Autoantibodies

AUTOANTIBODIES Editors" Dr. James B. Peter Specialty Laboratories, Inc. 2211 Michigan Avenue Santa Monica, California 9...

Author: J.B. Peter | Y. Shoenfeld

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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


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


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

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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

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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


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

XXV


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)

xxix


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 1000 nmol/L. Sera giving low-positive values (e.g., 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.

ANTI-AChR ANTIBODY IN MG 100000-

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ANTI-AChR ANTIBODY AND THYMIC PATHOLOGY 100000-

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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

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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

REFERENCES Alpers JH, Steward MD, Soothill JF. Differences in immune elimination in inbred mice. The role of low affinity antibody. Clin Exp Immunol 1972;12:121--132. Berek C, Berger A, Apel M. Maturation of the immune response in germinal centers. Cell 1991;67:1121-1129. Costello PB, Green FA. Binding affinity of serum IgG to cardiolipin and other phospholipids in patients with systemic lupus erythematosus and syphilis. Infect Immun 1988;56: 1738-1742. Farr RS. A quantitative immunochemical measure of the primary interaction between I-BSA and antibody. J Infect Dis 1958;103:239-245. Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME. Measurement of the true affinity constant in solution of antigen antibody complexes by enzyme linked immunoassay. J Immunol Methods 1985;77:305-319. 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;335:15441547. Gershwin ME, Steinberg AD. Quantitative characteristics of anti-DNA antibodies in lupus nephritis. Arthritis Rheum 1974; 17:947--954. Gharavi AE, Harris EN, Asherson RA, Hughes GR. Anticardiolipin antibodies: isotype distribution and phospholipid specificity. Ann Rheum Dis 1987;46:1--6. Gharavi AE, Chu JL, Elkon KB. Antibodies to intracellular proteins in human systemic lupus erythematosus are not due to random polyclonal B cell activation. Arthritis Rheum 1988;31:1337-- 1345. Gharavi AE. Antiphospholipid cofactor. Stroke 1992;23:$7S10. Gharavi AE, Sammaritano LR, Wen J, Elkon KB. Induction of antiphospholipid autoantibodies by immunization with 132 glycoprotein I (apolipoprotein H). J Clin Invest 1992;90:

1105--1109. Gharavi AE, Sammaritano LR, Wen J, Miyawaki N, Morse JH, Zarrabi MH, Lockshin MD. Characteristics of human immunodeficiency virus and chlorpromazine induced antiphospholipid antibodies: effect of beta 2-glycoprotein I on binding to phospholipid. J Rheumatol 1994;21:94--99. Harris EN, Gharavi AE, Tincani A, Chan JK, Englert H, Mantelli P, Allegro F, Ballestrieri G, Hughes GR. Affinity purified anticardiolipin and anti-DNA antibodies. J Clin Lab Immunol 1985;17:155-162. Hedman K, Lappalainen M, Sepp~il~i I, M~ikel~i O. Recent primary toxoplasma infection indicated by a low avidity of specific IgG. J Infect Dis 1989;159:736--740. Helyer B J, Howie JB. Renal disease associated with positive lupus erythematosus tests in a cross bred strain of mice. Nature (Lond) 1963;197:197-198. Hughes GR. Significance of anti DNA antibodies in systemic lupus erythematosus. Lancet 1972;2:861--863. Kasp E, Whiston R, Dumonde D, Graham E, Stanford M, Saunders M. Antibody affinity to retinal S-antigen in patients

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with retinal vasculitis. Am J Ophthalmol 1992; 113:697--701. Kokolina E, Noel LH, Nusbaum P, Geffriaud C, Grunfeld JP, Halwachs-Mecarelli L, Lesavre P. Isotype and affinity of antimyeloperoxidase autoantibodies in systemic vasculitis. Kidney Int 1994;46:177-184. Leanderson T, K~illiberg E, Gray D. Expansion, selection and mutation of antigen-specific B cells in germinal centers. Immunol Rev 1992;126:47--61. Levy RA, Gharavi AE, Sammaritano LR, Habina L, Qamar T, Lockshin MD. Characteristics of IgG antiphospholipid antibodies in patients with SLE and syphilis. J Rheum 1990;17:1036-1041. Lockshin MD. Antiphospholipid antibody syndrome. Rheum Dis Clin North Am 1994;20:45--59. Marriott JB, Oliveira DB. Serial functional affinity of autoantibodies in antiglomerular basement membrane disease. Clin Exp Immunol 1994;95:498--501. McGrath H Jr, Biundo JJ Jr. A longitudinal study of high and low avidity antibodies to double stranded DNA in systemic lupus erythematosus. Arthritis Rheum 1985;28:425--430. McNeil HP, Krilis SA, Chesterman CN. Purification of antiphospholipid antibodies using a new affinity method. Thromb Res 1988;52:641--648. McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-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 Y, Yasuda T, Triplett DA, Kioke T. Anticardiolipin antibodies recognize beta 2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med 1994;179:457--462. Miller FW, Waite KA, Biswas T, Plotz PH. The role of an autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity. Proc Natl Acad Sci USA 1990;87:9933--9937. Nossal GJ. The molecular and cellular basis of affinity maturation in the antibody response. Cell 1992;68:1--2. Nossent JC, Huysen V, Smeenk RJ, Swaak AJ. Low avidity antibodies to double stranded DNA in systemic lupus erythematosus: a longitudinal study of their clinical significance. Ann Rheum Dis 1989;48:677--682. Oldstone MB, Dixon FJ. Pathogenesis of chronic disease associated with persistent lymphocytic choriomeningitis virus infection. Relationship of the antibody production to disease in neonatally infected mice. J Exp Med 1969;129:483--492. Perelson AS. Immune network theory. Immunol Rev 1989; 110: 5--36. Pincus T, Schur PH, Rose JA, Descker JL, Talal N. Measurement of serum DNA-binding activity in systemic lupus erythematosus. N Engl J Med 1969;281:701--705. Polanec J, Sepp~iRi I, Rousseau S, Hedman K. Evaluation of protein-denaturing immunoassay for avidity immunoglobulin G to rubella virus. J Clin Lab Analysis 1994;8:16--21. Qamar T, Levy RA, Sammaritano LR, Gharavi AE, Lockshin

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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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

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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

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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.


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 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.

57

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 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).

REFERENCES

schmeding R. Vasculitis and antineutrophil cytoplasmic autoantibodies associated with propylthiouracil therapy. Lancet 1993;342:651-652. Endo H, Hosono T, Kondo H. Antineutrophil cytoplasmic autoantibodies in 6 patients with renal failure and systemic sclerosis. J Reumatol 1994;21:864--870. Esnault VL, Short AK, Jones SJ, Skehel JM, Lockwood CM. Lactoferrin co-purifies with myeloperoxidase and is recognized by antineutrophil cytoplasmic antibodies. Adv Exp Med Biol 1993;336:101--104. Ewert BH, Jennette JC, Falk RJ. Antimyeloperoxidase antibodies stimulate neutrophils to damage human endothelial cells. Kidney Int 1992;41:375-383. 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. Falk RJ, Terrell RS, Charles LA, Jennette JC. Antineutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA 1990;87:4115-4119. Falk RJ, Jennette JC. Immunofluorescence and ELISA determination of ANCA with description of a sub-class with antimyeloperoxidase activity. APMIS 1989;97($6):45. Goldschmeding R, Tervaert JW, van der Schoot CE, van der Veen C, Kallenberg CG, von dem Borne AE. ANCA, antimyeloperoxidase and antielastase: three members of a novel class of autoantibodies against myeloid lysosomal enzymes. APMIS 1989; 97($6):48--49. Guillerin L, Viser H, NoEl LH, Pourrat J, Vernier I, Gayraud M, Oksman F, Lesavre P. Antineutrophil cytoplasm anibodies in systemic polyarteritis nodosa with and without hepatitis B virus infection and Churg-Strauss syndrome. J Rheumatol 1993 ;20:1345-1349. Hagen EC, Stegeman CA, D'Amaro J, Schreuder GM, van Es LA, Tervaert JW, Kallenberg CG, van der Woude FJ. Decreased frequency of HLA-DR6/DR13 in patients with Wegener's granulomatosis. Kidney Int 1995;48:801--805. H~ssberger L, SjOholm AG, Jonsson H, Sturfelt G, Akesson A. Autoantibodies against neutrophil cytoplasm components in systemic lupus erythematosus and in hydralazine-induced

Agnes K. Verdoperoxidase. A ferment isolated from leukocytes. Acta Physiol Scand 1941;2(Suppl 8):1-62. Bosch X, Mirapeix E, Font J, Borrellas X, Rodriguez R, LopezSoto A, Ingelmo M, Revert L. Prognostic implication of antineutrophil cytoplasmic autoantibodies with myeloperoxidase specificity in antiglomerular basement membrane disease. Clin Nephrol 1991;36:107-113. Brouwer E, Huitema MG, Klok PA,de Weerd H, Tervaert JW, Weening JJ, Kallenberg CG. Antimyeloperoxidase-associated proliferative glomerulonephritis: an animal model. J Exp Med 1993;177:905--914. Brouwer E, Tervaert JW, Horst G, Huitema MG, van der Giessen M, Limburg PC, Kallenberg CG. Predominance of IgG1 and IgG4 subclass of antineutrophil cytoplasmic autoantibodies in patients with Wegener's Granulomatosis and clinically related disorders. Clin Exp Immunol 1991;83: 379-386. Brouwer E, Stegeman CA, Huitema MG, Limburg PC, Kallenberg CG. T cell reactivity to proteinase 3 and myeloperoxidase in patients with Wegener's granuloma tosis (WG). Clin Exp Immunol 1994;98:448--453. Cambridge G, Wallace H, Bernstein RM, Leaker B. Autoantibodies to myeloperoxidase in idiopathic and drug-induced lupus erythematosus and vasculitis. Br J Rheumatol 1994;33: 109-114. Cohen Tervaert JW, Goldschmeding R, von dem Borne AEGKr, Kallenberg CGM. Antimyeloperoxidase antibodies in the Churg-Strauss syndrome. Thorax 1991;46:70--71. Cohen Tervaert JW, Goldschmeding R, Elema JD, Limburg PC, van der Giessen M, Huitema MG, Koolen MI, Hene RJ, The TH, van der Hem GK, von dem Borne AE, Kallenberg CG. Association of autoantibodies to myeloperoxidase with different forms of vasculitis. Arthritis Rheum 1990;33:1264-1272. Davies DJ, Moran JE, Niall JF, Ryan GB. Segmental necrotizing glomerulonephritis with antineutrophil antibody: possible arbovirus aetiology? Br Med J 1982;285:606. Dolman KM, Gans RO, Vervaat ThJ, Zevenbergen G, Maingay D, Nikkels RE, Donker AJ, von den Borne AE, Gold-

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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.


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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vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 1988;318:1651-1657. Falk RJ, Terrell RS, Charles LA, Jennette JC. Antineutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA 1990;87:4115-4119. Finnern R, Lockwood CM, Ouwehand W. Antineutrophil cytoplasm autoantibody (ANCA) fragments from a human Vgene library. Clin Exp Immunol 1993;93:21. Gabay JE, Scott RW, Campanelli D, Griffith J, Wilde C, Marra MN, Seeger M, Nathan CF. Antibiotic proteins of human polymorphonuclear leukocytes. Proc Natl Acad Sci USA 1989;86:5610--5614. Gross WL, Hauschild S, Mistry N. The clinical relevance of ANCA in vasculitis. Clin Exp Immunol 1993;93(Suppl 1):7-11. Gross WL, Rasmussen N. Treatment of Wegener's granulomatosis: the view from two nonnephrologists. Nephrol Dial Transplant 1994;9:1219-1222. Gross WL, Csernok E. Immunodiagnostic and pathophysiologic aspects of antineutrophil cytoplasmic antibodies in vasculitis. Curr Opin Rheumatol 1995;7:1--19. Hagen EC, Andrassy K, Csernok E, Daha MR, Gaskin W, Lesavre P, Ludemann J, Rasmussen N, Savage CO, Sinico A, Wiik A, van der Woude FJ. The value of indirect immunofluorescence and solid phase techniques for ANCA detection. A report on the first phase of an international cooperative study on the standardization of ANCA assays. EEC/BCR Group for ANCA Assay Standardization. J Immunol Methods 1993;159:1-16. Handrock K, Gross WL. Relapsing polychondritis as a secondary phenomenon of primary systemic vasculitis. Ann Rheum Dis 1993;52:895--897. Hauschild S, Schmitt WH, Csernok E, Flesch BK, Rautmann A, Gross WL. ANCA in systemic vasculitis, collagen vascular diseases, rheumatic disorders and inflammatory bowel disease. In: Gross WL, ed. ANCA-Associated Vasculitides. London: Plenum Press, 1993:245--251. Hauschild S, Csernok E, Schmitt WH, Gross WL. Antineutrophil cytoplasmic antibodies in systemic polyarteritis nodosa with and without hepatitis B virus infection and Churg-Strauss syndrome-62 patients. J Rheumatol 1994;21: 1173--1174. Jayne DR, Esnault VL, Lockwood CM. ANCA anti-idiotype antibodies and the treatment of systemic vasculitis with intravenous immunoglobulin. J Autoimmun 1993;6:207--219. Jennette JC, Falk RJ. Pathogenic potential of antineutrophil cytoplasmic autoantibodies. Adv Exp Med Biol 1993;336:7-15. Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL, Hagen EC, Hoffman GS, Hunder GG, Kallenberg CG, et al. Nomenclature of systemic vasculitides. Proposal of an International Consensus Conference. Arthritis Rheum 1994;37:187-192. Kao RC, Wehner NG, Skubitz KM, Gray BH, Hoidal JR. Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 1988;82:1963-1973.

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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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-

69

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.

REFERENCES

Elsbach P. Cloning of the cDNA of a human neutrophil bactericidal protein. Structural and functional correlations. J Biol Chem 1989;264:9505--9509. Halbwachs-Mecarelli L, Nusbaum P, Noel LH, Reumaux D, Erlinger S, Grunfeld JP, Lesavre P. Antineutrophil cytoplasmic antibodies (ANCA) directed against cathepsin G in ulcerative colitis, Crohn's disease and primary sclerosing cholangitis. Clin Exp Immunol 1992;90:79--84. Hauschild S, Schmitt WH, Csernok E, Flesch BK, Rautmann A, Gross WL. ANCA in systemic vasculitides, collagen vascular diseases, rheumatic disorders and inflammatory bowel diseases. Adv Exp Med Biol 1993;336:245--251. Heumann D, Gallay P, Betz-Corradin S, Barras C, Baumgartner JD, Glauser MP. Competition between bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein for lipopolysaccharide binding to monocytes. J Infect Dis 1993;167:1351--1357. Iyer S, Lonnerdal B. Lactoferrin, lactoferrin receptors and iron metabolism. Eur J Clin Nutr 1993;47:232--241. Jones SJ, Zhao MH, Elliott JD, Lockwood CM. The antigenicity of bactericidal/permeability-increasing protein (BPI) is threatened by serine proteinases. Proceedings of the Sixth International ANCA Workshop. Paris: 1995. Kain R, Matsui K, Exner M, Binder S, Schaffner G, Sommer EM, Kerjaschki D. A novel class of autoantigens of antineutrophil cytoplasmic antibodies in necrotizing and crescentic glomerulonephritis: the lysosomal membrane glycoprotein h-lamp-2 in neutrophil granulocytes and a related membrane protein in glomerular endothelial cells. J Exp Med 1995; 181 : 585--597. Konstan MW, Byard PJ, Hoppel CL, Davis PB. Effect of highdose ibuprofen in patients with cystic fibrosis. N Engl J Med 1995;332:848-854. Mannion BA, Kalatzis ES, Weiss J, Elsbach P. Preferential binding of the neutrophil cytoplasmic granule-derived bactericidal/permeability-increasing protein to target bacteria. Implications and use as a means of purification. J Immunol 1989; 142:2807-2812. Moodie FD, Leaker B, Cambridge G, Totty NF, Segal AW. Alpha-enolase: a novel cytosoloic autoantigen in ANCA positive vasculitis. Kidney Int 1993;43:675--681. Mulder AH, Horst G, van Leeuwen MA, Limburg PC, Kallenberg CG. Antineutrophil cytoplasmic antibodies in rheumatoid arthritis. Characterisation and clinical correlations.

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.

73


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


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


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


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, 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

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

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.


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

94

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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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

96

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).

97

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

98

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 (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

monoclonal immunoglobulin A(Kappa) factor VIII: C inhibitor associated with primary amyloidosis: identification and characterization. J Lab Clin Med 1988;113:269--277. Green D, Lechner K. A survey of 215 nonhemophilic patients with inhibitors to factor VIII. Thromb Haemostas 1981;45: 200--203. Green D. Cytotoxic suppression of acquired factor VIII:C inhibitors. Am J Med 1991 ;91:14S-- 19S. Green D, Rademaker AW, Briet E. A prospective, randomized trial of prednisone and cyclophosphamide in the treatment of patients with factor VIII autoantibodies. Thromb Haemost 1993;70:753--757. Hauser I, Schneider B, Lechner K. Postpartum factor VIII inhibitors. A review of the literature with special reference to the value of steroid and immunosuppressive treatment. Thromb Haemost 1995;73:1--5. Herbst KD, Rapaport SI, Kenoyer DG, Stanton W, Feinstein DI. Syndrome of an acquired inhibitor to factor VIII responsive to cyclophosphamide and prednisone. Ann Intern Med 1981;95:575--578. Hoyer LW. Immunochemical properties of factor VIII and factor IX inhibitors. Blood Coag Fibrinolysis 1991;2:11S15S. Hultin MB. Acquired inhibitors in malignant and nonmalignant disease states. Am J Med 1991 ;91:9S- 13S. Kasper CK, Aledort LM, Aronson D, Counts R, Edson JR, van Eys J, Fratantoni J, Green D, Hampton J, Hilgartner M, Levine P, Lazerson J, McMillan C, Penner J, Shapiro S, Shulman NR. Proceedings: a more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh 1975;34:612. Kaufman RJ, Wasley LC, Dorner AJ. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 1988;263:6352--6362. Kavanagh ML, Wood CN, Davidson JF. The immunological characterization of human antibodies to factor VIII isolated by immunoaffinity chromatography. Thromb Haemost 1981;45:60-64. Kessler CM. An introduction to factor VIII inhibitors: the detection and quantitation. Am J Med 1991 ;91:1 S-5S. Lamphear B J, Fay PJ. Proteolytic interactions of factor IXa with human factor VIII and factor VIIIa. Blood 1992;80: 3120-3126. Langdell RD, Wagner RH, Brinkhous KM. Effect of antihemophilic factor on one-stage clotting tests: a presumptive test for hemophilia and a simple one-stage antihemophilic

Algiman M, Dietrich G, Nydegger UE, Boieldieu D, Sultan Y, Kazatchkine MD. Natural antibodies to factor VIII (antihemophilic factor) in healthy individuals. Proc Natl Acad Sci USA 1992;89:3795--3799. Berrut G, Shoaay I, Dupuis JM, Maigre M, Fressinaud P, Fressinaud E. Responses of autoimmune factor VIII inhibitors to a combination of cyclophosphamide and prednisone. Blood Coag Fibrinolysis 1994;5:145-146. Burnouf T, Burnouf-Radosevich M, Huart JJ, Goudemand M. A highly purified factor VIII: concentrate prepared from cryoprecipitate by on-exchange chromatography. Vox Sang 1991;60:8--15. Castaldi PA, Penny R. A macroglobulin with inhibitory activity against coagulation factor VIII. Blood 1970;35:370-376. Castenskiold EC, Colvin BT, Kelsey SM. Acquired factor VIII inhibitor associated with chronic interferon-alpha therapy in a patient with haemophilia A. Br J Haematol 1994;87:434-436. Eaton DL, Rodriguez HR, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavage by thrombin, factor Xa and activated protein C with activation and inactivation of FVIII coagulant activity. Biochemistry 1986;25:505-512. Fay PJ, Smudzin TM. Characterization of the interaction between the A2 subunit and A1/A3-C1-C2 dimer in human factor VIIIa. J Biol Chem 1992:267:13246--13250. Fulcher CA, de Graaf Mahoney S, Zimmerman TS. FVIII inhibitor IgG subclass and FVIII polypeptide specificity determined by immunoblotting. Blood 1987;69:1475--1480. Gawryl MS, Hoyer LW. Inactivation of factor VIII coagulant activity by two different types of human antibodies. Blood 1982;60:1103-- 1109. Gilles JG, Arnout J, Vermylen J, Saint-Remy J-M. Anti-Factor VIII antibodies of hemophiliac patients are frequently directed towards nonfunctional determinants and do not exhibit isotypic restriction. Blood 1993;82:2452-2461. Gilles JG, Saint-Remy JM. Healthy subjects produce both antifactor VIII and specific anti-idiotypic antibodies. J Clin Invest 1994:1496-- 1505. Gitschier J, Wood WI, Goralka TM, Wion KL, Chen EY, Eaton DH, Vehar GA, Capon DJ, Lawn RM. Characterization of the human factor VIII gene. Nature 1984;312:326--330. Glueck HI, Coots MC, Benson M, Dwulet FE, Hurtubise PE. A

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factor assay procedure. J Lab Clin Med 1953;41:637--647. Lian EC, Larcada AF, Chiu AY. Combination immunosuppressive therapy after factor VIII infusion for acquired factor VIII inhibitor. Ann Intern Med 1989;11:774-778. Lionnet F, Gouault-Heilmann M, Azoulay C, Lacorte JM, Schaeffer A. Autoimmune factor VIIII: C inhibitor durably responsive to immunoglobulin: a new case. Thromb Haemost 1990;64:488-489. Loftus LS, Arnold WD. Acquired hemophilia in a patient with myeloma. West J Med 1994;160:173--176. Marwaha N, Sarode R, Chauhan AP, Varma JS, Dash S. Simultaneous occurrence of factor VIIIC inhibitor and antinuclear antibody in postpartum period. Am J Hematol 1991;37:49--50. Pittman DD, Marquette KA, Kaufman RJ. Role of the B domain for factor VIII and factor V expression and function. Blood 1994;84:4214-4225. Rizza CR, Biggs R. The treatment of patients who have FactorVIII antibodies. Br J Haematol 1973;24:65. Scandella D, Mattingly M, de Graaf S, Fulcher CA. Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization. Blood 1989;74: 1618--1626. Scandella D, Timmons L, Mattingly M, Trabold N, Hoyer LW. A soluble recombinant factor VIII fragment containing the A2 domain binds to some human anti-Factor VIII antibodies that are not detected by immunoblotting. Thromb Haemost 1992;67:665--671.

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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.


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

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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.

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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.


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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arthritis and osteoarthritis. Arthritis Rheum 1985;28:241--248. Kerwar SS, Oronsky AL. Passive collagen arthritis induced by anticollagen IgG. Int Rev Immunol 1988;4:17--23. Klemperer P, Pollack AD, Baehr G. Diffuse collagen disease. Acute disseminated lupus erythematosus and diffuse scleroderma. JAMA 1942;119:331-334. Lawrence JM, Moore TL, Osborn TG, Nesher G, Madson KL, Kinsella MB. Autoantibody studies in juvenile rheumatoid arthritis. Semin Arthritis Rheum 1993;22:265--274. Lotz M, Vaughan JH. Rheumatoid arthritis. In: Talmage DW, Frank MM, Austin KF, Claman HN, eds. Immunological Disease, Volume II. Boston: Little Brown and Company, 1988;1365--1416. Mackel AM, DeLustro F, Harper FE, LeRoy EC. Antibodies to collagen in scleroderma. Arthritis Rheum 1982;25:522--532. Menzel J, Steffen C, Kolarz G, Kojer M, Smolen J. Demonstration of anticollagen antibodies in rheumatoid arthritis synovial fluids by 14C-radioimmunoassay. Arthritis Rheum 1978;21:243--248. Miller EJ. Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 1972;11:4903--4909. Moreland LW, Gay RE, Gay S. Collagen autoantibodies in patients with vasculitis and systemic lupus erythematosus. Clin Immunol Immunopathol 1991 ;60:412--418. Morgan K, Clague RB, Collins I, Ayad S, Phinn SD, Holt PJ. Incidence of antibodies to native and denatured cartilage collagens (types II, IX, and XI) and to type I collagen in rheumatoid arthritis. Ann Rheum Dis 1987;46:902--907. Morgan K, Buckee C, Collins I, Ayad S, Clague RB, Holt PJ. Antibodies to type II and XI collagens: evidence for the formation of antigen specific as well as cross reacting antibodies in patients with rheumatoid arthritis. Ann Rheum Dis 1988;47:1008--1013. Morgan K, Clague RB, Collins I, Ayad S, Phinn SD, Holt JL. A longitudinal study of anticollagen antibodies in patients with rheumatoid arthritis. Arthritis Rheum 1989;32:139--145. Morgan K. What do anticollagen antibodies mean? Ann Rheum Dis 1990;49:62--65. Morgan K, Clague RB, Reynolds I, Davis M. Antibodies to type II collagen in early rheumatoid arthritis. Br J Rheumatol 1993;32:333--335. Moro L, Smith BD. Identification of collagen alphal(I) trimer and normal type I collagen in a polyomavirus-induced mouse tumor. Arch Biochem Biophys 1977;182:33--41. Mundlos S, Spranger J. Genetic disorders of connective tissues. Curr Opin Rheumatol 1991 ;3:832-837. Petty RE, Hunt DW, Rosenberg AM. Antibodies to type IV collagen in rheumatic diseases. J Rheumatol 1986;13:246253. Poole AB, Glant TT, Mikecz K. Autoimmunity to cartilage collagen and proteoglycan and the development of chronic inflammatory arthritis. In: Dingle JT, Gordon JL, eds. Research Monographs in Cell and Tissue Physiology, Volume 15. New York: Elsevier, 1988:55-65. Ronnelid J, Lysholm J, Engstrom-Laurent A, Klareskog L, Heyman B. Local antitype II collagen antibody production in

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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.

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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.

REFERENCES

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Abdelmoula M, Spertini F, Shibata T, Gyotoku Y, Luzuy S, Lambert PH, Izui S. IgG3 is the major source of cryoglobulins in mice. J Immunol 1989;143:526-564. Abdou NI, Wall H, Lindsley HB, Halsey JF, Suzuki T. Network theory in autoimmunity. In vitro suppression of serum anti-DNA antibody binding to DNA by anti-idiotypic antibody in systemic lupus erythematosus. J Clin Invest 1981 ;67:1297-1304. Abe T, Takeuchi T, Kiyotaki M, Koide J, Hosono O, Homma M, Otake T, Kano S. Anti-idiotypic antibodies in a patient with monoclonal rheumatoid factor after pneumococcal bacteremia. J Immunol 1984;132:2381-2385. Blank M, Manosroi J, Tomer Y, Manosroi A, Kopolovic J, Charcon-Polak S, Shoenfeld Y. Suppression of experimental systemic lupus erythematosus (SLE) with specific antiidiotypic antibody-saporin conjugate. Clin Exp Immunol 1994 ;98:434--441. Bona C, Paul WE. Cellular basis of regulation of expression of idiotype. I. T-suppressor cells specific for MOPC 460 idiotype regulate the expression of cells secreting anti-TNP antibodies beating 460 idiotype. J Exp Med 1979;149:592-600. Bona CA, Victor-Kobrin C, Manheimer AJ, Bellon B, Rubinstein LJ. Regulatory arms of the immune network. Immunol Rev 1984;79:25--44. Bonomo L, Casato M, Afeltra A, Caccavo D. Treatment of idiopathic mixed cryoglobulinemia with alpha interferon. Am J Med 1987;83:726-730. 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. Brown JC, Rodkey LS. Autoregulation of an antibody response via network-induced auto-anti-idiotype. J Exp Med 1979; 150:67--85. Chen PP, Gofii F, Houghten RA, Fong S, Goldfien R, Vaughan JH, Frangione B, Carson DA. Characterization of human rheumatoid factors with seven antiidiotypes induced by synthetic hypervariable region peptides. J Exp Med 1985; 162:487-500. D'Amico G, Ferrario F, Colasanti G, Bucci A. Glomerulonephritis in essential mixed cryoglobulinemia (EMC). In: Davison PJ, Giullou PJ, eds. Proceedings of the XXI congress of the European Dialysis and Transplant Association. London: Pitman, 1984:527--548. Dammacco F, Sansonno D. Antibodies to hepatitis C virus in

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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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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

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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.


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

90%

*MAG-negative refers to the 50% of cases of IgM paraproteinemic neuropathy who are anti-MAG/anti-SGPG antibody-negative.

281

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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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

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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.


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 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-

REFERENCES Avila JL, Rojas M, Carrasco H. Elevated levels of antibodies against sulphatide are present in all chronic chagasic and dilated cardiomyopathy sera. Clin Exp Immunol 1993;92: 460-465. Brouet JC, Mariette X, Chevalier A, Hauttecouer B. Determination of the affinity of monoclonal human IgM for myelinassociated glycoprotein and sulfated glucuronic paragloboside. J Neuroimmunol 1992;36:209-215. Costallat LT, de Oliveira RM, Santiago MB, Cossermelli W, Samara AM. Neuropsychiatric manifestations of systemic lupus erythematosus: the value of anticardiolipin, antigangliosides and antigalactocerebrosides antibodies. Clin Rheum 1990;9:489--497. Cunningham MT, Olson JD, Koerner TA. Glycosphingolipid inhibition of the adhesion of thrombin-activated platelets to surfaces is potentiated by albumin. Glycobiology 1993;3: 331--337. Garci~i-Monc6 JC, Wheeler CM, Benach JL, Furie RA, Lukehart SA, Stanek G, Steere AC. Reactivity of neuroborreliosis patients (Lyme disease) to cardiolipin and gangliosides. J Neurol Sci 1993;117:206--214. Hakomori SI, Murakami WT. Glycolipids of hamster fibroblasts and derived malignant-transformed cell lines. Proc Natl Acad Sci USA 1968;59:254-261. Hakomori SI. Glycosphingolipids in cellular interaction, differentiation, and oncogenesis. Annu Rev Biochem 1981; 50:733--764. Hakomori SI. Chemistry of glycosphingolipids. In: Kanfer JN, Hakomori S, eds. Handbook of Lipid Research, Volume 3. Sphingolipid Biochemistry. New York: Plenum Press, 1983:1-165.

motor neuropathies such as sensory polyneuropathy and chronic inflammatory demyelinating polyradiculoneuropathy (Hughes, 1994). Other glycolipid autoantigens of interest include blood group A chain glycolipids, sulfolactoceramides, globosides, sphingomyelin and psychosine. The utility of many glycolipid-specific antibodies, particularly those found in cancer and hematologic disorders, is not well established. Expanded panels of glycolipid autoantigens may improve the differential diagnoses of motor neuropathy diseases such as lower motor neuron disease, multifocal motor neuropathy, amyotrophic lateral sclerosis and other demyelinating neuropathies. Antiglycolipid antibodies, especially antisulfatide, antitumor, and antiblood group glycolipids are clinically important for understanding the etiopathogenesis of autoimmunity and cancer. They also are useful in aiding diagnoses of neuroinfections and neurologic overlap syndromes, and in differentiating neuropathy subtypes and types of neuropsychiatric involvement. See also GANGLIOSIDE AUTOANTIBODIES.

Hakomori SI, Young WW Jr. Glycolipid antigens and genetic markers. In: Kanfer JN, Hakomori S, eds. Handbook of Lipid Research, Volume 3. Sphingolipid Biochemistry. New York: Plenum Press, 1983:381-436. Harada R, Takahashi N, Owaki I, Kannagi R, Endo N, Morita N, Inoue M. Study of anti-idiotype antibodies to human monoclonal antibody. Igaku Kenkyu 1992;62:1--18. Hughes RA. Inflammatory neuropathies. Bailli~res Clin Neurol 1994;3:45--72. Ishizuka I, Yamakawa T. Glycoglycerolipids. In: Weigandt H, ed. New Comprehensive Biochemistry, Volume 10. Glycolipids. Amsterdam: Elsevier, 1985:101-197. IUPAC-IUB Commission on Biochemical Nomenclature. The nomenclature of lipids. Eur J Biochem 1977;79:11--21. Kaise S, Yasuda T, Kasukawa R, Nishimaki T, Watarai S, Tsumita T. Antiglycolipid antibodies in normal and pathologic human sera and synovial fluids. Vox Sang 1985;49: 292--300. Kannagi R, Levery SB, Hakomori S. Sequential change of carbohydrate antigen associated with differentiation of murine leukemia cells: i-I antigenic conversion and shifting of glycolipid synthesis. Proc Natl Acad Sci USA 1983;80: 2844-2848. Kasai N, Pachner AR, Yu RK. Antiglycolipid antibodies and their immune complexes in multiple sclerosis. J Neurol Sci 1986;75:33-42. Kaye EM, Ullman MD, Kolodny EH, Krivit W, Rischert JC. Possible use of CSF glycosphingolipids for the diagnosis and therapeutic monitoring of lysosomal storage disease. Neurology 1992;42:2290--2294. Kirschning E, Rutter G, Uhlig H, Dernick R. A sulfatidereactive human monoclonal antibody obtained from a

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multiple sclerosis patient selectively binds to the surface of oligodendrocytes. J Neuroimmunol 1995;56:191-200. Ladisch S, Li R, Olson E. Ceramide structure predicts tumor ganglioside immunosuppressive activity. Proc Natl Acad Sci USA 1994;91:1974-1978. Lichey J, Zuberbier T, Luck W, Lau S, Wahn U. Influence of glycosphingolipids on the release of histamine and sulfidopeptide leukotrienes from human basophils. Int Arch Allergy Immunol 1994;103:252--259. Lindstrom K, Von Dem Borne AE, Breimer ME, Cedergren B, Okubo Y, Rydberg L, Teneberg S, Samuelsson BE. Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja antibodies. Glycoconj J 1992;9:325-329. Matsuda K, Taki T, Hamanaka S, Kasama T, Rokukawa C, Handa S, Yamamoto N. Glycosphingolipid compositions of human T-lymphotropic virus type I (HTLV-I) and human immunodeficiency virus (HIV)-infected cell lines. Biochim Biophys Acta 1993;1168:123-129. Minasian LM, Szatrowski TP, Rosenblum M, Steffans T, Morrison ME, Chapman PB, Williams L, Nathan CF, Houghton AN. Hemorrhagic tumor necrosis during a pilot trial of tumor necrosis factor alpha and anti-GD3 ganglioside monoclonal antibody in patients with metastatic melanoma. Blood 1994;83:56--64. Miyake M, Taki T, Kannagi R, Hitomi S. First establishment of a human monoclonal antibody directed to sulfated glycosphingolipids SM4s_Ga1 and SM4g from a patient with lung cancer. Cancer Res 1992;52:2292--2297. Miyatani N, Saito M, Ariga T, Yoshino H, Yu RK. Glycosphingolipids in the cerebrospinal fluid of patients with multiple sclerosis. Mol Chem Neuropathol 1990; 13:205--216. Murakami H, Lam Z, Furie BC, Reinhold VN, Asano T, Furie B. Sulfated glycolipids are the platelet autoantigens for human platelet-binding monoclonal anti-DNA autoantibodies. J Biol Chem 1991;266:15414-15419. Needham LK, Schnaar RL. Carbohydrate recognition in the peripheral nervous system: a calcium-dependent membrane binding site for HNK-1 reactive glycolipids potentially involved in Schwann cell adhesion. J Biol Chem 1993;121: 397-408. Pathak S, Illavia SJ, Khalili-Shirazi A, Webb HE. Immunoelectron microscopical labelling of a glycolipid in the envelopes of brain cell-derived budding viruses, Semliki Forest, influenza and measles, using a monoclonal antibody directed chiefly against galactocerebroside resulting from Semliki Forest virus infection. J Neurol Sci 1990;96:293302.

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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


golgin 97

97

glomerulonephritis

coiled-coil

n/s


golgin 180

180

SjOgren's syndrome

coiled-coil

n/s


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, 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

345

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-

346

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

347

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

348

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.

REFERENCES Amiral J, Bridey F, Dreyfus M, Vissoc AM, Fressinaud E, Wolf M, Meyer D. Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia. Thromb Haemost 1992;68:95--96. Amiral J, Bridey F, Wolf M, Boyer-Neuman C, Fressinaud E, Vissac AM, Peynaud-Debayle E, Dreyfus M, Meyer D. Antibodies to macromolecular platelet factor 4-heparin complexes in heparin-induced thrombocytopenia: a study of 44 cases. Thromb Haemost 1995;73:21--28. Aotsuka S, Okawa-Takatsuji M, Kinoshita M, Yokohari R. Analysis of negatively charged dye-binding antibodies reactive with double-stranded DNA and heparin sulfate in serum from patients with rheumatic diseases. Clin Exp Immunol 1988;73:436--442. Arepally G, et al. Comparison of the 14C-serotonin release assay with the PF4/heparin ELISA in heparin-induced thrombocytopenia. Am J Clin Pathol 1995;in press. Bell WR, Tomasulo PA, Alving BM, Duffy TP. Thrombocytopenia occurring during the administration of heparin. A prospective study in 52 patients. Ann Intern Med 1976;85: 155--160. Blajchman MA, Austin RC, Fernandez-Rachubinski F, Sheffield WP. Molecular basis of inherited antithrombin deficiency. Blood 1992;80:2159--2171. Boshkov LK, Warkentin TE, Hayward CP, Andrew M, Kelton JG. Heparin-induced thrombocytopenia and thrombosis: clinical and laboratory studies. Br J Haematol 1993;84:322-328. Capitanio AM, Niewiarowski S, Rucinski B, Tuszynski GP, Cierniewski CS, Hershock D, Kornecki E. Interaction of platelet factor 4 with human platelets. Biochim Biophys Acta 1985;839:161--173. Chong BH. Heparin-induced thrombocytopenia. Br J Haematol 1995;89:431--439. Chong BH, Grace CS, Rozenberg MC. Heparin-induced thrombocytopenia: effect of heparin platelet antibody on platelets. Br J Haematol 1981;49:531--540. Cines DB, Kaywin P, Bina M, Tomaski A, Schreiber AD. Heparin-associated thrombocytopenia. N Engl J Med 1980; 303:788-795. Cines DB, Tomaski A, Tannenbaum S. Immune endothelial-cell injury in heparin-associated thrombocytopenia. N Engl J Med 1987;316:581--589. Conley CL, Hartmann RC, Lalley JS. The relationship of heparin activity to platelet concentration. Proc Soc Exp Biol Med 1948;69:284--287. Coon WW, Willis PW 3rd. Some side effects of heparin, heparinoids, and their antagonists. Clin Pharmacol Ther 1966;7:379-398. Deckmyn H, De Reys S. Functional effects of human antiplatelet antibodies. Semin Thromb Hemost 1995;21:46--59. Denomme GA, Warkentin TE, Horsewood P, Smith JW, Hayward CP, Kelton JG. Evaluation of the Fc receptor IIa genotype frequencies among patients with heparin induced thrombocytopenia. Blood 1994;84:661. Denton J, Lane DA, Thunberg L, Slater AM, Lindahl U.

Binding of platelet factor 4 to heparin oligosaccharides. Biochem J 1983;209:455--460. Files JC, Malpass TW, Yee EK, Ritchie JL, Harker LA. Studies of human platelet s-Granule release in vivo. Blood 1981;58: 607--618. Fratantoni JC, Pollet R, Gralnick HR. Heparin-induced thrombocytopenia: confirmation of diagnosis with in vitro methods. Blood 1975;45:395--401. Greinacher A, Alban S, Dummel V, Franz G, Mueller-Eckhardt C. Characterization of the structural requirements for a carbohydrate-based anticoagulant with a reduced risk of inducing the immunological type of heparin-associated thrombocytopenia. Thromb Haemost 1995;74:886-892. Greinacher A, Amiral J, Dummel V, Vissac A, Kiefel V, Mueller-Eckhardt C. Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of platelet aggregation test, heparin-induced platelet activation test, and platelet factor 4/heparin enzyme-linked immunosorbent assay. Transfusion 1994a;34:381-385. Greinacher A, Liebenhoff U, Kiefel V, Presek P, MuellerEckhardt C. Heparin-associated thrombocytopenia: the effects of various intravenous IgG preparations on antibody mediated platelet activation- a possible new indication for high dose i.v. IgG. Thromb Haemost 1994b;71:641-645. Greinacher A, Potzsch B, Amiral J, Dummel V, Eichner A, Mueller-Eckhardt C. Heparin-associated thrombocytopenia: isolation of the antibody and characterization of a multimolecular PF4-heparin complex as the major antigen. Thromb Haemost 1994c;71:247-251. Greinacher A, Michels I, Mueller-Eckhardt C. Heparin-associated thrombocytopenia: the antibody is not heparin specific. Thromb Haemost 1992;67:545--549. Handin RI, Cohen HJ. Purification and binding properties of human platelet factor four. J Biol Chem 1976;251:42734282. Hirsh J, Dalen JE, Deykin D, Poller L. Heparin: mechanism of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and safety. Chest 1992;102:$337-$351. Jacobsson KG, Riesenfeld J, Lindahl U. Biosynthesis of heparin. Effects of n-butyrate on cultured mast cells. J Biol Chem 1985;260:12154-12159. Kelton JG, Sheridan D, Santos A, Smith J, Steeves K, Brown C, Murphy WG. Heparin-induced thrombocytopenia: laboratory studies. Blood 1988;72:925--930. Klein HG, Bell WR. Disseminated intravascular coagulation during heparin therapy. Ann Intern Med 1974;80:477--481. Kosfeld RE, Lansing AM, Masri Z, Liu YK. Heparin-induced thrombocytopenia and recurrent thromboembolism in siblings. Am J Hematol 1985;18:421--423. Laster J, Elfrink R, Silver D. Re-exposure to heparin of patients with heparin-associated antibodies. J Vasc Surg 1989;9:677681. Linhardt RJ, Ampofo SA, Fareed J, Hoppensteadt D, Mulliken JB, Folkman J. Isolation and characterization of human heparin. Biochemistry 1992;31:12441--12445. Maccarana M, Lindahl U. Mode of interaction between platelet factor 4 and heparin. Glycobiology 1993;3:271-277. Mast AE, Enghild JJ, Pizzo SV, Salvesen G. Analysis of the

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plasma elimination kinetics and conformational stabilities of native, proteinase-complexed, and reactive site cleaved serpins: comparison of ~l-proteinase inhibitor, c~2-antichymotrypsin, antithrombin III, c~2-antiplasmin, angiotensinogen, and ovalbumin. Biochemistry 1991;30:1723-1730. Park KS, Rifat S, Eck H, Adachi K, Surrey S, Poncz M. Biologic and biochemic properties of recombinant platelet factor 4 demonstrate identity with the native protein. Blood 1990;75:1290-1295. Roberts B, Rosato FE, Rosato EF. Heparin- a cause of arterial emboli? Surgery 1964;55:803--808. Rosenberg RD, Bauer KA. The heparin-antithrombin system: a natural anticoagulant mechanism. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Philadelphia: J.B. Lippincott Company, 1994:837-860. Schmitt BP, Adelman B. Heparin-associated thrombocytopenia: a critical review and pooled analysis. Am J Med Sci 1993; 305:208--215. Sheridan D, Carter C, Kelton JG. A diagnostic test for heparininduced thrombocytopenia. Blood 1986;67:27-30. Stuckey JA, St. Charles R, Edwards BF. A model of the platelet factor 4 complex with heparin. Proteins 1992;14:277-287. Visentin GP, Ford SE, Scott JP, Aster RH. Antibodies from patients with heparin-induced thrombocytopenia/thrombosis

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are specific for platelet factor 4 complexed with heparin or bound to endothelial cells. J Clin Invest 1994a;93:81-88. Visentin GP, Malik MI, Menden K, Aster RH. A prospective study of the formation of antibodies reactive with heparin: PF4 complexes in patients treated with heparin. Blood 1994b;84:80. Walton PL, Ricketts CR, Bangham DR. Heterogeneity of heparin. Br J Haematol 1966;12:310-325. Warkentin TE, Kelton JG. Heparin-induced thrombocytopenia. Annu Rev Med 1989;40:31--44. Warkentin TE, Levine MN, Hirsh J, Horsewood P, Roberts RS, Gent M, Kelton JG. Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. N Engl J Med 1995;332:1330-1335. Wolf H, Nowack H, Wick G. Detection of antibodies interacting with glycosaminoglycan polysulfate in patients treated with heparin or other polysulfated glycosaminoglycans. Int Arch Allergy Appl Immunol 1983;70:157--163. Zhang X, Chen L, Bancroft DP, Lai CK, Maione TE. Crystal structure of recombinant human platelet factor 4. Biochemistry 1994;33:8361-8366. Zucker MB, Katz IR. Platelet factor 4: production, structure, and physiologic and immunologic action. Proc Soc Exp Biol Med 1991;198:693--702.


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





-

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.


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 0.10).

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.

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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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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

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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

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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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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 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).

REFERENCES Czaja AJ. Autoimmune hepatitis. Evolving concepts and treatment strategies. Dig Dis Sci 1995;40:435-456. Gerken G, Manns M, Ramadori G, Poralla T, Dienes HP, Meyer zum Btischenfelde KH. Liver membrane antibodies in chronic active hepatitis. Studies on mechanically and enzymatically isolated rabbit hepatocytes. J Hepatol 1987;5: 65--74. Harford J, Ashwell G. The hepatic receptor for asialoglycoproteins. In: Horowitz MI, ed. The Glycoconjugates. New York: Academic Press, 1982;4:27-55. Hopf U, Meyer zum Btischenfelde KH, Arnold W. Detection of a liver-membrane autoantibody in HBsAg-negative chronic hepatitis. N Engl J Med 1976;294:587--582. Hopf U, Jahn H-U, Moiler B, Stemerowicz R, Writtenbrink C, Klein R, Berg PA. Liver membrane antibodies (LMA) recognize a 26 kD protein on the hepatocellular surface. Clin Exp Immunol 1990;79:54--61. Huppertz HI, Treichel U, Gassel AM, Jeschke R, Meyer zum Btischenfelde KH. Autoimmune hepatitis following hepatitis A virus infection. J Hepatol 1995;23:204--208. Johnson PJ, McFarlane IG. Meeting report: International Autoimmune Hepatitis Group. Hepatology 1993;18:998-1005.

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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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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

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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.

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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.

477


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.

483


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 (p95%) 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% .

(,~

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.

599


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

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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-

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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

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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.


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.

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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

612

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

614

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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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.,

618

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


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.


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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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

625

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.

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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-

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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


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

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

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

658

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.

659


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

662

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.


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

REFERENCES Aho K, Steiner G, Kurki P, Paimela L, Leirisalo-Repo M, Palosup T, Smolen JS. Anti-RA33 as a marker antibody of rheumatoid arthritis in Finnish population. Clin Exp Rheumatol 1993;11:645--647.

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.

Astaldi-Ricotti GC, Bestagno M, Cerino A, Negri R, Caporali R, Cobianchi F, Longhi M, Maurizio Montecucco C. Antibodies to hnRNP core protein A1 in connective tissue diseases. J Cell Biochem 1989;40:1--5. Barnett SF, Northington SJ, LeSturgeon WM. Isolation and in vitro assembly of nuclear ribonucleoprotein particles and

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purification of core protein particles. Methods Enzymol 1990; 181:293--307. Beyer AL, Christensen ME, Walker BW, LeStourgeon WM. Identification and. characterization of the packaging proteins of core 40S hnRNP particles. Cell 1977;11:127-138. Biamonti G, Ruggiu M, Saccone S, Della Valle G, Riva S. Two homologous genes, originated by duplication, encode the human hnRNP proteins A2 and A1. Nucleic Acids Res 1994 ;22:1996--2002. Burd CG, Swanson MS, Gorlach M, Dreyfuss G. Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B 1, and C2 proteins: A diversity of RNA binding proteins is generated by small peptide inserts. Proc Natl Acad Sci USA 1989;86"9788--9792. Buvoli M, Cobianchi F, Bestagno MG, Mangiarotti A, Bassi MT, Biamonti G, Riva S. Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein. EMBO J 1990;9:1229--1235. Cobianchi F, SenGupta DN, Zmudzka BZ, Wilson SH. Structure of rodent helix-destabilizing protein revealed by cDNA cloning. J Biol Chem 1986;261:3536-3543. Cobianchi F, Karpel RL, Williams KR, Notario V, Wilson SH. Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. J Biol Chem 1988;263:1063-1071. Cordonnier C, Meyer O, Palazzo E, de Brandt M, Hayem G, Elias A, Nicaise P, Haim T, Kahn M-F. A prospective longitudinal study of anti-RA 33, antikeratin (AKA) antibodies, antiperinuclear factor (APF) and antinuclear antibodies (ANA) as early serological markers of rheumatoid arthritis (RA) (Abstract). Arthritis Rheum 1994;37(Suppl)" $329. Dangli A, Guialis A, Vretou E, Sekeris CE. Autoantibodies to the core proteins of hnRNPs. FEBS Lett 1988;231" 118--124. Dreyfuss G, Matunis MJ, Pifiol-Roma S, Burd C. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem 1993;62:289--321. Hassfeld W, Steiner G, Hartmuth K, Kolarz G, Scherak O, Graninger W, Thumb N, Smolen JS. Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis. Arthritis Rheum 1989;32:1515--1520. Hassfeld W, Steiner G, Smolen JS. Anti-RA33: a new antinuclear antibody in rheumatoid arthritis. In: Smolen JS, Kalden JR, Maini RN, eds. Rheumatoid Arthritis- Recent Research Advances. Berlin: Springer-Verlag, 1992:319--327. Hassfeld W, Steiner G, Graninger W, Witzmann G, Schweitzer H, Smolen JS. Autoantibody to the nuclear antigen RA33: a marker for early rheumatoid arthritis. Br J Rheumatol 1993;32:199--203. Hassfeld W, Steiner G, Studnicka-Benke A, Skriner K, Graninger W, Fischer I, Smolen JS. Autoimmune response to the spliceosome. An immunological link between rheumatoid arthritis, mixed connective tissue disease and systemic lupus erythematosus. Arthritis Rheum 1995;38:777-785. Isenberg DA, Steiner G, Smolen JS. Clinical utility and serological connections of anti-RA33 antibodies in systemic lupus erythematosus. J Rheumatol 1994;21"1260-1263. Jensen LA, Kuff EL, Wilson SH, Steinberg AD, Klinman DM. Antibodies from patients and mice with autoimmune diseases

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react with recombinant hnRNP core protein A1. J Autoimmun 1988;1:73-83. Kumar A, Williams KR, Szer W. Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins. J Biol Chem 1986;261:11266-11273. Lamm GM, Lamond AI. Non-snRNP protein splicing factors. Biochim Biophys Acta 1993;1173:247--265. Lt~hrmann RL, Kastner B, Bach M. Structure of spliceosomal RNPs and their role in pre-mRNA splicing. Biochim Biophys Acta 1990:1087:265--292. Matunis EL, Matunis MJ, Dreyfuss G. Characterization of the major hnRNP proteins from Drosophila melanogaster. J Cell Biol 1992;116:257--269. Mayeda A, Krainer AR. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell 1992; 68:365-375. Mayeda A, Munroe SH, Caceres JF, Krainer AR. Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J 1994;15:5483--5495. Meyer O, Tauxe F, Fabregas D, Gabay C, Goycochea M, Haim T, Elias A, Kahn MF. Anti-RA33 antinuclear autoantibody in rheumatoid arthritis and mixed connective tissue disease: comparison with antikeratin and antiperinuclear antibodies. Clin Exp Rheumatol 1993;11:473--478. Montecucco C, Caporali R, Negri C, de Gennaro F, Cerino A, Bestagno M, Cobianchi F, Astaldi-Ricotti GC. Antibodies from patients with rheumatoid arthritis and systemic lupus erythematosus recognize different epitopes of a single heterogeneous nuclear RNP core protein. Arthritis Rheum 1990;33:180-186. Pifiol-Roma S, Choi YD, Matunis MJ, Dreyfuss G. Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. Genes Dev 1988;2:215--227. Pifiol-Roma S, Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature 1992;355: 730-732. Riva S, Morandi C, Tsoulfas P, Pandolfo M, Biamonti G, Merrill B, Williams KR, Multhaup G, Beyreuther K, Werr H, Henrich B, Sch~fer KP. Mammalian single-stranded DNA binding protein UP 1 is derived from the hnRNP core protein A1. EMBO J 1986;5:2267--2273. Sharp PA. Split genes and RNA splicing. Cell 1994;77:805-815. Skriner K, Steiner G, Sommergruber WH, Sinski A, Smolen JS. Anti-RA33 autoantibodies may recognize epitopes in the Nterminal region of hnRNP-A2 (RA33)'. Clin Exp Rheum 1994a;12(Suppl 11):79--82. Skriner K, Steiner G, Sommergruber WH, Smolen JS. Epitope Mapping of RA33/hnRNP-A2: a major epitope is located in the second RNA binding domain [Abstract]. Arthritis Rheum 1994b;37(Suppl):S393. Steiner G, Hartmuth K, Skriner K, Maurer-Fogy I, Sinski A, Thalman E, Hassfeld W, Barta A, Smolen JS. Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex. J Clin

Invest 1992;90:1061-- 1066. Steiner G, Skriner K, Sommergruber WH, Smolen J. RA33 the A2 protein of the heterogeneous nuclear ribonucleoprotein: ELISA and epitope mapping [Abstract]. Arthritis Rheum 1993 ;36(Suppl):D64. Steiner G. The A2 protein of the heterogeneous nuclear ribonucleoprotein. In: Maini RN, van Venrooij W, eds. Handbook of Biological Markers of Disease. Amsterdam: Kluwer Academic Publishers, 1994:B. 1.3:1--9. van Venrooij WJ, Sillekens PTG. Small nuclear RNA associated proteins: autoantigens in connective tissue diseases.

Clin Exp Rheumatol 1989;7:635--645. Wilk HE, Werr H, Friedrich D, Kiltz HH, Schaefer KP. The core proteins of 35S hnRNP complex: characterization of nine different species. Eur J Biochem 1985;146:71-81. Williams RC Jr, Byres LC, Malone CC, Scornik JC, Steiner G, Lindstr6m FD. Studies on identical twins discordant for rheumatoid arthritis. Submitted. Yang X, Bani MR, Lu SJ, Rowan S, Ben-David Y, Chabot B. The A1 and A1B proteins of heterogeneous nuclear ribonucleoprotein particles modulate 5' splice site selection in vivo. Proc Natl Acad Sci USA 1994;91:6924-6928.

667


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.

glycosylated proteins. J Biol Chem 1995;270:1543--1549. 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. Proc Natl Acad Sci USA 1988;85:7317-7321. D'Arpa P, Machlin PS, Rattle H 3rd, Rothfield NF, Cleveland DW, Earnshaw WC. cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl Acad Sci USA 1988;85:2543--2547. de Rooij DJ, Habets WJ, van de Putte LB, Hoet MH, Verbeek AL, van Venrooij WJ. Use of recombinant RNP peptides 70K and A in an ELISA for measurement of antibodies in mixed connective tissue disease: a longitudinal follow up of 18 patients. Ann Rheum Dis 1990;49:391--395. 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. Deutscher SL, Harley JB, Keene JD. Molecular analysis of the 60-kDa human Ro ribonucleoprotein. Proc Natl Acad Sci USA 1988;85:9479-9483. Earnshaw WC, Machlin PS, Bordwell BJ, Rothfield NF, Cleveland DW. Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B. Proc Natl Acad Sci USA 1987;84:4979--4983. Elkon KB, Hines JJ, Chu JL, Parnassa A. Epitope mapping of recombinant HeLa SmB and B' peptides obtained by the

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combinant proteins. J Immunol Methods 1992;151:177--189. Verheijen R, de Jong BA, van Venrooij WJ. A recombinant topoisomerase I ELISA: screening for IgG, IgM, and IgA antitopo I autoantibodies in human sera. Clin Exp Immunol 1992;89:456--460. Wagatsuma M, Asami N, Miyachi J, Uchida S, Watanabe H, Amann E. Antibody recognition of the recombinant human nuclear antigens RNP 70 kD, SS-A, SS-B and Sm-D by autoimmune sera. Mol Immunol 1993;30:1491-1498. Yamamoto K, Miura H, Moroi Y, Yoshinoya S, Goto M, Nishioka K, Miyamoto T. Isolation and characterization of a complementary DNA expressing human U1 small nuclear ribonucleoprotein C polypeptide. J Immunol 1988;140:311317.


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


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

REFERENCES

Abdel-Khalik A, Paton L, White AG, Urbaniak SJ. Human leucocyte antigens A, B, C and DRW in idiopathic "warm" autoimmune haemolytic anaemia. Br Med J 1980;280:760-761. Cartron JP, Agre P. Rh blood groups and Rh-deficiency syndrome. In: Cartron JP, Rouger P, eds. Blood Cell Biochemistry. Molecular Basis of Major Human Blood Group Antigens. New York: Plenum Press, 1995;6:189-225. Donath J, Landsteiner K. Ueber paroxysmale Hamoglobinurie. Muench Med Wschr 1904;51:1590--1593. Engelfriet CP, Overbeeke MA, von dem Borne AE. Autoimmune hemolytic anemia. Semin Hematol 1992;29:3--12. Feizi T, Kunkel HG, Roelcke D. Cross idiotypic specificity among cold agglutinins in relation to combining activity for blood group-related antigens. Clin Exp Immunol 1974;18: 283-293. Garratty G. Autoimmune hemolytic anemia. In: Garratty G, ed. Immunobiology of Transfusion Medicine. New York: Marcel Dekker, 1994;18:493--521. Hawkes CH, Nourse CH. Familial autoimmune haemolytic anaemia. Br Med J 1977;1:1392--1395.

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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


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).

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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

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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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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).

694

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).

695

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-

696

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

697

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.

REFERENCES

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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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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

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(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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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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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

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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#

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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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.


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.

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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

786

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.


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

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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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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.


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)

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


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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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.

829


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.

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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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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

838

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

840

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.

841


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


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 (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



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)



Centromere autoantibodies

CENPs A, B and C, centromererelated proteins

SSc (30%) RA and SLE ( 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%)



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 (

Autoantibodies

Autoantibodies and Autoimmunity: Molecular Mechanisms in Health and Disease

Autoantibodies and Autoimmunity: Molecular Mechanisms in Health and Disease

Autoantibodies

Autoantibodies and Autoimmunity: Molecular Mechanisms in Health and Disease

Autoantibodies and Autoimmunity: Molecular Mechanisms in Health and Disease

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