989; Wooley et al., 1989) and vasculitis (Reininger et al., 1990). As in humans, there is no strong association between RF production and lupuslike symptoms in mice (Andrews et al., 1978). Nonautoimmune mice transgenically expressing a human IgM RF associated with vasculitis show specific deletion of reactive B cells upon injection of deaggregated human IgG (Tighe et al., 1995). This indicates that RFs in normal healthy animals are subject to a regulatory control that might be weaker in animals susceptible to autoimmunity.

Genetics The concordance rate of RA in monozygotic seropositive twins is substantially higher than in monozygotic seronegative twins (Lawrence, 1970). This correlation may be influenced by the strong association between HLA-DR4 and seropositive RF in RA (Nelson et al., 1994). Studies of the genetic origins of the Ig V regions of isolated RF clones show that Via and V L family usage varies in RFs of different origin. RFs from healthy individuals have a V gene family pattern more similar to RFs found in patients with lymphoproliferative diseases and peripheral blood lymphocytes, as compared to RFs from RA synovial tissue (Thompson et al., 1994) (Figure 1). There is a similar, although not so clear, bias in germline gene usage (BCrretzen et al., 1995). The current evidence does not indicate that the inheritance of particular V-genes is responsible for generating pathogenic RFs in RA. The differences in V-gene use between RA peripheral blood lymphocytes and RA synovial tissues suggest

708

that unique, local mechanisms operate in the rheumatoid synovial tissues.

Factors in Pathogenicity and Etiology RFs of the IgM isotype are predominant in serum and the most studied RFs, but IgA, IgG, IgD and IgE RFs are also found in RA and other disease conditions (Table 3) (Gioud Paquet et al., 1987; Banchuin et al., 1992). IgG RF can self-associate to make large complexes; such complexes, which can be found in the synovial fluid and tissue of RA patients (Munthe and Natvig, 1972; Winchester et al., 1971), might contribute to the disease process by activating complement (Brown et al., 1982). IgA RFs in patients with RA are reportedly associated with bone erosions and symptoms originating from mucosal membranes and secretory organs (Jonsson and Valdimarsson, 1993). Elevation in IgA RF may precede the increase in IgM RF titer, but the measuring of IgA RFs is still limited. In normal immune responses in healthy individuals, RFs of the IgM isotype are the most prevalent, but some evidence of IgG and IgA RFs is found (Otten et al., 1992; Jonsson et al., 1995). High concentrations of

Figure 1. V gene family usage in RF of different origins.

Table 3.

Disease

RF Isotype Frequencies IgM

IgA

IgG

IgE

Rheumatoid arthritis*

92

65

66

68

Systemic lupus erythematosust

59

36

27

9

Sj6gren's syndromes

55

55

9

9

Polymyalgia rheumatica#

12

12

24

Mixed connective tissue diseases#

26

22

26

Normals#

<5

<5

<5

These analyses were done by ELISA or RIA. Frequencies of RF determined by LFT/Waaler and nephelometry are lower, see Table 1. *Gioud-Paquet et al., 1987; tPope et al., 1981; Gripenberg et al., 1988; SMuller et al., 1989; #van Leeuwen et al., 1988.

IgM and IgG RFs may precede the development of RA and are thus considered a risk factor for RA (del Puente et al., 1988; Walker et al., 1986). By definition, RFs react with the Fc part of the IgG molecule. The predominant binding area is localized to the CH2/CH3 interface region. Many RFs react with IgG1, IgG2, IgG4 but not IgG3 (Jefferis et al., 1984; Nardella et al., 1988; Recht et al., 1981). This reactivity pattern, called Ga reactivity, is similar to the binding of the Staphylococcus aureus protein A to Fc, indicating that RFs bear the conformational internal image of this protein. This suggests that RFs could arise as antibodies to the idiotypic determinants on antibodies to microbial Fc-binding proteins (Nardella et al., 1988). Monoclonal RFs from patients with RA, however, show broader reactivity, including binding to IgG3 (Bonagura, 1993). Analyses of the fine specificity of monoclonal RFs from various disease conditions shows that some RA RFs bind outside the CH2/CH3 interface (Natvig et al., 1972; Bonagura et al., 1993; Robbins et al., 1993); this difference in reactivity apparently reflects differences

in the primary structure of RFs found in RA and other conditions. The first structural studies on RFs utilized idiotypic markers and sequencing of monoclonal RFs from patients with lymphoproliferative diseases (Carson et al., 1987). Recent analyses of human hybridoma cells from RF-producing B cells of synovial tissue and peripheral blood of RA patients and from healthy immunized donors have been produced (Thompson et al., 1994; Randen et al., 1992). mAb RFs show that in addition to the differences in V gene usage, the mutational patterns and the affinity are subject to stricter control in RFs of healthy immunized donors than in RA RFs (Table 4) (BCrretzen et al., 1994). Somehow, the RA RFs show affinity maturation and mutational patterns more typical of antigen-driven processes where normal tolerance mechanisms are apparently broken or bypassed. Following infections or immunizations, RFs are produced transiently in healthy subjects. Immune complexes are probably the stimulus for the production of such normal RFs (Coulie and Van Snick, 1985; Nemazee, 1985; Stanley et al., 1987). The

Table 4. Mean number of mutations, dissociation constant, (kd) and ratio between replacement (R) and silent (S) mutations in the frameworks (FR) and complementarity-determining regions (CDR) in the variable heavy chain region of rheumatoid factors (RFs) from healthy immunized donors and patients with rheumatoid arthritis (RA). The RFs summarized are of IgM isotype, and the DP-10 and Kv325 germline genes. This shows that there is a selection against replacement mutations in the heavy chain CDR1+2 of RFs from immunized healthy donors compared to RFs from patients with RA.

Rheumatoid factors from:

Mutations (mean)

Kd (pM)

R/S ratio FRl+2+3

R/S ratio CDRI+2

Healthy donors

12

2.00

1.1

0.4

RA patients

16.1

0.30

0.9

2.4

709

kinetics of appearance and disappearance of normal RFs following immunization with tetanus toxoid closely follows the kinetics of the antigen-specific antibodies with a peak value at day six (Tarkowski et al., 1985). The RFs produced in a normal immune response hypermutate and apparently obtain T-cell help (BCrretzen, 1994). Normally, T cells specific for the IgG Fc region are tolerated (Bogen, 1993), but RF-producing B cells may obtain help by presenting peptides from the antigen part of the immune complex to specific T-helper cells (Roosnek and Lanzavecchia, 1991). In the rheumatoid synovium, there is a strong T-cell help to the B cells (Chattopadhyay et al., 1979). Other mechanisms for RF production, such as polyclonal activation and molecular mimicry, have also been suggested. Pokweed mitogen can stimulate PBL from RA patients, but not from healthy donors to produce RFs; Epstein-Barr virus can stimulate RF synthesis in normal peripheral blood lymphocytes (Williams, 1994). Produced as paraproteins in patients with lymphoproliferative diseases, RFs can yield pathogenic effects. For example, the idiopathic hypergammaglobulinemic purpura of Waldenstr6m is a rare benign disease (Finder et al., 1990) characterized by the presence of RFs. This led to the hypothesis that circulating immune complexes might be responsible for the extravasation of blood in small vessels and immunoglobulin deposits in vasculitis.

Methods of Detection The first assay for RF utilized sheep red blood cells sensitized with rabbit IgG(Waaler, 1940; Rose et al., 1949). Later, the latex fixation test that has become a widely used test for RF was developed (Singer and Plotz, 1956). Both these assays primarily measure IgM RFs (Franklin et al., 1957; Svartz et al., 1958; Schrohenloher et al., 1964). Rabbit IgG used for the detection of RF in humans usually gives somewhat lower sensitivity and higher specificity than human IgG. However, for most practical diagnostic purposes, rabbit IgG is equivalent to that of human IgG. When studied at clonal levels, most human RFs react with human IgG, but several do not cross react with rabbit IgG (Randen et al., 1992). Both the Waaler-Rose test and the original latex fixation test are still used as simple agglutination tests for screening and semiquantitative titration of RF activity. However, in large laboratories, these tests have, to a large extent, been replaced by quantitative analyses. These methods are mainly nephelometry using latex particles coated with

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human IgG or nephelometric or turbidimetric analyses based on precipitation with soluble aggregated human IgG. Commercial kits for these quantitative analyses have gradually become well standardized, with calibrators based on international reference preparations. In addition, many laboratories use in-house or commercially developed quantitative ELISA tests. These are primarily used for IgM and IgA RFs, but kits for IgG RFs are also offered. The standardization for these tests is still not satisfactory, and it is not always clear whether the commercial kit producers are aware of the technical difficulties involved. For example, IgM RFs in serum samples can cause false-positive reactions for IgA and IgG RFs by binding both to the ELISA coat and to the IgG Fc in the anti-IgA and anti-IgG antibody-enzyme conjugates. To avoid this, the secondary antibodies should be in the form of F(ab') 2 fragments, i.e., they must be treated with pepsin before use. Furthermore, even with pepsintreated conjugates, IgM RFs in the samples can cause false-positive IgG RF reactions by binding to free IgG or IgG in soluble immune complexes. Controls with reducing agents to depolymerize the IgM molecules may be necessary to avoid this. Finally, false-negative reactions for IgG RFs are easily obtained by the "hidden" RFs. The RF activity is masked due to binding to serum IgG, but assays developed for hidden RFs are elaborate, and are, therefore, not widely used. The major advantage of the new quantitative tests is the increased reproducibility of the results. Traditionally, a methodological variation of + one twofold titration step is accepted for agglutination tests. In contrast, for a nephelometric latex RF test in our laboratory, the coefficient of variation is below 10%.

CLINICAL UTILITY

Applications An elevated RF is included in the criteria for the classification of RA as accepted by the American College of Rheumatology (American Rheumatism Association) (Arnett et al., 1988). However, a positive or negative RF neither confirms nor excludes RA. If by other criteria, a patient is suspected to have RA, a positive RF strengthens the diagnosis and subclassifies the patient as seropositive. A negative RF does not exclude RA; rather, if the other criteria are strong, it subclassifies the patient as seronegative. Until recent-

ly, testing of RF suffered strongly from the lack of accuracy and precision. Titers obtained in different laboratories could not be compared, and within laboratories there were also large variations of the methods. The precision is now improved because of the increasing use of international reference calibrators, giving results in comparable international units, (IU)/mL. The introduction of quantitative tests has reduced the variation of the analyses considerably. A remaining problem is the question of cut-off points, i.e., the distinction between a normal and an elevated result. Usually the cut-off will be determined by the reference material used in the various laboratories. This may be misleading, because although the frequency of RFs in healthy persons is claimed not to increase with age, it is in fact increased in many types of nonrheumatoid, chronically ill elderly (Juby et al., 1994), and age-adjusted reference ranges are needed for the general population.

Disease Associations Concentrations of circulating RFs vary with ethnic groups, but in most populations the ratio of men to women is --1:1 (Hooper et al., 1972). In nonsymptomatic subjects, elevated levels of IgM and IgG RF are considered risk factors for developing RA, and the risk is reportedly related to RF titer (del Puente et al., 1988; Walker et al., 1986). RA is about three times more frequent in women than in men. The probability that an RF-positive patient has RA, therefore, is increased if the patient is a woman. Other factors that increase the probability include a family history of RA and the age of onset (35--40 years) (Shmerling and Delbanco, 1991).

Frequencies Elevated RFs are found in 70--90% of RA patients, but also in considerable frequencies in patients with other rheumatic as well as infectious and pulmonary diseases (Table 1) (Shmerling and Delbanco, 1991). In RA, there is a correlation between RF titer and disease activity. Seropositive patients are more likely to have severe disease than seronegative patients, and a higher RF titer also correlates with more severe disease and poorer long-term prognosis (Shmerling and Delbanco, 1991). In contrast, a protective effect against nephritis is suggested for RFs in SLE (Howard et al., 1991). In some studies, successful treatment of RA

patients correlates with a decrease in RF titer. IgM and IgG RFs decrease significantly in patients who improve following treatment with gold, but there is no correlation between RF decrease and clinical improvement in patients treated with nonsteroidal anti-inflammatory drugs alone or with D-penicillamine (Pope et al., 1986). In contrast, a significant correlation between decrease in RF and several clinical activity parameters is reported to follow treatment with Dpenicillamine but not with piroxicam (Thoen et al., 1988). Chimeric CD4 mAbs reduce CD4-positive T cells but not RF concentrations (van der Lubbe et al., 1994). In contrast, treatment with low dosage methotrexate can decrease RF concentrations without a corresponding improvement of clinical variables (Spadaro et al., 1993). That some therapies do influence RF titers and some d o not suggests different regulatory effects of different treatments. RFs are found in about 5% of children with juvenile rheumatoid arthritis (JRA). The age of onset, as defined by the ACR, is <16 years but is often lower, with a peak at 1 to 3 years of age (Sullivan et al., 1975). Older children, children with late onset and polyarticular JRA, more frequently have RFs. Because RFs are seldom found in younger children with JRA but can be found in many other connective tissue diseases of childhood, RF tests are of limited diagnostic aid. Children with high titer RF may represent a polyarticular form of JRA. However, 65--75% of children with seronegative JRA have hidden 19S IgM RFs (Moore and Dorner, 1993). Titers of these hidden RFs reportedly correlate with disease activity (Moore et al., 1980), but because of difficulties in establishing assays, detection of hidden RFs has not been widely used. As in RA, seropositive children with JRA have poorer prognosis than seronegative children and are associated with rheumatoid nodule, and HLA-DR4 (Cassidy and Valkenburg, 1967; Vehe et al., 1990).

Sensitivity and Specificity The diagnostic utility of an RF test is determined by many elements. The sensitivity of a test is the true positive rate, i.e., how many of the patients with the disease the test will identify. The false-positive rate is the rate of positive RF tests in panels of control subjects. IgM RF are found in 2--10% of normal adults, giving a specificity > 90% (del Puente et al., 1988; Hooper et al., 1972). From these numbers and the prevalence of the disease in the population, positive predictive value (PPV) and negative predic-

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tive values (NPV), i.e., the probability of disease, based on a positive or negative test result, can be calculated. The frequency of RF in RA patients is between 5 0 - 9 0 % , giving a sensitivity in the same range. The positive and negative predictive values will vary according to the selection criteria of the subjects. In population screening tests where 0.5 to 3 % of the subjects have RA, the positive predictive value varies from 2 0 - 3 0 % and the negative predictive value ranges from 93--95% (Shmerling and Delbanco, 1991). These values are improved in panels of selected patients with symptoms or other predisposing factors (i.e., sex, age, familial history) indicating RA, and in such patients the RF test can be a useful diagnostic tool (Shmerling and Delbanco, 1991). In JRA, the sensitivity is much lower than in RA, only 5%, suggesting that RF is of little diagnostic aid in this disease (Eichenfield et al., 1986). In nonrheumatic diseases, the RF latex test may be useful in infants with clin-

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ical signs suggestive of congenital syphilis (Meyer, 1993).

CONCLUSION RFs are among the most thoroughly investigated autoantibodies. They are primarily associated with RA but are also found in many other disease conditions and in healthy immunized normals. In the normal immune response they have physiological roles, but there are many implications of pathogenic effects in various diseases. Analyses have revealed that RFs in health and in various disease conditions have different structural and functional properties. These differences may result from abnormal regulation of RFs, involving the interplay of genetic and environmental factors (infectious agents or antigens).

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RIBOSOMAL AUTOANTIBODIES Edward Dwyer, M.D. a and Robert G. Lahita, M.D., Ph.D. b

aDepartment of Medicine, Columbia University College of Physicians & Surgeons, New York, NY 10032; and bDivision of Rheumatology, St. Luke's~Roosevelt Hospital, New York, NY 10019, USA

HISTORICAL NOTES Although classically characterized as the prototype of an autoimmune disease with intranuclear macromolecules as principal targets, systemic lupus erythematosus (SLE) is also characterized in a significant subset of patients by autoantibodies to intracytoplasmic constituents (Asherson, 1959). Many of these anticytoplasmic autoantibodies are directed against ribosomal components (Sturgill and Carpenter, 1965; Lamon and Bennett, 1970); during the last decade, the primary antigenic targets of the antiribosomal humoral immune response in SLE were defined.

tains a single 18S species of RNA (1874 bases) and at least 33 different basic proteins. The larger, more complex 60S subunit incorporates three distinct species of ribosomal RNA (rRNA) termed 28S (4718 bases), 5.8S (160 bases), and 5S (120 bases); there are approximately 46 different basic proteins associated with the 60S subunit, and an additional three acidic phosphoproteins, designated P0, P1, and P2. Each distinct ribosomal protein species is designated numerically and the prefix "S" or "L" is added to indicate whether the protein is a constituent of the small or large subunit, respectively (McConkey et al., 1979); the only exception to this rule is the nomenclature for the three aforementioned phosphoproteins of the 60S subunit.

AUTOANTIGENS Methods of Purification Definition

Ribosomes are complex macromolecular structures incorporating both protein and ribonucleic acid (RNA) elements; the principal function of the ribosome is the template-directed translation of messenger RNA (mRNA) into a mature protein through a repetitive catalytic process of elongation whereby individual amino acids are sequentially added to a lengthening polypeptide chain (Figure 1). Characteristics

The mammalian 80S ribosome has a molecular mass of 4.2 x 103 kd and is formed from two distinct subunits: a larger 60S subunit and a smaller 40S subunit (Figure 2). The 40S subunit, which initially combines with mRNA before interacting with the 60S subunit, is a ribonucleoprotein complex which con-

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Intact human 80S ribosomes are typically isolated by ultracentrifugation of cytoplasmic extracts of various cell lines (e.g., Hela cells) through a discontinuous sucrose gradient. Nonprimate mammalian 80S ribosomes are generally obtained from homogenates of mouse or rat liver; alternatively, rabbit reticulocytes as a source of 80S microsomes have the additional advantage of avoiding contamination of cytoplasmic extracts with nuclear components. The 40S and 60S subunits are separated from intact 80S ribosomes by high salt (e.g., 500 mM KC) sucrose gradient centrifugation. Proteins are obtained from the isolated 40S and 60S subunits by extraction with 67% acetic acid followed by acetone precipitation; separation of the rRNA species by phenol/chloroform extraction is followed by ethanol precipitation. Individual protein species are isolated by 2-D gel electrophoresis with isoelectric-focusing in the first

Figure 1. Ribosomal protein synthesis, mRNA is translated into protein by the sequential addition of specific amino acids complexed to tRNA. dimension and SDS-PAGE in the second dimension. Nearly pure, individual proteins can be extracted from the corresponding gel "spots". Pure preparations of individual rRNA species from total rRNA are obtained by sucrose gradient centrifugation.

AUTOANTIBODIES

Pathogenetic Role Antiphosphoprotein Antibodies. The most common antiribosomal autoantibodies are targeted against the phosphoproteins P0 (38 kd), P1 (19 kd), and P2 (17 kd), which are present in approximately 12--19% of SLE sera (Francoeur et al., 1985; Elkon et al., 1985);

reactivity of these anti-P antibodies is directed against a single common epitope present on the carboxyl terminus of each of these three proteins. This reactivity is quite infrequent in other autoimmune diseases and is altogether absent in normal populations. The three phosphoproteins form a pentameric complex (e.g., [P1 P212P0 ) which is physically associated with the "GTPase domain" of the 60S subunit (Figure 3); the function of this domain in the interaction of the ribosome with the two elongation factors EF-I~ and EF-2 is a requisite step in the process of protein synthesis. Sera with anti-P reactivity prevent the interaction of the ribosome with EF-I~ and EF-2 in vitro and thereby interrupts protein synthesis (Uchiumi et al., 1991).

Figure 2. Structural composition of 80S eukaryotic ribosome. 717

Figure 3. Structural relationships of autoantigens of the GTPase domain of 60S ribosomal subunit. Anti-L12 Protein Antibodies. A considerably less frequent target of the antiribosomal immune response is that directed against the basic 20 kd L 12 protein in less than 1% of SLE patients (Sato et al., 1990). This protein, a component of the "GTPase domain" of the 60S subunit, is physically associated with the ribosomal P proteins. To date, all SLE patients with an anti-L12 response have also exhibited an anti-P response; anti-L12 autoantibodies are absent in other autoimmune diseases as well as normal controls. Anti-L5/SS Protein Complex Antibodies. An exceedingly rare antigenic target of the SLE antiribosomal immune response, described in only four case reports, is that directed against the L5/5S rRNA ribonucleoprotein complex (Steitz et al., 1988; Guialis et al., 1994). This reactivity typically demonstrates strong nucleolar staining but weak cytoplasmic staining by indirect immunofluorescence. This pattern may reflect immunogenicity of the RNP complex prior to its incorporation into intact cytoplasmic ribosomes; indeed, sera possessing this reactivity react quite weakly with intact ribosomes, probably reflect-

ing a structural position of the L5/5S complex in the fully assembled ribosome which is relatively inaccessible. Ribosomal autoantibody activity is probably directed to the protein moiety of the complex because treatment of the L5/5S particle with trypsin abolishes activity; whereas, exposure to RNase has no effect (Steitz et al., 1988). Anti-S10 Protein Antibodies. The basic 20 kd S10 protein is the only definitive protein autoantigen of the smaller 40S subunit and is present in approximately 11% of SLE patients (Bonfa et al., 1989). This protein functions during initiation of protein synthesis, interacting with mRNA and the initiation factor elF-3. In a study of 89 SLE patients with active disease, 50% of those with anti-S10 reactivity also demonstrated anti-P reactivity but in the remaining 50% anti-S10 was the sole autoantibody identified (Sato et al., 1991). This reactivity is similarly absent in patients with other autoimmune diseases or in normal control populations (Table 1). In the murine MRL model of SLE, anti-S10 activity occurs spontaneously in 10% of the diseased

Table 1. Frequency and Specificity of Various Antiribosomal Autoantibodies in SLE Frequency Anti-P

12--19%

Anti-L12

<1%

Anti-L5/5S

<1%

Anti-S 10

11%

Anti-Ja Anti-rRNA

718

Antibody Associations 75% + anti-rRNA 100% + anti-P

Specificity >99% >99% ND

50% + anti-P

>99%

100% + anti-P

>99%

8% 15%

population; other murine models of SLE (e.g., NZW/NZB) do not demonstrate this reactivity (Bonfa et al., 1989). Anti-Ja Antibodies. Anti-Ja specificity in SLE is characterized by reactivity to 26 and 30 kd ribosomal proteins as detected by conventional SDS-PAGE immunoblotting (Bonfa et al., 1992). This specificity is present in 8% of SLE patients and is absent in other autoimmune disease and normal control populations. The presence of anti-Ja is strongly correlated with anti-P and anti-dsDNA reactivity. Anti-28S Antibodies. The autoimmune response directed against the rRNA species of ribosomes in patients with antiribosomal autoantibodies is exceedingly selective with response to a single epitope of 28S rRNA in all patients expressing this reactivity. This epitope incorporates approximately 58 nucleotides from bases 1944--2002 and is also a component of the "GTPase domain" of the 60S subunit (Uchiumi et al., 1991; Chu et al., 1991); cross-linking studies reveal a close physical proximity of the epitope to the P proteins as well as the L12 protein. This particular epitope demonstrates remarkable evolutionary conservation, being 97% identical in amino acid sequence to homologous ribosomal sequences of other eukaryotic species as compared to approximately 65% similarity in adjacent flanking regions; in addition, this epitope exhibits only 58% sequence similarity to the rRNAassociated "GTPase domain" of prokaryotes. The specificity of antibody binding to this epitope is demonstrated by mutagenesis experiments whereby alterations of less than four nucleotides can abrogate reactivity (Chu et al., 1991). All SLE patients demonstrating this reactivity also express anti-P (Sato et al., 1994), and conversely, 75% of individuals with anti-P also demonstrate reactivity to this rRNA epitope (Chu et al., 1991) (Table 1). This reactivity is similarly absent in other diseases or normal controls. Methods of Detection

Immunoblotting assays are used to detect and characterize the fine specificity of autoantibodies directed against ribosomal components. In general, extracts of total protein are run on 2-D gels with each unique protein species identified by its characteristic migratory pattern or "spot" on the gel; subsequent electrophoretic transfer to nitrocellulose and incubation with individual sera allows determination of the antigenic

specificity of each sample (Sato et al., 1990; Bonfa et al., 1989). A more focused investigation concentrating on a single antigenic species, or an admixture of a limited number of proteins, allows a more simplified and straightforward 1-D SDS-PAGE gel immunoblotting (Sato et al., 1991). Reactivity against individual rRNA species is detected by immunoprecipitation of total rRNA with patient sera followed by application of Sepharoseprotein A chromatography; the precipitated rRNA is then extracted with phenol/chloroform and its identity is defined by migration through an ethidium bromidestained polyacrylamide gel (Chu et al., 1991; Sato et al., 1994). The further delineation of the specific immunoreactive epitopes of individual rRNA molecules is achieved through RNase protection assays whereby the protected fragment or epitope is identified by either Northern analysis with specific rRNA probe's or, alternatively, the identity of the protected fragment is obtained through direct sequencing. A more focused assessment of reactivity directed against discrete linear rRNA epitopes utilizes in vitro-derived rRNA transcripts of approximately 100 bases as the target species; reactivity of serum samples is determined through the use of 32p-labeled transcripts and subsequent quantitation of radioactivity present in immunoprecipitated rRNA.

CLINICAL UTILITY The autoimmune response to ribosomal components is quite specific to SLE, and therefore, its detection may be quite valuable in establishing the diagnosis of SLE. Significantly, there is a positive correlation between the presence of antiribosomal reactivity and another autoantibody quite specific to SLE, the anti-Sm antibody; conversely, approximately 20% of patients with anti-Sm reactivity also exhibit antiribosomal reactivity (Francoeur et al., 1985). Although only 10% of randomly selected SLE patients demonstrate antiribosomal antibodies, the frequency significantly increases to approximately 40% in those individuals with active disease, regardless of the specific clinical features or the organ system involved (Sato et al., 1991) (Table 1). Although antiribosomal antibodies are more frequent in active disease, for the most part, antibodies directed against non-P ribosomal components are not generally associated with specific clinical symptoms or syndromes. Although anti-P antibodies were initially associated with lupus-induced

719

psychosis (Bonfa et al., 1987), subsequent reports did not entirely confirm this association; comprehensive, prospective clinical studies are needed (Teh and Isenberg, 1994).

CONCLUSION As with nucleosome and spliceosome antibodies, autoantibodies to ribosomes frequently target several protein or nucleic acid species within a single subcellular particle. The specificity of the response to particular molecular species of the complex demonstrates considerable commonality and overlap among patients with the same disease. Further, these protein-

REFERENCES Asherson GL. Antibodies against nuclear and cytoplasmic cell constituents in systemic lupus erythematosus and other diseases. Br J Exp Pathol 1959;40:209--215. Bonfa E, Tavares AV, Viana VS, Levy-Neto M, Kumeda CA, Cossermelli W. Antibodies to new cytoplasmic autoantigens: anti-JA, a potential marker for disease activity in systemic lupus erythematosus. Braz J Med Biol Res 1992;25:601--609. Bonfa E, Golombek SJ, Kaufman LD, Skelly S, Weissbach H, Brot N, Elkon KB. Association between lupus psychosis and antiribosomal P protein antibodies. N Engl J Med 1987;317: 265-271. Bonfa E, Parnassa AP, Rhoads DD, Roufa DJ, Wool IG, Elkon KB. Antiribosomal S 10 antibodies in humans and MRL/lpr mice with systemic lupus erythematosus. Arthritis Rheum 1989;32:1252-- 1261. Chu J-L, Brot N, Weissbach H, Elkon K. Lupus antiribosomal P antisera contain antibodies to a small fragment of 28S rRNA located in the proposed ribosomal GTPase center. J Exp Med 1991;174:507-514. Elkon KB, Parnassa AP, Foster CL. Lupus autoantibodies target ribosomal P proteins. J Exp Med 1985;162:459--471. Francoeur AM, Peebles CL, Heckman KJ, Lee JC, Tan EM. Identification of ribosomal protein autoantigens. J Immunol 1985;135:2378-2384. Guialis A, Patrinou-Georgoula M, Tsifetaki N, Aidinis V, Sekeris CE, Moutsopoulos HM. Anti-5S RNA/protein (RNP) antibody levels correlate with disease activity in a patient with systemic lupus erythematosus (SLE) nephritis. Clin Exp Immunol 1994;95:385--389. Lamon EW, Bennett JC. Antibodies to ribosomal ribonucleic

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nucleic acid targets are often essential functional components of the cell and the target within the particle is frequently an essential functional or catalytic domain of the complex; for the antiribosomal response, the catalytic domain that is the principal target of the humoral autoimmune response is the "GTPase domain", although infrequently, other domains may also occasionally be involved. The clinical utility of antiribosomal antibodies in SLE awaits the development of standardized assay techniques that can be readily applied to well-defined patient populations. See also NUCLEOSOME-SPECIFIC AUTOANTIBODIES, RIBOSOMAL P PROTEIN AUTOANTIBODIES and SPLICESOMAL SNRNP AUTOANTIBODIES.

acid (rRNA) in patients with systemic lupus erythematosus (SLE). Immunology 1970;19:439--442. McConkey EH, Bielka H, Gordon J, Lastick SM, Lin A, Ogata K, Reboud JP, Traugh JA, Traut RR, Warner JR, Welfle H, Wool IG. Proposed uniform nomenclature for mammalian ribosomal proteins. Mol Gen Genet 1979;169:1-6. Sato T, Uchiumi T, Ozawa T, Kikuchi M, Nakano M, Kominami R, Arakawa M. Autoantibodies against ribosomal proteins found with high frequency in patients with systemic lupus erythematosus with active disease. J Rheumatol 1991 ;18:1681--1684. Sato T, Uchiumi T, Kominami R, Arakawa M. Autoantibodies specific for the 20-KDal ribosomal large subunit protein L 12. Biochem Biophys Res Commun 1990;172:496-502. Sato T, Uchiumi T, Arakawa M, Kominami R. Serological association of lupus autoantibodies to a limited functional domain of 28S ribosomal RNA and to the ribosomal proteins bound to the domain. Clin Exp Immunol 1994;98:35--39. Steitz JA, Berg C, Hendrick JP, La Branche-Chabot H, Metspalu A, Rinke J, Yario T. A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells. J Cell Biol 1988;106:545--556. Sturgill BC, Carpenter RR. Antibody to ribosomes in systemic lupus erythematosus. Arthritis Rheum 1965;8:213--218. Teh LS, Isenberg DA. Antiribosomal P protein antibodies in systemic lupus erythematosus. Arthritis Rheum 1994;37: 307--335. Uchiumi T, Traut RR, Elkon K, Kominami R. A human autoantibody specific for a unique conserved region of 28 S ribosomal RNA inhibits the interaction of elongation factors 1 alpha and 2 with ribosomes. J Biol Chem 1991;266:20542062.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RIBOSOMAL P PROTEIN AUTOANTIBODIES Eloisa Bonfa, M.D., Ph.D. a, Herbert Weissbach, Ph.D. b, Nathan Brot, Ph.D. b and Keith B. Elkon, M.D. c

aDivision of Rheumatology, University of Sao Paulo, S(to Paulo, Brazil; bRoche Institute of Molecular Biology, Roche Center, Nutley, NJ 07110, USA; and CThe Hospital for Special Surgery, Cornell University Medical Center, New York, NY 10021, USA

H I S T O R I C A L NOTES The major immunological specificity of autoantibodies directed against cytoplasmic constituents in systemic lupus erythematosus (SLE) is the three ribosomal phosphoproteins P0, P1 and P2 (Francoeur et al., 1985; Elkon et al., 1985). Anti-P antibodies are highly specific for SLE (Bonfa and Elkon, 1986) and have been associated with psychiatric manifestations (Schneebaum et al., 1991). Experimental models and other forms of clinical activity (Sato et al., 1991; Hulsey et al., 1995; Hines et al., 1991) and the characterization of these autoantigens (Elkon et al., 1988) permitted considerable progress concerning the mechanism underlying the induction of these autoantibodies. In addition, recent evidence for expression of a P protein epitope on the surface of cells suggests a mechanism for pathogenicity of these autoantibodies (Koren et al., 1992).

THE AUTOANTIGEN(S) Definition/Structure

Located in the 60S ribosomal subunit, three phosphoproteins called P0, P 1 and P2 have apparent molecular sizes as determined by SDS gel electrophoresis of 38, 19 and 17 kd, respectively (Elkon et al., 1985). In contrast to the majority of the ribosomal proteins which are basic, P1 and P2 are acidic, rich in alanine and poor in arginine. The spatial arrangement of these proteins on the ribosomes as suggested by immune electron microscopy and cross-linking studies is the presence of two dimers of P1 and P2 anchored by the amino terminal

portion of P2 to P0 (Uchiumi et al., 1987). This pentameric complex is located in a highly accessible region on the stalk of the ribosome. Biochemical studies suggest that P1/P2 play a fundamental role in all three phases of ribosomal polypeptide formation (initiation, translocation and termination) (MacConnel and Kaplan, 1982). P1 and P2 are required for protein synthesis and guanosine triphosphatase activity as demonstrated when ribosomal cores in which P1 and P2 are selectively removed lose these two functions (MacConnel and Kaplan, 1982). In addition, Fab fragments of high titer anti-P sera completely inhibit globin mRNA translation in an in vitro protein synthesis system (Stacey et al., 1988). Inhibition of protein synthesis is also achieved by microinjection of antiribosome P antibodies into cultured human cells (Stacey et al., 1988). The specificity of anti-P antibodies is restricted to a common antigenic determinant located on the highly conserved carboxyl-terminal portion of the three P proteins (Elkon et al., 1986). Fine epitope mapping reveals a single linear epitope within the carboxylterminal 11 amino acids of the P autoantigen (Elkon et al., 1988). Evidence for additional antibodies recognizing conformation determinants is inferred by the inability of the P peptide to reverse anti-P inhibition of protein synthesis in vitro. Furthermore, preincubation of anti-P sera with the same peptide has a minimal effect on anti-P reactivity to native protein, emphasizing the presence of nondenatured epitopes on the P protein (Elkon et al., 1986). Sources

The 60S ribosomal subunits may be isolated by sucrose gradient centrifugation from almost any 721

eukaryotic cell. The usual source of this preparation is rat liver, rabbit reticulocytes and Artemia salina. Although crude ribosomal preparations are useful for immunoblotting, synthetic P peptide and P fusion protein are the best alternatives for ELISA or radioimmunoassay to detect anti-P antibodies (Bonfa et al., 1994).

Sequence Information The comparison of the primary structure of these ribosomal proteins in several eukaryotes shows that the amino-(N) and carboxyl-(C) termini are the most highly conserved regions of the P proteins (Elkon et al., 1986). In addition, there is considerable similarity of amino acid sequences in the carboxyl, but not the amino terminus of P0, P1 and P2 (Elkon et al., 1988).

AUTOANTIBODIES

Animal Models. Recognition of the same epitope by anti-P antibodies present in humans and in approximately 10% of MRL/Ipr mice (but not in NZB/W F1) with spontaneous murine lupus suggests a similar mechanism for induction of this immune response (Bonfa et al., 1988b). Immunization of normal and autoimmune strains of mice with large amounts of self ribosomes is insufficient to break tolerance. In contrast, heterologous ribosomes are potent immunogens. Although the specificities of the induced antibodies are almost restricted to P proteins, fine epitope mapping differs from spontaneously occurring anti-P. That both P1 and P2 homodimers are essential to induce anti-P reactivity (Hines et al., 1991) supports previous suggestions that repetitive epitopes are important for autoantibody production (Lafer et al., 1981). The anomalously and sustained anti-P response in immunized MRL mice, compared with normal mice, suggests that B cells are presensitized to this specific autoantigen (Hines et al., 1991).

Terminology Factors in Pathogenicity Recognition of the major ribosomal antigen targeted by lupus sera as the three ribosomal proteins P0, P1 and P2, prompted the nomination of this group of autoantibodies as anti-P0, P1 and P2, respectively (Elkon et al., 1985; Francoeur et al., 1985).

Pathogenetic Role Anti-P and anti-Sm antibodies are associated both in human SLE and in MRL/lpr mice, since it has been demonstrated that a significantly higher proportion of anti-P positivity in sera of patients (28%) and MRL/ Ipr mice (21%) positive for anti-Sm compared to humans (11%) and mice without this antibody (6%) (Elkon et al., 1989). This is probably not due to generalized B-cell activation, because mice positive for both antibodies do not have higher concentrations of total IgG, anti-DNA or antichromatin (Elkon et al., 1989). Rather, the relationship between anti-P and anti-Sm suggests a role for these antigens in the initiation and perpetuation of antibody synthesis, even though they are located in distinct subcellular compartments. The apoptosis-associated physical clustering of lupus autoantigens and the resultant predisposition to oxidative modification might provide a mechanism by which the same immunogenic structure could generate an immune response against both proteins (Casciola-Rosen et al., 1994). 722

Anti-P antibodies in SLE demonstrated a moderate restriction in isotype, with a prevalent IgM and IgG response and a near absence of IgA (Bonfa et al., 1987a; Schneebaum et al., 1991). The dominant IgG subclasses are IgG1 and IgG2 (Bonfa et al., 1987a). All these findings indicate that multiple heavy chain gene segments are utilized in the anti-P peptide response. However, the abnormal class switching (IgM, IgG1 and IgG2 but not IgA nor IgG4) (Honjo and Kataoka, 1978) and the distinct subclass profile from most foreign proteins (Yount et al., 1968) indicates that either T cells or the nature of the antigen selectively drive heavy chain expression. Moreover, the preferential selection of kappa light chain and the restricted spectrotype of the anti-P response suggest that the limited heterogeneity of antiP peptide antibodies may reflect affinity maturation of these immune responses (Bonfa et al., 1987a).

Methods of Detection Antibodies to ribosomal P proteins are often missed by routine immunofluorescence using HEp-2 cells. Indeed, only 30--60% of anti-P sera produce cytoplasmic staining, which is also associated with other antibody specificities. Counterimmunoelectrophoresis, although highly specific, also lacks sensitivity. Im-

munoblotting, ELISA and radioimmunoassay (RIA) are currently the most specific and sensitive techniques for detection of anti-P antibodies. The ELISA and RIA using a fusion P protein or synthetic peptides are superior to immunoblotting using a crude ribosomal fraction, because they are quantitative and simple to perform (Bonfa et al., 1994).

CLINICAL UTILITY Disease Association

Anti-P antibodies are highly specific serologic markers for SLE since they are not detected in other autoimmune diseases (Bonfa and Elkon, 1986). Moreover, these autoantibodies are not found in infectious disorders such as malaria and leprosy (Bonfa et al., 1987c) even though numerous reports document the presence of other autoantibodies in these diseases (Masala et al., 1979; Adu et al., 1982). In addition, the target in antigens in melanoma sera, a neoplastic disease in which antibodies are thought to play an important role in host defense against cancer (Lewis, 1967), are also distinct from P proteins (Bonfa et al., 1988a). It should also be emphasized that this antibody may be present in lupus sera even when other "ANA-positive" antibodies are not detected, therefore, in these circumstances they are useful to confirm the diagnosis of SLE (Bonfa and Elkon, 1986). The frequency of anti-P antibodies is 10--20% in randomly selected SLE populations (Francouer et al.,

1985; Bonfa and Elkon, 1986). The higher frequencies observed in Japanese and Malaysian Chinese patients indicate ethnic differences in the capacity to produce anti-P (Sato et al., 1991; Teh et al., 1993). By contrast, no difference in anti-P occurrence was found in four American ethnic groups (Amett et al., 1994). Although there are some discrepancies on the clinical correlation of anti-P antibodies (Teh and Isenberg, 1994), an association with psychiatric disease was demonstrated in three independent studies (Bonfa et al., 1987b; Schneebaum et al., 1991; Nojima et al., 1992) (Table 1). The first report showed that these antibodies were present in 18 of 20 (90%) patients with lupus psychosis and absent in psychotic patients without SLE (Bonfa et al., 1987b). The association between anti-P and psychosis was enhanced by the 5-fold to 30-fold increases in levels of this antibody in four of five patients with active psychosis from whom paired samples were available. Moreover, fluctuations of anti-P activity were selective in that they did not occur during other exacerbations of SLE such as arthritis, myositis, rash or lymphadenopathy and were not paralleled by antiDNA elevations. The strong association of elevated anti-P with psychiatric disease in lupus, particularly psychosis and severe depression, were confirmed by a prospective multicenter study with 269 lupus patients (Schneebaum et al., 1991). These authors have observed that the frequency of anti-P in lupus patients with severe depression (n = 8) and psychosis (n = 29) was 88 and 45%, respectively, compared with only 9% in patients with other manifestation of neuropsychiatric SLE (n = 45). Furthermore, in a large

Table 1. Frequency of Anti-P Antibodies in SLE Patients with Neuropsychiatric Disorder as Described by Different Investigators Investigators

SLE patients (n)

Anti-P antibody # (%) Psychosis

Bonfa et al., 1987b Schneebaun et al., 1991 van Dam et al., 1991 Sato et al., 1991

75

18(90)*

269

13(45)

54**

7(88)

2(17)***

138

3(50)

91

9(24)*

Teh et al., 1992

116

4(31)

West et al., 1995

52

6(60)*

Nojima et al., 1992

Depression

5(100)

2(20)

Study Other CNS 3(15)

Retrospective

4(9)

Prospective

4(25)

Prospective

-

Prospective

3(8)

Retrospective

4(25)

Retrospective

3(7)

Prospective

*Depression was included in psychosis group; **only patients with monosymptomatic exacerbations of SLE; ***included five cases with dementia and no cases with depression.

723

Japanese population anti-P was a marker for SLEinduced psychiatric disease, since anti-P was detected in nine of 10 patients who developed lupus psychosis. Likewise, the occurrence of lupus psychosis was significantly higher in patients with anti-P (9/38) than those without this antibody (1/53) (Nojima et al., 1992). These findings diverge with those of Sato et al., in which only three (50%) patients with active psychosis had anti-P. In this study, however, all five patients with depression were positive for this antibody. Therefore, once again, anti-P was helpful in identifying this subset of psychiatric disorder due to lupus (Sato et al., 1991) (Table 1). In contrast to these findings, other investigators failed to find any correlation of anti-P and lupus psychosis (van Dam et al., 1991; Teh et al., 1992) (Table 1). Prominent among the possible explanations for these conflicting data are the different criteria used to select the patients. In two studies showing correlation, only patients with severe psychiatric disturbance were selected (Bonfa et al., 1987b; Schneebaum et al., 1991); whereas, other studies included patients with mild psychiatric abnormalities, cognitive impairment or dementia (van Dam et al., 1991; Teh et al., 1992; Hanly et al., 1993). In addition, the retrospective nature of some studies, the time at which serum is collected and the ethnic origins of the patients are other factors possibly relevant to the discrepant findings (van Dam et al., 1991; Teh et al., 1992). Despite these discrepancies, longitudinal studies revealed striking elevations of anti-P levels before and during active phases of psychosis; whereas, no fluctuation is observed in patients with activity of other manifestations of SLE (Bonfa et al., 1987b; Schneebaum et al., 1991). Although others have not confirmed this correlation (van Dam et al., 1991; Teh et al., 1992) serial assays may be useful in monitoring the response to therapy in a subset of patients with anti-P antibodies and psychosis. Moreover, elevations of anti-P levels in the context of behavioral disturbance in SLE may help to distinguish SLE-induced psychiatric disease from that caused by other processes since the frequency of anti-P was not increased in SLE patients with transient behavioral disturbances thought to be due to corticosteroids (Bonfa et al., 1987b). A recent prospective study confirmed that anti-P is particularly useful diagnostically in those patients presenting primarily psychiatric disease due to SLE. Furthermore, antibody levels decreased coincident with therapy and clinical improvement of psychiatric

724

symptoms (Table 1) (Sterling et al., 1995). Further, a combination of abnormal CSF IgG index/oligoclonal bands, elevated CSF antineuronal antibodies and/or serum antiribosomal-P antibodies, yield a sensitivity of 100%, specificity of 86% and a PPV of 95% for diffuse neuropsychiatric SLE. However, it must be emphasized that at least half of the patients with antiP will not present psychosis (Bonfa et al., 1987b). Although a selective enrichment of IgG anti-P in the CSF of one patient with active psychosis was demonstrated (Golombek et al., 1986) the determination of cerebrospinal fluid (CSF) anti-P antibody levels does not seem to be a better predictor of lupus psychosis. In this regard, two studies with paired serum and CSF from a total of nine patients with active psychosis demonstrated several hundred-fold lower anti-P levels than in serum, suggesting that antiP production is probably not occurring within the CNS (Schneebaum et al., 1991; Teh et al., 1992). The authors speculated that this could be due to the binding of the antibody to the nerve tissue, but further studies are necessary to confirm this hypothesis (Schneebaum et al., 1991). Other clinical associations of anti-P antibodies awaiting confirmation include a correlation with multisystem disease activity (Sato et al., 1991) and with discoid rashes, photosensitivity and oral ulcerations (van Dam et al., 1991). An association of liver involvement with anti-P antibodies was suggested by the concurrent development of chronic active hepatitis and anti-P in a patient with SLE (Teh et al., 1992; Koren et al., 1993b) and by a case-control study correlating anti-P with hepatic disease and with kidney involvement (Hulsey et al., 1995). Other investigators did not confirm this correlation with kidney disease (Bonfa et al., 1987b; van Dam et al., 1991). Finally, the fundamental and unanswered question regarding these clinical associations is the mechanism by which anti-P antibodies might cause disease. The P proteins are cytoplasmic and therefore not characteristically accessible to these antibodies. On the other hand, the expression of P peptide on the surface of cultured human neuroblastoma and hepatoma cells makes possible a direct effect of anti-P antibodies on the development of neuropsychiatric and liver disease (Koren et al., 1992). In vitro studies also demonstrate that anti-P antibodies can directly injure hepatic cells in a complement-independent fashion (Koren et al., 1993a). Moreover, the finding of a significant lower anti-P activity in cerebrospinal fluid than in serum even when expressed as a fraction of the total IgG,

supports the notion of anti-P binding to the cells of central nervous system (Schneebaum et al., 1991).

CONCLUSION Antibodies to the three 60S ribosomal phosphoproteins P0, P1 an P2 are found in approximately 10% of randomly selected SLE patients and in up to 40% of Oriental lupus patients. Remarkably, the frequency of anti-P in MRL/lpr mice is also 10% and all mouse and SLE anti-P-positive sera recognize the same epitope which is confined within the 22 carboxylterminal amino acids of the P proteins. These antibodies appear to be highly specific diagnostic markers for SLE because they are not detected in other autoimmune disorders, infectious and neoplastic diseases. Anti-P antibodies are detected more frequently in lupus patients with severe psychiatric manifestations, particularly depression. Although there are some controversies in the literature, rising titers of anti-P at the onset of behavioral disturbances suggest the

REFERENCES Adu D, Williams DG, Quakyi IA, Voller A, Anim-Addo Y, Bruce-Tagoe AA, Johnson GD, Holborow EJ. Anti-ssDNA and antinuclear antibodies in human malaria. Clin Exp Immunol 1982;49:310--316. Arnett FC, Reveille JD, Elkon KB. Autoantibodies to ribosomal P in systemic lupus erythematosus. Arthritis Rheum 1994; 37:S167. Bonfa E, Elkon KB. Clinical and serological associations of the antiribosomal P protein antibody. Arthritis Rheum 1986;29: 981--985. Bonfa E, Chu JL, Brot N, Elkon KB. Lupus antiribosomal P peptide antibodies show limited heterogeneity and are predominantly of the IgG 1 and IgG subclasses. Clin Immunol Immunopathol 1987a;45:129-138. Bonfa E, Golombek SJ, Kaufman LD, Skelly S, Weissbach H, Brot N, Elkon K. Association between lupus psychosis and antiribosomal P protein antibodies. N Engl J Med 1987b;30: 317:265--271. Bonfa E, Llovet R, Scheinberg M, de Souza JM, Elkon KB. Comparison between autoantibodies in malaria and leprosy with lupus. Clin Exp Immunol 1987c;70:529-537. Bonfa E, Bystryn JC, Elkon KB. Detection of immunoglobulin G antibodies in melanoma sera reactive with intracellular proteins. J Invest Dermatol 1988a;90:207--212. Bonfa E, Marshak-Rothstein A, Weissbach H, Brot N, Elkon K. Frequency and epitope recognition of antiribosome P antibodies from humans with systemic lupus erythematosus and MRL/lprmice are similar. J Immunol 1988b;140:3434-3437.

diagnosis of lupus psychosis. In addition, other organ involvement including renal and hepatic disease might be correlated with the presence of anti-P antibodies. Although these antibodies are associated with general disease activity, the findings deserve further investigations. The limited heterogeneity of anti-P autoantibodies, the serologic association with anti-Sm, and the terminal and exposed location of the P autoantigen, emphasize the role of ribosomes in inducing and/or maintaining autoantibody production. However, in experimental models autologous ribosomes are ineffective in breaking tolerance; this supports the notion that a modification of the self protein is essential to promote anti-P response. The recent evidence that P protein is expressed on the surface of cells raises some attractive possibilities for direct involvement of these antibodies in the key process of cell damage. See also AUTOANTIBODIES THAT PENETRATE INTO LIVING CELLS, NEURONAL AUTOANTIBODIES and RIBOSOMAL AUTOANTIBODIES.

Bonfa E, Gaburo Junior N, Tavares AV, Cossermelli W. Comparison of five methods for the detection of antiribosomal P protein antibody. Braz J Med Biol Res 1994;27: 637-643. Casciola-Rosen LA, Anhalt G, Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 1994;179:1317--1330. Elkon KB, Parnassa AP, Foster CL. Lupus autoantibodies target ribosomal P protein. J Exp Med 1985;162:459-471. Elkon K, Skelly S, Parnassa A, Moller W, Danho W, Weissbach H, Brot N. Identification and chemical synthesis of a ribosomal protein antigenic determinant in systemic lupus erythematosus. Proc Natl Acad Sci USA 1986;83:7419--7423. Elkon KB, Bonfa E, Llovet R, Danho W, Weissbach, Brot N. The properties of the ribosomal P2 protein autoantigen are similar to those of foreign protein antigens. Proc Natl Acad Sci USA 1988;85:5186--5189. Elkon KB, Bonfa E, Llovet R, Eisenberg RA. Association between anti-Sm and ribosomal P protein autoantibodies in human systemic lupus erythematosus and MRL/lpr mice. J Immunol 1989;143:1549--1554. Francouer AM, Peebles CL, Heckman KJ, Lee JC, Tan EM. Identification of ribosomal protein autoantigens. J Immunol 1985; 135;2378-2384. Golombek SJ, Graus F, Elkon KB. Autoantibodies in the cerebrospinal fluid of patients with systemic lupus erythematosus. Arthritis Rheum 1986;29:1090--1097. Hanly JG, Walsh NM, Fisk JD, Eastwood B, Hong C, Sherwood G, Jones JV, Elkon K. Congnitive impairment and

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autoantibodies in systemic lupus erythematosus. Br J Rheumatol 1993;32:291--296. Hines JJ, Weissbach H, Brot N, Elkon K. Anti-P autoantibody production requires P1/P2 as immunogens but is not driven by exogenous self-antigen in MRL mice. J Immunol 1991; 146:3386--3395. Honjo T, Kataoka T. Organization of immunogobulin heavy chain genes and allelic deletion model. Proc Natl Acad Sci USA 1978;75:2140--2144. Hulsey M, Goldstein R, Scully L, Surbeck W, Reichlin M. Antiribosomal P antibodies in systemic lupus erythematosus: a case-control study correlating hepatic and renal disease. Clin Immunol Immunopathol 1995;74:252-256. Koren E, Reichlin MW, Koscec M, Fugate RD, Reichlin M. Autoantibodies to the ribosomal P proteins react with a plasma membrane related target on human cells. J Clin Invest 1992;89:1236-1234. Koren E, Koscec M, Reichlin M, Fugate R. Possible role of autoantibodies to ribosomal P proteins in development of liver disease in patients with systemic lupus erythematosus. Arthritis Rheum 1993a;36:$72. Koren E, Schnitz W, Reichlin M. Concomitant development of chronic active hepatitis autoantibodies to ribosomal P proteins in a patient with systemic lupus erythematosus. Arthritis Rheum 1993b;36:1325-1328. Lafer EM, Rauch J, Andrzejewski C, Mudd D, Furie M, Furie B, Schwartz RS, Stollar BD. Polyspecific monoclonal lupus autoantibodies reactive with both polynucleotides and phospholipids. J Exp Med 1981;153:897-909. Lewis MG. Possible immunological factors in human malignant melanoma. Lancet 1967;ii:921--922. MacConnel WP, Kaplan NO. The activity of the acidic phosphoproteins from the 80S rat ribosomes. J Biol Chem 1982;257:5359--5366. Masala C, Amendolea MA, Nuti M, Riccar-Ducci R, Tarabini CGL, Tarabini CG. Autoantibodies in leprosy. Int J Lepr 1979;47:171--175. Nojima Y, Minota S, Yamada A, Takaku F, Aotsuka S, Yokohari R. Correlation of antibodies to ribosomal P protein with psychosis in patients with systemic lupus erythematosus. Ann Rheum Dis 1992;51:1053--1055. Sato T, Echiumi T, Ozawa T, Kikuchi M, Nakano M, Komi-

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nami R, Arakawa M. Autoantibodies against ribosomal proteins found with high frequency in patients with systemic lupus erythematosus with active disease. J Rheumatol 1991;18:1681--1684. Schneebaum AB, Singleton JD, West SG, Blodgett JK, Allen LG, Cheronis JC, Kotzin BL. Association of psychiatric manifestations with antibodies to ribosomal P proteins in systemic lupus erythematosus. Am J Med 1991;90:54--62. Stacey DW, Skelly S, Watson T, Elkon K, Weissbach H, Brot N. The inhibition of protein synthesis by IgG containing antiribosome P autoantibodies from systemic lupus erythematosus patients. Arch Biochem Biophys 1988;267:398--403. Sterling GW, Woodruff E, Wener MH, Kotzin BL. Neuropsychiatric lupus erythematosus: a ten-year prospective study on the value of diagnostic tests. Am J Med 1995;99:153--163. Teh LS, Bedwell AE, Isenberg DA, Gordon C, Emery P, Chaeles PJ, Harper M, Amos N, Williams B D. Antibodies to protein P in systemic lupus erythematosus. Ann Rheum Dis 1992;51:489--494. Teh LS, Lee MK, Wang F, Manivasagar M, Charles PJ, Nicholson GD, Hay EM, Isenberg DA, Amos A, Williams B D. Antiribosomal P protein antibodies in different populations of patients with systemic lupus erythematosus. Br J Rheumatol 1993;32:663--665. Teh LS, Isenberg DA. Antiribosomal P protein antibodies in systemic lupus erythematosus. Arthritis Rheum 1994;37: 307-315. Uchiumi T, Wahba AJ, Traut RR. Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by cross-linking. Proc Natl Acad Sci USA 1987;84:5580--5584. van Dam A, Nossent H, de Jong J, Meilorf J, ter Borg E-J, Swaak T, Smeenk R. Diagnostic value of antibodies of antibodies against ribosomal phosphoproteins. J Rheumatol 1991 ;18:1026-1034. West SG, Emlen W, Wener MH, Kotzin BL. Neuropsychiatric lupus erythematosus: a 10-year prospective study on the value of diagnostic tests. Am J Med 1995;99:153--163. Yount WJ, Dorner MM, Kunkel HG et al. Studies on human antibodies. VI. Selective variations in subgroup composition and genetic marker. J Exp Med 1968;127:633--646.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

RNA P O L Y M E R A S E I-III A U T O A N T I B O D I E S Yutaka Okano, M.D. a and Thomas A. Medsger, Jr., M.D. b

aDepartment of Medicine, Nippon Kokan Hospital, Kawasaki 210, Japan; and bDivision of Rheumatology/Clinical Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

HISTORICAL NOTES First reported in 1982 with biochemically purified RNA polymerase (RNAP) class I as antigen (Stetler et al., 1982), the existence of serum autoantibodies specific to RNAP I was confirmed in 1987 (Reimer et al., 1987). In the 1990s, radioimmunoprecipitation assays showed that RNAP class II and class III, like RNAP class I, are targets for autoantibodies and suggested that these antibodies are associated with systemic sclerosis (SSc), especially SSc with diffuse cutaneous involvement (Okano et al., 1993; Kuwana et al., 1993; Hirakata et al., 1993).

THE AUTOANTIGEN(S)

Nomenclature The molecular structure and function of RNAPs are widely studied (Sentenac, 1985); however, RNAPs are most frequently classified based on enzymatic activity rather than structure. The three classes of RNAP, class I, II and III (or class A, B and C, respectively) catalyze the transcription of different sets of genes into RNA (Table 1). Although their precise subunit composition remains somewhat controversial, each of the eukaryotic RNAPs is known to be a multiprotein complex composed of eight to 14 protein subunits ranging in size from 10 to 220 kd. Features common to the three classes include the presence of two large distinct proteins with molecular weights of greater than 100 kd (Table 2) and a collection of several smaller proteins, some of which are shared by all three or sometimes two classes of the enzymes. Two largest

subunits of each class are readily distinguishable by their characteristic mobilities on SDS-polyacrylamide gel electrophoresis (PAGE); whereas, the smaller RNAP subunits are more difficult to visualize by SDS-PAGE after radiolabeling and remain less well defined.

Origin RNAPs play a central role in basic genetic processes responsible for producing and maintaining the proteins and nucleic acids of a cell. RNAPs exist in all eukaryotic cells, tissues and organs. Amino acid sequence similarities between RNA polymerase subunits in bacteria and eukaryocytes suggest a common evolutionary origin. However, there are no reports testing the reactivity of anti-RNAP autoantibodies to bacterial RNAPs.

Methods of Purification In the initial studies on autoantibodies to RNAP I, biochemically purified RNAP I from rat tumor cells was used as antigen in a solid-phase radioimmunoassay (Stetler et al., 1982; Stetler and Jacob, 1984). Recently, human cell lines including HeLa, HEp-2 and K562 cells were utilized for detecting anti-RNAP antibodies in patients' sera and characterizing the antigen specificities (Okano et al., 1993; Kuwana et al., 1993; Hirakata et al., 1993, Satoh et al., 1994a; 1994b). A monoclonal antibody (referred to as 8WG16 or anti-CTD monoclonal antibody) reactive with the highly conserved carboxyl terminal repeat of the 220kd subunit (Thompson et al., 1989) is now commercially available (Promega Corporation, Madison, WI,

727

Table 1. Characteristics of the Three Classes of Eukaryotic RNA Polymerase Class

Localization

Sensitivity to c~-amanitin

Product

I

Nucleolus

Unaffected

Large ribosomal RNA precursors

II

Nucleoplasm

Very sensitive

Messenger RNA precursors and most of the small nuclear RNA found in ribonucleoprotein particles that mediate premessenger RNA splicing

III

Nucleoplasm

Moderately sensitive

Small RNAs, e.g., 5S ribosomal RNA and tRNAs

Table 2. Largest Subunits of Three Classes of Eukaryotic RNA Polymerase Class

Designation

Molecular weight (kd)

I

IA IB

190 126

IIO (phosphrylated form of IIA)* IIA* IIB (proteolytic derivative of IIA) IIC

240 220 180 145

IIIA IIIB

155 138

III

*These two subunits contain a unique sequence (Ser-Pro-Ser-Tyr-Ser-Pro-Thr), which is repeated 26 times at the carboxyl terminus of yeast subunits and 52 times in mammalian subunits.

catalog #E3800 and #E3801). Through immunoaffinity chromatography with the anti-CTD monoclonal antibody, RNAP II can be purified from HeLa cells (Thompson et al., 1990); this antigen is also commercially available (Promega #E3810). The cDNAs encoding almost all subunit proteins of eukaryocytic RNAPs have been isolated. However, there are no reports using recombinant antigens for detecting autoantibodies in sera.

AUTOANTIBODIES Pathogenetic Role There is no evidence for a pathogenic role of antiRNAP antibodies in human disease. In animal models, there are several studies published. Anti-RNAP I antibodies are detectable in sera of all lupus mice tested from three different strains (MRL/Mp-lpr/lpr, MRL/Mp-+/+ and NZB/W) (Stetler et al., 1985; Stetler and Cavallo, 1987). Because anti-RNAP I antibodies are concentrated to as much as 70 times the serum concentration in the kidneys of lupus mice, and

728

because this concentration is directly proportional to disease severity, (Stetler and Cavallo 1987) antiRNAP I antibodies might have an important role in the pathogenesis of murine lupus nephritis. High titers of anti-RNAP I antibodies are spontaneously produced by the tight skin (TSK) mouse, an experimental model for SSc (Shibata et al., 1993). Genetics In one preliminary study on HLA class II of SSc patients with anti-RNAP antibodies (Clawson et al., 1994), the frequency of DR4 was significantly higher in anti-RNAP positive patients than in normal controls, primarily attributable to the DRB 1"0402 allele. There are no published reports on other immunogenetic aspects (family, twins or VH and V L gene usage) of patients with anti-RNAP antibodies. Methods of Detection In early studies, biochemically purified rabbit liver RNAP I was used to detect anti-RNAP I autoantibodies (Stetler et al., 1982; 1987; Stetler and Jacob,

1984). In the immunofluorescence antinuclear antibody test, anti-RNAP I antibodies produce speckled staining of the nucleolus of interphase cells and punctate staining of the nucleolar organizing regions of mitotic chromosomes (Reimer et al., 1987); this is compatible with the known nucleolar location of RNAP I. Almost all sera with anti-RNAP I antibodies also contain anti-RNAP III antibodies or anti-RNAP II and RNAP III antibodies (Okano et al., 1993; Kuwana et al., 1993; Satoh et al., 1994b). Some of these sera produce the characteristic "anti-RNAP I" staining. Other anti-RNAP I-containing-sera fail to produce nucleolar staining but yield speckled nuclear staining resembling that of other ANA specificities, e.g., antiU 1 small nuclear ribonucleoprotein antibodies (Okano et al., 1993; Kuwana et al., 1993). The speckled nuclear staining is probably produced by anti-RNAP II or anti-RNAP III antibodies because of the extranucleolar localization of these molecules (Hirakata et al., 1993; Okano et al., 1993). Therefore, it is impossible to differentiate anti-RNAP I, as well as antiRNAP II and III antibodies, from other ANA specificities based solely on the staining pattern by indirect immunofluorescence. Radioimmunoprecipitation assays with HeLa cell extract are used to identify the antigen specificities of anti-RNAP antibodies. Sera with anti-RNAP I antibodies were initially described to immunoprecipitate 13 proteins from [35S]-methionine-labeled HeLa cell extract with molecular weights ranging from 210 kd to 14 kd (Reimer et al., 1987). These proteins had molecular weights identical to those precipitated by rabbit anti-RNAP I antisera obtained through immunization with biochemically purified RNAP I proteins. In addition, their common identities were confirmed in immunoabsorption experiments. Because mammalian RNAP I has only eight well-defined subunits, concern remains whether the additional proteins represent true subunits of RNAP I, components of RNAP II and III which share subunits with RNAP I or coimmunoprecipitating RNAP I-associated proteins. Indeed, that some of the additional proteins are subunits of RNAP II and III (Okano et al., 1993; Hirakata et al., 1993; Kuwana et al., 1993) is manifest by reactivity with the distinctive pairs of large subunits with molecular weights greater than 100 kd in each class of RNAP. Based on these reactivities, sera containing antibodies to RNAPs can be classified into three groups (Figure 1), including those which im-

munoprecipitate six proteins corresponding to the three pairs of large subunits of all three classes of RNAP (lanes 2 and 3), those which precipitate four large proteins corresponding to the two pairs of large subunits of RNAP I and III (lanes 4 and 5) and those which precipitate only the two large RNAP III subunit proteins (lanes 6 and 7). In addition, autoimmune sera were reported to preferentially immunoprecipitate the 240 kd phosphorylated (IIO) form of the large RNAP II subunit (Satoh et al., 1994a; 1994b). With whole-cell extracts as substrates for immunoblots, anti-RNAP sera do not produce reliable reactivity to subunit proteins of RNAPs, presumably due to insufficient copies of RNAP molecules in a cell. Purified or partially purified RNAPs are good substrates for the identification of immunoreactive subunit proteins in several studies. Immunoblots suggest two types of autoimmune sera reactive with RNAPs: autoantibodies reactive with the distinctive large subunits in each class and those reactive with the small subunits that are shared among two or three RNAPs (Okano et al., 1993; Kuwana et al., 1993; Hirakata et al., 1993). However, systematic analyses of immunoreactive subunit proteins using large numbers of anti-RNAP-positive sera and purified antigens are needed. RNAP-reactive antibodies can inhibit or deplete RNAP enzymatic activity (Reimer et al., 1987; Okano et al., 1993; Kuwana et al., 1993).

CLINICAL UTILITY Prevalence and Clinical Associations

In initial reports (Stetler et al., 1982; 1987), antiRNAP I antibodies were detected at a high frequency in patients with various connective tissue diseases. However, these original results have not been confirmed by other investigators. Subsequent studies reveal that anti-RNAP I antibodies are found in SSc patients at a frequency of 4 to 33%, but are observed rarely in other autoimmune diseases (Table 3). One study suggests that anti-RNAP I-positive patients have significantly more extensive cutaneous thickening than the antinucleolar antibody-negative patients (Reimer et al., 1988). First observed in 4% of SSc patients (but not in 210 patients with SLE or polymyositis), autoantibodies to RNAP II are usually accompanied by antibodies to RNAP I and III (Hirakata et al., 1993).

729

Figure 1. Immunoprecipitated proteins of a [35S]-methionine-labeled HeLa cell extract obtained using serum specimens from patients with systemic sclerosis. Lane 1: proteins precipitated by normal human serum (NHS). Lanes 2, 3, 4, 5, 6 and 7: proteins precipitated from serum specimens of six patients with systemic sclerosis. Antigen specificity (noted on the top) to which serum antibodies are directed was determined based on observations of the largest subunits of RNA polymerase I, II and III in immunoprecipitates. Positions of the largest subunits of RNA polymerase I (IA and IB), RNA polymerase II (IIO, IIA and IIC) and RNA polymerase III (IIIA and IIIB) are indicated by arrows on the right. Positions of molecular weight standards[kd] are shown at the left of lane 1.

However, subsequent studies revealed that antibodies to RNAP II were also detected in SLE or overlap syndrome patients (Satoh et al., 1994a). In this study the authors identified autoantibodies to R N A P IIO that were previously unrecognized. Anti-RNAP II antibodies were detected in SLE patients of all races; whereas, in SSc patients anti-RNAP II were seen in Japanese and African Americans, but never in Caucas-

730

ians. The striking association of anti-RNAP IIO and anti-DNA topoisomerase I (topo I) antibodies found in Japanese and African American SSc patients (but not in U.S. Caucasian patients) is associated with a significantly higher frequency of diffuse cutaneous involvement, pigmentation changes, flexion contractures, and acro-osteolysis, compared with patients having autoantibodies to antitopo I alone.

Table 3. Prevalence of Autoantibodies to RNA Polymerases, According to the Reactive RNA Polymerase Class, Region (Race) and Diagnosis Authors (Year)

Method of Detection*

Polymerase Class

Region (Race)

Diagnosist

Patients Positive/Studied

Frequency (%)

Stetler et al., 1982

RIA

RNAP I

USA

SLE MCTD RA

12/12 4/4 7/9

100 100 78

Stetler et al., 1987

RIA

RNAP I

USA

SS SSc SLE

8/9 11/19 19/19

88 58 100

Reimer et al., 1987

IF, RIP

RNAP I

USA

SSc SLE, MCTD or RA

9/208 0/100+

4 0

Kipnis et al., 1990

RIP

RNAP I, II and III

USA

SSc PRP

6/69 0/40

9 0

Okano et al., 1993

RIP

RNAP RNAP RNAP RNAP

USA USA USA USA

SSc SSc SSc SLE, PM, PRP

13/252 27/252 17/252 0/150

5 10 7 0

Kuwana et al., 1993

RIP

RNAP I, II, and III RNAP I and III RNAP I, II or III

Japan (Japanese)

SSc SSc SLE, PM, PRP, SLE-PM Overlap

2/275 12/275 0/286

4 0

13/336 0/317

I, II and III I and III III I, II or III

1

Hirakata et al., 1993

RIP

RNAP II

USA

SSc SLE, PM, MCTD

Satoh et al., 1994a

RIP

RNAP II

Japan (Japanese)

SLE MCTD/Overlap SSc PM, SS

7/76 6/42 7/35 0/39

9 14 20 0

USA (Caucasian)

SLE SSc MCTD/Overlap, PM

2/29 1/9 0/3

7 ll 0

6/33 2/3 1/3

18 67 33

USA (African- American) SLE MCTD/Overlap Ssc

(continued)

Table 3.

(continued) Method of Detection*

Polymerase Class

Region (Race)

Diagnosist

Satoh et al., 1994a

RIP

RNAP I and III

Japan (Japanese)

SSc SLE, MCTD/Overlap, PM, SS

1/35 0/157

3 0

USA (Caucasian)

SSc SLE, MCTD/Overlap, PM

2/9 0/32

22 0

USA (African-American) SLE SSc MCTD/Overlap

1/33 1/3 0/3

3 33 0

Japan (Japanese)

SSc

3/71

4

USA (Caucasian)

SSc

1/39

3

USA (African-American) SSc

2/19

11

Japan (Japanese)

SSc

2/71

3

USA (Caucasian)

SSc

2/39

5

USA (African-American) SSc

0/19

0

(continued)

Satoh et al., 1994b

RIP

RNAP I, II and III

RNAP I and III

RNAP II

Kuwana et al., 1994b

RIP

RNAP I, II and III

Japan (Japanese)

SSc

15/71

21

USA (Caucasian)

SSc

0/39

0

USA (African-American) SSc

1/19

5

Japan (Japanese)

SSc

14/275

5

USA (Caucasian)

SSc

50/207

24

2/14

14

USA (African-American) SSc =

Patients Positive/Studied

Frequency (%)

Authors (Year)

solid-phase radioimmunoassay with biochemically purified RNAP I; IF = immunofluorescence tests for antinuclear antibodies" RIP = radioimmunoprecipitation assay

*

RIA

t

with [35S]-labeled cell extract as antigen SLE = systemic lupus erythematosus; MCTD = mixed connective tissue disease; RA = rheumatoid arthritis; SS = Sj6gren's syndrome; SSc = systemic sclerosis (scleroderma); PRP = primary Raynaud's phenomenon; PM = polymyositis

Autoantibodies to RNAP III were found in 23% of consecutive unselected SSc patients, but never in 150 comparison patients (Okano et al., 1993). Anti-RNAP III antibody was significantly more frequent in patients with diffuse cutaneous involvement than in those with limited cutaneous involvement or those with SSc overlap syndrome. Among patients with diffuse cutaneous involvement, anti-RNAP III antibody was more common than anti-topo I antibody. Patients with anti-RNAP III had a significantly higher mean maximum skin thickness score, but significantly lower frequencies of telangiectasias, inflammatory myopathy, restrictive lung disease and serious cardiac abnormalities than did patients with antitopo I antibody. Subsequently, a publication on Japanese patients showed that the presence of antibodies to RNAP I, II and III (Kuwana et al., 1993) was highly specific for SSc, particularly the diffuse cutaneous variant. Those having anti-RNAP antibody were significantly more often male and older at onset, had higher frequencies of renal and cardiac involvement and had a reduced five-year cumulative survival.

Racial Differences. Several recent studies confirm that autoantibody recognition of all three classes of RNAPs is influenced by race (Kuwana et al., 1994a;

REFERENCES Clawson K, Okano Y, Fertig N, Medsger TA Jr, Morel PA. Human leukocyte antigen (HLA) associations in systemic sclerosis (SSc) patients with autoantibodies to RNA polymerase (POI). Arthritis Rheum 1994;37(6):R27. Hirakata M, Okano Y, Pati U, Suwa A, Medsger TA Jr, Hardin JA, Craft J. Identification of autoantibodies to RNA polymerase II. Occurrence in systemic sclerosis and association with autoantibodies to RNA polymerases I and III. J Clin Invest 1993 ;91:2665--2672. Kipnis RJ, Craft J, Hardin JA. The analysis of antinuclear and antinucleolar autoantibodies of scleroderma by radioimmunoprecipitation assays. Arthritis Rheum 1990;33:1431--1437. Kuwana M, Kaburaki J, Mimori T, Tojo T, Homma M. Autoantibody reactive with three classes of RNA polymerases in sera from patients with systemic sclerosis. J Clin Invest 1993;91:1399--1404. Kuwana M, Kaburaki J, Okano Y, Tojo T, Homma M. Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis. Arthritis Rheum 1994a;37:75--83. Kuwana M, Okano Y, Kaburaki J, Tojo T, Medsger TA Jr. Racial differences in the distribution of systemic sclerosisrelatedserumanlinuclearanlibc~es.ArthritisRtetn~ 1994b;37:902-906. Okano Y, Steen VD, Medsger TA Jr. Autoantibody reactive

1994b; Satoh et al., 1994a; 1994b). However, close association of anti-RNAP and the diffuse cutaneous subset of SSc was consistent across different racial groups.

CONCLUSION Radioimmunoprecipitation assays with [35S]-methionine-labeled cell extract as the antigen source is the preferred method for detecting RNAP I, II and III antibodies. A practical technique suitable for a screening assay is not available. Autoantibodies to RNAP I and III seem to be specific for SSc, especially for SSc with diffuse cutaneous involvement and serious internal organ involvement. Autoantibodies to RNAP II are found in SSc as well as SLE and overlap syndromes. The variable disease associations of antibodies to RNAPs among Japanese, U.S. Caucasians and African Americans suggests that genetic factors ins production of these autoantibodies. The precise specificities of subunit proteins carrying antigen determinants recognized by autoantibodies to RNAPs are unknown as are the pathogenetic mechanisms that might link these autoimmune responses to clinical manifestations of disease.

with RNA polymerase III in systemic sclerosis. Ann Intern Med 1993;119:1005--1013. Reimer G, Rose KM, Scheer U, Tan EM. Autoantibody to RNA polymerase I scleroderma sera. J Clin Invest 1987;79:65-72. Reimer G, Steen VD, Penning CA, Medsger TA Jr, Tan EM Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma). Arthritis Rheum 1988;31:525--532. Satoh M, Ajmani AK, Ogasawara T, Langdon JJ, Hirakata M, Wang J, Reeves WH. Autoantibodies to RNA polymerase II are common in systemic lupus erythematosus and overlap syndrome. Specific recognition of the phosphorylated (IIO) form by a subset of human sera. J Clin Invest 1994a;94: 1981--1989. Satoh M, Kuwana M, Ogasawara T, Ajmani AK, Langdon JJ, Kimpel D, Wand J, Reeves WH. Association of autoantibodies to topoisomerase I and the phosphorylated (IIO) form of RNA polymerase II in Japanese scleroderma patients. J Immunol 1994b;153:5838-5848. Sentenac A. Eukaryotic RNA polymerases. CRC Crit Rev Biochem 1985;18:31--90. Shibata S, Muryoi T, Saitoh Y, Brumeanu TD, Bona CA, Kasturi KN. Immunochemical and molecular characterization of anti-RNA polymerase I autoantibodies produced by tight skin mouse. J Clin Invest 1993;92:984-992 Stetler DA, Rose KM, Wenger ME, Berlin CM, Jacob ST.

733

Antibodies to distinct polypeptides of RNA polymerase I in sera from patients with rheumatic autoimmune disease. Proc Natl Acad Sci USA 1982;79:7499--7503. Stetler DA, Jacob ST. Phosphorylation of RNA polymerase I augments its interaction with autoantibodies of systemic lupus erythematosus patients. J Biol Chem 1984:259:13629-13632. Stetler DA, Sipes DE, Jacob ST. Anti-RNA polymerase I antibodies in sera of MRL lpr/lpr and MRL +/+ autoimmune mice. Correlation of antibody production with delayed onset of lupus-like disease in MRL +/+ mice. J Exp Med 1985; 162:1760--1770. Stetler DA, Cavallo T. Anti-RNA polymerase I antibodies: potential role in the induction and progression of murine lupus nephritis. J Immunol 1987;138:2119--2123.

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Stetler DA, Reichlin M, Berlin CM, Jacob ST. Autoantibodies against RNA polymerase I in scleroderma and Sj6gren's syndrome sera. Biochem Biophys Res Commun 1987;144: 1296-- 1302. Thompson NE, Steinberg TH, Aronson DB, Burgess RR. Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. J Biol Chem 1989;264:11511-11520. Thompson NE, Aronson DB, Burgess RR. Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody. J Biol Chem 1990;265:7069-7077.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

SIGNAL RECOGNITION PARTICLE AUTOANTIBODIES Frederick W. Miller, M.D., Ph.D.

Molecular Immunology Laboratory, Division of Cellular and Gene Therapies, Centerfor Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA

HISTORICAL NOTES

Origin/Function

Human autoantibodies to the signal recognition particle (anti-SRP) were first described in a polymyositis patient in 1986 (Reeves et al., 1986) and shortly thereafter independently identified in several additional patients (Okada et al., 1987). Thirteen individuals with polymyositis and anti-SRP were reported in 1990; their similar clinical presentations and disease course suggested that these autoantibodies may identify a distinct group of myositis patients (Targoff et al., 1990). Subsequent studies, as part of a larger investigation of the etiology, classification, and treatment of myositis, confirmed that these myositisspecific autoantibodies (MSA) identify an epidemiologically (Left et al., 1991), clinically (Love et al., 1991; Miller, 1993; 1994) and immunogenetically (Miller, 1991) distinct form of idiopathic inflammatory myopathy.

Composed of six polypeptides and a tRNA-like molecule known as 7SL RNA, SRP can undergo selfassembly in solution from the isolated components, each of which is fully characterized and sequenced (Zwieb, 1989; Lutcke and Dobberstein, 1993; Zwieb and Larsen, 1994). The six SRP polypeptides (9, 14, 19, 54, 68 and 72 kd) are present in the particle either as monomers (SRP19 and SRP54) or as heterodimers (SRP9/14 and SRP68/72). SRP can be divided into two major domains by the use of nuclease digestion: the Alu domain consists of SRP9/14 attached to the Alu sequences at the 5' and 3' ends of the 7SL RNA and the S domain is made of the other four proteins attached to the central S-fragment of 7SL RNA (Figure 2). The first step mediated by SRP is the binding of one of its proteins, SRP54, to a region known as the signal sequence of newly synthesized secretory and membrane polypeptides as they emerge from the ribosome (Lutcke and Dobberstein, 1993). SRP54 can be proteolytically digested into two fragments: the Cterminal region that is rich in methionine residues (M domain) and the N-terminal portion (called SRP54G or SRP54N+G) that contains a GTPase domain (Figure 2). Nascent polypeptide binding is accomplished by SRP54M with the SRP54G domain enhancing the affinity of this interaction (Luirink and Dobberstein, 1994). The Alu domain of SRP plays a role in retarding protein elongation as SRP chaperones nascent polypeptides to the ER (Rapoport, 1990). The protein in the ER that binds SRP is called the SRP-binding protein or docking protein. SRP itself does not move the nascent polypeptide into the ER, but rather brings

THE AUTOANTIGENS

Nomenclature The process of protein synthesis (called translation) is an extremely complex and regulated orchestration of hundreds of compartmentalized proteins and RNAs (Trachsel, 1991). An important part of translation involves directing some of the newly made proteins to the endoplasmic reticulum where they are further modified or exported. The signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein complex that directs this movement of newly synthesized proteins from the polysome to the endoplasmic reticulum (ER) (Figure 1).

735

Figure 2. Schematic structure of the mammalian signal recognition particle (SRP). The 7SL RNA is depicted as a thick line; the proteins are indicated by circles and identified by their molecular masses (in kd). The 54 kd SRP protein, SRP54, the major target of anti-SRP autoantibodies, is shaded and the GTPase (G) and methionine-rich (M) domains are indicated. Taken from (Lutcke et al., 1993). Figure 1. The signal recognition particle (SRP) cycle of protein translocation from the ribosome to the endoplasmic reticulum (ER). The upper left portion shows SRP binding to a ribosome free in the cytoplasm and to the signal sequence (indicated as a thicker region) of the newly synthesized polypeptide to induce elongation slowing or arrest. After this binding, the SRPribosome-nascent polypeptide chain complex is brought to the ER, and the SRP binds to the SRP-receptor or docking protein that is embedded in the ER membrane and has bound guanosine 5'-diphosphate (GDP) (shown in the upper fight part of the figure). Next the docking protein binds guanosine 5'-triphosphate (GTP), replacing the previously bound GDP (seen in the lower fight). Finally, the nascent polypeptide and ribosome are released from the SRP, and the signal sequence binds to the signal sequence binding protein in the ER membrane (lower left). The SRP is then released from the SRP-receptor to allow the cycle to be repeated. Taken from (Rapoport, 1990). it into close proximity to another protein embedded in the ER known as the signal sequence receptor that binds the signal sequence after it is released from SRP54 (Figure 1).

Sequence Similarity SRP appears to be a ubiquitous particle with homologues in life forms as diverse as insects, plants, yeast and bacteria (Zwieb et al., 1994; Lutcke and Dobberstein, 1993). These SRP homologues, which are generally smaller and simpler ribonucleoprotein complexes, serve similar functions as signal-specific chaperones in the posttranslational translocation of proteins (Poritz et al., 1990; Lutcke and Dobberstein, 1993).

736

AUTOANTIBODIES

Pathogenetic Role Autoantibodies to the SRP (anti-SRP) immunoprecipitate the entire ribonucleoprotein complex, but bind primarily to SRP54 (Reeves et al., 1986; Okada et al., 1987; Targoff et al., 1990). Some anti-SRP also target SRP68 and SRP72 (unpublished observations). There is no direct evidence that anti-SRP autoantibodies have a pathologic role, and they are not found spontaneously in any animals nor have they been induced in any animal model of myositis to date. They do appear, however, to be one of a group of autoantibodies found only in myositis patients (Plotz et al., 1995) and thus are known as myositis-specific autoantibodies or MSA. Although their genesis is unknown, an appealing hypothesis is that SRP, in binding to and processing foreign proteins, become structurally altered and are then targeted for immune attack in certain individuals.

Genetics Little is known about the genes associated with antiSRP, but HLA-DR5 and DRw52 were found in the majority of the small number of patients identified to date with these antibodies (Goldstein et al., 1990; Love et al., 1991). The epitopes for anti-SRP are poorly studied, but preliminary data suggest that the primary epitope is within the G domain of SRP54

(Karin Romisch, personal communication). The antiSRP epitopes also appear to be conserved in a wide range of species including human, dog, mouse and rat (Targoff et al., 1990).

Methods of Detection The simplest method of screening for MSAs and most other autoantibodies is indirect immunofluorescence testing using HEp-2 cells as a substrate. Although this is often performed for the purpose of detecting ANAs, if the laboratory is evaluating other patterns of reactivity, a homogeneous cytoplasmic pattern may be observed in patients with antibodies to SRP. Protein-A assisted immunoprecipitation of proteins and RNA, from cultured HeLa cells, is the most sensitive and specific method of detecting the MSAs including antiSRP (Rider et al., 1995) (Figure 3). Enzyme-linked immunosorbent assays using purified dog pancreas SRP also detect anti-SRP (Targoff et al., 1990); these techniques are not available outside a few research centers.

classic "anti-SRP syndrome" is a form of polymyositis, affecting mainly black females, in whom the myositis is acute and severe in onset and occurs in the fall of the year. Myalgias and cardiac involvement are common. The muscle biopsy often demonstrates severe myonecrosis with minimal inflammation.

Antibody Frequencies in Disease Anti-SRP autoantibodies are rare. They are present in only about 4% of U.S. myositis patients and in only 18% of those with anticytoplasmic antibodies other than anti-Jo- 1 (Targoff et al., 1990). The distribution throughout the world and frequency and prevalence of anti-SRP outside the U.S. are unknown. These autoantibodies are identified, however, in sera of adult and juvenile polymyositis patients from North and South America, Japan and India (Hirakata et al., 1992; Rider et al., 1994; Rider and Miller, 1994 and unpublished observations). The few non-U.S, patients identified to date appear to share similar clinical characteristics with those in the U.S.

Effect of Therapies CLINICAL UTILITY

Disease Associations Anti-SRP are only found in patients with myositis syndromes. This is a group of disorders characterized by chronic muscle weakness resulting from muscle inflammation of unknown cause. The etiology and pathogenesis of these heterogeneous diseases, of which polymyositis and dermatomyositis are the most common forms, are poorly understood. Current data suggest, however, that they may develop after exposure of genetically susceptible individuals to environmental agents that induce immune activation and subsequent chronic muscle inflammation (Love and Miller, 1993; Plotz et al., 1995). Patients with these disorders have systemic connective tissue diseases and often demonstrate cutaneous, cardiac, pulmonary and gastrointestinal manifestations which can complicate diagnosis and adversely affect prognosis (Plotz et al., 1989; Miller, 1994). Anti-SRP autoantibodies, like the other MSAs, are useful in classifying the myositis syndromes into serologic groups with more homogeneous epidemiologic, clinical, serologic and prognostic features (Love et al., 1991; Left et al., 1991; Joffe et al., 1993; Miller, 1994; Plotz et al., 1995). Although there are some differences from patient to patient, the

The disease is resistant to corticosteroid therapy, requiring the early addition of cytotoxic agents. As a group, patients with anti-SRP have a particularly poor prognosis with a mean 5-year mortality of 75%, primarily as a result of cardiac disease and progressive weakness. Anti-SRP patients often present with serum creatine kinase levels of more than 100 times the upper limits of normal and can be bed-bound and require nasogastric feeding very early in their disease course. They tend to have prolonged periods of significant disability and may require a wheelchair, a walker and other rehabilitation aids and therapy. Thus, although clinical utility is limited by the restricted availability of assays, patients known to have antiSRP antibodies should be considered for early and aggressive therapy (Plotz et al., 1995).

CONCLUSION Human autoantibodies directed at the signal recognition particle are a rare form of myositis-specific autoantibodies. Anti-SRP antibodies primarily target and bind to SRP54, the 54 kd protein of this ribonucleoprotein complex that is involved in facilitating the translocation of nascent polypeptides from the poly-

737

Figure 3. Confirmation of anti-SRP autoantibodies by RNA immunoprecipitation (showing the common 7SL RNA, upper panel) and protein immunoprecipitation (showing the predominant SRP54 protein, lower panel) analyses. The upper panel shows the results of silver staining of a 7M urea-10% polyacrylamide gel after electrophoresis of phenol extracts from immunoprecipitates of HeLa cells. The lower panel shows results of autoradiography after sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis of 35-S-labeled HeLa cell proteins immunoprecipitated by sera. Lanes 1--13 are from sera of anti-SRP-positive polymyositis patients; NL is normal serum; aJo (anti-Jo-1), aPL-7, aPL-12, Ro, La, aSm and aSRP are reference sera containing the indicated autoantibodies; TNA is total nucleic acid. The positions of migration of 5.8S, 5.0S and tRNA (upper panel) or molecular weight marker proteins (lower panel) are indicated. Taken from (Targoff et al., 1990). 738

Table 1. Summary of Characteristics of Signal Recognition Particle (SRP) Autoantibodies

Discovery Date

1986

Autoantigen

S R P - a ribonucleoprotein complex of six polypeptides and 7SL RNA

Function of SRP

Chaperones nascent polypeptides to the endoplasmic reticulum for further processing

Primary Autoantibody Target

SRP54 (the 54 kd polypeptide of S R P ) - reactivity known to human, dog, rodent SRP

Origin and pathogenetic role

Unknown

Genetic associations

HLA DR5 and DRw52

Detection

Indirect immunofluorescence shows diffuse cytoplasmic staining - confirmation by immunoprecipitation or immunoblotting

Clinical associations

A rare myositis-specific autoantibody- seen in only 4% of myositis patients in the U . S . - often found in black females with an acute severe onset of polymyositis in the fall; myalgias, cardiac involvement, poor response to therapy and 5-year survival of only 25%, with deaths primarily due to cardiac disease.

some to the endoplasmic reticulum. Characterized by a diffuse cytoplasmic staining pattern after indirect immunofluorescence on HEp-2 cells, anti-SRP are confirmed by immunoprecipitation of the characteristic six SRP proteins and 7SL RNA. As is the case for all myositis-specific autoantibodies studied in detail to date, anti-SRP delineate a relatively homogenous group of patients with similar clinical features, immunogenetics and responses to therapy. Patients in the U.S. with the "anti-SRP syndrome" tend to be black females with an acute onset of severe polymyositis in

the fall of the year, with a high incidence of cardiac involvement, resistance to corticosteroid and other therapy, H L A - D R 5 and D R w 5 2 and a 5-year mortality of 75%, primarily as a result of cardiac involvement and progressive muscle weakness and atrophy. How these autoantibodies arise and whether they have any etiopathologic role in polymyositis remain unclear. See also AMINOACYL-tRNA HISTIDYL (JO-1) SYNTHETASE AUTOANTIBODIES, AMINOACYL-tRNA (OTHER THAN HISTIDYL) SYNTHETASE AUTOANTIBODIES and MI-2 AUTOANTIBODIES.

REFERENCES

Love LA, Left RL, Fraser DD, Targoff IN, Dalakas M, Plotz PH, Miller FW. A new approach to the classification of idiopathic inflammatory myopathy: myositis-specific autoantibodies define useful homogeneous patient groups. Medicine 1991;70:360--374. Love LA, Miller FW. Noninfectious environmental agents associated with myopathies. Curr Opin Rheumatol 1993;5: 712--718. Luirink J, Dobberstein B. Mammalian and Escherichia coli signal recognition particles. Mol Microbiol 1994;11:9--13. Lutcke H, Dobberstein B. Structure and function of signal recognition particle (SRP). Mol Biol Rep 1993;18:143--147. Miller FW. Humoral immunity and immunogenetics in the idiopathic inflammatory myopathies. Curr Opin Rheumatol 1991;3:902--910. Miller FW. Myositis-specific autoantibodies. Touchstones for understanding the inflammatory myopathies. JAMA 1993; 270:1846--1849. Miller FW. Classification and prognosis of inflammatory muscle disease. Rheum Dis Clin North Am 1994;20:811-826. Okada N, Mimori T, Mukai R, Kashiwagi H, Hardin JA.

Goldstein R, Duvic M, Targoff IN, Reichlin M, McMenemy AM, Reveille JD, Warner NB, Pollack MS, Arnett FC. HLAD region genes associated with autoantibody responses to histidyl-transfer RNA synthetase (Jo-1) and other translationrelated factors in myositis. Arthritis Rheum 1990;33:1240-1248. Hirakata M, Mimori T, Akizuki M, Craft J, Hardin JA, Homma M. Autoantibodies to small nuclear and cytoplasmic ribonucleoproteins in Japanese patients with inflammatory muscle disease. Arthritis Rheum 1992;35:449--456. Joffe MM, Love LA, Left RL, Fraser DD, Targoff IN, Hicks JE, Plotz PH, Miller FW. Drug therapy of the idiopathic inflammatory myopathies: predictors of response to prednisone, azathioprine, and methotrexate and a comparison of their efficacy. Am J Med 1993;94:379--387. Left RL, Burgess SH, Miller FW, Love LA, Targoff IN, Dalakas MC, Joffe MM, Plotz PH. Distinct seasonal patterns in the onset of adult idiopathic inflammatory myopathy in patients with anti-Jo-1 and antisignal recognition particle autoantibodies. Arthritis Rheum 1991 ;34:1391--1396.

739

Characterization of human autoantibodies that selectively precipitate the 7SL RNA component of the signal recognition particle. J Immunol 1987;138:3219--3223. Plotz PH, Dalakas M, Left RL, Love LA, Miller FW, Cronin ME. Current concepts in the idiopathic inflammatory myopathies: polymyositis, dermatomyositis, and related disorders. Ann Intern Med 1989;111:143--157. Plotz PH, Rider LG, Targoff IN, Raben N, O'Hanlon TP, Miller FW. NIH Conference. Myositis: immunologic contributions to understanding cause, pathogenesis, and therapy. Ann Intern Med 1995;122:715--724. Poritz MA, Bernstein HD, Strub K, Zopf D, Wilhelm H, Walter P. An E. coli ribonucleoprotein containing 4.5S RNA resembles mammalian signal recognition particle. Science 1990;250:1111-1117. Rapoport TA. Protein transport across the ER membrane. Trends Biochem Sci 1990;15:355--358. Reeves WH, Nigam SK, Blobel G. Human autoantibodies reactive with the signal-recognition particle. Proc Natl Acad Sci USA 1986;83:9507-9511.

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Rider LG, Miller FW, Targoff IN, Sherry DD, Samayoa E, Lindahl M, Wener MH, Pachman LM, Plotz PH. A broadened spectrum of juvenile myositis. Myositis-specific autoantibodies in children. Arthritis Rheum 1994;37:1534-1538. Rider LG, Miller FW. New perspectives on the idiopathic inflammatory myopathies of childhood. Curr Opin Rheumatol 1994;6:575--582. Rider LG, Miller FW. Laboratory evaluation of the inflammatory myopathies. Clin Diagn Lab Immunol 1995;2:1--9. Targoff IN, Johnson AE, Miller FW. Antibody to signal recognition particle in polymyositis. Arthritis Rheum 1990; 33:1361--1370. Trachsel H. Translation in eukaryotes. Boston: CRC Press, 1991. Zwieb C. Structure and function of signal recognition particle RNA. Prog Nucleic Acid Res Mol Biol 1989;37:207--234. Zwieb C, Larsen N. The signal recognition particle database (SRPDB). Nucleic Acids Res 1994;22:3483--3484.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

SILICATE AND SILICONE ANTIBODIES Guo-Qiu Shen, M.D. and Emmanuel A. Ojo-Amaize, Ph.D.

Specialty Laboratories Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES Silicone first became commercially available in 1943, and the first experimentation for subdermal implantation began in the late 1940s (Braley, 1964; Sergott et al., 1986). Silicone plastic polymers are used in many medical fields such as for breast implants in cosmetic or reconstructive surgery, vascular prostheses, joint repair and replacement. In 1964, silicone-induced human adjuvant disease was reported as an ailment characterized by autoimmune disease-like symptoms, granulomas and serological abnormalities (Miyoshi et al., 1964). A delayed hypersensitivity reaction to silicone plastic as defined by the presence of refractile particles in phagocytes and passage between lymphocytes and macrophages has been reported in some patients with breast and joint implants (Gower et al., 1984).

THE ANTIGEN(S) Silicone is a surface-reactive organic (carbon-containing) form of silicon; silica is a highly fibrogenic inorganic (mineral) form of silicon. The molecular formula is SiO 2. Silica can dissolve in NaOH, which becomes sodium silicate (Wells, 1975). Silicone is a polymer with a Si-O backbone and primarily methyl sidegroups (Figure 1). Oxygen is a highly electronegative element involved in the generation of polarized and ionized organic molecules. The hypothesis is that the numerous oxygen atoms may affect the silicon and sidegroups, resulting in the formation of electrostatic charges within the polymer, and indeed the major electronegative static charge of the silicone surface may cause silicone to be more antigenic since electrostatic forces are important in antigen-antibody binding

(Hebal, 1984). Silicone could act as a specific immunogen leading to cross-reacting immune responses toward self or through adjuvant properties which could activate natural host autoreactive systems (Shons and Schubert, 1992; Yoshida et al., 1993).

ANTIBODIES Pathogenetic Role The mechanism of induction of antibodies or cellmediated immunity by silicone is unknown. Recent studies show that specific immune responses can be generated against silicone (polydimethylsiloxane) solutions (Heggers et al., 1983; Kossovsky et al., 1987; Goldblum et al., 1992; Kossovsky et al., 1993; Wolf et al., 1993; Ojo-Amaize et al., 1994). Increased amounts of silicone-specific antibodies were detected in two patients with unusual complications of ventriculoperitoneal (VP) shunt placement (Goldblum et al., 1992). In that study (Goldblum et al., 1992) silastic tubing served as the solid-phase antigen to test serum from the patients and controls. IgG binding to silastic tubing was consistently higher in the two patients than in healthy controls. These findings demonstrate that a specific humoral response to silicone can develop in some patients after exposure to silicone antigen. High concentrations of silicone are found in women with either frank implant ruptures or leakage of their silicone gel implants using siliconecoated plates in EIA (Wolf et al., 1993). IgG binding to fibronectin-laminin adsorbed to silicone was detected in 3.6% of 249 patients with silicone breast implants (SBI); IgG binding to silicone alone was detected in 1.6%. These small numbers and differences may be due to the low sensitivity of the assay o

741

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Figure1. Silicones and silicates structure, a: Silicones. b: Silicates. method utilized. Nevertheless, this observation demonstrates the presence of antibodies against macromolecules, especially fibronectin and laminin denatured by silicone in patients with medical exposure (Kossovsky et al., 1993). Silicone may induce autoimmune disease by acting as a specific immunogen leading to cross-reacting immune responses to self tissues (Yoshida et al, 1993; Kossovsky and Freiman, 1994). IL-1, TNF-t~ and IL-6 can be produced spontaneously in cultured PBMCs of individuals with positive silicate-reactive T-cells. The results showed that the levels of IL-I~, IL-lra and antisilicate antibodies (both IgG and IgM) were elevated in 27 symptomatic women with SBI. Evidence from human and animal models suggests that polydimethylsiloxanes (silicone), the analogues of the silicon-oxygen ions found in silicates, are not immunologically inert, but can elicit specific humoral and cell-mediated immune reactions (Heggers et al., 1983; Kossovsky et al., 1987; Goldblum et al., 1992; Kossovsky et al., 1993; Wolf et al., 1993; Ojo-Amaize et al., 1994). Although elevated silicon concentrations are found in sera from some women with SBI (OjoAmaize et al., 1994), in uremic and dialysis patients (Indrapraset et al., 1974; Dobbie and Smith, 1986), in the brains of patients with senile dementia and in neurofibrillary tangles of patients with Alzheimer's disease (Candy et al., 1986; Edwardson et al., 1986), no reports of silicate-reactive antibodies (as opposed to silicone antibodies) in humans or animals are available. Silicate is known to induce interleukin-1 (IL-1) production in antigen-presenting monocytes and macrophages (APC) (Dinarello, 1978). It is thus tempting to postulate that silicate can interact directly or indirectly (by binding to exogenous or endogenous peptides) with APC and can then be presented to B cells and T cells in conjunction with major histocompatibility complex molecules for induction of a specific immune response. A similar hypothesis has been proposed for Be 2+ (Newman, 1993). Therefore, if a haptenic stimulus like silicate consistently triggers the production of cytokines such as IL-1 and IL-6, 742

deregulation of self-tolerance may occur which could lead to the production of autoantibodies (silicate + self MHC-peptide complex ~ IL-6 --~ Th2 T cells ~ IL4 ~ humoral immune response) or to the induction of antiself cellular immune response (silicate + self MHC-peptide complex ~ IL-1 --~ Thl T cells ~ IL2/IFN7 --~ cellular immune response). The intracellular cytokines may contribute to inflammatory reaction and lead to tissue injury in autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), insulin-dependent diabetes, and multiple sclerosis. Each of these diseases is associated to one degree or another with abnormal cytokine production (Brennan and Feldmann, 1992). Interestingly, an association has been found between the presence of silicone breast implants and elevated concentrations of IL-I~ and IL-lra in women with silicone breast implants (Ojo-Amaize et al., 1996).

Factors in Pathogenicity Isotypes.

An enzyme immunoassay (EIA) method can detect and quantitate specific IgG and IgM antibodies to BSA-bound silicate in the plasma of different groups of women. Of 40 symptomatic women with silicone breast implants, 30% produced silicate-reactive IgG antibodies, but 9% (8/91) asymptomatic women with SBI also produced low levels of IgG silicate antibodies (Shen et al., 1996). The 30% (12/40) who tested positive had silicate antibodies restricted to IgG isotype (2 13 units __ 22.6 SD) and four (10%) had both IgG and IgM antibodies (~ 8.1 units + 23.2 SD). In a comparison of 91 asymptomatic women with SBI, only eight patients (9%) had IgG antisilicate antibodies (2 5.4 units _+5.2 SD; p < 0.001), two patients had antibodies of both IgG and IgM isotypes (2 5.1 units + 4.8 SD; p > 0.2). One of 50 healthy women without SBI (2%) had low levels of IgG and IgM antisilicate antibodies (Figure 2). None of the non-SBI women with Sj6gren's syndrome, scleroderma or rheumatoid arthritis had silicate-reactive antibodies of either IgG or IgM isotype (Figure 2a,b). Of 20 non-SBI patients with SLE tested, only one had low amounts of both IgG and IgM silicate-reactive antibodies (Figure 2a,b).

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Figure 2. Determination of disease specificity of silicate-reactive antibodies. A: Silicate-reactive IgG antibodies in healthy women, symptomatic women with breast implants and in women without implants but with different autoimmune diseases: SLE, systemic lupus erythematosus; SS, Sj6gren's syndrome; Scl, scleroderma; and RA, rheumatoid arthritis. B: Silicate-specific IgM antibodies in the different groups of women as in A. Methods of Detection Antibodies to sodium silicate can be assayed by EIA (Shen et al., 1996). Briefly, Immulon I microtiter plates are precoated with 200 gL of 0.5% fat-free bovine serum albumin (BSA) in distilled water (dHzO)/well. Half of each plate is coated with 200 laL of 1 mg NazSiO3/mL dH20, and the other half left uncoated and washed with dH20 served as control

wells without added silicate. 200 jaL of plasma, diluted 1:100 in 1% BSA inPBS-Tween is added to the appropriate wells. Following incubation, the standard EIA procedure is followed. Indeed, the frequency (30%) of antibodies detected is far greater than that (1.7--3%) when silicone gel adsorbed to a variety of proteins are employed (Kossovsky et al., 1993). This new test for silicate-reactive antibodies might prove valuable for the accurate

743

assessment of silicone effects on the immune system. There is no close correlation between the presence of silicate-reactive antibodies described and the T-cell responses to silicate, SiO 2 and silicone gel described elsewhere (Ojo-Amaize et al., 1994). Of 300 silicone breast implant patients tested for T-cell reactivity with silicate, 140 (47%) were positive against either SiO 2, silicate or silicone (or a combination of these antigens) in the T-cell proliferation assay, but only 10% of the 140 patients had silicate-specific IgG antibodies (Ojo-Amaize et al., in preparation). Therefore, a combined cellular/humoral test for silicone reactivity is likely to increase the sensitivity of laboratory tests for silicone reactivity.

CLINICAL UTILITY The clinical relevance of the presence of silicate antibodies or silicate/silicon(e)-specific T-cells in women with silicone breast implants is yet to be determined.

Disease Associations Many patients are reported to develop clinical syndromes resembling scleroderma, RA, SLE, and other rheumatic diseases after augmentation mammoplasty (Kumagai et al., 1984; van Nunen et a1.,1982; Varga et al., 1989; Kaiser et al., 1990; Marik et al., 1990; Baldwin and Kaplan, 1983; Freundlich et al., 1994). In a recent report of 50 women with silicone breast implants (SBI) who developed Sj6gren's syndrome (Freundlich et al., 1994), clinical examination revealed

REFERENCES Baldwin CM Jr, Kaplan EN. Silicone-induced human adjuvant disease? Ann Plast Surg 1983;10:270-273. Braley SA Jr. The medical silicones. Trans Am Soc Artif Intern Organs 1964;10:240-243. Brennan FM, Feldmann M. Cytokines in autoimmunity. Curr Opin Immunol 1992;4:754-759. Candy JM, Oakley AE, Klinowski J, Carpenter TA, Perry RH, Attack JR, Perry EK, Blessed G, Fairbairn A, Edwardson JA. Aluminosilicates and senile plaque formation in Alzheimer's disease. Lancet 1986;1:354--357. Cuellar ML, Scopelitis E, Tenenbaum SA, Garry RF, Silveira LH, Cabrera G, Espinoza LR. Serum antinuclear antibodies in women with silicone breast implants. J Rheumatol 1995; 22:236--240.

744

fibromyalgia tender points in 65% of the women, with 31% satisfying the ACR criteria for fibromyalgia (Freundlich et al., 1994; Martin, 1995). Other less clearly defined clinical syndromes have been termed "human adjuvant disease" (Sergott et al., 1986; Endo et al., 1987; Varga et al., 1989; Kaiser et al., 1990; Marik et al., 1990). In published studies, antinuclear antibodies (ANA) are found in about 26% of the SBI patient populations. All but two patients, one with SS-A and one with RNP antibodies, had a nonspecific speckled ANA pattern on HEp-2 slides (Freundlich et al., 1994; Vasey et al., 1994; Martin, 1995). In another study, 58% of (470/813) patients with SBI had ANA; a speckled pattern (341/813; 42%) was the most common followed by homogeneous pattern in 113/813; but centromere (5/813) and nucleolar (63/813) antibody patterns were also present (Cuellar et al., 1995). Also, some patients with silicone breast implant were found to have antisilicone IgG antibodies which bound to silicone tubing and silicone-coated to plates (Kossovsky et al., 1987; 1993; Goldblum et al., 1992). The symptomatic women with silicone breast implants had T-cell responses and antibodies to silicate (OjoAmaize et al., 1994; Shen et al., 1996).

CONCLUSION Silicate-specific antibodies of IgG and IgM isotypes can be detected in the circulation of women with silicone gel breast implants. The clinical relevance of the presence of these antibodies is yet to be determined.

Dinarello CA. Interleukin-1. Rev Infect Dis 1978;6:51. Dobbie JW, Smith MB. Urinary and serum silicon in normal and uraemic individuals. Ciba Found Symp 1986;121:194-213. Edwardson JA, Klinowski J, Oakley AE, Perry RH, Candy JM. Aluminosilicates and the ageing brain: implications for the pathogenesis of Alzheimer's disease. Ciba Found Symp 1986;121:160--179. Endo LP, Edwards NL, Longley S, Corman LC, Panush RS. Silicone and rheumatic diseases. Semin Arthritis Rheum 1987;17:112-118. Freundlich B, Altman C, Snadorfi N, Greenberg M, ~Tomaszewski J. A profile of symptomatic patients with silicone breast implants: a Sj6gren's-like syndrome. Semin Arthritis Rheum 1994;24:44--53. Goldblum RM, Pelley RP, O'Donell AA, Pyron D, Heggers JP.

Antibodies to silicone elastomers and reactions to ventriculoperitoneal shunts. Lancet 1992;340:510--513. Gower DJ, Lewis JC, Kelly DL Jr. Sterile shunt malfunction. A scanning electron microscopic perspective. J Neurosurg 1984 ;61:1079-- 1084. Hebal MB. The biological basis for the clinical application of the silicones. A correlate to their biocompatibility. Arch Surg 1984;119:843--848. Heggers JP, Kossovsky N, Parson S, Robson MC, Pelley RP, Raine TJ. Biocompatibility of silicone implants. Ann Plastic Surg 1983;11:38-45. Indraprasit S, Alexander GV, Gonick HC. Tissue composition of major and trace elements in uremia and hypertension. J Chronic Dis 1974;27:135--161. Kaiser W, Biesenbach G, Stuby U, Grafinger P, Zazgornik J. Human adjuvant disease: remission of silicone induced autoimmune disease after explanation of breast augmentation. Ann Rheum Dis 1990;49:937--938. Kossovsky N, Heggers JP, Robson MC. Experimental demonstration of the immunogenicity of silicone-protein complexes. J Biomed Mater Res 1987;21:1125--1133. Kossovsky K, Zeidler M, Chun G, Papasian N, Nguyen A, Rajguru S, Stassi J, Gelman A, Sponsler E. Surface dependent antigens identified by high binding avidity of serum antibodies in a subpopulation of patients with breast prostheses. J Applied Biomaterils 1993;4:281--288. Kossovsky N, Freiman CJ. Silicone breast implant pathology. Clinical data and immunologic consequences. Arch Pathol Lab Med 1994;118:686-693. Kumagai Y, Shiokawa Y, Medsger TA Jr, Rodnan GP. Clinical spectrum of connective tissue disease after cosmetic surgery. Observations on eighteen patients and a review of the Japanese literature. Arthritis Rheum 1984;27:1--12. Marik PE, Kark AL, Zambakides A. Scleroderma after silicone augmentation mammoplasty. A report of 2 cases. S Afr Med J 1990;77:212-213. Martin L. Silicone breast implants and connective tissue diseases: an ongoing controversy. J Rheumatol 1995;22:198-200. Miyoshi K, Miyramura T, Kobayashi Y. Hypergammaglobulinemia by prolonged adjuvanticity in man. Disorders devel-

oped after augmentation mammoplasty. Jpn J Med 1964; 2122:9-14. Newman LS. To Be2 + or not to Be2+: immunogenetics and occupational exposure. Science 1993;262:197--198. Ojo-Amaize, EA, Conte V, Lin H-C, Brucker RF, Agopian MS, Peter JB. Silicone-specific blood lymphocyte response in women with silicone breast implants. Clin Diagn Lab Immunol 1994;1:689--695. Ojo-Amaize EA, Lawless OJ, Peter JB. Elevated plasma concentrations of interleukin- 113 and interleukin- 1 receptor antagonist in women with silicone breast implants. Clin Diagn Lab Immunol 1996;in press. Sergott TJ, Limoli JP, Baldwin CM Jr, Laub DR. Human adjuvant disease, possible autoimmune disease after silicone implantation: a review of the literature, case studies and speculation for the future. Plastic Reconstr Surg 1986;78: 104--114. Shen GQ, Ojo-Amaize EA, Agopian MS, Peter JB. Silicate antibodies in women with breast implants: development of an assay for detection of humoral immunity. Clin Diagn Lab Immunol 1996;in press. Shons AR, Schubert W. Silicone breast implants and immune disease. Ann Plastic Surg 1992;28:491--499. van Nunen SA, Gatenby PA, Basten A. Postmammoplasty connective tissue disease. Arthritis Rheum 1982;25:694--697. Varga J, Schumacher HR, Jimenez SA. Systemic sclerosis after augmentation mammoplasty with silicone implants. Ann Intern Med 1989; 111:377--383. Vasey FB, Havice DL, Bocanegra TS, Seleznick MJ, Brideford PH, Martinez Osuna P, Espinoza LR. Clinical findings in symptomatic women with silicone breast implants. Semin Arthritis Rheum 1994;24:22-28. Wells AF, ed. Structural Inorganic Chemistry, 5th edition. Oxford: Clarendon Press, 1975. Wolf LE, Lappe M, Peterson RD, Ezrailson EG. Human immune response to polymethylsiloxane (silicone): screening studies in a breast implant population. FASEB J 1993;7: 1265--1268. Yoshida SH, Chang CC, Teuber SS, Gershwin ME. Silicon and silicone: theoretical and clinical implications of breast implants. Reg Toxicol Pharmacol 1993;17:3-18.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

SKIN DISEASES AUTOANTIBODIES Luis A. Diaz, M.D., Agustin Espafia-Alonso, M.D., Janet A. Fairley, M.D., George J. Giudice, Ph.D., Jos6 M. Mascar6 Jr., M.D. and Zhi Liu, Ph.D.

Department of Dermatology, Medical College of Wisconsin, Milwaukee, WI 53226-0509, USA

OVERVIEW

Luis A. Diaz, M.D. and George J. Giudice, Ph.D. Acquired autoimmune blistering skin diseases specific for squamous epithelia are characterized by spontaneous blister formation (Table 1). Histological examination reveals intraepidermal (e.g., pemphigus vulgaris [PV], pemphigus foliaceus [PF]) or subepidermal (bullous pemphigoid [BP], herpes gestationis [HG]) fluid-filled spaces (Figure 1). The skin diseases included in this group can be severely disabling. Mortality rates for certain of these diseases, e.g., PV and PF, were extremely high prior to the advent of corticosteroid and immunosuppressive therapies. Little was known about the pathogenesis of blister formation and the etiology of the bullous diseases until the early 1960s. In that decade a hallmark in the immunopathogenesis of these disorders was established when pioneering work with immunofluorescence (IF) techniques demonstrated that these patients with PV and PF produce autoantibodies that react with cell-surface antigens within the epidermis and that patients with B P have autoantibodies specific for antigens of the dermal-epidermal junction. The IF staining patterns produced by these autoantibodies are diagnostic of the respective disease.

THE AUTOANTIGENS

Immunochemical characterization of the relevant epidermal antigens was accomplished in the 1980s (Table 1). PF and PV were shown to react with components of the desmosome, an adhesion-related structure associated with the keratinocyte cell surface

746

(see Figure 2). PF autoantibodies specifically react with the 160 kd glycoprotein, desmoglein 1; whereas, PV autoantibodies recognize a related 130 kd glycoprotein, desmoglein 3 (Dsg 3). The sera of patients with BP and HG react with 2 proteins, one of 230 kd (BP230) and the other of 180 kd (BP180). These two BP antigens were localized to the epidermal hemidesmosome, a cell structure involved in anchoring basal keratinocytes to the basement membrane (see Figure 3). During the last 10 years, the PV and PF desmosomal antigens and the BP hemidesmosomal antigens have been cloned and the molecular structure of each of these proteins has been determined. The PV and PF antigens are desmosomal transmembrane glycoproteins known as desmoglein 3 (Dsg3) and desmoglein 1 (Dsgl), respectively (Figure 4). These glycoproteins belong to the cadherin family of calcium-dependent cell adhesion molecules. Moreover, Dsgl and Dsg3 share extensive amino acid similarity in both the intracellular and extracellular domains. The extracellular domain of these molecules is now thought to contain epitopes that bind pathogenic autoantibodies from the sera of these patients. Paraneoplastic pemphigus is a recently described autoimmune bullous dermatosis associated with circulating autoantibodies directed against a complex of five epidermal polypeptides (250, 230, 210, 190 and 170 kd). This form of pemphigus has been described in patients with different types of neoplasia, although lymphoproliferative neoplasms appear to be more common.

Table 1. Autoimmune Blistering Diseases Associated with Antidesmosomal and Antihemidesmosomal Antibodies Bullous Disease

Autoantibody Systems

Autoantigens

Blister Location

Pemphigus vulgaris

Antidesmosomes

Desmoglein 3

Intraepidermal (suprabasilar acantholysis)

Nonendemic pemphigus foliaceus

Antidesmosomes

Desmoglein 1

Intraepidermal (subcorneal acantholysis)

Endemic pemphigus foliaceus (fogo selvagem)

Antidesmosomes

Desmoglein 1

Intraepidermal (subcorneal acantholysis)

Paraneoplastic pemphigus

Antidesmosomes Antihemisdesmosomes Anti-PNP 170

Desmoplakin 1 BP230 antigen PNP 170 antigen

Intraepidermal (epidermal necrosis)

Bullous pemphigoid

Antihemidesmosome

BP230 and BP 180

Subepidermal

Herpes gestationis

Antihemidesmosome

BP180

Subepidermal

Cicatricial pemphigoid

Antihemidesmosome Antilaminin 5

BP 180 Laminin 5

Subepithelial

The two major BP antigens are polypeptides that are structurally unique i.e., B P230 is intracellular and immunolocalized to the plaque of the hemidesmosome; whereas, the BP180 antigen is a transmembrane protein with its amino-terminus located in the hemidesmosomal plaque and its carboxy-terminus projecting into the basal lamina (see Figure 4). The extracellular domain of the BP180 antigen shows

interrupted collagenous sequences and bears an immunodominant antigenic region (MCW-1) which is recognized by autoantibodies of patients with B P and HG. In the following sections, the specific autoantibodies and the latest developments in the immunopathology of these unique autoimmune diseases of the skin will be reviewed.

Figure 1. Schematic diagram of human skin depicting the locations of lesions associated with various autoimmune bullous diseases. In bullous pemphigoid, herpes gestationis and cicatricial pemphigoid, basal keratinocytes detach from the basement membrane, forming a subepidermal blister. Keratinocyte cell-cell detachment (acantholysis) is seen in the suprabasal and subcorneal layers of the epidermis in pemphigus vulgaris and pemphigus foliaceus, respectively. 747

Figure 3. Schematic diagram of the hemidesmosome and Figure 2. Schematic diagram of the desmosome. Depicted in this figure are two epidermal keratinocytes held together via the adhesive properties of the desmosome. The intracellular desmosomal "plaque" is a plasma membrane-associated platelike structure that functions as an insertion site for the keratin intermediate filaments. Multiple transmembrane glycoproteins (putative cell adhesion molecules) are present within the desmosome. Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) autoantibodies react with antigens located within the intercellular region of the desmosome (large arrow).

associated structures. The hemidesmosome consists of an electron-dense "plaque" located at the dermal pole of basal keratinocytes. The hemidesmosomal plaque, like that of the desmosome, acts as an insertion site for keratin intermediate filaments. Fine filamentous structures (anchoring filaments) extend from the keratinocyte plasma membrane to the lamina densa (LD) The thicker anchoring fibrils span between the LD and anchoring plaques. Autoantibodies in bullous pemphigoid (BP), herpes gestationis (HG) and cicatricial pemphigoid (CP) react with antigens located within the lamina lucida (large arrow) and the hemidesmosomal plaque (not indicated).

Figure 4. Schematic diagram of the epidermal autoantigens associated with autoimmune bullous diseases. Desmoglein 1 (Dsgl) and desmoglein 3 (Dsg3) are cell adhesion molecules located within the epidermal desmosome and are recognized by pemphigus foliaceus and pemphigus vulgaris sera, respectively. The extracellular domains of Dsg 1 and Dsg3 are composed of four repeating modules (E 1 through E4) and contain six calcium binding sites (black vertical bars). The tripeptide (R-A-L) is the putative cell-cell recognition site. The BP180 antigen is a transmembrane hemidesmosomal protein with an extracellular domain that contains 15 interrupted collagen triple helical domains (black boxes) and an immunodominant antigenic site (MCW- 1) recognized by the majority of bullous pemphigoid and herpes gestationis sera. Cicatricial pemphigoid sera also react with the MCW-1 region as well as a site near the carboxy-terminus.

ACKNOWLEDGEMENTS This work was supported in part by U.S. Public Health Service Grants R 2 9 - A R 4 0 4 1 0 (G.J.G.), R01-

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A R 32599, R37-AR32081 (L.A.D.) and training grant T 3 2 - A R 0 7 5 7 7 from the National Institutes of Health and by a V A Merit Review Grant (L.A.D).

AUTOANTIBODIES IN PEMPHIGUS VULGARIS Jos6 M. Mascar6 Jr., M.D., Janet A. Fairley, M.D., George J. Giudice, Ph.D. and Luis A. Diaz. M.D.

H I S T O R I C A L NOTES Pemphigus vulgaris (PV), the most common form of pemphigus worldwide, was first described in 1860 (Hebra, 1860; Korman, 1988; Stanley, 1993). This chronic autoimmune blistering disease of the skin is characterized by a loss of adhesion between keratinocytes (acantholysis) that induces the formation of intraepidermal blisters.

of normal human epidermis, bovine desmosome preparations and extracts of cultured human squamous carcinoma cells (Hashimoto et al., 1990). This molecule is located in the desmosomes of epidermal keratinocytes. Rabbit antibodies raised against recombinant Dsg3 antigen and immunoelectron microscopy showed that the antibodies bound to the desmosomes of cultured keratinocytes (Karpati et al., 1993).

Origin/Sources THE AUTOANTIGENS Definition The major target of autoantibodies in PV is a 130 kd glycoprotein, named desmoglein 3 (Dsg3), a constituent of the desmosome (Figure 4). Dsg3 is a member of the cadherin family of cell adhesion molecules (Amagai, 1995). Certain members of the cadherin family (known as the classic cadherins) are found in adherens junctions, while others, such as the desmogleins and desmocollins, are constituents of the desmosome (Garrod, 1993; Amagai, 1995). The desmoglein subfamily contains three members: Dsgl, which is the antigen recognized by autoantibodies in pemphigus foliaceus (PF); Dsg2, present in a variety of epithelia (e.g., colon); and Dsg3, the autoantigen in PV (Buxton et al., 1993). The desmosomal cadherins, like many of the other members of this superfamily, contain four cadherin repeats and six calcium-binding sites within their extracellular domain. Classic cadherins contain a highly conserved cytoplasmic region, and desmogleins are distinguished from other cadherins in having an extra carboxy terminal domain. Early studies of the antigen in PV utilizing 14Clabeled keratinocyte cell extracts showed that PV patients' sera immunoprecipitated a 210 kd complex which, upon electrophoretic analysis under reducing conditions, consisted of two polypeptides of 130 and 85 kd (Stanley et al., 1984). The 130 kd polypeptide is highly glycosylated and is the antigen recognized by autoantibodies in PV. The 85 kd molecule in the antigenic complex is plakoglobin (Korman et al., 1989). In addition, the 130 kd PV antigen can be detected by immunoblot analysis using SDS extracts

Antibodies affinity-purified against the 130 kd PV antigen were used to isolate cDNA clones encoding the antigen from a human keratinocyte expression library (Amagai et al., 1991). Northern blot analysis indicated that the antigen in PV is encoded by a 6 kb mRNA that is restricted in distribution to stratified squamous epithelia. Furthermore, Southern blot analysis demonstrated that the antigen is encoded by a single human gene located on chromosome 18 (Wang et al., 1994). Using bacterial fusion proteins encoding different domains of the antigen, pathogenically relevant epitopes of the antigen in PV were localized to the amino-terminal region of the Dsg3 extracellular domain (Amagai et al., 1992). Antidesmosome antibodies, affinity purified using the amino-terminal fusion proteins, induce acantholysis when injected into neonatal mice. However, adsorption of PV sera with these fusion proteins fails to remove all of the pathogenic activity. This suggests that, although some pathogenic epitopes are found in the amino-terminal extracellular domain, other pathogenic epitopes are located outside of this region or are conformational epitopes. As bacterial fusion proteins fail to form the proper conformation of the authentic antigen in PV, a baculovirus expression system was used to produce a soluble form of the antigen as a chimeric molecule termed PVIg that encodes the extracellular domain of Dsg3 and the constant region of IgG1 (Amagai et al., 1994). This recombinant protein was recognized by all 35 of the PV sera tested, but not by control sera. Reactivity against the keratinocyte cell surfaces was completely removed in some of the sera by incubation with the PVIg while in other PV sera, it only lowered the IIF titers. In addition, PVIg adsorbed some of the

749

pathogenic antibodies from patients' sera and prevented gross blister formation in the neonatal mouse model of PV.

employed with reasonable results. The use of calciumsupplemented buffers produces a significant increase in the sensitivity of the indirect IF assay for pemphigus (Matis et al., 1987).

AUTOANTIBODIES

Pathogenetic Role

Methods of Detection

Several clinical and experimental observations strongly support the notion that PV autoantibodies are pathogenic. For example, neonates of mothers with PV may develop a transient disease due to transplacental passage of maternal antiepidermal autoantibodies. Furthermore, IgG from PV patients induces acantholysis of human skin in the organ culture system (Schiltz and Michel, 1976).

Patients with PV have autoantibodies directed against the intercellular spaces of the epidermis. These autoantibodies can be detected in vivo, bound to lesional epidermis and circulating in the serum by direct and indirect immunofluorescence (IIF), respectively (Figure 5). The IIF staining pattern produced by the autoantibodies in PV is similar to that seen in PF. Direct IF demonstrates deposition of IgG, with or without complement (C3), on the cell surface of the epidermal keratinocytes of perilesional skin biopsies in 100% of the patients with active disease. However, if the biopsy specimens are obtained from lesional skin, the results may be negative. IgG autoantibodies directed against the cell surface of keratinocytes can be detected in the sera of approximately 80--85% of pemphigus patients by IIF analysis against stratified squamous epithelia (Beutner and Jordon, 1964). PV autoantibodies are mainly of the IgG4 subclass, but other subclasses, such as IgG1, can also be present (Dmochowski et al., 1992). Monkey esophagus is an ideal substrate for IIF detection of PV antibodies, but other substrates such as guinea pig and rabbit lip or esophagus can also be

Figure 5a. Direct immunofluorescence study of biopsy specimen obtained from lesional skin of a pemphigus vulgaris patient. C3 deposits are observed in the intercellular spaces of the keratinocytes (fluorescein-conjugated goat antiserum to human C3, original magnification, x40). 750

Animal Model. The pathogenicity of antidesmosome antibodies can be demonstrated in vivo by passive transfer of the IgG fraction from the sera of PV patients (Anhalt et al., 1982). In these passive-transfer experiments, PV IgG injected into the peritoneum of n~onatal BALB/c mice rapidly enter the circulation of the animals and shortly thereafter are bound to the intercellular regions of the epidermis. Within 18--24 hours after injection, the animals develop an extensive

Figure 5b. Indirect immunofluorescence study using serum from a pemphigus vulgaris patient on a monkey esophagus substrate. IgG autoantibodies stain the intercellular spaces of the epithelium in a similar way as in the direct immunofluorescence (fluorescein-conjugated goat antiserum to human IgG, original magnification, •

cutaneous blistering disease, showing the characteristic suprabasilar acantholysis. There is a good correlation between the extent of disease produced in the mice and the titer of autoantibodies in the injected IgG fractions. There is also a clear correlation between disease severity in the animals and the titers of circulating antidesmosome autoantibodies attained in the serum of the mice (Anhalt et al., 1982). F(ab')2 fragments of PVIgG injected into neonatal mice also bind to the epidermis and produce typical acantholytic lesions without detectable C3 deposition. Moreover, similar acantholytic lesions are caused by injection of whole IgG into genetically C5-deficient mice or into mice depleted of complement by pretreatment with cobra venom factor. These studies suggest that complement activation is not required for the production of PV lesions (Anhalt et al., 1986). The mechanism by which pemphigus autoantibodies induce acantholysis is unclear. An increase in protease activity is found in PV lesions (Baird et al., 1990), and the addition of pemphigus IgG to keratinocyte cultures stimulates the synthesis of plasminogen activator and produces acantholysis (Hashimoto et al., 1983). However, in the PV neonatal mouse model, it has been shown that mice previously treated with dexamethasone (which is known to inhibit plasminogen-activator activity) continue to develop skin lesions after passive transfer of PV IgG. Genetics

Genetic factors might play a role in the development of PV. Although it occurs in all ethnic and racial groups, the disease has a higher incidence in the Jewish population and is associated with HLA-26 (p = 0.0001), HLA-B38 (p = 0.004), SC21 (p = 0.02), HLA-DR4 (p = 0.0001) in Ashkenazi Jews and HLADR4 and HLA-DRw6 in non-Ashkenazi Jews; these markers were absent in less than 5% of patients (Ahmed et al., 1990; Szafer et al., 1987; Scharf et al., 1988; 1989" Sinha et al., 1988). PV-specific restriction fragment length polymorphisms as detected by the DQ beta probe: DQw 1 (2.5kb Bam HI) and DQw3 (6.9 kb Pvu II) were reported. In studies of PV patients (Ashkenazi Jews), 92% (n:26) expressed the major histocompatibility complex haplotypes DR4 and DQw3, and all were DR4, DQw8 [DQ"3.2"] (Ahmed et al., 1990; 1991); 75% of these patients expressed extended haplotypes" HLA-B38, SC21, DR4 and HLA-B35, SC31, DR4. A potential susceptibility gene mapped to the DR4, DQw8 region, and this PV

susceptibility trait appears to be inherited in a dominant fashion (Ahmed et al., 1993). Among healthy relatives of PV patients, a high percentage have low amounts of circulating PV antidesmosome antibodies (Mohimen et al., 1993; Ahmed et al., 1993). Furthermore, the inheritance of low levels of antibodies in relatives was almost always linked to the major histocompatibility complex haplotypes DR4 or DR6 of the patients (Ahmed et al., 1993).

CLINICAL UTILITY

The autoantibody response in these patients is highly specific and autoantibodies are present in the sera of all patients with active disease. In general, antibody titers correlate with disease activity and may be used to monitor therapy. The staining of the epidermal intercellular spaces by DIF in both PV and PF is identical, although sometimes the fluorescence is limited to or predominantly in the site of bulla formation (suprabasilar in PV and upper epidermis in PF). The exact diagnosis is based on a correlation between clinical, histological and immunopathological criteria. A diagnosis of pemphigus should not be made when the only finding is the presence of C3 deposits in the intercellular spaces of the skin, since many nonspecific inflammatory dermatoses can be associated with this pattern. Circulating IgG autoantibodies directed against the intercellular spaces of the stratified squamous epithelia are characteristic of all types of pemphigus (PV, PF and PNP). As mentioned before, monkey esophagus is the best substrate for IIF studies of serum from patients with PV. In a small percentage of patients with clinically active disease, no circulating antiepithelial cell surface antibodies can be demonstrated by IIF. In these cases, DIF must be employed to demonstrate the antibody already bound to the patient's skin, but not circulating in his blood. The different types of pemphigus cannot be distinguished on the basis of IIF, although PNP has a characteristic reactivity with other epithelia and tissues. IIF is very specific for pemphigus and false-positives are infrequent (Ahmed and Workman, 1983). Circulating antibodies mimicking the PV pattern have been reported in bums, penicillin allergy, toxic epidermal necrolysis, systemic lupus erythematosus, myasthenia gravis, bullous pemphigoid, cicatricial pemphigoid, lichen planus and in patients with antibodies directed against blood groups A and B (Becker and Gaspari,

751

1993). Such antibodies tend to be present in low titers and again, clinical, histological and immunopathological correlation is mandatory for the diagnosis of PV. In cases where there is a problem in differentiating the type of pemphigus, other techniques such as immunoblotting and immunoprecipitation can be employed, although they are not routinely used. The production of recombinant forms of Dsg3 (Amagai et al., 1994) offers the possibility of developing new diagnostic tests (such an ELISA) in the near future.

however, the precise mechanism of cell detachment (acantholysis) in PV is unknown. Acantholysis induced by pemphigus autoantibodies might be caused by activation of proteases such as plasminogen activator. The etiology of this disease is presently unknown, although genetic factors might play a role as suggested by the association of certain major histocompatibility complex haplotypes with the disease and the presence of circulating autoantibodies in healthy relatives.

CONCLUSIONS

ACKNOWLEDGEMENTS

PV is a disease of cell adhesion of epidermal keratinocytes. The sera of PV patients contain circulating antibodies directed against the cell surface of keratinocytes. By immunoblotting and immunoprecipitation, these autoantibodies recognize a 130 kd polypeptide now known to be Dsg3, a desmosomal core protein and a member of the cadherin family of cell adhesion molecules. The autoantibodies in PV are pathogenic;

This work was supported in part by U.S. Public Health Service Grants R29-AR40410 (G.J.G.), R01AR 32599, R37-AR32081 (L.A.D.) and training grant T32-AR07577 from the National Institutes of Health and by a VA Merit Review Grant (J.A.F. & L.A.D.). Dr. Mascaro is the recipient of grant 94/5590 from the Fondo de Investigaciones, Sanitarias, Madrid, Spain.

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plasminogen activator activity of human epidermal cells. A mechanism for the loss of epidermal cohesion and blister formation. J Exp Med 1983;157:259--272. Hashimoto T, Ogawa MM, Konohana A, Nishikawa T. Detection of pemphigus vulgaris and pemphigus foliaceous antigen by immunoblot analysis using different antigen sources. J Invest Dermatol 1990;94:327--331. Hebra F. Acute Exantheme und Hautkrankheiten. In: Virchow R, ed. Handbuch der speciellen Pathologie und Therapie. Erlangen: F. Enke, 1860;3:572--601. Karpati S, Amagai M, Prussick R, Cehrs K, Stanley JR. Pemphigus vulgaris antigen, a desmoglein type of cadherin, is located within keratinocyte desmosomes. J Cell Biol 1993; 122:409--415. Korman NJ. Pemphigus. J Am Acad Dermatol 1988;18:12191238. Korman NJ, Eyre RW, Klaus-Kovtun V, Stanley JR. Demonstration of an adhering junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceous and pemphigus vulgaffs. N Engl J Med 1989;321:631--635. Matis WL, Anhalt GJ, Diaz LA, Rivitti EA, Martins CR, Berger RS. Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies. J Invest Dermatol 1987;89:302304. Mohimen A, Narula M, Ruocco V, Pisani M, Ahmed AR. Presence of the autoantibody in healthy relatives of Italian patients with pemphigus vulgaris. Arch Dermatol Res 1993;285:176--177. Scharf SJ, Freidmann A, Brautbar C, Szafer F, Steinman L, Horn G, Gyllensten U, Erlich HA. HLA class II allelic

variation and susceptibility to pemphigus vulgaris. Proc Natl Acad Sci USA 1988;85:3504-3508. Scharf SJ, Freidmann A, Steinman L, Brautbar C, Erlich HA. Specific HLA-DQB and HLA-DRB 1 alleles confer susceptibility to pemphigus vulgaris. Proc Natl Acad Sci USA 1989;86:6215-6219. Schiltz JR, Michel B. Production of epidermal acantholysis in normal human skin in vitro by the IgG fraction from pemphigus serum. J Invest Dermatol 1976;67:254--260. Sinha AA, Brautbar C, Szafer F, Friedmann A, Tzfoni E, Todd JA, Steinman, McDevitt HO. A newly characterized HLA DQb allele associated with pemphigus vulgaris. Science 1988;239:1026-1029. Stanley JR, Koulu L, Thivolet C. Distinction between epidermal antigens binding pemphigus vulgaris and pemphigus foliaceous autoantibodies. J Clin Invest 1984;74:313-320. Stanley JR. Cell adhesion molecules as targets of autoantibodies in pemphigus and pemphigoid, bullous diseases due to defective epidermal cell adhesion. Adv Immunol 1993;53: 291325. Szafer F, Brautbar C, Tzfoni E, Frankel G, Sherman L, Cohen I, Hacham-Zadeh S, Aberer W, Tappeiner G, Holubar K, Steinman L, Friedmann A. Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQw 1 and DQw3 alleles of the HLAD region. Proc Natl Acad Sci USA 1987;84:6542-6545. Wang Y, Amagai M, Minoshima S, Sakai K, Green KJ, Nishikawa T, Shimizu N. The human genes for desmogleins (DSG1 and DSG3) are located in a small region on chromosome 18q12. Genomics 1994;20:492-495.

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AUTOANTIBODIES IN PEMPHIGUS FOLIACEUS Agustin Espafia-Alonso, M.D., Janet A. Fairley, M.D., George J. Giudice, Ph.D. and Luis A. Diaz, M.D.

HISTORICAL NOTES Pemphigus foliaceus (PF) was described in 1844 as a disease producing superficial cutaneous blisters and erosions; mucosal surfaces are not involved (Cazenave, 1844). The classic intraepidermal blisters localized to the subcorneal regions of the epidermis were described in 1943 (Civatte, 1943). In the 1960s, antiepidermal autoantibodies detected by IF techniques were demonstrated in patients with PF; these autoantibodies were found to be deposited at the keratinocyte cell surface in lesional epidermis and also circulating in the patients' sera (Beutner et al., 1968). An endemic variant of PF, also known as Fogo Selvagem (FS), occurs in certain rural regions of Brazil (Diaz et al., 1989b). Patients with the endemic and nonendemic forms of PF possess the same antiepidermal autoantibodies. These autoantibodies are disease specific and their serum titers correlate well with disease extent and activity (Squiquera et al., 1988). Relatives of FS patients are seronegative by IIF unless they show clinical signs of skin disease (Squiquera et al., 1988). In contrast, relatives of pemphigus vulgaris patients have detectable antiepidermal autoantibodies (Ahmed et al., 1993).

THE AUTOANTIGEN The antiepidermal autoantibodies in patients with nonendemic PF and FS specifically recognize the desmosomal glycoprotein Dsgl (Stanley, 1993). By immunoblotting techniques, the sera of at least 30% of FS patients recognize a 160 kd protein that comigrates with Dsgl (Koulu et al., 1984; Stanley et al., 1984). By immunoprecipitation techniques under nondenaturing conditions, sera of all PF patients precipitates a desmosomal complex consisting of Dsg 1 and plakoglobin, another desmosomal protein (Eyre and Stanley, 1987; Korman et al., 1988). Calcium is essential in these immunoprecipitation techniques, suggesting that the Dsgl epitopes recognized by PF autoantibodies are calcium-dependent and conformational. In similar studies with proteolytic fragments of bovine epidermal Dsgl radiolabeled with 125I, all PF sera precipitated a major 80 kd amino-terminal fragment of 754

Dsg 1 (Olague-Alcala et al., 1994). Cloned, sequenced Dsgl is a transmembrane glycoprotein belonging to the cadherin family of cell adhesion molecules (Koch et al., 1990; Wheeler et al., 1991) (Figure 4). The Dsgl molecule shares with the rest of cadherin proteins an extracellular domain with three pairs of calcium-binding sites that are crucial in homophilic cell-cell adhesion. The intracellular domain of Dsgl interacts with the keratinocyte cytoskeleton. Several segments of the Dsgl ectodomain were expressed in a prokaryote system and tested by immunoblot for reactivity with a panel of PF and control sera (Allen et al., 1993); 30-40% of PF sera recognized one or more of these recombinant forms of Dsgl, but these proteins were unable to adsorb pathogenic autoantibodies from PF sera. More recently, a recombinant form of Dsgl was generated using the COS-1 expression system, a mammalian epithelial cell line (Emery et al., 1995). Using an indirect IF detection system, all endemic and nonendemic PF sera showed reactivity with the COS/ Dsgl transfectants. This reactivity against the Dsgl ectodomain was predominantly an IgG4-restricted response and was calcium-dependent. The epitopes recognized by pathogenic autoantibodies are localized to the extracellular domain of Dsgl (Allen et al., 1993; Amagai et al., 1995; Emery et al., 1995) (Figure 4, Table 1). Soluble, immunoreactive baculovirus-generated peptides encompassing the extracellular domain of Dsg 1 remove pathogenic autoantibodies from the sera of PF patients (Amagai et al., 1995). These studies will facilitate the identification of epitopes recognized by pathogenic PF autoantibodies on the extracellular domain of Dsgl.

AUTOANTIBODIES Methods of Detection

Autoantibodies associated with PF were first identified by direct and indirect IF techniques, which showed that circulating autoantibodies from PF patients bind to the intercellular regions of human and monkey esophageal squamous epithelia and produce a charac-

teristic staining pattern that has since become a diagnostic finding for all of the clinical variants of pemphigus (Beutner et al., 1968) (Figure 6). The antiepidermal autoantibodies belong to the IgG class, and their serum titers correlate well with the activity and the extent of the cutaneous disease (Squiquera et al., 1988). Autoantibodies are also detected bound in vivo to lesional epidermis. In addition, detection of complement proteins bound to lesional epidermis from patients with PF suggests that the skin lesions in these patients may result from, or be amplified by, local activation of the complement cascade (Kawana et al., 1988; 1989). As shown by immunoelectron microscopy with gold-labeled probes, these autoantibodies bind predominantly to the extracellular region of the epidermal desmosomes (Rappersberger et al., 1992).

Pathogenetic Role The pathogenic role of PF antiepidermal autoantibodies was demonstrated in vitro when patients' IgG, introduced into the growth medium of epidermal cell cultures and skin organ cultures, caused acantholysis (cell-cell detachment) and increased activity of plasminogen activator (Morioka et al., 1987). Accord-

ingly, plasminogen activator was considered responsible for inducing cell detachment in the lesions of PF patients. The pathogenicity of autoantibodies in PF in vivo was demonstrated when IgG from FS patients was passively transferred into neonatal B ALB/c mice and induced the classic clinical, histological and immunological findings of human PF in these experimental animals (Roscoe et al., 1985). Although pemphigus vulgaris in pregnant women can be associated with a transient neonatal skin disease thought to be precipitated by transplacental passage of pathogenic autoantibodies, this characteristic is not observed in nonendemic and endemic PF (Rocha-Alvarez et al., 1992). In a series of 19 mother/ neonate pairs, mothers with FS had antiepidermal autoantibodies in their sera, but the babies' cord blood was negative or showed only low titers of these autoantibodies (Rocha-Alvarez et al., 1992). Cutaneous disease was also absent in a neonate whose mother had nonendemic PF (Eyre and Stanley, 1988). It is proposed that PF autoantibodies do not cross the placenta because this organ may contain desmosomes rich in Dsgl which immunoadsorb pathogenic autoantibodies.

Factors in Pathogenicity The IgG response in FS patients is predominantly IgG4 and in some patients only IgG4 antibodies are detected. Furthermore, IgG4 from these patients can induce disease in injected mice (Rock et al., 1989). In addition to intact IgG protein, both the F(ab')2 and

Figure 6a. Direct immunofluorescence study of biopsy specimen obtained from perilesional skin of a pemphigus foliaceus patient. IgG4 deposits are observed in the intercellular spaces of the epidermis (mouse monoclonal antibodies to human IgG4, original magnification, x 160).

Figure 6b. Indirect immunofluorescence study using serum from a pemphigus foliaceus patient. Circulating autoantibodies bind to the intercellular spaces of normal human skin (mouse monoclonal antibodies to human IgG4, original magnification, x80). 755

Fab' fragments are pathogenic in a mouse model system (Rock et al., 1990). Simple binding of Fab' from IgG antidesmosome antibodies is proposed as the trigger for acantholysis by impairing the function of a cell adhesion molecule. PF autoantibodies show an apparent clonal restriction as evidenced by isoelectric focusing analysis. The IgG4 oligoclonal bands are distributed throughout the pH range; whereas, the IgG1 banding is prominent in the more basic region. The wide distribution of IgG4 banding suggests that this response follows the IgG1 response (Calvanico et al, 1993). Because these studies were carried out using partially purified antigen from human and bovine epidermal extracts, confirmation and expansion with recombinant immunoreactive fragments of Dsg 1 containing pathogenic epitopes are needed. Genetics

The immunogenetics of PF is documented mainly in FS, because nonendemic PF is sporadic and rare. A remarkable feature of FS is the relatively high frequency in multiple blood-line-related family members (Diaz et al., 1989a; Friedman et al., 1995); this suggests that endemic PF develops in genetically predisposed individuals. FS is strongly associated with Class II HLA alleles. Of 42 patients tested, 88% had one or both of the HLA-DR1 and DR4 genes; whereas, these genes were present in only 34% of the controls (Peltz-Erler and Santamaria, 1989). Furthermore, the DR7 marker was present in only one of the 42 patients tested and in 29% of the controls. By serological and DNA typing analysis, a variant of HLA-DR1, an antigen common in the black population, DRB 1"0102 was present in 15 of 37 patients (41%) and in only 9% of controls (Moraes et al., 1991). Although this allele appears to confer susceptibility to the development of PF in Brazil, PF is not common in Blacks in other parts of the world. PF patients lacked the HLA-DQw2 allele (DQB 1"0201), which was detected in 22% of the control population, implicating this allele in resistance to PF in individuals in the endemic areas. In a class II HLA typing study of an Amerindian tribe (Xavante Indians) the allele frequency for DRB 1 *0404 (a variant of HLADR1) was significantly increased (relative risk, 9.6) in Xavante with FS compared with Xavante controls (Cerna et al., 1993). That both DRB 1" 102 and DRB 1"404 have an identical sequence in the third hypervariable region of the 756

first domain of the ~ chain of the class II antigen suggests that this hypervariable region of DRB 1 might be involved in susceptibility to developing endemic PF. This sequence of amino acids is also postulated to be the "shared epitope" that predisposes to the development of rheumatoid arthritis (Gregersen et al., 1987).

CLINICAL UTILITY The IF pattern (both by DIF and IIF) produced by PF antibodies is identical to that found in other forms of pemphigus. Serum titers correlate well with disease activity and healthy relatives of patients are seronegative by IIF (Squiquera et al., 1988). As mentioned in the section on PV, there may be false-positives in some cases. Diagnosis often relies on making a correlation between clinical, histological and immunopathological features. In some cases, it may be necessary to use more sophisticated techniques, such as immunoblotting and immunoprecipitation, to demonstrate serum reactivity against Dsgl. It must be noted that a high percentage of PV patients also have antibodies that recognize Dsgl, this makes it necessary to show reactivity with Dsgl but not with Dsg3 to diagnose PF. The production of recombinant molecules of Dsg 1 (Amagai et al., 1995) offers the possibility of performing diagnostic ELISA tests in the future.

CONCLUSION The endemic (FS) and nonendemic forms of PF are unique examples of human organ-specific humoral autoimmune diseases. Although the etiology of the nonendemic form of PF is unknown, epidemiological data strongly suggest an environmental agent (Lombardi et al., 1992). In both forms of the disease, patients produce highly specific antiepidermal autoantibodies, which recognize the extracellular domain of Dsgl, a desmosomal glycoprotein member of the cadherin family of cell adhesion molecules. Apparently, PF autoantibodies, binding to extracellular epitopes of Dsgl, impair its adhesive function and cause epidermal cell-cell detachment and intraepidermal blister formation. Binding of PF autoantibodies is postulated to activate plasminogen activator from epidermal cells, with resultant keratinocyte detachment and blistering.

The IgG autoimmune response in PF is predominantly IgG4, and these autoantibodies are indeed pathogenic as determined by passive transfer experiments. In addition, patients with the endemic form of PF possess certain susceptibility HLA-DR alleles, i.e., HLA-DRB 1 *0102, DRB 1 *0404, DRB 1 * 1402. These alleles share the same primary structure in the third hypervariable region of the DRB1 chain at amino acid position 67 to 74. All in all, it appears that individuals having the appropriate susceptibility HLA markers, and exposed to the precipitating environmental antigen, produce pathogenic antiepidermal autoantibodies that cause the clinical disease known as FS. Future investigations addressing these issues should

REFERENCES

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ACKNOWLEDGEMENTS This work was supported in part by U.S. Public Health Service Grants R29-AR40410 (G.J.G.), R01AR 32599, R37-AR32081 (L.A.D.) and training grant T32-AR07577 from the National Institutes of Health and by a VA Merit Review Grant (J.A.F. & L.A.D.). Dr. Espana is a postdoctoral research fellow supported by a grant from the Fundacion Ramon Areces, Madrid, Spain.

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AUTOANTIBODIES IN PARANEOPLASTIC PEMPHIGUS Jos6 M. Mascar6 Jr., M.D., Janet A. Fairley, M.D., George J. Giudice, Ph.D. and Luis A. Diaz. M.D.

H I S T O R I C A L NOTES Paraneoplastic pemphigus (PNP), a recently described autoimmune bullous disease, is characterized by prominent mucous membrane involvement with persistent painful blisters or erosions of the oral mucosa and a polymorphous skin eruption in the presence of an associated neoplasm (Anhalt et al., 1990) (Table 2). Cutaneous lesions can resemble those of pemphigus vulgaris, erythema multiforme, bullous pemphigoid or lichen planus. Histologic examination reveals the presence of suprabasilar acantholysis and intraepidermal clefts as in pemphigus vulgaris together with vacuolar degeneration of the basal cell layer and the presence of necrotic keratinocytes (Horn and Anhalt, 1992). Deposition of IgG and C3 in the intercellular regions of the epidermis and often granular deposition of complement along the basement membrane zone can be demonstrated by DIF. Circulating autoantibodies reactive with the cell surface of stratified and nonstratifying epithelia as well as some nonepithelial tissues (e.g., cardiac tissue) immunoprecipitate a characteristic complex of proteins from human keratinocyte extracts. Associated neoplasms are usually hematologic (non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Waldenstr6m' s macroglobulinemia, Castleman's tumor), but thymoma, sarcomas or squamous cell carcinomas are also reported. THE AUTOANTIGENS By immunoprecipitation with 14C-labeled human keratinocyte extracts, the sera of PNP patients recognize a complex of five different polypeptides of 250, 230, 210, 190 and 170 kd (Anhalt et al., 1990; Ousler et al., 1992). These proteins are not recognized by the sera of patients with other types of pemphigus. Although some PNP sera show weak or undetectable reactivity with the 250 and 230 kd antigens, there is consistent reactivity with the remaining protein bands of 210, 190 and 170 kd. The 250 and 210 kd bands correspond to desmoplakins I and II, respectively (Ousler et al., 1992). Desmoplakins are major structural proteins of the desmosome and are exclusively located intracellularly

(O'Keffe et al., 1989) (Figure 2). They contribute to the formation of the cytoplasmic desmosomal plaque in all epithelia and the presence of antibodies to these proteins may account for the ability of PNP sera to react with numerous types of epithelia by IIF. Because it is found in some patients with erythema multiforme major, reactivity against desmoplakins is not a specific finding of PNP sera (Foedinger et al., 1995). The 230 kd band corresponds to the BP230 bullous pemphigoid antigen. This protein is known to be the major component of the intracellular plaque of the hemidesmosome, a structure involved in cell-matrix adhesion, and is recognized by the majority of sera from patients with bullous pemphigoid (Mueller et al., 1989) (Figure 3). The identity of the 190 and 170 kd antigens is not yet established. Although variably detected and thought to be a degradation product of one of the higher molecular weight components, the 170 kd protein is now considered a distinct and integral component of the PNP antigenic complex. By immunoblot analysis with various antigen sources (human epidermal extracts, bovine muzzle desmosome preparations and extracts of cultured squamous cell carcinoma cells), all six PNP sera in a recent study reacted with the 210 and 190 kd protein bands (Hashimoto et al., 1995); the 250 and 230 kd antigens were detected at a lower frequency compared with immunoprecipitation studies, and the 170 kd band was not detected at all. Recognition of a 135 kd band by one patient's serum and of a recombinant pemphigus vulgaris antigen by two patients' sera suggests involvement of this protein in some cases of PNP. In another report using immunoblot analysis, 2 of 5 sera of PNP patients recognized a 130 kd band that comigrated with the 130 kd pemphigus vulgaris antigen (Joly et al., 1994). Nevertheless, the role of the pemphigus vulgaris antigen in the pathogenesis of PNP is still uncertain.

AUTOANTIBODIES Methods of Detection

PNP patients have circulating antibodies directed against the cell surface of keratinocytes. These auto759

Table 2. Diagnostic Criteria for PNP

Polymorphous skin eruption and mucositis Suprabasilar acantholysis and intraepidermal cleft on histology Deposition of IgG and C3 in the intercellular spaces of the epidermis Circulating antibodies reactive with the cell surface of epithelial as well as some nonepithelial tissues

ures do not permit a differentiation between PNP and either pemphigus vulgaris or pemphigus foliaceus. Autoantibodies in PNP can also bind to the cell surface of simple, columnar and transitional epithelia (Figure 7), as well as other nonepithelial tissues. Binding to the intercellular spaces is consistently seen in human respiratory tract, colon, small bowel, urinary bladder epithelium, thyroid epithelium, myocardium (intercalated discs), skeletal muscle and liver.

Immunoprecipitation of the PNP complex from human keratinocyte extracts Modified from (Anhalt et al., 1990). antibodies can be detected in vivo by DIF examination of biopsies obtained from the perilesional skin and mucosa of the patients. Deposits of IgG with or without C3 on the epidermal cell surfaces can usually be demonstrated. In addition, granular or linear distribution of immunoreactants at the basement membrane zone is also seen in some patients (Figure 7). The combination of staining of both the intercellular regions of the epidermis and the basement membrane zone is characteristic of PNP and was included in the original diagnostic criteria. The sera of PNP patients contain IgG class autoantibodies to the cell surface of a variety of stratified squamous epithelia. For example, 24 of 28 PNP patients' sera (86%) stained the intercellular regions of the epithelium of monkey esophagus; whereas, all of the PNP sera showed this pattern on mouse tongue sections (Helou et al., 1995). However, these meas-

Figure 7a. Direct immunofluorescence study of biopsy specimen obtained from perilesional skin of a paraneoplastic pemphigus patient. C3 deposits are observed in the intercellular spaces of the epidermis, as well as along the basement membrane zone (fluorescein-conjugated goat antiserum to human C3, original magnification, • 760

CLINICAL UTILITY The use of transitional epithelium (rat bladder) as a substrate for IIF was initially reported as highly specific (98.9%) in detecting the circulating autoantibodies of PNP patients and in differentiating them from other types of pemphigus (Liu et al., 1993). However, rat urinary bladder is not as sensitive as initially reported (Helou et al., 1995); indeed, in a group of 28 patients, only 75% showed reactivity with rat bladder epithelium; whereas, immunoprecipitation techniques detected reactivity with the characteristic PNP complex in all the sera. The sensitivity of the assay can be improved to 90% by combining complement-fixation with IIF techniques. In this procedure, the addition of fresh human serum as a source of~ complement amplifies the detection of complementbinding antibodies that are present in low titers in the serum, there may also be false-positive results, e.g., in some patients with erythema multiforme major, antidesmoplakin antibodies stain urinary bladder and other

Figure 7b. Indirect immunofluorescence study using rat bladder as a substrate. The serum from a patient with paraneoplastic pemphigus contains IgG antibodies that stain the intercellular spaces of the transitional epithelium (fluorescein-conjugated goat antiserum to human IgG, original magnification, •

tissues (Foedinger et al., 1995). In conclusion, rat bladder epithelium has a combined specificity (83%) and sensitivity (75%) that makes it a reasonable substrate for IIF screening of sera from patients suspected of having PNP. However, a negative result does not exclude the diagnosis, and immunoprecipitation remains the most reliable laboratory method for detection of PNP.

Pathogenetic Role The pathogenicity of PNP autoantibodies was demonstrated by passive transfer in the mouse model originally developed for pemphigus vulgaris (Anhalt et al., 1982). Purified IgG from PNP patients injected intraperitoneally into neonatal B ALB/c mice induced intraepidermal vesiculation. In addition, human IgG was detected by DIF in the intercellular spaces of lesional skin of these animals. Since both BP230 and desmoplakins are intracellular proteins, it is unlikely that antibodies directed against these proteins are involved in the initial stages of pathogenesis. Furthermore, antidesmoplakin antibodies present in patients with erythema multiforme major sera are apparently not pathogenic (Foedinger et al., 1995). Therefore, the 190 or 170 kd polypeptides are candidate PNP antigens for recognition by pathogenic autoantibodies.

REFERENCES Anhalt GJ, Labib RS, Voorhees, JJ, Beals TF, Diaz LA. Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease. N Engl J Med 1982; 306:1189--1196. Anhalt GJ, Kim SC, Stanley JR, Korman NJ, Jabs DA, Kory M, Izumi H, Rattle H 3rd, Mutasim D, Ariss-Abdo L, Labib RS. Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia. N Engl J Med 1990;323:1729--1735. Foedinger D, Anhalt GJ, Boecskoer B, Elbe A, Wolff K, Rappersberger K Autoantibodies to desmoplakin I and II in patients with erythema multiforme. J Exp Med 1995;181: 169-179. Hashimoto T, Amagai M, Watanabe K, Chorzelski TP, Bhogal BS, Black MM, Stevens HP, Boorsma DM, Korman NJ, Gamou S, Shimizu N, Nishikawa T. Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis. J Invest Dermatol 1995;104:829--834. Helou J, Allbritton J, Anhalt GJ. Accuracy of indirect immunofluorescence testing in the diagnosis of paraneoplastic

CONCLUSION In summary, PNP is an autoimmune blistering disease caused by circulating autoantibodies associated with different types of neoplasia. This disease is characterized by an IIF staining pattern in which circulating antibodies label both the cell surface of keratinocytes and the basement membrane zone. The antibodies bind to the cell surface of stratified squamous and other types of epithelia, as well as nonepithelial tissues. These antibodies are pathogenic and immunoprecipitate an antigenic complex of 250, 230, 210, 190 and 170 kd. The specificity of the autoantibodies directly responsible for PNP is presently unknown; the sensitizing antigens are thought to originate in the neoplastic tissue.

ACKNOWLEDGEMENTS This work was supported in part by U.S. Public Health Service Grants R29-AR40410 (G.J.G.), R01AR 32599, R37-AR32081 (L.A.D.) and training grant T32-AR07577 from the National Institutes of Health and by a VA Merit Review Grant (J.A.F. & L.A.D.). Dr. Mascaro is the recipient of grant 94/5590 from the Fondo de Investigaciones, Sanitarias, Madrid, Spain.

pemphigus. J Am Acad Dermatol 1995;32:441--447. Horn TD, Anhalt GJ. Histologic features of paraneoplastic pemphigus. Arch Dermatol 1992;128:1091-1095. Joly P, Thomine E, Gilbert D, Verdier S, Delpech A, Prost C, Lebbe C, Lauret P, Tron F. Overlapping distribution of autoantibody specificities in paraneoplastic pemphigus and pemphigus vulgaris. J Invest Dermatol 1994;103:65--72. Liu AY, Valenzuela R, Helm TN, Camisa C, Melton AL, Bergfeld WF. Indirect immunofluorescence on rat bladder transitional epithelium: a test with high specificity for paraneoplastic pemphigus. J Am Acad Dermatol 1993:28: 696-699. Mueller S, Klaus--Kovtun V, Stanley JR. A 230-kd basic protein is the major bullous pemphigoid antigen. J Invest Dermatol 1989;92:33--38. O'Keffe EJ, Erickson HP, Bennett V. Desmoplakin I and desmoplakin II purification and characterization. J Biol Chem 1989;264:8310-8318. Oursler J, Labib RS, Ariss-Abdo L, Burke T, O'Keefe EJ, Anhalt GJ. Human autoantibodies against desmoplakins in paraneoplastic pemphigus. J Clin Invest 1992;89:17751782.

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AUTOANTIBODIES IN BULLOUS PEMPHIGOID, HERPES GESTATIONIS AND CICATRICIAL PEMPHIGOID George J. Giudice, Ph.D. and Luis A. Diaz, M.D.

HISTORICAL NOTES Bullous pemphigoid (BP) was first described in 1953 as a subepidermal blistering disease seen predominantly in the elderly (Lever, 1953; Korman, 1993). In BP patients, basal keratinocytes of the epidermis detach from the underlying basement membrane, producing tense, fluid-filled vesicles with an inflammatory infiltrate. By indirect and direct immunofluorescence techniques, patients with B P exhibit circulating and tissue-bound autoantibodies directed against antigens of the cutaneous basement membrane zone (BMZ) (Jordon et al., 1967). Proteins of the complement system were also detected at the BMZ of perilesional skin from these patients (Jordon et al., 1975). Similar clinical and immunological findings were subsequently observed in herpes gestationis (HG), a vesiculobullous disease that occurs during pregnancy (Shornick, 1993). Also classified within this group of acquired, subepithelial bullous disorders is cicatricial pemphigoid (CP), which typically affects the mucous membranes and occasionally the skin (Mutasim et al., 1993a).

THE AUTOANTIGENS

BP autoantibodies exhibit a reactivity pattern restricted to epithelia. The major epithelial antigens reactive with BP autoantibodies include polypeptides of 230 and 180 kd, which are currently known as the BP230 (BPAG1) and the BP180 (BPAG2) antigens (Labib et al., 1986). Both of these proteins were recently cloned and characterized at the molecular level (Stanley et al., 1988; Diaz et al., 1990; Tanaka et al., 1991; Sawamura et al., 1991; Giudice et al., 1992). BP230, an intracellular protein localized to the hemidesmosomal plaque (Klatte et al., 1989; Tanaka et al., 1990) is encoded at chromosome locus 6p 12-p 11 (Tanaka et al., 1991; Sawamura et al., 1991). In contrast, BP180 is a transmembrane protein with a type II orientation encoded at chromosome locus 10q24.3 (Li et al., 1991). Its amino-terminal domain localizes to the intracellular hemidesmosomal plaque, and the car-

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boxy-terminal half projects into the extracellular milieu of the BMZ (Giudice et al., 1991; 1993; Hopkinson et al., 1992; Nishikawa et al., 1993). The extracellular domain of the human BP180 antigen contains an immunodominant epitope (designated MCW-1) that is recognized by the majority of B P and HG sera (Giudice et al., 1993) as well as CP autoantibodies (Balding et al., 1996).

AUTOANTIBODIES Methods of Detection

Autoantibodies to BP antigens are most frequently assayed by IIF using human skin cryostat sections as substrate (Mutasim et al., 1993b). The relative simplicity of the technique and its wide availability make this a powerful assay for the detection of circulating anti-BMZ IgG in BP, CP and HG. The major disadvantage of the immunofluorescence technique is its inability to distinguish between antibodies of BP180 and BP230 specificities, for which immunoblotting and immunoprecipitation are the primary methods of detection (Labib et al., 1986). An EL1SA for detecting reactivity of BP and HG sera with the MCW-1 antigenic region of BP180 is easy to perform, quantitative and, most importantly, highly specific (98.3%) (Giudice et al., 1994). Pathogenetic Role Human Disease. Several observations support the concept that the onset and evolution of the skin lesions of BP, CP and HG patients are caused by antiBMZ autoantibodies. Circulating and BMZ-bound autoantibodies produced by these patients localize to the site of blister formation, i.e., the upper lamina lucida for BP and HG (Karpati et al., 1991) and the lower lamina lucida and lamina densa for CP (Prost et al., 1987). In almost all HG sera and over 50% of B P sera, the anti-BMZ autoantibodies belong to the IgG1 or IgG3 subclass and avidly fix complement in vitro (Kelly et al., 1989; Suzuki et al., 1992). Well-docu-

mented, self-limited subepidermal blistering disease in neonates born to mothers with active HG suggests transplacental passage of pathogenic autoantibodies (Chorzelski et al., 1976; Katz et al., 1977). Plasmapheresis can induce clinical remission in some HG patients (Van de Wiel et al., 1980). The predominant IgG subclass of autoantibodies in CP is IgG4, followed by IgG1 which is able to fix complement (Bernard et al., 1991). Involvement of the complement system in the pathogenesis of B P, CP and HG is manifest by C3 bound to the BMZ of lesional skin in BP, CP and HG patients. Perilesional areas of B P and HG patients also contain other components of both the classical and alternative complement cascade including C lq, C3, C4, C5, factor B, properdin, B 1H globulin and the membrane attack complex (C5-9) (Korman, 1993). Current evidence suggests that various inflammatory cells and their secreted mediators may play an important role in the pathogenesis of B P, CP and HG. Cell lineages identified in these inflammatory infiltrates include eosinophils, neutrophils, lymphocytes, mast cells and monocyte/macrophages (Korman, 1993). Degranulated mast cells and eosinophils are also observed in BP lesions. Furthermore, several granular proteins including eosinophil cationic protein (ECP), eosinophil major basic protein (MBP), and neutrophilderived myeloperoxidase (MPO) can be detected in the lesional skin of BP patients. Granulocyte proteinases and by-products of the oxidative pathway, i.e., 02 radicals, might also be directly involved in the destruction of the cutaneous B MZ of these patients. Indeed, BP blister fluid contains several proteinases, including plasmin, collagenase, elastase and 92-kd gelatinase. Blister fluids of BP patients contain various inflammatory mediators, including C5a fragments (a potent chemoattractant for neutrophils) and chemoattractants from mast cells, including eosinophilic/neutrophilic chemotactic factors and histamine (Baba et al., 1976; Katayama et al., 1984). In addition, BP blister fluids contain leukotrienes, which are potent leukocyte chemotactic factors (Kawana et al., 1990), and various cytokines (e.g., interleukin-1,-2, -4,-5, tumor necrosis factors and interferon-y) (Grando et al., 1989; Takiguch et al., 1989; Endo et al., 1992; Zillikens et al., 1992; Tamaki et al., 1994). These observations support the hypothesis that inflammatory and biochemical mediators play key roles in autoantibodymediated disruption of the dermal-epidermal junction. However, the precise nature and origin of these

mediators and their role in blister formation in BP, CP and HG remain unknown due to the lack of an appropriate model system. Animal Models. Evaluation of the pathogenic relevance of anti-BMZ autoantibodies associated with BP, HG and CP using an in vitro organ culture system showed that circulating autoantibodies, complement and inflammatory cells are crucial in blister formation (Gammon et al., 1982). Attempts to demonstrate the pathogenic activity of autoantibodies produced by BP patients using passive transfer techniques have been largely unsuccessful (Anhalt and Diaz, 1987). Recombinant forms of murine B P180 encompassing the MCW-1 homologous site were recently used to produce polyclonal antibodies in rabbits. These rabbit antibodies, when passively transferred into neonatal BALB/c mice induced a subepidermal blistering disease in these experimental animals that duplicates the key immunopathological features of the human diseases, B P and HG (Liu et al., 1993). In this murine model of BP, complement activation is an essential step in the pathogenesis of blister formation (Liu, et al., 1995). In addition, neonatal mice depleted of neutrophils (by prior treatment with an antineutrophil antiserum) are resistant to the pathogenic effect of IgG antimurine BP180 (Liu et al., submitted). The absence of blister formation in these animals correlated with reduced neutrophilic infiltration into the skin sites show that neutrophils play a crucial role in the pathogenesis of blister formation in this experimental model of B P and HG. The subepidermal blistering disease seen in this BP/HG model is triggered by anti-BP 180 antibodies and is dependent upon complement activation and neutrophilic infiltration. This experimental model of BP should greatly facilitate future studies of the pathophysiology of autoantibodymediated diseases of the dermal-epidermal junction.

CLINICAL UTILITY In BP, HG and CP, biopsies for DIF show the deposition in vivo of anti-BMZ antibodies and complement. In BP, approximately 50 to 90% of patients demonstrate IgG deposition, while C3 is detected more often, in nearly 100% of patients (Figure 8). DIF findings in HG demonstrate C3 deposits in 100% of the patients while IgG is only detected in 25% of the cases. In CP, 80% of the patients have deposits of one or more immunoreactants (immunoglobulins, comple-

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Figure 8a. Direct immunofluorescence study of biopsy specimen obtained from lesional skin of a bullous pemphigoid patient. C3 deposits are observed along the basement membrane zone on the left side; while, on the right side, they can be observed on the roof of the blister (fluorescein-conjugated goat antiserum to human C3, original magnification, x40).

Figure 8b. Indirect immunofluorescence study of a bullous pemphigoid serum using normal human skin that has been split through the lamina lucida with 1 M sodium chloride (salt-split skin). The serum from the patient contains IgG autoantibodies that stain the roof of the separation (fluorescein-conjugated goat antiserum to human IgG, original magnification, •

ment, fibrin) at the BMZ, however, these findings may vary depending on the tissue that is being studied (i.e., skin, buccal mucosa, conjunctiva). Linear deposits of IgG along the BMZ are typical of these conditions, however, they are not specific as these findings may be found in other bullous diseases such as epidermolysis bullosa acquisita and bullous systemic lupus erythematosus. Skin biopsies from those two diseases may be differentiated by direct immunoelectron microscopy, immunohistochemical methods and DIF studies on sodium-chloride-separated skin (Gammon et al., 1990). IIF is used in B P, HG and CP to detect the presence of anti-BMZ autoantibodies in patients' sera. Approximately 70% of patients with active BP have circulating anti-BMZ IgG autoantibodies detectable by routine IIF. This proportion drops to 20-25% in the case of HG. However, using the complement-fixation IIF technique the percentage of sera positive by IIF can be increased to 90%. In CP, circulating anti-BMZ antibodies are detectable in approximately 10 to 30% of the patients, and they are usually detected at very low titers. In those sera negative by routine IIF, complement fixation assays also have been proven negative. In general, the titers of autoantibodies in these conditions correlate poorly with disease activity. Circulating anti-BMZ antibodies can also be observed in epidermolysis bullosa acquisita and bullous systemic lupus erythematosus. Those autoantibodies

can be differentiated either by indirect immunoelectron microscopy or by IIF on skin that has been split through the lamina lucida with 1 M sodium chloride (Gammon et al., 1984). In BP and HG, the autoantibodies will bind to the roof (epidermal side, Figure 8), or both to the roof and base of the separation. On the other hand, in epidermolysis bullosa acquisita and bullous systemic lupus erythematosus, circulating antibodies will only bind to the base (dermal side) of the separation. Most of the cases of CP where circulating antibodies can be demonstrated will show a pattern similar to BP using salt-split skin, although a subset of patients reacting with antigens different from BP180 (i.e., laminin 5) will bind the base of the split. Rarely, circulating anti-BMZ antibodies have been found in cases of psoriasis, leg ulcers and in burn patients. In some cases, it may be necessary to use immunoblotting techniques to differentiate diseases. Using epidermal extracts, patients with BP recognize the BP230 antigen and less frequently the BP180 antigen. On the other hand, patients with epidermolysis bullosa acquisita and bullous systemic lupus erythematosus react with 290 and 145 kd protein in dermal extracts (collagen VII). As mentioned above, an ELISA for the detection of reactivity against the MCW-1 antigenic region of BP180 has recently been reported as a promising tool in the diagnosis of BP and HG (Giudice et al., 1994).

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CONCLUSION

ACKNOWLEDGEMENTS

Autoantibodies to the hemidesmosomal protein B P 180 are a hallmark for the inflammatory subepidermal blistering d i s e a s e s - BP, CP and HG. In experimental BP/HG, separation at the dermal-epidermal junction is triggered by a binding of IgG anti-BP180 and is dependent on complement activation and neutrophilic infiltration. Further dissection of the molecular and immunopathologic mechanisms of subepidermal blister formation in these autoimmune skin disorders will have profound clinical i m p l i c a t i o n s - e. g., the establishment of more accurate diagnostic tools and the development of more effective therapeutic strategies for managing these diseases.

This work was supported in part by U.S. Public Health Service Grants R29-AR40410 (G.J.G.), R01AR 32599, R37-AR32081 (L.A.D.) and training grant T32-AR07577 from the National Institutes of Health and by a VA Merit Review Grant (L.A.D.). Dr. Liu is the recipient of a Dermatology Foundation Career Development Award sponsored by SmithKline Beecham Pharmaceuticals.

REFERENCES Anhalt GJ, Diaz LA. Animal models for bullous pemphigoid. Clin Dermatol 1987;5:117-- 125. Baba T, Sonozaki H, Seki K, Uchiyama M, Ikesawa Y, Toriisu M. An eosinophil chemotactic factor present in blister fluids of bullous pemphigoid patients. J Immunol 1976;116:112116. Balding SD, Prost C, Diaz LA, Bernard P, Bedane C, Aberdam D, Guidice GJ. Cicatricial pemphigoid antibodies react with multiple sites on the BP180 extracellular domain. J Invest Dermatol 1996;in press. Bernard P, Prost C, Aucouturier P, Durepaire N, Denis F, Bonnetblanc JM. The subclass distribution of IgG autoantibodies in cicatricial pemphigoid and epidermolysis bullosa acquisita. J Invest Dermatol 1991;97:259--263. Chorzelski TP, Jablonska S, Beutner EH, Maciejowska E, Jarzabek-Chorzelska M. Herpes gestations with identical lesions in the newborn. Passive transfer of the disease? Arch Dermatol 1976;112:1129-1131. Diaz LA, Rattle H 3rd, Saunders WS, Futamura S, Squiquera HL, Anhalt GJ, Giudice GJ. Isolation of a human epidermal cDNA corresponding to the 180-kd autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome. J Clin Invest 1990;86:1088--1094. Endo H, Iwamoto 1, Fujita M, Okamoto S, Yoshida S. Increased immunoreactive interleukin-5 levels in blister fluids of bullous pemphigoid. Arch Dermatol Res 1992;284:312-314. Gammon WR, Merritt CC, Lewis DM, Sams WM Jr., Carlo JR, Wheeler CE Jr. An in vitro model of immune complexmediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement. J Invest Dermatol 1982;78:285--290. Gammon WR, Briggaman RA, Inman AO III, Queen LL, Wheeler CE. Differentiating antilamina lucida and antisublamina densa anti-BMZ antibodies by indirect immuno-

fluorescence on 1.0 M sodium chloride separated skin. J Invest Dermatol 1984; 82:139--144. Gammon WR, Kowalewski, C, Chorzelski TP, Kumar V, Briggaman RA, Beutner EH. Direct immunofluorescence studies of sodium chloride-separated skin in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita. J Am Acad Dermatol 1990;22:664-670. Giudice GJ, Squiquera HL, Elias PM, Diaz LA. Identification of two collagen domains within the bullous pemphigoid autoantigen, BP180. J Clin Invest 1991;87:734--738. Giudice GJ, Emery DJ, Diaz LA. Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180. J Invest Dermatol 1992;99:243--250. Giudice GJ, Emery DJ, Zelickson BD, Anhalt GJ, Liu Z, Diaz LA. Bullous pemphigoid and herpes gestationis autoantibodies recognize a common noncollagenous site on the BP180 ectodomain. J Immunol 1993;151:5742--5750. Giudice GJ, Wilske KC, Anhalt GJ, Fairley JA, Taylor AF, Emery DJ, Hoffman RG, Diaz LA. Development of an ELISA to detect anti-BP180 autoantibodies in bullous pemphigoid and herpes gestationis. J Invest Dermatol 1994;102:878--881. Grando SA, Glukhenky BT, Drannik GN, Epshtein EV, Kostromin AP, Korostash TA. Mediators of inflammation in blister fluids from patients with pemphigus vulgaris and bullous pemphigoid. Arch Dermatol 1989;125:925--930. Hopkinson, SB, Riddelle KS, Jones JCR. Cytoplasmic domain of the 180-kd bullous pemphigoid antigen, a hemidesmosomal component: molecular and cell biologic characterization. J Invest Dermatol 1992;99:264--270. Gordon RE, Beutner EH, Witebsky E, Blumenthal G, Hale WL, Lever WF. Basement zone antibodies in bullous pemphigoid. JAMA 1967;200:751--756. Jordon RE, Nordby JM, Milstein H. The complement system in bullous pemphigoid. III. Fixation of C lq and C4 by pemphigoid antibody. J Lab Clin Med 1975;86:733-740. Karpati S, Stolz W, Meurer M, Braun-Falco O, Krieg T. Herpes gestationis: ultrastructural identification of the extracellular

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antigenic sites in diseased skin using immunogold techniques. Br J Dermatol 1991;125:317-324. Katayama I, Doi T, Nishioka K. High histamine level in the blister fluid of bullous pemphigoid. Arch Dermatol Res 1984;276:126--127. Katz A, Minto JO, Toole JWP, Medwidsky W. Immunopathologic study in herpes gestationis in mother and infant. Arch Dermatol 1977;13:1069-1072. Kawana S, Ueno A, Nishiyama S Increased levels of immunoreactive leukotriene B4 in blister fluids of bullous pemphigoid patients and effects of a selective 5-1ipoxygenase inhibitor on experimental skin lesions. Acta Derm Venereol 1990;70:281--285. Kelly SE, Cerio R, Bhogal BS, Black MM. The distribution of IgG subclasses in pemphigoid gestationis: PG factor is an IgG1 autoantibody. J Invest Dermatol 1989;92:695--698. Klatte DH, Kurpakus MA, Grelling KA, Jones JC. Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells. J cell Biol 1989; 109:3377-3390. Korman NJ. Bullous pemphigoid. Dermatol Clin 1993;11:483-498. Labib RS, Anhalt GJ, Patel HP, Mutasim DF, Diaz LA. Molecular heterogeneity of bullous pemphigoid antigens as detected by immunoblotting. J Immunol 1986;136:12311235. Lever, WF. Pemphigus. Medicine 1953;32:1-123. Li KH, Sawamura D, Giudice GJ, Diaz LA, Mattei MG, Chu ML, Uitto J. Genomic organization of collagenous domains and chromosomal assignment of human 180-kDa bullous pemphigoid antigen-2, a novel collagen of stratified squamous epithelium. J Biol Chem 1991;266:24064--24069. Liu Z, Diaz LA,Troy JL, Taylor AF, Emery DJ, Fairley JA, Giudice GJ. A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180. J Clin Invest 1993;92:2480-2488. Liu Z, Giudice GJ, Swartz SJ, Fairley JA, Till GO, Troy JL, Diaz LA. The role of complement in experimental bullous pemphigoid. J Clin Invest 1995;95:1539-1544. Mutasim DF, Pelc NJ, Anhalt GJ. Cicatricial pemphigoid. Dermatol Clinics 1993a;11:499-510. Mutasim DF, Pelc NJ, Supapannachart N. Established methods in the investigation of bullous diseases. Dermatol Clin 1993b; 11:399--418.

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Nishikawa, Y, Uematsu J, Owaribe K. HD4, a 180 kd bullous pemphigoid antigen, is a major transmembrane glycoprotein of the hemidesmosome. J Biochem 1993;113:493--501. Prost C, Labeille B, Chaussade V, Guillaume JC, Martin N, Dubertret L. Immunoelectron microscopy in subepidermal autoimmune bullous diseases: a prospective study of IgG and C3 bound in vivo in 32 patients. J Invest Dermatol 1987;89: 567--573. Sawamura D, Li K, Chu ML, Uitto J. Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains. J Biol Chem 1991;266: 1778417790. Shornick JK. Herpes gestationis. Dermatol Clin 1993;11:527533. Stanley JR, Tanaka T, Mueller S, Klaus-Kovtun V, Roop D. Isolation of complementary DNAfor bullous pemphigoid antigen by use of patient's autoantibodies. J Clin Invest 1988;82:1864-1870. Suzuki M, Harada S, Yaoita Y. Purification of bullous pemphigoid IgG subclasses and their capability for complement fixation. Acta Derm Venereol 1992;72:245--249. Takiguch Y, Kamiyama O, Saito E, Nagao S, Kaneko F, Minagawa T. Cell-mediated immune reaction in the mechanism of blister formation in bullous pemphigoid. Dermatologica 1989; 1:137. Tamaki K, So K, Furuya T, Furue M. Cytokine profile of patients with bullous pemphigoid. Br J Dermatol 1994;130: 128--129. Tanaka T, Korman NJ, Shimizu H, Eady RA, Klaus-Kovtun V, Cehrs K, Stanley JR. Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA. J Invest Dermatol 1990;94:617--623. Tanaka T, Parry DA, Klaus-Kovtun V, Steinert PM, Stanley JR. Comparison of molecularly cloned bullous pemphigoid antigen to desmoplakin I confirms that they define a newly family of cell adhesion junction plaque proteins. J Biol Chem 1991 ;266:12555-12559. Van de Wiel A, Hart HC, Flinterman J, Kerckhaert JA, Du Boeuff JA, Imhof JW. Plasma exchange in herpes gestationis. Br Med J 1980;281:1041--1042. Zillikens D, Ambach A, Schuessler M, Dummer R, Hartmann M, Burg G. The interleukin-2 receptor in lesions and serum of bullous pemphigoid. Arch Dermatol Res 1992;284:141-145.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

SMOOTH MUSCLE AUTOANTIBODIES Senga Whittingham, Ph.D. and Ian R. Mackay, M.D.

Centre for Molecular Biology and Medicine, Monash University, Victoria 3168, Australia

HISTORICAL NOTES Smooth muscle autoantibodies (SMA) were first observed by immunofluorescence on unfixed sections of rat stomach in the sera of patients with chronic active hepatitis (Johnson et al., 1965). Hitherto, the immunoserologic profile characteristic of chronic active hepatitis comprised a positive lupus erythematosus (LE) cell test, complement-fixing antibodies to a microsomal fraction of cell cytoplasm and a high concentration of serum gamma globulin (Mackay et al., 1956). The markers in this profile, except for the high level of gamma globulin, were also a feature of systemic lupus erythematosus (SLE); to acknowledge this, the liver disease was termed "lupoid" hepatitis (Mackay et al., 1956). When "antismooth muscle factor" was found in eight of 10 cases of "lupoid hepatitis" and in none of 16 cases of SLE (Johnson et al., 1965), a distinguishing marker was added to the profile, which clearly differentiated "lupoid" hepatitis from the other major autoimmune liver disease, primary biliary cirrhosis (PBC). "Lupoid" hepatitis was proposed as an autoimmune disease in 1956, leading eventually to the replacement of the term "lupoid" by "autoimmune" (Mackay et al., 1965). Subsequently the disease was classified as Type 1 autoimmune chronic hepatitis (Homberg et al., 1987) and more recently as autoimmune hepatitis (AH) (Johnson and McFarlane, 1993). Proof that the "antismooth muscle factor" was antibody gave rise to the abbreviation SMA. The association of SMA with autoimmune hepatitis is amply confirmed; the reactivity of SMA is not confined to smooth muscle (Whittingham et al., 1966). Actin was identified as the autoantigen (Gabbiani et al., 1973). The report of weakly reactive SMA in three of 47 patients with rheumatoid arthritis (Johnson et al., 1965) was con-

firmed (Kurki et al., 1983). Low-titer SMA also occur in a variety of diseases including acute and chronic infections, cancer and systemic autoimmune diseases (Brown et al., 1986). SMA is now known to represent a heterogeneous collection of antibodies of different specificities that react with various antigens in the cytoskeleton of smooth muscle cells. They are members of an extended family of anticytoskeletal antibodies whose specificity can be broadly classified according to the cytofilament with which they react (Kurki and Virtanen, 1984; Toh, 1991).

THE AUTOANTIGENS Definition

As SMA were originally identified by immunofluorescence, the. autoantigens are accordingly defined as those components of smooth muscle that react by immunofluorescence with these antibodies. There are three groups of cytoskeletal filaments on which the autoantigens reside. These are 6 nm microfilaments containing the contractile protein actin, 10 nm intermediate filaments which have been classified into five groups according to their cellular origin (Lazarides, 1980), and 25 nm microtubules containing tubulin (Table 1). The two major classes of intermediate filaments reactive with SMA are vimentin in mesenchymal cells and desmin in smooth muscle, skeletal muscle and cardiac muscle cells. Actin

Actin, a globular protein of molecular weight 46 kd, may exist as G-actin which is monomeric or as F-

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Table 1. Classification of Smooth Muscle Autoantigens Major Cytoskeletal Component

Autoantigen

Disease Association

Microfilaments (6 nm)

F-actin

AH AH-PBC overlap

G-actin

Alcoholic cirrhosis

Vimentin

Infections by viruses, protozoa, mycoplasm Systemic autoimmune diseases- systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, polymyositis Polymyalgia rheumatica Sjt~gren's syndrome Inflammatory bowel disease Behqet's syndrome Graft-versus-host disease

Desmin

Myocarditis

Tubulin

Infectious mononucleosis

Intermediate filaments (10 nm)

Microtubules (25 nm)

actin which is polymerized into filaments. F-actin is the biologically active form and is found in all eukaryotes. The energy generated in muscle by ATP hydrolysis allows the thin actin filaments and thick myosin filaments to slide past each other (Schr6der et al., 1993). Actin is also involved in a wide range of cellular processes such as cytokinesis, vesicular transport and ameboid movement. In evolutionary terms, actin is a highly conserved protein which is widely dispersed among cells. The polymerized form of actin, F-actin, is the specific autoantigen of AH. The reactivity of sera that contain autoantibodies to F-actin is highly dependent on the natural conformation of epitopes present in the native molecule. Some procedures like immunoblotting destroy native antigenic determinants. Thus, to maximize binding with antibody under assay conditions, care should be taken to preserve the conformational determinants. Untreated or lightly fixed tissues with the intact native microfilament are therefore favored as substrates.

Few studies have mapped the autoepitopes on vimentin. However, patients with SLE are reported to react preferentially with a 30 kd peptide located at the N-terminus of the molecule (Alcover et al., 1985). Monoclonal antibodies derived from patients with SLE and SLE-prone mice exhibit cross-reactivity with DNA and vimentin (Andre-Schwartz et al., 1984), but the determinant common to these antigens is undefined and the possibility that antigen bound to monoclonal antibodies could give spurious results cannot be excluded. Monoclonal antibodies raised against antigens of measles and herpes simplex viruses, cross-react with vimentin (Fujinami et al., 1983). In this case, the antigenic determinants appear to be virus-specific because the monoclonal antibody against measles virus phosphoprotein is nonreactive with herpes simplex protein and vice versa.

Vimentin

Desmin is a highly conserved molecule that may coexist with vimentin in the same cell. It is synonymous with skeletin and derives its name from the Greek word meaning link or bond (Lazarides, 1980). As an autoantigen it has not been studied intensively.

Vimentin is a major polypeptide of intermediate filaments that derives its name from its wavy (vimentus: Latin) arrangement of perinuclear coils and whorls (Franke et al., 1978). Conserved like other intermediate filaments as an m-helical coiled-coil rod flanked by two variable nonhelical domains, vimentin has a molecular weight of 57 kd and is present in mesenchymal cells and most laboratory cell lines. Its role in the cell is believed to be anchoring the nucleus during cell division or movement.

768

Desmin

THE AUTOANTIBODIES SMA of F-Actin Specificity SMA of anti-F-actin specificity is an important

autoantibody marker of AH (Gabbiani et al., 1973; Bottazzo et al., 1976; Lidman et al., 1976; Kurki et al., 1978a; Toh, 1979; Andersen et al., 1979) present in over 90% of patients in the acute phase of AH and in 20% of patients with the AH-PBC overlap syndrome, these SMA are present at high titer, polyvalent and predominantly class IgG (Table 2) although a higher component of IgM may be detected in the AHPBC overlap syndrome (Kurki et al., 1980).

Pathogenetic Role Aside from its use as a diagnostic marker, there are important questions on the immunopathogenic stimulus for the production of SMA and possible pathogenicity of this autoantibody for hepatocytes. The actual cause of hepatocellular damage in AH is unclear because a vulnerable molecular target on the liver cell surface has not been identified, although a possible candidate is the asialoglycoprotein receptor (McFarlane et al., 1986; Treichel et al., 1990). The liver cell is rich in submembranous actin which is the likely source of the "polygonal" staining recognized earlier by immunofluorescence using SMA-positive sera and liver substrates (Farrow et al., 1971). Liver destruction from any cause would release actin as a source of autoantigen, and it is conceivable that the copious submembranous actin of hepatocytes would be accessible as a target for a damaging immune response. There is no spontaneously occurring animal model and antibodies to F-actin, like antibodies to DNA, are difficult to induce experimentally. Moreover, the autoantigenic determinants on F-actin are not known, nor whether the reaction of the autoantibody with the determinants results in impairment of function of actin. Notwithstanding, SMA of anti-F actin specificity are an important diagnostic marker and their detection in the diagnostic laboratory is straightforward. The status of other specificities of SMA is less clear. These antibodies are relatively ubiquitous, low

titer and usually IgM, suggesting that immunologic tolerance to the relevant autoantigens is only partially broken, and homeostasis is readily restored once the virus or offending trigger that induces these antibodies comes under control.

Methods of Detection The source of tissues for screening for SMA is usually an unfixed frozen section of a composite block of stomach, kidney and liver obtained from a healthy rat or mouse. The sensitivity of the test can be increased if sections of tissues are prepared from rats injected repeatedly with phalloidin, a drug which greatly increases generation of microfilaments (Fusconi et al., 1990). The tissue block is cut and appropriately oriented for snap freezing using a mixture of isopentane and liquid nitrogen and stored a t - 7 0 ~ until sectioned at 46 nm in a freezing microtome. The sections are air-dried and either tested immediately or stored in an airtight container a t - 7 0 ~ SMA is detected in serum at a screening dilution of 1/10 by the standard immunofluorescence test. However, it is advisable to include a potent polyvalent antihuman immunoglobulin fluoresceinated conjugate to detect both immunoglobulin (Ig) G and IgM antibodies because SMA of antiactin specificity are predominantly IgG; whereas, SMA of other specificities are usually IgM (Table 2). It is also wise to titrate the serum because of the prozone phenomenon sometimes observed in the lower dilutions of strongly reactive SMA. The pattern of SMA on the frozen tissue sections of the composite block is a guide to the specificity of SMA and will determine which tests should be performed if more precise designation is necessary for clinical diagnosis. The characteristic reaction pattern of F-actin SMA is that of strong homogeneous staining of the cytoplasm of muscle fibers of the outer muscle layer of the stomach, the muscularis mucosa; the strands of intraglandular muscle coursing through the mucosa;

Table 2. Characteristics of SMA in Hepatitis Disease

Autoantigen

Ig class of autoantibody

Titer

AH

F-actin

Polyvalent but mainly IgG

High

AH-PBC overlap

F-actin

Polyvalent but mainly IgG and IgM

High

Alcoholic cirrhosis

G-actin

IgM

Low

Viral hepatitis

Nonactin

IgM

Low

769

Figure 1. SMA of anti-F actin specificity reacts with a the muscularis mucosa of rat stomach and strands of interglandular muscle coursing through the mucosa, b mesangial cells of renal glomeruli (arrows), c muscle of blood vessels and d the "stress" fibers of murine fibroblasts appearing as actin cables. the media of the blood vessels, SMA-V; the intracellular fibrils of the renal tubules, SMA-T; and the mesangial cells of the renal glomerulus, SMA-G (Bottazzo et al., 1976) (Figure 1). This staining is neutralized by F-actin. When the reactivity of the autoantibody is very strong, staining of other cells may occur because all cells contain actin and the small amount of actin within becomes evident. The polygonal pa~tern on liver sections is conferred by the staining of submembranous actin and is more obvious in livers from rats treated with phalloidin which results in increased deposition of actin (Fusconi et al.,

770

1990). However, the immunofluorescence pattern is not always typical of actin because it may be masked by the reactivity of other autoantibodies. These include antinuclear antibodies which are frequently present in AH and antimitochondrial antibodies which stain the mitochondrial-rich cytoplasm of the proximal renal tubules, gastric parietal cells and, to a lesser degree, all cells which contain mitochondria.

Further Specification of Anti-F-Actin When staining of smooth muscle is obtained in the

composite block, the preferred source of antigen for further characterization of the antibody as anti-F-actin is a monolayer of acetone-fixed fibroblasts prepared as follows. Monolayers of mammalian fibroblasts are grown on multiwell, Teflon-coated microscope slides in petri dishes containing appropriate tissue culture medium supplemented with fetal calf serum in a humidified atmosphere of CO 2 and air at 37~ Prior to harvest, the monolayer is exposed to 10 lag vinblastine/mL for 4 hours, rinsed with phosphate-buffered saline, fixed with acetone at-20~ for 5 minutes and air dried. Vinblastine, a microtubule-disrupting drug which depolymerizes tubulin to form paracrystals, causes collapse of the intermediate filaments to form a perinuclear coil. When examined by immunofluorescence, the antiactin reaction with fibroblasts is seen as staining of "stress" fibers which span the long axis of the cell in a cable-like fashion (Figure 1), while SMA of nonactin specificities binds to other filamentous structures. Some sera appear to contain an actindepolymerizing factor (Chaponnier et al., 1979; Harmer et al., 1983) that interferes with the staining of the "stress" fibers, and this possibly accounts for the prozone phenomenon sometimes seen at lower dilutions in the titration of SMA. If the presence of SMA is not suspected, a clue to its presence may be revealed by the staining of the cytoskeleton in the cytoplasm of the human epithelial cell monolayer employed in the frequently requested test for ANA. However, the human epithelial cell is an inappropriate substrate for testing for antiactin as the staining varies from discrete speckles to truncated cables or randomly distributed filaments (Figure 2) and is unreliable.

SMA of Nonactin Specificity SMA of antivimentin specificity are detected in many conditions, including viral infections, the multisystem autoimmune diseases, including SLE, rheumatoid arthritis, systemic sclerosis and polymyositis and in miscellaneous diseases (Table 1). The SMA of antivimentin, antidesmin and antitubulin (Dighiero et al., 1990) specificities as detected by immunofluorescence is usually of low titer and class IgM. The pattern on the composite block contrasts with that of anti-F actin in that there is no staining of the mesangial cells of the renal glomerulus, the intracellular fibrils of the renal tubules and other actin-rich areas (Kurki et al., 1978b). Instead the staining is that of smooth muscle only in the tissue chosen as substrate, usually stom-

ach, so reactivity with blood vessels features prominently. These antibodies may also be detected on monolayers of selected cell lines which, when treated with vinblastine, show collapse of the intermediate filaments to give the appearance of a perinuclear coil (antivimentin and antidesmin), and collapse of microtubules to give the appearance of paracrystals (antitubulin) (Toh, 1991). Antibodies to these antigens are also demonstrable by ELISA using purified preparations of the autoantigens (Dighiero et al., 1990). Patients with AH react with a broad range of microfilament-associated proteins such as filamin, myosin, r tubulin and tropomyosin (Dighiero et al., 1990; Girard and Senecal, 1995). However, the contribution that these reactions make to SMA is difficult to evaluate, because inhibition of the immunofluorescence by these antigens is variable, and in some cases the immunofluorescence test for SMA is negative.

CLINICAL UTILITY

Disease Association SMA is the standard diagnostic marker of AH, the classical expression of which includes an insidious onset of lethargy, malaise, loss of appetite, arthralgiamyalgia, amenorrhea, signs of hepatosplenomegaly, jaundice and an acneiform skin rash. It is distinguished from other liver diseases by a female preponderance. There is a bimodal age distribution with a peak at puberty associated with the HLA-B8, DR3 phenotype (tenfold risk), and a peak at menopause associated with the HLA-DR4 phenotype. The serologic features of hypergammaglobulinemia, antinuclear antibodies and SMA and the histologic evidence of lymphocytoplasmacytic infiltrate of the portal tract spilling into the periportal area with piecemeal necrosis of hepatocytes points to chronic intrahepatic inflammation of the autoimmune type. The antiactin component of SMA is the subject of a separate chapter. However, the rather unappreciated utility of SMA of antiactin specificity for the diagnosis of AH merits emphasis. The sensitivity is relatively high (at least 90%) and the specificity of a high titer reaction (>40) approaches 100% for the diagnosis of AH. Although the latter point was emphasized by many earlier writers, it appears that few routine diagnostic immunology laboratories provide an antiactin result in SMA-positive cases of

771

Figure 2. Immunofluorescent filamentous staining (arrow) may be observed in the human epithelial cell monolayer used for the frequently requested test for ANA. The serum needs to be tested against smooth muscle to confirm the reaction is SMA. The serum giving the staining here is the same as that giving the staining in Figure 1.

liver disease. Moreover, of three recently published sets of diagnostic guidelines for liver disorders in which there is reference to SMA as a diagnostic marker of AH (Johnson and McFarlane, 1993; Leevy et al., 1994; Ludwig et al., 1995), only one nominates antiactin as a specific marker for AH.

CONCLUSION The detection of SMA is an established marker for AH. Further confirmation can be attained by demon-

REFERENCES Alcover A, Hernandez C, Avila J. Human vimentin autoantibodies preferentially interact with a peptide of 30 kD tool. wt, located close to the amino-terminal of the molecule. Clin Exp Immunol 1985;61:24-30. Andersen P, Small JV, Andersen HK, Sobieszek A. Reactivity of smooth-muscle antibodies with F- and G-actin. Immunology 1979;37:705-709. Andre-Schwartz J, Datta SK, Shoenfeld Y, Isenberg DA, Stollar BD, Schwartz RS. Binding of cytoskeletal proteins by monoclonal anti-DNA lupus autoantibodies. Clin Immunol Immunopathol 1984;31:261--271. Bottazzo G-F, Florin-Christensen A, Fairfax A, Swana A, Doniach D, Groeschel-Stewart U. Classification of smooth muscle autoantibodies detected by immunofluorescence. J

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strating that the observed SMA has specificity for Factin. This is most conveniently done by immunofluorescence on monolayers of acetone-fixed fibroblasts whereby "stress" fibers are stained as actin cables. F-actin specificity is important because SMA includes nonspecific reactivities with other cytoskeletal filaments. While antibody to F-actin does not have a known cytopathogenic effect in AH, the copious submembranous actin of hepatocytes could represent an accessible target for hepatocellular damage. See also ACTIN AUTOANTIBODIES and LIVER MEMBRANE AUTOANTIBODIES.

Clin Pathol 1976:29:403-410. Brown C, Pedersen J, Underwood JR, Gust I, Toh BH. Autoantibodies to intermediate filaments in acute viral hepatitis A, B and non-A, non-B are directed against vimentin. J Clin Lab Immunol 1986:19:1--4. Chaponnier C, Borgia R, Rungger-Brandle E, Weil R, Gabbiani G. An actin-destablizing factor is present in human plasma. Experientia 1979;35:1039--1040. Dighiero G, Lymberi P, Monot C, Abuaf N. Sera with high levels of antismooth muscle and antimitohondrial antibodies frequently bind to cytoskeleton proteins. Clin Exp Immunol 1990:82:52--56. Farrow LJ, Holborow EJ, Brighton WD. Reaction of human smooth muscle antibody with liver cells. Nature New Biol 1971;232:186--187. Franke WW, Schmid E, Osborn M, Weber K. Different

intermediate-sized filaments distinguished by immunofluorescence microscopy. Proc Natl Acad Sci USA 1978;75:5034-5038. Fujinami RS, Oldstone MB, Wroblewska Z, Frankel ME, Koprowski H. Molecular mimicry in virus infection: crossreaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments. Proc Natl Acad Sci USA 1983;80:2346--2350. Fusconi M, Cassani D, Zauli D, Lenzi M, Ballardini G, Volta U, Bianchi FB. Antiactin antibodies: a new test for an old problem. J Immunol Methods 1990; 130:1--8. Gabbiani G, Ryan GB, Lamelin JP, Vassali P, Majno G, Bouvier CA, Cruchaud A, Luscher EF. Human smooth muscle autoantibody. Its identification as antiactin antibody and a study of its binding to nonmuscular cells. Am J Pathol 1973;72:473--478. Girard D, Senecal JI. Antimicrofilament IgG antibodies in normal adults and in patients with autoimmue diseases: immunofluorescence and immunoblotting analysis of 201 subjects reveals polyreactivity with microfilament-associated proteins. Clin Immunol Immunopathol 1995;74:193--201. Harmer JH, Lolait SJ, Toh BH, Pedersen JS, Chaponnier C, Gabbiani G. Actin depolymerizing factor and the organization and distribution of actin in astrocytomas and meningiomas. Br J Cancer 1983;48:89-93. Homberg JC, Abuaf N, Bernard O, Islam S, Alvarez F, Khalil SH, Poupon R, Darnis F, Levy VG, Grippon P, et al. Chronic active hepatitis associated with antiliver/kidney microsome antibody type 1: a second type of "autoimmune" hepatitis. Hepatology 1987;7:1333--1339. Johnson GD, Holborow EJ, Glynn LE. Antibody to smooth muscle in patients with liver disease. Lancet 1965;2:878--879. Johnson PJ, McFarlane IG. Meeting report. International Autoimmune Hepatitis Group. Hepatology 1993:18:9981005. Kurki P, Linder E, Miettinen A, Alfthan O. Smooth muscle antibodies of actin and "nonactin" specificity. Clin Immunol Immunopathol 1978a;9:443-453. Kurki P, Virtanen I, Stenman S, Linder E. Characterization of human smooth muscle autoantibodies reacting with cytoplasmic intermediate filaments. Clin Immunol Immunopathol 1978b;11:379--387. Kurki P, Miettinen A, Linder E, Pikkarainen P, Vuoristo M, Salaspuro MP. Different types of smooth muscle antibodies

in chronic active hepatitis and primary biliary cirrhosis: their diagnostic and prognostic significance. Gut 1980;21:878-884. Kurki P, Helve T, Virtanen I. Antibodies to cytoplasmic intermediate filaments in rheumatic diseases. J Rheumatol 1983;10:558--562. Kurki P, Virtanen I. The detection of human antibodies against cytoskeletal components. J Immunol Methods 1984;67:209-223. Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature 1980;283:249-256. Leevy CM, Sherlock S, Tygstrup N, Zetterman R. Diseases of the liver and biliary tract. Standardization of nomenclature, diagnostic criteria and prognosis. New York: Raven Press, 1994:58. Lidman K, Biberfeld G, Fagraeus A, Norberg R, Tortensson R, Utter G, Carlsson L, Luca J, Lindberg U. Antiactin specificity of human smooth muscle antibodies in chronic active hepatitis. Clin Exp Immunol 1976;24:266--272. Ludwig J, et al. Terminology of chronic hepatitis. International Working Party Report. Am J Gastroenterol 1995 ;90:181-189. Mackay IR, Taft LI, Cowling DC. Lupoid hepatitis. Lancet 1956;2:1323-1326. Mackay IR, Weiden S, Hasker J. Autoimmune hepatitis. Ann N Y Acad Sci 1965;124:767--780. McFarlane BM, McSorley CG, Vergani D, McFarlane IG, Williams R. Serum autoantibodies reacting with the hepatic asialoglycoprotein receptor protein (hepatic lectin) in acute and chronic liver disorders. J Hepatol 1986;3:196--205. Schr6der R, Manstein DJ, Jahn W, Holden H, Rayment I, Holmes KC, Spudich JA. Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1. Nature 1993;364:171-174. Toh BH. Smooth muscle autoantobodies and autoantigens. Clin Exp Immunol 1979;38:621--628. Toh BH. Anticytoskeletal autoantibodies: diagnostic significance for liver diseases, infections and systemic autoimmmune diseases. Autoimmunity 1991; 11:119-- 125. Treichel U, Poralla T, Hess G, Manns M, Meyer zum Biischenfelde KH. Autoantibodies to human asialoglycoprotein receptor in autoimmune-type chronic hepatitis. Hepatology 1990; 11:606--612. Wittingham S, Mackay IR, Irwin J. Autoimmune hepatitis. Immunofluorescence reactions with cytoplams of smooth muscle and renal glomerular cells. Lancet 1966;1:133--135.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

SPLICEOSOMAL snRNPs AUTOANTIBODIES Stanford L. Peng, B.A., B.S. a'b and Joseph E. Craft, M.D. b

aDepartment of Biology and bSection of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8031, USA

THE AUTOANTIGENS

mal, Sm protein-containing U1, U2, U4, U5, U6, U7, U11 and U12 snRNPs. The U1, U2 and U7 snRNPs exist as monomeric particles; the U4, U5 and U6 snRNPs exist physiologically as a tri-snRNP molecule but commonly purify as a U4/U6 di-snRNP and a single U5 snRNP. Hence, the references of most literature to U4AJ6 and U5 particles. The U11 and U12 snRNPs probably function as a dynamic disnRNP (Wassarman and Steitz, 1992). Each U snRNP is composed of its respective uridine-rich (thus U) small nuclear RNA (snRNA) and a set of polypeptides. The U snRNAs each possess a unique 2', 2', 7trimethylguanosine (m3G) cap, except for the U6 snRNA, which possesses a y-methyl phosphate cap. The snRNAs also contain several sites of RNA-RNA interaction and protein binding, including a uridinerich Sm site (or domain A) to which binds a common core of Sm polypeptides: B'/B, D! (usually referred to as simply D), D2, D3, E, F, G (perhaps actually representing two proteins) and a recently described 69 kd protein (Hackl et al., 1994). Each individual snRNP contains additional specific proteins essential for splicing function and/or particle integrity (Craft, 1992) (Tables 1 and 2). Together in the context of the spliceosome, these particles mediate the excision of introns from premessenger RNA via a complex series of RNA-RNA, RNA-protein and protein-protein interactions (Baserga and Steitz, 1993).

Nomenclature and Structure

Epitopes

Small nuclear ribonucleoprotein particles comprise a ubiquitous group of heterogeneous molecules that play varied roles in cellular metabolism. They include the nonspliceosomal, Sm-unrelated 7SK RNP, RNase P RNP and telomerase RNP, as well as the spliceoso-

Autoantibodies against the snRNP particles recognize both protein and RNA epitopes, including the trimethylguanosine cap (Gilliam and Steitz, 1993; Okano and Medsger, 1992) (Table 2). Sm antibodies typically bind B'/B, D, sometimes E and rarely F and G; U1-

HISTORICAL NOTES Antibodies against small nuclear ribonucleoprotein particles (snRNPs) cultivated a collaboration between clinical immunology and molecular biology. In the first description of these antibodies, sera of patients with systemic lupus erythematosus (SLE) demonstrated reactivity in immunodiffusion with a soluble nuclear specificity termed Sm, named after the prototype patient (Tan and Kunkel, 1966). Later, SLE sera were found to contain another precipitin (termed antiMo), whose target was thought to be a ribonucleoprotein (RNP) because of its sensitivity to ribonuclease and trypsin (Mattioli and Reichlin, 1973). At that same time, antibodies to "extractable nuclear antigen," which contained both the Sm and RNP antigens in physical association, were found in sera from patients with mixed connective tissue disease (MCTD) (Sharp et al., 1971). Later, these two antigens were discovered to be part of the spliceosomal complexes that play essential roles in RNA processing. Since then, anti-snRNP antibodies have been found in a variety of autoimmune diseases and have aided investigations in both autoimmunity and RNA metabolism (Craft, 1992).

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Table 1. Characteristics of the Abundant Spliceosomal snRNPs Particle

Particle Size

Abundance (Copies/cell)

RNA Length (bp)

RNA Polymerase

Cap Structure

Non-Sm Proteins

U 1 snRNP

12S

1 x 106

164

II

m3GpppA

70K, A, C

U2 snRNP

17S

5 • 105

188

II

m3GpPpA

A', B", 9 others

tri-snRNP

25S

U4/U6 snRNP: U4 snRNP U6 snRNP

12S

U5 snRNP

120 kd/150kd, 7 others 120 kd/150 kd 2 x 105 1 x 105

145 108

II III

m3GpppA mpppG

20S

2 x 105

115

II

m3GpppA

8 proteins

U7 snRNP

(249 kd)

5 x 103

63

II

m3GpppN

2-7 proteins?

U1 l/U12 snRNP: U 11 snRNP U12 snRNP

18S 1 x 104 5 x 103

132 150

II II

m3GpppA m3Gppp N

9 3 or more proteins 9

The U1 and U2 snRNPs exist as monomeric particles; whereas, the U11 and U12 snRNPs function as a di-snRNP; and the U4, U5 and U6 snRNPs function as a tri-snRNP. Initial characterization of the U4, U5 and U6 snRNPs, however, isolated a U4/U6 disnRNP and a monomeric U5 snRNP. All spliceosomal snRNPs possess the Sm core, which includes the B'/B, D1, D2, D3, E, F, G, and 69 kd proteins. The U2 snRNP contains eleven specific proteins, including A', B", and nine uncharacterized proteins (35, 53, 60, 66, 92, 110, 120, 150, 160 kd); the U4/U6 snRNP contains at least one specific protein of 120 or 150 kd; the U5 snRNP contains eight specific proteins (15, 40, 52, 100, 102, 116, 200, 205 kd); the tri-snRNP contains seven specific, uncharacterized proteins (15.5, 20, 27, 90 kd and a triplet at 60 kd) in addition to the U4/U6 and U5-specific proteins (Craft, 1992); the U7, U l l and U12 snRNPs are not well characterized, but serological and molecular studies suggest that the U7 snRNP may contain up to seven specific proteins (13.5, 18, 23, 30, 34, 44, 50 kd; Pironcheva and Russev, 1994), and that the U11 snRNP contains at least three specific proteins (62, 65, 140 kd) (Gilliam and Steitz, 1993). Table 2. Characteristics of the snRNP Proteins Protein

kd

Particle Specificity

RNP Motifs

A

34

U1

2

A'

31

U2

0

leucine-rich; acidic

9

B/B'

28/29

Sm

0

proline-rich; alanine-rich

PPGMRPP conformational throughout

B"

28.5

U2

2

lysine-rich

9

C

22

U1

0

proline-rich, zinc-finger

PAPGMRPP

D (D 1)

16

Sm

0

cationic, nucleic acid-binding

carboxy-terminus

E

12

Sm

0

ribosomal protein-like

9

70K

70

U1

1

arginine-rich

amino acids 267--350 amino acids 276-297 conformational throughout

Other Motifs

Epitopes conformational throughout

Characteristics of the snRNP proteins B, B', D and E proteins, the most well-characterized proteins, are listed. The Sm core also includes D2, D3, F and G. Ribonucleoprotein motifs consist of 70-80 amino acid regions that confer RNA binding activity.

s n R N P - s p e c i f i c antibodies usually r e c o g n i z e the U1specific 70K, A or C polypeptides; U2-specific

antibodies usually r e c o g n i z e the U2-specific A ' or B ' polypeptides. The two U 4 / U 6 - s p e c i f i c sera appear to

775

recognize the same polypeptide of 120 or 150 kd. One study described sera that bind U5-specific proteins of 100, 102 and occasionally 200 kd, although these antibodies were not U5-specific (Craft, 1992). Other isolated studies describe sera that recognize an 18 kd U7-specific protein (Pironcheva and Russev, 1994), and one serum that recognizes U11-specific proteins of 62, 65 and 140 kd (Gilliam and Steitz, 1993). At this time, U12-specific antisera are not described. Some epitopes of anti-snRNP antibodies bear homology to proteins of infectious organisms, such as the herpes simplex virus type 1 ICP4 protein (Misaki et al., 1993), the HIV-1 gp120/41 (Douvas and Takehana, 1994), the influenza B M1 matrix protein (Guldner et al., 1990) or the Plasmodium knowlesi circumsporozoite protein (Habets et al., 1987). Interestingly, the Sm B'/B, U1 A and C proteins share a common C-terminal epitope, PP/aPGMR/iPP (Misaki et al., 1993). Epitopes of all these proteins, however, typically include conformational and linear epitopes throughout the molecules; the significance of these homologies and motifs remains unclear. Native vs. Recombinant Antigens

The development of recombinant antigens promises to alleviate the need for tedious purification procedures. For example, the U1 70K, A and C proteins, as well as the Sm proteins B, D and E are expressed in E. coli and used as substrates in a variety of epitope mapping studies (Craft, 1992). Alternatively, other studies reconstitute whole snRNPs in vitro using synthetically transcribed snRNAs in conjunction with nuclear extracts (Sumpter et al., 1992). One study suggests that recombinant snRNP antigens provide a more sensitive substrate than cell extracts (Delpech et al., 1993). While none compare the clinical utility of these recombinant antigens with native antigens, assays using recombinant antigens for antibody detection generally do not sacrifice specificity in comparison to assays using crude extracts, such as immunodiffusion (Craft, 1992). The role of recombinant antigens in the study of autoimmune disease, therefore, remains speculative yet promising. Methods of Purification

Studies on anti-snRNP antibodies use snRNP antigens in a wide array of purity. On one end of the spectrum, crude or partially purified extracts from calf thymus remain a popular substrate for immunodiffusion as776

says. Commercially purified, crude Sm and RNP substrates are available from Baxter Scientific (McGraw Park, IL), Apotex Scientific, Inc. (Arlington, TX) or Immunovision (Springdale, AR). On the other hand, highly purified native snRNPs are prepared by nondenaturing affinity chromatography using a resinbound monoclonal anti-2', 2', 7-trimethylguanosine antibody (Fatenejad et al., 1993). Individual U snRNPs may be subsequently fractionated after fast protein liquid chromatography and glycerol gradient centrifugation (Behrens and Ltihrmann, 1991). Such latter techniques, although quite cumbersome, provide highquality snRNPs for immunology and molecular biology.

AUTOANTIBODIES Terminology

The most popular categorizations for anti-snRNP antibodies include Sm and RNP antibodies. The antiSm specificity includes autoantibodies that target proteins of the common Sm core, typically B'/B or D, and anti-RNP usually refers to anti-U 1 snRNP-specific autoantibodies that target the U1 RNA or the U1specific proteins 70K, A or C. These two specificities remain the most important distinction among antisnRNP antibodies; however, other anti-snRNP antibodies target proteins unique to the U2, U4/U6, U7 or U l l snRNPs (Craft, 1992; Gilliam and Steitz, 1993; Pironcheva and Russev, 1994). Therefore, references should use anti-snRNP to refer to these antibodies as a whole, anti-Sm to refer to antibodies that target the Sm protein core (of the U1, U2, U5, U4/U6, U7 and Ull/U12 snRNPs), and anti-U1, U2, U4/U6, U7 and U 11 (sn)RNP to refer to antibodies that bind epitopes on proteins unique to these particles. Methods of Detection

Detection of anti-snRNP antibodies typically includes a combination among several available tests. For initial screening, the indirect immunofluorescent antinuclear antibody test (ANA) provides rather nonspecific information as many non-snRNP reactivities also produce positive ANAs (Figure 1). However, fine-speckled staining with nucleolar sparing should suggest the presence of these antibodies (anti-Sm, anti-RNP, anti-U2 and anti-U4/U6 all produce such a pattern of fluorescence). One popular

Figure 1. Indirect immunofluorescent antinuclear antibody test. HEp-2 cell substrates stained by human anti-Sm demonstrate a finespeckled nuclear pattern against a diffuse nucleoplasmic staining with nucleolar sparing a. These speckles represent foci of spliceosomal components. Some studies report a more diffuse pattern for anti-U 1 snRNP reactivity, reflecting U 1 snRNP' s less focal nucleoplasmic distribution (Matera and Ward, 1993); routine distinction may be difficult, however, as demonstrated by the similarly speckled nuclear staining pattern of a U1 snRNP-specific serum b.

but relatively insensitive method for verifying the presence of Sm or RNP antibody activity is the double immunodiffusion Ouchterlony technique. This method does not require special instrumentation or highly purified antigen, but it remains unsatisfactory because it requires large quantities of immunoglobulin and up to two days for final test interpretation. Another test, the immunoprecipitation assay, offers increased sensitivity, but is limited by the use of radioactivity, the time commitment and an inability to distinguish among specific anti-snRNP activities.

Sensitive yet specific tests are necessary. The enzyme-linked immunosorbent assay (ELISA) and immunoblot combine the sensitivity of immunoprecipitation and the specificity of immunodiffusion. ELISA offers a particularly rapid, sensitive verification of ANA but requires individual substrate proteins or RNPs in order to distinguish snRNP specificities. Most laboratories now rely upon the production of recombinant and affinity-purified snRNP proteins for detection of anti-snRNP antibodies. The immunoblot technique also provides a reliable and sensitive 777

method for detecting specific epitopes, providing information regarding reactivities against individual snRNP proteins (Figure 2). Thus, most testing for anti-snRNP involves the initial screening by ANA, confirmation by ELISA and perhaps further verification and/or characterization by immunoblot.

Factors Involved in Pathogenicity Investigations into pathogenic roles for the anti-snRNP antibodies remain inconclusive on the whole. Some studies suggest that these antibodies may bind to stressed cells that express autoantigen on their surfaces, become internalized and then interact with intracellular antigens and modify various proliferative and/or functional responses (Golan et al., 1993). Other

studies suggest a pathogenic role for anti-snRNP antibodies of the IgG1 subclass, which seem to comprise the majority of the anti-snRNP antibody response (Craft, 1992). Later studies, however, use more sensitive detection assays and increasingly find responses unrestricted as to subclass (Meilof et al., 1992). Other circumstantial evidence for the pathogenicity of anti-snRNP antibodies includes correlation of anti-snRNP antibody responses with particular clinical manifestations in neonatal, child and adult human patients as well as spontaneous and experimentally induced mouse models of lupus (Craft, 1992; Horng et al., 1992). Overall, however, there is a lack of direct evidence that anti-snRNP antibodies produce tissue injury.

Genetics In efforts to gain further insight into the nature of these antibodies, numerous studies examined possible H L A correlations, and a few investigated the role of other genes like immunoglobulin V H or V L genes. Some studies report an association of anti-snRNP with the Gm (Abu-Shakrah et al., 1989; Barron et al., 1993; Behrens and Ltihrmann, 1991; James et al., 1995) immunoglobulin haplotype (Genth et al., 1987), but the significance of this finding remains uncertain. Sm antibodies are associated with particular MHC class II alleles, especially those of the HLA-DR2 and HLA-DR4 groups but also members of the DP, DQ and DR families. Likewise, anti-U1 snRNP and anti70K antibodies are associated with HLA-DR2 and DR4, as well as several other members in the D, DP, DQ and DR families (Barron et al., 1993; Craft, 1992; Hoffman et al., 1993). On the other hand, comprehensive family studies failed to show an association between the inheritance of anti-snRNP antibodies and HLA types (Shoenfeld et al., 1992). Thus, undefined polygenetic factors unrelated to HLA undoubtedly help shape the immune response to snRNPs.

Etiology Figure 2. Detection of anti-snRNP antibodies by immunoblot. Anti-Sm and anti-U1 snRNP antibodies detect Sm core and U 1specific proteins, respectively. Lane 1: normal serum. Lane 2: anti-U1 serum recognizing the U1 A protein. Lane 3: serum with anti-Sm and anti-U1 activity, recognizing 70K, A, B'/B and D. Lane 4: anti-Sm serum recognizing B'/B and D. The U1C, Sm- E, Sm F, and Sm G polypeptides are typically difficult to visualize by this method. This blot utilized purified human snRNPs as substrate (Fatenejad et al., 1993). 778

Not surprisingly, the etiology of the autoimmune response to anti-snRNP antibodies remains unclear, although a number of theories are proposed (Theofilopoulos, 1995). In one line of thought, stress or infection leads to the release and presentation to the immune system of anatomically sequestered autoantigen (Golan et al., 1993). A particularly popular model involves the molecular mimicry of autoantigens by

proteins of infectious agents. Some evidence suggests that the anti-snRNP antibody response begins with the U1 snRNP and then progresses to anti-Sm and other specificities (Craft, 1992; Fatenejad et al., 1993). Other investigators argue that anti-snRNP antibodies comprise a part of the normal repertoire and that cross-reactive idiotypes or epitopes may induce them (James et al., 1995; Shoenfeld and Mozes, 1990). Other theories, which have received more thorough attention in lupus-prone murine models, include induction by cryptic epitopes, activation of ignorant lymphocytes, induction by neo-self-determinants, defects in central or peripheral immune tolerance, polyclonal lymphocyte activation and immunoregulatory disturbances. No studies, however, have demonstrated physiologic relevance.

CLINICAL UTILITY Disease Associations

Sm antibodies offer a highly specific, but relatively insensitive, clinical marker for SLE (Craft, 1992). Indeed, their presence constitutes one of the revised American Rheumatism Association criteria for diagnosis, even though their overall prevalence ranges from approximately 20--30% in SLE (Table 3). Epidemiological studies generally describe the presence of anti-Sm antibodies in 10--20% of white SLE patients and 30-40% or more of Asian and black SLE patients, data which remain applicable to childhood

SLE (Barron et al., 1993). Anti-Sm reactivity is not described definitively in other diseases, although a few studies describe Sm antibodies in monoclonal gammopathies (Abu-Shakrah et al., 1989), schizophrenia (Sirota et al., 1993) and uveitis (Amital et al., 1992). Numerous studies suggest the association of antiSm antibodies with disease activity and particular disease manifestations (Craft, 1992). Some report associations with milder renal and/or central nervous system disease, organic brain syndrome (Hirohata and Kosaka, 1994), disease flares or more active disease. Other studies, however, do not uphold these findings, reporting no correlation with disease manifestations (Gulko et al., 1994). Therefore, while the presence of anti-Sm antibodies provides a substantial aid in the diagnosis of SLE and may identify a particular subset of patients prone to particular disease manifestations, their significance in terms of disease course and prognosis remains poorly defined. Anti-U 1 snRNP antibodies typically appear in both SLE and MCTD, but several differences distinguish their presentation (Craft, 1992). In MCTD, the presence of anti-U1 snRNP antibodies is required for diagnosis; whereas, anti-U1 snRNP antibodies occur in only 30--40% of SLE (Table 3). MCTD is typified by the high-titer U1 snRNP antibody activity in isolation; whereas, anti-U 1 snRNP antibody activity in SLE commonly accompanies anti-Sm antibodies, although isolated anti-U1 snRNP antibodies are described in SLE. In addition, nearly all MCTD patients demonstrate anti-70K activity; whereas, as

T a b l e 3. Prevalence of anti-snRNP Antibodies in Rheumatologic Disease Specificity

SLE

MCTD

Sm

20--30

0

U1

30--40

100

U2

15

15

overlap syndromes

U4/U6

9

9

Sjt~gren's syndrome, scleroderma

U5

9

9

9

U7

9

9

Ull

9

9

U12

9

9

Other Diseases

rheumatoid arthritis, polymyositis, scleroderma, Sj6gren's syndrome

scleroderma

Numbers indicate the best consensus on percent prevalences. U4/U6, U5, U7, Ull and U12 snRNP antibodies are not fully investigated in SLE or MCTD.

779

few as 10% to as many as 85% of anti-U1 snRNP antibody-positive SLE patients possess anti-70K, depending on the sensitivity of the assay. In SLE, anti-A antibodies appear to be twice as common as anti-70K, appearing in approximately 75% of anti-U1 snRNP antibody-positive SLE patients or 23% of SLE patients overall; but when patient sera are grouped and examined irrespective of disease, anti-70K, anti-A and anti-C antibodies appear to have similar prevalences. Blacks demonstrate a two- to threefold higher prevalence of this antibody than Caucasians and Asians. All these data also remain similar in childhood disease (Hoffman et al., 1993). Other diseases in which antiU1 snRNP activity is described include rheumatoid arthritis, polymyositis, scleroderma, and Sj6gren's syndrome, but the significance in these diseases is not fully investigated. The strong association between MCTD and anti-U 1 snRNP antibodies makes it difficult to interpret studies suggesting the association of antibodies with clinical manifestations (Craft, 1992). Several investigations report the association of anti-U1 snRNP antibodies with such signs or symptoms as myositis, esophageal hypomotility, Raynaud's phenomenon, arthralgias/arthritis, sclerodactyly and interstitial changes on chest radiographs in the absence of nephritis (anti-70K activity in particular) (Snowden et al., 1993), although each of these findings may simply reflect MCTD-like symptoms. Likewise, antibody levels are not clearly shown to reflect disease activity, although several studies report a correlation (Craft, 1992). Isolated studies also describe anti-U1 snRNP antibodies in monoclonal gammopathies (Abu-Shakrah et al., 1989) and uveitis (Amital et al., 1992). Thus, as for anti-Sm, the presence of anti-U 1 snRNP antibodies provides a helpful diagnostic adjunct, but their utility in the monitoring of disease remains unclear. Anti-U2 snRNP antibodies also appear in SLE and MCTD but with much lower frequencies than the antiU1 snRNP or anti-Sm specificities. First identified in a patient with scleroderma-polymyositis overlap syndrome, they are also described in other overlap syndromes: scleroderma with myositis, psoriasis, Raynaud's phenomenon and other sera without a specific disease association. Up to 15% of both MCTD and SLE demonstrate this activity (Craft, 1992). Detailed studies regarding clinical associations and epidemiology, however, have not been performed. Other anti-snRNP antibodies against the U4/U6 snRNP, U5 snRNP, U7 snRNP, U11 snRNP or the trimethylguanosine cap structure are rarely described.

780

Anti-U4/U6 snRNP-specific antibodies were uniquely described in one patient with Sj6gren syndrome and another with scleroderma; four patients with scleroderma were found to possess antitrimethylguanosine activity (Gilliam and Steitz, 1993; Okano and Medsger, 1992). One study described U5 snRNP activity in the majority of fifteen patients with SLE or MCTD beating high titer Sm or U 1 snRNP antibodies, but no one has reported anti-U5 snRNP antibody-specific responses (Craft, 1992). Anti-U7 snRNP antibodies were found in a small series of SLE patients (Pironcheva and Russev, 1994), and anti-Ull snRNP antibodies were found in one scleroderma serum in association with antitrimethylguanosine activity (Gilliam and Steitz, 1993). Due to the paucity of information about these antibodies, their clinical significance remains unknown. Cross-Reactions of Anti-snRNP Antibodies

Although anti-U1 snRNP and anti-Sm antibodies typically recognize particle-specific epitopes, several studies demonstrate cross-reactive epitopes among the snRNPs and other autoantigens. Anti-DNA antibodies, for example, exhibit strong cross-reactivity with denatured U1 A and Sm D proteins (Reichlin et al., 1994), and the anti-DNA idiotype 16/6 bears crossreactivity with U1 RNP, Sm and Ro (Kaburaki and Stollar, 1987). Such cross-reactivity between DNA and snRNP antibodies was demonstrated in the MRL mouse model for SLE (Bloom et al., 1993). Antibodies to 70K may cross-react with the p68 autoantigen (Netter et al., 1991). Anti-Sm antibodies may cross-react with RNA polymerase I (Morris and Stetler, 1989) and ribosomal proteins (Nojima et al., 1989), but the significance of these findings, if any, is unclear. In addition, autoantibodies may appear to demonstrate both anti-U1 snRNP-specific and antiSm-specific reactivities if they recognize the prolinerich PP/aGMR/iPP motif present in the C-terminal region of the U1 A, U1 C, and Sm B'/B proteins (Misaki et al., 1993). Such findings warrant critical analysis in the investigation of antibody specificities, especially in the interpretation of clinical correlations.

CONCLUSION The antispliceosomal snRNP autoantibodies are definitive specificities found in various rheumatologi-

cal diseases. Their targets include the proteins and RNAs of the U small nuclear ribonucleoprotein particles involved in the splicing of premessenger RNA. Anti-U snRNP-specific antibodies target U snRNP-specific proteins, such as the U1 A, C or 70K proteins or the U2 A' or B' proteins. Anti-Sm antibodies target the core Sm polypeptides common to all spliceosomal snRNPs. Detected via immunofluorescent ANA, immunodiffusion, ELISA, immunoprecipitation and/or immunoblot, the anti-snRNP antibodies are described in several connective tissue diseases, especially SLE and MCTD; anti-Sm antibodies provide a specific marker for SLE, and isolated antiU1 snRNP antibodies remain a hallmark of MCTD. Some studies report less prominent prevalences for U2, U4/U6, U5, U7, U11 snRNP and trimethylguanosine antibodies, although details about these rarer specificities remain largely unknown. While many studies investigated the clinical correlations of these

REFERENCES Abu-Shakrah M, Krupp M, Argov S, Buskila D, Slor H, Shoenfeld Y. The detection of anti-Sm-RNP activity in sera of patients with monoclonal gammopathies. Clin Exp Immunol 1989;75:349--353. Amital H, Klemperer I, Blank M, Yassur Y, Palestine A, Nussenblatt RB, Shoenfeld Y. Analysis of autoantibodies among patients with primary and secondary uveitis: high incidence in patients with sarcoidosis. Int Arch Allergy Immunol 1992;99:34--36. Barron KS, Silverman ED, Gonzales J, Reveille JD. Clinical, serologic, and immunogenetic studies in childhood-onset systemic lupus erythematosus. Arthritis Rheum 1993;36: 348--354. Baserga SJ, Steitz JA. The diverse world of small ribonucleoproteins. In: Gesteland RF, Atkins JF, eds. The RNA World: The Nature Of Modern RNA Suggests A Prebiotica RNA World. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1993. Behrens SE, Ltihrmann R. Immunoaffinity purification of a [U4N6.U5] tri-snRNP from human cells. Genes Dev 1991:5: 1439--1452. Bloom DD, Davignon J-L, Cohen PL, Eisenberg RA, Clarke SH. Overlap of the anti-Sm and anti-DNA responses of MRL/Mp-lpr/lpr mice. J Immunol 1993; 150:1579-1590. Craft J. Antibodies to snRNPs in systemic lupus erythematosus. Rheum Dis Clin North Am 1992;18:311--335. Delpech A, Gilbert D, Daliphard S, Le Loet X, Godin M, Tron F. Antibodies to Sm, RNP and SSB detected by solid-phase ELISAs using recombinant antigens: a comparison study with counter immunoelectrophoresis and immunoblotting. J Clin Lab Anal 1993;7:197--202.

antibodies, no particular associations were conclusively determined regarding specific disease manifestations, disease course, pathogenicity or genetic markers. Thus, future work with these autoantibodies must elucidate their role in the pathology of connective tissue disease and/or understand their genesis as a result of an underlying disorder.

ACKNOWLEDGEMENTS Supported in part by the National Institutes of Health (AR40072 and AR42475), the Arthritis and Lupus Foundations and donations to Yale Rheumatology in the memories of Albert L. Harlow and Chantal Marquis. SLP was supported by the Medical Scientist Training Program, Yale University School of Medicine.

Douvas A, Takehana Y. Cross-reactivity between autoimmune anti-U 1 snRNP antibodies and neutralizing epitopes of HIV-I and gp120/41. AIDS Res Hum Retroviruses 1994;10:253-262. Fatenejad S, Mamula MJ, Craft J. Role of intermolecular/intrastructural B- and T-cell determinants in the diversification of autoantibodies to ribonucleoprotein particles. Proc Natl Acad Sci USA 1993;90:12010-12014. Genth E, Zarnowski H, Mierau R, Wohltmann D, Hartl PW. HLA-DR4 and Gm(1,3;5,21) are associated with UI-nRNP antibody positive connective tissue disease. Ann Rheum Dis 1987;46:189--196. Gilliam AC, Steitz JA. Rare scleroderma autoantibodies to the U11 small' nuclear ribonucleoprotein and to the trimethylguanosine cap of U small nuclear RNAs. Proc Natl Acad Sci USA 1993;90:6781--6785. Golan TD, Gharavi AE, Elkon KB. Penetration of autoantibodies into living epithelial cells. J Invest Dermatol 1993: 100:316--322. Guldner HH, Netter HJ, Szostecki C, Jaeger E, Will H. Human anti-p68 autoantibodies recognize a common epitope of U1 RNA containing small nuclear ribonucleoprotein and influenza B virus. J Exp Med 1990;171:819--829. Gulko PS, Reveille JD, Koopman WJ, Burgard SL, Bartolucci AA, Alarc6n GS. Survival impact of autoantibodies in systemic lupus erythematosus. J Rheumatol 1994;21:224-228. Habets WJ, Sillekens PT, Hoet MH, Schalken JA, Roebroek AJ, Leunissen JA, van de Ven WJ, van Venrooij WJ. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNAassociated B antigen. Proc Natl Acad Sci USA 1987;84: 2421-2425.

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Hackl W, Fischer U, Luhrmann R. A 69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species. J Cell Biol 1994;124:261--272. Hirohata S, Kosaka M. Association of anti-Sm antibodies with organic brain syndrome secondary to systemic lupus erythematosus. Lancet 1994;343:796. Hoffman RW, Cassidy JT, Takeda Y, Smith-Jones EI, Wang GS, Sharp GC. U1-70-kD autoantibody-positive mixed connective tissue disease in children. A longitudinal clinical and serologic analysis. Arthritis Rheum 1993 ;36:1599-1602. Horng YC, Chou YH, Tsou Yau KI. Neonatal lupus erythematosus with negative anti-Ro and anti-La antibodies: report of one case. Acta Paediatr Sin 1992;33:372-375. James JA, Gross T, Scofield RH, Harley JB. Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B'-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity. J Exp Med 1995;181: 453-461. Kaburaki J, Stollar BD. Identification of human anti-DNA, antiRNP, anti-Sm, and anti-SS-A serum antibodies bearing the cross-reactive 16/6 idiotype. J Immunol 1987; 139:385--392. Mattioli M, Reichlin M. Physical association of two nuclear antigens and mutual occurrence of their antibodies: the relationship of the Sm and RNAprotein (Mo) systems in SLE sera. J Immunol 1973;110:1318--1324. Meilof JF, Hebeda KM, de Jong J, Smeenk RJ. Analysis of heavy and light chain use of lupus-associated anti-La/SS-B and anti-Sm autoantibodies reveals two distinct underlying immunoregulatory mechanisms. Res Immun 1992;143:711720. Misaki Y, Yamamoto K, Yanagi K, Miura H, Ichijo H, Kato T, Mato T, Welling-Wester S, Nishioka K, Ito K. B cell epitope on the U1 snRNP-C autoantigen contains a sequence similar to that of the herpes simplex virus protein. Eur J Immunol 1993;23:1064--1071. Morris P, Stetler DA. Monoclonal antibody against the lupus antigen Sm cross-reacts with RNA polymerase I. Autoimmunity 1989;2:241--251. Netter HJ, Will H, Szostecki C, Guldner HH. Repetitive P68autoantigen specific epitopes recognized by human anti-(U 1) small nuclear ribonucleoprotein autoantibodies. J Autoimmun 1991;4:651--663. Nojima Y, Minota S, Yamada A, Takaku F. Identification of an

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acidic ribosomal protein reactive with anti-Sm autoantibody. J Immunol 1989;143:1915--1920. Okano Y, Medsger Jr TA. Novel human autoantibodies reactive with 5'-terminal trimethylguanosine cap structures of U small nuclear RNA. J Immunol 1992;149:1093--1098. Pironcheva G, Russev G. Characterization of the protein moiety of U7 small nuclear RNP particles. Microbios 1994;77:41-46. Reichlin M, Martin A, Taylor-Albert E, Tsuzaka K, Zhang W, Reichlin MW, Koren E, Ebling FM, Tsao B, Hahn BH. Lupus autoantibodies to native DNA cross-react with the A and D snRNP polypeptides. J Clin Invest 1994;93:443-449. Sharp GC, Irvin WS, LaRoque RL, Velez C, Daly V, Kaiser AD, Holman HR. Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest 1971;50:350359. Shoenfeld Y, Mozes E. Pathogenic idiotypes of autoantibodies in autoimmunity: lessons from new experimental models of SLE. FASEB J 1990;4:2646-2651. Shoenfeld Y, Slor H, Shafrir S, Krause I, Granados J, Villareal GM, Alarc6n-Segovia D. Diversity and pattern of inheritance of autoantibodies in families with multiple cases of systemic lupus erythematosus. Ann Rheum Dis 1992;51:611--618. Sirota P, Firer M, Schild K, Zurgil N, Barak Y, Elizur A, Slor H. Increased anti-Sm antibodies in schizophrenic patients and their families. Prog Neuropsychopharmacol Biol Psychiatry 1993;17:793--800. Snowden N, Hay E, Holt PJL, Bernstein R. Clinical course of patients with anti-RNP antibodies. J Rheum 1993;20:1256-1258. Sumpter V, Kahrs A, Fischer U, Kornstadt U, Luhrmann R. In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA. Mol Biol Rep 1992;16:229--240. Tan EM, Kunkel HG. Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J Immunol 1966;96:464--471. Theofilopoulos AN. The basis of autoimmunity. Part I. Mechanisms of aberrant self-recognition. Immunol Today 1995;16: 90-98. Wassarman KM, Steitz JA. The low-abundance U l l and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex. Mol Cell Biol 1992;12:1276-1285.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

SS-A (Ro) AUTOANTIBODIES Morris Reichlin, M.D. and R. Hal Scofield, M.D.

Arthritis~Immunology Program, Oklahoma Medical Research Foundation, Department of Medicine, College of Medicine, Oklahoma University Health Sciences Center, Oklahoma City, OK 73104, USA

HISTORICAL NOTES Antibodies to the Ro/SS-A antigen were first reported in 1962, as antibodies reactive with the SjD antigen using the gel diffusion method with sera from patients with Sj6gren's syndrome (SS) (Anderson et al., 1962). Antibodies to the Ro/SS-A antigen were again detected by the gel diffusion method in 1969 in patients with systemic lupus erythematosus (SLE) and SS (Clark et al., 1969). In 1975, the antibodies were described a third time in SS sera and were designated "SS-A" after their detection in gel diffusion with Wil 2 extract (Alspaugh and Tan, 1975). In 1979, the antigenic identity of Ro and SS-A was demonstrated in an interlaboratory comparison (Alspaugh and Maddison, 1979). The clinical specificity of antiRo/SS-A for SS and SLE as well as their almost uniform presence in patients with both SS and SLE is now well recognized.

AUTOANTIGEN

Definition In 1981, Ro/SS-A was shown to be a ribonucleoprotein-containing small uridine-rich nucleic acids known as hY 1, hY 3, hY 4 and hY 5 (Lerner et al., 1981). The abbreviation "hY" stands for h_uman cytoplasmic. The major protein is a 60 kd molecule (Venables et al., 1983; Wolin and Steitz, 1984; Yamagata et al., 1984), and the Ro/SS-A particle contains 1 mol of protein and 1 mol of hY RNA (Yamagata et al., 1984). The different immunoblot patterns of precipitating antiRo/SS-A sera where the anti-Ro/SS-A precipitins are alone or accompanied by anti-U 1 RNP or anti-La/SS-

B, are seen in Figure 1. The erythrocyte form of the 60 kd protein is distinct from but related to the 60 kd protein of nucleated cells (Rader et al., 1989). When first described in nucleated cells, the 52 kd form of the Ro/SS-A protein was measurable only by immunoblotting (Ben-Chetrit et al., 1988). An analogous 54 kd Ro/SS-A protein was characterized by immunoblot of erythrocytes (Rader et al., 1989). In contrast to nucleated cells, erythrocytes contained only hY ~ and hY 4 bound to 60 kd Ro/SS-A (Rader et al., 1989). Human platelets contain only hY 3 and hY 4 Ro/SS-A RNAs associated with the Ro/SS-A proteins (Itoh and Reichlin, 1991).

Origin/Sources Most, if not all, sera with anti-Ro/SS-A precipitins preferentially react with the native 60 kd Ro/SS-A molecule (Boire et al., 1991; Itoh and Reichlin, 1992); whereas, most antibodies to 52 kd Ro/SS-A prefer the denatured 52 kd Ro/SS-A molecule (Itoh and Reichlin, 1992). Thus, 70% of anti-Ro/SS-A precipitinpositive sera react with the denatured 60 kd Ro/SS-A in immunoblots of MOLT-4 extract, and 89% of such sera react with a recombinant fusion protein of 60 kd Ro/SS-A 13-galactosidase (James et al., 1990). Because the autoimmune response is directed to the human protein (i.e., it is species-specific), human cells or tissue extracts must be utilized for maximal sensitivity and specificity (Reichlin et al., 1989). As many as 5--10% of anti-Ro/SS-A-positive sera react only with human Ro/SS-A (M. Reichlin, unpublished observations). Although ubiquitously present in all species and tissues, the Ro/SS-A antigen found in highest con-

783

AUTOANTIBODIES Pathogenetic Role

Figure 1. Immunoblotanalysis of 10 representative anti-Ro-SSA sera using MOLT-4 cell extract. Lanes 1--4 sera have precipitating anti-Ro/SS-A alone, while Lanes 5 and 6 sera have anti-Ro/SS-A anti-nRNP precipitins and Lanes 7--10 have antiRo/SS-A and anti-La/SS-B precipitatins.

centration in lymphocyte lines and spleen, is also present in high concentrations in kidney, liver and stomach but in lower concentrations in heart, brain, skeletal muscle and lung. Ro/SS-A is in lowest concentration in red blood cells (Itoh et al., 1990). Because of the species specificity, human spleen and/or cell lines (MOLT-4) are the preferred source for preparation of extracts for gel diffusion or for affinity purification (Yamagata et al., 1984).

784

Human Disease. Evidence for a pathogenic role for anti-Ro/SS-A antibodies in human disease comes from several sources. First, antibodies to Ro/SS-A detected by ELISA are almost uniformly present in the following SLE subsets: subacute cutaneous lupus erythematosus, neonatal lupus erythematosus, homozygous C2 and C4 deficiency, the vasculitis of SS, ANA-negative SLE, interstitial lung disease and photosensitive rash (Reichlin, 1994). Second, the antibodies to Ro/SS-A are enriched in acid eluates of saline extract of affected organs from SLE and SS patients. These include two cases of lupus nephritis (Maddison and Reichlin, 1979), a parotid gland from a patient with SS (Penner and Reichlin, 1982) and most recently an afflicted heart from a child dying with complete congenital heart block (Reichlin et al., 1994). Third, human IgG-containing anti-Ro/SS-A antibodies can induce repolarization abnormalities in neonatal rabbit hearts (Alexander et al., 1992) and induce conduction abnormalities with slowing of the rate and heart block in one-third of the Langendorf preparations of adult rabbit hearts (Garcia et al., 1994). Likewise, ventricular myocytes from young rabbit hearts studied by the patch clamp method show that inward currents are profoundly affected by antiRo/SS-A-containing IgG (Garcia et al., 1994). These experiments provide animal models for the heart disease induced by anti-Ro/SS-A autoantibodies. Finally, UV irradiation of keratinocytes increases the expression of Ro/SS-A antigen on the cell surface enhancing the possibility of direct injury of skin cells by anti-Ro/SS-A antibodies (Furukawa et al., 1990). Animal Models. Anti-Ro/SS-A were induced in mice immunized with fragments of the 60 kd Ro/SS-A or La/SS-B (Topfer et al., 1995). Rabbits immunized with the nucleocapsid protein of the vesicular stomatitis virus developed antibodies to 60 kd Ro/SS-A (Huang et al., 1995). In both mice and rabbits, the anti-Ro/SS-A produced were accompanied by a new and distinct autoimmunity to the entire Ro/SS-A ribonucleoprotein complex. Methods of Detection The most sensitive and specific methods for detection of anti-Ro/SS-A antibodies are: 1) a sandwich ELISA

for detection of antibody against the human 60 kd Ro/SS-A (Rader et al., 1989) or immunoprecipitation with detection of the hY Ro/SS-A RNAs (Manoussakis et al., 1993). However, because of ease of performance and reliability, counter immunoelectrophoresis (CIE) with human extracts is still probably the preferred method for clinical diagnosis with the original gel diffusion method a close second. Because these latter two methods exhibit 100% specificity and 85--90% sensitivity for detecting anti-Ro/SS-A antibodies, a serious argument can be made that these methods are still the preferred methods, because both the sandwich ELISA and the immunoprecipitation method are more analytically sensitive methods but detect small amounts of autoantibody in asymptomatic normals with resultant decrease in diagnostic specificity. Some 17% of normal sera have elevated amounts of anti-Ro/SS-A by anti-Ro/SS-A-specific ELISA (Gaither et al., 1987), and as many as 25% of asymptomatic first-degree relatives of anti-Ro/SS-A-positive patients with SLE and SS have elevated amounts of anti-Ro/SS-A by anti-Ro/SS-A-specific ELISA (Arnett et al., 1989). The sensitive ELISA method detects elevated anti-Ro/SS-A levels in an unacceptable proportion of normal persons. If the cut-off point is elevated the specificity is improved, but the sensitivity is virtually lost leaving little gain. None of these latter normals or first-degree relatives are positive for antiRo/SS-A by CIE or gel diffusion. The affinity of antiRo/SSA for antigen in normals is identical to that from patients (Gaither et al., 1987). On balance then, the simplest and somewhat less sensitive methods are still the best methods for clinical diagnosis.

Genetics Anti-Ro/SS-A antibodies are associated with HLADQ1/DQ2 heterozygosity (p = 0.0024) (Harley et al., 1986). After restriction length polymorphisms of DQlc~ and DQ2~ were found to be associated with anti-Ro/SS-A (Fujisaku et al., 1990), the specific HLA alleles were identified as DQB1 "0201 and DQA1 *0101, DQA1 "0102 or DQA1 "0103 (Scofield et al., 1994). At least one HLA-DQA1 allele with glutamine in position 34 or HLA-DQB1 allele with leucine in position 26 was reported but not sufficient for antiRo/SS-A (Reveille et al., 1991); analysis of these alleles in another cohort did not lead to the same conclusion but does concur that the presence of four such alleles is associated with anti-Ro/SS-A in SLE

(Scofield and Harley, 1994). These two HLA-DQ associations are apparently related but distinct markers. Binding of specific epitopes of 60 kd Ro/SS-A is associated with certain HLA haplotypes or alleles (Ricchiuti et al., 1994; Scofield et al., 1995). Anti-Ro/SS-A in SLE is associated with a polymorphism of the T-cell receptor [3 gene (Frank et al., 1990), which together with DQB 1 "0201 and one of the DQA1 *01 alleles is associated with higher amounts of anti-Ro/SS-A in the sera of SLE patients (Scofield et al., 1994). Analyses of T-cell receptor or immunoglobulin variable gene usage are not available.

Factors in Etiology Anti-Ro/SS-A autoantibodies are polyclonal but virtually nothing has been reported about anti-Ro/SSA antibodies of the IgA or IgM classes; all IgG subclasses are represented. The epitopes of 60 and 52 kd Ro/SS-A were characterized with a variety of techniques including short overlapping peptides (Scofield and Harley, 1991; Scofield et al., 1991; Routsias et al., 1994), large peptide fragments and partial gene clones (Frank et al., 1994; McCauliffe et al., 1994; Peek et al., 1994; Saitta et al., 1994; B lange et al., 1994). As expected given the variety of techniques, the details differ, but in a qualitative sense, these studies agree that anti-Ro/SS-A antibodies bind many epitopes on 60 kd Ro/SS-A. The 52 kd Ro/SS-A is bound in fewer places and perhaps contains an immunodominant region. The etiology of anti-Ro/SS-A is unknown. Polyclonal activation of B cells is an unlikely explanation, given the powerful associations with immunogenetics and clinical manifestations and evidence of an antigen-driven response. The 60 kd Ro/SS-A molecule shares six sites of 4--8 amino acids with a nucleocapsid protein of vesicular stomatitis virus. The epitopes of 60 kd Ro/SS-A align better (p = 0.00017) with these shared sequences (Scofield et al., 1991). AntiRo/SS-A is found in 42% of SLE patients who bind this viral protein (Hardgrave, et al., 1993) versus 21% who do not bind the viral protein. Finally, immunization with the nucleocapsid protein induces anti-Ro/SSA autoantibody (Huang et al., 1995). Thus, these observations show that Ro/SS-A autoimmunity can be induced by a protein that is selected on the basis of shared short sequence.

785

Table 1. Ro/SS-A Summary 9

Historically, antibodies to the Ro/SS-A antigen were discovered repeatedly by gel diffusion.

9

The Ro/SS-A antigen is an RNA protein particle with the protein carrying the major part of the antigenicity. There are two molecular types of the protein, a 60 kd and a 52 kd form.

9

The Ro/SS-A particle is ubiquitously distributed in all tissues. The autoimmune response is directed to the human protein.

9

Evidence is accumulating that immune complexes of Ro/SS-A- anti-Ro/SS-A are involved in pathogenicity in humans, especially in the heart disease of neonatal lupus.

9

While there are numerous sensitive ways to measure anti-Ro/SS-A, the simple methods of gel diffusion and counterimmunoelectrophoresis are preferred for clinical diagnosis.

9

HLA DQ and T cell receptor 13gene polymorphisms associate strongly with anti-Ro/SS-A production.

9

Multiple epitopes in 60 kd Ro/SS-A are reactive with autoimmune sera. There are several shared linear epitopes between 60 kd Ro/SS-A and the nucleocapsid protein of vesicular stomatitis virus.

9

Numerous clinical subsets within the spectrum of SLE and SS recognized to be strongly associated with autoantibodies to 60 kd Ro/SS-A.

9

Measurement of anti-Ro/SS-A is clinically useful and in some patients the only autoantibody detectable.

CLINICAL UTILITY

Disease Associations The clinical associations of anti-Ro/SS-A are well known; in certain subsets, the antibodies are invariably found and frequently are the only autoantibody detectable in high titer by the gel diffusion method. Thus, anti-Ro/SS-A are found in virtually all patients with subacute cutaneous LE and the vasculitis of SS. Anti-La/SS-B are also present in about 50% of these cases, but anti-Ro/SS-A are the only autoantibody detected in the other half of the cases. Overall, anti-Ro/SS-A precipitins occur in 4 0 - 5 0 % of SLE, 60--75% of primary SS and in a high proportion of secondary SS whether the associated disease is SLE, RA, PSS, polydermatomyositis or primary biliary cirrhosis. If sensitive ELISA methods are used for detection, an additional increment of patients with anti-Ro/SS-A is detected in all the clinical situations described above, albeit with a loss of diagnostic specificity (Reichlin, 1994).

Effect of Therapies Decline of the anti-Ro/SS-A levels sometimes occurs when patients are treated with cytotoxic drugs. In 2 0 - 2 5 % of patients so treated, the anti-Ro/SS-A levels become undetectable by gel diffusion (M. Reichlin, unpublished observations). The usual situation is that the anti-Ro/SS-A levels do not noticeably fluctuate

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with disease activity or with steroids and/or immunotherapy, but no formal studies of this type are available. In neonatal lupus erythematosus, the mothers invariably have precipitating anti-Ro/SS-A, and (by immunoprecipitation) antibodies to native 60 kd Ro/SS-A as well as autoantibodies to 52 kd Ro/SS-A and La/SS-B in about 80% of the cases. The particular antibody responsible for the heart disease (or the skin disease) is not known; but in the single case studied by acid elution of an affected child's heart, antibodies to native 60 kd Ro/SS-A predominated, but small amounts of antibody to denatured 52 kd Ro/SS-A were also present (Reichlin et al., 1994). As only about one in 50 children born to mothers with antiRo/SS-A develop heart block, much interest centers on the identification of a specific predictive profile of anti-Ro/SS-A and/or anti-La/SS-B, but none has been found.

CONCLUSION Detection of anti-Ro/SS-A is of interest and significance in the clinical diagnosis of SLE and Sj6gren's syndrome, but its highest utility is its tight association with disease subsets. Because it may be the only autoantibody present in many patients with SLE or SS, failure to measure anti-Ro/SS-A leaves a diagnostic void which cannot be filled by other tests (Table 1). Unraveling of the mechanism of the tight association

of anti-Ro/SS-A with its related clinical subsets is expected to provide insights into pathogenesis and

hopefully to prevention and/or therapy. See also SS-B (LA) AUTOANTIBODIES.

REFERENCES

Gaither KK, Fox OF, Yamagata H, Mamula MJ, Reichlin M, Harley JB. Implications of anti-Ro/Sj6gren's syndromeantigen autoantibody in normal sera for autoimmunity. J Clin Invest 1987;79:841--846. Garcia S, Nascimento JH, Bonfa E, Olivera SF, Tavares AV, de Carvalho AC. Cellular mechanisms of the conduction abnormalities induced by serum from anti-Ro/SSA-positive patients in rabbit hearts. J Clin Invest 1994;93:718-724. Hardgrave KL, Neas BR, Scofield RH, Harley JB. Antibodies to vesicular stomatitis virus proteins in systemic lupus erythematosus and in normal subjects. Arthritis Rheum 1993;36:962--970. Harley JB, Reichlin M, Arnett FC, Alexander EL, Bias WB, Provost TT. Gene interaction at HLA-DQ enhances autoantibody production in primary Sj6gren's syndrome. Science 1986;232:1145-1147. Huang SC, Pan Z, Kurien BT, James JA, Harley JB, Scofield RH. Immunization with vesicular stomatitis virus nucleocapsid protein induces autoantibodies to the 60 kD to ribonucleoprotein particle. J Invest Med 1995;43:151-158. Itoh Y, Kriet D, Reichlin M. Organ distribution of the Ro (SSA) antigen in the guinea pig. Arthritis Rheum 1990;33: 1815--1821. Itoh Y, Reichlin M. Ro/SS-A antigen in human platelets. Different distributions of the isoforms of Ro/SSA protein and the Ro/SS-A-binding RNAs. Arthritis Rheum 1991;34:888-893. Itoh Y, Reichlin M. Autoantibodies to the Ro/SSA autoantigen are conformation dependent. I. Anti-60 kD antibodies are mainly directed to the native protein; anti-52 kD antibodies are mainly directed to the denatured protein. Autoimmunity 1992;14:57--65. James JA, Dickey WD, Fujisaku A, O'Brien CA, Deutscher SL, Keene JD, Harley JB. Antigenicity of a recombinant Ro (SSA) fusion protein. Arthritis Rheum 1990;33:102--106. Lerner MR, Boyle JA, Hardin JA, Steitz JA. Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus. Science 1981 ;211:400-402. Maddison PJ, Reichlin M. Deposition of antibodies to a soluble cytoplasmic antigen in the kidneys of patients with systemic lupus erythematosus. Arthritis Rheum 1979;22:8. Manoussakis MN, Kistis KG, Liu X, Aidiuis V, Guialis A, Moutsopolous AM. Detection of anti-Ro(SSA) antibodies in autoimmune diseases: comparison of five methods. Br J Rheumatol 1993;32:449--455. McCauliffe DP, Yin H, Wang LX, Lucas L. Autoimmune sera react with multiple epitopes on recombinant 52 and 60 kDa Ro(SSA) proteins. J Rheumatol 1994;21:1073-1080. Peek R, Pruijn GJ, van Venrooij WJ. Epitope specificity determines the ability of anti-Ro52 autoantibodies to precipitate Ro ribonucleoprotein particles. J Immunol 1994;153: 4321-4329. Penner E, Reichlin M. Primary biliary cirrhosis associated with

Alspaugh MA, Tan EM. Antibodies to cellular antigens in Sj6gren's syndrome. J Clin Invest 1975;55:1067--1073. Alspaugh MA, Maddison PJ. Resolution of the identity of certain antigen-antibody systems in systemic lupus erythematosus and Sj6gren's syndrome: an interlaboratory collaboration. Arthritis Rheum 1979;22:796--798. Alexander E, Buyon JP, Provost TT, Gaurneri T. Anti-Ro/SSA antibodies in the pathophysiology of congenital heart block in neonatal lupus syndrome, an experimental model. In vitro electrophysiologic and immunocytochemical studies. Arthritis Rheum 1992;35:176-189. Anderson JR, Gray KG, Beck JS, Buchanan WU, McElhinney AJ, Precipitating antibodies in the connective tissue diseases. Ann Rheum Dis 1962;21:360-369. Arnett FC, Hamilton RG, Reveille JD, Bias WB, Harley JB, Reichlin M. Genetic studies of Ro (SS-A) and La (SS-B) autoantibodies in families with systemic lupus erythematosus and primary Sj6gren's syndrome. Arthritis Rheum 1989;32: 413--419. Ben-Chetrit E, Chan EK, Sullivan KF, Tan EM. A 52-kD protein is a novel component of the SS-A/Ro antigenic particle. J Exp Med 1988;167:1560-1571. Blange I, Ringertz NR, Pettersson I. Identification of antigenic regions of the human 52 kD Ro/SS-A protein recognized by patient sera. J Autoimmun 1994;7:263--274. Boire G, Lopez-Longo FJ, Lapointe S, Menard HA. Sera from patients with autoimmune disease recognize conformational determinants on the 60-kd Ro/SSA protein. Arthritis Rheum 1991:34:722--730. Clark G, Reichlin M, Tomasi TB Jr. Characterization of a soluble cytoplasmic antigen reactive with sera from patients with systemic lupus erythematosus. J Immunol 1969;102: 117--122. Frank MB, McArthur R, Harley JB, Fujisaku A. Anti-Ro (SSA) autoantibodies are associated with T cell receptor beta genes in systemic lupus erythematosus patients. J Clin Invest 1990;85:33--39. Frank MB, Itoh K, McCubbin V. Epitope mapping of the 52kD Ro/SSA autoantigen. Clin Exp Immunol 1994;95:390396. Fujisaku A, Frank MB, Neas B, Reichlin M, Harley JB. HLADQ gene complement and other histocompatibility relationships in man with the anti-Ro/SSA autoantibody response in systemic lupus erythematosus. J Clin Invest 1990;86:606611. Furukawa F, Kaslinhara-Sawami M, Lyons MB, Norris DA. Binding of antibodies to the extractable nuclear antigens SSA/Ro and SS-B/La is induced on the surface of human keratinocytes by ultraviolet (UVL): implications for the pathogenesis of photosensitive cutaneous lupus. J Invest Dermatol 1990;94:77--85.

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Sj6gren's syndrome: evidence for circulating and tissue deposited Ro/anti-Ro immune complexes. Arthritis Rheum 1982;25:1250-- 1253. Rader MD, O'Brien C, Liu YS, Harley JB, Reichlin M. Heterogeneity of the Ro/SSA antigen. Different molecular forms in lymphocytes and red blood cells. J Clin Invest 1989;83:1293--1298. Reichlin M, Rader MD, Harley JB. The autoimmune response to the Ro/SSA particle is directed to the human antigen. Clin Exp Immunol 1989;76:373--377. Reichlin M. Antibodies to ribonuclear proteins in systemic lupus erythematosus. In: McCune WJ, ed. Rheumatic Disease Clinics of North America. Philadelphia: W.B. Saunders Co., 1994;20:29-43. Reichlin M, Brucato A, Frank MB, Maddison PJ, McCubbin VR, Wolfson-Reichlin M, Lee LA. Concentration of autoantibodies to native 60-kd Ro/SS-A and 52-kd Ro/SS-A in eluates from the heart of a child who died with congenital complete heart block. Arthritis Rheum 1994;37:1698--1703. Reveille JD, MacLeod MJ, Whittington K, Arnett FC. Specific amino acid residues in the second hypervariable region of HLA-DQA 1 and DQB 1 chain genes promote the Ro (SSA)/La (SS-B) autoantibody responses. J Immunol 1991;146: 3871--3876. Ricchiuti V, Isenberg D, Muller S. HLA associations of antiRo60 and anti-Ro52 I antibodies in Sj6gren's syndrome. J Autoimmun 1994;7:611-621. Routsias JG, Sakarellos-Daitsiotis M, Detsikas E, Tzioufas AG, Sakarellos C, Moutsopoulus HM. Antibodies to EYRKK vesicular stomatitis virus-related peptide account only for a minority of anti-Ro 60kD antibodies. Clin Exp Immunol 1994 ;98:414--418. Saitta MR, Arnett FC, Keene JD. 60-kDa Ro protein autoepitopes identified using recombinant polypeptides. J Immunol 1994;152:4192-4202.

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Scofield RH, Harley JB. Auto~intigenicity of Ro/SSA antigen is related to a nucleocapsid protein of vesicular stomatitis virus. Proc Natl Acad Sci USA 1991;88:3343--3347. Scofield RH, Dickey WD, Jackson KW, James JA, Harley JB. A common autoepitope near the carboxyl terminus of the 60kD Ro ribonucleoprotein: sequence similarity with a viral protein. J Clin Immunol 1991;11:378-388. Scofield RH, Harley JB. Association of anti-Ro/SS-A autoantibodies with glutamine in position 34 of DQA1 and leucine in position 26 of DQB 1. Arthritis Rheum 1994;37:961--962. Scofield RH, Frank MB, Neas BR, Horowitz RM, Hardgrave KL, Fujisaku A, McArthur R, Harley JB. Cooperative association of T cell 13 receptor and HLA~-DQ alleles in the production of anti-Ro in systemic lupus erythematosus. Clin Immunol Immunopathol 1994;72:335-341. Scofield RH, Dickey WD, Hardgrave KL, Neas BR, Horowitz RM, McArthur RA, Fujisaku A, Frank MB, Harley JB. Immunogenetics of epitopes of the carboxyl terminus of the human 60-kD Ro autoantigen. Clin Exp Immunol 1995;99: 256--261. Topfer F, Gordon T, McCluskey J. Intra and intermolecular spreading of autoimmunity involving the nuclear self-antigens La(SS-B) and Ro(SS-A). Proc Natl Acad Sci USA 1995;92: 875--879. Venables PJ, Smith PR, Maini RN. Purification and characterization of the Sj6gren's syndrome A and B antigens. Clin Exp Immunol 1983;54:731--738. Wolin SL, Steitz JA. The Ro small cytoplasmic ribonucleoproteins: identification of the antigenic protein and its binding site on the Ro RNAs. Proc Natl Acad Sci USA 1984;81: 1996--2000. Yamagata H, Harley JB, Reichlin M. Molecular properties of the Ro/SSA antigen and enzyme-linked immunosorbent assay for quantitation of antibody. J Clin Invest 1984;74:625--633.

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

SS-B (La) AUTOANTIBODIES Catherine L. Keech, B.Sc., James McCluskey, M.D. and Tom P. Gordon, Ph.D.

Department of Clinical Immunology and Centre for Transfusion Medicine and Immunology, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia

HISTORICAL NOTES Precipitating autoantibodies designated anti-SJD and anti-SJT were reported in the sera of patients with Sj6gren' s syndrome in 1961 (Anderson et al., 1961). Two precipitin reactions in SLE sera were designated Ro and La based on the names of the patients in whom they were first identified (Clark et al., 1969). The anti-La precipitin was shown later to be identical to the anti-SS-B precipitin reported in sera from patients with Sj6gren's syndrome (Alspaugh and Tan, 1975). Although never confirmed by serum exchange, SJT is assumedto be identical to La and SS-B.

THE LA(SS-B) AUTOANTIGEN Definition The La molecule is a ubiquitously expressed phosphoprotein (Mr 47 kd) that associates with a variety of small RNAs including the precursors of cellular 5S RNA and tRNA, 7S RNA and the Ro cytoplasmic hY RNAs as well as some viral RNAs (Rinke and Steitz, 1982). The La protein binds to a short sequence of uridylate residues at the 3' termini of these RNAs via an 80 amino acid domain shared by many RNAbinding proteins and referred to as an RNA recognition motif (RRM) which contains ribonuc!eoprotein (RNP) 1 and RNP 2 consensus sequences (Pruijn et al., 1990). La, which probably is a transcription termination factor for RNA polymerase III (Gottlieb and Steitz, 1989), has ATPase activity capable of melting DNA/RNA hybrids in vitro (Bachmann et al., 1990). A potential ATP-binding motif may be involved in the ATP-dependent activity of melting

RNA/DNA hybrids (Topfer et al., 1993). Shuttling of La between the nucleus and cytoplasm may reflect the role for this protein in the maturation and/or transport of some of these cellular RNAs (Bachmann, 1989); a nuclear localization signal is present at the C terminus of the L protein (Pruijn, 1994) (Figure l b). Some La molecules exist as part of RNP particles containing the 60 kd Ro protein and the small hY RNAs (Figure la); however, the nature and function of these particles is not completely clear.

Origin/Sources The intracellular localization of La is predominantly nuclear by immunofluorescence (Figure 2a), but cytoplasmic localization is evident from the association of La with hY RNAs. Surface membrane expression of the La molecule and translocation of La to membrane surfaces follow ultraviolet irradiation, viral infection and serum starvation (Venables and Brookes, 1992). Transfection of the human La gene into a cultured human cell line reveals predominantly nuclear localization (Figure 2b). In murine cell lines transfected with a human La gene, the human La protein associates intracellularly with a mouse 60 kd Ro and the same RNAs as the mouse La, indicating that the human molecule is functionally conserved across species (Keech et al., 1993).

Methods of Purification Native La protein can be purified on poly(U) Sepharose (Stefano, 1984) and by immunoaffinity chromatography with monospecific antibodies against La antigen (Bachmann et al., 1986). Production of soluble recombinant La protein in milligram quantities in

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Figure 1. a: A model of the Ro/La ribonucleoprotein complex. La interacts via its RNA recognition motif with the poly-U tail of hY RNAs. Ro60 associates with the stem region of the hY RNAs (adapted from Slobbe et al., 1991). b: Major structural features and immunodominant epitopes of the La(SS-B) autoantigen. Discontinuous epitopes are located at the NH2-terminus (LaA) and RNA recognition motif (RRM) (LaC1) which contains consensus sequences for RNP1 and RNP2 RNA-binding protein. Precipitin-positive anti-La sera react with LaA, LaC 1 and the COOH-terminal epitope (LaL2/3), but the LaL2/3 epitope is not bound by precipitin-negative anti-La sera. A putative ATP binding site and a nuclear localization signal are located in the COOH-terminal region.

several bacterial expression systems utilizes the pGEX and pQE vectors (Topfer et al., 1993; McNeilage et al., 1990). The pGEX produces a glutathione-Stransferase fusion protein which can be purified on a glutathione affinity column, and in pQE a six-histidine fusion protein is produced allowing affinity purification by nickel ion chromatography. Both recombinant human and mouse La bind to poly(U) agarose (Topfer

790

et al., 1993). The functional integrity of recombinant La is shown by the ability of radiolabeled La translated in vitro to bind hY RNAs (Pruijn et al., 1991). Sequence Information

Complementary DNA clones encode the full-length La protein (Chambers et al., 1988; Chan et al., 1989)

Figure 2. Indirect immunofluorescence staining of a HEp-2 cells with a La-specific monoclonal antibody (A2, Chan, 1987) reveals a speckled nuclear distribution of La. Transfection and overexpression of La in these cells b results in intense nuclear staining with this monoclonal antibody. Original magnification x400. composed of 408 amino acids with a predicted molecular weight of 47 kd and the position of the RRM, putative ATP-binding site and nuclear localization signal (Figure lb). Comparison of sequences among human, mouse (Topfer et al., 1993), bovine (Chan et al., 1989) and rat (Semsei et al., 1993) reveals that La is well conserved during evolution. The human La gene encompasses 11 exons with a putative G/C-rich promoter upstream of the mRNA start site (Chambers et al., 1988). An alternative mRNA transcript encoding human La differs from the usual La mRNA due to an exchange of the first exon (Troster et al., 1994).

Commercial Sources Recombinant La is available commercially from AMRAD, Melbourne Australia. Purified native La is obtainable from Immuno Concepts, Sacramento, USA; Immunovision, Arkansas, USA; Shield Diagnostics, Dundee, UK and Immunodiagnostic Systems, Boldon, UK. Identification of autoepitopes on La is facilitated by the solubility and easy purification of the recombinant La fragments. Detailed B-cell epitope mapping by several groups (St. Clair, 1992) reveals immunodominant conformational epitopes located at the NH-2 terminus (McNeilage et al., 1992) and within the RRM (Rischmueller et al., 1995) and a third immunodominant epitope at the COOH-terminus (Figure 2b). The B-cell response to La is directed mainly against conformational epitopes; linear immunodominant epitopes are not convincingly demonstrated.

The majority of anti-La sera which produce precipitins on immunodiffusion react with the three major epitopes (Figure 2b), but nonprecipitating anti-La sera have a more restricted epitope recognition, reacting preferentially with the intact La protein and the NH-2 terminal epitope but never with the COOH-terminal (Gordon et al., 1992).

THE AUTOANTIBODY

Pathogenetic Role Human Disease. There is no direct evidence implicating a pathogenic role for anti-La in primary Sj6gren's syndrome (SS) or systemic lupus erythematosus (SLE). Anti-La are associated with particular clinical findings in these autoimmune diseases but because anti-La are invariably accompanied by anti-Ro, it is difficult to determine whether one or a combination of these autoantibodies is actually pathogenic. Patients with SS and anti-La tend to have more extraglandular disease such as cutaneous and vasculitic and hematologic cytopenias (Harley, 1989). Furthermore, serum anti-La activity correlates with the degree of salivary gland lymphocytic implying a possible role for anti-La in salivary pathology in SS (Atkinson et al., 1992). Additional indirect evidence for a pathogenic role of anti-La comes from the strong association of maternal anti-La (found in over 90% of mothers of these infants) and infants with neonatal lupus erythematosus 9(NLE) and congenital heart block (CHB) (Buyon and

791

Winchester, 1990). Both the La and Ro antigens are present on the surface of the fibers of an affected heart, suggesting that the heart block may be mediated by maternal anti-La antibodies binding to the surface of cardiac muscle cells (Horsfall et al., 1991).

Animal Models. Anti-La precipitins have not been identified in the sera of autoimmune strains of mice including MRL, NZB and DXSB mice (Treadwell et al., 1993), but anti-La reactivity by ELISA was found in MRL/lpr mice that spontaneously develop SLE. However, the fine specificity of the anti-La in MRL/ lpr sera differed from that of human anti-La, suggesting alternative mechanisms of autoantibody induction in the autoimmune mice (St. Clair, 1992). Anti-La can be induced by immunizing animals with native and recombinant heterologous and autologous La protein (St. Clair, 1992). Generally speaking, both monoclonal and polyclonal anti-La have different epitope profiles compared with human anti-La. AntiLa can also be produced in mice by immunization with a human monoclonal anti-DNA antibody bearing the 16/6 idiotype or with a mouse monoclonal antiidiotypic antibody specific for this idiotype. Furthermore, immunization with a monoclonal anti-La with similar specificity to human anti-La yields a SLE-like illness in mice, suggesting a role for anti-La in the induction of autoimmunity (Fricke et al., 1990). Immunization of mice with recombinant mouse La breaks tolerance to La in the B-cell compartment with subsequent intramolecular spreading of the autoimmune response to involve multiple regions of the La molecule (Topfer et al., 1995). Furthermore, intermolecular spreading of autoimmunity rapidly develops to include the 60 kd Ro component of the La/Ro RNP particle. This observation is consistent with a model of combined anti-La/Ro antibody production developing in La-immunized mice in which B cells of different anti-La and anti-Ro specificities can internalize La/Ro RNP complexes and present La determinants to primed La-specific CD4 + T cells (Topfer et al., 1995). In this model, stimulation of both La and Ro-specific B cells requires cognate T-helper cells recognizing processed antigen from only one component of the RNP but resulting in production of both sets of autoantibodies (Figure 3). Genetics The association of anti-La and anti-Ro with certain MHC class II alleles suggests that the production of

792

these autoantibodies is dependent on MHC-peptide interactions with T lymphocytes. Anti-La accompanied by anti-Ro are associated with serologically defined HLA-DR3, DQw2 haplotypes; whereas, anti-Ro without anti-La is associated with DR2, DQwl haplotypes. Higher levels of anti-La and anti-Ro occur in HLA-DQwl/DQw2 heterozygotes. Recent studies using RFLP analysis and oligonucleotide typing reveal that the highest relative risk for these autoantibodies is conferred by HLA/DQw2.1 (in linkage disequilibrium with HLA-DR3) and DQw6 which is a subtype of DQwl. Sequencing of the relevant DQA1 and DQB 1 alleles shows specific amino acid residues in both the ~ and 13 chains of DQ which correlate with the La/Ro autoantibody responses. These residues (a glutamine residue at position 34 of the DQA1 chain and a leucine residue at position 26 of the DQB1 chain) are located in the antigen binding cleft of the HLA molecule, raising the possibility that residues 34 and 26 are involved in the preferential presentation of La or Ro peptides to autoreactive T cells (Reveille and Arnett, 1992). Sera of healthy relatives of patients with SS often contain antinuclear antibodies. Family studies of SS and SLE patients reveal anti-La measured by ELISA in relatives with SS or SLE; whereas, anti-Ro occur more frequently both in relatives with autoimmune disease and in healthy relatives (Arnett et al., 1989). Genetic factors clearly play a significant role in the production of anti-La/Ro antibodies, but in the absence of twin studies, it is not possible to rule out shared environmental factors (Reveille and Arnett, 1992).

Factors in Pathogenicity Isotypes, Subclasses and Idiotypes. Serum anti-La are predominantly of the IgG isotype, although enrichment of IgA anti-La is reported in saliva (Horsfall et al., 1989). The kappa/lambda ratio determined following an anti-La ELISA showed strong skewing toward the use of kappa light chains for anti-La and serum electrophoresis revealed an oligoclonal anti-La response (Meilof et al., 1992). The IgG subclass distribution of anti-La shows a restriction primarily to the IgG1 subclass (Meilof et al., 1992), although another study showed more variability in the anti-La subclass distribution (Maran et al., 1993). These studies provide further evidence that La is a T-celldependent antigen in which isotype switching of antiLa antibodies may be driven by CD4 + T cells of the

Figure 3. Model of linked anti-La and anti-Ro autoantibody production. Following immunization with La protein, La-specific CD4+ T cells are activated by La peptide-MHC class II complexes on antigen-presenting cells (APC). La-specific B cells capturing La/Ro RNPs can present La determinants to the primed CD4+T cells leadingto production of anti-La. Similarly, cognate interactions between Ro-specific B cells (which can present La peptide-MHC class II complexes) and La-specific CD4+T cells can potentially lead to antiRo production.

TH2 subtype. An indirect ELISA using immobilized rabbit antiidiotype antibodies raised in rabbits by immunizing with affinity-purified IgG reveals cross-reactive idiotypes present on IgG in sera containing anti-Ro/La and anti-Ro antibodies (Horsfall et al., 1989).

Autoepitopes on La. The majority of precipitating anti-La autoantisera react with conformational epitopes spread throughout the La molecule. Because intramolecular spreading of the B-cell response follows initiation of immunity to a single La/Ro RNP component (Topfer et al., 1995), epitope mapping data from patients with established autoantibody responses are generally unhelpful in elucidating the initial events which initiated autoimmunity. However, the anti-La response appears to be more restricted in patients with anti-La precipitin-negative sera (Gordon et al., 1992) and may represent an early autoantibody response to dominant conformational determinants. In the subset

of patients with nonprecipitating anti-La, the response to La appears arrested at this early stage and does not spread to other regions of the La molecule. Similarly, studies of serial serum samples suggest that the antiLa response is initially directed against the NH2terminal epitope but extends over time to involve the middle and COOH-terminal regions of the molecule (McNeilage et al., 1990). The subclass restriction, MHC class II allele associations and polyclonality of the anti-La response, together with the targeting of epitopes present on the native La/Ro RNP particle (Rischmueller et al., 1995) provides strong evidence for the hypothesis that the response to La is generated by self-immunization. More direct evidence for this model is provided by the identification of humanspecific epitopes within the RRM and COOH-terminal fragments of La (Kohsaka et al., 1990).

Molecular Mimicry and Polyclonal Activation. Although data from epitope mapping studies indicate

793

that the mature anti-La response is largely self antigen-driven, they do not rule out a temporal model whereby molecular mimicry might initiate the immune response with subsequent autoimmunization. Indeed, a homology between amino acids 88--101 in the NH2 terminal region of La and a feline retroviral gag polypeptide was identified by sequence analysis (Kohsaka et al., 1990), but a lack of reactivity of antiLa with this sequence argues against molecular mimicry for this region at the B-cell level (McNeilage et al., 1992). Nevertheless, linear regions of sequence similarity between self and viral antigens may be important in initiating autoimmunity in the T-cell compartment where activation of specific T cells might then direct spreading of the response throughout the B-cell compartment as shown in the model for intermolecular spreading (Figure 3). Anti-La are generally associated with hypergammaglobulinemia and the anti-La often contribute to the elevated serum IgG concentrations. Anti-La are not, however, merely a consequence of polyclonal activation, because anti-La levels do not correlate with total serum IgG concentrations and fluctuations in anti-La do not parallel changes in serum IgG levels (Gordon et al., 1991).

Methods of Detection Anti-La antibodies were originally detected by double immunodiffusion and then by counterimmunoelectrophoresis (CIE) (Nakamura et al., 1985). With the advent of immunoblotting and ELISA, the relative insensitivity of immunodiffusion for detection of antiLa (Meilof, 1992) became clear. Thus, approximately 30% of sera which show Ro precipitins alone on CIE are positive on a recombinant La ELISA (Gordon et al., 1992) and immunoblotting increases the sensitivity of detection from 75% on immunodiffusion to 94% (van Venrooij et al., 1991), i.e., comparable to that of RNA-precipitation and 35S-methionine radiolabeling immunoprecipitation assays. Recombinant La protein or affinity-purified native La protein can be used as an antigen source for ELISA. Recombinant La has the advantages of monospecificity and ease of purification when expressed as a protein fused to glutathione-S-transferase or six histidines. Sera should be absorbed with bacterial lysate prior to recombinant La ELISA to avoid background reactions with contaminating bacterial protein. The purity of affinity-purified native La preparations will depend on the specificity of the

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antibody used for purification and co-purification of Ro proteins may occur if Ro/La RNP complexes are present in the antigen preparation (Meilof, 1992). There is no role for synthetic La peptides in detection of anti-La because of the lack of immunodominant linear epitopes on the La molecule. Immunoblotting of recombinant La protein or whole cell extract is the most sensitive and specific method for the detection of anti-La but not practical for many diagnostic laboratories. ELISA is technically simpler and of similar sensitivity to immunoblotting. However, the significance of a borderline positive result on ELISA remains unclear and its specificity for anti-La should be confirmed by immunoblotting. Although CIE is a sensitive assay for detection of anti-Ro, false-negative anti-La results are common on CIE because of the existence of nonprecipitating antiLa. By definition this population of autoantibodies can be detected only by immunoblotting or ELISA.

CLINICAL UTILITY

Frequencies in Disease The reported frequencies of anti-La in clinical syndromes will depend on the methods of detection and the referral bias of the center performing the study. Although reported in SS, SLE and in asymptomatic individuals, anti-La remain the serologic hallmark of Sj6gren's syndrome, with early estimates of their frequency ranging from 10 to 40% for primary Sj6gren's syndrome and 5--20% for secondary Sj6gren's syndrome using immunodiffusion assays. These frequencies are considerably higher when anti-La are measured by ELISA or immunoblotting (up to 90% for primary and 50% for secondary Sj6gren's syndrome (Harley, 1989). Reported in 6--15% of sera from SLE, anti-La precipitins are associated with a lower prevalence of renal disease and anti-DNA in these patients (Reichlin, 1986; Venables et al., 1989). Although better known for their association with antiRo, 25--35% of subacute cutaneous lupus erythematosus patients also have anti-La (Sontheimer and McCauliffe, 1990). Anti-La are a criterion for the classification of Sj6gren's syndrome. The association of antibodies to La or Ro with symptoms of dry eyes, xerostomia and a positive Rose bengal staining or Schirmer test, has a sensitivity and specificity of 94% for primary SS (Vitali et al., 1993). In another study, 83% of patients

with anti-La and anti-Ro precipitins fulfilled criteria for SS compared with 42% of those with anti-Ro precipitins alone, confirming the high diagnostic specificity of anti-La for SS (Venables et al., 1989).

Disease Associations Anti-La in SS are associated with a higher frequency of palpable purpura, leukopenia, lymphopenia and hypergammaglobulinemia (Harley, 1989) and possibly with more severe glandular involvement (Atkinson et al., 1992). Anti-La precipitins have rarely been reported in asymptomatic individuals and in patients who do not fulfill sufficient diagnostic criteria for Sj6gren's syndrome. Many patients with anti-La and no clinical features of SS will eventually develop sicca symptoms over time (Venables et al., 1989; Isenberg et al., 1982). Neonatal lupus erythematosus (NLE) is an autoimmune disease characterized by cutaneous lupus lesions resembling subacute cutaneous lupus erythematosus, CHB (approximately 50% of infants), or both. Although a strong association of NLE with antiRo was recognized first, the majority (around 90%) of mothers of babies with NLE are now known to have serum anti-La antibodies as well. Indeed, maternal anti-La are the antibody species most strongly associated with affected offspring, particularly when combined with anti-RoS2 antibodies (Buyon, 1992). Presumably, these autoantibodies are not sufficient to induce NLE or CHB because only a minority of offspring of mothers with anti-Ro or anti-La develop this syndrome.

Antibody Correlation with Disease Activity Whether the titer of anti-La correlates with disease activity in Sj6gren's syndrome or SLE is unknown. Detection p e r s e of anti-La precipitins is a stable serological finding which does not fluctuate during the course of disease. Likewise, patients whose sera

REFERENCES Alspaugh MA, Tan EM. Antibodies to cellular antigens in Sj6gren's syndrome. J Clin Invest 1975;55:1067-1073. Anderson JR, Gray KG, Beck JS. Precipitating autoantibodies in Sj6gren's syndrome. Lancet 1961;2:456-460. Arnett FC, Hamilton RG, Reveille JD, Bias WB, Harley JB, Reichlin M. Genetic studies of Ro (SS-A) and La (SS-B) autoantibodies in families with systemic lupus erythematosus

contain nonprecipitating anti-La do not appear to develop anti-La precipitins despite follow-up over several years (T. Gordon, unpublished observation). Measurement of sequential serum anti-La activities by ELISA in a small number of patients with Sj6gren's syndrome and SLE shoWs that the antibody responses to different antigenic La fragments vary in parallel over time and are independent of other autoantibodies such as anti-ssDNA. The fall in anti-La levels observed in some patients receiving immunosuppressive therapy did not permit any firm conclusions regarding the role of prednisolone and cytotoxic drugs on antiLa responses (St. Clair et al., 1990). Another study showed considerable variation in binding of antibody to different La epitopes, but the different patterns were not correlated with specific clinical manifestations of Sj6gren's syndrome (St. Clair et al., 1989).

CONCLUSION SS-B (La) is one of the best characterized nuclear autoantigens with respect to elucidation of its function and the mapping of B,cell autoepitopes. Anti-La are the serological hallmark of SS but their pathogenic role remains uncertain and a small proportion of SS patients remain anti-La negative. Recent studies are shedding light on the extent of immunological tolerance to La and other sequestered autoantigens and the mechanism of spreading of the autoimmune response to different components of the La/Ro RNP. The production of anti-La is driven largely by self-immunization and on preliminary evidence is most likely to be orchestrated by autoreactive T cells; however, the initial events which lead to initiation of autoimmunity to La are unknown. An important area for future research on La will be to identify the T-cell determinants on this molecule which are involved in the activation of La-specific T cells and subsequent generation of anti-La in both animal models and humans. See also SS-A (Ro) AUTOANTIBODIES.

and primary Sj6gren's Syndrome. Arthritis Rheum 1989;32: 413-419, Atkinson JC, Travis WD, Slocum L, Ebbs WL, Fox PC. Serum anti-SS-B/La and IgA rheumatoid factor are markers of salivary gland disease activity in primary Sj6gren's syndrome. Arthritis Rheum 1992;35:1368-1372. Bachmann M. The La antigen shuttles between the nucleus and the cytoplasm in CV-1 cells. Mol Cell Biochem 1989;85: 103-114.

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Bachmann M, Mayet WJ, Schroder HC, Pfeifer K, Meyer zum Btischenfelde KH, Muller WE. Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells. Proc Natl Acad Sci USA 1986;83:7770--7774. Bachmann M, Pfeifer K, Schroder HC, Muller WE. Characterization of the autoantigen La as a nucleic acid-dependent ATPase/dATPase with melting properties. Cell 1990;60:8593. Buyon JP, Winchester R. Congenital complete heart block. A human model of passively acquired autoimmune injury. Arthritis Rheum 1990;33:609--614. Buyon JP. Neonatal lupus syndromes. Am J Reprod Immunol 1992;28:259-263. Chambers JC, Kenan D, Martin BJ, Keene JD. Genomic structure and amino acid sequence domains of the human La autoantigen. J Biol Chem 1988;263:18043--18051. Chan EK, Sullivan KF, Tan EM. Ribonucleoprotein SS-B/La belongs to a protein family with consensus sequences for RNA-binding. Nucleic Acids Res 1989;17:2233--2244. Clark G, Reichlin M, Tomasi TB Jr. Characterization of a soluble cytoplasmic antigen reactive with sera from patients with systemic lupus erythematosus. J Immunol 1969;102: 117-122. Fricke H, Often D, Mendlouic S, Schoenfeld Y, Bakimer R, Sperling J, Mozes E. Induction of experimental systemic lupus erythematosus in mice by immunization with a monoclonal anti-La autoantibody. Int Immunol 1990;2:225--230. Gordon TP, Greer M, Reynolds P, Guidolin A, McNeilage LJ. Estimation of amounts of anti-La(SS-B) antibody directed against immunodominant epitopes of the La(SS-B) autoantigen. Clin Exp Immunol 1991;85:402-406. Gordon T, Mavrangelos C, McCluskey J. Restricted epitope recognition by precipitin-negative anti-La/SS-B-positive sera. Arthritis Rheum 1992;35:663--666. Gottlieb E, Steitz JA. Function of mammalian La protein: evidence for its action in transcription termination by RNA polymerase III. EMBO J 1989;8:851-861. Harley JB. Autoantibodies in Sj~3gren's syndrome. J Autoimmun 1989;2:283--394. Horsfall AC, Rose LM, Maini RN. Autoantibody synthesis in salivary glands of Sj6gren' s syndrome patients. J Autoimmun 1989;2:559-568. Horsfall AC, Venables PJ, Taylor PV, Maini RN. Ro and La antigens and maternal anti-La idiotype on the surface of myocardial fibres in congenital heart block. J Autoimmun 1991;4:165--176. Isenberg DA, Hammond L, Fisher C, Griffiths M, Stewart J, Bottazzo GF. Predictive value of SS-B precipitating antibodies in SjOgren's syndrome. Br Med J (Clin Res Ed) 1982;284:1738--1740. Keech CL, Gordon TP, Reynolds P, McCluskey J. Expression and functional conservation of the human La(SS-B) autoantigen in murine cell lines. J Autoimmun 1993;6:543-555. Kohsaka H, Yamamoto K, Fujii H, Miura H, Miyasaka N, Nishioka K, Miyamoto T. Fine epitope mapping of the human SS-B/La protein. Identification of a distinct autoepitope homologous to a viral gag polyprotein. J Clin Invest 1990;85:1566--1574.

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Maran R, Dueymes M, Pennec Y-L, Cesburn-Budd R, Shoenfeld Y, Youinou P. Predominance of IgG1 subclass of antiRo/SS-A, but not anti-La/SS-B antibodies in primary Sj~3gren's syndrome. J Autoimmun 1993;6:379--387. McNeilage LJ, MacMillan EM, Whittingham SF. Mapping of epitopes on the La(SS-B) autoantigen of primary Sj6gren's syndrome: identification of a cross-reactive epitope. J Immunol 1990;145:3829-3835. McNeilage LJ, Umapathysivam K, Macmillan E, Guidolin A, Whittingham S, Gordon T. Definition of a discontinuous immunodominant epitope at the NH2 terminus of the La/SSB ribonucleoprotein autoantigen. J Clin Invest 1992;89: 1652-1656. Meilof JF. Autoantibodies against small cytoplasmic ribonucleoproteins: the anti-Ro/SS-A and anti-La/SS-B autoimmune response. A review of autoantibody detection, autoantigen composition, autoantibody-disease associations and possible etiologic mechanisms. Rheumatol Int 1992;12:129--140. Meilof JF, Hebeda KM, de Jong J, Smeenk RJ. Analysis of heavy and light chain use of lupus-associated anti-La/SS-B and anti-Sm autoantibodies reveals two distinct underlying immunoregulatory mechanisms. Res Immunol 1992;143: 711--720. Nakamura RM, Peebles CL, Rubin RL, Malden DP, Tan EM. Autoantibodies to nuclear antigens (ANA). 2nd edition. Chicago American Society of Clinical Pathologists Press, 1985. Pruijn GJ. The La(SS-B) antigen. In: van Venrooij WJ, Maini RN, eds. Manual of Biological Markers of Disease, Section B4.2. Dordrecht: Kluwer Academic Publishers, 1994:1-- 14. Pruijn GJ, Slobbe RL, vanVenrooij WJ. Analysis of proteinRNA interactions within Ro ribonucleoprotein complexes. Nucleic Acids Res 1991;19:5173--5180. Pruijn GJM, Slobbe R, vanVenrooij WJ. Structure and function of La and Ro RNPs. Mol Biol Rep 1990;14:43--48. Reichlin M. Significance of the Ro antigen system. J Clin Immunol 1986;6:339-348. Reveille JD, Arnett FC. The immunogenetics of Sj~3gren's syndrome. Rheum Dis Clin North Am 1992;18:539--550. Rinke J, Steitz JA. Precursor molecules of both human 5S ribosomal RNA and transfer RNAs are bound by a cellular protein reactive with anti-La lupus antibodies. Cell 1982;29: 149--159. Rischmueller M, McNeilage LJ, McCluskey J, Gordon T. Human autoantibodies directed against the RNA recognition motif of La(SS-B) bind to a conformational epitope present on the intact La(SS-B) ribonucleoprotein particle. Clin Exp Immunol 1995;101:39-44. Semsei I, Troster H, Bartsch H, Schwemmle M, Igloi GL, Bachmann M. Isolation of rat cDNA clones coding for the autoantigen SS-B/La detection of species-specific variations. Gene 1993;126:265-268. Sontheimer RD, McCauliffe DP. Pathogenesis of anti-Ro/SS-A autoantibody-associated cutaneous lupus erythematosus. Dermatol Clin 1990;8:751--758. St. Clair EW. Anti-La antibodies. Rheum Dis Clin North Am 1992;18:359-377. St. Clair EW, Burch JA Jr, Ward MM, Keene JD, Pisetsky DS.

Temporal correlation of antibody responses to different epitopes of the human La autoantigen. J Clin Invest 1990; 85:515-521. St. Clair EW, Talal N, Moutsopoulos HM, Ballester A, Zerva L, Keene JD, Pisetsky DS. Epitope specificity of anti-La antibodies from patients with Sj6gren's syndrome. J Autoimmun 1989;2:335-344. Stefano JE. Purified lupus antigen La recognizes and oligouridylate stretch common to the 3' termini of RNA polymerase III transcripts. Cell 1984;36:145-154. Topfer F, Gordon T, McCluskey J. Characterisation of the mouse autoantigen La(SS-B): identification of conserved RNA-binding motifs, a putative ATP binding site and reactivity of recombinant protein with poly(U) and human autoantibodies. J Immunol 1993; 150:3091-3100. Topfer F, Gordon T, McCluskey J. Intra- and intermolecular spreading of autoimmunity involving the nuclear self-antigens La(SS-B) and Ro(SS-A). Proc Natl Acad Sci USA 1995;92: 875-879. Treadwell EL, Cohen P, Williams D, O'Brien K, Volkman A, Eisenberg R. MRL mice produce anti-Su autoantibody, a specificity associated with systemic lupus erythematosus. J Immunol 1993;150:695-699. Troster H, Metzger TE, Semsei I, Winterpacht A, Zabel B, Bachman M. One gene, two transcripts: isolation of an

alternative transcript encoding for the autoantigen La/SS-B from a cDNA library of a patient with primary SjOgrens syndrome. J Exp Med 1994;180:2059--2067. van Venrooij WJ, Charles P, Maini RN. The consensus workshops for the detection of autoantibodies to intracellular antigens in rheumatic diseases. J Immunol Methods 1991; 140:181-189. Venables P, Brookes S. Membrane expression of nuclear antigens: a model for autoimmunity in SjOgren's syndrome? Autoimmunity 1992;213:321--325. Venables PJ, Shattles W, Pease CT, Ellis JE, Charles PJ, Maini RN. Anti-La (SS-B): a diagnostic criterion for Sj6gren's syndrome? Clin Exp Rheumatol 1989;7:181--184. Vitali C, Bombardieri S, Moutsopoulos HM, Balestrieri G, Bencivelli W, Bernstein RM, Bjerrum KB, Braga S, Coll J, de Vita S, Drosos AA, Ehrenfeld M, Hatron PY, Hay EM, Isenberg DA, Janin A, Kalden JR, Kater L, Konttinen YT, Maddison PJ, Maini RN, Manthorpe R, Meyer O, Ostuni P, Pennec Y, Prause JU, Richards A, Sauvezie B, SchiCdt M, Sciuto M, Scully C, Shoenfeld Y, Skopouli FN, Smolen JS, Snaith ML, Tishler M, Todesco S, Valesini G, Venables PJW, Wattiaux MJ, Youinou P. Preliminary criteria for the classification of SjOgren' s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum 1993;36:340--347.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

STEROID CELL AUTOANTIBODIES A. Hoek, M.D., Ph.D. a, Nico M. Wulffraat, M.D., Ph.D. b and Hemmo A. Drexhage, M.D., Ph.D. a

aDepartment of Immunology, Erasmus University, 3000 DR Rotterdam; and bDepartment of Immunology, University Hospital for Children, 3501 CA Utrecht, The Netherlands

HISTORICAL NOTES Addison's disease is an uncommon disorder (30-60 per million) caused by a deficiency of adrenocortical hormones. The frequency is highest in the fourth decade of life and there is a female preponderance (2.5:1 male). Worldwide, Addison's disease is mostly due to infection with Mycobacterium tuberculosis or HIV; however, in developed countries, the majority of cases of idiopathic Addison's disease is now regarded as autoimmune in origin. The discovery of adrenal autoantibodies in the late 1950s initially pointed to an autoimmune origin of idiopathic Addison's disease. The first report (Anderson et al., 1957) described the presence of antibodies to a saline extract of human adrenal in two of ten patients with Addison's disease. Subsequently, these findings were confirmed in indirect immunofluorescence (IIF) using cryostat sections of human or monkey adrenal glands, (Blizzard and Kyle, 1963) and it soon became apparent that the presence of cytoplasmic adrenal antibodies was a good diagnostic tool to differentiate autoimmune Addison's disease from tuberculous adrenal insufficiency (Kamp et al., 1974). In further experiments using IIF, a proportion of cytoplasmic adrenal antibodies was found to crossreact with cytoplasmic antigens of other steroidproducing cells, including those of ovary, testis and placenta (Sotsiou et al., 1980). These cross-reacting antibodies were called steroid-cell antibodies. Except for patients who have suffered from spermatic cord torsion (Zanchetta et al., 1984), there is almost invariably an association between the presence of steroid cell antibodies and occurrence of cytoplasmic adrenal antibodies. Furthermore, absorption of steroid

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cell antibodies by adrenal homogenates confirms their cross-reactivity with adrenal cytoplasmic antigens. Originally found in two males affected by Addison's disease but without clinically overt hypogonadism, steroid cell antibodies were detected in almost all female Addisonian patients with primary amenorrhea and in around 60% of those with secondary amenorrhea (Betterle et al., 1993). A clinical association between failure of the adrenal and of the ovary is not surprising, since autoimmune Addison's disease seldom develops in isolation. Apart from the gonads, several other endocrine glands and organs can be affected in Addison's patients (Muir and MacLaren, 1991), and two major autoimmune polyglandular syndromes (APGS) are now identified. APGS type 1 (or autoimmune polyendocrinopathy candidiasis -- ectodermal dystrophy: APECED) mainly affects children (Ahonen et al., 1990); the diagnosis is made by the presence of two of the three major components (adrenal failure, mucocutaneous candidiasis and hypoparathyroidism). Ovarian failure is often present in females with APGS type 1. APGS type 2 mainly occurs in the fourth decade of life, has a female preponderance and is characterized by adrenal failure in association with hypothyroidism. In this syndrome, only a minority of women have a primary or secondary amenorrhea.

THE AUTOANTIGENS Definition Considerable progress has been made with regard to

the identification of the target antigens of cytoplasmic adrenal antibodies and possibly of steroid cell antibodies (Smith and Furmaniak, 1995). The adrenal cytochrome P450 enzyme 21 hydroxylase (21-OH, which converts 17-o~,progesterone and progesterone into 11-deoxycortisol and deoxycorticosterone), is the major autoantigen recognized by autoantibodies present in patients with Addison's disease (Winqvist et al., 1992; Baumann-Antczak et al., 1992), either in the form of isolated adrenal failure or associated with hypothyroidism (type 2 APGS). In type 1 APGS, autoantibodies can be directed to other members of the cytochrome P450 enzyme family, namely, to the P450 side-chain cleavage enzyme (P450 scc) and to 17-c~-hydroxylase (17-o~OH) (Krohn et al., 1992; Winqvist et al., 1993; Uibo et al., 1994a; 1994b), and to an ill-defined 51-kd protein (Velloso et al., 1994). However, there is some confusion and not all investigators could confirm the presence of these autoantibodies in type 1 APGS (negative results P450-scc: Song et al., 1994; 17-o~OH: Winqvist et al., 1993; Song et al., 1994). Of the steroidogenic P450 enzymes, 21-OH is adrenalspecific and 17-o~-OH is expressed in both adrenals and gonads; whereas, P450 scc is present in adrenal, gonads and placenta. The 51 kd protein is present in islets, granulosa cells and placenta. Possible targets of the steroid cell antibodies are thus 17-o~-OH and the P450-scc enzyme. However, in one patient with steroid cell antibodies, 17-c~-OH was not recognized (Winqvist et al., 1992). Studies are not available on correlations between the presence and activity of steroid cell antibodies in patients without APGS type 1 and autoantibodies to either 17-c~-OH or P450-scc. Also, studies of the adsorption of steroid cell antibodies activity with the enzymes 17-(x-OH or P450-scc are needed. Adrenal and steroid cell autoantibodies react with a major conformational epitope formed by the central and C-terminal tgarts of 21-OH (Wedlock et al., 1993). The reactivity of autoantibodies to 21-OH fragments expressed in yeast differs from that observed with fragments expressed in an in vitro transcription/translation system suggesting that the conformation of the molecule is important in autoantibody recognition (Asawa et al., 1994). Other autoantibodies to enzymes, including those to thyroid peroxidase also recognize conformational epitopes.

AUTOANTIBODIES Terminology/Methods of Detection A commonly used synonym for the term "steroid cell autoantibodies" is "autoantibodies to steroid-producing cells". The technique for detecting steroid cell antibodies is indirect immunofluorescence (IIF) with cryostat sections of human or monkey adrenal, ovary and testis. These cryostat sections are commercially available (see Figure 1).

Pathogenetic Role Human Disease. Steroid-cell autoantibodies are an indication of an existing autoimmune adrenalitis and oophoritis in females or herald such a condition (Betterle et al., 1993), but evidence for a direct pathogenic role of the antibodies in ovarian and adrenal failure is weak. Sera of patients with APGS type 1 and Addison's disease, positive for cytoplasmic adrenal autoantibodies and steroid cell autoantibodies (in high titer), are cytotoxic for cultured granulosa cells in the presence of complement (MacNatty et al., 1975). Such complement-dependent antibody cytotoxicity could be one of the immune mechanisms leading to autoimmune adrenal and ovarian failure. The antibodies may, however, also be the consequence of endocrine cellular destruction rather than their cause. This is, for instance, seen with as yet illdefined ovarian antibodies detectable in ELISA after iatrogenically induced premature ovarian failure (Wheatcroft et al., 1994). Antibody preparations from addisonian patients can inhibit the activity of 21-OH in the conversion of progesterone to deoxyprogesterone (Furmaniak et al., 1994), and likewise, antibodies to 17-~-OH and P450scc (Winqvist et al., 1993) can inhibit enzyme activity in gonadal cells. However, it remains difficult to envisage how the autoantibodies gain access to the intracytoplasmic enzymes in the living patient cells. That T cells might be involved in the ovarian and adrenal autoimmune reaction is supported by a case report on a patient with autoimmune thyroiditis, adrenalitis and secondary amenorrhea in whom T cells produced migration inhibiting factor (MIF) in response to ovarian and testicular antigens (Edmonds et al., 1973). Moreover, the marker pattern of peripheral blood T cells of such patients suggests activation (Mignot et al., 1989; Hock et al., 1995). By analogy, in insulin-dependent diabetes mellitus (IDDM), 799

Figure 1. Indirect immunofluorescence pattern of a steroid cell antibody positive serum preparation reacting with a cryostat section of a primate ovary. Note the positivity of mainly the theca layer. Courtesy of INOVA-Diagnostic (San Diego). autoimmune hypothyroidism, and more importantly in existing models for autoimmune oophoritis/adrenalitis (Sakagushi et al., 1982; Smith et al., 1991), T cells are probably more important than antibodies in the destruction of steroid-producing cells. The autoantibodies serve as convenient markers of the disease. Animal Models. The animal model most closely resembling human oophoritis in the presence of steroid cell antibodies is the neonatal thymectomy model in certain strains of mice (Smith et al., 1992). In the vast majority of cases of human autoimmune oophoritis, the primordial follicles are unaffected. Developing follicles are infiltrated by mononuclear inflammatory cells, and there is a clear pattern of increasing density of the infiltration with more mature follicles. Preantral follicles are only surrounded by small rims of lymphocytes and plasma cells; whereas, larger follicles have a progressively denser infiltrate, usually in the external and internal theca. The granulosa layer is usually spared in this process until luteinization of the degenerating follicle occurs. C o r p o r a l u t e a ~ when present are infiltrated as well. Mild infiltration can also be seen in the medulla and hilar regions of the ovaries (Sedmak et al., 1987; Bannatyne et al., 1990). This pattern of infiltration

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indicates that, indeed, the steroid-producing cells are the main target for the autoimmune attack. Immunohistochemical analysis reveals that the inflammatory cells are mainly T lymphocytes (CD4 + and CD8+), with a few B cells, together with large numbers of plasma cells. Macrophages and natural killer cells can also be found. The plasma cells mainly secrete IgG, but also IgA or IgM (Gloor and Hurliman, 1984), which likely indicates the local production of ovarian autoantibodies. Neonatal thymectomy in Balb/C mice at day three after birth (but hardly in DBA/2 mice) results in oophoritis, among other organ-specific autoimmune manifestations such as thyroiditis, gastritis and parotitis (Taguchi et al., 1980; Miyake et al., 1988). That the animal oophoritis is directly due to autoimmune T cells is shown by induction of oophoritis by transfer of CD4 + T cells from thymectomized donors to young recipients (Sakaguchi et al., 1982; Smith et al., 1991). This transfer of oophoritis could be prevented by infusion of CD4 + CD5 + T cells from normal adult mice in an early stage after the transfer of the CD4 + cells of the thymectomized donors. The histologic and serologic manifestations of the murine autoimmune oophoritis are hence comparable to the histological and serologic picture of human autoimmune.oophoritis

in association with Addison's disease. It is, however, remarkable that the adrenal glands are unaffected in mice, even in the presence of antibodies to steroidproducing cells. As the inflammation of the ovaries subsides, serum antioocyte and antizona antibodies also decrease to sometimes undetectable levels at day 150--360 when oocytes have completely disappeared from the atrophic ovary (Tung et a l., 1987). The absence of serum autoantibodies, therefore, does not exclude an autoimmune etiology of the ovarian disease. This finding may be of relevance in patients with adrenalitis and/or ~ amenorrhea; steroid cell antibodies should be sought at an early stage of the disease. Genetics

The immunogenetics of steroid-cell antibody-positive oophoritis is not known. One might speculate that such autoimmune oophoritis is genetically associated with the Addison susceptibility genes HLA-B8/DR3 (MacLaren and Riley, 1986). Alleles in the MHCclass II region which confer susceptibility for Addison's disease are DQ A1-0501, DR B1-0301 and DQ B 1-0201 (Bottazzo et al., 1995). However, autoimmune o0phoritis/adrenalitis in the context of type 1 APGS does not display this association. In fact, the only HLA association reported so far in the APGS affecting children is with HLA-A28 (Ahonen et al., 1988). In patients with APGS type 1

including ovarian failure, HLA-A3 is increased, and HLA-A9 decreased. In general, APGS type 1 is transmitted by autosomal recessive inheritance and the responsible gene maps to the long arm of chromosome 21. The putative defective gene remains to be identified.

CLINICAL UTILITY Disease Association

Steroid-cell antibodies are found almost exclusively in individuals or patients with Addison's disease who are serologically positive for cytoplasmic adrenal antibodies. Clinically, the presence of steroid cell antibodies correlates with gonadal deficiency, particularly in females (Table 1). Autoimmune endocrine failure of the testis is extremely rare. Although the original description of steroid cell antibodies was in a clinically normal man with Addison's disease (Anderson et al., 1968), this 'is a rare phenomenon. Only three of 79 males with autoimmune Addison's disease were found to have steroid cell antibodies reacting with testicular interstitial cells, and of these only one patient, a 15 year old, had some clinical evidence of testicular failure (Irvine and Barnes, 1975). Since 1975, only a few male cases of steroid cell antibody positivity, lacking a clinical picture of hypergonadotrophic hypogonadism (Ahonen et al., 1987; Betterle et al.,

Table 1. Prevalence of Steroid-cell Autoantibodies in Disease and Controls Ovarian failure Unselected infertility/amenorrhea With autoimmune thyroid disease or, IDDM With Addison's disease -- primary amenorrhea -- secondary amenorrhea Addison's disease (without ovarian failure) Isolated cases With hypoparathyroidism/candidiasis (type 1 APGS) With autoimmune thyroid disease (type 2 APGS)

<1% 5-10% 100% 60%

10-20% 60-80% 25-40%

Type 1 APGS, mucocutaneous candidiasis and hypoparathyroidism without Addison disease

10%

Autoimmune thyroid disease or IDDM

<1%

Healthy controls

<1%

Adapted from Sotsiou et al (1980), Betterle et al (1993), Ahonen et al (1987) and own data. Note: Autoimmune testicular failure is predominantly due to sperm antibodies" cases of steroid cell antibody-positive and -negative males with testicular endocrine failure have too rarely been described to give reliable data on prevalence.

801

1993) have been described; hence, autoimmune orchitis due to steroid cell antibodies is not a welldefined entity.

Antibody Frequency in Disease Almost all patients with a primary amenorrhea and Addison's disease have detectable serum steroid adrenal antibodies; 60% of patients with a secondary amenorrhea and Addison's disease have these antibodies (Table 1). In the absence of clinically overt gonadal failure (Table 1), steroid cell antibodies are found in about 15--20% of patients with clinical or latent Addison's disease (Betterle et al., 1993). Of steroid cell antibody-positive Addisonian patients, about 40% of females develop ovarian failure in a period of 10-15 years; in males, the steroid cell antibodies do not herald gonadal failure, but may be considered as markers of potential Addison' s disease (Betterle et al., 1993). Heterogeneity exists between type 1 and Type 2 APGS in relation to steroid cell antibodies (Table 1), 60--80% of the patients with hypoparathyroidism and Addison's disease (type I APGS) and 25--40% of patients with type 2 APGS have these antibodies (Table 1). In type 1 APGS without Addison's disease, 10% of patients show steroid cell antibodies. The high prevalence of steroid cell antibodies in patients with APGS type 1 probably explains the common association with gonadal failure seen in this group; the appearance of the steroid cell antibodies in a female patient with APGS type 1 without adrenocortical or ovarian failure signals a high risk of their development. The sensitivities/specificities/positive predictive values in females with type 1 APGS who initially had normal adrenocortical and ovarian function were 1.0/0.56/0.50 for steroid cell antibodies for predicting ovarian failure and 0.86/0.83/0.86 for steroid cell antibodies for predicting adrenocortical failure (Ahonen et al., 1987). Though seldom spontaneous, recovery of ovarian function has been described in women with amenorrhea and steroid cell antibodies (Finer et al., 1985; Taylor et al., 1989); this contrasts with genetically

REFERENCES Ahonen P, Miettinen A, Perheentupa J. Adrenal and steroidal cell antibodies in patients with autoimmune polyglandular

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determined cases. In the future, remissions might be enhanced by short courses of steroids in combination with ovulation-inducing therapies (Blumenfeld et al., 1993; Corenblum et al., 1993). If there is no recovery, oocyte donation is the only way to achieve pregnancy. Substitution with estrogen and progesterone should be given to treat the symptoms of sex hormone deficiency. Though there is no formal agreement, monitoring for other endocrine autoimmune diseases, particularly for the thyroid and IDDM, is advisable because of the strong associations of these endocrine autoimmune diseases with adrenal/ovarian autoimmunity.

CONCLUSION Autoantibodies to steroid-producing cells form a subgroup of the adrenal cytoplasmic antibodies. Steroid cell antibodies are markers, particularly in females, for a gonadal dysfunction due to autoimmune oophoritis. The frequency of steroid cell antibodies is virtually undetectable in unselected cases of ovarian failure (<1%), low in ovarian dysfunction when in combination with autoimmune thyroid disease or IDDM (5--10%), but high in ovarian dysfunction in combination with Addison's disease (>60%). In the absence of ovarian dysfunction, steroid cell antibodies can frequently be found in APGS type 1; in this syndrome, the steroid cell antibodies have a high predictive value in females for the development of gonadal dysfunction. Steroid cell antibodies are assayed via standard indirect immunofluorescence on commercially available cryostat sections of human or monkey ovary and testis. Autoantigens are still unknown, but strong candidates are the cytochrome P450 enzymes 17-c~hydroxylase and the side-chain cleavage (scc) enzyme. New, specific assays (ELISA, RIA) can be developed making use of these antigens manufactured via molecular biological techniques (Colls et al., 1995). See also AUTOANTIBODIES THAT PENETRATE INTO LIVING CELLS and THYROID PEROXIDASE AUTOANTIBODIES.

disease type 1 and risk of adrenocortical and ovarian failure. J Clin Endocrinol Metab 1987;64:494--500. Ahonen P, Koskimies S, Lokki ML, Tiilikainen A, Perheentupa J. The expression of autoimmune polyglandular disease type

1 appears associated with several HLA-A antigens but not with HLA-DR. J Clin Endocrinol Metab 1988;66:1152-1157. Ahonen P, Myll~irniemi S, Sipil~i I, Perheentupa J. Clinical variation of autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med 1990;322:1829--1836. Anderson JR, Goudie RB, Gray KG, Timbury GC. Autoantibodies in Addison's disease. Lancet 1957;i:1123-1124. Anderson JR, Gouidie RB, Gray K, Stuart-Smith DA. Immunological features of idiopathic Addison's disease: an antibody to cells producing steroid hormones. Clin Exp Immunol 1968;3:107--117. Asawa T, Wedlock N, Baumann-Antczak A, Rees Smith B, Furmaniak J. Naturally occurring mutations in human steroid 21-hydroxylase influence adrenal autoantibody binding. J Clin Endocrinol Metab 1994;79:372--376. Bannatyne P, Russell P, Shearman RP. Autoimmune o~3phoritis: a clinicopathologic assessment of 12 cases. Int J Gynaecol Pathol 1990;9:191-207. Blizzard RM, Kyle M. Studies of the adrenal antigens and antibodies in Addison's disease. J Clin Invest 1963;42:16531660. Baumann-Antczak A, Wedlock N, Bednarek J, Kiso Y, Krishnan H, Fowler S, Smith BR, Furmaniak J. Autoimmune Addison's disease and 21-hydroxylase (Letter). Lancet 1992 ;340:429--430. Betterle C, Rossi A, Dalla Priat S, Artifoni A, Pedini B, Gavasso S, Caretto A. Premature ovarian failure: autoimmunity and natural history. Clin Endocrinol 1993;39:35-43. Blumenfeld Z, Halachmi S, Peretz BA, Shmuel Z, Golan D, Makler A, Brandes JM. Premature ovarian f a i l u r e - the prognostic application of autoimmunity on conception after ovulation induction. Fertil Steril 1993;59:750-755. Bottazzo GF, Mirakian R, Drexhage HA. Adrenalitis, oophoritis and autoimmune polyglandular disease. In: Rich RR, ed. Clinical Immunology: Principles and Practice. St. Louis: Mosby, 1995;100:1523--1536. Colls J, Betterle C, Volpato M, Prentice L, Rees Smith B, Furmaniak J. Immunoprecipitation assay for autoantibodies to steroid 21-hydroxylase in autoimmune adrenal diseases. Clin Chem 1995;41:367--374. Corenblum B, Rowe T, Taylor PJ. High-dose, short-term glucocorticoids for the treatment of infertility resulting from premature ovarian failure. Fertil Steril 1993;59:988--991. Edmonds M, Lamki L, Killinger DW, Volpe R. Autoimmune thyroiditis, adrenalitis and oophoritis. Am J Med 1973;54: 782-787. Finer N, Fogelman I, Bottazzo GF. Pregnancy in a post menopausal woman. Postgrad Med J 1985;61:1079--1080. Furmaniak J, Kominami S, Asawa T, Wedlock N, Colls J, Rees Smith B. Autoimmune Addison' s disease: evidence for a role of steroid 21-hydroxylase autoantibodies in adrenal insufficiency. J Clin Endocrinol Metab 1994;79:1517--1521. Gloor E, Hurlimann J. Autoimmune oophoritis. Am J Clin Pathol 1984;81:105-109. Hoek A, van Kasteren Y, de Haan-Meulman M, Hooijkaas H,

Schoemaker J, Drexhage HA. Analysis of peripheral blood lymphocyte subsets, NK cells and delayed type hypersensitivity skin test in patients with.premature ovarian failure. Am J Reprod Immunol 1995;33:495--502. Irvine WJ, Barnes EW. Addison's disease, ovarian failure and hypoparathyroidism. Clin Endocrinol Metab 1975 ;4:379-434. Kamp P, Platz P, Nerup J. "Steroid-cell" antibody in endocrine diseases. Acta Endocrinol 1974;76:729-740. Krohn K, Uibo R, Aavik E, Peterson P, Savilahti K. Identification by molecular cloning of an autoantigen associated with Addison's disease as steroid 17-~-hydroxylase. Lancet 1992;339:770-773. MacLaren NK, Riley WJ. Inherited susceptibility to autoimmune Addison's disease is linked to human leukocyte antigens- DR3 and/or DR4, except when associated with type 1 autoimmune polyglandular syndrome. J Clin Endocrinol Metab 1986;62:455--459. McNatty KP, Short RV, Barnes EW, Irvine WJ. The cytotoxic effect of serum from patients with Addison's disease and autoimmune ovarian failure on human granulosa cells in culture. Clin Exp Immunol 1975;22:378--384. Mignot MH, Drexhage HA, Kleingeld M, et al. Premature ovarian failure. II: Considerations of cellular immunity defects. Eur J Obstet Gynecol Reprod Biol 1989;30:67-72. Miyake T, Taguchi O, Ikeda H, Sato Y, Takeuchi S, Nishizuka Y. Acute oocyte loss in experimental autoimmune oophoritis as a possible model of premature ovarian failure. Am J Obstet Gynecol 1988;158:186-192. Muir A, Mac Laren NK. Autoimmune diseases of the adrenal glands, parathyroid glands, gonads, and hypothalamicpituitary axis. Endocrinol Metab Clin North Am 1991;20: 619--645. Sakaguchi S, Takahashi T, Nishizuka Y. Study on cellular events in postthymectomy autoimmune oophoritis in mice. I. Requirement of Lyt-1 effector cells for oocytes damage after adoptive transfer. J Exp Med 1982:1565--1576. Sedmak DD, Hart WR, Tubbs RR. Autoimmune o6phoritis: a histopathological study involved ovaries with immunologic characterization of the mononuclear cell infiltrate. Int J Gynaecol Pathol 1987;6:73--81. Smith BR, Furmaniak J. Adrenal and gonadal autoimmune diseases (Editorial). J Clin Endocrinol Metab 1995;80:1502-1505. Smith H, Lou Y-H, Lacy P, Tung KSK. Tolerance mechanism in experimental ovarian and gastric autoimmune diseases. J Immunol 1992;149:2212-2218. Smith Y, Sakamoto Y, Kasai K, Tung KSK. Effector and regulatory cells in autoimmune oophoritis elicited by neonatal thymectomy. J Immunol 1991;147:2928-2933. Song Y-H, Connor EL, Muir JA, She JX, Zorovich B, Derovanesian D, Maclaren N. Autoantibody epitope mapping of the 21-hydroxylase antigen in autoimmune Addison's disease. J Clin Endocrinol Metab 1994;78:1108--1112. Sotsiou F, Bottazzo GF, Doniach D. Immunofluorescence studies on autoantibodies to steroid-producing cells, and to germline cells in endocrine disease and infertility. Clin Exp Immunol 1980;39:97-111. Taguchi O, Nishizuka Y, Sakakura T, Kojima A. Autoimmune

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oophoritis in thymectomized mice: detection of circulating antibodies against oocytes. Clin Exp Immunol 1980;40:540-553. Taylor R, Smith NM, Angus B, Home CHW, Dunlop W. Return of fertility after twelve years of autoimmune ovarian failure. Clin Endocrinol 1989;31:305--308. Tung KSK, Smith S, Kasai K, Oliver J, Feuchter F, Anderson RE. Murine autoimmune oophoritis, epididymitis and gastritis induced by day 3 thymectomy, Autoantibodies. Am J Pathol 1987;126:303--314. Uibo R, Perheentupa J, Ovod V, Krohn KJE. Characterization of adrenal autoantigens recognized by sera from patients with autoimmune polyglandular syndrome (APG) type 1. J Autoimmun 1994a;7:399--411. Uibo R, Aavik E, Peterson P, Perheentupa J, Aranko S, Pelkonen R, Krohn KJ. Autoantibodies to cytochrome P450 enzymes P450scc, P450c17, and P450c21 in autoimmune polyglandular disease types I and II and in isolated Addison' s disease. J Clin Endocrinol Metab 1994b;78:323--328. Velloso LA, Winqvist O, Gustafsson J, K~impe O, Karlsson FA.

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Autoantibodies against a novel 51 kDa islet antigen andglutamate decarboxylase isoforms in autoimmune polyendocrine syndrome type 1. Diabetologia 1994;37:61--69. Wedlock N, Asawa T, Baumann-Antczak A, Rees Smith B, Furmaniak J. Autoimmune Addison's disease: analysis of autoantibody binding sites on human steroid 21-hydroxylase. FEBS Lett 1993;332:123--126. Wheatcroft NJ, Toogood A, Li TC. Detection of antibodies to ovarian antigens in women with premature ovarian failure. Clin Exp Immunol 1994;26:122--128. Winqvist O, Karlsson FA, K~impe O. 21-hydroxylase, a major autoantigen in idiopathic Addison's disease. Lancet 1992; 339:1159--1562. Winqvist O, Gustafsson J, Rorsman F, Karsllon FA, K~impe O. Two different cytochrome P450 enzymes are the adrenal antigens in autoimmune polyendocrine syndrome type 1 and Addison's disease. J Clin Invest 1993;92:2377-2385. Zanchetta R, Mastrogiacomo I, Graziotti P, Foresta C, Betterle C. Autoantibodies against Leydig cells in patients after spermatic cord torsion. Clin Exp Immunol 1984;55:49-57.

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

STRIATIONAL AUTOANTIBODIES Herminio Reyes, Ph.D.

Specialty Laboratories, Inc., Santa Monica, CA 90404-3900, USA

HISTORICAL NOTES

In 1892, the apparently coincidental association of myasthenia gravis (MG) and a mediastinal tumor mass (thymoma) was described at the meeting of the Society for Psychiatry and Neurological Diseases in Berlin (Hoppe, 1892). That this association was more than coincidental is now clear as manifest by the occurrence of thymoma in -~10% of MG patients and of MG in 15--80% of thymoma patients (MtillerHermelink et al., 1993). The presence of muscle-binding, complementfixing immunoglobulins as detected by immunofluorescence in sera from myasthenic patients was reported in 1960 (Strauss et al., 1960). Striational antibodies (StrAb), which are reactive with proteins in the contractile elements of skeletal muscle, differ from myocardial autoantibodies, which are found in patients with acute rheumatic fever, postviral myocarditis and idiopathic dilated cardiomyopathy and rheumatic carditis in Dressler's syndrome (Limas et al., 1990; Neumann et al., 1990; Schulze et al., 1990; Caforio et al., 1992).

THE AUTOANTIGENS

The identity of all of the relevant antigens reactive with StrAb, which are heterogenous (Lanska, 1991; Mtiller-Hermelink et al., 1993; Ohta et al., 1991; Kuks et al., 1993; Williams et al., 1992; Victor et al., 1992; Aarli et al., 1990; Mygland et al., 1992a; 1992b; 1993; 1994), remains to be elucidated. Identification of their target autoantigens is expected to clarify their biologic significance. StrAb-reactive autoantigens include actin, ~-actinin, myosin, titin (connectin) and the ryanodine receptor (sarcoplasmic reticulum cal-

cium release channel protein), which regulates contraction and relaxation of skeletal muscle by the rapid release and reuptake of calcium (Aarli et al., 1990; Williams et al., 1992; Mygland et al., 1992a). Both polyclonal sera (Gilhus et al., 1984) and monoclonal antibodies (Dardenne et al., 1987) recognize epitopes common to both skeletal muscle and thymic epithelial cells. Although a very immunogenic cytoplasmic epitope of the acetylcholine receptor (AChR) ~-subunit (VICE-s) is expressed in fast troponin I (Marx et al., 1992; Osborn et al., 1992), a major protein in skeletal muscle, definitive proof that AChR autoimmunity in "paraneoplastic" (thymomaassociated) MG is due to an autoimmune response to an epitope present in skeletal muscle proteins is lacking.

THE AUTOANTIBODIES

StrAb are detectable by indirect immunofluorescence (IFA) using rodent or human muscle tissue as substrate (Kuks et al., 1993), by enzyme immunoassay using a crude mixture of proteins extracted from human skeletal muscle or purified skeletal muscle proteins (Williams and Lennon, 1986) or by solidphase radioimmunoassay or immunoblotting using proteins purified from rabbit human skeletal muscle (Ohta et al., 1991; Mygland et al., 1992a; 1992b; Williams et al., 1992). Because StrAb are heterogeneous (Lanska, 1991; Mtiller-Hermelink et al., 1993; Ohta et al., 1990; Kuks et al., 1993; Williams et al., 1992; Victor et al., 1992; Aarli et al., 1990; Mygland et al., 1992a; 1992b; 1993; 1994) and the proteins of the skeletal muscle contractile apparatus are diverse and arranged in a complex fashion, IFA detection using cryostat sections of skeletal muscle can be used

805

to screen for StrAb; this method enhances the likelihood of detecting a diversity of autoantibodies to skeletal muscle but is of limited utility for studies designed to identify the relevant antigens. The stimulus for production of StrAb, the basis for their prominent association with MG and their pathogenic significance are unknown. StrAb from different patients stain different regions of the sarcomere in stretched myofibrils (Williams et al., 1992), reflecting the heterogeneous nature and diverse reactivities of StrAb. Antibodies to titin constitute at least part of the StrAb which react with the I band near the A-I junction (Aarli et al., 1990); such autoantibodies are highly associated with presence of thymoma in MG (Williams et al., 1992). Recent investigations indicate that there is a correlation between the presence of ryanodine receptor autoantibodies and a severe form of thymoma-associated MG with a poor prognosis (Mygland et al., 1994). The binding of StrAb to thymic myoid and epithelial cells (Williams et al., 1992) reflects the presence of skeletal muscle components in the thymus (Ohta et al., 1991) and suggests that the thymus plays a central role in the development of StrAb. StrAb in spontaneously acquired MG patients are commonly of the IgG class; whereas, the StrAb that can arise in rheumatoid arthritis patients treated with D-penicillamine are reportedly IgM (Carrano et al., 1982). Autoantibodies to the ryanodine receptor in MG patients with thymoma are predominantly of the IgG1 and/or IgG3 subclasses; however, sera from some of these patients also contain IgG2, IgG4, IgA or IgM ryanodine receptor autoantibodies (Mygland et al., 1993). Human monoclonal antibody cell lines from patients with generalized myasthenia gravis and thymoma preferentially utilize VH and VL gene segments that are overrepresented in the autoimmune repertoire (Victor et al., 1992), suggesting that antigenic selection and somatic mutation could play a role in autoantibody production in MG. Hypotheses for the association of thymoma with MG include: 1) a common genetic defect predisposing to development of both MG and thymoma; 2) thymoma resulting from progression of MG-associated thymic alterations (thymic lymphofollicular hyperplasia) to thymoma; 3) a paraneoplastic autoimmune response to tumor-associated antigens; and 4) lack of negative T-cell selection or false-positive T-cell selection or autosensitization in thymoma (MtillerHermelink et al., 1993). Thymic tumors associated with MG are all charac806

terized by the presence of CDl-positive immature thymocytes interacting with neoplastic cortical epithelial cells; the latter are involved in the process of positive T-cell selection (Mtiller-Hermelink et al., 1993; Mygland et al., 1992b). These observations suggest that immunogenic peptides related to the acetylcholine receptor (AChR) might be present on neoplastic thymic epithelial cells. Restriction fragment length polymorphism (RFPL) analysis of the AChR subunit genes in thymic epithelial tumors reveals that the genomic organization of the AChR subunit genes is identical to that in normal tissues (Geuder et al., 1989; 1992). Although dot-blot and Northern blot analyses failed to establish that thymic epithelial tumor cells transcribe AChR subunit genes (Geuder et al., 1992), PCR analysis and cDNA cloning techniques revealed that thymoma epithelial cells transcribe the AChR c~-subunit gene (Geuder et al., 1992; Hara et al., 1991; Mtiller-Hermelink et al., 1993). However, functional acetylcholine receptors are not demonstrable on thymoma epithelial cells by sensitive electrophysiologic techniques (Mtiller-Hermelink et al., 1993). Studies utilizing monoclonal antibodies which recognize residues 373--380 of the AChR c~-subunit demonstrated the presence of VICE-~ (an immunogenic cytoplasmic epitope of the cz subunit of AChR) in normal thymic medullary epithelial cells and neoplastic epithelial cells from cortical thymomas and well-differentiated thymic carcinomas (Kirchner et al., 1987; 1988; Tzartos and Remoundos, 1992). VICE-~ expression is correlated to the clinical presence or absence of MG (Table 1). Further RFLP studies using an oligonucleotide probe corresponding to AChR c~-subunit residues 371--378 and immunoblot analysis of thymoma extracts provided evidence that there are other lociencoding proteins with VICE-cx that are distinct from AChR and suggest that thymoma associated with MG is characterized by the expression of AChR epitopes (Geuder et al., 1992; Mtiller-Hermelink et al., 1993). These epitopes are detectable at low levels in normal medullary epithelial cells but are overexpressed by cortical epithelial cells adjacent to immature thymocytes in MG-associated thymoma (Mtiller-Hermelink et al., 1993) suggesting that abnormal expression of these epitopes activate AChR-specific T cells or that "false-positive" AChR-sensitive T-cell selection results in the development of MG (Marx et al., 1991).

Table 1. (Modified from Mtiller-Hermelink et al., 1993) Type of Tumor

MG association

VICE-o~ Positive

Negative 1/1

Low

1/14

13/14

Low

7/13

6/13

Intermediate

14/15

1/15

High

Well-differentiated thymic carcinoma

8/8

0/8

High

Moderately differentiated thymic carcinoma

0/1

1/1

None

29/35

6/35

1/17

16/17

Medullary thymoma

0/1

Mixed thymoma Predominantly cortical thymoma Cortical thymoma

Thymic epithelial tumors with MG Thymic epithelial tumors without MG

CLINICAL UTILITY StrAb are most frequently (55%) found in MG patients >60 years of age but are rarely found in MG patients <20 years old or in patients with other conditions (Sano et al., 1993; Williams and Lennon 1986). StrAb are present in 80-90% of patients with both MG and thymoma (Williams et al., 1987; Lanska, 1991; Cikes et al., 1988; Carrano et al., 1982; 1983), but are present in only 6% of MG patients <40 years of age without thymoma (Cikes et al., 1988). StrAb are absent in 70--100% of MG patients without thymoma (Lanska, 1991; Cikes et al., 1988; Carrano et al., 1982; 1983; Keesey et al., 1980). Hence, StrAb are very sensitive and specific for thymoma in MG patients, and their absence virtually rules out a diagnosis of thymoma in MG, especially in patients aged 20--60, an age interval where thymoma is most common and the prevalence of false-positives of the test is relatively low (Hawkins et al., 1979; Lanska, 1991). StrAb are detectable infrequently in primary lung cancer patients with paraneoplastic neurologic disorders (Lennon, 1994); StrAb are also useful when monitoring immunosuppressive therapy for detection of autoimmune complications of bone marrow transplantation, including graft-versus-host disease (Cikes, et al., 1988; Dighiero et al., 1987). Nine percent (15/174) of patients with suspected MG were positive

REFERENCES Aarli JA, Stefansson K, Marton LSG, Wollmann RL. Patients with myasthenia gravis and thymoma have in their sera IgG

for StrAb; StrAb were detected in 34% (14/41), 30% (14/46) and 48% (13/27) of patients with AChRbinding, AChR-modulating and both AChR-binding and AChR-modulating autoantibodies, respectively (Reyes, unpublished). In a group of generalized MG patients (<10 years disease) selected for AChR seropositivity and no treatment with immunosuppressive drugs, the negative predictive value for thymoma of StrAb was 98%; whereas, the positive predictive value in this same group of patients was 31% (Kuks et al., 1993).

CONCLUSION Although the presence of a mediastinal mass in an MG patient is generally considered a strong indication for thymectomy to ameliorate myasthenic symptoms (Lanska, 1991), StrAb have definite diagnostic utility, especially in MG patients aged 20--60. Definitive proof that StrAb result from an immune response directed at cytoplasmic proteins associated with AChR in thymic epithelial cells in the process of neoplastic transformation to thymoma requires further studies of abnormal protein expression in the cortical microenvironment of thymomas. See also ACETYLCHOLINE RECEPTOR AUTOANTIBODIES.

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binding, complement-fixing serum globulin fraction in myasthenia gravis. Proc Soc Exp Biol Med 1960;105:184--191. Sano M, Lennon VA. Enzyme immunoassay of antihuman acetylcholine receptor autoantibodies in patients with myasthenia gravis reveals correlation with striational autoantibodies. Neurology 1993:43:573--578. Schulze K, Becker BF, Schauer R, Schultheiss HP. Antibodies to ADP-ATP c a r r i e r - an autoantigen in myocarditis and dilated cardiomyopathy- impair cardiac function. Circulation 1990;81:959--969. Strauss AJ, Seegal BC, Hsu KC, Burkholder PM, Nastuk WL, Osserman KE. Immunofluorescence demonstration of a muscle binding, complement-fixing serum globulin fraction in myasthenia gravis. Proc Soc Exp Biol Med 1960;105: 184--191. Tzartos S, Remoundos S. Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor ~-subunit. Eur J Biochem 1992;207:915--922.

Victor KD, Pascual V, Williams CL, Lennon VA, Capra JD. Human monoclonal striational autoantibodies isolated from thymic B lymphocytes of patients with myasthenia gravis use V n and VL gene segments associated with the autoimmune repertoire. Eur J Immunol 1992;22:2231--2236. Williams CL, Lennon VA. Thymic B lymphocyte clones from patients with myasthenia gravis secrete monoclonal striational autoantibodies reacting with myosin, ~-actinin, or actin. J Exp Med 1986;164:1043--1059. Williams CL, Lennon VA, Momoi MY, Howard FM. Serum antibodies and monoclonal antibodies secreted by thymic Bcell clones from patients with myasthenia gravis define striational antigens. Ann NY Acad Sci 1987;505:168-179. Williams CL, Hay JE, Huiatt TW, Lennon VA. Paraneoplastic IgG striational autoantibodies produced by clonal thymic B cells and in serum of patients with myasthenia gravis and thymoma react with titin. Lab Invest 1992;66:331--336.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

THYROGLOBULIN AUTOANTIBODIES C. Lynne Burek, Ph.D and Noel R. Rose, M.D., Ph.D.

Departments of Pathology, Molecular Microbiology and Immunology, The Johns Hopkins Medical Institutions, Baltimore, MD 21205-2196, USA

HISTORICAL NOTES In 1956, injection of rabbit thyroglobulin (Tg) into other rabbits was shown to elicit antibodies reactive with rabbit Tg (Witebsky and Rose, 1956). The infiltration of the thyroid glands of the immunized rabbits with mononuclear cells denoted the presence Of autoimmune thyroiditis (Rose and Witebsky, 1956). Subsequent studies showing antibodies to human Tg in Hashimoto's disease and related disorders led to the proposal that human thyroiditis is an example of a human autoimmune disease (Witebsky et al., 1957). Independent, almost simultaneous studies of the hypergammaglobulinemia commonly present in patients with Hashimoto' s thyroiditis revealed precipitating antibodies to human Tg in many patients with this disease (Roitt et al., 1956). Since these seminal discoveries, a smaller proportion of patients with Graves' disease, thyroid cancer and certain other thyroid diseases was shown to have thyroglobulin autoantibodies (Weetman and McGregor, 1994). In addition, some individuals with normal thyroids possess autoantibodies to Tg. In 1959 (Belyavin and Trotter, 1959), autoantibodies to other thyroidspecific antigens (especially the enzyme thyroperoxidase [TPO]), formerly called microsomal antigen, were discovered in thyroiditis patients. Unlike Tg, however, TPO does not initiate an autoimmune process in most experimental animals (Weetman, 1990). A time-course investigation in primates showed that Tg antibodies appeared first, followed by the microsomal antibodies, suggesting that the former may b e the consequence of initial autoimmune damage of the gland (Andrada et al., 1968). If this is so, the autoantibodies to Tg are better markers of thyroid autoimmune induction while antibodies to thyroperoxi-

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dase are likely to be better indicators of active thyroid inflammation. This concept is further supported by an investigation in families of juveniles with thyroiditis (Burek et al., 1982). This study indicated that while virtually all patients had both Tg and microsomal antibodies, the majority of their siblings had Tg antibodies alone. Those siblings with both antibodies were significantly more associated with subclinical disease (elevated TSH but normal T4) (Burek et al., 1982). These findings point out that Tg autoantibodies are likely to be the first autoimmune markers followed by the microsomal response.

THE AUTOANTIGENS Definition

Thyrog!obulin, the main component of the colloid in the thyroid follicle, plays an essential role in the storage of iodine and the synthesis of iodinated thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Tg is a 660 kd protein, consisting of two 330 kd homodimers; the gene for human Tg is on chromosome 8 (de Vijlder et al., 1985). Molecular cloning from cDNA prepared from human thyroid glands indicates that the mRNA is composed of 8,448 base pairs (Malthiery and Lissitzky, 1987) expressed as a molecule of 2,767 amino acids (Malthiery and Lissitzky, 1987). Of these amino acids, 67 are tyrosines (134 per molecule) which, when iodinated, are the unique amino acids that make up the thyroid hormones. In addition to extensive glycosylation (10% carbohydrate by weight) (Dunn, 1991), Tg undergoes posttranslation modifications including phosphorylation, sulfation and iodination.

Structure/Sequence Information The amino acid structure shows three types of domains (Dunn, 1991). The N-terminal portion of the molecule contains a sequence of about 60 amino acids repeated ten times; cystine, proline and glycine are highly represented in this domain, possibly maintaining a rigid confirmation to promote homonogenesis. A second domain, found in the central part of the molecule, consists of three segments of 14--17 amino acids. The third domain appears five times between residues 1,603 and 2,186. The C-terminal part of Tg shares no sequence similarity with the N-terminal part of the molecule. There is considerable sequence similarity among species, including about 70% of the amino acid sequences of human and bovine Tg (Mercken et al., 1985). As expected, therefore, antibodies to Tg show some species cross-reactivity. The iodine content of Tg varies considerably with the diet of the individual. Normally, no more than 25% of the tyrosines are iodinated. However, only four of the tyrosines per chain (positions 5; 2,553; 2,567 and 2,746) are believed to play a role in homogenesis (Rawitch et al., 1983); these four tyrosines have high affinity for iodine (Palumbo et al., 1982; Gavaret et al., 1977). Evidence that dietary iodine plays a role in modulating autoimmune thyroiditis is largely based on studies of thyroiditis-prone experimental animals, such as OS chickens, BB/W rats and NOD mice (Bagchi et al., 1985; Allen et al., 1986; Wicker et al., 1992). In these animals, increased dietary iodine enhances the frequency and severity of thyroiditis. How dietary iodine increases thyroiditis is controversial; heavily iodinized Tg is probably more antigenic than Tg with lesser amounts of iodine (Sundick et al., 1987).

containing serum from diseased OS birds (Jaroszewski et al., 1978). Direct perfusion of purified Tg antibodies into the thyroid gland of rabbits causes pathogenetic changes within the thyroid (Inoue et al., 1993). Circumstantial evidence for a pathogenetic role of antibodies includes reports of immune complexes deposited in the thyroids of patients with Hashimoto's disease (Aichinger et al., 1985) Antibody-dependent, cell-mediated cytotoxicity can cause injury to thyroglobulin-coated target cells in vitro (Bogner et al., 1984). Presently, chronic thyroiditis in the human is best considered a cell-mediated autoimmune disease in which antibodies play a contributory role.

Factors in Pathogenicity

AUTOANTIBODIES

Antibody Isotypes. Tg antibodies are primarily IgG (Torrigiani et al., 1969). Whether the IgG subclass distribution of the Tg antibodies is skewed, however, is not clear. Comparison of the relative reactivities of the Tg antibody subclass distribution using an uncharacterized "standard" serum suggests an IgG1 and IgG4 restriction in patients with autoimmune thyroiditis (Parkes et al., 1984). By contrast, isolation of particular IgG subclasses of Tg autoantibodies from individual serum samples (Weetman et al., 1989) and assay of patient sera against a well-characterized, affinity-purified, quantitated standard (Kuppers et al., 1993) showed no particular restriction. Absolute quantitation against a defined population of antibodies of known quantity in autoimmune thyroid disease revealed Tg autoantibodies in all four subclasses with roughly equal quantities of IgG1 and IgG3 (Caturegli et al., 1994a). Compared with Graves' disease, differentiated carcinoma and nontoxic goiter, thyroiditis patients showed a higher IgG2 and a lower IgG4. The overrepresentation of IgG2 in the thyroiditis patients may reflect the type of T-helper cell population associated with this disease (Mosmann et al., 1986).

Pathogenetic Role

Methods of Detection

Thyroi'ditis can be adoptively transferred to experimental animals, using T lymphocytes, but transfer of disease by injection of serum is difficult to show (Kuppers et al., 1988). On the basis of this evidence, the disease is often considered cell-mediated, despite persistent evidence that thyroiditis can be transferred by serum under special conditions. For example, in OS chickens genetically predisposed to thyroiditis, the disease can be augmented by injection of antibody-

The methods used to detect autoantibodies to Tg range from precipitation in fluid medium, to indirect immunofluorescence, hemagglutination, ELISA and radiometric assays (Bigazzi et al., 1992). Currently, the two most commonly used procedures are hemagglutination and ELISA. A high degree of sensitivity without loss of specificity is achieved by tanned cell hemagglutination or the chromic chloride hemagglutination test; both utilize modified human group O 811

erythrocytes coated with Tg. Because the chromic chloride method does not change the surface of the red cell membrane (Burek and Rose, t980), there is little or no background hemagglutination at high serum concentrations due to reactivity to the altered cell membranes found when using tanned red cells. This means a reliable evaluation of low titers of Tg antibody can be made, as frequently found in the case of juvenile thyroiditis (Loeb et al., 1973; Burek et al., 1982). A further advantage is that the cells can be used for many days without a decrease in sensitivity, unlike tanned cells coated with Tg. Tanned cells must be prepared freshly each day. Commercially available kits using fixed tanned cell erythrocytes coated with Tg were found in our laboratory to be substantially less sensitive (unpublished data). Titers of 512 by the chromic chloride method are negative by thecommercial tanned cell kit in a comparison study (unpublished data). The sensitivity of ELISA is reported to be higher than the tanned cell hemagglutination (commercial sources) (Toh, 1991; Kohno et al., 1989), but on par with the chromic chloride hemagglutination (Voller et al., 1980; Caturegli et" al., 1994). A further advantage of ELISA is that specific IgG isotypes of Tg can be determined (Caturegli et al., 1994). A recent report utilized an affinity-purified Tg antibody that was quantitated for individual subclasses (Caturegli et al., 1994b). In addition, some sera that are negative in the hemagglutination tests are positive by ELISA. This phenomenon may have more to do with

the epitope or subclass specificity of antibody rather than the sensitivity of the assay, as the assays are equally sensitive (Kuppers et al., 1993; Caturegli et al., 1994). Radioimmunoassays for Tg antibodies are as sensitive as ELISA (Ewins and Wilkens, 1983), but are seldom used due to the inherent disadvantages of radioisotopes. A radiometric test using 125I-labelled Tg provides sensitive assay for Tg antibodies (Beever et al., 1989), but has no advantages over ELISA. The highly sensitive assays for Tg autoantibodies and the frequency in patients with thyroiditis are much higher and virtually equal to the frequency of the TPO antibodies (Burek et al., 1982; Tamaki et al., 1992; Phillips et al., 1991) (Table 1). With the advent of the highly sensitive assays for Tg, specificity associated with disease may be reduced. Studies conducted to evaluate the fine specificity of thyroglobulin autoantibodies reveal a qualitative difference in the immune response between healthy euthyroid individuals and those with autoimmune thyroid disease (Bouanani et al., 1989; 1992; Bresler et al., 1990a). Normal individuals commonly produce antibodies to the shared or cross-reactive epitopes (Bresler et al., 1990b), some of which are closely related to the homonogenic regions, i.e., those containing T4. Patients with chronic thyroiditis, on the other hand, develop additional autoantibodies to the species-restricted portion of the thyroglobulin molecule (Bresler et al., 1990). Recently, one epitope of Tg that is recognized significantly more frequently by thyroiditis patients than

Table 1. Frequency of Tg Autoantibodies in Different Investigations Group

Method

anti-Tg%

a n t i - M % Comments

Name

Hashimoto's thyroiditis

RIA

100

n.d.

Peake et ai., 1974

Hashimoto's thyroiditis Graves' disease NTG

TC-HA

55 38 5

100 80 27

Thyroiditis in juveniles

TgCC-HA M-IIF

95

Hashimoto's thyroiditis Graves' disease Blood donors

RIA

Hashimoto's thyroiditis Graves' disease Normals

EIA

Hashimoto's thyroiditis

TC-HA

Commercial kit

Abreau et al., 1977

80

Prepared in house

Burek et al., 1982

97 51 18

97 72 18

Used 125I-Tgor M

Prenticeet al., 1990

100 98 91

n.d.

36

99

10000 x more sensitive Tamaki et al., 1992 than commercial HA Commercial k i t s

Nordykeet al., 1993

n.d. = not done. NTG = nontoxic goiter, Tg = thyroglobulin, M = microsomal antigen, TC-HA = tanned cell hemagglutination, TgCC-HA = chronic chloride hemagglutination, EIA = enzyme immunoassay, RIA = radioimmunoassay.

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by those with different thyroid disorders was identified (Caturegli et al., 1994b); it may have clinical application in the future. Current research is aimed at defining these disease-associated epitopes in order to better understand the induction of autoimmune thyroid disease and to improve the specificity of the tests for Tg autoantibodies.

CLINICAL UTILITY

Application Autoantibodies to the two major thyroid antigens (Tg and thyroid peroxidase) provide a valuable exclusionary test in patients with thyroid disease, because over 98% of thyroiditis patients have autoantibodies directed to either or both of these antigens. Thus, negative tests can virtually exclude a diagnosis of thyroiditis. A positive test, however, does not eliminate the diagnosis of such conditions as adenocarcinoma of the thyroid or hyperthyroidism, because many of these patients have autoantibodies to thyroid antigens. In addition, a certain number of normal individuals have such antibodies, depending largely upon age and gender. The titers, however, are generally lower than in patients with chronic thyroiditis. As discussed previously, work is currently being conducted to determine fine specificities of Tg antibodies to help differentiate thyroid conditions and determine subsets of thyroid disorders (Bresler et al., 1990; Caturegli et al., 1994). A recent follow-up to a 20-year community study in England concludes that a primary risk factor to future autoimmune thyroid dysfunction was a positive thyroid autoantibody test (Vanderpump et al., 1995). Therefore, these antibodies have predictive value for determining those at risk of future disease. Miscellaneous uses of testing for Tg antibodies are described below. Tg antibodies may also help in assessing potential cases of postpartum thyroiditis (Tamaki et al., 1992). A change in Tg antibody levels may provide useful information in postoperative patients with differentiated thyroid carcinoma (Rubello et al., 1990); an elevation of these antibodies could indicate recurrence or metastases. ?

.

Disease Associations Reports on the frequency of Tg antibodies found in individuals with thyroid disorders or other conditions

are highly variable depending upon the method used for detection and upon the population studied. Most of the published frequencies of Tg antibodies utilize the traditional tanned cell hemagglutination methods (Bigazzi et al., 1992; McKenzie and Zakarija, 1991; Rose and Bigazzi, 1978). Using the traditional tanned cell test, antibodies to Tg are found in the sera of most patients with chronic lymphocyte thyroiditis (76%), primary myxedema (72%), hyperthyroidism (33%), colloid goiter (8%), nodular goiter (8%), adenoma (28%) and thyroid cancers (13--65%) (depending upon histological type). Antibodies to Tg are also found in patients with other autoimmune endocrinopathies, such as pernicious anemia (27%), Addison's disease of the adrenal gland (28%), and diabetes mellitus (20%). In a random population of nearly 700 healthy women subjects, 18% were found to have autoantibodies to Tg (Prentice et al., 1990). However, the incidence of Tg antibodies varies greatly with the age of normal subjects. The highest instances are found in older females, of whom as many as 30% may be positive (Prentice et al., 1990). In males of the same age range, the incidence of thyroglobulin antibodies was reported as much lower, approximately 3--6% (Safran et al., 1987). Besides age, geographical location and environmental influences, such as dietary iodine may also influence the prevalence of Tg antibodies (Safran et al., 1987).

CONCLUSIONS Autoantibodies to Tg can be a valuable marker of thyroid autoimmunity and can aid in the diagnosis of autoimmune thyroid disease. Lack of Tg antibodies in conjunction with microsomal/TPO autoantibodies can virtually exclude a diagnosis of autoimmune thyroiditis. However, presence of Tg antibodies does not by itself make a diagnosis without pertinent clinical or biochemical findings, as these autoantibodies can be found in a number of other diseases and even in normals. Future work is directed toward determining disease-specific autoimmune responses. Autoantibodies to Tg in individuals with thyroiditis are directed primarily to species-restricted determinants of human Tg and are markers associated with pathogenesis. Naturally occurring autoantibodies are directed to species-conserved sites of the molecule and are markers of nonpathogenic responses to Tg. These findings suggest that epitope specificities may be used as predictors in predisposed populations to

813

identify those at risk for future autoimmune thyroid disease and distinguish them from those who have

"benign" autoimmune responses. See also THYROID PEROXIDASE AUTOANTIBODIES.

REFERENCES

Burek CL, Rose NR. Detection of autoantibodies. In: Sonnenwirth AC, Jarett L, eds. Gradwohl's Clinical Laboratory Methods and Diagnosis. 8th Edition. St. Louis: C.V. Mosby Co., 1980:1257-1278. Burek CL, Hoffman WH, Rose NR. The presence of thyroid autoantibodies in children and adolescents with autoimmune thyroid disease and in their siblings and parents. Clin Immunol Immunopathol 1982;25:395--404. Caturegli P, Kuppers RC, Mari0tti S, Burek CL, Pinchera A, Ladenson PW, Rose NR. IgG subclass distribution of thyroglobulin antibodies in patients with thyroid disease. Clin Exp Immunol 1994a;98:464--469. Caturegli P, Mariotti S, Kuppers RC, Pinchera A, Rose NR. Epitopes on thyroglobulin: a study of patients with thyroid disease. Autoimmunity 1994b;18:41--49. de Vijlder JJ, Baas F, Kok K, van Dijk JE, Geurts van Kessel A, van Ommen GJB, Tegelaers WHH. Molecular basis of thyroglobulin synthesis defects. In: Eggo MC, Burrow GN, eds. Thyroglobulin- The Prothyroid Hormone. New York: Raven Press, 1985:69--76. Dunn JT. Thyroglobulin: chemistry and biosynthesis. In: Braverman LE, Utiger RD, eds. The Thyroid. 6th Edition. New York: J.B. Lippincott Company, 1991:98-110. Gavaret JM, Deme D, Nunez J, Salvatore G. Sequential reactivity of tyrosyl residues of thyroglobulin upon iodination catalyzed by thyroid peroxidase. J Biol Chem 1977;252: 3281--3285. Inoue K, Niesen N, Milgrom F, Albini B. Transfer of ex= perimental autoimmune thyroiditis by in situ perfusion of thyroids with immune sera. Clin Immunol Immunopathol 1993;66:11-17. Jaroszewski J, Sundick RS, Rose NR. Effects of antiserum containing thyroglobulin antibody on the chicken thyroid gland. Clin Immunol Immunopathol 1978;10:95--103. Kohno T, Mitsukawa T, Matsukura S, Tsunetoshi Y, Ishikawa E. More sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum. J Clin Lab Anal 1989;3:163-- 168. Kuppers RC, Neu N, Rose NR. Animal models of autoimmune thyroid disease. In: Farid NR, ed. Immunogenetics of endocrine disorders. New York: Alan R. Liss, Inc., 1988: 111--131. Kuppers RC, Outschoorn IM, Hamilton RG, Burek CL, Rose NR. Quantitative measurement of human thyroglobulinspecific antibodies by use of a sensitive enzyme-linked immunoassay. Clin Immunol Immunopathol 1993;67:68-77. Loeb PB, Drash AL, Kenny FM. Prevalence of low-titer and negative antithyroglobulin antibodies in biopsy-proved juvenile Hashimoto's thyroiditis. J Pediatr 1973;82:17-21. Malthiery Y, Lissitzky S. Primary structure of human thyroglobulin deduced from the sequence of its 8,448-base complementary DNA. Eur J Biochem 1987;165:491--498. McKenzie JM, Zakarija M. Antibodies in autoimmune thyroid

Abreau CM, Vagenakis AG, Roti E, Braverman LE. Clinical evaluation of a hemagglutination method for microsomal and thyroglobulin antibodies in autoimmune thyroid disease. Ann Clin Lab Sci 1977;7:73--78. Aichinger G, Fill H, Wick G. In situ immune complexes, lymphocyte subpopulations, and HLA-DR-positive epithelial cells in Hashimoto' s thyroiditis. Lab Invest 1985;52:132-140. Allen EM, Appel MC, Braverman LE. The effect of iodide ingestion on the development of spontaneous lymphocytic thyroiditis in the diabetes-prone BB/W rat. Endocrinology 1986;118:1977--1981. Andrada JW, Rose NR, Kite JR. Experimental thyroiditis in the Rhesus monkey. IV. The role of thyroglobulin and cellular antigens. Clin Exp Immunol 1968;3:133--151. Bagchi N, Brown TR, Urdanivia E, Sundick RS. Induction of autoimmune thyroiditis in chickens by dietary iodine. Science 1985;230:325-327. Beever K, Bradbury J, Phillips D, McLachlan SM, Pegg C, Goral A, Overbeck W, Feifel G, Smith BR. Highly sensitive assays of autoantibodies to thyroglobulin and to thyroid peroxidase. Clin Chem 1989;35:1949-1954. Belyavin G, Trotter WR. Investigations of thyroid antigens reacting with Hashimoto sera. Lancet 1959;1:648--652. Bigazzi PE, Burek CL, Rose NR. Antibodies to tissue-specific endocrine, gastrointestinal, and surface-receptor antigens. In: Rose NR, Conway de Macario E, Fahey JL, Friedman H, Penn GM, eds. Manual of Clinical Laboratory Immunology. 4th Edition. Washington, D.C.: American Society for Immunology, 1992:765--774. Bogner U, Schleusener H, Wall JR. Antibody-dependent cell mediated cytotoxicity against human thyroid cells in Hashimoto's thyroiditis but not Graves' disease. J Clin Endocrinol Metab 1984;59:734--738. Bouanani M, Piechaczyk M, Pau B, Bastide M. Significance of the recognition of certain antigenic regions on the human thyroglobulin molecule by natural autoantibodies from healthy subjects. J Immunol 1989;143:1129-1132. Bouanani M, Hanin V, Bastide M, Pau B. New antigenic clusters on human thyroglobulin defined by an expanded panel of monoclonal antibodies. Immunol Lett 1992;32:259-264. Bresler HS, Burek CL, Hoffman WH, Rose NR. Autoantigenic determinants on human thyroglobulin: II. Determinants recognized by autoantibodies from patients with chronic autoimmune thyroiditis compared to autoantibodies from healthy subjects. Clin Immunol Immunopathol 1990a;54:7686. Bresler HS, Burek CL, Rose NR. Autoantigenic determinants on human thyroglobulin: I. Determinant specificities of murine monoclonal antibodies. Clin Immunol Immunopathol 1990b;54:64--75.

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disease. In: Braverman LE, Utiger RD, eds. The Thyroid. 6th Edition. New York: J.B. Lippincott, 1991:506--524. Mercken L, Simons MJ, Swillens S, Massaer M, Vassart G. Primary structure of bovine thyroglobulin deduced from the sequence of its 8,431-base complementary DNA. Nature 1985;316:647--651. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348--2357. Nordyke RA, Gilbert FI Jr, Miyamoto LA, Fleury KA. The superiority of antimicrosomal over antithyroglobulin antibodies for detecting Hashimoto' s thyroiditis. Arch Intern Med 1993;153:862--865. Palumbo G, Tecce MF, Formisano S. Evidence that early iodination of thyroglobulin occurs at specific sites [Abstract]. Ann Endocrinol (Paris) 1982;43:68A. Parkes AB, McLachlan SM, Bird P, Rees-Smith B. The distribution of microsomal and thyroglobulin antibody activity among the IgG subclasses. Clin Exp Immunol 1984;57:239-243. Phillips D, Prentice L, Upadhyaya M, Lunt P, Chamberlain S, Roberts DF, McLachlan S, Smith BR. Autosomal dominant inheritance of autoantibodies to thyroid peroxidase and thyroglobulin- studies in families not selected for autoimmune thyroid disease. J Clin Endocrinol Metab 1991;72: 973--975. Prentice LM, Phillips DI, Sarsero D, Beever K, McLachlan SM, Smith BR. Geographical distribution of subclinical autoimmune thyroid disease in Britain: a study using highly sensitive direct assays for autoantibodies to thyroglobulin and thyroid peroxidase. Acta Endocrinol (Copenh) 1990;123: 493--498. Rawitch AB, Chernoff SB, Litwer MR, Rouse JB, Hamilton JW. Thyroglobulin structure-function. The amino acid sequence surrounding thyroxine. J Biol Chem 1983;258: 2079-2082. Roitt IM, Doniach D, Campbell PN, Hudson RV. Autoantibodies in Hashimoto's disease. Lancet 1956;ii:820--821. Rose NR, Witebsky E. Studies on organ specificity. V: Changes in the thyroid glands of rabbit following active immunization with rabbit thyroid extracts. J Immunol 1956;76:417--427. Rose NR, Bigazzi PE. The Autoimmune diseases. In: Baumgarten A, Richards F, eds. CRC Handbook Series in Clinical Laboratory Science, Section F: Immunology. West Palm Beach: CRC Press, 1978:305-344. Rubello RD, Girelli ME, Casara D, Piccolo PM, Perin A, Busnardo B. Usefulness of the combined antithyroglobulin antibodies and thyroglobulin assay in the follow-up of patients with differentiated thyroid cancer. J Endocrinol Invest 1990;13:737--742. Safran M, Paul TL, Roti E, Braverman LE. Environmental

factors affecting autoimmune thyroid disease. Endocrinol Metab Clin North Am 1987;16:327--342. Sundick RS, Herdegen DM, Brown TR, Bagchi N. The incorporation of dietary iodine into thyroglobulin increases its immunogenicity. Endocrinology 1987; 120:2078--2084. Swillens S, Ludgate M, Mercken L, Dumont JE, Vassart G. Analysis of sequence and structure homologies between thyroglobulin and acetylcholinesterase: possible functional and clinical significance. Biochem Biophys Res Commun 1986;137:142--148. Tamaki H, Katsumaru H, Amino N, Nakamoto H, Ishikawa E, Miyai K. Usefulness of thyroglobulin antibody detected by ultrasensitive enzyme immunoassay: a good parameter for immune surveillance in healthy subjects and for prediction of postpartum thyroid dysfunction. Clin Endocrinol (Oxf) 1992;37:266-273. Toh B. Anticytoskeletal autoantibodies: diagnostic significance for liver diseases, infections and systemic autoimmune diseases. Autoimmunity 1991; 11:119-- 125. Torrigiani G, Doniach D, Roitt IM. Serum thyroglobulin levels in healthy subjects and in patients with thyroid disease. J Clin Endocrinol Metab 1969;29:305-314. Vanderpump MP, Tunbridge WM, French JM, Appleton D, Bates D, Clark F, Grimley Evans J, Hasan DM, Rodgers H, Turnbridge F, Young ET. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol (Oxf) 1995;43:55--68. Voller A, Bidwell DE, Burek CL. An enzyme-linked immunosorbent assay (ELISA) for antibodies to thyroglobulin. Proc Soc Exp Biol Med 1980;163:402--405. Weetman AP, Black CM, Cohen SB, Tomlinson R, Banga JP, Reimer CB. Affinity purification of IgG subclasses and the distribution of thyroid autoantibody reactivity in Hashimoto' s thyroiditis. Scand J Immunol 1989;30:73-82. Weetman AP. Thyroid peroxidase as an antigen in autoimmune thyroiditis. Clin Exp Immunol 1990;80:1--3. Weetman AP, McGregor AM. Autoimmune thyroid disease: further developments in our understanding [Review]. Endocr Rev 1994; 15:788-830. Wicker LS, Appel MC, Dotta F, Pressey A, Miller B J, DeLarato NH, Fischer PA, Boltz RC Jr, Peterson LB. Autoimmune syndromes in major histocompatibility complex (MHC) congenic strains of nonobese diabetic (NOD) mice. The NOD MHC is dominant for insulitis and cyclophosphamideinduced diabetes. J Exp Med 1992;176:67--77. Witebsky E, Rose NR. Studies on organ specificity. IV. Production of rabbit thyroid antibodies in the rabbit. J Immunol 1956;76:408--416. Witebsky E, Rose NR, Terplan K, Paine JR, Egan RW. Chronic thyroiditis and autoimmunization. JAMA 1957;164:1439-1447.

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9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

THYROID PEROXIDASE AUTOANTIBODIES Basil Rapoport, M.D. and Sandra M. McLachlan, Ph.D.

Thyroid Molecular Biology Unit, V.A. Medical Center, San Francisco, CA 94121, USA

HISTORICAL NOTES First noted in 1958 in sera from patients with Hashimoto's thyroiditis (Belyavin and Trotter, 1959), autoantibodies to thyroid microsomal preparations are predominantly of IgG class. The specific antigen remained unidentified until 1985 when sera containing microsomal autoantibodies were found to recognize thyroid peroxidase (TPO), the major enzyme involved in multiple steps of thyroid hormone synthesis (Czarnocka et al., 1985; Portmann et al., 1985). Molecular cloning of the cDNA in 1987 for the thyroid microsomal antigen showed it to be the same as that of thyroid peroxidase, which was cloned the previous year (Seto et al., 1987; Libert et al., 1987).

THE AUTOANTIGEN(S) Human thyroid peroxidase, commonly known by its abbreviation "TPO", is a large (933 amino acid residue, -- 105 kd), membrane-associated glycoprotein with a heme prosthetic group (Table 1). TPO is expressed only in thyrocytes, where it is present primarily on the apical surface. The protein contains a single membrane-spanning region near its carboxyl terminus; the protein is oriented primarily toward the extracellular compartment (Kaufman et al., 1989). Native vs. Recombinant Antigen Performance

Recombinant TPO produced in eukaryotic cells is recognized by human autoantibodies in an identical manner to TPO of thyroid cell origin (Kaufman et al., 1990). In vivo, TPO exists as a dimer. Purification of antigen leads to recovery of monomers which are

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recognized as well as the dimeric form (Nishikawa et al., 1994c). Sources

TPO can be obtained in limited amounts from human thyroid tissue. TPO from other species, including pig, is not recognized by autoantibodies as well as the human form. Therefore, recombinant human TPO is supplanting human thyroid tissue as the primary source of antigen. Although recombinant TPO can be expressed in prokaryotic cells, yeast, insect cells and in a variety of mammalian cells using different plasmid or viral vectors (McLachlan and Rapoport, 1992), TPO of eukaryotic cell (Chinese hamster ovary; CHO) or insect cell origin is required, because the autoantibodies primarily recognize conformational epitopes. Methods of Purification

As reviewed elsewhere (McLachlan and Rapoport, 1992), the classical approach to purification of membrane-associated TPO involves detergent solubilization and limited tryptic digestion of a thyroid particulate fraction. The same procedure can be used for TPO generated in CHO cells and insect cells. An important ancillary step is affinity-purification using murine monoclonal antibodies to human TPO (Nakagawa et al., 1985). Membrane-bound TPO can be converted into a secreted protein by the introduction of Stop codons upstream of the membrane-spanning region. Amplification of the transgenome can enhance expression and secretion of soluble TPO (McLachlan and Rapoport, 1992) which can then be purified by affinity chromatography from conditioned culture medium.

Table 1. Thyroid Peroxidase: Autoantigen and Autoantibodies AUTOANTIGEN Properties

933 amino acids; 105 kd; glycoprotein; heine prosthetic group

Tissue distribution

Thyroid specific; thyroid cell apical surface

Source Natural Recombinant

Primate thyroid Eukaryotic (CHO cell or Baculovirus)

AUTOANTIBODIES Isotypes

IgG predominantly; some IgA IgG1 > IgG2 > IgG4 >> IgG3

Light Chains

Kappa > Lambda

Affinities

High (--10-1~ kd)

Assays

ELISA; precipitation of 125I-TPO; competition for TPO binding to immobilized murine monoclonal anti-TPO; detection with chemiluminescent TPO

Epitopes Conformational Linear Immunodominant region Human monoclonal antibodies

Predominant in all individuals Some individuals All individuals; >80% of autoantibodies in an individual serum Recombinant F(ab); IgG1 and IgG4; kappa and lambda; high affinities; conformational epitopes

DISEASE ASSOCIATIONS

Hashimoto's thyroiditis; Graves' disease

PREDICTIVE VALUE

Autoimmune hypothyroidism in general population; postpartum thyroid dysfunction

Commercial Sources Although no commercial sources of purified TPO are known, many companies market diagnostic kits for TPO autoantibodies, including some with suboptimal specificity and sensitivity due to continued use of crude thyroid microsomal extracts (even though they are labeled as TPO). The origin and purity of TPO in the more modern and specific TPO autoantibody ELISA and radioimmunoassay kits (e.g., Kronus, Dana Point, California, and Henning, Berlin) are generally trade secrets. Nichols Coming (San Juan Capisti:ano, California) manufactures a chemiluminescent assay using purified recombinant CHO-cell derived human TPO.

Sequence Information As reported by three groups (Magnusson et al., 1987; Libert et al., 1987; Kimura et al., 1987), the amino sequence of human TPO includes 933 amino acids. Of

the two TPO linear epitopes recognized by autoantibodies, one (mAb47/C21; amino acid residues 7 1 3 721) lies outside the autoantibody immunodominant region (Chazenbalk et al., 1993b) and, therefore, represents only a minor component in the autoantibody repertoire. The proportion of antibodies in an individual serum to the C2 linear epitope (amino acid residues 590--622) and the relationship of this epitope to the immunodominant region is unknown (McLachlan and Rapoport, 1992). The closely associated cluster of epitopes in the immunodominant region was mapped (Chazenbalk et al., 1993a) but its precise location on the TPO molecule is unknown. The epitopes within the immunodominant region are highly conformational and probably discontinuous and cannot be localized by peptide or polypeptide fragmentscreening techniques. Mutagenesis of the entire TPO molecule is currently being used to localize this region (Nishikawa et al., 1994a). Definitive information will require crystallization of TPO-human autoantibody complexes.

817

AUTOANTIBODIES

Nomenclature Thyroid peroxidase (TPO) autoantibodies is the preferred terminology; the term "thyroid microsomal antibody" should be discarded.

Pathogenetic Role TPO autoantibodies of IgG class are invariably present in Hashimoto' s thyroiditis; concentrations correlate with the active phase of the disease (Weetman and McGregor, 1994). Autoantibodies can damage thyroid cells directly by activating the complement cascade as well as by antibody-dependent cell cytotoxicity. This evidence notwithstanding, cytotoxic T-cells could be the primary inducers of thyrocyte damage and the damage by autoantibodies could reflect a secondary amplification of the process. Circumstantial evidence for this point of view is that autoantibodies may not have primary access to TPO which is expressed mainly on the apical surface of thyroid follicular cells within the follicular lumen. There are no animal models that precisely correspond to Hashimoto's thyroiditis. Spontaneous autoimmune thyroiditis, best studied in the obese strain chicken, buffalo rat and NOD mouse, is associated primarily with autoantibodies to thyroglobulin rather than TPO. The TPO-induced murine model of thyroiditis has an accentuated cell-mediated response relative to human autoimmune thyroiditis (Kotani et al., 1990).

Genetics Inheritance of the ability to produce TPO autoantibodies is complex; the location and nature of the gene or gene cluster responsible for what appears to be a dominant component (Pauls et al., 1993) are not known. A number of candidate genes, notably including those coding for MHC antigens (Roman et al., 1992), are excluded. A panel of TPO autoantibodies, obtained from combinatorial libraries from intrathyroidal B cells (Rapoport and McLachlan, 1994), should help answer the question of thyroid autoantibody V region gene inheritance. Thus, some TPO autoantibodies are encoded by a V H gene 91% similar to germline gene hv1263 (Chazenbalk et al., 1993a) which is closely related to 5 l pl, a member of a highly polymorphic group of VH1 genes (Sasso et al., 1993).

818

V H gene polymorphism may play a role in the inheritance of TPO autoantibodies.

Isotypes, Subclasses, Affinity and Epitopes TPO autoantibodies are predominantly IgG with much lower levels of IgA (Prummel et al., 1993). As reviewed elsewhere (McLachlan and Rapoport, 1992), autoantibodies are represented by IgG1 > IgG2 > IgG4 >> IgG3, kappa > lambda, and have high affinities for TPO (-~10-1~ kd). An immunodominant region of TPO comprising conformational epitopes was mapped using a large repertoire of monoclonal human, IgG class TPO autoantibodies generated by the combinatorial library approach (Rapoport et al., 1995). Like their counterparts in human serum, these TPO human monoclonal autoantibodies are of subclasses IgG1 and IgG4 and are of very high affinity. The majority (44) are of kappa light chain type. Four lambda TPO human autoantibodies were recently cloned (Portolano et al., 1995). The immunodominant region contains two partly overlapping domains (A and B) on the surface of native TPO that are recognized by -80% of TPO autoantibodies in individual patient sera (Chazenbalk et al., 1993a; 1993b). Use of recombinant human F(ab) monoclonal TPO autoantibodies to "fingerprint" the profiles of TPO epitopes reactive with polyclonal TPO autoantibodies in individual sera (Nishikawa et al., 1994b), shows the epitope profiles are not related to disease activity and are conserved over long periods of time (Jaume et al., 1995a; 1995b). Some sera containing autoantibodies to TPO crossreact with myeloperoxidase and/or lactoperoxidase, and there is evidence that some TPO autoantibodies cross-react with thyroglobulin and that some may inhibit, in part, TPO enzymatic activity (Rapoport and McLachlan, 1994). The cloned autoantibodies to the immunodominant region on TPO have none of these properties (Nishikawa et al., 1995). There is no information on molecular mimicry and disease is unrelated to gammopathy. Polyclonal activation is unlikely because a high degree of somatic mutation in many TPO autoantibody V H genes, as well as their high affinity for TPO, suggest an antigen-driven process (Chazenbalk et al., 1993a). A role for TPO-specific cytotoxic T cells in spontaneous human autoimmune thyroid disease is possible, but supporting evidence for this phenomenon is lacking.

Methods of Detection Although still very prevalent, assays using thyroid microsomal extracts are outdated. Specific assays for TPO autoantibodies include: ELISA, precipitation of 125I-labeled TPO with protein A, competition for TPO binding to immobilized murine monoclonal antibodies, autoantibody capture by TPO-coated beads and detection with chemiluminescent TPO. All are good assays. The ELISA and chemiluminescent assays have the advantage of not using radioactivity.

CLINICAL UTILITY

Application Not only does the presence of TPO autoantibodies unequivocally confirm autoimmune thyroiditis, but they are frequently the presenting indication of underlying disease. There is a good association between presence of TPO autoantibodies and histological thyroiditis (Yoshida et al., 1978). However, because of the extensive regenerative capacity of the thyroid under the influence of thyroid stimulating hormone (TSH), chronic thyroid damage can be present for years before the clinical manifestations of hypothyroidism are evident, if ever. Thus, most individuals with autoimmune (Hashimoto's) thyroiditis are asymptomatic. The detection of TPO autoantibodies is evidence against goiter or hypothyroidism of a nonautoimmune variety, for example colloid or "simple" goiter. Because TPO autoantibodies are present in all forms of autoimmune thyroid disease, including Graves' disease with hyperthyroidism, they cannot be used to subclassify or differentiate among different diseases.

Disease Associations As in autoimmune thyroid disease, TPO autoantibodies are present predominantly in women (female: male approximately 7:1). TPO autoantibodies can occur in neonates (beyond the placental transfer period), in young children and in adolescents. Through at least the sixth decade, their prevalence increases with age in women (Prentice et al., 1990). Family members are more likely to be TPO autoantibodypositive. TPO autoantibodies can be present in all races and in all regions of the world, perhaps being less common in Africa.

TPO autoantibodies correlate with histological thyroiditis. Quantitatively, there is a tendency for individuals with higher autoantibody levels to be more susceptible to hypothyroidism. In the general population, this relationship is not very close because other variables, including the regenerative capacity of the thyroid and dietary iodine intake contribute to the development of hypothyroidism (Weetman and McGregor, 1994). The annual risk of developing hypothyroidism was raised from 2.6 to 4.3% per year if TPO autoantibodies were present in addition to raised serum TSH levels (Vanderpump et al., 1995). The clearest correlation between TPO autoantibodies and disease is in pregnancy and in the postpartum period. Asymptomatic women who in the first trimester of pregnancy have the highest levels of TPO autoantibodies are most likely to develop hypothyroidism in the postpartum period (Amino et al., 1978). Further, the extent of the rebound in TPO autoantibodies after delivery, particularly of IgG1 subclass (Jansson et al., 1986), correlates well with the development of hypothyroidism. Pregnancy is, arguably, the most important indication for measurement of TPO autoantibodies. The hypothyroidism of Hashimoto's thyroiditis is fully corrected by administration of synthetic thyroxine. There is no clear evidence that this therapy has any influence on TPO autoantibody levels. Thionamide drugs used to inhibit thyroid hormone synthesis in Graves' hyperthyroidism do reduce TPO autoantibody levels; however, this effect is unlikely to be of clinical significance. Patients with underlying autoimmune thyroiditis who are treated for unrelated conditions with immunosuppressive agents, such as glucocorticoids, have decreases in their TPO autoantibody levels. Conversely, cytokine therapy for unrelated conditions may enhance TPO autoantibody levels and some individuals may develop thyroid dysfunction. Unorthodox therapies (IV immunoglobulin and plasmapheresis) may influence antibody levels, but have no role in the treatment of autoimmune thyroid disease. TPO autoantibodies, except those of IgG3 subclass, can cross the placenta but are less likely to cause hypothyroidism than TSH receptor antibodies which can induce neonatal thyroid dysfunction. This difference is presumably because metabolic clearance of TPO autoantibodies in the neonate reduces the period over which thyroid damage occurs. In contrast, TSH receptor autoantibodies can affect thyroid function on a more acute basis.

819

TPO autoantibodies are present in a large proportion of patients with newly diagnosed, untreated Graves' disease. Further, TPO autoantibodies (and hence autoimmune thyroiditis) are also more frequently present in individuals with other organ-specific autoimmune diseases, including type I diabetes mellitus, autoimmune adrenalitis (Addison's disease), pernicious anemia and polyglandular endocrine failure syndromes.

Sensitivity, Specificity, Predictive Values Experience is only now accumulating with new assays of greater sensitivity and specificity for TPO autoantibodies. Almost all have analytical sensitivities and specificities of > 95%. The use of recombinant TPO of nonthyroidal origin has eliminated the rare falsepositive results observed previously with thyroglobulin-contaminated thyroid microsomes (Kaufman et al., 1990). It remains to be seen whether or not reported cross-reactivities of polyclonal serum TPO autoantibodies with thyroglobulin and myeloperoxidase, for example, are valid or of clinical significance. Because most autoimmune thyroid disease is subclinical, the positive predictive value for hypothyroidism is not very high. Most individuals with TPO autoantibodies (and, incidentally, thyroglobulin autoantibodies) remain euthyroid as defined by serum TSH levels. The greatest positive predictive value in measuring TPO autoantibodies is in pregnancy.

REFERENCES Amino N, Kuro R, Tanizawa O, Tanaka, F, Hayashi C, Kotani K, Kawashima M, Miyai K, Kumahara Y. Changes of serum antithyroid antibodies during and after pregnancy in autoimmune thyroid diseases. Clin Exp Immunol 1978;31:30-37. Belyavin G, Trotter WR. Investigations of thyroid antigens reacting with Hashimoto's. sera. Lancet 1959;i:648--652. Chazenbalk GD, Portolano S, Russo D, Hutchison JS, Rapoport B, McLachlan SM. Human organ-specific autoimmune disease: molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ. J Clin Invest 1993a;92:62-74. Chazenbalk GD, Costante G, Portolano S, McLachlan SM, Rapoport B. The immunodominant region on human thyroid peroxidase recognized by autoantibodies does not contain the monoclonal antibody 47/c21 linear epitope. J Clin Endocrinol Metab 1993b;77:1715-1718. Czarnocka B, Ruf J, Ferrand M, Carayon P, Lissitzky S. Purification of the human thyroid peroxidase and iden820

CONCLUSIONS TPO autoantibodies are proven markers of the immune response to the thyroid in Hashimoto's thyroiditis. The formerly elusive microsomal antigen is now known to be TPO. The cloning and eukaryotic expression of the cDNA for this glycoprotein provides a source of pure, conformationally intact antigen. The availability of this antigen is now used in new assays of enviable sensitivity and specificity for the clinical evaluation of autoimmune diseases. Pure recombinant antigen enabled the molecular cloning and expression of a large repertoire of human monoclonal TPO autoantibodies, which for the first time, permit detailed epitopic fingerprinting of polyclonal TPO autoantibodies in an individual patient's serum. Further, new insights into the genes coding for organspecific autoantibodies may be of value in understanding the genetic background and in predicting the disease. Finally, the availability of recombinant human TPO autoantibodies permits investigation of the process of TPO-specific capture by B cells and presentation to antigen-specific T cells. These studies may lead to new information for helper T-cell regulation of autoantibody production and, ultimately, to specific therapeutic intervention. See also THYROGLOBULIN AUTOANTIBODIESand THYROTROPINRECEPTOR AUTOANTIBODIES.

tification as the microsomal antigen involved in autoimmune thyroid diseases. FEBS Lett 1985;109:147--152. Jansson R, Thompson PM, Clark F, McLachlan SM. Association between thyroid microsomal antibodies of subclass IgG1 and hypothyroidism in autoimmune postpartum thyroiditis. Clin Exp Immunol 1986;63:80--86. Jaume JC, Parkes AB, Lazarus JH, Hall R, Costane G, McLachlan SM, Rapaport B. Thyroid peroxidase autoantibody fingerprints. II. A longitudinal study in postpartum thyroid, itis. J Clin Endocrinol Metab 1995a;80:1000-1005. Jaume JC, Costante G, Nishikawa T, Phillips DI, Rapoport B, McLachlan SM. Thyroid peroxidase autoantibody fingerprints in hypothyroid and euthyroid individuals. I. Cross-sectional study in elderly women. J Clin Endocrinol Metab 1995b;80: 994-999. Kaufman KD, Rapoport B, Seto P, Chazenbalk GD, Magnusson RP. Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis. J Clin Invest 1989;84:394-403. Kaufman KD, Filetti S, Seto P, Rapoport B. Recombinant

human thyroid peroxidase generated in eukaryotic cells: a source of specific antigen for the immunologic assay of antimicrosomal antibodies in the sera of patients with autoimmune thyroid disease. J Clin Endocrinol Metab 1990;70:724--728. Kimura S, Kotani T, McBride OW, Umeki K, Hiai K, Nakayama T, Ohtaki S. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs. Proc Natl Acad Sci USA 1987;84:5555-5559. Kotani T, Umeki K, Hirai K, Ohtaki S. Experimental murine thyroiditis induced by porcine thyroid peroxidase and its transfer by the antigen-specific T cell line. Clin Exp Immunol 1990;80:11-- 18. Libert F, Ruel J, Ludgate M, Swillens S, Alexnader N, Vassart G, Dinsart C. Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules. EMBO J 1987;6:4193--4196. Magnusson RP, Chazenbalk GD, Gestautas J, Seto P, Filetti S, Rapoport B. Molecular cloning of the complementary deoxyribonucleic acid for human thyroid peroxidase. Mol Endocrinol 1987; 1:856--861. McLachlan SM, Rapoport B. The molecular biology of thyroid peroxidase: cloning, expression and role as autoantigen in autoimmune thyroid disease. Endocr Rev 1992;13:192-206. Nakagawa H, Kotani T, Ohtaki S, Nakamura M, Yamazaki I. Purification of thyroid peroxidase by monoclonal antibodyassisted immunoaffinity chromatography. Biochem Biophys Res Comm 1985;127:8-14. Nishikawa T, Nagayama Y, Seto P, Rapoport B. Human thyroid peroxidase-myeloperoxidase chimeric molecules: tools for the study of antigen recognition by thyroid peroxidase autoantibodies. Endocrinology 1994a; 133:2496--2501. Nishikawa T, Costante G, Prummel MF, McLachlan SM, Rapoport B. Recombinant thyroid peroxidase autoantibodies can be used for epitopic "fingerprinting" of thyroid peroxidase autoantibodies in the sera of individual patients. J Clin Endocrinol Metab 1994b;78:944--949. Nishikawa T, Rapoport B, McLachlan SM. Exclusion of two major areas on thyroid peroxidase from the immunodominant region containing the conformational epitopes recognized by human autoantibodies. J Clin Endocrinol Metab 1994c;79: 1648-- 1654. Nishikawa T, Jaume JC, McLachlan SM, Rapoport B. Human monoclonal autoantibodies against the immunodominant region on thyroid peroxidase: lack of cross-reactivity with related peroxidases or thyroglobulin and inability to inhibit thyroid peroxidase enzymatic activity. J Clin Endocrinol Metab 1995;80:1461--1466. Pauls DL, Zakarija M, McKenzie JM, Egeland JA. Complex segregation analysis of antibodies to thyroid peroxidase in Old

Order Amish families. Am J Med Genet 1993;47:375-379. Portmann L, Hamada N, Heinrich G, DeGroot LJ. Antithyroid peroxidase antibody in patients with autoimmune thyroid disease: possible identity with antimcrosomal antibody. J Clin" Endocrinol Metab 1985:61:1001-1003. Portolano S, Prummel MF, Rapoport B, McLachlan SM. Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type. Mol Immunol 1995;in press. Prentice LM, Phillips DIW, Sarsera D, Beever K, McLachlan SM, Rees Smith B. Georgrahical distribution of subclinical autoimmune thyroid disease in Britain: a study using highly sensitive direct assays for autoantibodies to thyroglobulin and thyroid peroxidase. Acta Endocrinol 1990;123:493--498. Prummel MF, Wiersinga WM, Rapoport B, McLachlan SM. IgA class thyroid peroxidase and thyroglobulin autoantibodies in Graves' disease: association with the male sex. Autoimmunity 1993;16:153--155. Rapoport B, McLachlan SM. Thyroid peroxidase as an autoantigen in autoimmune thyroid disease: update 1994. In: NegroVilar A, Braverman LE, Refetoff S, eds. Endocrine Review Monographs 3. Clinical and Molecular Aspects of Diseases of the Thyroid. Bethesda: The Endocrine Society, 1994:96-102. Rapoport B, Portolano S, McLachlan SM. Combinatorial libraries: new insights into human organ-specific autoantibodies. Immunol Today 1995;16:43--49. Roman SH, Greenberg D, Rubinstein P, Wallenstein S, Davies TF. Genetics of autoimmune thyroid disease: lack of evidence for linkage to HLA within families. J Clin Endocrinol Metab 1992;74:496-503. Sasso EH, illms van Dijk, Bull AP, Milner EC. A fetally expressed immunoglobulin VH1 gene belongs to a complex set of alleles. J Clin Invest 1993;91:2358--2367. Seto P, Hirayu H, Magnusson RP, Portman L, DeGroot LJ, Rapoport B. Isolation of a cDNA clone for the thyroid microsomal antigen. Homology with the gene for thyroid peroxidase. J Clin Invest 1987;80:1205-1208. Vanderpump MPJ, Tunbridge WMGT, French JM, Appleton D, Bates D, Clark F, Grimley Evans J, Hasan DM, Rodgers H, Tunbridge F, Young ET. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Wicldaam survey. Clin Endocrinol 1995;43:55--68. Weetman AP, McGregor AM. Autoimmune thyroid disease: further developments in our understanding. Endocr Rev 1994;15:788--830. Yoshida H, Amino N, Yagawa K, Uemura K, Satoh M, Miayi K, Kumahara Y. Association of serum antithyroid antibodies with lymphocytic infiltration of the thyroid gland: studies of seventy thousand autopsied cases. J Clin Endocrinol Metab 1978;46:859--862.

821

9 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

THYROTROPIN RECEPTOR AUTOANTIBODIES Robert Volp6, M.D.

Division of Endocrinology, Department of Medicine, University of Toronto, Endocrinology Research Laboratory, Wellesley Hospital, Toronto, Ontario M4Y 1J3 Canada

HISTORICAL NOTES

Before their discovery, thyrotropin (TSH) receptor (TSHR) antibodies were the subject of speculation regarding the cause of Graves' disease (GD). As late as 1962, arguments for and against TSH per se as the cause of GD were elaborated in a well-known textbook (Bakke and Werner, 1962). In 1956, an extrapituitary origin for GD was argued on the basis of an abnormal thyroid stimulator of extrapituitary origin (Adams and Purves, 1956); this was later termed longacting thyroid stimulator, LATS (Bakke and Werner, 1962). In 1964, LATS was shown to be an immunoglobulin G (IgG), i.e., an antibody capable of stimulating the thyroid (Kriss et al., 1964). Because LATS mimicked most, if not all, of the effects of TSH, it seemed likely that both TSH and LATS-IgG stimulated thyroid function by interacting with the same TSHR (McKenzie, 1968). The dose-dependent inhibition of 125I-labeled TSH binding to its receptor on thyroid membranes by Graves' IgG confirmed this concept (Solomon and Chopra 1972; Manley et al., 1974). An explosion of knowledge ensued regarding the autoantigen and the antibodies which respond to it. Because it could be detected in the serum of only a variable proportion of Graves' patients, LATS was considered by some to be merely an epiphenomenon (Solomon and Chopra, 1972; Adams et al., 1975); whereas, a "LATS protector" was the true stimulator (Adams et al., 1975). However, even at the time, LATS was thought to stimulate thyroid function by interacting with the same cell surface receptor as TSH, but this was not immediately established. Human thyroid cells in vitro were soon found to be more effective as assay systems than the intact mouse (Onaya et al., 1973), and problems of cross-

822

reactivity and sensitivity were overcome (Zakarija and McKenzie, 1987). Antibodies capable of stimulating human thyroid cells were subsequently detected in virtually all patients with Graves' disease (Zakarija and McKenzie, 1987; Rees et al., 1988). Not" all TSHR antibodies, however, stimulated the thyroid cells. Rather, some could block the effect of TSH and contribute to hypothyroidism (Rees et al., 1988).

THE AUTOANTIGEN

The TSHR, thoroughly studied over the past few years (Rees et al., 1988; Weetman and McGregor, 1994), is present on the thyroid cell surface in very small amounts (103 to 104 sites per cell) and consists of an and [3 subunit linked by a disulfide bridge. The subunit (50 kd) is water soluble and forms the binding site for TSH on the outside surface of the cell membrane. The ~ subunit (30 kd) penetrates the liquid bilayer. The greatest recent stimulus to the field came from the molecular cloning of the TSHR (Parmentier et al., 1989) and the subsequent cloning of TSHR from human and rat thyroid tissue (Nagayama and Rapoport, 1992). Sequence similarity between TSHR and the previously cloned luteinizing hormone (LH) (cG receptor, a member of the G protein-coupled receptor family) was assumed; primers derived from transmembrane sequences of the LH/cG receptor allowed successful cloning of the human TSHR (Nagayama and Rapoport, 1992; Weetman and McGregor, 1994; Lefkowitz, 1995). Recent evidence suggests that the TSHR exists as a single polypeptide chain without subunits (Nagayama and Rapoport, 1992). Human TSHR is encoded by a single gene located on chro-

mosome 14q31 and spans more than 60 kilobases. The generally hydrophilic amino-terminal half of the receptor encodes the large extracellular region with sequence similarity to the leucine-rich glycoprotein family. The carboxyl-terminal half of the receptor contains the characteristic seven hydrophobic membrane-spanning segments. A segment between amino acids 30 and 50 in the extracellular domain is involved either in TSH binding, or is related to maintaining the correct conformation of the molecule. There is considerable variation in T-lymphocyte activation and antibodies binding to different epitopes on the receptor (Weetman and McGregor, 1994). The epitopes for antibodies span the entire TSHR extracellular domain and are not usually linear (Weetman and McGregor, 1994; Vlase et al., 1995). Although the amino acid sequence of the transmembrane domain of human TSHR resembles hLH/ cG receptor, the similarity is much less within the extracellular domain (Figures 1 and 2). Solubilized TSHR circulates in small amounts and thus is available to the immune system (Murakami et al., 1993). The extracellular domain of TSHR is highly immunogenic, at least in terms of inducing strong, specific proliferative T-cell responses (Carayanniotis et al., 1995). A 1.3 kilobase variant of TSHR mRNA is found not only in the thyroid but in low amounts in extraocular muscle and, to a lesser extent, in fat and fibroblasts (Paschke et al., 1994b). TSHR transcripts or cDNA fragments are found in many tissues including human peripheral lymphocytes, fatty tissues and muscles (Davies, 1994). Quite possibly, the list will be expanded to include other tissues such as the adrenal glands and gonads, which also have high affinity, low capacity TSH binding sites (Trokoudes et al., 1979). Recently, somatic and germ-line mutations of the TSHR genes (generally within the transmembrane domain) were found to cause either constitutive activation or resistance (Paschke et al., 1994a; Kopp et al., 1995; Sunthornthepvarakul et al., 1995). These mutations have not changed the immunogenicity of the receptor (Watson et al., 1995).

THE AUTOANTIBODIES Terminology While some TSHR antibodies stimulate the thyroid, others are inhibitory. Thus, the definitions currently

utilized for the various TSHR antibodies include terms describing assays or functions. The term "thyrotropinbinding inhibitory immunoglobulin (TBII)" refers to antibodies which bind to the TSHR, thereby preventing the binding of labeled TSH. Thyroid stimulating antibodies (TSAb), on the other hand, are those which stimulate thyroid cells resulting in increased thyrocyte cAMP in bioassay. Finally, thyroid stimulation blocking antibodies (TSBAb) refers to a similar bioassay in which inhibition of TSH-generated cAMP is demonstrated in thyrocytes (Volp6, 1990).

Pathogenetic Role Human Disease Model. Thyroid stimulating antibodies (TSAb) are the proximate cause of hyperthyroidism in GD as is readily demonstrated in neonatal GD in which passive transfer of TSAb cause the fetus and neonate to be hyperthyroid for several weeks after delivery (Volp6, 1990). In addition, when TSAb are still present in a GD patient at the end of a course of antithyroid drugs, cessation of the medication will almost invariably lead to relapse. Conversely, when TSAb disappear following treatment, there is a greater probability that the patient will remain in remission for at least a short time. The prevalence of TSAb in GD is --95% (Volp6, 1990). Although occasionally found in patients with Hashimoto's thyroiditis who do not manifest hyperthyroidism (usually because of thyroid parenchymal cell damage), TSAb are not found in normal persons. In GD during pregnancy, TSAb and thyroid activity decline in the third trimester but rebound in the postpartum state. The TSAb found in patients recovering from yersiniosis, are not accompanied by thyroid dysfunction (Wolf et al., 1991). Conversely, in destructive thyroiditis, TSAb may appear transiently as a consequence of antigen shedding (Volpe, 1990). Thyroid Stimulation Blocking Antibodies (TSBAb) are reported in large numbers of patients with atrophic thyroiditis and severe hypothyroidism (Weetman and McGregor, 1994). Transplacental transfer of TSBAb can produce transient neonatal hypothyroidism (Weetman and McGregor, 1994). Although some patients with goitrous hypothyroidism have TSBAb, the role of the antibodies is not clear, because in these cases TSBAb do not correlate with the severity of the hypothyroidism and the goiter would be expected to regress. Atrophic thyroiditis also occurs commonly in the absence of TSBAb; hence, the role of TSBAb in atrophic thyroiditis should be re-evaluated (Volp6, 1990). 823

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TSHR

761 OTVL

Figure 1. Amino acid sequence of the human TSHR. Amino acid numbering includes the 20-residue signal peptide. Sequence similarity to human LH/cG and FSH receptors is shown. Amino acids are shown in the single amino acid code - - -, identical amino acids .... gap. The exons are indicated as 1-10. Membrane-spanning regions are boxed and designated I-VII. The entire transmembrane region of the receptor is shown within the large box. A number of features in the extracellular region of the receptor are boxed. These are the cysteine residues (C), the potential N-linked glycosylation sites (N-X-S/T), and the two unique tracts in the TSHR (amino acid residues 38-45 and 317-366). The thyrotropin receptor 25 years after discovery: new insight after molecular cloning (Nagayama and Rapoport, 1992).

A n i m a l Model. No spontaneous or induced animal models for TSHR antibodies are reported. Immunization of mice with TSHR results in T-cell responses, but unaltered thyroid function (Carayanniotis et al., 1995). Genetics The disturbances in immunoregulation which lead to the development of TSHR antibodies are partly

8 2 4

genetic and partly environmental in nature (Volp6, 1990). Although GD and other organ-specific autoimmune diseases with which it is associated tend to aggregate in families, the modes of inheritance do not follow simple genetic rules. Environmental factors such as stress, infection, trauma, drugs, nutrition, smoking and aging might distort penetrance and expression by acting on the immune system (Volp6, 1990). The strong female preponderance in most may

Figure 2. Structure of the TSHR and locations of known mutations. The amino acids are indicated by the single-letter code and numbered consecutively starting with the transcription-initiation codon. The Y on asparagine residues (N) identifies potential sites of glycosylation. The vertical lines indicate exon boundaries (Sunthornthepvarakul et al., 1995).

be partially related to the effect of one gene upon another and partially on hormonal factors. The concordant of GD in dizygotic twins is reported to be --3--9%, and in monozygotic twins, --30--60% (Volp6, 1990). The higher concordance rate in monozygotic twins is strong evidence for a genetic basis for GD; but genetic factors alone do not explain the lack of concordance in 40-70%. The age of initiation of the disease varies widely between twins, even as much as

10 years (Volp6, 1990). Moreover, one identical twin may have GD, while the other has Hashimoto's thyroiditis. However, it may not follow that environmental factors are necessarily responsible for this lack of concordance in monozygotic twins. The immune system generates its enormous diversity of IgG genes and T-lymphocyte receptor genes so that identical twins are unlikely to be identical for key immunological genes.

825

In addition to an increased frequency of HLADR3, in Caucasians with GD (Volp6, 1990), HLADQAl*0501 is greatly increased among GD patients with a relative risk of 3.35 even after exclusion of DR3-positive subjects (Yanagawa et al., 1993). Studies of T-cell receptor genes and thyroid peroxidase antibody genes do not show any association (Weetman and McGregor, 1994). Reduced variability of T-lymphocyte receptor Va and Vf~ gene usage in intrathyroidal cells in GD was reported (Davies et al., 1991), but not confirmed (Mclntosh et al., 1993). T-cell gene rearrangements show no oligoclonality in the majority of patients with GD (Weetman and McGregor, 1994). The binding of TSH and TSHR antibodies is closely related, and indeed, mutually exclusive (Nagayama and Rapoport, 1992). The 50 kd Fab fragments of TSHR antibodies compete effectively with TSH in receptor-binding studies and are powerful TSH agonists; whereas, Fab fragments from TSBAb act as powerful TSH antagonists. The light chain restriction of TSHR antibodies, usually lambda, is unexpected and unexplained (Zakarija and McKenzie, 1987). Molecular mimicry between the TSHR antigen and microbial antigens is suggested in respect to Yersinia enterocolitica, but other organisms are also mentioned (Weetman and McGregor, 1994; Volp6, 1990). Crossreactivity between antigens from certain micro-organisms, especially, Y. enterocolitica and thyroid cell membrane antigens (including TSHR) is reported (Weetman and McGregor, 1994) but not confirmed (Arscott et al., 1992; Resetkova et al., 1994). Such cross-reactivity, although possibly representing "molecular mimicry", does not p e r se imply a significant role in the induction of GD; indeed, the evidence suggests otherwise. For example, in patients recovering from active Y. enterocolitica infections, TSHR antibodies (some with thyroid-stimulating properties) are frequently detectable in the absence of thyroid dysfunction (Wolf et al., 1991). Furthermore, although immunization of experimental animals with Y. enterocolitica leads to production of TSHR antibodies, the histology of the animal thyroid glands is normal (Sakata et al., 1988). Antigen presentation, even if the antigen is of similar sequence to TSHR, is apparently insufficient to induce GD. The possibility of an anti-idiotype that functions as an agonist to the original antigen has to be considered (Zakarija and McKenzie, 1988). Experimentally induced anti-Id antibodies to TSH autoantibodies do bind to the TSHR and stimulate the thyroid (Zakarija 826

and McKenzie, 1988). However, were the idiotypic network the explanation for TSAb, more examples of anti-TSH in autoimmune disease would be expected.

Methods of Detection Receptor Assay (Thyrotropin Binding Inhibitory Immunoglobulin, (TBII)). Inhibition of binding of 125I-TSH to thyroid membranes forms the basis of a simple assay for TSHR antibodies. Detergent-solubilized thyroid membranes show virtually no nonspecific interference with normal IgG (Rees et al., 1988). The inhibition of labeled TSH binding to the detergentsolubilized TSHR by TSHR antibodies shows a steeper dose-response relationship than that using particulate membrane preparations. The 125I-labeled bovine TSH should have a high specific biological activity. To achieve this, the labeled TSH is reacted with TSHR; after separation of bound and free, the receptor-bound "active" hormone is dissociated and further purified by gel filtration. This receptor-purified, 125I-labeled TSH combined with a detergentsolubilized TSHR provides the basis of a rapid, sensitive, specific and inexpensive assay (TBII) for TSHR antibodies in unextracted serum.

Bioassays. Assays for LATS are insensitive and only poorly reproducible (Volp6, 1990). Interaction of serum with isolated thyroid cells in culture, measuring the release of cAMP into a hypotonic medium vastly improves assays for TSAb. IgG concentrates are usually employed, although serum dialyzed against a hypotonic medium can be used directly. Cells from human or porcine tissue or the rat thyroid cell line FRTL5 can be utilized (Rees et al., 1988). There is a close correlation between results of the TSAb bioassay and receptor assay results for TSHR antibodies (Rees et al., 1988; Volp6, 1990). Discrepancies often reflect the presence of TSBAb. This blocking activity is also measured by a bioassay with incubation performed in the presence of 100 mla/L of bovine TSH.

CLINICAL UTILITY

Application/Disease Association The major use of TSHR antibodies is in the management and occasionally the diagnosis of patients with GD (Table 1). The bioassay is not frequently em-

ployed, as the TBII can be considered almost synonymous with TSAb in patients with hyperthyroidism. Patients suffering from hyperthyroidism with a diffuse goiter can be readily diagnosed as GD without such assays. However, when there is no exophthalmos, assay may prove useful. As mentioned above, TSAb which are detectable in 95% of patients, correlate with disease activity, being highest in those with large goiters, severe exophthalmos and pretibial myxedema. TSAb decrease with large doses of corticosteroid. The assay can be transiently positive in subacute and silent thyroiditis, and in yersiniosis, and is thus not completely specific (Volp6, 1990). The assays are most valuable in relation to antithyroid drug therapy (Volp6, 1994a). The declines in TSHR antibodies which usually occur, do not reflect direct immunosuppression, but rather modulation of thyroid cell functions, including thyroid hormone synthesis, with consequent reduction of thyrocyteimmunocyte signaling (Volp6, 1994b). Assays for TSH receptor antibodies can predict relapse of hyperthyroid GD following antithyroid drug therapy, i.e., assays positive at the end of treatment portend relapse. However, a negative result at the end of treatment does not preclude relapse. After 131I therapy for GD, the titers rise for several months (Volp6, 1990). Assays for TSHR antibodies are very useful for monitoring GD during pregnancy. Because the antibodies tend to decline in the third trimester (sometimes to normal), only to rebound in the postpartum period (Volp6, 1990), the dosage of antithyroid drugs can usually be reduced to low levels in the third trimester without difficulty. In that small proportion of pregnant GD patients in whom the antibodies are still very high in the third trimester, fetal and neonatal

hyperthyroidism may ensue due to the transplacental passive transfer of the TSAb with severe complications in the infant, such as craniosynostosis and even fatalities (Zakarija and McKenzie, 1987; Volp6, 1990). Occasionally, the blocking activity of the antibodies dominates over stimulating activity, and transient hypothyroidism results (Zakarija and McKenzie, 1987; Volp6, 1990). Being due to passive transfer of the antibody, these conditions last only several weeks, but their severity often requires prompt treatment. In patients with exophthalmos, but no evidence of hyperthyroidism, the presence of TSAb suggests euthyroid ophthalmic Graves' disease (Volp6, 1990). For the diagnosis of hypothyroidism, particularly in patients with atrophic thyroiditis and in transient neonatal hypothyroidism, assays for TSBAb are sometimes useful (Zakarija and McKenzie, 1987; Rees et al., 1988).

CONCLUSION Antibodies to the TSHR have clear-cut functional consequences and are disease-producing when they arise. GD is an antibody-mediated disorder with a direct relationship to the presence and amount of TSAb. TSBAb, on the other hand, may be a cause or at least a factor in hypothyroidism. The determination of TSHR antibodies is occasionally useful in the diagnosis of GD, but is more valuable in the management during antithyroid drug treatment as well as during and after pregnancy. TSHR antibodies in the absence of hyperthyroidism can be present transiently in subacute and silent thyroiditis and in convalescent yersiniosis.

Table 1. Significance of TSAb* 9

Positivein -95% of patients with GD.

9

Transientlypositive in some patients with subacute or silent thyroiditis and after acute yersiniosis (cross-reactivity).

9

Risesfurther for several months after 131I therapy for GD.

9

Usually(not invariably) decline with antithyroid drug therapy.

9

If positive after antithyroid drug course, relapse of GD almost invariable.

9

Declinein 3rd trimester of pregnancy, rebound thereafter.

9

If high in late pregnancy, can cause fetal and neonatal GD.

9

Positivetest [aelps to diagnose euthyroid exophthalmos.

827

REFERENCES Adams DD, Dirmikis S, Doniach D, E1 Kabir DJ, Hall R, Ibbertson HK, Irvine WJ, Kendall-Taylor P, Manley SQ, Mehdi SW, Munro DS, Purves HD, Smith BR, Stewart RD. Nomenclature of thyroid-stimulating antibodies [Letter]. Lancet 1975;1:1201. Adams DD, Purves HD. Abnormal responses to the assay of thyrotrophin. Proceedings of the University of Otago Medical School 1956;34:11-12. Arscott P, Rosen EC, Koenig RJ, Kaplan MM, Ellis T, Thompson N, Baker JR Jr. Immunoreactivity to Yersinia enterocolitica antigens in patients with autoimmune thyroid disease. J Clin Endocrinol Metab 1992;75:295--300. Bakke J, Werner SC. Etiology of toxic diffuse goiter. In: Werner SC, ed. The Thyroid, a Fundamental and Clinical Text, 2nd edition. New York: Harper and Row, 1962:506514. Carayanniotis G, Huang GC, Nicholson LB, Scott T, Allain P, Mcgregor AM, Banga JP. Unaltered thyroid function in mice responding to a highly immunogenic thyrotropin receptor: implications for the establishment of a mouse model for Graves' disease. Clin Exp Immunol 1995;99:294-302. Davies TF. The thyrotropin receptors spread themselves around (Editorial). J Clin Endocrinol Metab 1994;79:1232-1233. Davies TF, Martin A, Concepcion ES, Graves P, Cohen L, BenNun A. Evidence of limited variability of antigen receptors on intrathyroidal T cells in autoimmune thyroid disease. N Engl J Med 1991;325:238--244. Kopp P, van Sande J, Parma J, Duprez L, Gerber H, Joss E, Jameson JL, Dumont JE, Vassart G. Brief report: congenital hyperthyroidism caused by a mutation in the thyrotropinreceptor gene. N Engl J Med 1995;332:150-154. Kriss JP, Pleshakov V, Chien JR. Isolation and identification of the long-acting thyroid stimulator and its relation to hyperthyroidism and circumscribed pretibial myxedema. J Clin Endocrinol Metab 1964;24:1005-1028. Lefkowitz RJ. G proteins in medicine. N Engl J Med 1995; 332:186--187. Manley SW, Bourke JR, Hawker RW. The thyrotrophin receptor in guinea-pig thyroid homogenate: interaction with the long-acting thyroid stimulator. J Endocrinol 1974;61: 437--445. McIntosh RS, Watson PF, Pickerill AP, Davies R, Weetman. No restriction of intrathyroidal T cell receptor V~ families in the thyroid of Graves' disease. Clin Exp Immunol 1993;91: 147-152. McKenzie JM. Humoral factors in the pathogenesis of Graves' disease. Physiol Rev 1968;48:252-310. Murakami M, Miyashita K, Monden T, Yamada M, Iriuchijima T, Moil T. Evidence that a soluble form of TSH receptor is present in the peripheral blood of patients with Graves' disease. In: Nagataki S, Mori T, Torizuka K, editors. 80 Years of Hashimoto Disease. Amsterdam: Elsevier, 1993: 683--685. Nagayama Y, Rapoport B. The thyrotropin receptor 25 years after its discovery: new insight after its molecular cloning. Mol Endocrinol 1992;6:145-- 156.

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Onaya T, Kotani M, Yamada T, Ochi Y. New in vitro tests to detect the thyroid stimulator in sera from hyperthyroid patients by measuring colloid droplet formation and cyclic AMP in human thyroid slices. J Clin Endocrinol Metab 1973;36:859--866. Parmentier M, Libert F, Maenhaut C, Lefort A, Gerard C, Perret J, Van Sande J, Dumont JE, Vassart G. Molecular cloning of the thyrotropin receptor. Science 1989;246:16201622. Paschke R, Tonacchera M, Van Sande J, Parma J, Vassart G. Identification and functional characterization of two new somatic mutations causing constitutive activation of the thyrotropin receptor in hyperfunctioning autonomous adenomas of the thyroid. J Clin Endocrinol Metab 1994a;79:1785-1789. Paschke R, Metcalfe A, Alcalde L, Vassart G, Weetman A, Ludgate M. Presence of nonfunctional thyrotropin receptor variants transcripts in retroocular and other tissues. J Clin Endocrinol Metab 1994b;79:1234-1238. Rees SB, McLachlan SM, Furmaniak J. Autoantibodies to the thyrotropin receptor. Endocr Rev 1988;9:106--121. Resetkova E, Notenboom R, Arreaza G, Mukuta T, Yoshikawa N, Volp6 R. Seroreactivity to bacterial antigens is not a unique phenomenon in patients with autoimmune thyroid diseases in Canada. Thyroid 1994;4:269--274. Sakata S, Matsuda M, Komaki T, Kojima N, Yabuuci E, Miura K. Production of anti-TSH receptor antibodies in rats by immunization with Yersinia enterocolitica (Abstract). Proceeding of the 8th International Congress on Endocrinology. Kyoto, July 17--23, 1988. Solomon DH, Chopra IJ. Graves' disease- 1972. Mayo Clin Proc 1972;47:803-813. Sunthornthepvarakul T, Gottschalk ME, Hayashi Y, Refetoff S. Brief report: resistance to thyrotropin caused by mutations in the thyrotropin receptor gene. N Engl J Med 1995;332:155-160. Trokoudes KM, Sugenoya A, Hazani E, Row W, Volpe R. Thyroid-stimulating hormone (TSH) binding to extrathyroidal human tissues: TSH and thyroid-stimulating immunoglobulin effects on adenosine 3',5'-monophosphate in testicular and adrenal tissues. J Clin Endocrinol Metab 1979;48:919--923. Vlase H, Graves PN, Magnusson RP, Davies TF. Human autoantibodies to the thyrotrophin receptor: recognition of linear, folded, and glycosylated recombinant extracellular domain. J Clin Endocrinol Metab 1995;80:46--53. Volp6 R. Evidence that the immunosuppressive effects of antithyroid drugs are mediated through actions on the thyroid cell, modulating thyrocyte-immunocyte signaling: a review. Thyroid 1994a;4:217--223. Volp6 R. Autoimmune endocrinopathies: aspects of pathogenesis and the role of immune assays in investigation and management. Clin Chem 1994b;40:2132-2145. Volp6 R. Immunology of the thyroid. In: Volpe R, ed. Autoimmune Diseases of the Endocrine System. Boca Raton: CRC Press, 1990:73-240. Watson PF, French A, Pickerill AP, McIntosh RS, Weetman AP. Lack of association between a polymorphism in the coding region of the thyrotropin receptor gene and Graves'

disease. J Clin Endocrinol Metab 1995;80:1032-1035. Weetman AP, McGregor AM. Autoimmufie thyroid disease: further developments in our understanding. Endocr Rev 1994;15:788--830. Wolf M, Misaki T, Bech K, Tvede M, Silva JE, Ingbar SH. Immunoglobulins of patients recovering from Yersinia enterocolitica infections exhibit Graves' disease-like activity in human thyroid membranes. Thyroid 1991:1:315--320.

Yanagawa T, Mangklabruks A, Chang YB, Okamoto Y, Fisfalen ME, Curran PG, DeGroot LJ. Human histocompatibility leukocyte antigen-DQA*0501 allele associated with genetic susceptibility to Graves' disease in a Caucasian population. Clin Endocrinol Metab 1993;76:1569--1574. Zakarija M, McKenzie JM. The spectrum and significance of autoantibodies reacting with the thyrotropin receptor. Endocrinol Metab Clin North Am 1987;16:343--364.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

TOPOISOMERASE-I (Scl-70) AUTOANTIBODIES Dolores Vazquez-Abad, M.D. and Naomi F. Rothfield, M.D.

Department of Medicine, Division of Rheumatic Diseases, University of Connecticut Health Center, Farmington, CT 06030-1310, USA

HISTORICAL NOTES

Sera from scleroderma patients containing anti-topoisomerase-I antibodies (anti-topo-I) as originally described in 1979 (Douvas et al., 1979) reacted in double immunodiffusion with a component of calf thymus nuclei. Because the reaction was highly specific for scleroderma and because the reactive protein had a molecular weight of 70 kd, theantigen was named "Scl-70" (Douvas et al., 1979). Double immunodiffusion in agar against calf thymus extract containing Scl-70 became the standard method for detecting anti-topo-I. In 1986, Scl-70 was identified as topoisomerase-I (Shero et al., 1986) with almost simultaneous confirmation by other groups (Guldner et al., 1986; Maul et al., 1986). Although anti-topo-I were first known as anti-Scl-70 (Douvas et al., 1979), the preferred terminology is anti-topoisomerase-I antibodies (anti-topo-I) (Shero et al., 1987).

THE AUTOANTIGEN(S) Definition

Topoisomerase-I (topo-I), catalyzes the breaking/rejoining of single-stranded DNA and relaxes supercoiled DNA in vitro (Shero et al., 1986; D'Arpa et al., 1988; Heck et al., 1988; Hoffman et al., 1989). The 67.7 kd carboxyl-terminal fragment expresses the enzymatically active site (D'Arpa et al., 1988) which is located between amino acids 344 and 483 (D'Arpa et al., 1988; Hildebrandt et al., 1991). Although native topo-I has a molecular mass of 100 kd, smaller (60-90) proteolytic fragments are functionally active

830

(Shero et al., 1986; D'Arpa et al., 1988; 1990; Heck et al., 1988; Hoffman et al., 1989; Oddou et al., 1988; Juarez et al., 1988; Kosovsky and Soslau, 1993; Hildebrandt et al., 1991; Tsay et al., 1990). Native vs. Recombinant Antigen

The standard native topo-I used for detection of the autoantibodies is derived from calf thymus. Many analyses of the performance of calf thymus extracts and chromatographically purified calf thymus topo-I have been published (Hildebrandt et al., 1991; Tsay et al., 1990). The techniques for antibody detection include double immunodiffusion, ELISAs, functional enzyme assay and immunoblots. Comparison of these methods using native calf thymus topo-I shows that the direct binding ELISA with the chromatographically purified calf thymus topo-I is more sensitive than double immunodiffusion with calf thymus extract and more specific than immunoblots with extracts from human cell lines (HeLa) as another source of native topo-I (Hildebrandt et al., 1991). Autoantibodies to topo-I also cross-react with enzymatically active mitochondrial topo-I from human platelets (Kosovsky and Soslau, 1993). After recombinant human topo-I first was cloned and expressed (D'Arpa et al., 1988), different recombinant fragments spanning the complete sequence of human topo-I were used to describe a major epitope near the topo-I active site (D'Arpa et al., 1988). With human recombinant topo-I, anti-topo-I from nonrelated scleroderma patients bind a "universal" epitope, suggesting a similar humoral immune response to topo-I (Seelig et al., 1993; Kuwana et al., 1993a; Kato et al., 1993; Cram et al., 1993). An ELISA for the

detection of human anti-topo-I using an E. coli recombinant protein containing the C-terl;ninal 695 residues of human topo-I showed a sensitivity of 61% and a specificity of 98% (Verheijen et al., 1990). Comparable sensitivity for detection of autoantibodies is found with a baculovirus-expressed human topo-I (provided by W.C. Earnshaw, Ph.D.) and with chromatographically pure calf thymus topo-I (unpublished observations). The performance of chromatographically pure calf thymus topo-I was less sensitive than the baculovirus-expressed human topo-I. The recombinant topo-I was 1.2 times more sensitive by ELISA and 3.36 times more active in the functional assay (unpublished observations.) Origin/Sources

Human topo-I is transcribed from a single-copy human gene that encodes a 4.1 kb mRNA (D'Arpa et al., 1988). The encoded polypeptide from a 765 amino acid open reading frame has a molecular mass of 90,649 kd and a calculated isoelectric point of 10.05 with 26% basic and 18% acidic residues (D'Arpa et al., 1988). As mentioned, the majority of the screening for anti-topo-I is performed using calf thymus topo-I. This antigen is either commercially available as nuclear extracts, or chromatographically purified enzyme (Hildebrandt et al., 1991; Tsay et al., 1990). That the structure of topo-I is highly conserved is suggested by reports of human anti-topo-I cross reacting with mitochondrial and plant topo-I (Kosovsky and Soslau, 1993; Agris et al., 1990). Methods of Purification

Purified native topo-I is obtained from calf thymus homogenized in a buffer containing KPO 4, followed by chromatographic separation with hydroxyl apatite and Biorex 70 columns (Hildebrandt et al., 1991; Tsay et al., 1990). Commercial Sources

Native topo-I is commercially available from Gibco BRL (Grand Island, NY, catalog number: 38042), from ICN laboratories (Costa Mesa, CA, catalog number 152311), and from Promega (Madison, WI, catalog number M285 !).

THE AUTOANTIBODIES

Anti-topo-I are the predominant antibodies in silicaassociated scleroderma, an environmental toxin model for scleroderma. These anti-topo-I recognize the same epitopes as in non-silica-associated scleroderma. Silica particles seem to act as an adjuvant and trigger disease and anti-topo-I in genetically susceptible individuals (McHugh et al., 1994) Pathogenetic Role Animal Model. Histopathological changes of the skin similar to human scleroderma with collagen deposition causing cutaneous hyperplasia develop in tight skin mice which also have increased serum anti-topo-I with aging. (Muryoi et al., 1991). Monoclonal antibodies to topo-I from these mice bear a cross-reactive idiotype that is also present in non-anti-topo-I immunoglobulin from tight skin mice, but not in immunoglobulin from normal mice. Most mouse anti-topo-I derive from the VHJ558 family with random light chain associations (Muryoi et al., 1992). The epitopes identified by mouse MoAb to topo-I are at the amino terminal end of the enzyme, close to a similar sequence in the UL70 protein of cytomegalovirus. Human Disease. B cells are hypothesized to proliferate in response to a virus and the anti-topo-I develop due to molecular mimicry between the virus and topoI (Muryoi et al., 1992). Amino acid sequence similarity between a region of topo-I close to the universal C-terminal epitope and a retroviral protein suggests that molecular mimicry might play an important role in the production of anti-topo-I in humans (Jimenez and Batuman, 1993). Genetics

Anti-topo-I in Caucasians are associated with DRw 11 (Morel et al., 1994). The presence of tyrosine at position 30, alanine at position 38 or threonine at position 77 of the DQB 1 alleles correlates highly with anti-topo-I in Caucasians (Reveille et al., 1992). In a Japanese population, the same DQB1 locus with tyrosine at position 26 is associated with anti-topo-I. The association of a major B-cell epitope is with a sequence at the [31 domain of the DRB gene; whereas, the association of other epitopes with HLA-DR52 suggests that together HLA-DR and DQ genes control the autoimmune response to topo-I in Japanese 831

patients with scleroderma (Kuwana et al., 1993b). Autoreactive germline gene VH4-2 1 JH4 DXP1 is used in human IgM anti-topo-I (Vazquez-Abad et al., 1993). Use of the same germline gene in 25 clones from one patient suggests that an oligoclonal expansion might be responsible for the presence of antitopo-I (Vazquez-Abad et al., 1993).

Factors in Pathogenicity Human anti-topo-I is mainly IgG and IgA, and to a lesser extent IgM (Hildebrandt et al., 1990a; VazquezAbad et al., 1995). In two studies evaluating the isotypes of anti-topo-I over time. IgG and IgA antitopo-I were found from the beginning of the disease (Vazquez-Abad et al., 1995; Hildebrandt et al., 1993). Although a clinical association between IgM and IgA anti-topo-I was suggested (Hildebrandt et al., 1993), a subsequent study including more samples and precise follow-up failed to find an association between antitopo isotypes and clinical features (Vazquez-Abad et al., 1995). Studies to date support early B-cell selection and maturation as responsible for the IgG and IgA anti-topo-I in scleroderma patients. Exposed idiotypes of human anti-topo-I are both private and cross-reactive idiotypes. The immunodominant idiotypes from scleroderma anti-topo-I are close to the antigen-binding site and are stable after class switch, suggesting that they are mainly associated with the CDR1 or CDR2 (Vazquez-Abad et al., 1993). Human anti-topo-I identify two or more epitopes (Seelig et al., 1993; Kuwana et al., 1993a; Kato et al., 1993; Cram et al., 1993) including a "universal" epitope close to the active site, which probably explains the inhibition of the DNA-topo-I assay in vitro by human anti-topo-I (Piccini et al., 1991; Seelig et al., 1993). Taken together the results suggest that human antitopo-I result from an oligoclonal restricted B-cell response probably regulated through a particular V H gene utilization or rearrangement, and triggered by either autoantigen, an external antigen or by an environmental toxin acting as an adjuvant.

Methods of Detection Anti-topo-I are commonly detected by double immunodiffusion using calf thymus extract (Douvas et al., 1979; Hildebrandt et al., 1991)and by ELISAs using calf thymus nuclear extract or recombinant 832 "

topo-I (Hildebrandt et al., 1991; Tsay et al., 1990; Verheijen et al., 1990). Immunoblotting techniques using human nuclear extracts (HeLa cells), calf thymus nuclear extract, or recombinant proteins can be useful for confirmation of results using crude preparations of antigens. The topo-I functional enzyme assay measures the mobility of supercoiled DNA as visualized in UV light after washing the DNA agarose gel in ethidium bromide (Figure 1). When the DNA is incubated with topo-I, the DNA is decoiled and migrates slower than the supercoiled DNA. Anti-topo-I inhibits the decoiling of DNA when incubated with topo-I before adding to the DNA sample. This research assay identifies anti-topo-I that bind topo4 close to the active site, thus inhibiting the decoiling of DNA in vitro (Hildebrandt et al., 1991; Verheijen et al., 1990).

CLINICAL UTILITY

Application The presence of antitopoisomerase I antibodies confirms the diagnosis of scleroderma but does not exclude an additional diagnosis, e.g., scleroderma and systemic lupus erythematosus or scleroderma and Sj6gren's syndrome. Although the presence of antitopo-I is more common in scleroderma patients with diffuse cutaneous involvement than in those with limited cutaneous involvement, the antibody is not helpful in differentiating between diseases.

Disease Associations Anti-topo-I are present in 20--40% of scleroderma patients irrespective of age (Batuman and Jimenez, 1993; Bona and Rothfield, 1994) and in the same percentage of males and females (Rothfield and Vazquez, unpublished observation). It is more common in Japanese patients than in Caucasians (Reveille and Arnett, 1993). In American patients with proximal scleroderma, anti-topo-I are more common in Blacks than in Caucasians (Reveille and Arnett, 1993). Antitopo-I are not present in relatives of scleroderma patients (Barnett and McNeilage, 1993; Maddison et al., 1993).

Antibody Frequencies in Diseases The amount of anti-topo-I does not vary with disease

Figure 1. DNA-topoisomerase-I functional assay. The samples are run in a 1% agarose gel and bands visualized after washing the gel with ethidium bromide. All lanes contain 0.25 pg of ~X174 DNA (Gibco BRL). Lane 1 shows the DNA running alone, the upper band is decoiled and the lower band is supercoiled DNA. Lane 2 shows the effect of adding 0.5 U topoisomerase-I to the DNA, supercoiled DNA has been decoiled, migrating to the upper band. Lanes 3-10 show the results of testing sera for the presence of human sera. Lanes 4, 6, 8 and 10 show that sera has no effect on the migration of the DNA. Lanes 3, 5, 7 and 9 show the effect of incubating the sera with topoisomerase-I, lane 4 shows how the decoiling of DNA expected by adding topoisomerase-I is abolished by this serum sample Which contains antitopoisomerase-I; whereas, the other samples did not abolish the function of topo-I, and thus are negative for antitopoisomerase-I. activity or duration (Vazquez-Abad et al., 1995) nor have the antibodies been detected in patients without scleroderma except for a few patients (4/65) with primary R a y n a u d ' s syndrome (Weiner et al., 1991). Two of four patients with primary R a y n a u d ' s syndrome subsequently developed tight skin, (p < 0.004) (Weiner et al., 1991). The effect of various therapies on anti-topo-I and the transplacental transfer of IgG

anti-topo-I are not defined (Hildebrandt et al., 1990b). Patients with anti-topo-I are more likely to have facial skin involvement and heart involvement than patients without the antibody (Weiner et al., 1988). Anti-topo-I are associated with kidney involvement, pulmonary fibrosis and ischemic ulcers of the finger tips (Steen et al., 1988). The association between antitopo-I and pulmonary fibrosis is confirmed (Giordano

Table 1. Topoisomerase-I Antibodies Summary Disease associations

Scleroderma, 20--40%; rare in primary Raynaud's syndrome

Sex

Males = Females

Race

Japanese > Caucasians American Blacks > American Caucasians

Relatives of patients

Antibodies not present

Fluctuation with disease

Not with duration, activity or severity

Predictor

Tight skin in Raynaud's syndrome

Clinical associations

Facial skin, heart, kidney, pulmonary fibrosis, ischemic fingertip ulcers. Cancer association very strong.

833

et al., 1986; Kuwana et al., 1 9 9 4 ) a s is a strong association of anti-topo-I and cancer in patients with scleroderma (Weiner et al., 1988; Rothfield et al., 1992).

Sensitivity and Specificity All investigators agree that the anti-topo-I test is very specific; anti-topo-I are reported in only one normal healthy individual who did not have scleroderma. The individual originally studied as a normal healthy control for studies has been followed for 8 years.

REFERENCES Agris PF, Parks R, Bowman L, Guenther RH, Kovacs SA, Pelsue S. Plant DNA topoisomerase I is recognized and inhibited by human scl-70 sera autoantibodies. Exp Cell Res 1990; 189:276--279. Barnett AJ, McNeilage LJ. Antinuclear antibodies in patients with scleroderma (systemic sclerosis) and in their blood relatives and spouses. Ann Rheum Dis 1993:52:365-368. Batuman OA, Jimenez SA. Systemic sclerosis in the molecular pathology of autoimmune disease. In: Bona C, Siminovitch KA, Zanetti M, Theofilopoulos AN, eds. The Molecular Pathology of Autoimmune Diseases. Switzerland-Harwood: Academic Publications, 1993:377-399. Bona C, Rothfield N. Autoantibodies in scleroderma and tightskin mice. Curr Opin Immunol 1994;6:931-937. Cram DS, Fisicaro N, McNeilage LJ, Coppel RL, Harrison LC. Antibody specificities of Thai and Australian scleroderma sera with topoisomerase-I recombinant fusion proteins. J Immunol 1993;151:6872-6881. D'Arpa P, Machlin PS, Rattle H 3rd, Rothfield NF, Cleveland DW, Earnshaw WC. cDNA cloning of human topoisomerase I. catalytic activity of a 67.7 kDa carboxyl-terminal fragment. Proc Natl Acad Sci USA 1988;85:2543--2547. Douvas AS, Achten M, Tan EM. Identification of a nuclear protein (Scl-70) as a unique target of human antinuclear antibodies in scleroderma. J Biol Chem 1979;254:1051410522. Giordano M, Valentini IG, Migliaresi S, Picillo U, Vatti M. Different antibody patterns and different prognoses in patients with scleroderma with various extent of skin sclerosis. J Rheumatol 1986;13:911-916. Guldner H, Scostecki C, Vosberg HP, Lakomek HJ, Pennr E, Bautz FA. Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein as DNA topoisomerase I. Chromosoma 1986;94:132-138. Heck MM, Hittleman WR, Earnshaw WC. Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle. Proc Nat Acad Sci USA 1988;85:1086-1090. Hildebrandt S, Weiner E, Senecal JL, Noell S. The IgG, IgM, and IgA isotypes of antitopoisomerase I and anticentromere autoantibodies. Arthritis Rheum 1990a;33:724--727.

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CONCLUSION In summary, the presence of antitopoisomerase I antibodies confirms the diagnosis of scleroderma (Table 1). The antibody predicts the development of tight skin in patients with primary R a y n a u d ' s syndrome. In scleroderma patients, the antibody is associated with the presence of diffuse skin thickening, lung involvement and the development of cancer.

Hildebrandt S, Weiner ES, Senecal JL, Noell S, Earnshaw WC, Rothfield NF. Autoantibodies to topoisomerase I (Scl-70): analysis by gel diffusion, immunoblot, and enzyme-linked immunosorbent assay. Clin Immunol Immunopathol 1990b; 57:399-410. Hildebrandt S, Weiner ES, Senecal J-I, Noell GS, Earnshaw WC, Rothfield NF. Autoantibodies to topoisomerase I (Scl70) analysis by gel diffusion, immunoblot, and enzymelinked immunosorbent assay. Clin Immunol Immunopathol 1991;57:399-410. Hildebrandt S, Jackh G, Weber S, Peter HH. A long-term longitudinal isotypic study of antitopoisomerase I autoantibodies. Rheumatol Int 1993;12:231--234. Hoffman A, Heck MM, Bordwell BJ, Rothfield NF, Earnshaw WC. Human autoantibody to topoisomerase II. Exp Cell Res 1989;180:409--418. Jimenez SA, Batuman O. Immunopathogenesis of systemic sclerosis: possible role of retroviruses. Autoimmunity 1993;16:225--233. Juarez C, Vila JL, Gelpi C, Agusti M, Amengual MJ, Martinez MA, Rodriguez JL. Characterization of the antigen reactive with anti-sc 1-70 antibodies and its application in an enzymelinked immunosorbent assay. Arthritis Rheum 1988;31:108-115. Kato T, Yamamoto K, Takeuchi H, Okubo M, Hara E, Nakada S, Oda K, Ito K, Nishioka K. Identification of a universal B cell epitope on DNA topoisomerase I, an autoantigen associated with scleroderma. Arthritis Rheum 1993;36:1580-1587. Kosovsky MJ, Soslau G. Immunological identification of human platelet mitochondrial DNA topoisomerase I. Biochim Biophys Acta 1993;1164:101--107. Kuwana M, Kaburaki J, Mimori T, Tojo T, Homma M. Autoantigenic epitopes on DNA topoisomerase I - clinical and immunogenetic associations in systemic sclerosis. Arthritis Rheum 1993a;36:1406-1413. Kuwana M, Kaburaki J, Okano Y, Inoko H, Tsuji K. The HLADR and DQ genes control the autoimmune response to DNAtopoisomerase I in systemic sclerosis. J Clin Invest 1993b; 92:1296--1301. Kuwana M, Kaburaki J, Okano Y, Tojo T, Homma M. Clinical and prognostic associations based on serum antinuclear

antibodies in Japanese patients with systemic sclerosis. Arthritis Rheum 1994;37:75--83. Maddison PJ, Stephens C, Briggs D, Welsh KI, Harvey G, Whyte J, McHugh N. Connective tissue disease and autoantibodies in the kindreds of 63 patients with systemic sclerosis. The United Kingdom Systemic Sclerosis Study Group. Medicine (Baltimore) 1993;72:103--112. Maul GG, French BT, van Venrooij WJ, Jimenez SA. Topoisomerase I identified by scleroderma 70 antisera: enrichment of topoisomerase I at the centromere in mouse mitotic cells before anaphase. Proc Natl Acad Sci USA 1986;83:51455149. McHugh NJ, Whyte J, Harvey G, Hausten UF. Antitopoisomerase I antibodies in silica-associated systemic sclerosis. Arthritis Rheum 1994;37:1198-1205. Morel PA, Chang HJ, Wilson JW, Conte C, Saidman SL, Bray JD, Tweady DJ, Medsger TA Jr. Severe systemic sclerosis with antitopoisomerase-I antibodies is associated with an HLS-DRwl 1 allele. Hum Immunol 1994;40:101--110. Muryoi YT, Kasturi KN, Kafina MJ, Saitoh Y, Usuba O, Perlish JS, Fleischmajer R, Bona CA. Self reactive repertoire of tight skin mouse: immunochemical and molecular characterization of antitopoisomerase I autoantibodies. Autoimmunity 1991;9:109--117. Muryoi T, Kasturi KN, Kafina MJ, Cram DS,Harrison LC, Sasaki T, Bona CA. Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes. J Exp Med 1992;175: 1103--1109.

Oddou P, Schmidt U, Knippers R, Richter A. Monoclonal antibodies neutralizing mammalian DNA topoisomerase I activity. Eur J Biochem 1988;177:523--529. Piccini G, Cardellini E, Reimer G, Arnett FC, Durban E. An antigenic region of topoisomerase I in DNA polymerase chain reaction-generated fragments recognized by autoantibodies from scleroderma patients. Mol Immunol 1991;28: 333-339. Reveille JD, Durban E, McLeod-St. Clair MJ, Goldstein R, Moreda R, Ahman RD, Arnett FC. Association of amino acid sequences in the HLA-DQB1 first domain with antitopoisomerase I autoantibody response in scleroderma (progressive systemic sclerosis). J Clin Invest 1992;90:973-980. Reveille JD, Arnett FC. Frequencies of scleroderma-related

autoantibodies in patients meeting the American College of Rheumatology criteria for systemic sclerosis: reply. Arthritis Rheum 1993;36:1333--1336. Rothfield N, Kurtzman S,.Vazques-Abad D, Charron C, Daniel L, Greenberg B. Association of antitopoisomerase I with cancer [Letter]. Arthritis Rheum 1992;35:724. Seelig HP, Schroter H, Ehrfeld H, Renz M. Autoantibodies against topoisomerase I detected with the natural enzyme and overlapping recombinant peptides. J Immunol Methods 1993;165:241--252. Shero JH, Bordwell B, Rothfield NF, Earnshaw WC. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science 1986;231:737-740. Shero JH, Bordwell B, Rothfield NF, Earnshaw WC. Antibodies to topoisomerase I in sera from patients with scleroderma. J Rheumatol 1987;14:138--140. Steen VD, Powell DL, Medsger TA. Clinical correlations and prognosis based on serum autoantibodies in patients with systemic sclerosis. Arthritis Rheum 1988;31:196--203. Tsay GJ, Fann RH, Hwang J. Specificity of anti-Scl-70 antibodies in scleroderma: increased sensitivity of detection using purified DNA topoisomerase I from calf thymus. J Rheumatol 1990;17:1314-1319. Vazquez-Abad D, Pascual V, Zanetti M, Rothfield NF. Analysis of human antitopoisomerase-I idiotypes. J Clin Invest 1993 ;92:1302-1313. Vazquez-Abad D, Russell CA, Cusick SM, Earnshaw WC, Rothfield NF. Longitudinal study of anticentromere and antitopoisomerase-I isotypes. Clin Immunol Immunopathol 1995;74:257-270. Verheijen R, Van den Hoogen F, Beijer R, Richter A, Penner E, Habets WJ, van Venrooij WJ. A recombinant topoisomerase I used for autoantibody detection in sera from patients with systemic sclerosis. J Clin Exp Immunol 1990;80:38--43. Weiner ES, Earnshaw WC, Senecal JL, Bordwell B, Johnson P, Rothfield NF. Clinical associations of anticentromere and antibodies topoisomerase I. A study of 355 patients. Arthritis Rheum 1988;31:378--385. Weiner ES, Hildebrandt S, Senecal JL, Daniels L, Noell S, Joyal F, Roussin A, Earnshaw WC, Rothfield NF. Prognostic significance of anticentromere antibodies and antitopoisomerase I antibodies in Raynaud's disease. A prospective study. Arthritis Rheum 1991 ;34:68--77.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

TUBULAR BASEMENT MEMBRANE AUTOANTIBODIES Ralph Butkowski, Ph.D. a, Todd Nelson b and Aristidis Charonis, M.D. Ph.D. b

alNCSTAR Corporation, Stillwater, MN 55455; and ;'Department of Laboratory Medicine and Pathology, University of Minnesota Medical School Minneapolis, MN 55082, USA

HISTORICAL N O T E S

THE AUTOANTIGENS

Antibodies to kidney tubular basement membrane (TBM) are occasionally present in tubulointerstitial nephritis (TIN), a common disease leading to renal insufficiency (Brentjens et al., 1982). With a sensitive assay employing TIN antigen, antibodies to TBM (anti-TBM) are detected more frequently in TIN than previously observed by immunofluorescent microscopy (Lindqvist et al., 1994). These antibodies are useful tools for identification of antigens that may play a role in pathogenesis of TIN. Several hallmark studies provided insight to antiTBM and/or their target antigens, including the original animal model of antibody-mediated TIN developed in guinea pigs (Steblay and Rudofsky, 1971). Reviews of experimental TIN (Rudofsky and Pollara, 1983) and of the histopathology of human TIN associated with TBM antibodies are available (McCluskey et al., 1983), as are more recent reviews of the roles of humoral and cell-mediated immunity and the pathological mechanisms in human TIN (Colvin and Fang, 1989; McCluskey and Colvin, 1989; Neilson, 1989; Wilson, 1989). Nephritogenic antigens recognized by TBM antibodies (Wilson, 1991) as well as the molecular characterization of a specific 58 kd antigen (TINantigen), and its induction of TIN in the Brown Norway rat were reviewed (Crary et al., 1993). Of the multiple antigens recognized by anti-TBM, most are not characterized in detail. The various forms of TINantigen have in common their unique anatomic distribution in the kidney and the absence of directly detectable antigen in the Lewis rat.

Definitions

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Tubulointerstitial nephritis antigen is a basement membrane component that reacts with TBM antibodies present occasionally in human TIN and commonly present in animal models of TIN. This article uses the terminology "TIN-antigen" in referring to TBM components meeting the criterion of having the characteristic tissue distribution, absence of directly detectable antigen in the Lewis rat, reactivity with human anti-TBM, and ability of the pure antigen to induce TIN in animals. Due to the absence of a standard nomenclature for such antigens, various terminology is used in the literature. Of several molecular weight forms described in varying detail, the 58 kd TIN-antigen is characterized most extensively at the biochemical level, including an amino acid sequence deduced from a cDNA (Nelson, 1995). Immunoblot analysis of several species using antibodies to the 58 kd TIN-antigen shows some heterogeneity (Figure 1). A 48 kd antigen, designated 3M-l, has a potential role in pathogenesis of TIN (Neilson et al., 1991). Another molecule partially characterized by amino terminal sequencing induces TIN (Yoshioka et al., 1992). The latter two molecules apparently differ from the 58 kd TIN-antigen because their partial sequences are not found in 58 kd TIN-antigen. Gel filtration of collagenase-digested rabbit TBM reveals a high molecular weight complex reactive with various anti-TBM specific for 58 kd TIN-antigen. The complex resolved into major 58 kd, and minor components of 160 kd, 175 kd and 300 kd; each com-

and monoclonal antibodies to TIN-antigen (Butkowski et al., 1991). TIN-antigen is present in basement membranes of kidney cortex, small intestine, skin and cornea, but not in the renal medulla. Proximal TBM shows highest concentrations of TIN-antigen, which is also present in Bowman's capsule, distal TBM, peritubular capillaries and focally in the renal interstitium, but not in the glomerulus (Figure 2). TINantigen is detected in epithelial basement membranes of small intestine, with highest concentrations in the ileum and trace amounts in the duodenum and jejunum. In skin, TIN-antigen is detected along epithelial basement membranes. Native vs. Recombinant Antigen Performance

Native TIN-antigen and synthetic peptides can be used in assay systems (Crary, 1993; Neilson et al., 1991). Purified TIN-antigen as well as synthetic P1, the nephritogenic domain of 3M-1, induce TIN in animals (Crary, 1993; Neilson et al., 1991). Studies of recombinant TIN-antigen are in progress. Figure 1. Immunoblot analysis of TIN-Antigen with a monoclonal antibody. Binding of antibody to purified 58 kd TINAntigen (Lane 1); solubilized TBM from rabbit (Lane 2); bovine (Lane 3); human (Lane 4), mouse (Lane 5) and Brown Norway rat (Lane 6). Lewis rat TBM was not reactive (Lane 7). Prominent 58 kd bands (indicated by 58K) are seen in each reactive species except bovine TBM which shows 52, 45 and 35 kd bands. Weakly staining high molecular weight components (arrows, Lanes 5 and 6) are also detected.

ponent reacts with anti-TBM. Both 58 kd TIN-antigen and the high molecular weight forms induce TIN in the Brown Norway rat (Crary, 1993). TIN-antigen interacts in vitro with laminin and type IV collagen and inhibits laminin polymerization but does not selfassociate or interact with heparin, suggesting lack of significant electrostatic interactions with negatively charged molecules (Kalfa et al., 1994). TIN-antigen also promoted cell adhesion of kidney epithelial cells and aortic endothelial cells. TIN-antigen apparently is crucial to the ultrastructure of specific basement membranes and their adhesive interactions with overlying cells. The characteristic distribution of TIN-antigen in the kidney was defined by direct and indirect immunofluorescent microscopy with human autoantibodies (Brentjens et al., 1989) as was the tissue binding of human autoantibodies and of polyclonal

Origin and Sources

TIN-antigen can be purified in milligram quantities from rabbit kidney cortex. Similar quantities from human and other species are difficult to obtain due to apparent degradation during purification. Although detected in cell culture extracts and in organs other than kidney by immunofluorescence and immunoblot analysis, TIN-antigen has been prepared only from kidney tissue. Methods of Purification

Of the two methods used for purification of the 58 kd TIN-antigen from rabbit kidney, the first utilizes denaturing extraction of isolated TBM in guanidineHCL followed by ion-exchange, gel filtration and reverse-phase chromatography (Butkowski et al., 1990). In the second method, which was adopted to avoid conformational changes in TIN-antigen due to chaotropic agents, isolated rabbit TBM is digested with collagenase and the solubilized mixture is purified by gel filtration and ion-exchange chromatography (Butkowski et al., 1991). Other investigators use variations of these methods to purify native antigen from enzyme digests and extracts of TBM. For preparation of small quantities, TIN-antigen can be extracted from polyacrylamide gels.

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preprocathepsin B and other members of the cysteine proteinase family. By partial sequence analysis, the 3M-1 protein is related to the intermediate filamentassociated proteins.

AUTOANTIBODIES

Nomenclature

Figure 2. Distribution of TIN-Antigen in the kidney cortex revealed by immunofluorescent microscopy. Monoclonal antiTIN-Ag was reacted with sections of bovine (A--D) and human (E,F) kidney sections. Reactivity is observed with Bowman's capsule (arrow inside glomerulus, g) and TBM, but not with GBM A. Fluorescence of TBM varies from intense to weak (arrows, B). Reactivity is observed with peritubular capillary basement membranes (arrows, C), and weakly in undefined interstitial sites (arrow, D). Similar staining characteristics are seen on human kidney E. For comparison, similar staining of human autoantibody is illustrated on human kidney F.

Commercial Sources TIN-antigen is not commercially available.

Sequence Information The deduced sequence of 58 kd rabbit TIN-antigen reveals 474 amino acids, including seven potential glycosylation sites, as expected from the manose-rich oligosaccharides found in TIN-antigen. Cystine residues are clustered in two discrete regions from amino acids 55--152 and 238--349. The amino terminal region of TIN-antigen contains a sequence similar to an EGF-like repeat found in several classes of extracellular molecules including laminin A and S chains, von Willebrand's factor, mucin and the alpha1 chain of type I collagen. The carboxy terminal two. thirds of TIN-antigen is 30% identical to human

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The terms "TBM antibodies" or "anti-TBM antibodies" (abbreviated "anti-TBM) are used here to described antibodies that bind TBM in TIN. In pure forms of anti-TBM-mediated human TIN and in animal models, kidney-bound antibody detected by immunofluorescence exhibits the characteristic distribution of TIN-antigen as described above. An exception is a case of anti-TBM from serum and renal eluate which bind basement membrane of collecting tubules (Klassen et al., 1973). These results suggest other TBM antigens can be involved in TIN (Klassen et al., 1973).

Pathogenetic Role Human Disease. Direct proof of a pathogenic role of TBM antibodies in TIN is lacking, as transfer of antibody to animal models has not resulted in disease induction. Since disease activity is related to antibody concentration, it is possible that insufficient quantities of antibody were available (Wilson, 1989). However transfer experiments using antibodies generated in guinea pig and rat models indicated a pathogenic role for TBM antibodies. The pathology of TIN includes macrophages, multinucleated cells, and T and B cells in the interstitium as well as degenerative and proliferative lesions of tubules (Brentjens et al., 1989). Evidence for both cellular and humoral mechanisms in human TIN is based upon correlations with animal models. It seems likely that the relative role of cell- and antibodymediated pathogenic mechanisms in human TIN may vary depending on antibody titers (Lindqvist et al., 1994). Anti-TBM antibody-dependent T-cell immune response was established in models (Neilson, 1989). Cell-mediated events result in chronic interstitial lesions and expansion of extracellular matrix into the interstitium in the models (Neilson, 1989). Parallel pathological changes are observed in human TIN, supporting the concept of similar operative mechanisms (Neilson, 1989).

Animal Models. Most of what is assumed about the immunopathogenesis of TIN is extrapolated from guinea pig, rat and mouse models (Wilson, 1989), which are produced by immunization with kidney homogenates, purified TBM or TBM extracts and more recently with purified TIN-antigen. Spontaneous anti-TBM are also reported in the New Zealand black/white mouse and in the Samoyed dog (Wilson, 1989). In the guinea pig model as first induced by immunization with rabbit kidney cortex basement membrane or bovine TBM, TIN can be transferred with antibody, but not with immune cells, suggesting a major role for the anti-TBM (Wilson, 1989). Antibody production and TIN was decreased with antiidiotypic antibodies (Wilson, 1989). Furthermore, TIN is not inducible in animals depleted of C3 and infused with TBM antibodies (Wilson, 1989). Evidence for cellular immunity in the guinea pig model includes the demonstration of cellular sensitivity to immunogen, and gold salt-induced cellular sensitivity associated with anti-TBM. In the rat model, Brown Norway (BN) rats are immunized with bovine TBM. Induction of TIN in BN rats with bovine TBM is related quantitatively to antibody binding to the kidney. However, correlation with serum anti-TBM concentration and disease is less well defined. Lack of an autologous anti-idiotypic antibody response related to suppressor T-cell mechanisms is believed to result in the antibody-mediated TIN (Neilson, 1989). However, in contrast to the guinea pig model, transfer of TIN with antibodies produces only mild disease. Sensitized immune cells can transfer TIN and generate anti-TBM. In the Lewis rat where TIN-antigen is not detectable, cellular immune mechanisms cause TIN (Wilson, 1989). This model is transferred by lymph node cells of immunized rats but not by antibody. TIN induced in mice exhibits predominantly cellular immune mechanisms (Neilson, 1989). Pathologic lesions develop after 6--7 weeks; whereas, antiTBM appear early, supporting the concept of a major role of cellular immunity in this model. Lesions that result from cell transfer occur earlier and are more significant than those observed with serum transfer alone. The T cells are thought to induce Lyt 2 + effector cells, which are apparently inhibited by contrasuppressive mechanisms in nonsusceptible strains of mice. Inhibition of TIN by suppressor cell mechanisms was demonstrated by injection of spleen cells sensitized with 3M-1 TIN-antigen. In spon-

taneous TIN in kdkd mice, inactivation of suppressor T cells is believed to be mediated by antigen-specific contrasuppressor T cells.

Genetics In the few reports of primary TIN associated with anti-TBM, endstage renal failure usually ensued (Katz et al., 1992). A genetic component to these cases was not reported. Familial membranous glomerulonephritis with anti-TBM is recognized (Colvin and Fang, 1989). A report of three cases of anti-TBM nephritis associated with membranous nephropathy revealed antibodies reactive by ELISA and immunoblotting with 58 and 175 kd bands, which correspond to the 58 kd TIN-antigen and a molecular aggregate (Katz et al., 1992). Human susceptibility to TIN might be HLArelated. In the guinea pig, rat and mouse models of antiTBM-associated TIN, strain differences in susceptibility segregate with the major histocompatibility complex (Wilson, 1989). TIN-antigen, which does not segregate with the major histocompatibility complex genes, is variably found in different strains of rats.

Factors Involved in Pathogenicity and Etiology As with other organ-specific autoimmune diseases, little is known about the initiating events in primary anti-TBM-associated TIN. Favored theories include defective regulatory mechanisms and induction of cross-reactive autoimmune responses by infectious agents (McCluskey and Colvin, 1989). Drug-induced TIN possibly results from molecular complex formation between TIN-antigen and the drug. An instance of transplantation-induced anti-TBM resulted when an antigen-negative patient received an antigenpositive kidney (Wilson, 1989). Patients with renal allografts develop anti-TBM at a frequency of 0.9-6.1% (Colvin and Fang, 1989), perhaps due to antigen exposure resulting from transplantation, or secondary exposure resulting from rejection or toxicity. TBM antibodies occurring in transplantation are believed to have minimal effect on allograft survival.

Methods of Detection Because anti-TBM are rare, there are few standardized quantitative immunoassays. Detection usually is by direct and indirect immunofluorescence. A sensitive radioimmunoassay was used in studies of TBM anti-

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bodies in patients with anti-GBM nephritis (Graindorge and Mahieu, 1978). An ELISA method detected 15 TIN-antigen-positive sera among 69 individuals with TIN but without glomerulonephritis (Lindqvist et al., 1994). ELISA methods will likely prevail for convenience of use in research. Immunofluorescent detection is likely to remain the method of choice for general use unless greater prevalence and significance of anti-TBM are demonstrated.

Table 1. Occurrence of Anti-TBM Antibodies Primary anti-TBM disease Transplantation induced Disease associated Drug induced Membranous glomerulonephritis Anti-GBM disease Nephronopthisis Lupus nephritis Poststreptococcal glomerulonephritis

CLINICAL UTILITY

Application Quantitative immunoassays are not generally done for anti-TBM, except for research purposes. The antibodies are usually detected in the clinical setting by direct immunofluorescence during evaluation of renal biopsy specimens.

established in most situations. These include occasional cases of primary idiopathic interstitial nephritis, anti-TBM nephritis, systemic lupus and Sj6gren's syndrome, infections, several forms of glomerulonephritis and IgA nephropathy.

CONCLUSION

Disease Associations/Frequency Anti-TBM are generally associated with another renal disease or are induced by drugs or renal transplantation. The common renal disease associations with antiTBM disease include membranous glomerulonephritis and antiglomerular basement membrane disease (Colvin and Fang, 1989) (Table 1). Anti-TBM occur frequently in cases of nephronopthisis, infrequently in lupus nephritis and rarely in poststreptococcal glomerulonephritis. A pathogenic role for anti-TBM is not established at this time. When anti-TBM occur in membranous glomerulonephritis or in drug-induced interstitial nephritis, the patients are predominantly male. In drug-induced TIN with TBM antibodies the ratio of males to females is 5:1 (Colvin and Fang, 1989). Systemic diseases can involve immune complex deposition in the interstitium and along TBM. The antibody specificity in these deposits remains to be

REFERENCES Brentjens JR, Noble B, Andres GA. Immunologicallymediated lesions of kidney tubules and interstitium in laboratory animals and man. In: Thomas HC, Miescher PA, MuellerEberhard HJ, eds. Immunological Aspects of Liver Disease. New York: Springer-Verlag, 1982;7:357--378. Brentjens JR, Matsuo S, Fukatsu A, Min I, Kohli R, Anthone

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Further research is needed to determine whether or not anti-TBM could serve as serum markers of renal status in glomerulonephritis, transplantation and/or systemic autoimmune diseases. Because severity of renal disease appears to correlate with the presence of these antibodies, there may be value in testing for them. The sensitivity and specificity of anti-TBM determination for various diseases has not been explored. It is currently not known whether or not anti-TBM detection will be of prognostic or diagnostic value; however, the literature cited in the text suggests detection of anti-TBM may be useful prognostically. Further research is needed to make this determination. Additional research is needed to establish the identity of the TBM antigen involved in various conditions where TBM antibodies are detected, and to establish the relationships among the several TBM components reported to react with TBM antibodies. See GLOMERULAR BASEMENT MEMBRANE AUTOANTIBODIES.

R, Anthone S, Biesecker G, Andres G. Immunologic studies in two patients with antitubular basement membrane nephritis. Am J Med 1989;86:603--608. Butkowski RJ, Langeveld JPM, Wieslander J, Brentjens JR, Andres GA. Characterization of a tubular basement membrane componentreactive with autoantibodies associated with tubulointerstitial nephritis. J Biol Chem 1990;265:2109121098.

Butkowski RJ, Kleppel MM, Katz A, Michael AF, Fish AJ. Distribution of tubulointerstitial nephritis antigen and evidence for multiple forms. Kidney Int 1991;40:838--846. Colvin RB, Fang LST. Interstitial nephritis. In: Tisher CC, Brenner BM, eds. Renal Pathology: With Clinical and Functional Correlations. Philadelphia: Lippincott, 1989;1: 728--776. Crary GS, Katz A, Fish AJ, Michael AF, Butkowski RJ. Role of a basement membrane glycoprotein in antitubular basement membrane nephritis. Kidney Int 1993;43:140--146. Graindorge PP, Mahieu PR. Radioimmunologic method for detection of antitubular basement membrane antibodies. Kidney Int 1978;14:594--606. Kalfa TA, Thull JD, Butkowski RJ, Charonis AS. Tubulointerstitial nephritis antigen interacts with laminin and type IV collagen and promotes cell adhesion. J Biol Chem 1994;269: 1654-1659. Katz A, Fish AJ, Santamaria P, Nevins T, Kim Y, Butkowski RJ. Role of antibodies to tubulointerstitial nephritis antigen in human antitubular basement membrane nephritis associated with membranous nephropathy. Am J Med 1992;93:691--698. Klassen J, Kano K, Milgrom F, Menno AB, Anthone S, Anthone R, Sulveda M, Sepulveda M, Elmwood CM, Andres GA. Tubular lesions produced by autoantibodies to tubular basement membrane in human renal allografts. Int Arch Allergy Appl Immunol 1973;45:675--689. McCluskey RT, Bahn AK, Colvin RB. Experimental and human antitubular basement membrane nephritis. In: Cummings NB, Michael AF, Wilson CB, eds. Immune Mechanisms in Renal Disease. New York: Plenum Medical Book Co., 1983;279294. McCluskey RT, Colvin RB. Immunopathogenetic mechanisms of tubulointerstitial injury. In: Tisher CC, Brenner BM,

eds. Renal Pathology: With Clinical and Functional Correlations. Philadelphia: Lippincott, 1989; 1:642--655. Lindqvist B, Lundberg L, Wieslander J, eds. The prevalence of circulating antitubular basement membrane-antibody in renal diseases, and clinical observations. Clin Nephrol 1994;41: 199--204. Neilson EG. Pathogenesis and therapy of interstitial nephritis. Kidney Int 1989;35:1257--1270. Neilson EG, Sun MJ, Kelly CJ, Hines WH, Haverty TP, Clayman MD, Cooke NE. Molecular characterization of a major nephritogenic domain in the autoantigen of antitubular basement membrane disease. Proc Natl Acad Sci USA 1991 ;88:2006-2010. Nelson TR, Charonis AS, Mclvor RS, Butkowski RJ. Identification of a cDNA encoding tubulointerstitial nephritis antigen. J Biol Chem 1995;270:16265--16270. Rudofsky UH, Pollara B. Experimental autoimmune renal tubulointerstitial disease. In: Cummings NB, Michael AF, Wilson CB, eds. Immune Mechanisms in Renal Disease. New York: Plenum Medical Book Co., 1983;261--278. Steblay RW, Rudofsky U. Renal tubular disease and autoantibodies against tubular basement membrane induced in guinea pigs. J Immunol 1971;107:589-594. Wilson CB. Study of the immunopathogenesis of tubulointerstitial nephritis using model systems. Kidney Int 1989;35:938-953. Wilson CB. Nephritogenic tubulointerstitial antigens. Kidney Int 1991;39:508--517. Yoshioka K. Hino S, Takemura T, Miyasoto H. Honda E, Maki S. Isolation and characterization of the tubular basement membrane antigen associated with human tubulo-interstitial nephritis. Clin Exp Immunol 1992;90:319--325.

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9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

TYROSINASE AUTOANTIBODIES Pnina Fishman, Ph.D. a, Ofer Merimsky, M.D. b, Ehud Baharav, M.D. a and Yehuda Shoenfeld, M.D. c

aResearch Laboratory of Clinical Immunology, The Basil and Gerald Felsenstein Medical Research Center, Bellinson Campus, Petach-Tiqva, 49100; bDepartment of Oncology, Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicines, Tel Aviv University; and CDepartment of Medicine "B", Research Unit of Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel

HISTORICAL NOTES Tyrosinase autoantibodies (antityrosinase antibodies) can be detected in the sera of patients with melanoma, vitiligo and melanoma-associated hypopigmentation (Merimsky et al., 1995a; 1994; Baharav et al., 1995a; Song et al., 1994). Because melanomas are known to be immunogenic tumors, some patients generate antibodies against melanoma cell antigens and the antibodies are thought to lead to tumor regression (Merimsky et al., 1994). Tyrosinase, an enzyme involved in the synthesis of melanin, is one antigen to which antibodies can be generated in both melanoma and vitiligo (Merimsky et al., 1995a; 1995b; Baharav et al., 1995a). Because several autoimmune diseases are characterized by autoantibodies to autoantigens which are enzymes (e.g., proteinase 3 in Wegener's granulomatosis and pyruvate dehydrogenase in primary biliary cirrhosis, etc.), tyrosinase was postulated to be an autoantigen in vitiligo and melanoma, and indeed high titers of autoantibodies against tyrosinase are typical of patients with vitiligo and may also appear in patients with melanoma at several stages of the disease (Merimsky et al., 1995a; Baharav et al., 1995a; Song et al., 1994).

THE AUTOANTIGENS By a process termed "melanogenesis", mature melanocytes produce melanin in their melanosomes. Tyrosinase, also known as molecule T4, is a 75 kd, coppercontaining enzyme which is essential for melanogene-

842

sis, is synthesized by epithelial, mucosal, retinal and ciliary body melanocytes (Fitzpatrick et al., 1979) and is stored in cytoplasmic organelles called melanosomes (Hearing et al., 1973). Melanin is metabolically derived from the tyrosinase-catalyzed oxidation of the amino acid L-tyrosine. The first step is ring hydroxylation followed by dehydrogenation, resulting in L-phenylalanine-3,4orthoquinone, a substance that undergoes polymerization to form melanin (Riley, 1991). Production of melanin is regulated by a subtle balance among tyrosinase, tyrosinase-related protein 1, DOPAchrome tautomerase and melanogenic inhibitor (Kameyama et al., 1993; Tsukamoto et al., 1992). Two enzymes demonstrating tyrosinase activity have been isolated from melanocytes. Both are expressed in pigmented tissues (Shibahara et al., 1986; Jimenez et al., 1980; Yamamoto et al., 1989) and show substantial similarity in their amino acid sequences. The abundant type is an intracellular membrane-bound enzyme, whereas the other is a soluble form (Wittbjer et al. 1990). How tyrosinase might be exposed to the immunological system is unknown. The membrane soluble forms of tyrosinase from malignant or normal pigmented cells might serve as a target for the production of autoantibodies. Melanoma cells in culture release tyrosinase to the growth medium (Karg et al., 1990), and tyrosinase activity can be detected in the sera of metastatic melanoma patients (Sohn et al., 1969; Nishioka et al., 1979; Agrup et al., 1989). Melanocytes have phagocytic capacity and express MHC class II molecules (A1Badri et al., 1993) and thus can serve as antigen-presenting cells.

2 . 5 ...........

"-O--T

Diffuse Vitiligo

---'O""" Localized Vitiligo

2....... 0

.......

Control

1.5-

9

1-

O

0o5

-

l

I

I

I

I

I

1/5

1/10

1/25

1/50

1/100

1/200

T

11400

Sera Dilutions

Figure 1. Serum dilution curve of antityrosinase antibodies measured by ELISA using mushroom tyrosinase as an antigen. High titers of antityrosinase antibodies are seen in patients with diffuse vitiligo (n = 7; p < 0.001) in comparison with the control group (n = 25) and patients with localized vitiligo (n - 11).

Disease Association

pathogenesis of vitiligo, titers of antityrosinase antibodies were measured in sera of patients with vitiligo and healthy volunteers (Merimsky et al., 1995a; 1995b; Baharav et al., 1995a). Antityrosinase antibodies (characterized as IgG) are present in high titers in sera of patients with vitiligo in comparison to healthy volunteers (Figure 1). Pseudo antityrosinase antibodies (i.e., immune complexes binding nonspecifically to tyrosinase) detected in the serum of patients with connective tissue diseases, including SLE rheumatoid arthritis, Sj6gren's syndrome and Wegener's granulomatosis disappeared once the circulating immune complexes were precipitated from the sera using polyethylene glycol. In similar experiments with sera of patients with vitiligo, antityrosinase antibodies were still detected after circulating immune complexes were removed (Baharav et al., 1995b). Among 26 patients with vitiligo and associated endocrine disease whose sera were examined by immunoblotting, 77% reacted with a tyrosinase-like protein, 61% reacted with recombinant tyrosinase and none reacted with the tyrosinase-related protein (Song et al., 1994). None of 31 normal controls, four patients with alopecia or four patients with SLE had tyrosinase autoantibodies, but 12% of 42 patients with autoimmune endocrine disease without a history of vitiligo had the autoantibodies.

u

Melanoma. As reported by several groups, tyrosinase

AUTOANTIBODIES Methods of Detection Serum antityrosinase antibodies can be detected by ELISA using commercially available mushroom tyrosinase (Baharav et al., 1995a). Tyrosinase antibody-containing sera do not cross-react with cellular autoantigens that play a major role in other autoimmune disorders as assessed by ELISA, including myeloperoxidase, proteinase-3, pyruvate dehydrogenase or the NC-1 fraction of type IV collagen (Baharav et al., 1995b). Antityrosinase autoantibodies recovered from vitiligo patients' sera by affinity purification have relatively high functional affinity to tyrosinase but do not block the enzymatic activity of tyrosinase (unpublished data). In addition to the ELISA method, autoantibodies to tyrosinase but not to tyrosinase-related protein can also be detected in patients with vitiligo by immunoblotting with recombinant human tyrosinase expressed in Escherichia coli (Song et al., 1994).

CLINICAL UTILITY

To assess autoimmunity to tyrosinase in the

843

........

1.2

1 "

O.8-

0.6

"

8 O

o 0

O

0.4--

0.2-

O O

o

0

0 ......

Normal Control

Diffuse Vitiligo

Localized Vitiligo

o ~

Melanoma

.....

Melanoma+ Vitiligo

Figure 2. Distribution of antityrosinase antibodies in different groups of patients. Anti-tyrosinase antibodies were examined in a serum dilution of 1:50 using mushroom tyrosinase as the antigen. The mean value of patients with diffuse vitiligo, metastatic melanoma and melanoma positive vitiligo is significantly higher than the control values (p < 0.0009, p < 0.0001, p < 0.01, respectively).

activity is present in the sera of patients with metastatic melanoma (Sohn et al., 1969; Nishioka et al., 1979; Agrup et al., 1989). Melanoma cells in culture release tyrosinase into the medium (Karg et al., 1990). The tyrosinase found in the sera of patients with melanoma might reflect release from either malignant or normal pigmented cells and probably serves as an immune target for the production of antityrosinase antibodies. Sera of 56 patients with malignant melanoma were examined to see whether antityrosinase antibodies might serve as a marker for disease progression (Merimsky et al., 1995a; 1995b; Baharav et al., 1995a). With an ELISA using mushroom tyrosinase as the coating antigen, antibodies were higher in patients with metastatic melanoma (mean = 0.37 + 0.03) and in patients with melanoma that developed vitiligo (mean = 0.37 +0.09) in comparison to healthy volunteers (mean = 0.11 + 0.008) (Figure 2). Melanoma is a highly immunogenic tumor and the patients are capable of raising different types of antibodies against the melanoma cells. Since these

REFERENCES

Agrup P, Carstam R, Wittbjer A, Rorsman H, RosengrenE. Tyrosinase activity in serum from patients with malignant melanoma. Acta Derm Venereol (Stockholm) 1989;69:120-124. 844

antibodies are also potent against normal melanocytes, some patients tend to develop vitiligo and are considered to have a better prognosis (Bystryn and Naughton, 1984; Donaldson et al., 1974; Laucius and Mastrangelo, 1979; Lemer and Cage, 1973). It is conceivable that antityrosinase antibodies participate in the immune-mediated destruction of normal melanocytes in patients with melanoma.

CONCLUSION Antityrosinase antibodies are found in the sera of patients with diffuse vitiligo, metastatic melanoma and with melanoma and vitiligo. The autoantigen is tyrosinase itself, the enzyme which participates in pigment (melanin) formation by both melanocytes and melanoma cells. The production of autoantibodies in both diseases is associated with the development of white patches on the patient's skin.

A1 Badri AM, Foulis AK, Todd PM, Gariouch JJ, Gudgeon JE, Stewart DG, Garcie JA, Goudie RB. Abnormal expression of MHC class II and ICAM-1 by melanocytes in vitiligo. J Pathol 1993;169:203--206. Baharav E, Merimsky O, Altomonte M, Shoenfeld Y, Pavlovic

M, Malo M, Ferrone S, Fishman P. Antityrosinase antibodies participate in the immune response to vaccination with antiidiotypic antibodies mimicking the high-molecular-weight melanoma-associated antigen. Melanoma Res 1995a;5:337343. Baharav E, Dueymes M, Bendaoud B, Merimsky O, Fishman P, Shoenfeld Y, Youinou P. Immune complexes from patients with connective tissue disease bind to tyrosinase nonspecifically. Int Arch Allergy Appl Immunol 1995b;106:in press. Bystryn JC, Naughton GK. Immunity to pigmented cells in vitiligo and melanoma. Fed Proc 1984;43:1664--1665. Donaldson RC, Canaan SA Jr, McLean RB, Ackerman LV. Uveitis and vitiligo associated with BCG treatment for malignant melanoma. Surgery 1974;76:771--778. Fitzpatrick TB, Eisen AZ, Waliff K, Freedberg I, Austen KF. Dermatology In General Medicine. New York: McGraw Hill, 1979. Hearing VJ, Nicholson JM, Montague PM, Ekel TM, Tomecki KJ. Mammalian tyrosinase. Structural and functional interrelationship to isoenzymes. Biochem Biophys Acta 1973; 522:327--339. Jimenez M, Lee Maloy WL, Hearing VJ. Specific identification of an authentic clone for mammalian tyrosinase. J Biol Chem 1980;264:3397-3403. Kameyama K, Takemura T, Hamada Y, Sakai C, Kondoh S, Nishiyama S, Urabe K, Hearing VJ. Pigment production in murine melanoma cells is regulated by tyrosinase, tyrosinaserelated protein 1 (TRP1), DOPAchrome tautomerase (TRP2) and a melanogenic inhibitor. J Invest Dermatol 1993;100: 126-131. Karg E, Hultberg B, Isaksson A, Rosengren E, Rorsman H. Enzyme release from cultured human melanoma cells. Acta Derm Venereol (Stockh) 1990;70:286-290. Laucious JF, Mastrangelo MJ. Cutaneous depigmentary phenomena in patients with malignant melanoma. In: Clark WH, Goldman II, Mastrangelo MJ, eds. Human Malignant Melanoma. Philadelphia: Grune and Stratton, 1979:209-225. Lerner AB, Cage CW. Melanoma in horses. Yale Biol Med 1973 ;46:646-650.

Merimsky O, Shoenfeld Y, Yecheskel G, Chaitchik S, Azizi E, Fishman P. Vitiligo- and melanoma-associated hypopigmentation: a similar appearance but a different mechanism. Cancer Immunol Immunother 1994;38:411-416. Merimsky O, Baharav E, Shoenfeld Y, Tsigelman R, Chaitchik S, Fishman P. Antityrosinase antibodies in malignant melanoma. 1995a;submitted. Merimsky O, Fishman P, Feldman I, Shafir R, Rapapport Y, Shoenfeld Y, Chaitchik S. Malignant melanoma of the head and neck: clinical and immunological considerations. Am J Clin Oncol 1995b;in press. Nishioka K, Romasdahl MM, McMlurtrey MJ. Adaptation of triatiated tyrosinase assay to serum tyrosinase and its specific elevation in melanoma. In: Klaus SN, ed. Pigmented Cell. Basel: Karger, 1979;15:300--304. Riley PA. Melanogenesis: a realistic target for antimelanoma therapy? Eur J Cancer 1991;27:1172-1177. Shibahara S, Tomita Y, Sakahura T, Nager C, Chaudhuri B, Muller R. Cloning and expression of cDNA encoding mouse tyrosinase. Nucleic Acids Res 1986;14:2413-2427. Sohn N, Gang H, Gumport SL, Goldstein M, Deppisch LM. Generalized melanosis secondary to malignant melanoma. Report of a case with serum and tissue tyrosinase studies. Cancer 1969;24:897-903. Song Y-H. Conor E, Li Y, Zorovich B, Balducci P, Maclaren N. The role of tyrosinase in autoimmune vitiligo. Lancet 1994;344:1049-- 1052. Tsukamoto K, Jackson IJ, Urabe K, Montague PM, Hearing VJ. A second tyrosinase related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO J 1992; 11:519--526. Wittbjer A, Odh G, Rosengren AM, Rosengren E, Rorsman H. Isolation of soluble tyrosinase from human melanoma. Acta Derm Venereol (Stockholm) 1990;70:291--294. Yamamoto H, Takeuchi S, Kudo T, Sato C, Takeuchi T. Melanin production in cultured albino melanocytes transfected with mouse tyrosinase cDNA. Jpn J Genet 1989;64: 121--135.

845

9 1996 Elsevier Science B.V. All rights reserved. Autoantibodies. J.B. Peter and Y. Shoenfeld, editors.

XENOREACTIVE HUMAN NATURAL ANTIBODIES William Parker, Ph.D. a and Jeffrey L. Platt, M.D. a'b

Departments ofaSurgery, blmmunology and bPediatrics, Duke University Medical Center, Durham, NC 27110, USA

HISTORICAL NOTES

XENOANTIGENS

Natural antibodies which bind to the cells of other species are called xenoreactive natural antibodies. The earliest description of such antibodies is attributed to Landois (Landsteiner, 1962) who noted that transfusion of the blood of one species into another species caused a fatal transfusion reaction and determined that the agglutination and lysis of xenogeneic cells were due to components of the recipient's blood (Boyden, 1964). Xenoreactive natural antibodies were first thought to be highly diverse in specificity, because the antibodies were able to distinguish the cells of one species from another species and one strain of bacteria from another strain. This idea was based on reports that a goat serum adsorbed with pigeon, rabbit, or human erythrocytes lost the ability to agglutinate erythrocytes from the species used for adsorption but retained the ability to agglutinate erythrocytes from other species, suggesting that xenoreactive natural antibodies are species specific (Malkoff, 1900). Later, however, xenoreactive natural antibodies were found to be broadly reactive, some because they recognize molecules expressed broadly in phylogeny and some because they bind to a variety of antigenic determinants (Thompson and Mandel, 1990). A much simpler view of xenoreactive natural antibodies recently emerged from experiments demonstrating that xenoreactive natural antibodies in humans and higher primates recognize predominantly one carbohydrate determinant (Galal-3Gal) expressed on the cells of nonprimate mammals. Much of this recent progress in the study of xenoreactive natural antibodies was sparked by the realization that these antibodies initiate the rejection of vascularized xenografts (Platt et al., 1990c), thereby preventing successful transplantation of animal organs into humans.

Definition

846

The antigens recognized by human xenoreactive natural antibodies on porcine cells are well characterized because of the clinical interest in xenotransplantation. Here the focus is mostly on antiporcine xenoreactive natural antibodies, although the same principles probably apply to antibodies against antigens from lower mammals. The saccharide which is the major target of human xenoreactive natural antibodies, Galo~I-3Gal[31-4GlcNAc-R, is related structurally and biosynthetically to blood group A [GalNAc~l-3(Fucocl-2)Gal-R] and blood group B [Galo~l-3(Fuco~l-2)Gal-R] (Platt, 1995a). The xenogeneic saccharide is termed the "linear B type 2" epitope. The immunodominant region of the linear B type 2 saccharide appears to consist of the terminal two residues (Galo~l-3Gal), and therefore, the determinant is sometimes described as Galo~l-3Gal (linear B) or "aGal". Human IgG antibodies specific for Galo~l-3Gal are autoreactive because they bind to damaged human hematopoietic cells (Galili et al., 1983; 1986). However, human cells are unable to synthesize Galo~l-3Gal, and the nature of the determinants on human cells which are recognized by antiGalo~l-3Gal IgG is unknown. Up to 80% of human xenoreactive natural IgM recognize Galo~l-3Gal (Parker et al., 1994; Sandrin et al., 1993; Collins et al., 1994). The specificity of the remaining 20% of xenoreactive antibodies which do not bind to Galo~l-3Gal is uncertain. Binding of human IgM to epitopes other than Galo~l-3Gal on cultured porcine endothelial cells is blocked 50--100% by prior incubation of the cells with porcine serum, suggesting that the xenoreactive human IgM which

does not recognize Gal~l-3Gal may be autoreactive or may interact with porcine cells nonspecifically. While the specificities and properties of xenoreactive IgM are well characterized because of the important role of xenoreactive IgM in xenotransplantation, the specificities and properties of xenoreactive IgG are not well understood. Binding of Xenoreactive Natural Antibodies to Synthetic vs. Natural Antigens While the epitope recognized by xenoreactive natural antibodies consists largely of a simple disaccharide, the conditions which allow xenoreactive natural antibodies to utilize that epitope for binding to cells are complex. The immunodominant sugar is clearly ~linked galactose; however, there is some evidence that one or two residues toward the reducing end also contribute to the binding of xenoreactive natural antibodies. Although xenoreactive natural antibodies bind to target cells with high avidity, at least three lines of evidence show that the affinity of each antigen combining site for Galc~l-3Gal is very low. First, monovalent xenoreactive IgM fragments do not bind to antigen (Parker et al., 1994). Second, whereas 5 mM synthetic Gal~l-3Gal is needed to inhibit the binding of xenoreactive IgM to porcine cells (Holzknecht and Platt, 1995), only 0.1 mM of that sugar structure is required for comparable inhibition when the determinants are displayed on porcine thyroglobulin, which contains six Galc~l-3Gal determinants per molecule (Thall and Galili, 1990). Third, the infusion into baboons of large quantities of Gal~l-6Glu, which is recognized by anti-Gal antibodies, fails to prevent hyperacute rejection in four of five porcine-to-baboon xenografts (Ye et al., 1994). These observations that xenoreactive antibodies do not bind to monovalent ligands with high affinity suggest that antibody binding depends on polyvalent interactions. Factors other than the density of Gal~l-3Gal determine the amount of binding of xenoreactive IgM to Gal~l-3Gal on cell surfaces (Collins et al., 1994; Alvarado et al., 1995). One factor may be the spatial distribution of those determinants. Other factors may include stearic hindrance or other unfavorable interactions such as charge-charge repulsion, limiting the binding of xenoreactive IgM. Several observations point toward factors other than the density of Gal~l3Gal being involved in the binding of xenoreactive IgM. First, as little as 470 pM of purified porcine platelet integrins (a family of membrane glycopro-

teins) inhibits by 70% the binding of the IgM to porcine cells (Platt and Holzknecht, 1994); whereas, 1 mM porcine thyroglobulin is required to achieve a comparable level of inhibition (Turman et al., 1991), even though each molecule of thyroglobulin contains six Gal~l-3Gal substitutions (Thall and Galili, 1990). This difference in the reactivity of the platelet integrins and porcine thyroglobulin with xenoreactive natural antibodies is not likely due to greater expression of Gal~l-3Gal on the integrins, but rather to preferential recognition of Gal~l-3Gal determinants in the tertiary conformation in which they are expressed on the integrins. A second line of evidence that the specificity of xenoreactive natural antibodies is determined by the spatial distribution or local environment of the Gal~l-3Gal determinants derives from the demonstration that although porcine platelets contain the same number of Gal~l-3Gal determinants, they vary over a seven-fold range in the amount of human IgM they bind (Alvarado et al., 1995). Phylogeny of Gal~l-3Gal Gal~l-3Gal is synthesized in all mammals except apes, Old World monkeys and humans, owing to expression of ~l-3galactosyltransferase which catalyzes a reaction between UDP-galactose and Gal~I4GlcNAc-R. Humans, apes and Old World monkeys do not have al-3galactosyltransferase, and instead have a pseudogene (Galili et al., 1987) (Table 1). In those species capable of synthesizing the sugar, Gal~l-3Gal is found on a wide variety of cell types such as erythrocytes and other hematopoietic cells, endothelial cells, fibroblasts and islet cells and on such diverse biomolecules as laminin, fibrinogen, thyroglobulin, IgG, integrins and glycolipids (Thall and Galili, 1990; Hamadeh et al., 1992; Galili et al., 1984). Commercial Sources There are two commercial sources of synthetic Gal~l3Gal: Toronto Research Chemicals (Downsview, Ontario Canada) produces Gal~l-3Gal (~1-3 galactobiose); Dextra Laboratories Ltd (Reading, UK) produces Gal~l-3Gal, Galc~l-3Gall] 1-4Gal (c~1-3, 1314 galactotriose), Gal~l-3Gall31-4Gal~l-3Gal (c~1-3, [31-4, al-3 galactotetraose), Gal~l-3Gall31-4GlcNAc and Gal~I-3Gall31-4GlcNAc covalently linked to bovine serum albumin. Proteins such as murine laminin, porcine thyroglobulin and bovine thyr0globu847

Table 1. Phylogeny of Gal~l-3Gal Species

Galc~l-3Gal Synthesis

al-3galactosyl Transferase

Mouse

+

+

Pig

+

-I-

New World Monkey

+

-t-

Anti-Gal Antibodies

Old World Monkey Human Mammalian species which do not synthesize Galc~l-3 Gal produce antibodies which react with that determinant. *pseudogene.

lin which contain naturally occurring Galo~l-3Gal determinants are available from various suppliers of biochemicals.

XENOREACTIVE NATURAL ANTIBODIES

Terminology Xenoreactive human natural antibodies are sometimes called "heterophile antibodies" (to denote their binding to heterologous cells). The terms "heteroantibodies" or "xenoantibodies"; however, do not distinguish the origin from the specificity of the antibodies and are unnecessarily vague. While the term "natural" is still used, these antibodies are thought to be elicited in response to environmental stimuli such as gut bacteria (Springer and Horton, 1969; Springer and Schuster, 1964). Antibodies isolated by affinity chromatography with columns containing immobilized c~-galactosyl residues are xenoreactive and are termed "anti-Gal" antibodies (Galili, 1993).

Pathogenetic Role of Xenoreactive Natural Antigens Xenoreactive natural antibodies initiate hyperacute rejection of vascularized organ xenografts (Platt, 1995a). Xenoreactive natural antibodies do so mainly by activating complement on the endothelial cell lining of donor blood vessels. Natural IgM or IgG antibodies might activate complement by the classical and/or alternative pathways. There is compelling evidence that the reaction of human xenoreactive antibodies with porcine cells, which may be a good model for the most clinically relevant combination of different species, initiates the activation of comple-

848

ment through the classical complement pathway (Dalmasso et al., 1992; Dalmasso and Platt, 1993). Anti-Galo~l-3Gal antibodies are postulated to contribute to several physiologic functions, including binding to the surface of damaged or senescent erythrocytes to help clear those cells from the circulation and binding to bacteria to aid in host defense against those microbes.

Xenoreactive Natural Antibodies in Xenograft Rejection There are several functional properties of xenoreactive natural antibodies which affect the ability of the antibodies to mediate hyperacute rejection. Perhaps foremost is the high functional avidity of xenoreactive natural antibodies for porcine cells. High functional avidity facilitates the binding of antibodies to cell surfaces even when the antibodies are present in low concentrations. Xenoreactive IgG appears to bind with approximately 10-fold less avidity to porcine cells, which may contribute to the inability of xenoreactive IgG to mediate hyperacute rejection. Another factor which contributes to the effectiveness of xenoreactive natural IgM antibodies in mediating hyperacute rejection is their ability to activate complement efficiently after binding to a cell. In contrast, the IgG specific for xenogeneic targets is predominantly IgG2 (Ross et al., 1993) which activates complement inefficiently. Yet another factor which influences the biological properties of xenoreactive natural antibodies is the occurrence of these IgM antibodies in all normal sera tested to date and at high concentrations in most individuals. The concentrations of these antibodies vary over a 20-fold range in the population (Parker et al., 1994).

Assays for Xenoreactive Natural Antibodies The first assays for xenoreactive natural antibodies involved hemagglutination (Hammer, 1989). Lymphocytotoxicity was also used extensively to assay for xenoreactive antibodies (Strober et al., 1989; Edwards et al., 1990). Another assay utilizes fluorescence activated cell sorting to measure the binding of xenoreactive natural antibodies to lymphocytes (Edwards et al., 1990). While the use of blood cells for detecting natural antibodies is convenient, some antibodies that are not relevant for xenograft rejection may be detected and some relevant antibodies may not be detected. To avoid this problem, cultured endothelial cells, the presumed target of the rejection reaction, a r e used to measure xenoreactive natural antibody by ELISA (Platt et al., 1990b). With the identification of Gakzl-3Gal as a major target of xenoreactive natural antibodies, solid-phase assays which use proteins such as porcine thyroglobulin that contain Gal~l-3Gal are of use.

CLINICAL UTILITY Xenoreactive antibodies play a critical role in hyperacute xenograft rejection and perhaps in other types of xenograft rejection. Four lines of evidence show that xenoreactive natural antibodies initiate hyperacute rejection of porcine organs transplanted into primates. First, during perfusion of a xenogeneic organ, natural antibodies are rapidly depleted from blood (Platt et al., 1990a; Platt et al., 1990b; Platt and Holzknecht, 1994) and deposited in the xenogeneic organ (Figure 1) (Rose et al., 1991; Platt et al., 1991; Geller et al., 1992). Second, the depletion of natural antibodies by perfusion of blood through a xenogeneic organ (Rose et al., 1991; Platt et al., 1991) or by other techniques (Merkel et al., 1971; Moberg et al., 1971) prolongs the survival of xenogeneic organ grafts, although prolonged survival might in some cases reflect concomitant depletion of complement or other plasmff components. Third, administration of antidonor antibodies hastens the rejection process and in some cases causes hyperacute rejection (Chavez-Peon et al., 1971). Fourth, susceptibility to hyperacute rejection correlates with the presence of xenoreactive natural antibodies in the serum of xenograft recipients (Perper and Najarian, 1967). Xenoreactive natural antibodies are also thought to contribute to the rejection of xenografts when hyperacute rejection is averted; they

Figure 1. Xenoreactive IgM deposited in a porcine heartI transplanted into a baboon.

are implicated in the pathogenesis of acute vascular rejection and may contribute to cellular rejection (Plat, 1995b). Because xenoreactive natural antibodies are destructive to a xenograft and because all individuals except newborns have these antibodies, preventing or reverting the binding of xenoreactive natural antibodies to a donor organ are important therapeutic approaches which must be implemented before clinical xenotransplantation will become useful. Preventing the reaction between xenoreactive natural antibodies and target cells is achieved by blocking antibody binding and by immunoadsorption of the offending antibodies. Administration of soluble antigen to the organ recipient blocks the binding of xenoreactive antibodies to a xenogeneic organ. Unfortunately, this approach has met with only limited success (Ye et al., 1994) because the interaction of xenoreactive antibodies with monovalent ligands is weak. Xenoreactive natural antibodies can be adsorbed from the circulation of recipients by extracorporeal perfusion of the recipient's blood through a xenogeneic organ (Cooper et al., 1988; Platt et al., 1991). Currently several laboratories are testing the ability of columns bearing Galo~l-3Gal to adsorb xenoreactive natural antibodies.

CONCLUSION Based on several observations, xenoreactive natural antibodies like isohemagglutinins, are members of a

849

class of antibodies. First, xenoreactive natural antibodies and isohemagglutinins share such functional properties as high functional avidity for cells, low affinity for soluble saccharides, homogeneous binding to cells, more avid binding at lower temperatures and thermal lability (Parker et al., 1994). Second, xenoreactive natural antibodies, like isohemagglutinins, function in one individual in nearly the same manner as they function in other individuals. Third, based on (1) agglutination titers and (2) affinity isolation and quantitation of IgM antibodies, the level of xenoreactive natural antibodies in human serum is similar to the level of isohemagglutinins and these levels vary similarly in the population. Given the similarity of xenoreactive natural antibodies and isohemagglutinins and the similar density of blood group antigen and Gal~l-3Gal on the cell surface, transplantation of ABO-incompatible organs may be a model for transplantation of xenogeneic organs (Platt, 1995a)~ Of special reference to the

clinical applicability of xenotransplantation may be the experience of a number of groups in bypassing the ABO barrier to allotransplantation. Although antiblood group antibodies return to the circulation of graft recipients and the blood group antigen continues to be expressed, rejection does not occur. The phenomenon, in which temporary depletion of antidonor antibodies allows the "permanent" engraftment of an incompatible organ, is referred to as "accommodation" (Platt et al., 1990c) and evidence of accommodation is seen in xenografts. The concept of accommodation and the success of ABO-incompatible allografts provides an important impetus toward the clinical application of xenotransplantation. See also ALPHA-GALACTOSYL (ANTI-GAL) AUTOANTIBODIES.

REFERENCES

Galili U, Rachmilewitz EA, Peleg A, Flechner I. A unique natural human IgG antibody with anti-~-galactosyl specificity. J Exp Med 1984;160:1519--1531. Galili U, Flechner I, Knyszynski A, Danon D, Rachmilewitz EA. The natural anti-c~-galactosyl IgG on human normal senescent red blood cells. Br J Haematol 1986:62:317--324. Galili U, Clark MR, Shohet SB, Buehler J, Macher BA. Evolutionary relationship between the natural anti-Gal antibody and the Gal ~l-3Gal epitope in primates. Proc Natl Acad Sci USA 1987;84:1369-1373. Galili U. Interaction of the natural anti-Gal antibody with c~galactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol Today 1993;14:480--482. Geller RL, Turman M, Bach FH, Platt JL. Deposition of polyreactive antibodies in xenograft rejection: detection using anti-idiotype monoclonal antibodies. Transpl Proc 1992;24: 595. Hamadeh RM, Jarvis GA, Galili U, Mandrell RE, Zhou P, Griffiss JM. Human natural anti-gal IgG regulates alternative complement pathway activation on bacterial surfaces. J Clin Invest 1992;89:1223-1235. Hammer C. Preformed natural (PNAB) and possibilities of modulation of hyperacute xenogeneic rejection (HXAR). Transpl Proc 1989;21:522-523. Holzknecht ZE, Platt JL. Identification of porcine endothelial cell membrane antigens recognized by human xenoreactive antibodies. J Immunol 1995;154:4565-4575. Landsteiner K. The Specificity of Serological Reaction. New York: Dover Publications, 1962. Malkoff GM. Beitrag zur Frage der Agglutination der rothen Blutkorperchen. Deutsche Medicinische Wohlenschrift 1900;26:229-231. Merkel FK, Bier M, Beavers CD, Merriman WG, Wilson C,

Alvarado CG, Cotterell AH, McCurry KR, Collins BH, Magee JC, Berthold J, Logan JS, Platt JL. Variation in the level of xenoantigen expression in porcine organs. Transplantation 1995;59:1589-1596. Boyden SV. Natural antibodies and the immune response. Adv Immunol 1964;5:1-28. Chavez-Peon B, Monchik G, Winn HJ, Russell PS. Humoral factors in experimental renal allograft and xenograft rejection. Transpl Proc 1971;3:573-576. Collins BH, Parker WR, Platt JL. Characterization of porcine endothelial cell determinants recognized by human natural antibodies. Xenotransplantation 1994;1:36--46. Cooper DK, Human PA, Lexer G, Rose AG, Rees J, Keraan M, DuToit E. Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J Heart Transpl 1988;7:238--246. Dalmasso AP, Vercellotti GM, Fischel RJ, Bolman RM, Bach FH, Platt JL. Mechanism of complement activation in the hyperacute rejection of porcine organs transplanted into primate recipients. Am J Pathol 1992;140:1157-1166. Dalmasso AP, Platt JL. Prevention of complement-mediated activation of xenogeneic endothelial cells in an in vitro model of xenograft hyperacute rejection by C1 inhibitor. Transplantation 1993;56:1171-1176. Edwards N, Ott G, Berger C, He X, Teppler I, Copey L, Smith C, Reemtsma K, Rose E. Incidence of preformed antibodies against potential xenodonors in human sera. Transplantation 1990;49:1022--1024. Galili U, Korkesh A, Kahane I, Rachmilewitz EA. Demonstration of a natural antigalactosyl IgG antibody on thalassemic red blood cells. Blood 1983;61:1258-1264.

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ACKNOWLEDGEMENTS Supported by NIH grants HL50988 and HL52297.

Starzl TE. Modification of xenograft response by selective plasmapheresis. Transplant Proc 1971;3:534-537. Moberg AW, Shons AR, Gewurz H, Mozes M, Najarian JS. Prolongation of renal xenografts by the simultaneous sequestration of preformed antibody, inhibition of complement, coagulation and antibody synthesis. Transplant Proc 1971;3: 538--541. Parker WR, Bruno D, Holzknecht ZE, Platt JL. Characterization and affinity isolation of xenoreactive human natural antibodies. J Immunol 1994;153:3791-3803. Perper RJ, Najarian JS. Experimental renal heterotransplantation. I. In widely divergent species. Transplantation 1966;3:377-388. Perper RJ, Najarian JS. Experimental renal heterotransplantation. III. Passive transfer of transplantation immunity. Transplantation 1967;5:514--533. Platt JL, Lindman BJ, Chen H, Spitalnik SL, Bach FH. Endothelial cell antigens recognized by xenoreactive human natural antibodies. Transplantation 1990a;50:817--822. Platt JL, Turman MA, Noreen HJ, Fischel RJ, Bolman RM, Bach FH. An ELISA assay for xenoreactive natural antibodies. Transplantation 1990b;49:1000-1001. Platt JL, Vercellotti GM, Dalmasso AP, Matos AJ, Bolman RM, Najarian JS, Bach FH.. Transplantation of discordant xenografts: a review of progress. Immunol Today 1990c;11:450456. Platt JL, Fischel RJ, Matas A1, Reif SA, Bolman RM, Bach FH. Immunopathology of hyperacute xenograft rejection in a swine-to-primate model. Transplantation 1991 ;52:214--220. Platt JL, Holzknecht ZE. Porcine platelet antigens recognized by human xenoreactive natural antibodies. Transplantation 1994;57:327--335. Platt J L. Hyperacute Xenograft Rejection. Austin: R.G. Landes Company, 1995a. Platt JL. Xenotransplantation: the need, the immunologic hurdles and the prospects for success. ILAR J 1995b;37:22-31. Rose AG, Coopel DK, Human PA, Reichenspurner H, Reichart B. Histopathology of hyperacute rejection of the heart: experimental and clinical observations in allografts and xeno

graft. J Heart Lung Transplant 1991;10:223--234. Ross JR, Kirk AD, Ibrahim SE, Howell DN, Baldwin WM, III, Sanfilippo FP. Characterization of human antiporcine natural antibodies recovered from ex v i v o perfused hearts - predominance of IgM and IgG2. Transplantation 1993;55:1144-1150. Sandrin MS, Vaughall HA, Dabkowski PL, McKenzie IFC. Antipig IgM antibodies in human serum react predominantly with Galo~(1,3)Gal epitopes. Proc Natl Acad Sci USA 1933;90:11391-11395. Springer GF, Schuster R. Stimulation of isohemolysins and isohemagglutinins by influenza virus preparations. Vox Sang 1964;9:589-598. Springer GF, Horton RE. Blood group isoantibody stimulation in man by feeding blood group-active bacteria. J Clin Invest 1969;48:1280-1291. Strober S, Dejbachsh-Jones S, Van Vlasselaer P, Duwe G, Salimi S, Allison JP. Cloned natural suppressor cell lines express the CD3+CD4-CD8-surface phenotype and the a, b heterodimer of the T cell antigen receptor. J Clin Invest 1969;48:1280-- 1291. Thall A, Galili U. Distribution of Galc~l->3Gall31->4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay. Biochemistry 1990;29:3959--3965. Thompson SC, Mandel TE. Fetal pig pancreas. Preparation and assessment of tissue for transplantation, and its in vivo development and function in athymic (nude) mice. Transplantation 1990;49:571--581. Turman MA, Casali P, Notkins AL, Bach FH, Platt JL. Polyreactivity and antigen specificity of human xenoreactive monoclonal and serum natural antibodies. Transplantation 1991;52:710-717. Ye Y, Neethling FA, Niekrasz M, Koren E, Richards SV, Martin M, Kosanke SI Oriol R, Cooper DK. Evidence that intravenously administered ~-Galactosyl carbohydrates reduce baboon serum cytotoxicity to pig kidney cells (PK15) and transplanted pig hearts. Transplantation 1994;58:330-337.

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AUTOANTIBODIES Critical Characteristics

The table on the following pages was compiled by James B. Peter, M.D., Ph.D. based on the overall literature with a special emphasis on data ~resented in the related chapter(s) in this text NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Acetylcholine receptor autoantibodies (Nicotinic AChR autoantibodies)

Skeletal muscle high affinity receptor for a-bungarotoxin

Myasthenia gravis (90%) Thymoma (30%) Primary lung carcinoma (5--10%) LES (5--10%)

IP of 125I-alpha-bungarotoxin-labeled AChR

Postsynaptic effector of impaired neuromuscular transmission in MG Positive distinguishes acquired MG from congenital MG Reversibly induced by D-penicillamine Marker of autoimmune liver disorders (33%)

Actin autoantibodies

G-actin

Autoimmune CAH (40--90%) Primary biliary cirrhosis (<22%) Connective tissue disease (CTD) (60%)

ELISA

Increased prevalence of HLA-B8, DR3

Adrenal (cytoplasmic) autoantibodies

21-hydroxylase 17-c~ hydroxylase (APGS I?) p-450 side-chain cleavage enzyme (APGS I?)

Addison's disease in western societies: recent (70--80%) longstanding (20--30%) with ovarian failure (90%) APGS I (>90%)

IIF

In adrenal cell destruction, cell-mediated immune mechanisms (T cellular) are probably more important

Alpha-galactosyl autoantibodies (Anti-gal)

Cell surface carbohydrate epitopes

Graves' disease Senescent red cells ~-thalassemia Sickle cell anemia Xenotransplantation Guillain-B~irre syndrome

ELISA with alpha-galactosyl 1% of circulating immunoglobulins epitopes on mouse laminin or produced throughout life are anti-gal linked to BSA autoantibodies

Threonyl-tRNA synthetase

"Antisynthetase syndrome" myositis (80--90%, 60--70% with anti-PL-12); interstitial lung disease (ILD) (80%); arthritis (60--90%); Raynaud's (60%); mechanic's hands (70%). Almost all have myositis and/or ILD; rarely, arthritis alone. -5% of PM/DM have one or another non-Jo-1 antisynthetase

Currently IP is preferred for all. ID good for PL-7; CIE for PL-7, PL-12; IB for EJ, some PL- 12. Inhibition of enzyme activity by antibody can be used for any

Aminoacyl-tRNA synthetase autoantibodies (other than histidyl-tRNA) PL-7 PL-12

Alanyl-tRNA synthetase and tRNA ala

EJ

Glycyl-tRNA synthetase

OJ

Multienzyme complex of synthetases; common antigen is isoleucyl-tRNA synthetase, sometimes also other components

Role in pathogenesis unknown.; associated with HLA-DR52; sera inhibit synthetase activity and precipitate rRNA for antigenic enzyme

oo NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

IIF IB

Significant neurologic improvement may accompany tumor ablation/immunosuppression

Amphiphysin autoantibodies

128 kd synaptic vesicle cytoplasmic protein

Paraneoplastic stiff-man syndrome with breast carcinoma

Antineutrophil cytoplasmic autoantibodies (ANCA) in inflammatory bowel diseases

Unknown nuclear neutrophil antigen yields P-ANCA pattern; P-ANCA is a misnomer

Ulcerative colitis (60--80%) Crohn' s disease (15 %) Primary sclerosing cholangitis (65%)

IIF: for pattern screening; Methanol-fixed neutrophil ELISA; No definitive method

Stratifies HLA associations in ulcerative colitis Produced in intestinal mucosa

Antineutrophil cytoplasmic autoantibodies (ANCA) with specificity for myeloperoxidase

P-ANCA pattern Myeloperoxidase (140 kd)

Microscopic polyangiitis (45%) Idiopathic crescentic glomerulonephritis (65%) Churg-Strauss Syndrome (60%) Wegener' s granulomatosis (10%)

Screening: IIF for ANCA Confirmation: ELISA with purified human myeloperoxidase

In vitro activation of primed neutrophils In vivo possibly pathogenic for crescentic

Antineutrophil cytoplasmic autoantibodies (ANCA) with specificity for proteinase 3

C-ANCA pattern Proteinase 3 (29 kd)

Wegener's granulomatosis (80--95%) Churg-Strauss syndrome (30%) Microscopic polyangiitis (20%) Polyarteritis nodosa (11%) Rapidly progressive glomerulonephritis (8%) '

Screening: IIF Confirmation: ELISA with highly purified native PR3

Useful marker of disease activity Sensitivity: 53--80% Specificity: 97%

Potential marker of disease activity for systemic vasculitis; marker of disease activity in cystic fibrosis; role in GI disease unknown

ELISA IB

Antineutrophil cytoplasmic autoantibodies (ANCA) with specificity other than proteinase 3 and myeloperoxidase (X-ANCA)

Bactericidal/permeability increasing protein (BPI) (55 kd)

glomerulonephritis (animal studies)

ANCA can activate neutrophils and granulocytes in vitro BPI-ANCA may be involved in LPSinduced inflammation. Lactoferrin-ANCA associated with rheumatoid vasculitis

Serological markers for certain rare Lactoferrin (78 kd) forms of systemic vasculitis Cathepsin G (29 kd) Elastase (29 kd) h-lamp-2 (gp 170/80--110; ? 130)

h-lamp-2-ANCA associated with necrotizing crescentic glomerulonephritis

Antinuclear autoantibodies (ANA)

Nuclear antigens in situ

Screen for SLE (>95%) Screen for individual nuclear antibodies Preliminary identification of antibodies

IIF

Detectable in some heathy individuals; Best quantified in IU/mL using WHO standard

Autoantibody penetration into cells

DNA, nRNP, SS-A (Ro), SS-B (La), proteinase 3, Hu, ribosomal protein P

May be observed by direct immunofluorescence of skin in SLE and MCTD

Immunofluorescence, electron microscopy, flow cytometry

May cause immune dysregulation, cell damage and apoptosis; by causing apoptosis of lymphocytes, natural autoantibodies may participate in tolerance

[32-Glycoprotein I ([~2-GPI) autoantibodies

[32-glycoprotein I (apolipoprotein H) (50 kd)

Antiphospholipid phenomena, e.g., thrombosis, fetal loss and thrombocytopenia

ELISA

Thrombotic events in young people See also Phospholipid autoantibodies

NAMES

AUTOANTIGENS

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Bromelain-treated erythrocyte autoantibodies

Phosphatidylcholine

Hemolytic anemia in SLE "Idiopathic" Associated with hematologic malignancies

ELISA Plaque-forming cells

Cause hemolytic anemia in NZB mice. Murine antiphosphatidylcholine antibodies (aPTC) are encoded by five V H gene families. Most human and murine aPTC are IgM

C1 inhibitor autoantibodies

C 1 inhibitor

Acquired angioneurotic edema, type II

ELISA Inhibition of C1 Inhibitor function

Decrease of functional C1 inhibitor associated with secondary decrease of C lq, C4 and C2

C lq autoantibodies

Collagen-like region (CLR) of Clq (176 kd)

Hypocomplementemic urticarial vasculitis syndrome (>90%) SLE (17--46%) Some other glomerulonephritides

ELISA using solid-phase CLR as antigen; anti-Clq also detected by ELISA with C lq in high-salt buffer

Associated with proliferative forms of lupus nephritis

C3 nephritic factor (C3NeF) autoantibodies

Neoantigen on the Bb portion of C3bBb

Membranoproliferative glomerulonephritis Partial lipodystrophy

Prevention of decay/ B cells capable of producing C3NeF are dissociation of C3bBb present in normals. C3 consumption when mixed May be controlled by idiotypic network with normal human serum

Calcium channel autoantibodies (L-type)

Alpha 1 subunit of skeletal muscle voltage-gated calcium channel

Amyotrophic lateral sclerosis (75%)

ELISA with purified alpha 1 subunit

Passive transfer causes altered release of calcium and of acetylcholine from motor neuron terminals

Calcium channel autoantibodies (N-type)

Neuronal high affinity receptor for c0-conopeptide GvIA

Lambert-Eaton myasthenic syndrome (LES) with primary lung carcinoma (73%) LES with no carcinoma (36%) Paraneoplastic encephalomyeloneuropathies (27%) Primary lung carcinoma (22%)

IP

Marker of autoimmune cerebellar ataxia Putative presynaptic effector of impaired CNS/autonomic transmission Positive distinguishes autoimmune from hereditary neuropathies

Calcium channel autoantibodies (P/Q-type)

Neuronal high affinity receptor for c0-conopeptide MvIIC

Lambert-Eaton myasthenic syndrome (LES) (95%) Paraneoplastic encephalomyeloneuropathies (40%) Small cell lung carcinoma (18%)

IP

Positive distinguishes LES from MG Putative presynaptic effector of impaired neuromuscular transmission in LES Positive distinguishes autoimmune from degenerative neurologic disorders

Centriole/centrosome autoantibodies

Centriole/centrosome antigens

Scleroderma spectrum of diseases

IIF

More clinical studies required

Enolase

L~

CLINICAL UTILITY (frequency in specific diseases)

IB of purified enolase

L~

NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency. in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Centromere autoantibodies

CENPs A, B and C, centromererelated proteins

SSc (30%) RA and SLE (<5%) Marker for limited form of SSc

IIF IB

Microinjection into cells disrupts kinetochore assembly and progression through mitosis.

Chromo autoantibodies

Heterochromatin proteins p23 and p25

10% of patients with centromere autoantibodies

IB

Autoantibodies recognize a conserved structural motif that may be responsible for suppressing gene transcription.

Coagulation factor autoantibodies (excluding factor VIII)

Factor II through Factor XII

Bleeding disorders

Bethesda method

Immune-mediated coagulopathy

Coagulation factor VIII autoantibodies

Factor VIII

Idiopathic (46%) Autoimmune diseases (18%) Malignancies (7%) Postpartum (7%)

Inhibition of coagulation assay; Inhibition of chromogenic substrate conversion; ELISA

Can result in life-threatening bleeding disorders

Collagen autoantibodies

Collagen Type I

RA (20--60%) SLE (20--40%) Vasculitis (20%) Scleroderma (86%) Thromboangiitis obliterans (44%)

ELISA RIA

-5% of normal population have autoantibodies to denatured collagen Type I.

Collagen Type II

RA (20--80%) SLE (15--35%) Relapsing polychondritis (44%) Meniere' s disease

ELISA RIA

Denatured collagen Type II (oq chain) is biochemically similar to collagen Type XI (or3 chain); autoantibodies may crossreact

Collagen Type IV

SLE (85%) Scleroderma (54--68%) Juvenile RA (5%) RA (16%) Vasculitis (55%) MCTD (5%)

ELISA RIA

C lq contains a collagen-like sequence that can cross-react with collagen autoantibodies

NAMES

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Cryoglobulins Rheumatoid factor (IgM) autoantibodies Anti-Fc activity Anti-idiotypic anti-F(ab')2 activity? RF arising as internal image anti-idiotypic antibodies?

IgG with antiviral activity (antiHCV, anti-HTLV-I gag p19). IgG with antiglomerular activity against a 50 kd renal antigen?

Mixed essential cryoglobulinemia (16--57%) Multiple myeloma (3--12%) Sj6gren's syndrome (3--8%) Waldenstr6m' s macroglobulinemia (8--19%) SLE (4--89%) Rheumatoid arthritis (20-25%) Scleroderma (50%) Polyarteritis nodosa (5%) Henoch-Sch6nlein purpura (47--60%) Chronic infections (2--90%) Chronic lymphocytic leukemia (9%) Chronic liver disease (7--90%)

ELISA

In experimental models, glomerulonephritis can be induced by a cryoprecipitating monoclonal IgG3,.but for vasculitis to occur, the RF activity of the monoclonal must be present. In man, there is morphological identity between glomerular deposits and serum cryoprecipitate. Moreover, the crossreacting idiotype present on circulating cryo IgM RF can also be detected in the Ig of renal biopsies.

Cryoglobulins secondary to hepatitis C virus infection (Monoclonal IgM rheumatoid factors [mRF] bearing the WA cross-idiotype [Xid])

Hepatitis C virus ( H C V ) - IgG complexes? H C V - lipoprotein (VLDL)

Type II cryoglobulinemia associated with HCV infection

ELISA assay for WA Xid

Mixed cryoglobulins containing HCV, VLDL, WA mRF and IgG mediate vasculitis. See also Cryoglobulins

Cytokine autoantibodies

IL-I~ IL-6 IL-10 IFN-c~ LIF (leukemia inhibitory factor)

Absent in Crohn's disease Decreased in atopic disease?

Double antibody precipitation radioimmunoassay (ELISA for screening)

High affinity; Naturally occurring; Neutralizing (in vitro) autoantibodies; Underlie the anti-inflammatory effect of high-dose IVIg?

Actin

Chronic liver diseases (40--90%) CTD (10--60%) Parasitic infections Potential marker of cell destruction

ELISA

Also found in some normal individuals (about 10%); can be produced by nonspecific stimulation of immune system

Cytokeratin

CTD (25--75%) Infectious diseases

ELISA

Vimentin

CAH CTD (25--60%) Chronic liver disease (25--35%) Lymphoproliferative diseases Infectious diseases

Cytoskeletal autoantibodies Actin autoantibodies

Intermediate filament autoantibodies

(continued)

OO ",-3

AUTOANTIGENS

NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Cytoskeletal autoantibodies

(continued)

Desmin

Chronic liver disease (40-45%) Potential marker of cell destruction

Neurofilaments

Prion diseases Alzheimer' s disease ALS

ELISA, IB

Neuronal, intermediate filamentPhosphorylated in axons NF triplet = 200, 160, 70 kd

Tubulin

Chronic liver diseases ( 3 0 - 6 0 % ) Guillain-B~irre syndrome Viral, parasite infections Potential marker of cell destruction

ELISA

Found in normal individuals (-15%) Might be produced by nonspecific stimulation of immune system

dsDNA autoantibodies

DNA

SLE (60-90%) Potential marker of disease severity

Farr assay preferred ELISA and Crithidia less reliable

Farr is diagnostic and predictive of SLE. Increasing Farr levels predict exacerbation.

Endomysial autoantibodies

Antigenic determinants on Celiac disease (68--100%) noncollagenous proteins produced Dermatitis herpetiformis (70-- 100%) by fibroblasts

IIF with cryostat sections of monkey esophagus

High specificity for active gluten-sensitive enteropathy (99.7--100%)

Endothelial cell autoantibodies (AECA)

Constitutive endothelial proteins (ranging from 15 to 200 kd)

Cell-ELISA IP Cytofluorometry IB

Cytotoxicity (Ab-, C' and ADCCmediated)

Tubulin autoantibodies

Nonconstitutive adherent molecules (DNA histones, ~2-GPI)

56-kd nuclear RNP protein autoantibodies

Primary autoimmune vasculitis (WG, MPA up to 80%, Kawasaki up to 60%), vasculitis associated with autoimmune diseases (SL'E up to 80%, Antiphospholipid syndrome up to 60%, RA up to 80%)

Cytokine upregulation in Kawasaki disease and downregulation in hemolytic-uremic syndrome

Allografis

56-kd nRNP

Myositis (85%) PM (85%) DM (85%) JDM (85%) Myositis + cancer (75%) Myositis + CTD (85%) Marker for differential diagnosis of myositis; potential marker for disease severity in adults; potential marker for a subgroup of JDM

IB ELISA with purified 56 kd protein

Endothelial activation (adhesion molecule expression, cytokine secretion, PGI 2 metabolism)

Component of large nuclear RNP complex -- the nuclear RNA processing apparatus of eukaryotic cells

NAMES

Lpl

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Fibrillarin autoantibodies

Fibrillarin (34 kd nucleolar antigen)

Marker antibody for SSc (3--6%)

IIF on cultured cells (screening & reactivity with native autoAg) IB (confirmatory in combination with IIF) IP with radiolabeled fibrillarin (confirmatory)

Pathogenetic role in SSc unknown. In genetically susceptible mice, mercury and silver induce fibrillarin autoantibodies. Systemic deposits of immune complexes containing fibrillarin autoantibodies and glomerulonephritis.

Fibronectin autoantibodies

Fibronectin (440 kd)

SLE (29--34%) RA (14%) Viral, bacterial infections Potential marker of disease severity

ELISA with bovine fibronectin Capture ELISA with human fibronectin

Immune complex-associated renal damage in animal studies

Filaggrin autoantibodies "Antikeratin autoantibodies"

Cornified layers of rat and human squamous epithelia Filaggrin and profilaggrin-related proteins

High diagnostic value for RA: sensitivity -50% specificity -99% early marker, can precede the onset of disease Prognostic value: correlation with active and/or severe forms to be confirmed by prospective studies

IIF on cryosections of rat esophagus IB on extract of rat esophagus epithelium IB on the neutral-acidic variant of human epidermal filaggrin

Closely related to antiperinuclear factor (APF). Antiperinuclear factor, antikeratin and antifilaggrin are largely the same RAspecific autoantibodies.

Ganglioside autoantibodies

Branched and unbranched oligosaccharides containing sialic (Nacetyl or N-glycolyl neuraminic) acid residues

PBC neuropathy (15%) Sudden deafness (40%) Multiple sclerosis (20%) Neuropsychiatric SLE (10--50%) Miller-Fisher syndrome (95%) Sensory polyneuropathy with monoclonal gammopathy (5--50%) Chronic inflammatory demyelinating polyradiculoneuropathy (20%) Guillain-Barr6 syndrome (15%) Motor neuron disease (20--50%) Amyotrophic lateral sclerosis (25%) Multifocal motor neuropathy (35 %)

ELISA High-performance TLC Liposome immune lysis assay (LILA)

Autoantibodies can cross-react with related carbohydrate residues on glycoproteins. Reflecting molecular mimicry, autoantibodies to bacterial polysaccharides might bind to peripheral nerve glycoconjugates and induce neuropathy.

Gliadin antibodies (AGA)

Gliadin

Celiac disease (85--95%)

ELISA

IgG and IgA AGA are valuable for diagnosis of atypical and silent forms of celiac disease.

Glomerular basement membrane autoantibodies

tx3 chain of type IV collagen

Goodpasture' s syndrome (100%) Pulmonary-renal syndrome (15-20%)

ELISA with purified antigen

Positive test heralds urgent need for treatment. Negative test rules out active anti-GBM disease

o

NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Glutamic acid decarboxylase autoantibodies

GAD 65 & GAD 67

Stiff-man syndrome (linear & conformation-dependent epitopes) (60-- 100%) IDDM (conformation-dependent) (-80%)

IP

Marker of pancreatic [3-cell autoimmunity; associated with HLA in IDDM. Relevance of autoantibodies to pathogenesis of stiff-man syndrome and IDDM is unknown. IgG subclasses denote level of risk of IDDM (in relatives of people with IDDM).

Glycolipid autoantibodies (excluding Ganglioside autoantibodies)

Primarily branched and unbranched oligosaccharides, with or without sulfate and without neuraminic acid; lipid moieties can also be antigenic determinants

Neurological autoimmune diseases (5-88%) Neuropsychiatric SLE (50--81%) Leprosy (100%) RA/Overlap syndrome (55%) Leukemia/lymphoma (33 %) ITP (31%); Lung cancer (17%) MCTD (17%)

ELISA High performance TLC Liposome immune lysis assay

Autoantibodies can cross-react with related carbohydrate residues on glycoproteins.

Golgi apparatus autoantibodies

Golgin 95 Golgin 160 Golgin 97 Golgin 180 Macrogolgin/giantin (376 kd) GCP372

Systemic diseases (e.g., Sj6gren's syndrome, SLE) Cerebellar dysfunction; malignancies Miscellaneous Viral infections Indicate particular subsets of patients?

IIF on adherent cells (HEp-2 or IMR-33)

Induced by Lactate Dehydrogenase Elevating Virus infections in mice

Granulocyte-specific antinuclear autoantibodies (GS-ANA)

Unknown

RA (50--75%) Felty's syndrome (FS) (_>90%) Potential marker of erosive disease

IIF

Involved in local and circulating immune complexes, especially pronounced in FS where GS-ANA strongly fix complement

Heat shock protein autoantibodies

Hsp 90, Hsp 70, Hsp 60

No clear disease associations; may reflect IB with cell lysates tissue damage or stress ELISA with recombinant proteins

Possible role in antigen presentation

Heparin-associated autoantibodies

Platelet factor 4 (PF4)heparin complex

Heparin-induced thrombocytopenia (HIT) (>90%)

ELISA with complexes of PF4/heparin

Reactivity with endothelial cells and platelet activation

Heterophile antibodies

Xeno-antigens

Confound autoantibody detection on nonhuman substrates

IIF on rodent tissue

No diagnostic role in autoimmunity

Histidyl-tRNA synthetase autoantibodies (Jo-1 autoantibodies)

Histidyl-tRNA synthetase

Polymyositis (2%) Disease specific Marker for clinical subset: "antisynthetase syndrome"

ID IB IP of tRNA

Associated with HLA class II genes related to HLA-DRw52 specificity

Histone (H2A-H2B)-DNA autoantibodies

(H2A-H2B)-DNA subunit of chromatin

Drug-induced lupus (33--95%) SLE (50--75%) SSc (20%)

ELISA with (H2A-H2B)DNA complexes or with chromatin

Only IgG antibodies are useful for evaluation of drug-induced lupus.

NAMES Histone autoantibodies [other than Histone (H2A-H2B)DNA autoantibodies] (AHA)

AUTOANTIGENS Histones Nucleosomes

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTI(~N METHODS

SLE, DIL, RA, JRA, other rheumatic disease, PBC, infectious diseases, neurological diseases

IB

SLE (35--80%) depending on histone subfraction considered DIL (>90%) RA (30--75%) JRA (30--75%) PBC (40--60%) Systemic sclerosis (30%) Localized scleroderma (45%) Felty's syndrome (80%) Only in some cases have AHA been reported to correlate with disease activity in SLE

ELISA

OTHER IMPORTANT FEATURES Possible relevance to the pathogenesis of SLE glomerulonephritis

IgA-fibronectin (Fn) aggregates

Antigen-antibody complexes or nonspecific binding of Fn to IgA

IgA nephropathy (48--93%) Other glomerular disease (12%) Possible serological marker for differentiating IgA nephropathy

ELISA

IgA-Fn aggregates not responsible for renal injury See also Fibronectin autoantibodies

IgE receptor autoantibodies

High affinity IgE receptor (Fcs ~-subunit

Chronic urticaria (25%)

Basophil histamine release assay

Activation of mast cells by cross-linkage of Fcs

Insulin autoantibodies

Insulin

IDDM (- 10%) Insulin autoimmune syndrome

RBA

Inversely associated with age of onset of IDDM

Interferon-inducible protein IFI 16 autoantibodies

IFI16 (80 kd nucleolar and nucleoplasmic protein) Expression inducible by IFN7

SLE (29%) Anti-dsDNA-positive SLE (35%) SSc/PM (4%)

ELISA with recombinant IF116 protein

No significant association with nephritis or sicca symptoms

Islet cell autoantibodies (ICA)

Several, but glutamic acid decarboxylase (GAD65) predominant

IDDM (75--86%) NIDDM (10%) Potential marker for insulin-dependent diabetes

IIF on frozen sections of blood group 0 human pancreas

Predictive marker for IDDM in immune intervention research

Islet cell surface autoantibodies (ICSA)

Unknown

IDDM (30--70%)

IIF, RBA or complementdependent cytotoxicity on insulin-producing cells

Unclear role in immune-mediated 13-cell destruction

Ki autoantibodies

Ki antigen (32 kd)

SLE (12%) Overlap syndrome (20%)

ID, IP, ELISA

Autoantibodies may cross-react with SV40 large T-cell antigen in some cases

Always associated with positive ANA

Oo

bo

NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES Marker for scleroderma-polymyositis overlap syndrome in Japanese patients

Connective autoantigen in inner ear diseases

Ku autoantibodies

Ku70, Ku80

SLE (5--6%) Overlap syndrome (30%) Other systemic autoimmune diseases (<3%) "

IP, Antigen-capture ELISA

Laminin autoantibodies

Proteoglycan

Sensorineural hearing loss (14--68%)

ELISA IB

Liver/kidney microsomal (LKM) autoantibodies

LKM-I: P450 II D6

Autoimmune hepatitis

L K M - 2 : P 4 5 0 II C9

Drug-induced hepatitis

LKM-3: UGT 1

Chronic hepatitis D

IB with recombinant P450 II D6 is most sensitive and specific for LKM-1 autoantibodies

LM: P450 I A2

Halothane hepatitis

IIF for LKM pattern

Other enzymes

Chronic hepatitis C

ASGPR

Autoimmune hepatitis (80%)

ELISA, RIA, IB

LMA autoantibodies

Liver membrane antigen (LMA), 26 kd protein

Autoimmune hepatitis (up to 100%); Viral hepatitis C (90%)

IIF

LSP autoantibodies

Liver-specific protein (LSP) 43 kd protein

Autoimmune hepatitis (up to 100%); Chronic viral hepatitis (50%)

RIA, ELISA

PM autoantibodies

Hepatocyte plasma membrane (PM)

Autoimmune hepatitis

IB

Lupus anticoagulant autoantibodies

Prothrombin [32-Glycoprotein I Other phospholipid-binding proteins

SLE (34%) Drugs: chlorpromazine (37--87%) Normal blood donors (-1%) Viral, protozoal, bacterial infections Potential marker for thrombotic predisposition

Screen: activated partial thromboplastin time (APTT) Confirmation: platelet neutralization procedure (PNP)

Can be pathogenic by inhibiting protein C system Arterial/venous thrombosis Recurrent fetal loss Thrombocytopenia

Lymphocytotoxic autoantibodies (LCA)

T cells Possible target antigens: CD45, ~2M, TCR, MHC

In SLE with lymphopenia, CNS and spontaneous abortion Viral infections

Microcytotoxicity assays, FACS

Influence on T-cell function is still not conclusive

Liver membrane autoantibodies Asialoglycoprotein receptor (ASGPR) autoantibodies

Linked to inflammatory activity

Proved to be pathogenic in animal models; linked to inflammatory activity

NAMES Mi-2 autoantibodies

All react with 240 kd nuclear protein, probably helicase; smaller proteins may react

Mitochondrial autoantibodies

E2 subunits of: pyruvate dehydrogenase complex

Mitotic spindle apparatus autoantibodies

Myelin-associated glycoprotein (MAG) autoantibodies

Myelin basic protein (MBP) autoantibodies

taO

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

97% with Mi-2 have DM; 15--25 % of adult DM (10-- 15 % JDM) have Mi-2; high disease specificity makes useful for diagnosis

Currently IP or ID Promising initial results with ELISA/IB of recombinant protein

PBC (>95%)

ELISA and IB

branched chain 2-oxo-acid dehydrogenase complex

PBC (53--55%)

ELISA and IB

2-oxo-glutarate dehydrogenase complex

PBC (39--88%)

ELISA and IB

PBC (41--66%)

ELISA and IB

E1 alpha subunit of pyruvate dehydrogenase complex Protein X

PBC (>95%)

NUMA (236 kd)

Sj6gren's syndrome (SS)

MSA 35 (35 kd)

Variety of other rheumatic and infectious IB diseases

The carbohydrate antigenic epitope (sulfate-3 glucuronate) of MAG is the predominant epitope. It is shared by the peripheral nerve system-specific P0-protein and by the two acidic glycolipids SGPG and SGLPG.

Polyneuropathy associated with IgM monoclonal gammopathy occurring in monoclonal gammopathy of unknown significance

MBP (18.5 kd)

IIF

OTHER IMPORTANT FEATURES High frequency of HLA-DR7; role in pathogenesis unknown

Diagnostic marker of PBC Role in pathogenesis unknown Titers do not correlate with disease stage.

More clinical studies required

IB ELISA HPTLC

Plasma exchange, i.v. immunoglobulins and/or immunosuppression are beneficial in polyneuropathy associated with antiMAG IgM paraproteinemia

ELISA RIA

Associated with demyelinating diseases

Waldenstr6m's macroglobulinemia (50%)

Multiple sclerosis (CSF) (79%) Optic neuritis (CSF) (89%) AIDS dementia complex (CSF) (74%) Alzheimer's disease (serum) (89%)

Potential marker for disease activity

4:~

NAMES Myocardial autoantibodies Cardiac

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS IIF with human cardiac tissue

Fibrillary and sarcolemmal proteins

IDCM (40--70%) Myocarditis (>70%) Dressier' s syndrome Chagas disease Rheumatic carditis Serologic marker for differentiating above conditions from coronary disease

Adenine nucleotide translocator (ANT) autoantibodies

ANT (32 kd)

IDCM (70%) Myocarditis (75%)

Branched-chain ctketoacid dehydrogenase (BCKD) autoantibodies

BCKD-E2 (52 kd)

Cardiac myosin heavy chain autoantibodies

Cardiac myosin heavy chain (200 kd)

IDCM (70%) Myocarditis (70%)

Neuronal autoantibodies

Several neuronal, lymphocytic and intracellular specificities (including 52 kd)

Neuroblastoma binding or SLE (25--50%) Other connective tissue diseases (5--10%) cytotoxicity assay IB Potential CSF marker for SLE Neuropsychiatric SLE

Neuronal nuclear autoantibodies, Type 1 (ANNA- 1; Hu autoantibodies)

35--40 kd CNS and PNS neuronal Small cell lung carcinoma-related neuropathies, including sensoriRNA-binding proteins (HuD, neuropathy, GI dysmotilities and HuC, Hel-N1 family) encephalomyeloradiculopathies (>80%) Small cell lung carcinoma (10--20%)

Neuronal nuclear autoantibodies, Type 2 (ANNA-2; Ri autoantibodies)

55 kd (Nova) and 80 kd CNS neuronal RNA-binding proteins

IDCM (70%) Myocarditis (70%)

Breast or small cell lung carcinomarelated midbrain/cerebellar encephalopathy, myelopathy and, less often, neuropathy

OTHER IMPORTANT FEATURES Autoantibodies may bind to myocyte cell surfaces and disturb Ca ++homeostasis and energy metabolism Autoantibodies may bind to heart valve matrix in rheumatic disease and initiate valve injury

ELISA with human skeletal muscle ANT

ELISA with human cardiac enzyme

ELISA with human cardiac myosin May be consequence of neural injury May bind directly to central neurons in specific conditions (e.g., chorea)

IIF IB with native or recombinant antigen

Female > male (2:1) Prevalence relatively low when small cell lung carcinoma-related limbic or cerebellar syndromes are not accompanied by a neuropathy A marker in children of autoimmune encephalopathies/neuropathies (sometimes associated with neuroblastoma) Calcium channel autoantibodies (N>P/Q) may coexist

IIF IB with native or recombinant antigen

Female > male (3:2) Significant neurologic recovery may accompany tumor ablation/ immunosuppression Calcium channel autoantibodies (N>P/Q) may coexist

NAMES

oo

AUTOANTIGENS

CLINICAL U T I L I T Y (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Neutrophil autoantibodies

l~c 7-RIII (CD 16) CR3 (CD1 lb)

Autoimmune neutropenia

IIF and flow cytometry

May be a secondary finding in SLE, RA Also infectious or drug-induced

Nuclear envelope autoantibodies

Nuclear lamins LAP1 LAP2 p58 gp210 Tpr

CTD (up to 30%)

IB using NE-enriched fractions

No known influence on classification or prognosis.

PBC (up to 30%)

Nucleolar autoantibodies

Several proteins and small ribonucleoprotein complexes of the nucleolus

SSc (18--43%) Potential marker of severity Occasionally present in other autoimmune diseases and in cancer patients

Screening: IIF Autoantigen identification: IP or IB

Nucleolar autoantibodies can be induced by heavy metals in certain mouse strains

Nucleosome-specific autoantibodies

Nucleosomes, chromatin, DNA/histone-structures

SLE (common, but frequencies not yet known)

ELISA after absorption of histone and DNA autoantibodies

Mediate nephritis

p53 autoantibodies

The tumor suppressor gene p53

Only in cancer patients (5-40% depending on the type of cancer); Absent in normal population; Serological marker for cancer

ELISA

Can be used to monitor response to treatment. Can be used to detect precancerous lesions in individuals at high cancer risk.

Parietal cell autoantibodies

Gastric H+/K+ ATPase

Pernicious anemia (90%)

IIF ELISA

Autoantibodies probably not responsible for gastric lesion

Perinuclear factor autoantibodies

Perinuclear kerato-hyalin granules in buccal mucosal cells (Profilaggrin)

RA (60--80%)

IIF

Probably related to "antikeratin antibodies" (Filaggrin autoantibodies) Portend active/severe RA

Phospholipid autoantibodies cardiolipin (aCL)

Cardiolipin ~2-GPI Cardiolipin-13zGPI

SLE (30--40%) Associated with thrombosis, thrombocytopenia and recurrent fetal loss

ELISA

aCL: most frequent cause of acquired thrombophilia See also Phospholipid autoantibodies Phosphatidylserine

Phospholipid autoantibodies phosphatidylserine (aPS)

Phosphatidylserine

SLE (35%) SLE with thrombosis and/or thrombocytopenia (54%) Recurrent spontaneous abortion (7--15%) RSA with positive lupus anticoagulant or anticardiolipin antibodies (87%) Cardiac failure or transplant (20--37%) Healthy blood donors (4%)

ELISA

aPS autoantibodies are found in up to 10% of SLE patients in the absence of anticardiolipin antibodies See also Phospholipid autoantibodies Cardiolipin

NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Monoclonal antibody immobilization of platelet antigen (MAIPA)

Immune-mediated platelet destruction

Complex of 11-- 16 proteins (most Myositis (8%) SSc (3%) prominent band at 100 kd) 50% of anti-PM-Scl-positive patients have myositis/scleroderma overlap

ID IP is specific

Positive ANA with nucleolar patterns DR3-associated

Proliferating cell nuclear antigen (PCNA) (36 kd) Major epitope: VSDYEMKLMDLVEQ (aa 111--125) Minor epitope: (aa 181--195)

IIF/ID ELISA/IB IP for conformationdependent epitopes

Often very responsive to steroids PCNA crucial in: Cell cycle DNA replication DNA repair PCNA estimate of: Cell proliferation

IIF IB with native or recombinant antigen

>99% female 6% present with peripheral neuropathy Tumor identifiable in 90% Not found with primary lung carcinoma Calcium channel autoantibodies (P/Q or N) may coexist

Platelet autoantibodies

Glycoprotein IIb/IIIa, Ib-IX, V

PM-Scl autoantibodies

Proliferating cell nuclear antigen autoantibodies IgG/IgM (Anti-PCNA)

Idiopathic thrombocytopenia purpura (ITP) (80--90%) Autoimmune thrombocytopenia purpura (AITP) (>80%)

SLE (2--10% by IIF/ID) SLE (7% by ELISA/IB) Defined or evolving SLE No specific clinical subset associations

Purkinje cell cytoplasmic autoantibodies, Type 1 (PCA1; Yo autoantibodies)

52--62 kd cytoplasmic proteins of Carcinoma of ovary > breast > other mtillerian tissue; usually with subacute large neurons cerebellar ataxia

RA33 (Heterogeneous nuclear ribonucleoprotein complex autoantibodies)

The proteins A2, B 1, B2 of the heterogeneous nuclear RNP (hnRNP)

RA (35%) SLE (20%) MCTD (40%) Serological marker for early RA; Differentiate RA from other arthritides

IB

Detectable in early undifferentiated RA; dsDNA-anti-dsDNA immune complexes can bind (in a nonspecific manner) to the blotted antigen giving rise to falsepositive results.

Red cell autoantibodies Warm-type Cold-type Donath-Landsteiner antibodies

Red cells Rh I, i, Pr P

Autoimmune hemolytic anemia

Hemagglutination, hemolysis

Transient: postinfectious forms Persistent: chronic B-lymphocyte proliferation

Reticulin autoantibodies (IgA)

Unknown

Celiac disease (50--70%) Dermatitis herpetiformis (25%) Marker of untreated severe gluten enteropathy

IIF on composite block of frozen rat liver and kidney or frozen monkey esophagus

No evidence for primary pathogenetic significance See also Endomysial autoantibodies

NAMES Retinal autoantibodies

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

IRBP (140 kd) S-antigen (50 kd) Rhodopsin (40 kd) Phosducin (33 kd)

Possible retinal hypersensitivity Uveitis Possible retinal hypersensitivity Possible retinal hypersensitivity

CAR antigen (recoverin) (23 kd)

Marker for cancer (poor prognosis) and cancer-associated retinopathy

Retroviral autoantibodies

A variety of retroviral proteins (mainly gag)

SLE, SS, MCTD, multiple sclerosis, IDDM

IB with native or recombinant antigens

Retroviruses may cause autoimmune reaction by molecular mimicry or insertion mutagenesis.

Rheumatoid factors (RFs): IgM, IgA and IgG

IgG Fc region

RA: IgM (92%), IgG (66%), IgA( 65%); marker for more severe disease SEE: IgM (59%), IgG (27%), IgA (36%) SS: IgM (55%) IgA (55%)

Nephelometry ELISA/RIA Agglutination

Seropositive RA patients may have local immune complexes that can activate complement. RF in RA are associated with HLA-DR4.

Ribosomal P protein autoantibodies

Ribosomal phosphoproteins P0, P1 and P2

Specific marker for SLE (10--40%) Associated with neuropsychiatric manifestations of SLE and generalized disease activity

ELISA (synthetic P peptides or fusion protein) IB (Crude ribosomes)

P peptides are expressed on the surface of cultured human neuroblastoma cells

RNA polymerase I, II, III autoantibodies

RNA polymerase enzymes of classes I, II, III

Anti-RNA polymerase I, III: SSc (4--33%) Anti-RNA polymerase II: SSc (20%); SLE/MCTD/Overlap syndrome: (10%)

IP with 35S-methioninelabeled cell extract

Anti-RNA polymerase I: pathogenesis of murine lupus nephritis HLA-DR4 association

S-100 autoantibodies

o~ monomer = 10.4 kd 13 monomer = 10.5 kd

GBS Alzheimer's disease

ELISA, IB

Cytoplasmic neuronal antigen

Signal recognition particle (SRP) autoantibodies

SRP proteins

Polymyositis specific (5%)

IP of HeLa lysates

Associated with very poor prognosis (5-year survival: 25%)

Bullous pemphigoid 180 (BP180) autoantibodies

BP180 (180 kd)-epidermal hemidesmosomes

Bullous pemphigoid (50%) Herpes gestationis (75%) Cicatricial pemphigoid

IIF with sodium chlorideseparated normal human skin IB with total human epidermal extracts

Autoantibodies directed against mouse BP180 are pathogenic by passive transfer to neonatal mice.

Bullous pemphigoid 230 (BP230) autoantibodies

BP230 (230 kd)-epidermal hemidesmosomes

Bullous pemphigoid (90%) Herpes gestationis (rare) Cicatricial pemphigoid Paraneoplastic pemphigus

IIF with sodium chlorideseparated normal human skin IB with total human epidermal extracts

Intracellular autoantigen Autoantibodies are probably not pathogenic.

IB

Autoantibodies to these four antigens occur in normals; higher titers can be found in retinal disease

Small cell lung carcinoma

Skin autoantibodies

(continued)

OO

oo NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Skin autoantibodies

(continued) Desmoglein 1 autoantibodies

Desmoglein 1 (160 kd)-epidermal desmosomes

Pemphigus follaceus (100%) Pemphigus vulgaris (50%) Correlate with disease activity

IIF with stratified squamous epithelium (normal human skin) IB with total human epidermal extracts IP with radiolabeled human keratinocyte extracts or bovine muzzle epidermis

Pathogenic by passive transfer to neonatal mice

Desmoglein 3 autoantibodies

Desmoglein 3 (130 kd)-epidermal desmosomes

Pemphigus vulgaris (100%) Correlate with disease activity

IIF with stratified squamous epithelium (monkey esophagus) IB with total human epidermal extracts IP with radiolabeled human keratinocyte extracts

Pathogenic by passive transfer to neonatal mice

Desmoplakins I and II autoantibodies

Desmoplakin I (250 kd) and Desmoplakin II (210 kd)epidermal desmosomes

Paraneoplastic pemphigus (100%) Some patients with erythema multiforme maj or

IIF with stratified squamous Intracellular autoantigens epithelium and other types of Autoantibodies are probably not pathogenic. epithelia (rat bladder) IB with total human epidermal extracts IP with radiolabeled human keratinocyte extracts

F-actin

Autoimmune hepatitis (AH) (-97%)

IIF on cultured fibroblasts

Vimentin, desmin, tubulin

Viral Infections, SLE, RA, SS

IIF on mouse stomach

snRNP particles

SLE (20--40%) MCTD (100%)

ELISA, IP, IB

Sm

Sm core proteins (B', B, D, E)

SLE (20--30%); highly specific

U1 snRNP (RNP)

U1 snRNP (70 K, A, C)

SLE (30--40%); MCTD (100%); RA, PM, SSc, SS (rare)

Smooth muscle autoantibodies

Spliceosomal autoantibodies [Small nuclear ribonucleoprotein (snRNP) autoantibodies]

(continued)

HLA-B8, DR3 female with AH Immunopathogenic effect unknown Associations with clinical manifestations are inconsistent; roles in pathogenesis are unknown

NAMES

AUTOANTIGENS

CLINICAL UTILITY (frequency in specific diseases)

Spliceosomal autoantibodies [Small nuclear ribonucleoprotein (snRNP) autoantibodies]

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

ELISA, IP, IB

Associations with clinical manifestations are inconsistent; roles in pathogenesis are unknown

(continued) U2 snRNP

U2 snRNP (A', B")

SLE (15%); MCTD (15%); Overlap syndromes (occasional)

Other

U4/U6 snRNP, U5 snRNP

SS, SSc (rare)

U7 snRNP, U11 snRNP, U12 snRNP

No comprehensive studies available

SS-A (Ro) autoantibodies

SS-A (Ro) (60 kd, 52 kd)

Primary SS (60-75%) SLE: (40-5O%) Present in virtually all mothers who have children with neonatal LE and patients with subacute cutaneous LE

ID ELISA

Strong MHC associations; pathogenic role in lupus nephritis, complete congenital heart block

SSzB (La) autoantibodies

SS-B (La) phosphoprotein (47 kd)

Primary SS (10-40%) SLE (6-- 15%) Subacute cutaneous LE (25--35%) Neonatal LE (maternal anti-La -90%)

ELISA with recombinant or native SS-A (La) IB with cell extracts

HLA-DR3, DQw2

Steroid cell autoantibodies

17-c~-hydroxylase? P-450 side-chain cleavage enzyme?

Ovarian failure unselected (<1%) with Addison's disease (60-100%)

IIF

Steroid cell autoantibodies occur always in combination with adrenal cytoplasmic antibodies

IIF ELISA

Suggests thymoma in MG when coexisting with high value for acetylcholine receptor modulating antibodies Very uncommon in juvenile MG (age < 20), unless with graft-versus-host disease Autoimmune and chronic hepatitis (40%) Graft versus host disease (<10%)

Addison's disease with adrenal antibodies but without ovarian failure (10--20%) Striational autoantibodies (Skeletal muscle autoantibodies)

or ~,o

Proteins of skeletal muscle sarcoma, including titin, actin, c~-actinin, myosin and ryanodine receptor

MG and thymoma (80--90%) Absent in over 70% of MG without thymoma Adult-onset MG (30%) MG onset > age 60 (55%) Thymoma without MG (24%) Primary lung carcinoma (5--10%) LES (5--10%)

OO --O

o

NAMES Thyroglobulin autoantibodies

AUTOANTIGENS

Thyroglobulin

CLINICAL UTILITY (frequency in specific diseases) Autoimmune thyroid disease Lymphocytic thyroiditis (Hashimoto' s thyroiditis [36-- 100%]) Primary myxedema (72%) Graves' disease (50--98%)

PREFERRED DETECTION METHODS

OTHER IMPORTANT FEATURES

Hemagglutination (chronic chloride) ELISA

Rarely positive in the absence of thyroid peroxidase antibodies

Autoimmune endocrinopathies Diabetes (20%) Addison's disease (28%) Pernicious anemia (27%) Other: Thyroid carcinoma (13--65%) Nontoxic goiter (8%) Thyroid peroxidase autoantibodies

Thyroid peroxidase (107 kd)

Hashimoto's thyroiditis, including postpartum thyroiditis (95--100%) Graves' disease (-70%)

ELISA

Best marker for human autoimmune thyroiditis Pathogenetic importance uncertain

Thyrotropin receptor autoantibodies (includes Thyroid- stimulating autoantibodies and TSHblocking autoantibodies)

Extracellular domain of the thyrotropin receptor

Graves' disease (-95%) (decline with antithyroid drug treatment) Occasionally in subacute and silent thyroiditis, and in Yersiniosis TSH-blocking antibodies in atrophic thyroiditis with hypothyroidism (15--40%)

TSH receptor autoantibodies detected by a radioligand assay Thyroid- stimulating antibodies by bioassay (cAMP generation in thyroid cells) Thyrotrophin-blocking antibodies by bioassay

Thyroid-stimulating antibodies are the proximate cause of the hyperthyroidism of Graves' disease; also responsible for passive transfer of fetal and neonatal Graves' disease. TSH-blocking autoantibodies cause some cases of hypothyroidism

Topoisomerase I autoantibodies

Topoisomerase I (100 kd)

SSc (25%) Marker of tight skin

ID, ELISA

High relative risk of cancer

Tubular basement membrane autoantibodies

Tubulointerstitial nephritis antigen

Tubulointerstitial nephritis (<1%)

ELISA

Tyrosinase autoantibodies

Tyrosinase

Vitiligo, melanoma and melanoma associated hypopigmentation

ELISA with mushroom tyrosinase

Higher titer of IgG antibodies in patients with diffuse vitiligo compared to patients with localized disease

ABBREVIATIONS ANT APGS I CAH CAR CIE CTD DIL DM ELISA GVH HBV HPTLC

-...a

Admine nucleotide translocator Autoimmune polyglandular syndrome type I Chronic active hepatitis Carcinoma-associated retinopathy Counterimmunoelectrophoresis Connective tissue disease Drug induced lupus Dermatomyositis Enzyme-linked immunosorbent assay Graft-versus-host Hepatitis B virus High-performance thin layer chromatography

IB ID IDCM IIF IP IRBP ITP JDM JRA LE LES

Immunoblot Immunodiffusion Idiopathic dilated cardiomyopathy Indirect immunofluorescence Immunoprecipitation Interphotoreceptor retinol-binding Idiopathic thrombocytopenic purpura Juvenile diabetes mellitus Juvenile rheumatoid arthritis Lupus erythematosus Lambert-Eaton myasthenic syndrome

MCTD MG NCGN PBC PM RA RBA RNP SLE SS SSc TLC

Mixed connective tissue disease Myasthenia gravis Necrotizing crescentic glomerulonephritis Primary biliary cirrhosis Polymyositis Rheumatoid arthritis Radiobinding assay Ribonucleoprotein Systemic lupus erythematosus Sj6gren' s syndrome Systemic sclerosis Thin layer chromatography

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SUBJECT INDEX 28S antibodies 719 56-kd nuclear protein and Jo-1 autoantibodies 269-270 56-kd nuclear protein autoantibodies 266--270 o~-actinin 805 ~-enolase 68--69 ~-galactosyl epitope 24--28 c~l-antitrypsin 62 Acantholysis 755--756, 759 Acetylcholine receptor (AChR) autoantibodies 1-7, 21,805-806 Acquired angioedema 126, 128 Actin 767--772, 805 Actin autoantibodies 10-12, 217, 222, 224 Adaptins 325 Addison's disease 391,798-799, '813, 820 Adenine nucleotide translocator (ANT) 527--532 Adrenal autoantibodies 798 Adrenal autoimmune reaction 799, 800 Adrenal cytoplasmic antibodies 800-802 Adrenal cytoplasmic antigens 798 Adrenalitis 820 Adrenocorticotropic hormone (ACTH) autoantibodies 390-392 Affinity and avidity of autoantibodies 13-21 Affinity maturation 13 AIDS dementia complex 523-525 Allografts 93 Alpha-galactosyl (anti-gal) autoantibodies 24-28 Alu RNA protein 583 Alzheimer's disease and MBP 523--525 Amenorrhea, secondary 798 Aminoacyl-tRNA synthetase (Jo- 1) autoantibodies 31-34 Aminoacyl-tRNA synthetase (non-Jo-1) autoantibodies 36-45 Amphiphysin 142 Amyotrophic lateral sclerosis (ALS) 148--150 ANCA 47--72, 613 (see also Antineutrophil cytoplasmic autoantibodies) ANCA-associated vasculitides 61, 64--65 Anenxin XI 583 Ankylosing spondylitis and molecular mimicry 509 anti-DNA (see dsDNA) Anti-idiotypic antibodies and EMC 199--200 Anti-PL-12 36, 43 Anti-PL-7 36 Antisynthetase syndrome 33 Antibody affinity (see Affinity and avidity of autoantibodies) Antibody-dependent cell cytotoxicity (ADCC) 27, 441,444, 608 Antidesmoplakin antibodies 760-761 Antigen-presenting monocytes and macrophages (APC) 742 Antineutrophil cytoplasmic antibodies (ANCA) 47--72, 613 Antineutrophil cytoplasmic antibodies (ANCA) in inflammatory bowel disease 47-51

Antineutrophil cytoplasmic antibodies (ANCA) with specificity for myeloperoxidase 53--59 Antineutrophil cytoplasmic antibodies (ANCA) with specificity for proteinase 3 61--65 Antineutrophil cytoplasmic antibodies (ANCA) with specificity other than PR3 and MPO 68--72 Antinuclear antibodies (ANA) 47--49, 74-88, 331,561--565, 582--592, 777 Antiphospholipid syndrome 20, 247, 250 (see also Phospholipid autoantibodies) Antiphospholipid syndrome and AITP 639 Antiphospholipid syndrome and heat shock proteins 340 Antiphospholipid syndrome and IgG subclasses 103 Apoptosis 576 Arthrogryposis multiplex congenital (AMC) 5 Asialo-GM 1 278 Asialoglycoprotein receptor (ASGPR) 467, 469--472 Ataxia telangiectasia 419 Atrophic thyroiditis 823, 827 Autoantibodies, definitions 607 Autoantibodies in therapeutic preparations of human IgG (IVIg) 91-94 Autoantibodies that penetrate living cells 96-- 100, 613 Autoantibody subclasses 103-106 Autoimmune disease, criteria xxix Autoimmune disease, definition xxvii Autoimmune hemolytic anemia (AIHA) 123, 478, 677, 680--681 Autoimmune hepatitis 460-468, 470-472, 767, 769, 771-772 Autoimmune hepatitis and mitotic spindle 504--505 Autoimmune hepatitis type 2 457, 462--464 Autoimmune oophoritis/adrenalitis 799-800 Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy 798 Autoimmune polyglandular syndromes (APGS) 798 Autoimmune skin diseases 746--65 Autoimmune thyroiditis 810-814, 816--820 Avidity 21 [3-thalassemia 24, 27 [~2-glycoprotein I (~2-GPI) autoantibodies 21, 109--113, 614, 624, 626, 632-633 l]2-microglobulin 479 Bacterial endocarditis 262 Bactericidal/permeability-increasing protein (BPI) 68-72 Basement membrane zone 760, 762-764 Beta- 1-adrenoceptor 115 Beta-adrenergic receptor autoantibodies 115-117 Binding to extracellular molecules 614 Breast carcinoma 139-140 Breast carcinoma and p53 595, 597 Breast carcinoma and PCA-1 657 Breast implants 741

873

Bromelain-treated erythrocyte autoantibodies 120-123 Bullous pemphigoid (BP) 746-747, 762--765 Burkitt lymphoma and p53 595 C-ANCA 53-59 c-myc oncogene product 587 C 1 inhibitor (C 1 INH) autoantibodies 126-130 C 1 INH deficiency 129-130 Clq autoantibodies 129, 132-137 Clr and Cls 126 C3 nephritic factor 540-543 C a 2+ channel proteins 141 C a 2+ channel binding autoantibodies 143-145 Cadherin family, cell adhesion molecules 749-750 Calcium channel and related paraneoplastic disease autoantibodies 139-146 Calcium channel autoantibodies and amyotrophic lateral sclerosis (ALS) 148--50 Cancer and p53 autoantibodies 595-598 Cancer-associated retinopathies 694 Cardiolipin 109-113, 121,475-476, 569, 624-628, 630-633 Cardiomyopathy 116, 320 Cardiomyopathy, alcoholic 117 Cardiomyopathy, ischemic 117 Cathepsin G 47, 68-71 CB antigen 583 Celiac disease (CD) 237--242, 285--289, 684--692 Celiac disease, silent 286 Cell adhesion molecules 749 Cell surface binding and lysis 607 Cell surface receptors and pathogenic mechanisms 607--614 Centriole and centrosome autoantibodies 153-- 160 Centromere autoantibodies (ACA) 161-166, 170-171 Centrosome autoantibodies 153-160 Cerebellar ataxia and Golgi complex autoantibodies 326, 328 Chagas disease 116, 320, 527 Charcot-Marie-Tooth disease 516 Cholangitis, autoimmune 457 Chromatin 364, 370, 373, 574 Chromo autoantibodies 168-171 Chronic active hepatitis 10-12, 456, 462, 767 Chronic fatigue syndrome 320 Chronic graft-versus-host disease 209 Chronic heart failure 116 Chronic liver diseases and cytoskeletal autoantibodies 224 Chronic lymphocytic leukemia 123, 197 Chronic lymphocyte thyroiditis 813 Chronic urticaria 426-427 Churg-Strauss syndrome 57, 59, 65 Cicatricial pemphigoid 747, 763-765 Cirrhosis 459 Cirrhosis, alcoholic 768-769 Cirrhosis and cytoskeletal autoantibodies 224 CNS lupus (see also Neuropsychiatric SLE) CNS lupus and lymphocytotoxic autoantibodies 481 Coagulation factor (excluding factor VIII) autoantibodies 179-183 Coagulation factor VIII autoantibodies 172-177

874

Cold agglutinins 677-672 Collagen autoantibodies 185--193 Collagen-like region 132-- 137 Collagen, type IV 291 Complement, C2 and C4 deficiency 784 Complement C3 751,760, 763 Complement-mediated cytotoxicity 608 Connective tissue disease 44 Coxsackie virus 302 CREST (Calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia) 76, 156--157, 161, 165, 170, 438 CREST syndrome and IFI 16 438 Crithidia test 230-233 Crohn's disease 47-48, 50 Cross-reactive idiotypes 197, 205 Cryoglobulins 195-202 Cryoglobulins secondary to hepatitis C virus infection 205-207 Cystic fibrosis (CF) 70 Cytokine autoantibodies 209-215 Cytopenias 791 Cytoplasmic ANA pattern 74--88 Cytoskeletal autoantibodies 217-224, 767 Cytoskeletal filaments 10, 767 Cytotoxicity 607--614 DA1/DA2 584 Demyelination 516, 522--523 Dermatitis herpetiformis (DH) 237--242, 289, 684, 688, 690 Dermatomyositis (DM) 33--36, 42--45, 268--270, 484, 490-492, 642--650 Dermatomyositis and PM-Scl antibodies 568 Dermatomyositis, childhood 269-270 Desmin 218-221,224, 767, 771 Desmoglein 1 (Dsgl) 746--747, 754, 756 Desmoglein 3 (Dsg3) 746--747, 749, 759 Desmosome 746, 748, 750 Desmosome antibodies 750 Diabetes mellitus, insulin-dependent (IDDM) 150, 289, 299--305, 308, 310-311,430, 441--446, 702, 813, 820 Diabetes mellitus and ACTH 391 Dilated cardiomyopathy 115-- 117 DNA-dependent ATPase 584 DNA polymerase ~ 584 DNA topoisomerase, Scl-70 257 Donath-Landsteiner antibodies 677-682 Double-stranded DNA (see dsDNA) Down's syndrome 289 Dressler's syndrome 527, 532 Drug-associated neutropenia 558 Drug-induced hepatitis (tienilic acid) 462-464 Drug-induced SLE 104 Drug-induced SLE and histone antibodies 366-370, 378-379 dsDNA autoantibodies 18, 227-234 dsDNA autoantibodies, hidden 361 dsDNA avidity 231

Elastase 47, 53, 68-71 Endomysial and heterophile antibodies 352, 354, 356 Endomysial autoantibodies 237--242, 286, 688 Endoplasmic reticulum (ER) 735--736 Endothelial cell autoantibodies (AECA) 245--251 Endplate potential (EPP) 1 Epstein-Barr virus and APF in mononucleosis 620 Erythema multiforme major 760-761 Essential mixed cryoglobulinemia (EMC) 32, 195-202, 205-207 Eukaryotic expression systems 671 Evans' syndrome 558 F-actin 768-770, 772 Factor (see also Coagulation factor) Factor II (prothrombin) autoantibodies 179, 182 Factor V autoantibodies 179, 181 Factor VII autoantibodies 179, 181 Factor VIII 172-177 Factor IX autoantibodies 180-181 Factor X autoantibodies 180-- 181 Factor XI autoantibodies 180-182 Factor XII autoantibodies 180, 182 Factor XIII autoantibodies 180, 182 Familial membranous glomerulonephritis 839 Farr assay 19, 230--233 FceRI 423--427 Felty's syndrome 135, 332, 378 Fibrillarin autoantibodies 253-258, 568-569 Fibromyalgia 744 Fibronectin (Fn) autoantibodies 260-264 56-kd nuclear protein autoantibodies 266-270 Filaggrin (keratin) autoantibodies 271-275 ' (see also Perinuclear factor autoantibodies) Fogo selvagem (FS) 754-755 Follicle stimulating hormone and luteinizing hormone (FSH/LH) autoantibodies 390, 394-395 Functional affinity 21 G-actin 768 GABA 299, 308 Gal~l-3Gal 846--850 Galc~1-3Gall31-4GlcNAc-R 24--28 Ganglioside autoantibodies 277--282 Gastric parietal cells 600-605 Gastritis, chronic atrophic type A 602-604 GDIa 277, 280-281 GDlb 277, 281 Ge- 1 584 Gliadin antibodies (AGA) 285-289 Glomerular basement membrane (GBM) autoantibodies 21, 291-297 Glomerular basement membrane disease 839 Glomerulonephritis 18, 57, 59, 71, 199-200, 295, 326, 328, 358, 540-543, 839-840 Glomerulonephritis and Golgi complex autoantibodies 326, 328 Glomerulonephritis and lung hemorrhage 295

Glomerulonephritis, rapidly progressive 295 Glutamic acid decarboxylase (GAD) autoantibodies in diabetes mellitus 299--305 Glutamic acid decarboxylase (GAD) autoantibodies in stiff-man syndrome 308--311 Gluten-sensitive enteropathy 239, 242, 287, 687, 690 Glycolipid (excluding ganglioside) autoantibodies 314-323 Glycosphingolipids (GSL) 314-323 Glycyl-tRNA synthetase 36 GM 1antibodies 277 GN-1 584 Goiter 813 Golgi apparatus autoantibodies (AGAA) 25, 325-329 Gonadotropin autoantibodies 394-395 Goodpasture's syndrome 21, 58, 291-297 Graft-versus-host disease 92-93, 209 Granulocyte-specific antinuclear antibodies (GS-ANA) 331-334 Graves' disease 27, 388, 610, 81 0-812, 819-820, 822-823, 827 Graves' disease and neutropenia 556, 558 Growth hormone (hGH) autoantibodies 390, 396-397 GTlb 277, 281 Guillain-Barr6 syndrome 277, 280--282, 320 Gynecologic cancer and PCA-1 657 (H2A-H2B)-DNA complex 364--370, 575--579 H+/K+ ATPase 600-602, 605 HAMA and rheumatoid factors 404--406 HAMA, preventing induction 405 Hashimoto' s thyroiditis 387, 810-813, 816--820, 823 HCC-1 585 Heart and heterophile antibodies 352, 355-356 Heat shock protein autoantibodies 336-341 Hemolytic anemia 122-123 Hemolytis uremic syndrome 247, 250 Hemostasis 176, 180 Henoch-Sch6nlein purpura 63 Heparin 343-348 Heparin-associated autoantibodies 343--348 Heparin-associated thrombocytopenia 250 Heparin-induced thrombocytopenia (HIT) 343--348 Heparin-PF4 complex 345--347 Hepatitis B surface antigen 195--196 Hepatitis B virus, chronic 11 Hepatitis C virus and cryoglobulinemia 205-207 Hepatitis C virus, chronic 11 Hepatitis C virus infection 457 Hereditary angioedema (HAE) 126, 128 Herpes gestationis (HG) 746-747, 762-765 Heterochromatin 169 Heterogenous nuclear riboprotein complex autoantibodies 660-665 Heterophile antibodies 351-356 Hidden autoantibodies 357-362 Hirata's disease 430 Histamine release 423-426 Histidyl-tRNA synthetase antibodies (Jo-1) 21, 31

875

Histone 574--579 Histone antibodies, hidden 361 Histone autoantibodies other than (H2A-H2B)-DNA 373--384 Histone (H2A-H2B)-DNA autoantibodies 364-370, 575-579 HIV 700-703 HIV and AITP 639 HIV and neutropenia 558 HLA-A1, B8 and IgA antibodies 418 HLA-B8, DR3 and smooth muscle autoantibodies 771 HLA-DQ1, DQ2 and SS-A (Ro) autoantibodies 785 HLA-DQ6 and fibrillarin autoantibodies 255 HLA-DR/DQ and MS 522 HLA-DR1, DR4 and autoimmune skin diseases 756-757 HLA-DR1, DR4, Dw8 and centromere autoantibodies 164 HLA-DR2 and ANCA 48, 56, 63 HLA-DR2 and GBM disease 293 HLA-DR2, DQwl and SLE 229 HLA-DR2, DQwl and SS-B (La) autoantibodies 792 HLA-DR2, DR4 and spliceosomal autoantibodies 778 HLA-DR3 and Jo-1 autoantibodies 33 HLA-DR3 and non-Jo-1 autoantibodies 42 HLA-DR3 and PM-Scl antibodies 56"8, 646 HLA-DR3, B8 and actin autoantibodies 11 HLA-DR3, DQw2 and SS-B (La) autoantibodies 792 HLA-DR3/4; DQ2 and IDDM 303 HLA-DR4 and beta-adrenergic receptor autoantibodies 116 HLA-DR4 and filaggrin autoantibodies 273 HLA-DR4 and IDCM 529 HLA-DR4 and RA 192, 620, 708 HLA-DR4 and smooth muscle autoantibodies 771 HLA-DR4, DQw3/DQw8 and autoimmune skin diseases 751 HLA-DR5, DRw52 and SRP 736, 739 HLA-DR7 NS Mi-2 autoantibodies 488 HLA-DR15 and MBP 522 HLA-DRB 1, DR4 and insulin autoantibodies 431 HMG-1, HMG-2, HMG-17 (high mobility group) proteins 585 Homology vs. similarity 507 Hormone nonpeptide autoantibodies: thyroid 385--388 Hormone peptide autoantibodies 390-402 HTLV-1 702--703 Hu (see Neuronal nuclear autoantibodies) Human antimouse antibodies (HAMA) 403-406 Human lysosomal-associated membrane protein 2 (h-lamp-2) 68-69, 71 Hypergammaglobulinemia 794-795 Hyperprolactinemia 400--401 Hyperthyroidism 387, 813, 819 Hypocalcemia 398-399 Hypocomplementemic urticarial vasculitis syndrome (HUVS) 132, 134--137 Hypoparathyroidism 399 Hypophysitis, autoimmune 400 Hypopituitarism, idiopathic 396 Hypothyroidism 387, 798, 819

876

125I-o~-Bungarotoxin 1, 3

131I therapy 827 ICAM-1 48, 56 Idiopathic dilated cardiomyopathy (IDCM) 527--531 Idiotypes and anti-idiotypic antibodies 408-414 Idiotypic network 410 IgA autoantibodies 417-421 IgA deficiency 417 IgA fibronectin aggregates 263-264 IgA nephropathy 63, 263--264, 296 IgE receptor autoantibodies 423--427 IgG subclasses 103-106 IgM monoclonal gammopathy and MAG 516 Immune complex-mediated damage 611 Immune response and natural autoantibodies 534, 536-537 Immune thrombocytopenias 635-640 Immunofluorescence assay for ANA 74-88 Immunoglobulin class switching 106 Immunopancytopenia 559 Inflammatory bowel disease 47-51 Insulin autoantibodies 430-433 Insulin autoimmune syndrome 430 Insulin-dependent diabetes mellitus (see Diabetes mellitus) Interferon (IFN) 436--439 Interferon and cryoglobulins 201 Interferon-~ 210, 213-214 Interferon-inducible protein IFI 16 autoantibodies 436-439 Interleukin-1 (IL-1) 742 Interleukins 209-215 Intermediate filaments 217-224 Interstitial lung disease 32, 34, 44, 784 Interstitial nephritis, drug-induced 840 Intracellular microtubules 325 Islet cell autoantibodies (ICA) 441-446 Islet cell surface antibodies (ICSA) 444 Isoleucyl-tRNA synthetase 36 IVIg 65, 91--94 Ja antibodies 719 Jo-1 31--34 Juvenile chronic arthritis 378--379 Juvenile RA 707, 711 Juvenile RA and GS-ANA 333 Kawasaki's disease 247, 249-950 Keratin autoantibodies (see Filaggrin) Keratinocytes 748-749, 752, 754, 759, 762 Keratins 218--221,224 Kidney tubular basement membrane 836 Kinetochore 162, 166 Klotz plot 16-17 Ku and Ki autoantibodies 449--453 L-type VGCC antibodies 149--150 L5/5S protein complex antibodies 718 L7 585

L 12 protein antibodies 718 La (SS-B) 789--795 Lactoferrin 55, 68-71,332 Lactoperoxidase 818, 820 Lambert-Eaton myasthenic syndrome 139, 144, 149, 695-696 Lamins, nuclear 561-565 LATS 610 LE cell phenomenon 364, 367, 574 Legionella pneumophila 262 LIF 212, 214 Liver cytosol antigen type 1 autoantibodies 456-461 Liver/kidney microsomal (LKM) autoantibodies 462--464 Liver membrane autoantibodies 462-464, 467-472 Liver-specific membrane lipoprotein (LSP) 467 LKM-1 autoantibodies 456-457, 459, 462-464 LKM-2 autoantibodies 462-464 Locus 93D antigen 585 Long-acting thyroid stimulator (LATS) 822--823, 826 Lung carcinogenesis and p53 598 Lupoid hepatitis 767 Lupus (see also SLE and Systemic lupus erythematosus) Lupus anticoagulant 474-476, 624--626 Lupus glomerulonephritis 18 Lupus nephritis 229, 233, 481 Lupus nephritis and Clq 135 Lupus nephritis and nucleosome-specific autoantibodies 579 Lupus psychosis 724 Luteinizing hormone 822 Lymphocytotoxic autoantibodies 478-482 Lymphopenia 480 Lymphoproliferative disorders 129-130, 195 Lymphoproliferative disorders and neutropenia 558 Lysozyme 68-69, 71 MA protein 585 Mas 586 MaS (autoantigen pl50) 586 MCTD and Clq 135 MCTD and hnRNP complex 660, 664--665 MCTD and Ku autoantibodies 449, 452 MCTD and retroviral antibodies 702 Me (system C) 586 Mechanic's hands 34, 44 Melanin 842 Melanoma 842--844 Melanoma-associated hypopigmentation 842 Membranoproliferative glomerulonephritis (MPGN) 135, 199--200, 540-543 Mi-2 autoantibodies 484-492 Microfilaments 217 Microscopic polyangiitis (MPA) 57, 65 Microsomal antibodies 810, 818 Microtubule associated protein S 501 Microtubules 217 Mitochondrial and heterophile antibodies 351-352, 354 Mitochondrial autoantibodies 494-498 Mitotic spindle apparatus autoantibodies 156, 501-505

Mixed connective tissue disease (MCTD) 44, 76, 96, 730--733, 774, 779--780 (see also MCTD and...) Molecular mimicry 507--511 Monoclonal gammopathies 534 Monoclonal gammopathies of undetermined significance (MGUS) 279, 516 Mononuclear cells (MNC) 96 Multifocal motor neuropathy (MMN) 277, 281--282 Multiple sclerosis (MS) 320, 520--525 Multiple sclerosis and heat shock proteins 338--340 Multiple sclerosis and retroviral antibodies 702 Multivalency/avidity 15 Myasthenia gravis (MG) 1-7, 21, 141--146, 805--807 Myasthenia gravis and AChR idiotypes 412 Myasthenia gravis and hGH 396 Myasthenia gravis, experimental autoimmune 3 Mycoplasma pneumoniae 262, 504 Myelin basic protein (MBP) autoantibodies 520-525 Myelin oligodendrocyte glycoprotein (MOG) 520-523 Myelin-associated glycoprotein (MAG) autoantibodies 277, 513-517, 520 Myeloperoxidase (MPO) 21, 47, 53--59, 818 Myocardial autoantibodies 527--532 Myocarditis 528--532 Myoid cells 2 Myopathy 45 Myosin 805 Myositis 31, 33, 266--270, 484, 490-492, 642--650, 735--737 Myositis, penicillamine-induced 32 Myositis-specific antibodies (MSA) 31-34, 488, 490-492, 735-736 Myxedema, primary 813 Natural autoantibodies 357-362, 412, 534-537 Necrotizing and crescentic glomerulonephritis (NCGN) 57, 59, 71 Neonatal hypothyroidism 823, 827 Neonatal lupus erythematosus 784, 786, 791--792, 795 Neonatal neutropenia 558 Nephritic factor autoantibodies 540--543 Nephritogenic antigens 836 Nephronopthisis 840 Neuroblastoma 403, 553 Neuronal autoantibodies 546--548 Neuronal nuclear autoantibodies, (ANNA- 1) type 1 (Hu) 139, 141, 144, 551--553, 655 Neuronal nuclear autoantibodies (ANNA-2), type 2 (Ri) 655 Neuropsychiatric SLE 547--548, 723--725 (see also CNS lupus) Neutropenia, autoimmune 555-559 Neutrophil autoantibodies 555-559 Nicotinic acetylcholine receptors (AChR) 141, 145 Non-Goodpasture's GBM antibodies 295 NOR-90 586 NSPI, NSPII (nuclear speckled) 586 Nuclear ANA patterns 74-88 Nuclear antigens, other autoantibodies to 582-592

877

Nuclear envelope protein autoantibodies 561-565 Nuclear fine-speckled ANA pattern 74--88 Nuclear helicase 587 Nuclear homogeneous ANA pattern 74-88 Nuclear mitotic spindle ANA pattern 74-88 Nucleolar 128 p antigen 587 Nucleolar ANA pattern 74--88 Nucleolar autoantibodies (ANoA) 253, 567--571 Nucleolin antibodies 569 Nucleolus organizer regions (NOR) 569 Nucleophosmin/B23 antibodies 569 Nucleosides, nucleotides 587 Nucleosome-specific autoantibodies 574-579 Opsonization and natural autoantibodies 536 Optic neuritis 523-525 Ovarian cancer and PCA-1 657 Overlap syndromes 44, 780 Overlap syndromes and Clq 135 Overlap syndromes and PM-Scl 647 P-ANCA 47-51, 61--65, 331 p53 autoantibodies 595-598 P80 coilin 583 Paraneoplastic cerebellar degeneration (PCD) 655--658 Paraneoplastic disease 139-146, 150, 551-553, 655-658 Paraneoplastic encephalomyelitis 553 Paraneoplastic opsoclonus/myoclonus 553 Paraneoplastic pemphigus (PNP) 74--77, 759--761 Paraneoplastic sensory neuronopathy 553 Parathyroid hormone (PTH) autoantibodies 390, 398-399 Parietal cell autoantibodies 600-605 Parietal cell and heterophile antibodies 351-353 Pathogenic mechanisms 607--614 PEG assay 19, 230-233 Pemphigus foliaceus (PF) 746-747,~754--757 Pemphigus vulgaris (PV) 746--752 Perinuclear factor autoantibodies (APF) 618--622 (see also Filaggrin) Peripheral blood polymorphonuclear cells (PMN) 55, 61--62, 68, 70 Pernicious anemia 600-605, 813 Phagocytosis 26, 607--610 Phagocytosis and natural autoantibodies 536 Phosphatidylcholine 120-123 Phosphatidylserine 630-633 Phospholipid antibodies, natural 357-361 Phospholipid autoantibodies 20, 109-113, 474-476, 626--628, 630-633 (see also Antiphospholipid syndrome) Phospholipid autoantibodies, cardiolipin 624-628 Phosphoprotein antibodies 717, 721-725 Pituitary 400 Platelet-associated immunoglobulins 639 Platelet autoantibodies 635-640 Platelet factor 4 (PF4) 344--348 PM-Scl autoantibodies 568, 642-650 Poly(A) polymerase 588

878

Poly(ADP-ribose) 587 Poly(ADP-ribose) polymerase 588 Polyarteritis nodosa (PAN) 58--59 Polyarthritis 34 Polyglandular endocrine failure syndromes 820 Polymyositis (PM) 20-21, 36, 42--45, 268--270, 484, 490-492, 642-650, 735--739 Polymyositis and PM-Scl antibodies 568 Polymyositis, idiopathic 34 Polyneuropathy 513--517 Postpartum hypothyroidism 819 Postpartum thyroiditis 813 Poststreptococcal glomerulonephritis 840 Posttransfusion thrombocytopenic purpura 637 Pregnancy and AITP 639 Pregnancy and IgA deficiencies 419, 421 Pregnancy, hidden antibodies in 361 Pregnancy, TPO in 819--820 Pregnancy, TSAb in 823 Pregnancy, TSHR in 827 Primary biliary cirrhosis (PBC) 11--12, 76, 166, 472, 494--498, 504--505, 768--769 Primary biliary cirrhosis and IgG subclasses 104 Primary biliary cirrhosis and nuclear envelope protein autoantibodies 561,563 Primary sclerosing cholangitis 472 Procollagen 185 Profilaggrin 618-622 Prokaryotic expression systems 669 Prolactin 390, 400-402 Proliferating cell nuclear antigen autoantibodies (PCNA) 651--654 Prostate cancer and ANNA-1 (Hu) 553 Protein kinase NII 588 Proteinase 3 (PR3) 61--65 Prothrombin 179 Prothymosin ~ 588 Pseudomonas aeruginosa 70 Pulmonary hemorrhage and IgM ANCA 63 Pulmonary hypertension and U3/snoRNP antibodies 569 Purkinje cell cytoplasmic autoantibodies (PCA-1), type 1 (Yo) 139--142, 144, 655--658 Pyruvate dehydrogenase antibodies, hidden 361 RA RA RA RA RA RA RA RA RA RA RA RA RA RA

(see also Rheumatoid arthritis) and Clq 135 and chromo autoantibodies 170 and cryoglobulins 199 and cytoskeletal autoantibodies 224 and endothelial cell autoantibodies 245, 247 and factor VIII autoantibodies 176 and fibronectin autoantibodies 260, 262, 264 and filaggrin autoantibodies 274 and Golgi complex autoantibodies 326, 328 and GS-ANA 332-334 and heat shock proteins 338-340 and histone autoantibodies 378 and hnRNP complex 660-665

RA and IgA deficiency 420 RA and IgG subclasses 104 RA and lymphocytotoxic autoantibodies 480 RA and perinuclear factor autoantibodies 618-622 RA and type II collagen 186, 189--193 RA-33 (heterogeneous nuclear ribonucleoprotein complex) [hnRNP] autoantibodies 660-665 Raynaud's phenomenon 44, 156-157, 165, 833 Raynaud's phenomenon and mitotic spindle apparatus 504 Raynaud's phenomenon and NOR-90 569 RD gene product 589 Recombinant autoantigens 668--674 Recombinant DNA technology 668 Recurrent pregnancy loss 627 Recurrent spontaneous abortion 476 Red cell autoantibodies 677-682 Relapsing polychondritis 191 Renal allografts 839 Renal diseases and C lq 135 Replication protein A 589 Reticulin autoantibodies 684-692 Retinal autoantibodies 694--698 Retinal vasculitis 21 Retinopathies 694 Retroviral antibodies 700-703 Rheumatic heart disease 527, 532 Rheumatoid arthritis (see also RA and...) Rheumatoid arthritis (RA) and RF 706--712 Rheumatoid arthritis nuclear antigen (RANA) 588 Rheumatoid factor-negative RA 271 Rheumatoid factors (RF) 196-199, 205, 706-712 Rheumatoid factors, hidden 361 Ri and neuronal nuclear antibodies, type 2 141, 144 Ribonucleoprotein (RNP) 774, 776, 778 Ribosomal autoantibodies 716--720 Ribosomal P protein autoantibodies 362, 721--725 RNA polyribonucleotides 590 RNA polymerase I antibodies 568 RNA polymerase I-III (RNAP) autoantibodies 727-733 Ro (SS-A) 783-787, 792 S 10 protein antibodies 718 Scl-70 antibodies 830 Scleroderma 156, 160-161, 164-165, 170, 253, 257-258, 830-834 Scleroderma/systemic sclerosis 567-571 Senescent red cells 24 Sequence similarity 507 Serine proteinase inhibitors 126 Serum inhibitors, natural 362 SGLPG 315 SGPG 277, 280, 314-315, 513, 515 Sicca 795 Signal recognition particle autoantibodies 32, 735-739 Silica-associated scleroderma 831 Silicate and silicone antibodies 741-744 Silicate-reactive antibodies 743 Sjtigren's syndrome 76, 195, 707, 783-787, 789-795

Sj6gren's syndrome and Golgi complex 326--328 Sj6gren's syndrome and mitotic spindle 502, 504 Sjtigren's syndrome and neutropenia 558 Sj~3gren's syndrome and retroviral antibodies 701 Sjtigren's syndrome, secondary 786 Skeletal muscle 805 Skin diseases autoantibodies 746--765 SL/ki (sicca/lupus) 590 SLE (see also Systemic lupus erythematosus) SLE and ANA 74-88 SLE and antibody affinity 18 SLE and ~2-GPI 113 SLE and Clq 132, 134-147 SLE and C4 nephritic factor 540 SLE and cardiolipin 624, 627 SLE and collagen 190--191 SLE and cytoskeletal autoantibodies 224 SLE and dsDNA autoantibodies 227--234 SLE and endothelial cell autoantibodies 245, 247 SLE and factor II autoantibodies 181 SLE and factor VIII autoantibodies 176 SLE and factor XII autoantibodies 182 SLE and fibronectin autoantibodies 260, 262 SLE and glycolipid antibodies 320 SLE and Golgi complex autoantibodies 326, 328 SLE and heat shock proteins 338--340 SLE and hemolytic anemia 123 SLE and histone autoantibodies 366-370, 378-379, 382 SLE and hnRNP complex 660, 662, 664--665 SLE and IFI 16 436-439 SLE and IgA deficiency 420 SLE and IgG subclasses 103 SLE and Ku autoantibodies 449, 452 SLE and lymphocytotoxic autoantibodies 478--481 SLE and MPO 58 SLE and neuronal autoantibodies 546-548 SLE and neutropenia 558 SLE and nuclear lamins 563, 565 SLE and nucleosome autoantibodies 574--579 SLE and pathogenic idiotypes 412 SLE and PCNA 652--654 SLE and phosphatidylserine 620--633 SLE and retroviral antibodies 701 SLE and RF 707 SLE and ribosomal autoantibodies 716--720 SLE and ribosomal P protein autoantibodies 721-725 SLE and thrombocytopenia purpura 638--639 Sm antibodies 774, 776, 778 Small cell lung carcinoma (SCLC) 139, 141 Small cell lung cancer and ANNA-1 (Hu) 551--553 Small nuclear ribonucleoprotein particles (snRNPs) 774-781 Smooth muscle and heterophile antibodies 351-353 Smooth muscle autoantibodies 10-11, 218, 767-772 snoRNP antibodies 568 Spl00 591 Spliceosomal autoantibodies 774-781 SRP54 735-736, 739 SS-A (Ro) autoantibodies 783-787

879

SS-B (La) autoantibodies 789-795 Staphylococcus aureus 63

Steroid cell autoantibodies 798-802 Stiff-man syndrome 303--304, 308--311 Stress proteins (see Heat shock protein autoantibodies) Striational and heterophile antibodies 352, 355-356 Striational autoantibodies (StrAb) 142, 145, 805--807 Su 591 Subacute cutaneous lupus erythematosus 784, 786, 794-795 Systemic lupus erythematosus (SLE) 96, 474, 768, 774, 779--780, 783--785, 794 (see also SLE and...) Systemic lupus erythematosus, ANA-negative 784 Systemic lupus erythematosus, neuropsychiatric 320 Systemic lupus erythematosus, pathogenesis 576-577 Systemic sclerosis 58, 75-78, 157, 160-161,164-165, 170, 378, 647-649, 727, 729-733 Systemic vasculitis 21, 57, 63-64 (see also Vasculitis) T helper cells 106 Th, To, RNase P 591 Therapeutic role of anti-idiotypic antibodies 413-414 Thrombocytopenia 476, 627 Thrombocytopenia purpura, autoimmunemediated (AITP) 635-640 Thrombocytopenia, heparin-induced 343-348 Thrombosis and cardiolipin 627 Thrombosis and lupus anticoagulant 474-476 Thrombotic thrombocytopenic purpura 247, 250 Thymic epithelial cells 805 Thymic carcinoma 806--807 Thymoma 1, 5, 805--807 Thyroglobulin (Tg) autoantibodies 385, 387, 810-814, 818, 820 Thyroid carcinoma 813 Thyroid hormone autoantibodies 385-388 Thyroid peroxidase (TPO) autoantibodies 810, 813, 816--820 Thyroid stimulating antibodies (TSAb) 823 Thyroid stimulating hormone (TSH) 819--820 Thyroid stimulation blocking antibodies (TSBAb) 823 Thyroiditis 810-813 Thyrotropin receptor (TSHR) autoantibodies 822--827 Thyrotropin-binding inhibitory immunoglobulin (TBII) 823, 826 Thyroxine (T4) 385, 387, 810 Thyroxine-binding globulin (TBG) 385, 387 Topoisomerase-I (Scl-70) autoantibodies 730, 830-834 Transcription factor TFIIIA 591 Transfusion reactions and IgA antibodies 418-421

880

Translation (protein synthesis) 735 Transplantation (xenografts) 27--28, 93, 839, 849--850 Triiodothyronine (T3) 385, 387, 810 Trimethylguanosine (m3G) 774, 780 Trypanosoma cruz i 116, 527 Tuberculous adrenal insufficiency 798 Tubular basement membrane (TBM) autoantibodies 836-840 Tubulin 218-224, 767, 771 Tubulointerstitial nephritis (TIN) 836--840 Tumor suppressor gene p53 595--598 Tyrosinase autoantibodies 842-844 U snRNP (uridine-rich) 774 U1 774-781 U2 774-886 U3 254, 568-569 U4/U6 774-775 U5 774-775 U7 774-776 U11/U12 774-775 Ulcerative colitis 47-48, 50 Uridine 774 Varicella-zoster infection 209 Vasculitis 57, 205, 247, 250 (see also Systemic vasculitis) Vasculitis of Sj6gren's syndrome 784, 786 Vimentin 218-221,224, 767, 771 Viral illness and lymphocytotoxic autoantibodies 480 Vitamin B 12 deficiency 602, 604 Vitiligo 842--843 Voltage-gated calcium channel (VGCC) 148-150 von Willebrand Factor autoantibodies 180, 182 Waldenstr6m' s macroglobulinemia 127-130, 195, 516 Warm autoantibodies 677-682 Wegener's granulomatosis 21, 54, 56, 61-65, 247, 250 X-ANCA 68-72 Xenoantigens 846 Xenograft rejection 24, 27, 848 Xenografts 846-850 Xenoreactive human natural antibodies 846-850 Xenotransplantation 27 XH 592 XR 592 Yersinia enterocolitica 826 Yersiniosis 823, 826 Yo (see Purkinje cell cytoplasmic autoantibodies)

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